WO2025054202A1

WO2025054202A1 - Method of screening a sample comprising a transgene with a unique barcode

Info

Publication number: WO2025054202A1
Application number: PCT/US2024/045198
Authority: WO
Inventors: Adam James JOHNSON; Christie Anne CIARLO
Original assignee: Sana Biotechnology Inc
Current assignee: Sana Biotechnology Inc
Priority date: 2023-09-05
Filing date: 2024-09-04
Publication date: 2025-03-13
Anticipated expiration: 2026-03-05

Abstract

The present disclosure provides methods to detect a barcode sequence(s), wherein the barcode sequence indicates the presence of a transgene inserted into a cell or population of cells, methods of screening a sample for said barcode sequence(s), cells or populations of cells comprising one or more vectors encoding said transgene(s) and barcode(s), compositions thereof, and therapeutic uses and kits related thereto.

Description

MULTIPLEX METHOD OF SCREENING A POPULATION OF CELLS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of, and priority to, U.S. Provisional Application 63/580,663, filed on September 5, 2023, the contents of which is hereby incorporated herein by reference in its entirety.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (186152007640SEQLIST.xml; Size: 226,639 bytes; and Date of Creation: August 30, 2024) is herein incorporated by reference in its entirety.

FIELD

[0003] Provided herein is a method of screening a sample comprising one or more transgenes each with a unique barcode which are inserted into a vector for expression and subsequent detection, as well as compositions, methods of treatment, methods of monitoring, and kits related thereto.

BACKGROUND

[0004] The ability to express nucleic acids and proteins of interest in a reliable and safe manner in clinically relevant cells and tissues is key for the successful development of cell and gene therapies. Methods for characterizing or otherwise detecting the presence of nucleic acids and proteins of interest within a patient can include methods such as microscopic imaging, molecular analysis, gene expression analysis, and/or immunocytochemistry. These methods are often time-consuming, technically challenging, expensive, and low throughput. Accordingly, there is an unmet need for developing safe, efficient, and cost-effective methods for screening for such nucleic acids and proteins of interest used in cell and gene therapies administered to patients.

SUMMARY

[0005] In one aspect provided herein is a method of selecting a cell, population of cells, or therapy comprising detecting the presence or absence of a first barcode and/or a second barcode in the cell or population of cells, wherein the presence of the first barcode indicates the presence of a first transgene and the presence of the second barcode indicates the presence of a second transgene, and selecting the cell or population of cells as being suitable for: a) administration to a subject; b) manufacturing a drug product; c) gene editing or further gene editing; d) viral transduction or further viral transduction; e) creating a cell bank; f) differentiation into a cellular intermediate or a fully differentiated drug product; g) packaging for distribution; and/or h) cryopreservation or formulation, based on the presence or absence of the first barcode and/or second barcode.

[0006] In another aspect provided herein is a method of manufacturing a therapy, the method comprising detecting the presence or absence of a first barcode and/or a second barcode in a cell or population of cells wherein the presence of the first barcode indicates the presence of a first transgene and the presence of the second barcode indicates the presence of a second transgene, and wherein the therapy is determined to be suitable for any one or more uses for: a) administration to a subject; b) gene editing or further gene editing; c) viral transduction or further viral transduction; d) creating a cell bank; e) differentiation into a cellular intermediate or a fully differentiated drug product; f) packaging for distribution; and/or g) cryopreservation or formulation, based on the presence or absence of the first barcode and/or second barcode.

[0007] In some embodiments according to any of the methods provided herein, the therapy is a cell therapy. In some embodiments, the cell therapy is generated in vivo or ex vivo. In some embodiments, the cell therapy is generated in vivo in a subject in need thereof to treat a disease in the subject, wherein a vector comprising the first transgene and/or the second transgene and the first barcode and/or the second barcode is administered to the subject. In some embodiments, the vector is packaged in a fusosome for trafficking to the target cell or target population of cells in vivo within the subject.

[0008] In some embodiments according to any of the methods provided herein, the method further comprises administering to a subject in need thereof an effective dose of a cell, population of cells, or cell therapy to treat a disease in the subject. In some embodiments, the cell therapy comprises administering one or more doses of engineered immune cells expressing the first transgene and/or the second transgene.

[0009] In some embodiments according to any of the methods provided herein, the method further comprises monitoring a sample obtained from the subject administered the therapy for the presence or absence of the first barcode and/or the second barcode in the cell, population of cells, or therapy, the method comprising: (i) detecting the first barcode and/or the second barcode in the sample obtained from the subject receiving the cell, population of cells, or therapy, wherein detection of the first barcode indicates the presence of the first transgene, and/or detection of the second barcode indicates the presence of the second transgene in the sample; and (ii) determining the percentage, ratio, relative, or absolute number of cells expressing the first transgene and/or the second transgene.

[0010] In some embodiments according to any of the methods provided herein, the method further comprises determining the percentage, ratio, relative, or absolute number of cells expressing the first transgene and/or the second transgene at a second time point. In some embodiments, determining the percentage, ratio, relative, or absolute number of cells expressing the first transgene and/or the second transgene in a sample at a first time point and a second time point is used to monitor: i) cell therapy persistence; ii) cell therapy efficacy; iii) expansion of the cells expressing the first transgene and/or the second transgene; or iv) changes in a subject’s health or disease profile, in a subject receiving treatment.

[0011] In some embodiments according to any of the methods provided herein, the method further comprises one or more additional administrations of the cell, population of cells, or therapy to the subject comprising: i) the same or different dose as the initial dose of the cell, population of cells, or therapy administered to the subject; and/or ii) a cell, population of cells, or therapy comprising the same or different transgene encoded by a vector as the initial cell, population of cells, or therapy administered to the subject.

[0012] In another aspect provided herein is a method of monitoring a therapy administered to a subject, comprising obtaining a sample from the subject who was administered the therapy, detecting the presence or absence of a first barcode and/or a second barcode in the sample, wherein the presence of the first barcode indicates the presence of a first transgene, and the presence of the second barcode indicates the presence of a second transgene, and determining the percentage, ratio, relative, or absolute number of cells expressing the first transgene and/or the second transgene based on the presence or absence of the first barcode and/or second barcode. [0013] In some embodiments according to any of the method provided herein, the method comprises monitoring responsiveness to cell therapy in the subject. In some embodiments, if the monitoring indicates that the subject is responsive to the cell therapy, then there is no change in treatment. In some embodiments, if the monitoring indicates that the subject is not responsive to the cell therapy, then one or more actions are taken from the group consisting of rescreening or retesting of the sample, collecting and screening another sample from the subject, administering one or more additional therapeutic doses to the subject, changing the treatment of the subject, administering one or more additional therapeutic agents to the subject, and/or ceasing treatment of the subject.

[0014] In some embodiments according to any of the method provided herein, the cell therapy is monitored for its efficacy, optionally wherein the cell therapy is monitored for enhanced efficacy compared to the efficacy of a cell therapy administered to an individual wherein the cell therapy is not monitored. In some embodiments, if the monitoring indicates that the cell therapy is efficacious, then there is no change in treatment. In some embodiments, if the monitoring indicates that the cell therapy is not efficacious, then one or more actions are taken from the group consisting of rescreening or retesting of the sample, collecting and screening another sample from the subject, administering one or more additional therapeutic doses to the subject, changing the treatment of the subject, administering one or more additional therapeutic agents to the subject, and/or ceasing treatment of the subject.

[0015] In some embodiments according to any of the method provided herein, the cell therapy is monitored for its safety. In some embodiments, if the monitoring indicates that the cell therapy is safe for administration to the subject, then there is no change in treatment. In some embodiments, if the monitoring indicates that the cell therapy is not safe for administration to the subject, then one or more actions are taken selected from the group consisting of rescreening or retesting of the sample, collecting and screening another sample from the subject, changing the treatment of the subject, administering one or more additional therapeutic agents to the subject, and ceasing treatment of the subject.

[0016] In some embodiments according to any of the method provided herein, the cell therapy is monitored for cell persistence, cell activity, and/or cell expansion. In some embodiments, if the monitoring confirms cell persistence, cell activity, and/or cell expansion upon administration to the subject, then there is no change in treatment. In some embodiments, if the monitoring does not confirm cell persistence, cell activity, and/or cell expansion upon administration to the subject, then one or more actions are taken selected from the group consisting of rescreening or retesting of the sample, collecting and screening another sample from the subject, administering one or more additional therapeutic doses to the subject, changing the treatment of the subject, administering one or more additional therapeutic agents to the subject, and ceasing treatment of the subject. [0017] In some embodiments according to any of the method provided herein, the cell therapy is monitored for reduced or eliminated expression of the first transgene and/or the second transgene in the cell or population of cells. In some embodiments, the cell therapy is monitored for increased expression of the first transgene and/or the second transgene in the cell or population of cells. In some embodiments, the cell therapy is monitored for gene editing efficiency in the cell or population of cells. In some embodiments, if the monitoring confirms that there is no reduction or elimination of expression of the first transgene and/or the second transgene in the cell or population of cells, then there is no change in treatment. In some embodiments, if the monitoring confirms reduced or eliminated expression of the first transgene and/or the second transgene in the cell or population of cells, then one or more actions are taken selected from the group consisting of rescreening or retesting of the sample, collecting and screening another sample from the subject, changing the treatment of the subject, administering one or more additional therapeutic agents to the subject, and ceasing treatment of the subject. In some embodiments, if the monitoring confirms that there is a reduction or elimination of expression of the first transgene and/or the second transgene in the cell or population of cells, then there is no change in treatment. In some embodiments, if the monitoring confirms that there is no reduced or eliminated expression of the first transgene and/or the second transgene in the cell or population of cells, then one or more actions are taken selected from the group consisting of rescreening or retesting of the sample, collecting and screening another sample from the subject, changing the treatment of the subject, administering one or more additional therapeutic agents to the subject, and ceasing treatment of the subject. In some embodiments, if the monitoring confirms that there is increased expression of the first transgene and/or the second transgene in the cell or population of cells, then there is no change in treatment. In some embodiments, if the monitoring confirms no increase in expression of the first transgene and/or the second transgene in the cell or population of cells, then one or more actions are taken selected from the group consisting of rescreening or retesting of the sample, collecting and screening another sample from the subject, changing the treatment of the subject, administering one or more additional therapeutic agents to the subject, and ceasing treatment of the subject. [0018] In some embodiments according to any of the method provided herein, the cell therapy is monitored for its therapeutic kinetics. In some embodiments, if the monitoring confirms acceptable therapeutic kinetics, then there is no change in treatment. In some embodiments, if the monitoring does not confirm acceptable therapeutic kinetics, then one or more actions are taken selected from the group consisting of rescreening or retesting of the sample, collecting and screening another sample from the subject, administering one or more additional therapeutic doses to the subject, changing the treatment of the subject, administering one or more additional therapeutic agents to the subject, and ceasing treatment of the subject. [0019] In some embodiments according to any of the method provided herein, the cell therapy is monitored for controlling tumor growth, tumor expansion, and/or tumor recurrence. In some embodiments, the cell therapy is monitored for preventing antigen escape of a tumor. In some embodiments, if the monitoring indicates that the cell therapy controls tumor growth, tumor expansion, and/or tumor recurrence, and/or prevents antigen escape of a tumor, then there is no change in treatment. In some embodiments, if the monitoring indicates that the cell therapy does not control tumor growth, tumor expansion, and/or tumor recurrence, and/or does not prevent antigen escape of a tumor, then one or more actions are taken selected from the group consisting of rescreening or retesting of the sample, collecting and screening another sample from the subject, changing the treatment of the subject, administering one or more additional therapeutic agents to the subject, and ceasing treatment of the subject.

[0020] In some embodiments according to any of the method provided herein, the cell therapy is monitored for the presence or absence of a therapeutic viral vector. In some embodiments, if the monitoring confirms the presence of a therapeutic viral vector, then there is no change in treatment. In some embodiments, if the monitoring confirms the absence of a therapeutic viral vector, then one or more actions are taken selected from the group consisting of rescreening or retesting of the sample, collecting and screening another sample from the subject, administering one or more additional therapeutic doses to the subject, changing the treatment of the subject, administering one or more additional therapeutic agents to the subject, and ceasing treatment of the subject. In some embodiments, if the monitoring confirms the absence of a therapeutic viral vector, then there is no change in treatment. In some embodiments, if the monitoring confirms the presence of a therapeutic viral vector, then one or more actions are taken selected from the group consisting of rescreening or retesting of the sample, collecting and screening another sample from the subject, administering one or more additional therapeutic doses to the subject, changing the treatment of the subject, administering one or more additional therapeutic agents to the subject, and ceasing treatment of the subject. [0021] In some embodiments according to any of the method provided herein, the additional therapeutic agent is selected from the group consisting of a small molecule inhibitor, a chemotherapeutic agent, a cancer immunotherapy, an antibody, a cellular therapy, a nucleic acid, a surgery, a radiotherapy, an anti- angiogenic therapy, an anti-DNA repair therapy, an antiinflammatory therapy, an anti-neoplastic agent, a growth inhibitory agent, a cytotoxic agent, a hormonal therapy, a metabolic agent, a protein, a peptide, a pro-peptide, and any combination thereof.

[0022] In some embodiments according to any of the method provided herein, cessation of the therapy comprises triggering a safety switch in the cell, population of cells, or cell therapy administered to the subject.

[0023] In some embodiments according to any of the method provided herein, the cell therapy is monitored for vector copy number.

[0024] In some embodiments according to any of the method provided herein, the cell therapy is monitored for the number of cells with a first transgene, the number of cells with a second transgene, the number of cells with a first transgene and a second transgene, and/or the number of cells without a first transgene or a second transgene. In some embodiments, the cell therapy is monitored for the percentage of cells with a first transgene, the percentage of cells with a second transgene, the percentage of cells with a first transgene and a second transgene, and/or the percentage of cells without a first transgene or a second transgene.

[0025] In some embodiments according to any of the method provided herein, the cell therapy is monitored for the ratio of cells with a first transgene to cells without a transgene. In some embodiments, the cell therapy is monitored for the ratio of cells with a second transgene to cells without a transgene. In some embodiments, the cell therapy is monitored for the ratio of cells with a second transgene to cells with a first transgene. In some embodiments, the cell therapy is monitored for the ratio of cells with a first transgene to cells with a first transgene and a second transgene. In some embodiments, the cell therapy is monitored for the ratio of cells with a first transgene to cells with a first transgene and a second transgene to cells without a first transgene or second transgene. In some embodiments, the cell therapy is monitored for the ratio of cells with a second transgene to cells with a first transgene and a second transgene. In some embodiments, the cell therapy is monitored for the ratio of cells with a second transgene to cells with a first transgene and a second transgene to cells without a first transgene or second transgene. In some embodiments, the cell therapy is monitored for the ratio of cells with a first transgene to cells with a second transgene to cells with a first transgene and a second transgene. In some embodiments, the cell therapy is monitored for the ratio of cells with a first transgene to cells with a second transgene to cells with a first transgene and a second transgene to cells without a first transgene or second transgene.

[0026] In some embodiments according to any of the method provided herein, the method further comprises selecting a composition of cells or population of cells for cell therapy based upon the percentage of cells that have a first transgene, the percentage of cells that have a second transgene, the percentage of cells that have a first transgene and a second transgene, and/or the percentage of cells that do not have a first transgene or a second transgene. In some embodiments, the composition is selected when the percentage of cells that have a first transgene is at least 15%. In some embodiments, the composition is selected when the percentage of cells that have a second transgene is at least 15%. In some embodiments, the composition is selected when the percentage of cells that have a first transgene and a second transgene is at least 15%.

[0027] In some embodiments according to any of the method provided herein, the method further comprises selecting a composition of cells or population of cells for cell therapy based upon: i. the ratio of cells that have a first transgene to cells that have a second transgene, ii. the ratio of cells that have a first transgene to cells that have a first transgene and a second transgene, iii. the ratio of cells that have a second transgene to cells that have a first transgene and a second transgene, iv. the ratio of cells that have a first transgene to cells that have a first transgene and a second transgene to cells that do not have a first transgene or second transgene, v. the ratio of cells that have a second transgene to cells that have a first transgene and a second transgene to cells that do not have a first transgene or second transgene, and/or vi. the ratio of cells that have a first transgene to cells that have a second transgene to cells that have a first transgene and a second transgene to cells that do not have a first transgene or second transgene.

[0028] In some embodiments according to any of the method provided herein, the composition is selected when the ratio of cells with the first transgene to cells with no transgene is at least 1:6. In some embodiments, the composition is selected when the ratio of cells with the second transgene to cells with no transgene is at least 1:6. In some embodiments, the composition is selected when the ratio of cells with the first transgene and the second transgene to cells with no transgene is at least 1:6. In some embodiments, the composition is selected when the ratio of cells with the first transgene to cells with the second transgene is at least 1:1.

[0029] In some embodiments according to any of the method provided herein, the cell, population of cells, or cell therapy is capable of immune evasion. In some embodiments, the cell, population of cells, or cell therapy is capable of evading a host adaptive immune response. In some embodiments, the cell, population of cells, or cell therapy is capable of evading a host innate immune response. In some embodiments, the cell, population of cells, or cell therapy is capable of evading a T cell response, an NK cell response, a macrophage cell response, a microglial cell response, or a combination thereof.

[0030] In some embodiments according to any of the method provided herein, the cell, population of cells, or cell therapy is for use in treating a disease or disorder. In some embodiments, the disease is a cancer or an autoimmune disease. In some embodiments, the cancer is selected from the group consisting of ovarian cancer, gastric adenocarcinoma, pancreatic adenocarcinoma, glioblastoma, hepatocellular carcinoma, B-cell chronic lymphocytic leukemia (B-CLL), juvenile chronic myelogenous leukemia (CML), juvenile myelomonocytic leukemia (JMML), Acute Lymphoblastic Leukemia (ALL), Acute Myeloid Leukemia (AML), Adrenocortical Carcinoma, Kaposi Sarcoma (Soft Tissue Sarcoma), AIDS-Related Lymphoma (Lymphoma), Primary CNS Lymphoma (Lymphoma), Anal Cancer, Appendix Cancer, Astrocytomas, Atypical Teratoid/Rhabdoid Tumor, Basal Cell Carcinoma of the Skin, Bile Duct Cancer, Bladder Cancer, Bone Cancer (includes Ewing Sarcoma and Osteosarcoma and Malignant Fibrous Histiocytoma), Brain Tumors, Breast Cancer, Bronchial Tumors, Burkitt Lymphoma, Carcinoid Tumor, Carcinoma, Cardiac Tumors, Atypical Teratoid/Rhabdoid Tumor, Medulloblastoma, Germ Cell Tumor, Primary CNS Lymphoma, Cervical Cancer, Cholangiocarcinoma, Chordoma, Chronic Lymphocytic Leukemia (CLL), Chronic Myelogenous Leukemia (CML), Chronic Myeloproliferative Neoplasms, Colorectal Cancer, Craniopharyngioma, Cutaneous T-Cell Lymphoma, Ductal Carcinoma In Situ (DOS), Endometrial Cancer, Ependymoma, Esophageal Cancer, Esthesioneuroblastoma, Ewing Sarcoma, Extracranial Germ Cell Tumor, Extragonadal Germ Cell Tumor, Intraocular Melanoma, Retinoblastoma, Fallopian Tube Cancer, Fibrous Histiocytoma of Bone, Osteosarcoma, Gallbladder Cancer, Gastric Cancer, Gastrointestinal Carcinoid Tumor, Gastrointestinal Stromal Tumors (GIST) (Soft Tissue Sarcoma), Germ Cell Tumors, Central Nervous System Germ Cell Tumors, Extracranial Germ Cell Tumors, Extragonadal Germ Cell Tumors, Ovarian Germ Cell Tumors, Testicular Cancer, Gestational Trophoblastic Disease, Hairy Cell Leukemia, Head and Neck Cancer, Hepatocellular Cancer, Histiocytosis (Langerhans Cell), Hodgkin Lymphoma, Hypopharyngeal Cancer, Islet Cell Tumors, Pancreatic Neuroendocrine Tumors, Kaposi Sarcoma (Soft Tissue Sarcoma), Renal Cell Cancer, Langerhans Cell Histiocytosis, Laryngeal Cancer, Leukemia, Lip and Oral Cavity Cancer, Liver Cancer, Lung Cancer (Non-Small Cell, Small Cell, Pleuropulmonary Blastoma, and Tracheobronchial Tumor), Lung Squamous Cell Carcinoma, Lymphoma, Male Breast Cancer, Malignant Eibrous Histiocytoma of Bone and Osteosarcoma, Merkel Cell Carcinoma, Mesothelioma, Metastatic Cancer, Metastatic Squamous Neck Cancer with Occult Primary, Midline Tract Carcinoma With NUT Gene Changes, Oropharyngeal Cancer, Multiple Endocrine Neoplasia Syndromes, Multiple Myeloma/Plasma Cell Neoplasms, Mycosis Eungoides (Lymphoma), Myelodysplastic Syndromes, Myelodysplastic/Myeloproliferative Neoplasms, Chronic Myeloproliferative Neoplasms, acute B lymphoblastic leukemia (B-ALL), large B cell lymphoma (LBCL), diffuse large B cell lymphoma (DLBCL), high-grade B cell lymphoma (HGBCL), primary mediastinal B cell lymphoma (PMBCL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), or small lymphocytic lymphoma (SLL) Follicular Lymphoma, Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN), Systemic Mastocytosis, Nasal Cavity and Paranasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma, Non-Hodgkin Lymphoma, Pancreatic Cancer, Pancreatic Neuroendocrine Tumors (Islet Cell Tumors), Papillomatosis, Paraganglioma, Paranasal Sinus and Nasal Cavity Cancer, Parathyroid Cancer, Penile Cancer, Pharyngeal Cancer, Pheochromocytoma, Pituitary Tumor, Pleuropulmonary Blastoma, Primary Central Nervous System (CNS) Lymphoma, Primary Peritoneal Cancer, Prostate Cancer, Recurrent Cancer, Rhabdomyosarcoma, Salivary Gland Cancer, Vascular Tumors, Small Intestine Cancer, Soft Tissue Sarcoma, T-Cell Lymphoma, Thymoma and Thymic Carcinoma, Transitional Cell Cancer of the Renal Pelvis and Ureter, Vaginal Cancer, Vulvar Cancer, and Wilms Tumor. In some embodiments, the cancer is selected from the group consisting of NonHodgkin’s Lymphoma (NHL), Chronic Lymphocytic Leukemia (CLL), large B cell lymphoma (LBCL), diffuse LBCL (DLBCL), high-grade B cell lymphoma (HGBCL), primary mediastinal B cell lymphoma (PMBCL), mantle cell lymphoma (MCL), follicular lymphoma (FL), marginal zone lymphoma (MZL), and small lymphocytic lymphoma (SLL). In some embodiments, the method further comprises administration of one or more additional therapeutic agents. In some embodiments, the one or more additional therapeutic agents are selected from the group consisting of a chemotherapeutic agent, a gene therapy, a metabolite, a metabolic inhibitor, a cytokine, an immunotherapy, a radiotherapy, an oncolytic virus, an anti-cancer vaccine, a therapeutic antibody, a small molecule inhibitor, an additional cellular therapy, a nucleic acid, a surgery, an anti- angiogenic therapy, an anti-DNA repair therapy, an anti-inflammatory therapy, an anti-neoplastic agent, a growth inhibitory agent, a cytotoxic agent, and any combination thereof.

[0031] In some embodiments according to any of the methods provided herein, the autoimmune disease is selected from the group consisting of arthritis, rheumatoid arthritis, acute arthritis, chronic rheumatoid arthritis, gout or gouty arthritis, acute gouty arthritis, acute immunological arthritis, chronic inflammatory arthritis, degenerative arthritis, type II collagen- induced arthritis, infectious arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis, Still's disease, vertebral arthritis, juvenile-onset rheumatoid arthritis, osteoarthritis, arthritis chronica progrediente, arthritis deformans, polyarthritis chronica primaria, reactive arthritis, ankylosing spondylitis, inflammatory hyperproliferative skin diseases, psoriasis, plaque psoriasis, gutatte psoriasis, pustular psoriasis, psoriasis of the nails, atopy, atopic diseases, hay fever, Job's syndrome, dermatitis, contact dermatitis, chronic contact dermatitis, exfoliative dermatitis, exfoliative psoriatic dermatitis, allergic dermatitis, allergic contact dermatitis, dermatitis herpetiformis, nummular dermatitis, seborrheic dermatitis, non-specific dermatitis, primary irritant contact dermatitis, atopic dermatitis, x-linked hyper IgM syndrome, allergic intraocular inflammatory diseases, urticaria, chronic allergic urticaria, chronic idiopathic urticaria, chronic autoimmune urticaria, myositis, polymyositis/dermatomyositis, juvenile dermatomyositis, toxic epidermal necrolysis, scleroderma, systemic scleroderma, sclerosis, systemic sclerosis, multiple sclerosis (MS), MS associated with EBV infection, spino-optical MS, primary progressive MS (PPMS), relap sing-remitting MS (RRMS), progressive relapsing MS, secondary progressive MS (SPMS), progressive systemic sclerosis, atherosclerosis, arteriosclerosis, sclerosis disseminata, ataxic sclerosis, neuromyelitis optica spectrum disorder, inflammatory bowel disease (IBD), Crohn's disease, autoimmune-mediated gastrointestinal diseases, colitis, ulcerative colitis, colitis ulcerosa, microscopic colitis, collagenous colitis, colitis polyposa, necrotizing enterocolitis, transmural colitis, autoimmune inflammatory bowel disease, bowel inflammation, pyoderma gangrenosum, erythema nodosum, primary sclerosing cholangitis, respiratory distress syndrome, adult or acute respiratory distress syndrome (ARDS), meningitis, inflammation of all or part of the uvea, iritis, choroiditis, an autoimmune hematological disorder, rheumatoid spondylitis, rheumatoid synovitis, hereditary angioedema, cranial nerve damage, meningitis, herpes gestationis, pemphigoid gestationis, pruritis scroti, autoimmune premature ovarian failure, sudden hearing loss due to an autoimmune condition, IgE-mediated diseases, anaphylaxis, allergic and atopic rhinitis, encephalitis, Rasmussen's encephalitis, limbic and/or brainstem encephalitis, uveitis, anterior uveitis, acute anterior uveitis, granulomatous uveitis, nongranulomatous uveitis, phacoantigenic uveitis, posterior uveitis, autoimmune uveitis, glomerulonephritis (GN) with or without nephrotic syndrome, chronic or acute glomerulonephritis, primary GN, immune-mediated GN, membranous GN (membranous nephropathy), idiopathic membranous GN or idiopathic membranous nephropathy, membrano- or membranous proliferative GN (MPGN), Type I or Type II GN, rapidly progressive GN, proliferative nephritis, autoimmune polyglandular endocrine failure, balanitis including balanitis circumscripta plasmacellularis, balanoposthitis, erythema annulare centrifugum, erythema dyschromicum perstans, erythema multiform, granuloma annulare, lichen nitidus, lichen sclerosus et atrophicus, lichen simplex chronicus, lichen spinulosus, lichen planus, lamellar ichthyosis, epidermolytic hyperkeratosis, premalignant keratosis, pyoderma gangrenosum, allergic conditions and responses, allergic reaction, eczema, allergic or atopic eczema, asteatotic eczema, dyshidrotic eczema, vesicular palmoplantar eczema, asthma, asthma bronchiale, bronchial asthma, auto-immune asthma, conditions involving infiltration of T cells or chronic inflammatory responses, immune reactions against foreign antigens, immune reactions against fetal A-B-0 blood groups during pregnancy, chronic pulmonary inflammatory disease, autoimmune myocarditis, leukocyte adhesion deficiency, lupus, lupus nephritis, lupus cerebritis, pediatric lupus, non-renal lupus, extra-renal lupus, discoid lupus, discoid lupus erythematosus, alopecia lupus, systemic lupus erythematosus (SLE), cutaneous SLE, subacute cutaneous SLE, neonatal lupus syndrome (NLE), lupus erythematosus disseminatus, CNS lupus, anti- neutrophilic cytoplasmic autoantibody (ANCA) associated vasculitis, granulomatous polyangiitis, microscopic polyangiitis, autoimmune blistering skin diseases, anti-NMDA receptor neuropathy, stiff persons disease, anti-NMDA receptor encephalitis, anti-synthetase autoimmune syndromes, rapidly progressive glomerulopathy, Type I diabetes, Type II diabetes, latent autoimmune diabetes in adults, Type 1.5 diabetes, juvenile onset (Type I) diabetes mellitus, pediatric insulin-dependent diabetes mellitus (IDDM), adult onset diabetes mellitus (Type II diabetes), idiopathic diabetes, insipidus, diabetic retinopathy, diabetic nephropathy, diabetic large-artery disorder, immune responses associated with acute or delayed hypersensitivity mediated by cytokines or T-lymphocytes, tuberculosis, sarcoidosis, granulomatosis, lymphomatoid granulomatosis, Wegener's granulomatosis, agranulocytosis, vasculitides, vasculitis, large- vessel vasculitis, polymyalgia rheumatica, giant cell (Takayasu's) arteritis, medium-vessel vasculitis, Kawasaki's disease, polyarteritis nodosa/periarteritis nodosa, microscopic polyarteritis, immunovasculitis, CNS vasculitis, cutaneous vasculitis, hypersensitivity vasculitis, necrotizing vasculitis, systemic necrotizing vasculitis, ANCA- associated vasculitis, Churg-Strauss vasculitis, syndrome (CSS), ANCA-associated small-vessel vasculiti, temporal arteritis, aplastic anemia, autoimmune aplastic anemia, Coombs positive anemia, Diamond Blackfan anemia, hemolytic anemia, immune hemolytic anemia, autoimmune hemolytic anemia (AIHA), pernicious anemia (anemia pemiciosa), Addison's disease, pure red cell anemia, aplasia (PRCA), Factor VIII deficiency, hemophilia A, autoimmune neutropenia, pancytopenia, leukopenia, diseases involving leukocyte diapedesis, CNS inflammatory disorders, Alzheimer's disease, Parkinson's disease, multiple organ injury syndrome, multiple organ injury syndrome secondary to septicemia, trauma, or hemorrhage, antigen-antibody complex-mediated diseases, anti-glomerular basement membrane disease, anti-phospholipid antibody syndrome, anti-phospholipid syndrome, allergic neuritis, Behcet's disease/syndrome, Castleman's syndrome, Goodpasture's syndrome, Reynaud's syndrome, Sjogren's syndrome, Stevens-Johnson syndrome, pemphigoid, pemphigoid bullous, skin pemphigoid, pemphigus, pemphigus vulgaris, pemphigus foliaceus, pemphigus mucus-membrane pemphigoid, pemphigus erythematosus, autoimmune polyendocrinopathies, Reiter's disease or syndrome, thermal injury, preeclampsia, an immune complex disorder, immune complex nephritis, antibody-mediated nephritis, polyneuropathies, chronic neuropathy, IgM polyneuropathies, IgM-mediated neuropathy, thrombocytopenia, thrombotic thrombocytopenic purpura (TTP), post-transfusion purpura (PTP), heparin-induced thrombocytopenia, autoimmune or immune-mediated thrombocytopenia, idiopathic thrombocytopenic purpura (ITP), chronic or acute ITP, acquired thrombocytopenic purpura, scleritis, idiopathic cerato-scleritis, episcleritis, autoimmune disease of the testis or ovary, autoimmune orchitis or oophoritis, primary hypothyroidism, hypoparathyroidism, autoimmune endocrine diseases, thyroiditis, autoimmune thyroiditis, Hashimoto's disease, chronic thyroiditis, Hashimoto's thyroiditis, subacute thyroiditis, autoimmune thyroid disease, idiopathic hypothyroidism, Grave's disease, polyglandular syndromes, autoimmune polyglandular syndromes, polyglandular endocrinopathy syndromes, paraneoplastic syndromes, neurologic paraneoplastic syndromes, Lambert-Eaton myasthenic syndrome, Eaton-Lambert syndrome, stiff-man or stiff-person syndrome, encephalomyelitis, allergic encephalomyelitis, encephalomyelitis allergica, experimental allergic encephalomyelitis (EAE), myasthenia gravis, thymoma-associated myasthenia gravis, cerebellar degeneration, neuromyotonia, opsoclonus, opsoclonus myoclonus syndrome (OMS), sensory neuropathy, multifocal motor neuropathy, Sheehan's syndrome, hepatitis, autoimmune hepatitis, chronic hepatitis, lupoid hepatitis, giant cell hepatitis, chronic active hepatitis, autoimmune chronic active hepatitis, lymphoid interstitial pneumonitis (LIP), bronchiolitis obliterans, Guillain-Barre syndrome, Berger's disease (IgA nephropathy), idiopathic IgA nephropathy, linear IgA dermatosis, acute febrile neutrophilic dermatosis, subcorneal pustular dermatosis, transient acantholytic dermatosis, cirrhosis, primary biliary cirrhosis, pneumonocirrhosis, autoimmune enteropathy syndrome, Celiac or Coeliac disease, celiac sprue (gluten enteropathy), refractory sprue, idiopathic sprue, cryoglobulinemia, amyotrophic lateral sclerosis (ALS; Lou Gehrig's disease), coronary artery disease, autoimmune ear disease, autoimmune inner ear disease (AIED), autoimmune hearing loss, polychondritis, refractory or relapsed or relapsing polychondritis, pulmonary alveolar proteinosis, Cogan's syndrome/nonsyphilitic interstitial keratitis, Bell's palsy, Sweet's disease/syndrome, rosacea autoimmune, zoster-associated pain, amyloidosis, a non-cancerous lymphocytosis, a primary lymphocytosis, monoclonal B cell lymphocytosis, benign monoclonal gammopathy or monoclonal gammopathy of undetermined significance, peripheral neuropathy, paraneoplastic syndrome, channelopathies, epilepsy, migraine, arrhythmia, muscular disorders, deafness, blindness, periodic paralysis, channelopathies of the CNS, autism, inflammatory myopathy, focal or segmental or focal segmental glomerulosclerosis (ESGS), endocrine ophthalmopathy, uveoretinitis, chorioretinitis, autoimmune hepatological disorder, fibromyalgia, multiple endocrine failure, Schmidt's syndrome, adrenalitis, gastric atrophy, presenile dementia, demyelinating diseases, autoimmune demyelinating diseases, chronic inflammatory demyelinating polyneuropathy, Dressier's syndrome, alopecia areata, alopecia totalis, CREST syndrome, calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, telangiectasia, male or female autoimmune infertility, anti-spermatozoan antibodies, mixed connective tissue disease, Chagas' disease, rheumatic fever, recurrent abortion, farmer's lung, erythema multiforme, post-cardiotomy syndrome, post myocardial infarction cardiotomy syndrome, Cushing's syndrome, bird-fancier's lung, allergic granulomatous angiitis, benign lymphocytic angiitis, Alport's syndrome, alveolitis, allergic alveolitis, fibrosing alveolitis, interstitial lung disease, transfusion reaction, leprosy, malaria, parasitic diseases, leishmaniasis, kypanosomiasis, schistosomiasis, ascariasis, aspergillosis, Samter's syndrome, Caplan's syndrome, dengue, endocarditis, endomyocardial fibrosis, diffuse interstitial pulmonary fibrosis, interstitial lung fibrosis, pulmonary fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis, endophthalmitis, erythema elevatum et diutinum, erythroblastosis fetalis, eosinophilic faciitis, Shulman's syndrome, Felty's syndrome, flariasis, cyclitis, chronic cyclitis, heterochronic cyclitis, iridocyclitis (acute or chronic), Fuch's cyclitis, Henoch-Schonlein purpura, human immunodeficiency virus (HIV) infection, SCID, acquired immune deficiency syndrome (AIDS), echovirus infection, sepsis, endotoxemia, pancreatitis, thyroxicosis, parvovirus infection, rubella virus infection, post-vaccination syndromes, congenital rubella infection, Epstein-Barr virus infection, mumps, Evan's syndrome, autoimmune gonadal failure, Sydenham's chorea, poststreptococcal nephritis, thromboangitis ubiterans, thyrotoxicosis, tabes dorsalis, chorioiditis, giant cell polymyalgia, chronic hypersensitivity pneumonitis, keratoconjunctivitis sicca, epidemic keratoconjunctivitis, idiopathic nephritic syndrome, minimal change nephropathy, benign familial and ischemia-reperfusion injury, transplant organ reperfusion, retinal autoimmunity, joint inflammation, bronchitis, chronic obstructive airway /pulmonary disease, silicosis, aphthae, aphthous stomatitis, arteriosclerotic disorders, aspermiogenese, autoimmune hemolysis, Boeck's disease, cryoglobulinemia, Dupuytren's contracture, endophthalmia phacoanaphylactica, enteritis allergica, erythema nodosum leprosum, idiopathic facial paralysis, chronic fatigue syndrome, febris rheumatica, Hamman-Rich's disease, sensoneural hearing loss, haemoglobinuria paroxysmatica, hypogonadism, ileitis regionalis, leucopenia, mononucleosis infectiosa, traverse myelitis, primary idiopathic myxedema, nephrosis, ophthalmia symphatica, orchitis granulomatosa, pancreatitis, polyradiculitis acuta, pyoderma gangrenosum, Quervain's thyreoiditis, acquired splenic atrophy, non-malignant thymoma, vitiligo, toxic-shock syndrome, food poisoning, conditions involving infiltration of T cells, leukocyte-adhesion deficiency, immune responses associated with acute or delayed hypersensitivity mediated by cytokines or T- lymphocytes, diseases involving leukocyte diapedesis, multiple organ injury syndrome, antigenantibody complex-mediated diseases, antiglomerular basement membrane disease, allergic neuritis, autoimmune polyendocrinopathies, oophoritis, primary myxedema, autoimmune atrophic gastritis, sympathetic ophthalmia, rheumatic diseases, mixed connective tissue disease, nephrotic syndrome, insulitis, polyendocrine failure, autoimmune polyglandular syndrome type I, adult-onset idiopathic hypoparathyroidism (AOIH), cardiomyopathy, dilated cardiomyopathy, epidermolisis bullosa acquisita (EBA), hemochromatosis, myocarditis, nephrotic syndrome, primary sclerosing cholangitis, purulent or nonpurulent sinusitis, acute or chronic sinusitis, ethmoid, frontal, maxillary, sphenoid sinusitis, an eosinophil-related disorder, eosinophilia, pulmonary infiltration eosinophilia, eosinophilia-myalgia syndrome, Loffler's syndrome, chronic eosinophilic pneumonia, tropical pulmonary eosinophilia, bronchopneumonic aspergillosis, aspergilloma, granulomas containing eosinophils, anaphylaxis, seronegative spondyloarthritides, polyendocrine autoimmune disease, sclerosing cholangitis, sclera, episclera, chronic mucocutaneous candidiasis, Bruton's syndrome, transient hypogammaglobulinemia of infancy, Wiskott-Aldrich syndrome, ataxia telangiectasia syndrome, angiectasis, autoimmune disorders associated with collagen disease, rheumatism, neurological disease, lymphadenitis, reduction in blood pressure response, vascular dysfunction, tissue injury, cardiovascular ischemia, hyperalgesia, renal ischemia, cerebral ischemia, disease accompanying vascularization, allergic hypersensitivity disorders, glomerulonephritides, reperfusion injury, ischemic re-perfusion disorder, reperfusion injury of myocardial or other tissues, lymphomatous tracheobronchitis, inflammatory dermatoses, dermatoses with acute inflammatory components, multiple organ failure, bullous diseases, renal cortical necrosis, acute purulent meningitis, central nervous system inflammatory disorders, ocular or orbital inflammatory disorders, granulocyte transfusion-associated syndromes, cytokine-induced toxicity, narcolepsy, acute serious inflammation, chronic intractable inflammation, pyelitis, endarterial hyperplasia, peptic ulcer, valvulitis, emphysema, alopecia areata, adipose tissue inflammation/diabetes type II, obesity- associated adipose tissue inflammation/insulin resistance, endometriosis, and pulmonary hemosiderosis. In some embodiments, the autoimmune disease is selected from the group consisting of systemic lupus erythematosus (SLE), lupus nephritis, CNS lupus, anti-neutrophilic cytoplasmic autoantibody (ANCA) associated vasculitis, granulomatous polyangiitis, microscopic polyangiitis, multiple sclerosis, pemphigus vulgaris, autoimmune blistering skin diseases, membranous nephropathy, anti-NMDA receptor neuropathy, neuromyelitis optica, idiopathic thrombocytopenic purpura, autoimmune hepatitis, type 1 diabetes mellitus, rheumatoid arthritis, juvenile rheumatoid arthritis, chronic inflammatory demyelinating polyneuropathy, polymyositis/dermatomyositis, stiff persons disease, anti-NMDA receptor encephalitis, anti-synthetase autoimmune syndromes, anti-phospholipid antibody syndrome, Sjogren’s syndrome, cryoglobulinemia, focal segmental glomerulosclerosis, rapidly progressive glomerulopathy, and autoimmune hemolytic anemia. In some embodiments, the autoimmune disease is diabetes. In some embodiments, the method further comprises administration of one or more additional therapeutic agents. In some embodiments, the one or more additional therapeutic agents are selected from the group consisting of insulin, amylinomimetic(s), dopamine-2 agonist(s), DPP-4 inhibitor(s), metformin, alpha-glucosidase inhibitor(s), SGLT2 inhibitor(s), statins, GLP-1 receptor agonist(s), incretin, meglitinide(s), sulfonylureas, thiazolidinediones, nonsteroidal anti-inflammatory drugs (NSAIDs), antimalarial drugs, corticosteroids, azathioprine, mycophenolate, methotrexate, cyclosporine, voclosporin, leflunomide, belimumab, anifrolumab, abatacept, rituximab, vitamin D supplementation, dehydroepiandrosterone (DHEA), and any combination thereof.

[0032] In some embodiments according to any of the methods provided herein, the cell, population of cells, or cell therapy comprising the first barcode and/or the second barcode comprises improved traceability compared to a cell, population of cells, or cell therapy that does not comprise the first barcode and/or the second barcode. In some embodiments, the cell, population of cells, or cell therapy comprising the first barcode and/or the second barcode comprises enhanced safety compared to a cell, population of cells, or cell therapy that does not comprise the first barcode and/or the second barcode.

[0033] In some embodiments according to any of the methods provided herein, the method further comprises selecting the cell, population of cells, or cell therapy based on information obtained from one or more assays. In some embodiments, the one or more assays are selected from the group consisting of a phenotypic assay, a functional assay, a genotypic assay, a viral assay, a safety assay, an identity assay, a purity assay, and a cell count assay. [0034] In some embodiments according to any of the methods provided herein, the phenotypic assay is selected from the group consisting of a FACS assay, an ELISA assay, and any combination thereof.

[0035] In some embodiments according to any of the methods provided herein, the functional assay is selected from the group consisting of a hormone secretion assay, a cell response assay, a cell killing assay, a T cell proliferation assay, a T cell activation assay, a T cell killing assay, an NK cell killing assay, a macrophage cell killing assay, a cell function assay, a mixed meal tolerance test, continuous blood glucose level monitoring, monitoring blood glucose levels after a period of fasting, glucose tolerance tests, glucose utilization and oxidation, insulin secretion by a U-PLEX® Meso Scale Discovery (MSD) assay, glucose- stimulated insulin secretion (GSIS) assays, insulin content and proinsulin-to-insulin ratio, flow cytometry to measure the percentages of the different hormone-producing cells, qRT-PCR and immuno staining for cell-specific markers. C-Peptide assays, IFN-y ELISpot assays, microglia killing assays, cell engraftment animal models, cytokine release assays, ELISAs, killing assays using bioluminescence imaging or chromium release assay or Xcelligence analysis, mixed- lymphocyte reactions, immunofluorescence analysis, complement-dependent cytotoxicity (CDC) assay, instant blood-mediated inflammatory reaction (IBMIR), and any combination thereof.

[0036] In some embodiments according to any of the methods provided herein, the genotypic assay is selected from the group consisting of PCR, whole genome sequencing, whole exome sequencing, gene-targeted sequencing, hybrid capture sequencing, epigenetic sequencing, methylation sequencing, qPCR, RT-qPCR, RNA sequencing, microarray analysis, in situ hybridization, serial analysis of gene expression, and any combination thereof.

[0037] In some embodiments according to any of the methods provided herein, the viral assay is selected from the group consisting of a Treponema pallidum antibody (Syphilis) test, a CMV antibody (Anti-CMV IgG and IgM) test, a Hepatitis B Core antibody (Anti-HBc) test, a Hepatitis B Surface Antigen (HBsAg) test, a Hepatitis C Virus antibody (Anti-HCV) test, a Human Immunodeficiency Virus antibody (HIV 1/2 plus O) test, a human T-Lymphotropic Virus antibody (HTLV-I/II) test, a Trypanosoma cruzi antibody test, a human Herpes Virus 6 DNA test, a human Herpes Virus 7 DNA test, an Epstein-Barr Virus (EBV) DNA test, a Parvovirus B19 DNA test, a human Herpes Virus 8 DNA test, a Hepatitis A PCR test, a Hepatitis E Virus (HEV) Quantitative RT-PCR test, and any combination thereof.

[0038] In some embodiments according to any of the methods provided herein, the safety assay is selected from the group consisting of mycoplasma testing, sterility testing, endotoxin testing, karyotyping, replication-competent lentivirus testing, vector copy number testing, virus screening, cytokine independent outgrowth testing, balanced translocation testing, and any combination thereof.

[0039] In some embodiments according to any of the methods provided herein, the identity assay is flow cytometry for the polypeptide(s) encoded by a first transgene and/or a second transgene.

[0040] In some embodiments according to any of the methods provided herein, the purity assay is selected from the group consisting of cell viability, mycoplasma testing, sterility testing, endotoxin testing, presence/absence of residual activation beads, presence/absence of residual TCRa/p, presence/absence of a chimeric antigen receptor (CAR), presence/absence of B2M expression, presence/absence of CIITA expression, presence/absence of HLA-A/B/C expression, presence/absence of HLA-DP/DQ/DR expression, presence/absence of tolerogenic factor expression, presence/absence of safety switch, and any combination thereof.

[0041] In some embodiments according to any of the methods provided herein, the first transgene and/or the second transgene are encoded by a vector. In some embodiments, the first transgene and the second are encoded by the same vector. In some embodiments, the first transgene and the second transgene are encoded by different vectors. In some embodiments, the vector comprising the first transgene and/or the second transgene further comprises an identifying region comprising the first barcode and/or the second barcode.

[0042] In some embodiments according to any of the methods provided herein, the detecting the presence or absence of a first barcode and/or a second barcode in the cell or population of cells comprises sequencing and/or probe binding. In some embodiments, the sequencing comprises one or more of Sanger sequencing, NGS, PCR, qPCR, RT-PCR, or digital droplet PCR (ddPCR). [0043] In some embodiments according to any of the methods provided herein, the method further comprises contacting the sample with i) a first probe, ii) a second probe, or iii) a first probe and a second probe, wherein the first probe has a different sequence from the second probe; and detecting binding of i) the first probe to the first barcode in a first vector, ii) the second probe to the second barcode in a second vector, or iii) the first probe to the first barcode sequence in the first vector and the second probe to the second barcode in a second vector.

[0044] In some embodiments according to any of the methods provided herein, the first probe and the second probe are each conjugated to a detection marker selected from the group consisting of a fluorophore, biotin, an enzyme, a radioisotope, and a non-radioactive heavy metal isotope. In some embodiments, the first probe is attached to a fluorophore selected from the group consisting of Brilliant Ultra Violet 395, Alexa Fluor 350, Brilliant Ultra Violet 496, Qdot 525 Probe, Brilliant Ultra Violet 563, Qdot 565 Probe, Qdot 605 Probe, Brilliant Ultra Violet 615, Qdot 655 Probe, Brilliant Ultra Violet 661, Qdot 705 Probe, Brilliant Ultra Violet 737, Qdot 800 Probe, Brilliant Ultra Violet 805, Alexa Fluor 405, Brilliant Violet 421, Super Bright 436, eFluor 450, Pacific Blue, Coumarin and Coumarin Derivatives, Brilliant Violet 480, Cyan Fluorescent Protein (CFP), eFluor 506, Pacific Green, Pacific Orange, Super Bright 600, Super Bright 645, Brilliant Violet 650, Super Bright 702, Brilliant Violet 711, Super Bright 780, Brilliant Violet 786, Green Fluorescent Protein (GFP), BODIPY FL, NovaFluor Blue 510, Fluorescein (FITC), Alexa Fluor 488, Oregon Green 488, NovaFluor Blue 530, NovaFluor Blue 555, NovaFluor Blue 585, NovaFluor Blue 610-30S, NovaFluor Blue 610-70S, NovaFluor Blue 660-40S, NovaFluor Blue 660-120S, PerCP-Cyanine5.5, PerCP-eFluor 710, Alexa Fluor 532, Cy3, NovaFluor Yellow 570, Alexa Fluor 555, Alexa Fluor 561, Alexa Fluor 546, R- phycoerythrin (R-PE), Tetramethylrhodamine (TRITC), Red Fluorescent Protein (RFP), NovaFluor Yellow 590, Alexa Fluor 568, PE-eFluor 610, Texas Red (and Texas Red-X), NovaFluor Yellow 610, Alexa Fluor 594, NovaFluor Yellow 660, NovaFluor Yellow 690, NovaFluor Yellow 700, NovaFluor Yellow 730, NovaFluor Yellow 755, PE-Cyanine7,

NovaFluor Red 660, Allophycocyanin (APC), Cy5, eFluor 660, Alexa Fluor 647, NovaFluor Red 685, NovaFluor Blue 690, Alexa Fluor 660, NovaFluor Red 700, Alexa Fluor 680, NovaFluor Red 710, Alexa Fluor 700, NovaFluor Red 725, NovaFluor Red 755, Alexa Fluor 750, APC- eFluor 780, FAM, HEX, Rhodamine Red-X, Tamara, YY, Atto 550, Atto 590, Atto 700, Rox, TruRed, Cy7, Red 613, Cy3.5 581, Cy5.5, DAPI, Hoechst, SYTOX blue, SYTOX green, SYTOX orange, YOYO-1, TOTO-1, TO-PRO-1, chromomycin A3, mithramycin, propidium iodide, ethidium bromide, SYBR Green, any KIRA VIA dyes (e.g., KIR VIA Blue 520), PE- Dazzle 594, PE-Fire 640, PE-Cy5, PE-Fire 700, PE-FIRE 810, PerCP, APC-Cyanine 7, APC- Fire 750, APC-Fire 810, Spark UV 387, Spark Violet 423, Spark Violet 500, Spark Violet 538, Spark Blue 550, Spark Blue 574, Spark YG 570, Spark YG 581, Spark YG 593, Spark NIR 685, Spark Red 718, Brilliant Violet 510, Brilliant Violet 570, Brilliant Violet 605, Brilliant Violet 750, 7-AAD, APC-H7, Apotracker Green, APC-R700, Brilliant Blue 515, Brilliant Blue 700, Calcein-AM, Calcein Red- AM, Calcein Violet- AM, CF 570, CytoPhase Violet, DRAQ5, DRAQ7, Helix NP Blue, Helix NP Green, Helix NP NIR, MitoSpy Green, MitoSpy NIR, MitoSpy Orange, MitoSpy Red, Tag-it Violet, VioBright FITC, Zombie Aqua, Zombie Green, Zombie NIR, Zombie Red, Zombie UV, Zombie Violet, and Zombie Yellow. In some embodiments, the second probe is attached to a fluorophore selected from the group consisting of Brilliant Ultra Violet 395, Alexa Fluor 350, Brilliant Ultra Violet 496, Qdot 525 Probe, Brilliant Ultra Violet 563, Qdot 565 Probe, Qdot 605 Probe, Brilliant Ultra Violet 615, Qdot 655 Probe, Brilliant Ultra Violet 661, Qdot 705 Probe, Brilliant Ultra Violet 737, Qdot 800 Probe, Brilliant Ultra Violet 805, Alexa Fluor 405, Brilliant Violet 421, Super Bright 436, eFluor 450, Pacific Blue, Coumarin and Coumarin Derivatives, Brilliant Violet 480, Cyan Fluorescent Protein (CFP), eFluor 506, Pacific Green, Pacific Orange, Super Bright 600, Super Bright 645, Brilliant Violet 650, Super Bright 702, Brilliant Violet 711, Super Bright 780, Brilliant Violet 786, Green Fluorescent Protein (GFP), BODIPY FE, NovaFluor Blue 510, Fluorescein (FITC), Alexa Fluor 488, Oregon Green 488, NovaFluor Blue 530, NovaFluor Blue 555, NovaFluor Blue 585, NovaFluor Blue 610-30S, NovaFluor Blue 610-70S, NovaFluor Blue 660-40S, NovaFluor Blue 660-120S, PerCP-Cyanine5.5, PerCP-eFluor 710, Alexa Fluor 532, Cy3, NovaFluor Yellow 570, Alexa Fluor 555, Alexa Fluor 561, Alexa Fluor 546, R-phycoerythrin (R-PE), Tetramethylrhodamine (TRITC), Red Fluorescent Protein (RFP), NovaFluor Yellow 590, Alexa Fluor 568, PE-eFluor 610, Texas Red (and Texas Red-X), NovaFluor Yellow 610, Alexa Fluor 594, NovaFluor Yellow 660, NovaFluor Yellow 690, NovaFluor Yellow 700, NovaFluor Yellow 730, NovaFluor Yellow 755, PE-Cyanine7, NovaFluor Red 660, Allophycocyanin (APC), Cy5, eFluor 660, Alexa Fluor 647, NovaFluor Red 685, NovaFluor Blue 690, Alexa Fluor 660, NovaFluor Red 700, Alexa Fluor 680, NovaFluor Red 710, Alexa Fluor 700, NovaFluor Red 725, NovaFluor Red 755, Alexa Fluor 750, APC-eFluor 780, FAM, HEX, Rhodamine Red-X, YY, Atto 550, Atto 590, Atto 700, Tamara, Rox, TruRed, Cy7, Red 613, Cy3.5 581, Cy5.5, DAPI, Hoechst, SYTOX blue, SYTOX green, SYTOX orange, YOYO-1, TOTO-1, TO-PRO-1, chromomycin A3, mithramycin, propidium iodide, ethidium bromide, SYBR Green, any KIRA VIA dyes (e.g., KIRVIA Blue 520), PE-Dazzle 594, PE-Fire 640, PE-Cy5, PE-Fire 700, PE-FIRE 810, PerCP, APC-Cyanine 7, APC-Fire 750, APC-Fire 810, Spark UV 387, Spark Violet 423, Spark Violet 500, Spark Violet 538, Spark Blue 550, Spark Blue 574, Spark YG 570, Spark YG 581, Spark YG 593, Spark NIR 685, Spark Red 718, Brilliant Violet 510, Brilliant Violet 570, Brilliant Violet 605, Brilliant Violet 750, 7-AAD, APC-H7, Apotracker Green, APC- R700, Brilliant Blue 515, Brilliant Blue 700, Calcein-AM, Calcein Red- AM, Calcein Violet- AM, CF 570, CytoPhase Violet, DRAQ5, DRAQ7, Helix NP Blue, Helix NP Green, Helix NP NIR, MitoSpy Green, MitoSpy NIR, MitoSpy Orange, MitoSpy Red, Tag-it Violet, VioBright FITC, Zombie Aqua, Zombie Green, Zombie NIR, Zombie Red, Zombie UV, Zombie Violet, and Zombie Yellow.

[0045] In some embodiments according to any of the methods provided herein, the first barcode sequence and/or the second barcode sequence is located outside of the first and/or second transgene sequence. In some embodiments, the first barcode sequence and/or the second barcode sequence is located within the first and/or the second transgene sequence. In some embodiments, a portion of the first barcode sequence and/or the second barcode sequence is located within the first/and or the second transgene sequence and a portion of the first barcode sequence and/or the second barcode sequence is located outside of the first and/or second transgene sequence.

[0046] In some embodiments according to any of the methods provided herein, the first barcode sequence and/or the second barcode sequence comprises a diverged nucleotide sequence within the first transgene and/or the second transgene. In some embodiments, the diverged nucleotide sequence within the first transgene and/or the second transgene encodes the same amino acid sequence as a non-diverged nucleotide sequence. In some embodiments, the diverged nucleotide sequence is located at the junction of one or more transgene domains.

[0047] In some embodiments according to any of the methods provided herein, the first barcode sequence and the second barcode sequence are each between about 6 to about 30 nucleotides in length. In some embodiments, the first barcode sequence and the second barcode sequence are about the same nucleotide length. In some embodiments, the barcode sequence comprises a nucleic acid sequence selected from the group consisting of CATCGGAA, GGACAATT, TGTCAACT, TTACAGTT, ATTCAAGG, and TACAGTTA.

[0048] In some embodiments according to any of the methods provided herein, the first barcode sequence and/or the second barcode sequence is located within a first identifying region and/or a second identifying region, wherein the first identifying region and/or the second identifying region comprises primer binding sites that flank the first barcode sequence and/or the second barcode sequence.

[0049] In some embodiments according to any of the methods provided herein, each primer binding site comprises: i) a forward primer binding site; and/or ii) a reverse primer binding site. In some embodiments, the forward primer binding sites of the first barcode sequence and the second barcode sequence comprise the same sequence. In some embodiments, the forward primer binding sites of the first barcode sequence and the second barcode sequence comprise different sequences. In some embodiments, the reverse primer binding sites of the first barcode sequence and the second barcode sequence comprise the same sequence. In some embodiments, the reverse primer binding sites of the first barcode sequence and the second barcode sequence comprise different sequences. In some embodiments, the first and second forward primer binding sites and the first and second reverse primer binding sites are complementary to universal primers. In some embodiments, the first forward primer binding site and the second forward primer binding site is each between about 10 to about 30 nucleotides in length. In some embodiments, the first reverse primer binding site and the second reverse primer binding site is each between about 10 to about 30 nucleotides in length.

[0050] In some embodiments according to any of the methods provided herein, the first identifying region is located in a non-coding region or a coding region of the vector comprising the first transgene. In some embodiments, the second identifying region is located in a second non-coding region or a second coding region of the vector comprising the second transgene. In some embodiments, the first identifying region and the second identifying region are located in the same region of the same vector that comprises the first transgene and the second transgene. [0051] In some embodiments according to any of the methods provided herein, the first identifying region is upstream of a first promoter and/or the second identifying region is upstream of a second promoter. In some embodiments, the first identifying region is downstream of the first promoter and/or the second identifying region is downstream of the second promoter. In some embodiments, the first identifying region is upstream of the first promoter and the second identifying region is downstream of the second promoter. In some embodiments, the first identifying region is downstream of the first promoter and the second identifying region is upstream of the second promoter.

[0052] In some embodiments according to any of the methods provided herein, the first identifying region and/or the second identifying region is upstream of one or more additional regulatory elements. In some embodiments, the first identifying region and/or second identifying region is downstream of one or more additional regulatory elements. In some embodiments, the one or more additional regulatory elements is selected from the group consisting of: promoter sequences, enhancer sequences, intron sequences, terminator sequences, translation initiation signal sequences, polyadenylation signal sequences, replication element sequences, RNA processing and export element sequences, transposon sequences, transposase sequences, insulator sequences, 5' UTR sequences, 3' UTR sequences, mRNA 3' end processing sequences, boundary element sequences, locus control region (LCR) sequences, matrix attachment region (MAR) sequences, ubiquitous chromatin opening elements, linker sequences, secretion signal sequences, anchoring peptide sequences, localization signal sequences, fusion tag sequences, affinity tag sequences, chaperonin sequences, protease sequences, posttranscriptional regulatory element sequences, and any combination thereof.

[0053] In some embodiments according to any of the methods provided herein, the first identifying region is located within the first transgene sequence and/or the second identifying region is located within the second transgene sequence. In some embodiments, the first identifying region is located outside of the first transgene sequence and/or the second identifying region is located outside of the second transgene sequence. In some embodiments, the first identifying region is located within the first transgene sequence and the second identifying region is located outside of the second transgene sequence. In some embodiments, the first identifying region is located outside the first transgene sequence and the second identifying region is located within of the second transgene sequence.

[0054] In some embodiments according to any of the methods provided herein, the first identifying region is located 5' to the first promoter within about 1 to about 200 base pairs and/or wherein the second identifying region is located 5' to the second promoter within about 1 to about 200 base pairs. In some embodiments, the first identifying region is located 3' to the first transgene within about 1 to about 200 base pairs and/or wherein the second identifying region is located 3' to the second transgene within about 1 to about 200 base pairs. In some embodiments, the first identifying region is located 5' to the first promoter within about 1 to about 200 base pairs and/or wherein the second identifying region is located 3' to the second promoter within about 1 to about 200 base pairs. In some embodiments, the first identifying region is located 3' to the first promoter within about 1 to about 200 base pairs and/or wherein the second identifying region is located 5' to the second promoter within about 1 to about 200 base pairs.

[0055] In some embodiments according to any of the methods provided herein, a) the first identifying region of the first vector is inserted i) 5' to the first promoter, 5' to the first transgene, and 5' to a Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE), or ii) 3' to the first promoter, within the first transgene, and 5' to a WPRE; and/or b) the second identifying region of the second vector is inserted i) 5' to the second promoter, 5' to the second transgene, and 5' to a Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE), or ii) 3' to the second promoter, within the second transgene, and 5' to a WPRE.

[0056] In some embodiments according to any of the methods provided herein, the first identifying region and the second identifying region are each between about 10 to about 100 nucleotides in length, optionally wherein the first identifying region and the second identifying region are each between about 18 to about 30 nucleotides in length. In some embodiments, the first identifying region and the second identifying region are about the same length.

[0057] In some embodiments according to any of the methods provided herein, the first identifying region and/or the second identifying region have more than one barcode sequence. In some embodiments, the first identifying region and/or the second identifying region have two or more probe binding sites. In some embodiments, the first identifying region and/or the second identifying region comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs:24-30. In some embodiments, the first identifying region or the second identifying region comprises a first barcode sequence or a second barcode sequence comprising the nucleotide sequence selected from the group consisting of CATCGGAA, GGACAATT, TGTCAACT, TTACAGTT, ATTCAAGG, and TACAGTTA. In some embodiments, the first barcode sequence and/or the second barcode sequence are randomly generated. In some embodiments, the first identifying region or the second identifying region comprises a first probe binding site or a second probe binding site comprising the nucleotide sequence selected from the group consisting of SEQ ID NOs: 18-23 and 31.

[0058] In some embodiments according to any of the methods provided herein, the first transgene and/or the second transgene encodes an antibody or an antibody fragment, a chimeric antigen receptor (CAR), a chimeric autoantibody receptor (CAAR), a B-cell autoantibody receptor (BAR), a T cell receptor (TCR), an immunomodulatory factor, a safety switch, one or more transcription factors, a genome-editing complex, one or more fusogens, one or more tolerogenic factors, or any combination thereof.

[0059] In some embodiments according to any of the methods provided herein, the first transgene and/or the second transgene encodes a protein selected from the group consisting of: enzymes, structural polypeptides, signaling polypeptides, regulatory polypeptides, transport polypeptides, sensory polypeptides, motor polypeptides, defense polypeptides, storage polypeptides, transcription factors, antibodies, cytokines, hormones, catabolic polypeptides, anabolic polypeptides, proteolytic polypeptides, metabolic polypeptides, kinases, transferases, hydrolases, lyases, isomerases, ligases, enzyme modulator polypeptides, protein binding polypeptides, lipid binding polypeptides, membrane fusion polypeptides, cell differentiation polypeptides, epigenetic polypeptides, cell death polypeptides, nuclear transport polypeptides, nucleic acid binding polypeptides, reprogramming polypeptides, DNA editing polypeptides, DNA repair polypeptides, DNA recombination polypeptides, transposase polypeptides, DNA integration polypeptides, targeted endonucleases, recombinases, transposases, DNA polymerases, RNA polymerases, and reverse transcriptase.

[0060] In some embodiments according to any of the methods provided herein, the first transgene and/or the second transgene encodes a therapeutic antibody. In some embodiments, the therapeutic antibody is an antibody that binds to an antigen selected from the group consisting of CD47, Sirpa, CD52, amyloid beta, angiopoietin-like protein 3 (ANGPTL3), B cell activating factor (BAFF), A proliferation-inducing ligand (APRIE), B cell maturation antigen (BCMA), B. anthracis protective antigen (B. anthracis PA), calcitonin gene-related peptide (CGRP), calcitonin gene-related peptide receptor (CGRP-R), C-C chemokine receptor type 4 (CCR4), CD147, CD19, CD3, CD2, CD20, CD22, CD25, epithelial cell adhesion molecule (EpCAM), Glycoprotein 100 (GP100), CD30, CD33, CD38, CD4, CD52, CD6, CD79b, CD80, CD86, Clostridium difficile Toxin B, coagulation factor IX (Factor IX), coagulation factor X (Factor X), complement Cis (Cis), complement C5 (C5), cytotoxic T-lymphocyte antigen 4 (CTLA4), dabigatran, DNA/Histone Hl, Ebola virus Glycoprotein, epidermal growth factor receptor (EGFR), C-Met, epithelial cell adhesion molecule (EpCAM), Factor IX, Factor VIII, fibroblast growth factor 23 (FGF23), ganglioside GM3 (GM3), GD2 ganglioside (GD2), human epidermal growth factor receptor 2 (HER2), immunoglobulin E (IgE), insulin-like growth factor 1 receptor (IGF-1R), integrin alpha lib beta 3 (Integrin allbp3), integrin alpha4 beta? (Integrin a4p7), integrin subunit alpha 4 (ITGA4), integrin subunit alpha L (ITGAL), interferon alpha receptor 1 (IFNAR-1), interferon gamma (IFN-y), interleukin 1 alpha (IL-la), interleukin 1 beta (IL-1 P), interleukin 12, (IL- 12), interleukin 23 (IL- 23), interleukin 13 (IL- 13), interleukin 17 receptor alpha (IL-17 RA), interleukin 17A (IL-17A), interleukin 17F (IL-17F), interleukin 23 pl9 (IL- 23pl9), interleukin 31 receptor subunit alpha (IL-31RA), interleukin 4 receptor alpha (IL-4RA), interleukin 5 (IL-5), interleukin 5 receptor subunit alpha (IL-5RA), interleukin 6 (IL-6), interleukin 6 receptor (IL-6R), interleukin 8 (IL-8), Kallikrein, Lipid A region of endotoxin, lymphocyte activation gene 3 (LAG-3), programmed cell death 1 (PD-1), nectin cell adhesion molecule 4 (Nectin 4), neonatal Fc Receptor (FcRn), platelet-derived growth factor receptor alpha (PDGFRA), programmed cell death 1 ligand 1 (PD-L1), proprotein convertase subtilisin/kexin Type 9 (PCSK9), P-selectin, rabies virus glycoprotein (Rabies virus GP), receptor activator of nuclear factor kappaB ligand (RANKL), respiratory syncytial virus F protein (RSV-F), SARS-Cov-2 spike protein (SARS-Cov-2 S protein), sclerostin, SLAM family member 7 (SLAMF7), thrombopoietin receptor (TPOR), thymic stromal lymphopoietin (TSLP), tissue factor (TF), transferrin receptor protein 1 (TFR1), transforming growth factor-beta (TGF- P), tumor necrosis factor-alpha (TNF-a), tumor-associated calcium signal transducer 2 (TACSTD-2), vascular endothelial growth factor (VEGF), vascular endothelial growth factor A (VEGF-A), angiopoietin 2 (ANG-2), vascular endothelial growth factor receptor 2 (VEGFR-2), or von Willebrand Factor (vWF), and any combination thereof.

[0061] In some embodiments according to any of the methods provided herein, the first transgene and/or the second transgene comprises a sequence encoding one or more fusogens. In some embodiments, the one or more fusogens are selected from the group consisting of NiV-F, NiV-G, Gag-Pol, and Rev. In some embodiments, the first transgene and/or the second transgene comprise a sequence encoding a retroviral vector. In some embodiments, the retroviral vector is a lentiviral vector. In some embodiments, the first transgene and/or the second transgene 1 comprises a sequence encoding an AAV vector. In some embodiments, the first transgene and/or the second transgene comprises a sequence encoding a virus-like particle.

[0062] In some embodiments according to any of the methods provided herein, the first transgene and/or the second transgene encodes a CAR. In some embodiments, the CAR encoded by the first transgene and/or the second transgene comprises a hinge domain, a transmembrane domain, and one or more signaling domains. In some embodiments, the hinge domain is selected from the group consisting of: CD8a hinge domain, CD28 hinge domain, IgG4 hinge domain, IgG4 hinge-CH2-CH3 domain, and any functional variant thereof. In some embodiments, the transmembrane domain is selected from the group consisting of: alpha, beta, or zeta chain of a T cell receptor, CD28, CD3s, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CD8a, CD8p, 4-1BB/CD137, CD28, CD34, CD4, FcsRIy, CD 16, OX40/CD134, CD3^, CD3s, CD3y, CD35, TCRa, TCRp, TCR^, CD32, CD64, CD64, CD45, CD5, CD9, CD22, CD37, CD80, CD86, CD40, CD40L/CD154, VEGFR2, FAS, FGFR2B, and any functional variant thereof. In some embodiments, the signaling domain is selected from the group consisting of: B7-1/CD80, B7-2/CD86, B7-H1/PD-L1, B7-H2, B7-H3, B7-H4, B7-H6, B7-H7, BTLA/CD272, CD28, CTLA-4, Gi24/VISTA/B7-H5, ICOS/CD278, PD-1, PD-L2/B7-DC, PDCD6, 4-1BB/TNFSF9/CD137, 4-1BB Ligand/TNFSF9, BAFF/BLyS/TNFSF13B, BAFF R/TNFRSF13C, CD27/TNFRSF7, CD27 Ligand/TNFSF7, CD30/TNFRSF8, CD30 Ligand/TNFSF8, CD40/TNFRSF5, CD40/TNFSF5, CD40 Ligand/TNFSF5, DR3/TNFRSF25, GITR/TNFRSF18, GITR Ligand/TNFSF18, HVEM/TNFRSF14, LIGHT/TNFSF14, Lymphotoxin-alpha/TNFp, OX40/TNFRSF4, 0X40 Ligand/TNFSF4, RELT/TNFRSF19L, TACI/TNFRSF I 3B, TL1A/TNFSF15, TNFa, TNF RII/TNFRSF I B, 2B4/CD244/SLAMF4, BLAME/SLAMF8, CD2, CD2F-10/SLAMF9, CD48/SLAMF2, CD58/LFA-3, CD84/SLAMF5, CD229/SLAMF3, CRACC/SLAMF7, NTB- A/SLAMF6, SLAM/CD150, CD7, CD53, CD82/Kai-1, CD90/Thyl, CD96, CD160, CD200, CD300a/LMIRl, HLA Class I, HLA-DR, Ikaros, Integrin alpha 4/CD49d, Integrin alpha 4 beta 1, Integrin alpha 4 beta 7/LPAM-l, LAG-3, TCL1A, TCL1B, CRTAM, DAP12, Dectin- 1/CLEC7A, DPPIV/CD26, EphB6, TIM- 1 /KIM- 1 /HA VCR, TIM-4, TSLP, TSLP R, lymphocyte function associated antigen- 1 (LFA-1), NKG2C, CD3^, an immunoreceptor tyrosine-based activation motif (ITAM), CD27, 4-1BB, CD134/OX40, CD30, CD40, PD-1, ICOS, LIGHT, NKG2C, a ligand that specifically binds with CD83, any functional variant thereof, and any combination thereof. In some embodiments, the CAR encoded by the first transgene and/or the second transgene comprises a CD8a hinge domain, a CD8a transmembrane domain, a 4- IBB domain, and a CD3-zeta domain. In some embodiments, the CAR encoded by the first transgene and/or the second transgene further comprises one or more co- stimulatory domain(s).

[0063] In some embodiments according to any of the methods provided herein, the CAR encoded by the first transgene and/or the second transgene is selected from the group consisting of: a CD5-specific CAR, a CD19-specific CAR, a CD20-specific CAR, a CD22-specific CAR, a CD23-specific CAR, a CD30-specific CAR, a CD33-specific CAR, CD38-specific CAR, a CD70-specific CAR, a CD 123 -specific CAR, a CD 138- specific CAR, a Kappa, Lambda, B cell maturation agent (BCMA)-specific CAR, a G-protein coupled receptor family C group 5 member D (GPRC5D)- specific CAR, a CD123-specific CAR, a LeY-specific CAR, a NKG2D ligand- specific CAR, a WTl-specific CAR, a GD2-specific CAR, a HER2-specific CAR, a EGFR-specific CAR, a EGFRv III- specific CAR, a B7H3-specific CAR, a PSMA-specific CAR, a PSCA-specific CAR, a CAIX-specific CAR, a CD 171 -specific CAR, a CEA-specific CAR, a CSPG4- specific CAR, a EPH A2- specific CAR, a FAP-specific CAR, a FRa-specific CAR, a IL- 13Ra-specific CAR, a Mesothelin- specific CAR, a MUCl-specific CAR, a MUC16-specific CAR, a RORl-specific CAR, a C-Met-specific CAR, a CD 133- specific CAR, a Ep-CAM- specific CAR, a GPC3-specific CAR, a HPV16-E6- specific CAR, a IL13Ra2-specific CAR, a MAGEA3- specific CAR, a MAGEA4- specific CAR, a MARTI -specific CAR, a NY-ESO-1- specific CAR, a VEGFR2- specific CAR, a a-Folate receptor- specific CAR, a CD24- specific CAR, a CD44v7/8-specific CAR, a EGP-2-specific CAR, a EGP-40-specific CAR, a erb-B2- specific CAR, a erb-B 2,3,4-specific CAR, a FBP-specific CAR, a Fetal acethylcholine e receptor- specific CAR, a GD2-specific CAR, a GD3-specific CAR, a HMW-MAA- specific CAR, a IL- HRa- specific CAR, a KDR-specific CAR, a Lewis Y-specific CAR, a Ll-cell adhesion molecule- specific CAR, a MAGE-A1 -specific CAR, a Oncofetal antigen (h5T4)- specific CAR, a TAG-72-specific CAR, and a CD19/CD22-bispecific CAR. In some embodiments, the CAR encoded by the first transgene and/or the second transgene is a CD 19 CAR or a CD22 CAR.

[0064] In some embodiments according to any of the methods provided herein, the CARs encoded by the first transgene and the second transgene and optionally additional transgenes bind specifically to antigens selected from the group consisting of CD 19 and CD20, CD 19 and BCMA, CD20 and BCMA, CD19 and CD22, CD19 and BAFFR, CD33 and CD123, HER2 and B7H3, HER2 and EGFR, HER2 and IL13Ra, HER2 and ROR1, B7H3 and EGFR, B7H3 and IL13Ra, B7H3 and ROR1, EGFR and IL13Ra, EGFR and ROR1, and Her2 and B7H3 and EGFR and IL13Ra2 and RORE In some embodiments, the CARs encoded by the first transgene and the second transgene bind specifically to autoimmune disease antigens selected from the group consisting of CD 19 and CD20, CD 19 and BCMA, CD20 and BCMA, and CD 19 and CD22. In some embodiments, the CARs encoded by the first transgene and the second transgene bind specifically to leukemia or lymphoma antigens and is selected from the group consisting of CD19 and CD20, CD19 and BAFFR, and CD19 and CD22. In some embodiments, the CARs encoded by the first transgene and the second transgene bind specifically to the acute myeloid leukemia antigens CD33 and CD123. In some embodiments, the CARs encoded by the first transgene and the second transgene and optionally additional transgenes bind specifically to the solid tumor antigens Her2 and B7H3 and EGFR and IL13Ra2 and RORl.

[0065] In some embodiments according to any of the methods provided herein, the diverged nucleotide sequence within the first transgene and/or within the second transgene is located at the junction of: i) the signaling domain and the co- stimulatory domain; or ii) the hinge domain and the transmembrane domain. In some embodiments, the diverged nucleotide sequence within the first transgene and/or within the second transgene is located at the junction of the 4- 1BB and CD3-zeta domains.

[0066] In some embodiments according to any of the methods provided herein, the one or more transgenes comprise a chimeric autoantibody receptor (CAAR). In some embodiments, the CAAR comprises an antigen selected from the group consisting of a pancreatic P-cell antigen, synovial joint antigen, myelin basic protein, proteolipid protein, myelin oligodendritic glycoprotein, MuSK, keratinocyte adhesion protein desmoglein 3 (Dsg3), Ro-RNP complex, La antigen, myeloperoxidase, proteinase 3, cardiolipin, citrullinated proteins, carbamylated proteins, and a3 chain of basement membrane collagen.

[0067] In some embodiments according to any of the methods provided herein, the one or more transgenes comprise a B-cell autoantibody receptor (BAR). In some embodiments, the BAR comprises an FVIII antigen.

[0068] In some embodiments according to any of the methods provided herein, the one or more transgenes comprise a T cell receptor (TCR). [0069] In some embodiments according to any of the methods provided herein, the one or more transgenes comprise a tolerogenic factor. In some embodiments, the tolerogenic factor is selected from the group consisting of: CD16, CD24, CD35, CD39, CD46, CD47, CD52, CD55, CD59, CD64, CD200, CCL22, CTLA4-Ig, Cl inhibitor, FASL, IDO1, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, IL-10, IL-35, PD-L1, SERPINB9, CCL21, MFGE8, DUX4, B2M-HLA-E, CD27, IL-39, CD16 Fc Receptor, IL15-RF, H2-M3 (HLA-G), A20/TNFAIP3, CR1, HLA-F, and MANF.

[0070] In some embodiments according to any of the methods provided herein, the one or more transgenes comprise a safety switch. In some embodiments, the safety switch is selected from the group consisting of herpes simplex virus thymidine kinase (HSV-tk), cytosine deaminase (CyD), nitroreductase (NTR), purine nucleoside phosphorylase (PNP), horseradish peroxidase, inducible caspase 9 (iCasp9), rapamycin-activated caspase 9 (rapaCasp9), CCR4, CD16, CD19, CD20, CD30, EGFR, GD2, HER1, HER2, MUC1, PSMA, and RQR8. In some embodiments, the safety switch is a system wherein, upon activation, cells downregulate expression of one or more tolerogenic factors and/or upregulate expression of one or more immune signaling molecules, thereby marking the cell for elimination by the host immune system. In some embodiments, the one or more tolerogenic factors are selected from the group consisting of: CD16, CD24, CD35, CD39, CD46, CD47, CD52, CD55, CD59, CD64, CD200, CCL22, CTLA4-Ig, Cl inhibitor, FASL, IDO1, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, IL-10, IL-35, PD-L1, SERPINB9, CCL21, MFGE8, DUX4, B2M-HLA-E, CD27, IL-39, CD16 Fc Receptor, IL15-RF, H2-M3 (HLA-G), A20/TNFAIP3, CR1, HLA-F, and MANF. In some embodiments, the one or more immune signaling molecules are selected from the group consisting of B2M, HLA-A, HLA-B, HLA-C, HLA-D, HLA-E, RFXANK, CIITA, CTLA-4, PD-1, RAET1E/ULBP4, RAET1G/ULBP5, RAET1H/ULBP2, RAET1/ULBP1, RAET1L/ULBP6, RAET1N/ULBP3, and ligands of NKG2D.

[0071] In some embodiments according to any of the methods provided herein, the one or more transgenes encode a genome editing complex. In some embodiments, the genome editing complex comprises a genome targeting entity and a genome modifying entity. In some embodiments, the genome targeting entity is a nucleic acid-guided targeting entity. In some embodiments, the genome targeting entity is selected from the group consisting of: a sequence specific nuclease, a nucleic acid programmable DNA binding protein, an RNA guided nuclease, RNA-guided nuclease comprising a Cas nuclease and a guide RNA (CRISPR-Cas combination), a ribonucleoprotein (RNP) complex comprising a gRNA and a Cas nuclease, a homing endonuclease, a zinc finger nuclease (ZF) nucleic acid binding entity, a transcription activatorlike effector (TALE) nucleic acid binding entity, a meganuclease, a Cas nuclease, a core Cas protein, a homing endonuclease, an endonuclease-deficient-Cas protein, an enzymatically inactive Cas protein, a CRIS PR-associated transposase (CAST), a Type II or Type V Cas protein, or a functional portion thereof. In some embodiments, the genome targeting entity is selected from the group consisting of Casl, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8a, Cas8b, Cas8c, Cas9, CaslO, Casl2, Casl2a (Cpfl), Casl2b (C2cl), Casl2c (C2c3), Casl2d (CasY), Casl2e (CasX), Casl2f (C2cl0), Casl2g, Casl2h, Casl2i, Casl2k (C2c5), Casl3, Casl3a (C2c2), Casl3b, Casl3c, Casl3d, C2c4, C2c8, C2c9, Cmrl, Cmr2, Cmr3, Cmr4, Cmr5, Cmr6, Csdl, Csd2, Cas5d, Csel, Cse2, Cse3, Cse4, Cas5e, Csfl, Csml, Csm2, Csm3, Csm4, Csm5, Csnl, Csn2, Cstl, Cst2, Cas5t, Cshl, Csh2, Cas5h, Csal, Csa2, Csa3, Csa4, Csa5, Cas5a, CsxlO, Csxl l, Csyl, Csy2, Csy3, Csy4, Mad7, SpCas9, eSpCas9, SpCas9-HFl, HypaSpCas9, HeFSpCas9, and evoSpCas9 high-fidelity variants of SpCas9, SaCas9, NmeCas9, CjCas9, StCas9, TdCas9, LbCasl2a, AsCasl2a, AacCasl2b, BhCasl2b v4, TnpB, dCas (D10A), dCas (H840A), dCasl3a, dCasl3b, or a functional portion thereof.

[0072] In some embodiments according to any of the methods provided herein, the genome modifying entity cleaves, deaminates, nicks, polymerizes, interrogates, integrates, cuts, unwinds, breaks, alters, methylates, demethylates, or otherwise destabilizes the target locus. In some embodiments, the genome modifying entity comprises a recombinase, integrase, transposase, endonuclease, exonuclease, nickase, helicase, DNA polymerase, RNA polymerase, reverse transcriptase, deaminase, flippase, methylase, demethylase, acetylase, a nucleic acid modifying protein, an RNA modifying protein, a DNA modifying protein, an Argonaute protein, an epigenetic modifying protein, a histone modifying protein, or a functional portion thereof. In some embodiments, the genome modifying entity is selected from the group consisting of: a sequence specific nuclease, a nucleic acid programmable DNA binding protein, an RNA guided nuclease, RNA-guided nuclease comprising a Cas nuclease and a guide RNA (CRISPR-Cas combination), a ribonucleoprotein (RNP) complex comprising the gRNA and the Cas nuclease, a homing endonuclease, a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a meganuclease, a Cas nuclease, a core Cas protein, a TnpB nuclease, an endonuclease-deficient-Cas protein, an enzymatically inactive Cas protein, a CRIS PR-associated transposase (CAST), a Type II or Type V Cas protein, base editing, prime editing, a Programmable Addition via Site-specific Targeting Elements (PASTE), or a functional portion thereof. In some embodiments, the genome modifying entity is selected from the group consisting of Casl, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8a, Cas8b, Cas8c, Cas9, Cas 10, Casl2, Casl2a (Cpfl), Casl2b (C2cl), Casl2c (C2c3), Casl2d (CasY), Casl2e (CasX), Casl2f (C2cl0), Casl2g, Casl2h, Casl2i, Casl2k (C2c5), Casl3, Casl3a (C2c2), Casl3b, Casl3c, Casl3d, C2c4, C2c8, C2c9, Cmrl, Cmr2, Cmr3, Cmr4, Cmr5, Cmr6, Csdl, Csd2, Cas5d, Csel, Cse2, Cse3, Cse4, Cas5e, Csfl, Csml, Csm2, Csm3, Csm4, Csm5, Csnl, Csn2, Cstl, Cst2, Cas5t, Cshl, Csh2, Cas5h, Csal, Csa2, Csa3, Csa4, Csa5, Cas5a, CsxlO, Csxll, Csyl, Csy2, Csy3, Csy4, Mad7, SpCas9, eSpCas9, SpCas9-HFl, HypaSpCas9, HeFSpCas9, and evoSpCas9 high-fidelity variants of SpCas9, SaCas9, NmeCas9, CjCas9, StCas9, TdCas9, LbCasl2a, AsCasl2a, AacCasl2b, BhCasl2b v4, TnpB, FokI, dCas (D10A), dCas (H840A), dCasl3a, dCasl3b, a base editor, a prime editor, a target-primed reverse transcription (TPRT) editor, APOBEC1, cytidine deaminase, adenosine deaminase, uracil glycosylase inhibitor (UGI), adenine base editors (ABE), cytosine base editors (CBE), reverse transcriptase, serine integrase, recombinase, transposase, polymerase, adenine-to-thymine or “ATBE” (or thymine-to-adenine or “TABE”) transversion base editor, ten-eleven translocation methylcytosine dioxygenases (TETs), TET1, TET3, TET1CD, histone acetyltransferase p300, histone methyltransferase SMYD3, histone methyltransferase PRDM9, H3K79 methyltransferase DOT1L, transcriptional repressor, or a functional portion thereof.

[0073] In some embodiments according to any of the methods provided herein, the genome targeting entity and the genome modifying entity are different domains of a single polypeptide. In some embodiments, the genome targeting entity and genome modifying entity are two different polypeptides that are operably linked together. In some embodiments, the genome targeting entity and genome modifying entity are two different polypeptides that are not linked together.

[0074] In some embodiments according to any of the methods provided herein, the genome editing complex comprises a guide nucleic acid having a targeting domain that is complementary to at least one target locus, optionally wherein the guide nucleic acid is a guide RNA (gRNA). In some embodiments, the genome editing complex is an RNA-guided nuclease. In some embodiments, the RNA-guided nuclease comprises a Cas nuclease and a guide RNA (CRISPR- Cas combination). In some embodiments, the CRISPR-Cas combination is a ribonucleoprotein (RNP) complex comprising the gRNA and the Cas nuclease. In some embodiments, the Cas nuclease is a Type II or Type V Cas protein. In some embodiments, the Cas nuclease is selected from the group consisting of Cas3, Cas4, Cas5, Cas8a, Cas8b, Cas8c, Cas9, Cas 10, Cas 12, Casl2a (Cpfl), Casl2b (C2cl), Casl2c (C2c3), Casl2d (CasY), Casl2e (CasX), Casl2f (C2cl0), Casl2g, Casl2h, Casl2i, Casl2k (C2c5), Casl3, Casl3a (C2c2), Casl3b, Casl3c, Casl3d, C2c4, C2c8, C2c9, Cmr5, Csel, Cse2, Csfl, Csm2, Csn2, CsxlO, Csxll, Csyl, Csy2, Csy3, and Mad7.

[0075] In some embodiments according to any of the methods provided herein, the one or more transgenes encode one or more transcription factors. In some embodiments, the one or more transcription factors are selected from the group consisting of OCT4, SOX2, NANOG, KLF4, LIN28, C-MYC, ECAT1, UTF1, ESRRB, SV40LT, HESRG, CDH1, TDGF1, DPPA4, DNMT3B, ZIC3, p53DD, and L1TD1.

[0076] In some embodiments according to any of the methods provided herein, the one or more transgenes encode a recombinant protein. In some embodiments, the one or more transgenes comprise a multi-cistronic construct comprising two or more sequences encoding two or more transgenes. In some embodiments, the one or more transgenes are operably linked to one or more regulatory elements. In some embodiments, the first transgene is operably linked to a first promoter. In some embodiments, the second transgene is operably linked to a second promoter. In some embodiments, the first promoter and the second promoter comprise the same sequence. In some embodiments, the first promoter is selected from the group consisting of: an EFla promoter, an EFla short promoter, a CAG promoter, a ubiquitin/S27a promoter, an SV40 early promoter, an adenovirus major late promoter, a mouse metallothionein-I promoter, an RSV promoter, an MMTV promoter, a Moloney murine leukemia virus Long Terminal repeat region, a CMV promoter, an actin promoter, an immunoglobulin promoter, a heat shock promoter, polyoma virus promoter, a fowlpox virus promoter, a bovine papilloma virus promoter, an avian sarcoma virus promoter, a retrovirus promoter, a hepatitis-B virus promoter, a PGK promoter, an adenovirus late promoter, a vaccinia virus 7.5K promoter, a SV40 promoter, a tk promoter of HSV, a mouse mammary tumor virus (MMTV) promoter, an LTR promoter of HIV, a promoter of moloney virus, an Epstein Barr virus (EBV) promoter, a Rous sarcoma virus (RSV) promoter, a U6 promoter, and an UBC promoter. In some embodiments, the second promoter is selected from the group consisting of: an EFla promoter, an EFla short promoter, a CAG promoter, a ubiquitin/S27a promoter, an SV40 early promoter, an adenovirus major late promoter, a mouse metallothionein-I promoter, an RSV promoter, an MMTV promoter, a Moloney murine leukemia virus Long Terminal repeat region, a CMV promoter, an actin promoter, an immunoglobulin promoter, a heat shock promoter, polyoma virus promoter, a fowlpox virus promoter, a bovine papilloma virus promoter, an avian sarcoma virus promoter, a retrovirus promoter, a hepatitis-B virus promoter, a PGK promoter, an adenovirus late promoter, a vaccinia virus 7.5K promoter, a SV40 promoter, a tk promoter of HSV, a mouse mammary tumor virus (MMTV) promoter, an LTR promoter of HIV, a promoter of moloney virus, an Epstein Barr virus (EBV) promoter, a Rous sarcoma virus (RSV) promoter, a U6 promoter, and an UBC promoter.

[0077] In some embodiments according to any of the methods provided herein, the first vector and/or the second vector is a plasmid, a phagemid, a cosmid, a transposon, or a viral vector. In some embodiments, the viral vector is selected from the group consisting of adenovirus, Chimpanzee adenovirus, lentivirus, alphavirus, retrovirus, poxvirus, flavivirus, adeno-associated virus, rhabdoviridae, herpes simplex virus, chimeric virus, arenavirus, fowlpox virus, vesicular stomatitis virus, vaccinia virus, modified vaccinia virus Ankara, human cytomegalovirus, Sendai virus, measles virus, and Newcastle disease virus.

[0078] In some embodiments according to any of the methods provided herein, the sample is a tissue biopsy sample, a liquid biopsy sample, a control sample, a blood sample, a plasma sample, a cerebrospinal fluid sample, a sputum sample, a stool sample, a urine sample, a saliva sample, a cell sample, a tissue sample, a nucleic acid sample, mRNA, DNA, cell-free DNA, or cell-free RNA. In some embodiments, the sample comprises a cell or population of cells, such as any of the cells or populations of cells provided herein.

[0079] In another aspect provided herein is a vector comprising an identifying region and a transgene sequence, wherein the identifying region comprises a barcode that identifies the vector, wherein the identifying region is located upstream of the transgene sequence within about 300 bp, downstream of the transgene sequence within about 300 bp, or within the transgene sequence. In some embodiments, the first barcode sequence and/or the second barcode sequence comprises primer binding sites, wherein the forward primer binding site is 5' to the barcode sequence, and wherein the reverse primer binding site is 3' to the barcode sequence. [0080] In some embodiments according to any of the vectors provided herein, the forward primer binding sites of the first barcode sequence and of the second barcode sequence comprise the same sequence. In some embodiments, the forward primer binding sites of the first barcode sequence and of the second barcode sequence comprise different sequences. In some embodiments, the reverse primer binding sites of the first barcode sequence and of the second barcode sequence comprise the same sequence. In some embodiments, the reverse primer binding sites of the first barcode sequence and of the second barcode sequence comprise different sequences. In some embodiments, the first and second forward primer binding sites and the first and second reverse primer binding sites are complementary to universal primers. In some embodiments, the first forward primer binding site and the second forward primer binding site is each between about 10 to about 30 nucleotides in length. In some embodiments, the first reverse primer binding site and the second reverse primer binding site is each between about 10 to about 30 nucleotides in length.

[0081] In some embodiments according to any of the vectors provided herein, the first barcode sequence and/or the second barcode sequence is located outside of the first and/or second transgene sequence. In some embodiments, the first barcode sequence and/or the second barcode sequence is located within the first and/or the second transgene sequence. In some embodiments, a portion of the first barcode sequence and/or the second barcode sequence is located within the first and/or the second transgene sequence, and a portion of the first barcode sequence and/or the second barcode sequence is located outside of the first and/or second transgene sequence. In some embodiments, the first barcode sequence and the second barcode sequence are each between about 6 to about 30 nucleotides in length. In some embodiments, the first barcode sequence and the second barcode sequence are about the same nucleotide length. [0082] In some embodiments according to any of the vectors provided herein, the vector further comprises a promoter operably linked to the first transgene and/or the second transgene. [0083] In some embodiments according to any of the vectors provided herein, the first identifying region is located in a non-coding region or a coding region of the vector comprising the first transgene. In some embodiments, the second identifying region is located in a second non-coding region or a second coding region of the vector comprising the second transgene. In some embodiments, the first identifying region and the second identifying region are located in the same region of the vector comprising the first transgene and the second transgene. In some embodiments, the first identifying region is upstream of the first promoter and/or the second identifying region is upstream of the second promoter.

[0084] In some embodiments according to any of the vectors provided herein, the first identifying region is located 5' to the first promoter within about 1 to about 200 base pairs and/or the second identifying region is located 5' to the second promoter within about 1 to about 200 base pairs. In some embodiments, the first identifying region is downstream of the first promoter and/or the second identifying region is downstream of the second promoter. In some embodiments, the first identifying region is located 3' to the first transgene within about 1 to about 200 base pairs and/or the second identifying region is located 3' to the second transgene within about 1 to about 200 base pairs. In some embodiments, the first identifying region is upstream of the first promoter and the second identifying region is downstream of the second promoter. In some embodiments, the first identifying region is downstream of the first promoter and the second identifying region is upstream of the second promoter.

[0085] In some embodiments according to any of the vectors provided herein, the first identifying region and/or the second identifying region is upstream of one or more additional regulatory elements. In some embodiments, the first identifying region and/or second identifying region is downstream of one or more additional regulatory elements. In some embodiments, the one or more additional regulatory elements is selected from the group consisting of: promoter sequences, enhancer sequences, intron sequences, terminator sequences, translation initiation signal sequences, polyadenylation signal sequences, replication element sequences, RNA processing and export element sequences, transposon sequences, transposase sequences, insulator sequences, 5' UTR sequences, 3' UTR sequences, mRNA 3' end processing sequences, boundary element sequences, locus control region (LCR) sequences, matrix attachment region (MAR) sequences, ubiquitous chromatin opening elements, linker sequences, secretion signal sequences, anchoring peptide sequences, localization signal sequences, fusion tag sequences, affinity tag sequences, chaperonin sequences, protease sequences, posttranscriptional regulatory element sequences, and any combination thereof.

[0086] In some embodiments according to any of the vectors provided herein, the first identifying region is located within the first transgene sequence and the second identifying region is located within the second transgene sequence. In some embodiments, the first identifying region is located outside of the first transgene sequence and the second identifying region is located outside of the second transgene sequence. In some embodiments, the first identifying region is located within the first transgene sequence and the second identifying region is located outside of the second transgene sequence. In some embodiments, the first identifying region is located outside of the first transgene sequence and the second identifying region is located within the second transgene sequence.

[0087] In some embodiments according to any of the vectors provided herein, the first barcode sequence and/or the second barcode sequence of the first identifying region and/or the second identifying region comprises a diverged nucleotide sequence. In some embodiments, the diverged nucleotide sequence within the first transgene and/or the second transgene encodes the same amino acid sequence as a non-diverged nucleotide sequence. In some embodiments, the diverged nucleotide sequence is located at a junction between one or more transgene domains. [0088] In some embodiments according to any of the vectors provided herein, the forward primer binding site and the reverse primer binding site are complementary to universal primers. In some embodiments, the forward primer binding sites and/or the reverse primer binding sites are each between about 10 to about 30 nucleotides in length.

[0089] In some embodiments according to any of the vectors provided herein, the first identifying region and/or the second identifying region is each between about 10 to about 100 nucleotides in length. In some embodiments, the first identifying region and/or the second identifying region is each between about 18 and about 30 nucleotides in length. In some embodiments, the first identifying region and/or the second identifying region has more than one barcode sequence. In some embodiments, the first identifying region and/or the second identifying region has more than one probe binding site. In some embodiments, the first identifying region and/or the second identifying region comprises a nucleotide sequence set forth in SEQ ID NOs:24-30. In some embodiments, the first identifying region and/or the second identifying region comprises a first barcode sequence and/or a second barcode sequence each comprising the nucleotide sequence selected from the group consisting of CATCGGAA, GGACAATT, TGTCAACT, TTACAGTT, ATTCAAGG, and TACAGTTA. In some embodiments, the first identifying region and/or the second identifying region comprises a first probe binding site and/or a second probe binding site comprising the nucleotide sequence set forth in SEQ ID NO: 18-23 and 31. [0090] In some embodiments according to any of the vectors provided herein, the vector comprises a plasmid, a phagemid, a viral vector, a cosmid, or a transposon. In some embodiments, the viral vector is selected from the group consisting of adenovirus, Chimpanzee adenovirus, lentivirus, alphavirus, retrovirus, poxvirus, flavivirus, adeno-associated virus, rhabdoviridae, herpes simplex virus, chimeric virus, arenavirus, fowlpox virus, vesicular stomatitis virus, vaccinia virus, modified vaccinia virus Ankara, human cytomegalovirus, Sendai virus, measles virus, and Newcastle disease virus.

[0091] In some embodiments according to any of the vectors provided herein, the first transgene and/or the second transgene encodes an antibody or an antibody fragment, a chimeric antigen receptor (CAR), a chimeric autoantibody receptor (CAAR), a B-cell autoantibody receptor (BAR), a T cell receptor (TCR), an immunomodulatory factor, a safety switch, one or more transcription factors, a genome-editing complex, one or more fusogens, or one or more tolerogenic factors.

[0092] In some embodiments according to any of the vectors provided herein, the first transgene and/or the second transgene encodes a protein selected from the group consisting of: enzymes, structural polypeptides, signaling polypeptides, regulatory polypeptides, transport polypeptides, sensory polypeptides, motor polypeptides, defense polypeptides, storage polypeptides, transcription factors, antibodies, cytokines, hormones, catabolic polypeptides, anabolic polypeptides, proteolytic polypeptides, metabolic polypeptides, kinases, transferases, hydrolases, lyases, isomerases, ligases, enzyme modulator polypeptides, protein binding polypeptides, lipid binding polypeptides, membrane fusion polypeptides, cell differentiation polypeptides, epigenetic polypeptides, cell death polypeptides, nuclear transport polypeptides, nucleic acid binding polypeptides, reprogramming polypeptides, DNA editing polypeptides, DNA repair polypeptides, DNA recombination polypeptides, transposase polypeptides, DNA integration polypeptides, targeted endonucleases, recombinases, transposases, DNA polymerases, RNA polymerases, and reverse transcriptase.

[0093] In some embodiments according to any of the vectors provided herein, the first transgene and/or the second transgene encodes a therapeutic antibody. In some embodiments, the therapeutic antibody is an antibody that binds to an antigen selected from the group consisting of CD47, Sirpa, CD52, amyloid beta, angiopoietin-like protein 3 (ANGPTL3), B cell activating factor (BAFF), A proliferation-inducing ligand (APRIL), B cell maturation antigen (BCMA), B. anthracis protective antigen (B. anthracis PA), calcitonin gene-related peptide (CGRP), calcitonin gene-related peptide receptor (CGRP-R), C-C chemokine receptor type 4 (CCR4), CD147, CD19, CD3, CD2, CD20, CD22, CD25, epithelial cell adhesion molecule (EpCAM), Glycoprotein 100 (GP100), CD30, CD33, CD38, CD4, CD52, CD6, CD79b, CD80, CD86, Clostridium difficile Toxin B, coagulation factor IX (Factor IX), coagulation factor X (Factor X), complement Cis (Cis), complement C5 (C5), cytotoxic T-lymphocyte antigen 4 (CTLA4), dabigatran, DNA/Histone Hl, Ebola virus Glycoprotein, epidermal growth factor receptor (EGFR), C-Met, epithelial cell adhesion molecule (EpCAM), Factor IX, Factor VIII, fibroblast growth factor 23 (FGF23), ganglioside GM3 (GM3), GD2 ganglioside (GD2), human epidermal growth factor receptor 2 (HER2), immunoglobulin E (IgE), insulin-like growth factor 1 receptor (IGF-1R), integrin alpha lib beta 3 (Integrin allbp3), integrin alpha4 beta7 (Integrin a4p7), integrin subunit alpha 4 (ITGA4), integrin subunit alpha L (ITGAL), interferon alpha receptor 1 (IFNAR-1), interferon gamma (IFN-y), interleukin 1 alpha (IL-la), interleukin 1 beta (IL-1 P), interleukin 12, (IL- 12), interleukin 23 (IL- 23), interleukin 13 (IL- 13), interleukin 17 receptor alpha (IL-17 RA), interleukin 17A (IL-17A), interleukin 17F (IL-17F), interleukin 23 pl9 (IL- 23pl9), interleukin 31 receptor subunit alpha (IL-31RA), interleukin 4 receptor alpha (IL-4RA), interleukin 5 (IL-5), interleukin 5 receptor subunit alpha (IL-5RA), interleukin 6 (IL-6), interleukin 6 receptor (IL-6R), interleukin 8 (IL-8), Kallikrein, Lipid A region of endotoxin, lymphocyte activation gene 3 (LAG-3), programmed cell death 1 (PD-1), nectin cell adhesion molecule 4 (Nectin 4), neonatal Fc Receptor (FcRn), platelet-derived growth factor receptor alpha (PDGFRA), programmed cell death 1 ligand 1 (PD-L1), proprotein convertase subtilisin/kexin Type 9 (PCSK9), P-selectin, rabies virus glycoprotein (Rabies virus GP), receptor activator of nuclear factor kappaB ligand (RANKL), respiratory syncytial virus F protein (RSV-F), SARS-Cov-2 spike protein (SARS-Cov-2 S protein), sclerostin, SLAM family member 7 (SLAMF7), thrombopoietin receptor (TPOR), thymic stromal lymphopoietin (TSLP), tissue factor (TF), transferrin receptor protein 1 (TFR1), transforming growth factor-beta (TGF- P), tumor necrosis factor-alpha (TNF-a), tumor-associated calcium signal transducer 2 (TACSTD-2), vascular endothelial growth factor (VEGF), vascular endothelial growth factor A (VEGF-A), angiopoietin 2 (ANG-2), vascular endothelial growth factor receptor 2 (VEGFR-2), or von Willebrand Factor (vWF), and any combination thereof. [0094] In some embodiments according to any of the vectors provided herein, the first transgene and/or the second transgene comprises a sequence encoding one or more fusogens. In some embodiments, the one or more fusogens are selected from the group consisting of NiV-F, NiV-G, Gag-Pol, and Rev. In some embodiments, the first transgene and/or the second transgene comprise a sequence encoding a retroviral vector. In some embodiments, the retroviral vector is a lentiviral vector. In some embodiments, the first transgene and/or the second transgene comprises a sequence encoding an AAV vector. In some embodiments, the first transgene and/or the second transgene comprises a sequence encoding a virus-like particle.

[0095] In some embodiments according to any of the vectors provided herein, the first transgene and/or the second transgene encodes a CAR. In some embodiments, the CAR encoded by the first transgene and/or the second transgene comprises a hinge domain, a transmembrane domain, and one or more signaling domains. In some embodiments, the hinge domain is selected from the group consisting of: CD8a hinge domain, CD28 hinge domain, IgG4 hinge domain, IgG4 hinge-CH2-CH3 domain, and any functional variant thereof. In some embodiments, the transmembrane domain is selected from the group consisting of: alpha, beta, or zeta chain of a T cell receptor, CD28, CD3s, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CD8a, CD8p, 4-1BB/CD137, CD28, CD34, CD4, FcsRIy, CD 16, OX40/CD134, CD3^, CD3s, CD3y, CD35, TCRa, TCRp, TCR^, CD32, CD64, CD64, CD45, CD5, CD9, CD22, CD37, CD80, CD86, CD40, CD40L/CD154, VEGFR2, FAS, FGFR2B, and any functional variant thereof. In some embodiments, the signaling domain is selected from the group consisting of: B7-1/CD80, B7-2/CD86, B7-H1/PD-L1, B7-H2, B7-H3, B7-H4, B7-H6, B7-H7, BTLA/CD272, CD28, CTLA-4, Gi24/VISTA/B7-H5, ICOS/CD278, PD-1, PD-L2/B7-DC, PDCD6, 4-1BB/TNFSF9/CD137, 4-1BB Ligand/TNFSF9, BAFF/BLyS/TNFSF13B, BAFF R/TNFRSF13C, CD27/TNFRSF7, CD27 Ligand/TNFSF7, CD30/TNFRSF8, CD30 Ligand/TNFSF8, CD40/TNFRSF5, CD40/TNFSF5, CD40 Ligand/TNFSF5, DR3/TNFRSF25, GITR/TNFRSF18, GITR Ligand/TNFSF18, HVEM/TNFRSF14, LIGHT/TNFSF14, Lymphotoxin-alpha/TNFp, OX40/TNFRSF4, 0X40 Ligand/TNFSF4, RELT/TNFRSF19L, TACI/TNFRSF I 3B, TL1A/TNFSF15, TNFa, TNF RII/TNFRSF I B, 2B4/CD244/SLAMF4, BLAME/SLAMF8, CD2, CD2F-10/SLAMF9, CD48/SLAMF2, CD58/LFA-3, CD84/SLAMF5, CD229/SLAMF3, CRACC/SLAMF7, NTB- A/SLAMF6, SLAM/CD150, CD7, CD53, CD82/Kai-1, CD90/Thyl, CD96, CD160, CD200, CD300a/LMIRl, HLA Class I, HLA-DR, Ikaros, Integrin alpha 4/CD49d, Integrin alpha 4 beta 1, Integrin alpha 4 beta 7/LPAM-l, LAG-3, TCL1A, TCL1B, CRTAM, DAP12, Dectin- 1/CLEC7A, DPPIV/CD26, EphB6, TIM- 1 /KIM- 1 /HA VCR, TIM-4, TSLP, TSLP R, lymphocyte function associated antigen- 1 (LFA-1), NKG2C, CD3(^, an immunoreceptor tyrosine-based activation motif (ITAM), CD27, 4-1BB, CD134/OX40, CD30, CD40, PD-1, ICOS, LIGHT, NKG2C, a ligand that specifically binds with CD83, any functional variant thereof, and any combination thereof. In some embodiments, the CAR encoded by the first transgene and/or the second transgene comprises a CD8a hinge domain, a CD8a transmembrane domain, a 4- IBB domain, and a CD3-zeta domain. In some embodiments, the CAR encoded by the first transgene and/or the second transgene further comprises one or more co- stimulatory domain(s).

[0096] In some embodiments according to any of the vectors provided herein, the CAR encoded by the first transgene and/or the second transgene is selected from the group consisting of: a CD5-specific CAR, a CD19-specific CAR, a CD20-specific CAR, a CD22-specific CAR, a CD23-specific CAR, a CD30-specific CAR, a CD33-specific CAR, CD38-specific CAR, a CD70-specific CAR, a CD 123 -specific CAR, a CD 138- specific CAR, a Kappa, Lambda, B cell maturation agent (BCMA)-specific CAR, a G-protein coupled receptor family C group 5 member D (GPRC5D)- specific CAR, a CD123-specific CAR, a LeY-specific CAR, a NKG2D ligand- specific CAR, a WTl-specific CAR, a GD2-specific CAR, a HER2-specific CAR, a EGFR-specific CAR, a EGFRv III- specific CAR, a B7H3-specific CAR, a PSMA-specific CAR, a PSCA-specific CAR, a CAIX-specific CAR, a CD 171 -specific CAR, a CEA-specific CAR, a CSPG4- specific CAR, a EPH A2- specific CAR, a FAP-specific CAR, a FRa-specific CAR, a IL- 13Ra-specific CAR, a Mesothelin- specific CAR, a MUCl-specific CAR, a MUC16-specific CAR, a RORl-specific CAR, a C-Met-specific CAR, a CD 133- specific CAR, a Ep-CAM- specific CAR, a GPC3-specific CAR, a HPV16-E6- specific CAR, a IL13Ra2-specific CAR, a MAGEA3- specific CAR, a MAGEA4- specific CAR, a MARTI -specific CAR, a NY-ESO-1- specific CAR, a VEGFR2- specific CAR, a a-Folate receptor- specific CAR, a CD24- specific CAR, a CD44v7/8-specific CAR, a EGP-2-specific CAR, a EGP-40-specific CAR, a erb-B2- specific CAR, a erb-B 2,3,4-specific CAR, a FBP-specific CAR, a Fetal acethylcholine e receptor- specific CAR, a GD2-specific CAR, a GD3-specific CAR, a HMW-MAA- specific CAR, a IL- HRa- specific CAR, a KDR-specific CAR, a Lewis Y-specific CAR, a Ll-cell adhesion molecule- specific CAR, a MAGE-A1 -specific CAR, a Oncofetal antigen (h5T4)- specific CAR, a TAG-72-specific CAR, and a CD19/CD22-bispecific CAR. In some embodiments, the CAR encoded by the first transgene and/or the second transgene is a CD 19 CAR or a CD22 CAR.

[0097] In some embodiments according to any of the vectors provided herein, the CARs encoded by the first transgene and the second transgene and optionally additional transgenes bind specifically to antigens selected from the group consisting of CD 19 and CD20, CD 19 and BCMA, CD20 and BCMA, CD19 and CD22, CD19 and BAFFR, CD33 and CD123, HER2 and B7H3, HER2 and EGFR, HER2 and IL13Ra, HER2 and ROR1, B7H3 and EGFR, B7H3 and IL13Ra, B7H3 and ROR1, EGFR and IL13Ra, EGFR and ROR1, and Her2 and B7H3 and EGFR and IL13Ra2 and ROR1. In some embodiments, the CARs encoded by the first transgene and the second transgene bind specifically to autoimmune disease antigens selected from the group consisting of CD 19 and CD20, CD 19 and BCMA, CD20 and BCMA, and CD 19 and CD22. In some embodiments, the CARs encoded by the first transgene and the second transgene bind specifically to leukemia or lymphoma antigens and is selected from the group consisting of CD19 and CD20, CD19 and BAFFR, and CD19 and CD22. In some embodiments, the CARs encoded by the first transgene and the second transgene bind specifically to the acute myeloid leukemia antigens CD33 and CD123. In some embodiments, the CARs encoded by the first transgene and the second transgene and optionally additional transgenes bind specifically to the solid tumor antigens Her2 and B7H3 and EGFR and IL13Ra2 and ROR1.

[0098] In some embodiments according to any of the vectors provided herein, the diverged nucleotide sequence within the first transgene and/or within the second transgene is located at the junction of: i) the signaling domain and the co- stimulatory domain; or ii) the hinge domain and the transmembrane domain. In some embodiments, the diverged nucleotide sequence within the first transgene and/or within the second transgene is located at the junction of the 4- IBB and CD3-zeta domains.

[0099] In some embodiments according to any of the vectors provided herein, the one or more transgenes comprise a chimeric autoantibody receptor (CAAR). In some embodiments, the CAAR comprises an antigen selected from the group consisting of a pancreatic P-cell antigen, synovial joint antigen, myelin basic protein, proteolipid protein, myelin oligodendritic glycoprotein, MuSK, keratinocyte adhesion protein desmoglein 3 (Dsg3), Ro-RNP complex, La antigen, myeloperoxidase, proteinase 3, cardiolipin, citrullinated proteins, carbamylated proteins, and a3 chain of basement membrane collagen.

[0100] In some embodiments according to any of the vectors provided herein, the one or more transgenes comprise a B-cell autoantibody receptor (BAR). In some embodiments, the BAR comprises an FVIII antigen.

[0101] In some embodiments according to any of the vectors provided herein, the one or more transgenes comprise a T cell receptor (TCR).

[0102] In some embodiments according to any of the vectors provided herein, the one or more transgenes comprise a tolerogenic factor. In some embodiments, the tolerogenic factor is selected from the group consisting of: CD16, CD24, CD35, CD39, CD46, CD47, CD52, CD55, CD59, CD64, CD200, CCL22, CTLA4-Ig, Cl inhibitor, FASL, IDO1, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, IL-10, IL-35, PD-L1, SERPINB9, CCL21, MFGE8, DUX4, B2M-HLA-E, CD27, IL-39, CD16 Fc Receptor, IL15-RF, H2-M3 (HLA-G), A20/TNFAIP3, CR1, HLA-F, and MANF.

[0103] In some embodiments according to any of the vectors provided herein, the one or more transgenes comprise a safety switch. In some embodiments, the safety switch is selected from the group consisting of herpes simplex virus thymidine kinase (HSV-tk), cytosine deaminase (CyD), nitroreductase (NTR), purine nucleoside phosphorylase (PNP), horseradish peroxidase, inducible caspase 9 (iCasp9), rapamycin-activated caspase 9 (rapaCasp9), CCR4, CD16, CD19, CD20, CD30, EGFR, GD2, HER1, HER2, MUC1, PSMA, and RQR8. In some embodiments, the safety switch is a system wherein, upon activation, cells downregulate expression of one or more tolerogenic factors and/or upregulate expression of one or more immune signaling molecules, thereby marking the cell for elimination by the host immune system. In some embodiments, the one or more tolerogenic factors are selected from the group consisting of: CD16, CD24, CD35, CD39, CD46, CD47, CD52, CD55, CD59, CD64, CD200, CCL22, CTLA4-Ig, Cl inhibitor, FASL, IDO1, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, IL-10, IL-35, PD-L1, SERPINB9, CCL21, MFGE8, DUX4, B2M-HLA-E, CD27, IL-39, CD16 Fc Receptor, IL15-RF, H2-M3 (HLA-G), A20/TNFAIP3, CR1, HLA-F, and MANF. In some embodiments, the one or more immune signaling molecules are selected from the group consisting of B2M, HLA-A, HLA-B, HLA-C, HLA-D, HLA-E, RFXANK, CIITA, CTLA-4, PD-1, RAET1E/ULBP4, RAET1G/ULBP5, RAET1H/ULBP2, RAET1/ULBP1, RAET1L/ULBP6, RAET1N/ULBP3, and ligands of NKG2D.

[0104] In some embodiments according to any of the vectors provided herein, the one or more transgenes encode a genome editing complex. In some embodiments, the genome editing complex comprises a genome targeting entity and a genome modifying entity. In some embodiments, the genome targeting entity is a nucleic acid-guided targeting entity. In some embodiments, the genome targeting entity is selected from the group consisting of: a sequence specific nuclease, a nucleic acid programmable DNA binding protein, an RNA guided nuclease, RNA-guided nuclease comprising a Cas nuclease and a guide RNA (CRISPR-Cas combination), a ribonucleoprotein (RNP) complex comprising a gRNA and a Cas nuclease, a homing endonuclease, a zinc finger nuclease (ZF) nucleic acid binding entity, a transcription activatorlike effector (TALE) nucleic acid binding entity, a meganuclease, a Cas nuclease, a core Cas protein, a homing endonuclease, an endonuclease-deficient-Cas protein, an enzymatically inactive Cas protein, a CRIS PR-associated transposase (CAST), a Type II or Type V Cas protein, or a functional portion thereof. In some embodiments, the genome targeting entity is selected from the group consisting of Casl, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8a, Cas8b, Cas8c, Cas9, CaslO, Casl2, Casl2a (Cpfl), Casl2b (C2cl), Casl2c (C2c3), Casl2d (CasY), Casl2e (CasX), Casl2f (C2cl0), Casl2g, Casl2h, Casl2i, Casl2k (C2c5), Casl3, Casl3a (C2c2), Casl3b, Casl3c, Casl3d, C2c4, C2c8, C2c9, Cmrl, Cmr2, Cmr3, Cmr4, Cmr5, Cmr6, Csdl, Csd2, Cas5d, Csel, Cse2, Cse3, Cse4, Cas5e, Csfl, Csml, Csm2, Csm3, Csm4, Csm5, Csnl, Csn2, Cstl, Cst2, Cas5t, Cshl, Csh2, Cas5h, Csal, Csa2, Csa3, Csa4, Csa5, Cas5a, CsxlO, Csxl l, Csyl, Csy2, Csy3, Csy4, Mad7, SpCas9, eSpCas9, SpCas9-HFl, HypaSpCas9, HeFSpCas9, and evoSpCas9 high-fidelity variants of SpCas9, SaCas9, NmeCas9, CjCas9, StCas9, TdCas9, LbCasl2a, AsCasl2a, AacCasl2b, BhCasl2b v4, TnpB, dCas (D10A), dCas (H840A), dCasl3a, dCasl3b, or a functional portion thereof.

[0105] In some embodiments according to any of the vectors provided herein, the genome modifying entity cleaves, deaminates, nicks, polymerizes, interrogates, integrates, cuts, unwinds, breaks, alters, methylates, demethylates, or otherwise destabilizes the target locus. In some embodiments, n the genome modifying entity comprises a recombinase, integrase, transposase, endonuclease, exonuclease, nickase, helicase, DNA polymerase, RNA polymerase, reverse transcriptase, deaminase, flippase, methylase, demethylase, acetylase, a nucleic acid modifying protein, an RNA modifying protein, a DNA modifying protein, an Argonaute protein, an epigenetic modifying protein, a histone modifying protein, or a functional portion thereof. In some embodiments, the genome modifying entity is selected from the group consisting of: a sequence specific nuclease, a nucleic acid programmable DNA binding protein, an RNA guided nuclease, RNA-guided nuclease comprising a Cas nuclease and a guide RNA (CRISPR-Cas combination), a ribonucleoprotein (RNP) complex comprising the gRNA and the Cas nuclease, a homing endonuclease, a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a meganuclease, a Cas nuclease, a core Cas protein, a TnpB nuclease, an endonuclease-deficient-Cas protein, an enzymatically inactive Cas protein, a CRISPR-associated transposase (CAST), a Type II or Type V Cas protein, base editing, prime editing, a Programmable Addition via Site-specific Targeting Elements (PASTE), or a functional portion thereof. In some embodiments, the genome modifying entity is selected from the group consisting of Casl, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8a, Cas8b, Cas8c, Cas9, Cas 10, Casl2, Casl2a (Cpfl), Casl2b (C2cl), Casl2c (C2c3), Casl2d (CasY), Casl2e (CasX), Casl2f (C2cl0), Casl2g, Casl2h, Casl2i, Casl2k (C2c5), Casl3, Casl3a (C2c2), Casl3b, Casl3c, Casl3d, C2c4, C2c8, C2c9, Cmrl, Cmr2, Cmr3, Cmr4, Cmr5, Cmr6, Csdl, Csd2, Cas5d, Csel, Cse2, Cse3, Cse4, Cas5e, Csfl, Csml, Csm2, Csm3, Csm4, Csm5, Csnl, Csn2, Cstl, Cst2, Cas5t, Cshl, Csh2, Cas5h, Csal, Csa2, Csa3, Csa4, Csa5, Cas5a, CsxlO, Csxll, Csyl, Csy2, Csy3, Csy4, Mad7, SpCas9, eSpCas9, SpCas9-HFl, HypaSpCas9, HeFSpCas9, and evoSpCas9 high-fidelity variants of SpCas9, SaCas9, NmeCas9, CjCas9, StCas9, TdCas9, LbCasl2a, AsCasl2a, AacCasl2b, BhCasl2b v4, TnpB, FokI, dCas (D10A), dCas (H840A), dCasl3a, dCasl3b, a base editor, a prime editor, a target-primed reverse transcription (TPRT) editor, APOBEC1, cytidine deaminase, adenosine deaminase, uracil glycosylase inhibitor (UGI), adenine base editors (ABE), cytosine base editors (CBE), reverse transcriptase, serine integrase, recombinase, transposase, polymerase, adenine-to-thymine or “ATBE” (or thymine-to-adenine or “TABE”) transversion base editor, ten-eleven translocation methylcytosine dioxygenases (TETs), TET1, TET3, TET1CD, histone acetyltransferase p300, histone methyltransferase SMYD3, histone methyltransferase PRDM9, H3K79 methyltransferase DOT1L, transcriptional repressor, or a functional portion thereof.

[0106] In some embodiments according to any of the vectors provided herein, the genome targeting entity and the genome modifying entity are different domains of a single polypeptide. In some embodiments, the genome targeting entity and genome modifying entity are two different polypeptides that are operably linked together. In some embodiments, the genome targeting entity and genome modifying entity are two different polypeptides that are not linked together. In some embodiments, the genome editing complex comprises a guide nucleic acid having a targeting domain that is complementary to at least one target locus, optionally wherein the guide nucleic acid is a guide RNA (gRNA). In some embodiments, the genome editing complex is an RNA-guided nuclease. In some embodiments, the RNA-guided nuclease comprises a Cas nuclease and a guide RNA (CRISPR-Cas combination). In some embodiments, the CRISPR-Cas combination is a ribonucleoprotein (RNP) complex comprising the gRNA and the Cas nuclease. In some embodiments, the Cas nuclease is a Type II or Type V Cas protein. In some embodiments, the Cas nuclease is selected from the group consisting of Cas3, Cas4, Cas5, Cas8a, Cas8b, Cas8c, Cas9, CaslO, Casl2, Casl2a (Cpfl), Casl2b (C2cl), Casl2c (C2c3), Casl2d (CasY), Casl2e (CasX), Casl2f (C2cl0), Cas 12g, Casl2h, Casl2i, Cas 12k (C2c5), Casl3, Casl3a (C2c2), Casl3b, Casl3c, Casl3d, C2c4, C2c8, C2c9, Cmr5, Csel, Cse2, Csfl, Csm2, Csn2, CsxlO, Csxl l, Csyl, Csy2, Csy3, and Mad7.

[0107] In some embodiments according to any of the vectors provided herein, the one or more transgenes encode one or more transcription factors. In some embodiments, the one or more transcription factors are selected from the group consisting of OCT4, SOX2, NANOG, KLF4, LIN28, C-MYC, ECAT1, UTF1, ESRRB, SV40LT, HESRG, CDH1, TDGF1, DPPA4, DNMT3B, ZIC3, p53DD, and L1TD1.

[0108] In some embodiments according to any of the vectors provided herein, the one or more transgenes encode a recombinant protein. In some embodiments, the one or more transgenes comprise a multi-cistronic construct comprising two or more sequences encoding two or more transgenes. In some embodiments, the one or more transgenes are operably linked to one or more regulatory elements.

[0109] In another aspect is provided a cell comprising any of the vectors provided herein. In some embodiments, the cell is a mammalian cell. In some embodiments, the mammalian cell is a human cell. In some embodiments, the cell is an immune cell. In some embodiments, the cell is selected from the group consisting of: islet cells, beta islet cells, pancreatic islet cells, immune cells, B cells, T cells, natural killer (NK) cells, natural killer T (NKT) cells, macrophage cells, endothelial cells, muscle cells, cardiac muscle cells, smooth muscle cells, skeletal muscle cells, dopaminergic neurons, retinal pigmented epithelium cells, optic cells, hepatocytes, thyroid cells, skin cells, glial progenitor cells, neural cells, cardiac cells, stem cells, hematopoietic stem cells, induced pluripotent stem cells (iPSCs), mesenchymal stem cells (MSCs), embryonic stem cells (ESCs), pluripotent stem cell (PSCs), blood cells, endothelial stem cells, epithelial stem cells, adipose stem or progenitor cells, germline stem cells, lung stem or progenitor cells, mammary stem cells, olfactory adult stem cells, hair follicle stem cells, multipotent stem cells, amniotic stem cells, cord blood stem cells, neural stem or progenitor cells, HKB-11, CAP, BOSC 23 cells, WI-38 cells, MRC5 cells, A549 cells, HT1080 cells, HEK293 cells, HEK293T cells, HepG2 cells, Saos-2 cells, Huh7 cells, HeLa cells, MSTO-211 cells, PerC6, and MSTO-211 A cells. In some embodiments, the cell is a primary human cell.

[0110] In some embodiments according to any of the cells provided herein, the primary human cell is a pluripotent stem cell. In some embodiments, the pluripotent stem cell is derived or differentiated into islet cells. In some embodiments, the pluripotent stem cell or the derived stem cells are engineered to be hypoimmunogenic islet cells, wherein the cells are engineered to comprise any of the vectors provided herein. In some embodiments, the engineered hypoimmunogenic islet cells comprise engineered beta islet cells. In some embodiments, the engineered hypoimmunogenic islet cells further comprise additional engineered islet cells, wherein the additional engineered islet cells comprise alpha cells and/or delta cells. In some embodiments, the additional engineered islet cells comprise cells that express the same vector as the engineered beta islet cells. In some embodiments, the engineered hypoimmunogenic islets is an islet cluster.

[0111] In some embodiments according to any of the cells provided herein, the pluripotent stem cell is derived or differentiated into an immune cell. In some embodiments, the pluripotent stem cell or the derived stem cells are engineered to be hypoimmunogenic immune cells, wherein the cells are engineered to comprise any of the vectors provided herein. In some embodiments, the engineered hypoimmunogenic immune cells comprise engineered hypoimmunogenic T cells. In some embodiments, the engineered hypoimmunogenic T cells further comprise additional engineered hypoimmunogenic immune cells, wherein the additional engineered hypoimmunogenic immune cells comprise B cells and/or NK cells. In some embodiments, the additional engineered hypoimmunogenic immune cells comprise cells that express the same vector as the engineered hypoimmunogenic T cells. In some embodiments, the engineered hypoimmunogenic T cell is a conventional T cell, a regulatory T cell, a CD4+ T cell, a CD8+ T cell, an a/p T cell, a y/8 T cell, a tissue resident T cell, a tumor infiltrating T cell, a naive T cell, a memory T cell, an effector T cell, a cytolytic T cell, a helper T cell, a primed T cell, an activated T cell, a proliferating T cell, or a combination of one or more of the above T cells.

[0112] In some embodiments according to any of the cells provided herein, the engineered hypoimmunogenic islet cells comprise any of the vectors provided herein, wherein the vector generates germline modifications that: (a) inactivate or disrupt one or more alleles of: (i) one or more major histocompatibility complex (MHC) class I molecules or one or more molecules that regulate expression of the one or more MHC class I molecules, and/or (ii) one or more MHC class II molecules or one or more molecules that regulate expression of the one or more MHC class II molecules; and/or (b) increase expression of one or more tolerogenic factors, wherein the increased expression is relative to a control or wild-type islet that does not comprise the modifications.

[0113] In some embodiments according to any of the cells provided herein, the one or more molecules that regulate expression of the one or more MHC class I molecules is B2M. In some embodiments, the modifications comprise a modification that regulates the expression of the one or more MHC class I molecules, and the modification inactivates or disrupts one or more alleles of B2M. In some embodiments, the modification that inactivates or disrupts one or more alleles of B2M reduces mRNA expression of the B2M gene. In some embodiments, the modification that inactivates or disrupts one or more alleles of B2M reduces protein expression of B2M. In some embodiments, the modification that inactivates or disrupts one or more alleles of B2M comprises: inactivation or disruption of one allele of the B2M gene; inactivation or disruption of both alleles of the B2M gene; or inactivation or disruption of all B2M coding alleles in the cell. In some embodiments, the inactivation or disruption comprises an indel in the B2M gene. In some embodiments, the inactivation or disruption comprises a frameshift mutation or a deletion of a contiguous stretch of genomic DNA of the B2M gene.

[0114] In some embodiments according to any of the cells provided herein, the modification is a modification that regulates expression of the one or more MHC class II molecules, and the modification inactivates or disrupts one or more alleles of CIITA. In some embodiments, the modification that inactivates or disrupts one or more alleles of CIITA reduces protein expression of CIITA. In some embodiments, the modification that inactivates or disrupts one or more alleles of CIITA comprises: inactivation or disruption of one allele of the CIITA gene; inactivation or disruption of both alleles of the CIITA gene; or inactivation or disruption of all CIITA coding alleles in the cell. In some embodiments, the inactivation or disruption comprises an indel in the CIITA gene. In some embodiments, the inactivation or disruption is a frameshift mutation or a deletion of a contiguous stretch of genomic DNA of the CIITA gene. [0115] In some embodiments according to any of the cells provided herein, expression of HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, and HLA-DR are reduced in the engineered hypoimmunogenic islets.

[0116] In some embodiments according to any of the cells provided herein, the vector comprises one or more transgenes comprising one or more tolerogenic factors selected from the group consisting of CD16, CD24, CD35, CD39, CD46, CD47, CD52, CD55, CD59, CD64, CD200, CCL22, CTLA4-Ig, Cl inhibitor, FASL, IDO1, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, IL-10, IL-35, PD-L1, SERPINB9, CCL21, MFGE8, DUX4, B2M-HLA-E, CD27, IL- 39, CD16 Fc Receptor, IL15-RF, H2-M3 (HLA-G), A20/TNFAIP3, CR1, HLA-F, and MANF. In some embodiments, at least one of the one or more tolerogenic factors is CD47. In some embodiments, at least one of the one or more tolerogenic factors is PD-L1. In some embodiments, at least one of the one or more tolerogenic factors is HLA-E. In some embodiments, at least one of the one or more tolerogenic factors is HLA-G.

[0117] In some embodiments according to any of the cells provided herein, the engineered hypoimmunogenic islet cells further comprise a modification to increase expression of an exogenous safety switch. In some embodiments, the engineered hypoimmunogenic islet cells have the genotype B2M^indel/indel ; ciITA'^ndel/indel ; CD47tg; safety switch transgene. In some embodiments, the safety switch is a system wherein upon activation, cells downregulate expression of the one or more tolerogenic factors and/or upregulate expression of one or more immune signaling molecules, thereby marking the cell for elimination by the host immune system. In some embodiments, the one or more immune signaling molecules is selected from the group consisting of B2M, HLA-A, HLA-B, HLA-C, HLA-D, HLA-E, RFXANK, CIITA, CTLA-4, PD-1, RAET1E/ULBP4, RAET1G/ULBP5, RAET1H/ULBP2, RAET1/ULBP1, RAET1L/ULBP6, RAET1N/ULBP3, and other ligands of NKG2D. In some embodiments, the safety switch is a suicide gene. In some embodiments, the suicide gene is selected from the group consisting of cytosine deaminase (CyD), herpesvirus thymidine kinase (HSV-Tk), an inducible caspase 9 (iCaspase9), and rapamycin-activated caspase 9 (rapaCasp9).

[0118] In some embodiments according to any of the cells provided herein, the one or more modifications in (a) reduce cell surface protein expression of the one or more MHC class I molecules, optionally wherein the one or more modifications in (a) reduce cell surface trafficking of the one or more MHC class I molecules. In some embodiments, the one or more modifications in (a) reduce a function of the one or more MHC class I molecules, optionally wherein the function is antigen presentation. In some embodiments, the one or more modifications comprise a modification that regulates cell surface protein expression of the one or more MHC class I molecules and the modification inactivates or disrupts one or more alleles of B2M.

[0119] Also provided herein is a population of cells comprising any of the vectors or the cells provided herein. In some embodiments, the population of cells is selected from the group consisting of immune cells, B cells, T cells, natural killer (NK) cells, natural killer T (NKT) cells, macrophage cells, monocytes, and dendritic cells. In some embodiments, the population of cells further comprises a first subpopulation of cells and a second subpopulation of cells. In some embodiments, the first subpopulation of cells and/or the second subpopulation of cells is a population of engineered T cells comprising a chimeric antigen receptor (CAR).

[0120] In some embodiments according to any of the population of cells provided herein: i. the first subpopulation of cells comprises a first vector comprising a first barcode and a first transgene encoding a CD 19 CAR, and/or ii. wherein the second subpopulation of cells comprises a second vector comprising a second barcode and a second transgene encoding a CD22 CAR. In some embodiments, the population of cells further comprises a third subpopulation of cells. In some embodiments, the third subpopulation of cells comprises: i) a vector comprising a first barcode and a first transgene encoding a CD 19 CAR and/or a second barcode and a second transgene encoding a CD22 CAR; ii) a first vector comprising a first barcode and a first transgene encoding a CD 19 CAR and/or a second vector comprising a second barcode and a second transgene encoding a CD22 CAR; or iii) a vector comprising a barcode and a transgene encoding a CD19/CD22-bispecific CAR.

[0121] In some embodiments according to any of the population of cells provided herein, the population of cells further comprises a fourth population of cells. In some embodiments the fourth subpopulation of cells does not comprise a CAR. [0122] In some embodiments according to any of the population of cells provided herein the first subpopulation of cells and the second subpopulation of cells comprise the same cell or the same population of cells provided herein. In some embodiments, the population of cells comprises three or more subpopulations of cells, wherein each subpopulation of cells comprises a vector comprising a unique barcode sequence associated with a sequence encoding a transgene, wherein each unique barcode sequence is used to identify a population of cells comprising the transgene. In some embodiments, the population of cells comprises a first vector comprising a first transgene encoding CD 19 chimeric antigen receptor (CAR), and a second vector comprising a second transgene encoding CD22 CAR.

[0123] Also provided herein is a composition comprising any the cells or populations of cells provided herein, and any of the cells, populations of cells, or composition provided herein for use in the method provided herein. In some embodiments, the cell or population of cells are engineered in vivo by administering a vector comprising the first transgene and the first barcode and/or the second transgene and the second barcode to a subject. In some embodiments, the vector is packaged in a fusosome for trafficking to the target cell or target population of cells in vivo within the subject.

[0124] In some embodiments, any of the methods, vectors, cells, or population of cells provided herein further comprises a third, fourth, fifth, or more transgene with a third, fourth, fifth or more barcode, optionally encoded by the same vector, different vectors, or a combination thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

[0125] The drawings illustrate certain features and advantages of this disclosure. These embodiments are not intended to limit the scope of the appended claims in any manner.

[0126] FIG. 1 depicts a schematic of a vector with a representative barcode sequence insert. The magnified inset shows the identifying region (including the barcode insert), containing forward and reverse primer binding sites and a probe binding site, which is located upstream of the EFl-alpha promoter. The barcode sequence is capitalized and encased within a black outline. Sequences containing nucleotides highlighted in grey at the bottom of the magnified inset show exemplary barcodes from clones carrying transgenes. (Sequences of FIG. 1: SEQ ID NO: 376: ACAATTACTGCTGATCGAGTGTAGCCTTATCGCAGCTAGACGAAACTCATCGGAAA GACGGAGAAGTAACGCAGGAATGAAAGACCCCACCTGTAGGTTTGGCAAGA;

SEQ ID NO: 377:

TCTTGCCAAACCTACAGGTGGGGTCTTTCATTCCTGCGTTACTTCTCCGTCTTTCCGA TGAGTTTCGTCTAGCTGCGATAAGGCTACACTCGATCAGCAGTAATTGT;

SEQ ID NO: 378:

ACAATTACTGCTGATCGAGTGTAGCCTTATCGCAGCTAGACGAAACTGGACAATTAG ACGGAGAAGTAACGCAGGAATGAAAGACCCCACCTGTAGGTTTGGCAAGA;

SEQ ID NO: 379:

ACAATTACTGCTGATCGAGTGTAGCCTTATCGCAGCTAGACGAAACTTGTCAACTAG ACGGAGAAGTAACGCAGGAATGAAAGACCCCACCTGTAGGTTTGGCAAGA;

SEQ ID NO: 380:

ACAATTACTGCTGATCGAGTGTAGCCTTATCGCAGCTAGACGAAACTTTACAGTTAG ACGGAGAAGTAACGCAGGAATGAAAGACCCCACCTGTAGGTTTGGCAAGA;

SEQ ID NO: 381:

ACAATTACTGCTGATCGAGTGTAGCCTTATCGCAGCTAGACGAAACTATTCAAGGAG ACGGAGAAGTAACGCAGGAATGAAAGACCCCACCTGTAGGTTTGGCAAGA;

SEQ ID NO: 382:

ACAATTACTGCTGATCGAGTGTAGCCTTATCGCAGCTAGACGAAACTTACAGTTAAG ACGGAGAAGTAACGCAGGAATGAAAGACCCCACCTGTAGGTTTGGCAAGA).

[0127] FIG. 2 depicts a schematic of barcodes located at the junction of the 4- IBB signaling domain and the CD3zeta domain of the CAR components within the CD 19 CAR plasmid and CD22 CAR plasmid. The target nucleic acid for the CD 19 CAR and the CD22 CAR are delineated by a white and grey box, respectively. Sequence differences are represented by dots between the sequences. (Sequences of FIG. 2: SEQ ID NO: 383:

GCAGACCACTCAAGAGGAAGATGGCTGTAGTTGTAGGTTCCCTGAGGAGGAGGAGG GCGGCTGCGAACTGAGAGTGAAGTTCAGCAGAT;

SEQ ID NO: 384:

ATCTGCTGAACTTCACTCTCAGTTCGCAGCCGCCCTCCTCCTCCTCAGGGAACCTAC AACTACAGCCATCTTCCTCTTGAGTGGTCTGC;

SEQ ID NO: 385: QTTQEEDGCSCRFPEEEEGGCELRVKFSRS; SEQ ID NO: 386: CTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGT GAACTGAGAGTGAAGTTCAGCAG;

SEQ ID NO: 387: CTCAAGAGGAAGATGGCTGTAGTTGTAGGTTCCCTGAGGAGGAGGAGGGTGGCTGC GAACTGAGAGTGAAGTTCAGCAG) .

[0128] FIGs. 3A-3K show results from the digital droplet PCR assay used to assess the specificity and efficiency of the barcodes as probe-binding sequences by singleplex detection. FIG. 3A depicts an experimental plate layout for a singleplex ddPCR assay. FIG. 3B depicts ddPCR results for the CD22CARvl probe used on CD22 CAR plasmids. The fluorescence intensity was measured for the specified well in the 96-well plate (e.g., A01, A02, etc.). FIG. 3C depicts ddPCR results for the CD22CARv2 probe used on CD22 CAR plasmids. FIG. 3D depicts ddPCR results for the CD19CAR probe used on CD22 CAR plasmids. FIG. 3E depicts a graph of the calculated number of copies of the CD22 CAR plasmid versus the actual number of copies per reaction when the CD22CARvl, CD22CARv2, or CD19CAR probe was used for the reaction. FIG. 3F depicts a bar graph of the recovery efficiency of the CD22 CAR plasmid when the CD22CARvl or CD22CARv2 probe was used. FIG. 3G depicts ddPCR results for the CD22CARvl probe used on CD 19 CAR plasmids. FIG. 3H depicts ddPCR results for the CD22CARv2 probe used on CD 19 CAR plasmids. FIG. 31 depicts ddPCR results for the CD19CAR probe used on CD 19 CAR plasmids. FIG. 3 J depicts a graph of the calculated number of copies of the CD 19 CAR plasmid versus the actual number of copies per reaction when the CD22CARvl, CD22CARv2, or CD19CAR probe was used for the reaction. FIG. 3K depicts a bar graph of the recovery efficiency of the CD 19 CAR plasmid when the CD19CAR probe was used.

[0129] FIGs. 4A-4Q show the result from the digital droplet PCR assay used to assess the specificity and efficiency of the barcodes as probe-binding sequences for rapid multiplex detection. FIG. 4A depicts an experimental plate layout for a multiplex ddPCR assay. FIG. 4B depicts ddPCR results for the CD19CAR-FAM probe used on CD 19 CAR plasmids. FIG. 4C depicts ddPCR results for the CD22CARvl-HEX probe used on CD 19 CAR plasmids. FIG. 4D depicts ddPCR results for the CD19CAR-FAM probe used on CD22 CAR plasmids. FIG. 4E depicts ddPCR results for the CD22CARvl-HEX probe used on CD22 CAR plasmids. FIG. 4F depicts ddPCR results for the CD19CAR-FAM probe used on a mixture of CD 19 CAR and CD22 CAR plasmids. FIG. 4G depicts ddPCR results for the CD22CARvl-HEX probe used on a mixture of CD 19 CAR and CD22 CAR plasmids. FIG. 4H depicts a graph of the calculated number of copies of the CD 19 CAR plasmid versus the actual number of copies per reaction in the CD 19 CAR only, CD22 CAR only, and CD 19 CAR/CD22 CAR mixture reactions. Probes labeled with FAM were used to detect CD 19 CAR. FIG. 41 depicts a graph of the calculated number of copies of the CD22 CAR plasmid versus the actual number of copies per reaction in the CD 19 CAR only, CD22 CAR only, and CD 19 CAR/CD22 CAR mixture reactions. Probes labeled with HEX were used to detect CD22CAR. FIG. 4J depicts ddPCR results for the CD19CAR-FAM probe used on CD 19 CAR plasmids. FIG. 4K depicts ddPCR results for the CD22CARv2-HEX probe used on CD 19 CAR plasmids. FIG. 4L depicts ddPCR results for the CD19CAR-FAM probe used on CD22 CAR plasmids. FIG. 4M depicts ddPCR results for the CD22CARv2-HEX probe used on CD22 CAR plasmids. FIG. 4N depicts ddPCR results for the CD19CAR-FAM probe used on a mixture of CD 19 CAR and CD22 CAR plasmids. FIG. 40 depicts ddPCR results for the CD22CARv2-HEX probe used on a mixture of CD 19 CAR and CD22 CAR plasmids. FIG. 4P depicts a graph of the calculated number of copies of the CD 19 CAR plasmid versus the actual number of copies per reaction in the CD 19 CAR only, CD22 CAR only, and CD19 CAR/CD22 CAR mixture reactions. Probes labeled with FAM were used to detect CD 19 CAR. FIG. 4Q depicts a graph of the calculated number of copies of the CD22 CAR plasmid versus the actual number of copies per reaction in the CD 19 CAR only, CD22 CAR only, and CD19 CAR/CD22 CAR mixture reactions. Probes labeled with HEX were used to detect CD22CAR.

DETAILED DESCRIPTION

[0130] The present disclosure relates to a method of screening cells for the presence of a transgene by detecting an associated barcode, as well as cells or cell populations comprising a vector comprising a transgene and associated barcode sequence, cell compositions, methods, therapeutic uses and kits or products related thereto. The inclusion of unique barcode sequences that are each associated with a specific transgene permits for the rapid and multiplex screening of cells for the presence of one or more transgenes. Whereas standard methods of screening can only test for the presence of a small number of transgenes in a single reaction (e.g., due to spectral overlap of fluorescein-conjugated probes or fluorescein-conjugated antibodies), the methods described in the present disclosure can detect significantly more transgenes, i.e., as many transgenes as unique barcode sequences within the cells or cell populations being tested. Accordingly, the methods described in the present invention are cheaper, faster, more efficient, and more accurate than the current methods in the field.

[0131] All publications, comprising patent documents, scientific articles and databases, referred to in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. If a definition set forth herein is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated by reference, the definition set forth herein prevails over the definition that is incorporated herein by reference.

[0132] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

I. DEFINITIONS

[0133] Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as is commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.

[0134] As used herein, including in the appended claims, the singular forms “a,” “or,” and “the” include plural referents unless the context clearly dictates otherwise. For example, “a” or “an” means “at least one” or “one or more.” It is understood that aspects and variations described herein include embodiments “consisting” and/or “consisting essentially of’ such aspects and variations.

[0135] As used herein, the term “and/or” includes any and all combinations of one or more of the associated listed items.

[0136] As used herein, the term “exogenous” with reference to a polypeptide or a polynucleotide is intended to mean that the referenced molecule is introduced into the cell of interest. The exogenous molecule, such as exogenous polynucleotide, can be introduced, for example, by introduction of an exogenous encoding nucleic acid into the genetic material of the cells such as by integration into a chromosome or as non-chromosomal genetic material such as a plasmid or expression vector. Therefore, the term as it is used in reference to expression of an encoding nucleic acid refers to introduction of the encoding nucleic acid in an expressible form into the cell. In some cases, an “exogenous” molecule is a molecule, construct, factor and the like that is not normally present in a cell, but can be introduced into a cell by one or more genetic, biochemical or other methods.

[0137] The term “endogenous” refers to a reference molecule, such as a polynucleotide (e.g., gene), or polypeptide, that is present in a native or non-engineered cell and may be present in an engineered cell or population of cells of the same cell type. For instance, the term when used in reference to expression of an endogenous gene refers to expression of a gene encoded by an endogenous nucleic acid contained within the cell (e.g., within the cell’s native genome) and not exogenously introduced.

[0138] A “gene,” includes a DNA region encoding a gene product, as well as all DNA regions which regulate the production of the gene product, whether or not such regulatory sequences are adjacent to coding and/or transcribed sequences. Accordingly, a gene includes, but is not necessarily limited to, promoter sequences, terminators, translational regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, replication origins, matrix attachment sites and locus control regions. The sequence of a gene is typically present at a fixed chromosomal position or locus on a chromosome in the cell.

[0139] The term “locus” refers to a fixed position on a chromosome where a particular gene or genetic marker is located.

[0140] The term “expression” with reference to a gene or “gene expression” refers to the conversion of the information, contained in a gene, into a gene product. A gene product can be the direct transcriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, structural RNA, or any other type of RNA) or can be a protein produced by translation of an mRNA. Gene products also include RNAs which are modified, by processes such as capping, polyadenylation, methylation, and editing, and proteins modified by, for example, methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristoylation, and glycosylation. Hence, reference to expression or gene expression includes protein (or polypeptide) expression or expression of a transcribable product of or a gene such as mRNA. The protein expression may include intracellular expression or surface expression of a protein. Typically, expression of a gene product, such as mRNA or protein, is at a level that is detectable in the cell.

[0141] As used herein, a “detectable” expression level, means a level that is detectable by standard techniques known to a skilled artisan, and include for example, differential display, RT (reverse transcriptase)-coupled polymerase chain reaction (PCR), quantitative PCR (qPCR) or real-time PCR, digital droplet PCR (ddPCR), Northern Blot, and/or RNase protection analyses as well as immunoaffinity-based methods for protein detection, such as flow cytometry, ELISA, fluorescence in situ hybridization (FISH), or western blot. The degree of expression levels need only be large enough to be visualized or measured via standard characterization techniques.

[0142] As used herein, the term “increased expression”, “enhanced expression” or “overexpression” means any form of expression that is additional to the expression in an original or source cell that does not contain the modification for modulating a particular gene expression, for instance a wild-type expression level (which can be absence of expression or immeasurable expression as well).

[0143] The term “diverged” as used herein means any changes to a target nucleotide sequence from the wild-type nucleotide sequence or a nucleotide sequence that is native to an individual’s or cell’s genome, wherein the coded polypeptide or protein sequence is not changed. This may include but does not necessarily require no change to the post-translational modification status of the coded polypeptide or protein. For example, a diverged nucleotide sequence can include one or more silent mutations, or one or more point mutations within an intronic coding region that does not alter mRNA splicing or translation.

[0144] The term “tolerogenic factor” as used herein include immunosuppressive factors or immune-regulatory factors that modulate or affect the ability of a cell to be recognized by the immune system of a host or recipient subject upon administration, transplantation, or engraftment. Typically a tolerogenic factor is a factor that induces immunological tolerance to an engineered primary cell so that the engineered primary cell is not targeted, such as rejected, by the host immune system of a recipient. Hence, a tolerogenic factor may be a hypoimmunity factor. Examples of tolerogenic factors include immune cell inhibitory receptors (e.g., CD47), proteins that engage immune cell inhibitory receptors, checkpoint inhibitors and other molecules that reduce innate or adaptive immune recognition.

[0145] A “vector” or “construct” is capable of transferring gene sequences to target cells. Typically, “vector construct,” “expression vector,” and “gene transfer vector,” mean any nucleic acid construct capable of directing the expression of a gene of interest and which can transfer gene sequences to target cells. Thus, the term includes cloning, and expression vehicles, as well as integrating vectors. Methods for the introduction of vectors or constructs into cells are known to those of skill in the art and include, but are not limited to, lipid-mediated transfer (e.g., liposomes, including neutral and cationic lipids), electroporation, direct injection, cell fusion, particle bombardment, calcium phosphate co-precipitation, DEAE-dextran-mediated transfer and viral vector-mediated transfer.

[0146] As used herein, the term “engineered cell”, “engineered population of cells”, or “engineered cell or population of cells” refers to one or more cells comprising one or more genetic modifications or transgenes. In some embodiments, the engineering is a genetic engineering that directly changes the gene or regulatory elements thereof encoding a protein product in a cell, such as by gene editing, mutagenesis, or genetic engineering of an exogenous polynucleotide or transgene. “Modulation” of gene expression refers to a change in the expression level of a gene. Modulation of expression can include, but is not limited to, gene activation and gene repression. Modulation may also be complete, i.e., wherein gene expression is totally inactivated or is activated to wild-type levels or beyond; or it may be partial, wherein gene expression is partially reduced, or partially activated to some fraction of wild- type levels. [0147] The term “operatively linked” or “operably linked” are used interchangeably with reference to a juxtaposition of two or more components (such as sequence elements), in which the components are arranged such that both components function normally and allow the possibility that at least one of the components can mediate a function that is exerted upon at least one of the other components. By way of illustration, a transcriptional regulatory sequence, such as a promoter, is operatively linked to a coding sequence if the transcriptional regulatory sequence controls the level of transcription of the coding sequence in response to the presence or absence of one or more transcriptional regulatory factors. A transcriptional regulatory sequence is generally operatively linked in cis with a coding sequence, but need not be directly adjacent to it. For example, an enhancer is a transcriptional regulatory sequence that is operatively linked to a coding sequence, even though they are not contiguous.

[0148] The terms “polypeptide” and “protein,” as used herein, may be used interchangeably to refer to a series of amino acid residues joined by peptide bonds (z.e., a polymer of amino acid residues), and are not limited to a minimum length. Such polymers may contain natural or non-natural amino acid residues, or combinations thereof, and include, but are not limited to, peptides, polypeptides, oligopeptides, dimers, trimers, and multimers of amino acid residues. Thus, a protein or polypeptide includes include those with modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs. Full-length polypeptides or proteins, and fragments thereof, are encompassed by this definition. The terms also include modified species thereof, e.g., post-translational modifications of one or more residues, for example, methylation, phosphorylation glycosylation, sialylation, or acetylation. [0149] The term “complementary” as used herein may refer two strands of nucleic acids that hybridize under stringent conditions. One strand may be a primer and the other strand may be a vector sequence.

[0150] Throughout this disclosure, various aspects of the claimed subject matter are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the claimed subject matter. For example, in some embodiments, it is described that a feature is greater than about 10 units, and it is described (such as in another sentence) that the feature is less than about 20 units, and thus, the range of about 10 units to about 20 units is described herein.

[0151] As used herein, a “subject” or an “individual,” which are terms that are used interchangeably, is a mammal. In some embodiments, a “mammal” includes humans, nonhuman primates, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, rabbits, cattle, pigs, hamsters, gerbils, mice, ferrets, rats, cats, monkeys, etc. In some embodiments, the subject or individual is human. In some embodiments, the subject is a patient that is known or suspected of having a disease, disorder, or condition.

[0152] As used herein, the term “treating” and “treatment” includes administering to a subject an effective amount of cells described herein so that the subject has a reduction in at least one symptom of the disease or an improvement in the disease, for example, beneficial or desired clinical results. For purposes of this technology, beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptoms, diminishment of extent of disease, stabilized (z.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. Treating can refer to prolonging survival as compared to expected survival if not receiving treatment. Thus, one of skill in the art realizes that a treatment may improve the disease condition, but may not be a complete cure for the disease. In some embodiments, one or more symptoms of a disease or disorder are alleviated by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% upon treatment of the disease.

[0153] For purposes of this technology, beneficial or desired clinical results of disease treatment include, but are not limited to, alleviation of one or more symptoms, diminishment of extent of disease, stabilized (z.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable.

IL METHODS OF SELECTING

[0154] The present disclosure provides a method of selecting a cell, population of cells, or therapy (e.g., T cells, B cells, NK cells, islet cells such as beta cells, or hypoimmunogenic cells thereof) comprising detecting the presence or absence of a first barcode and/or a second barcode in the cell or population of cells, wherein the presence of the first barcode indicates the presence of a first transgene and the presence of the second barcode indicates the presence of a second transgene, and selecting the cell or population of cells as being suitable for: (a) administration to a subject;(b) manufacturing a drug product; (c) further gene editing or genome editing; (d) creating a cell bank; (e) differentiation into a drug product; (f) packaging for distribution; and/or (g) cryopreservation, based on the presence or absence of the first barcode and/or second barcode. In some embodiments, the therapy is a cell therapy. In some embodiments, the cell therapy is generated ex vivo, for example in tissue culture. In some embodiments, the cell therapy is generated in vivo in a subject in need thereof to treat a disease in the subject, wherein a vector comprising the first transgene and/or the second transgene and the first barcode and/or the second barcode is administered to the subject. In some embodiments, the vector is packaged in a fusosome for trafficking to a target cell or target population of cells in vivo within the subject. This in vivo cell therapy can be used, for example, to transduce cells with a CAR in vivo, such as wherein the CAR is expressed by a vector that is packaged in a fusosome for delivery to the recipient cells (e.g., the T cells to be engineered into CAR T cells). This process can also be termed “in vivo cell engineering.”

[0155] In some embodiments, the method further comprises detecting a third or more barcode(s) in the sample, wherein detection of the third or more barcode(s) indicates the presence of a third or more transgene(s). In some embodiments, the presence or absence of the first and second transgene or the first, second, and third (or more) transgenes are detected simultaneously. In some embodiments, the presence or absence of the first and second transgene or the first, second, and third (or more) transgenes are detected concurrently. In some embodiments, the presence or absence of the first and second transgene or the first, second, and third (or more) transgenes are detected sequentially. In some embodiments, the detection of the transgene can be used to determine transgene abundance. In some embodiments, the detection of the transgene can be used to assess a sample’s therapeutic persistence and/or therapeutic potential.

[0156] In some embodiments, the detection of the first barcode and/or the second barcode indicates the presence of the first transgene and/or the second transgene.

/. Detection of Barcodes

[0157] The present disclosure provides methods for detecting barcodes in a sample, wherein detection of a barcode indicates the presence of a transgene, including components in a vector comprising an identifying region comprising the barcode by which the barcode can be detected. In some embodiments, the method further comprises detecting a second barcode in the sample, wherein detection of the second barcode indicates the presence of a second transgene. In some embodiments, the method further comprises detecting a third barcode in the sample, wherein detection of the third barcode indicates the presence of a third transgene. In some embodiments, the first and/or the second barcodes are detected using sequencing and/or probe binding. In some embodiments, one or more probes bind to a barcode. In some embodiments, a probe binding site is a barcode. In some embodiments, the sample is a blood sample, plasma sample, or tissue sample. In some embodiments, the sample comprises a cell or population of cells, e.g., T cells, B cells, NK cells, islet cells such as beta cells, or hypoimmunogenic cells thereof. z. Barcode Binding

[0158] In some embodiments, the identifying region comprises a barcode and primer binding sites. The primer binding sites flank the barcode region, thereby allowing for the amplification and/or detection of the barcode by any method known in the art. Amplification techniques can include a polymerase chain reaction (PCR) amplification technique or a non-PCR amplification technique, for example, an isothermal amplification technique. The primer binding sites can bind to nucleic acid primers that comprise sequences complementary to the primer bind site sequences. In some embodiments, the primers are about 10 to about 50 nucleotides in length, for example about any of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, or 50 nucleotides in length. In some embodiments, the primers are about 5-15, about 10-20, about 15-25, about 20-30, about 25-35, about 30-40, about 35-45, about 40-50, about 45-55 nucleotides in length. In some instances, the primer sequences may be longer than 50 nucleotides, for example about 50, about 60, about 70, about 80, about 90, or more nucleotides in length. In some embodiments, the primer sequences are about 10 to about 30 nucleotides in length.

[0159] In some embodiments, the first identifying region and/or the second identifying region comprises one or more probe binding sites. Probe binding sites are nucleic acid sequences to which specific probes recognize and hybridize. In some embodiments, the probes bind to specific nucleic acid sequences. In some embodiments, the probes bind to a barcode. In some embodiments, the probes comprise or are nucleic acid molecules, polypeptides, or chemical molecules. Antibodies can include primary or secondary antibodies, wherein secondary antibodies can bind to primary antibodies to amplify the probe binding signal. Nucleic acid molecules can comprise either RNA or DNA molecules that are complementary to the probe binding sites. In some instances, the probes (e.g., the nucleic acid molecules) can be further attached to a marker for detection as described below. In some embodiments, the probes are attached, e.g., conjugated, to a detection marker. In some embodiments, the detection molecule can include any one or more of fluorophores, biotin, enzymes, radioisotopes, non-radioactive heavy metal isotopes, etc. Additional examples of probes include, but are not limited to, two- photon fluorescent probes, Raman probes (based on alkyne or nitrile tags), Siderophore- Dioxetane Probes, or enzyme-activated fluorescent probes. [0160] Known fluorophores include, for example, any of Brilliant Ultra Violet 395, Alexa Fluor 350, Brilliant Ultra Violet 496, Qdot 525 Probe, Brilliant Ultra Violet 563, Qdot 565 Probe, Qdot 605 Probe, Brilliant Ultra Violet 615, Qdot 655 Probe, Brilliant Ultra Violet 661, Qdot 705 Probe, Brilliant Ultra Violet 737, Qdot 800 Probe, Brilliant Ultra Violet 805, Alexa Fluor 405, Brilliant Violet 421, Super Bright 436, eFluor 450, Pacific Blue, Coumarin and Coumarin Derivatives, Brilliant Violet 480, Cyan Fluorescent Protein (CFP), eFluor 506, Pacific Green, Pacific Orange, Super Bright 600, Super Bright 645, Brilliant Violet 650, Super Bright 702, Brilliant Violet 711, Super Bright 780, Brilliant Violet 786, Green Fluorescent Protein (GFP), BODIPY FL, NovaFluor Blue 510, Fluorescein (FITC), Alexa Fluor 488, Oregon Green 488, NovaFluor Blue 530, NovaFluor Blue 555, NovaFluor Blue 585, NovaFluor Blue 610-30S, NovaFluor Blue 610-70S, NovaFluor Blue 660-40S, NovaFluor Blue 660-120S, PerCP- Cyanine5.5, PerCP-eFluor 710, Alexa Fluor 532, Cy3, NovaFluor Yellow 570, Alexa Fluor 555, Alexa Fluor 561, Alexa Fluor 546, R-phycoerythrin (R-PE), Tetramethylrhodamine (TRITC), Red Fluorescent Protein (RFP), NovaFluor Yellow 590, Alexa Fluor 568, PE-eFluor 610, Texas Red (and Texas Red-X), NovaFluor Yellow 610, Alexa Fluor 594, NovaFluor Yellow 660, NovaFluor Yellow 690, NovaFluor Yellow 700, NovaFluor Yellow 730, NovaFluor Yellow 755, PE-Cyanine7, NovaFluor Red 660, Allophycocyanin (APC), Cy5, eFluor 660, Alexa Fluor 647, NovaFluor Red 685, NovaFluor Blue 690, Alexa Fluor 660, NovaFluor Red 700, Alexa Fluor 680, NovaFluor Red 710, Alexa Fluor 700, NovaFluor Red 725, NovaFluor Red 755, Alexa Fluor 750, APC-eFluor 780, FAM, HEX, Rhodamine Red-X, Tamara, YY, Atto 550, Atto 590, Atto 700, Rox, TruRed, Cy7, Red 613, Cy3.5 581, Cy5.5, DAPI, Hoechst, SYTOX blue, SYTOX green, SYTOX orange, YOYO-1, TOTO-1, TO-PRO-1, chromomycin A3, mithramycin, propidium iodide, ethidium bromide, SYBR Green, any KIRA VIA dyes (e.g., KIR VIA Blue 520), PE-Dazzle 594, PE-Fire 640, PE-Cy5, PE-Fire 700, PE-FIRE 810, PerCP, APC-Cyanine 7, APC-Fire 750, APC-Fire 810, Spark UV 387, Spark Violet 423, Spark Violet 500, Spark Violet 538, Spark Blue 550, Spark Blue 574, Spark YG 570, Spark YG 581, Spark YG 593, Spark NIR 685, Spark Red 718, Brilliant Violet 510, Brilliant Violet 570, Brilliant Violet 605, Brilliant Violet 750, 7-AAD, APC-H7, Apotracker Green, APC-R700, Brilliant Blue 515, Brilliant Blue 700, Calcein-AM, Calcein Red- AM, Calcein Violet- AM, CF 570, CytoPhase Violet, DRAQ5, DRAQ7, Helix NP Blue, Helix NP Green, Helix NP NIR, MitoSpy Green, MitoSpy NIR, MitoSpy Orange, MitoSpy Red, Tag-it Violet, VioBright FITC, Zombie Aqua, Zombie Green, Zombie NIR, Zombie Red, Zombie UV, Zombie Violet, and Zombie Yellow. [0161] In some embodiments, the first probe and the second probe are each labeled with a distinct fluorophore. In some embodiments, the first probe and the second probe are used to detect the first barcode and the second barcode simultaneously or concurrently. In some embodiments, the first probe and the second probe are used to detect the first barcode and the second barcode separately. In some embodiments, the two or more distinct fluorophores conjugated to the two or more probes used to detect two or more barcodes simultaneously or concurrently do not spectrally overlap. Spectral overlap occurs when two fluorophores share similar excitation and emission wavelengths, such that the excitation spectra and the emission spectra are not readily distinguishable by the instruments used in the art to detect the presence of fluorophores. In some embodiments, the emission spectra of two or more fluorophores do not overlap. In some embodiments, the excitation spectra of two or more fluorophores do not overlap. In some embodiments, neither the excitation spectra nor the emission spectra of two or more fluorophores overlap. In some embodiments, the first probe and/or the second probe is labeled with FAM and/or HEX.

[0162] Known radioisotopes (z.e., radioactive isotope, radionuclide, or radioactive nuclide) include, for example, Phosphorus-32, Hydrogen-3 (tritium), Carbon-14, Chlorine-36, Lead-210, Chromium-51, Manganese-54, Cobalt-60, Zinc-65, Technetium-99, Cesium-137, Ytterbium- 169, Iridium-192, Gold-198, Americium-241, Molybdenum-99, Iodine-123, Iodine-131, Samarium- 153, Lutetium-177, Carbon-11, Nitrogen-13, Oxygen-15, Fluorine-18, Copper-64, Gallium-67, Thallium- 201, Hydrogen-1, Phosphorus-31, Fluorine-19, Sodium-23, Carbon-13, Oxygen-17, Nitrogen- 15, 2D FBP, 3DRP, OSEM2D, and OSEM3D/MAP. Non-radioactive heavy metal isotopes include 38 isotopes of lanthanides, 2 isotopes of indium, 1 isotope of yttrium, 6 isotopes of palladium, 1 isotope of bismuth (see, e.g., Han et al., Nat Protoc. 2018, 13(10):2121-2148), for example (but not limited to): ⁸⁹Y, ¹¹³In, ¹¹⁵In, ¹³⁹La, ¹⁴⁰Ce, ¹⁴¹Pr, ¹⁴²Nd, ¹⁴³Nd, ¹⁴⁴Nd, ¹⁴⁵Nd, ¹⁴⁶Nd, ¹⁴⁷Sm, ¹⁴⁸Nd, ¹⁴⁹Sm, ¹⁵⁰Nd, ¹⁵¹Eu, ¹⁵²Sm, ¹⁵³Eu, ¹⁵⁴Sm, ¹⁵⁵Gd, ¹⁵⁶Gd, ¹⁵⁷Gd, ¹⁵⁸Gd, ¹⁵⁹Tb, ¹⁶⁰Gd, ¹⁶¹Dy, ¹⁶²Dy, ¹⁶³Dy, ¹⁶⁴Dy, ¹⁶⁵Ho, ¹⁶⁶Er, ¹⁶⁷Er, ¹⁶⁸Er, ¹⁶⁹Tm, ¹⁷⁰Er, ¹⁷¹Yb, ¹⁷²Yb, ¹⁷³Yb, ¹⁷⁴Yb, ¹⁷⁵LU, ¹⁷⁶Yb, ¹⁹¹Ir, ¹⁹³Ir, ¹⁹⁵Pt, and ²⁰⁹Bi. Known reporter enzymes include, for example, horseradish peroxidase (HRP), alkaline phosphatase (AP), glucose oxidase, P-galactosidase, P- glucuronidase (GUS), luciferase, and Chloramphenicol O-Acetyltransferase (CAT). [0163] Fluorescent molecules can be identified by multiple technologies including, for example, fluorescence microscopy, flow cytometry, spectral flow cytometry, microplate reader, immunohistochemistry, multiplexed immunofluorescence (e.g. PhenoCycler, Vectra Polaris), fluorescence spectroscopy, fluorescence in situ hybridization, PET/fluorescence imaging (e.g., CRi Maestro, IVIS Lumina, IVIS Spectrum, iThera MSOT), etc. Radioisotopes can be detected by, e.g., single-photon emission computed tomography (SPECT), positron emission tomography (PET), magnetic resonance imaging, scintillation detector/counter, laser spectroscopy, isotope ratio mass spectrometry, or autoradiography. Non-radioactive heavy metal isotopes can be detected by methods including cytometry by time of flight (CyTOF), mass spectrometry (e.g., LC-MS), reversed-phase high-performance liquid chromatography (RP-HPLC), etc.

[0164] In some embodiments, the probe is between 10 and 50 nucleotides in length. In some embodiments, the probe is at least 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides in length. In some embodiments, the probe is about 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides in length. In some embodiments, the probe is between 10 and 50, 15 and 45, 20 and 40, or 25 and 35 nucleotides in length. In some embodiments, the probe is about any of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or nucleotides in length. In some embodiments, the probe is between 18 and 30 nucleotide in length. In some embodiments, the probe binds to a probe binding site that comprises a sequence that is complementary to the barcode. In some embodiments, the probe and the barcode comprise a sequence of the same length. In some embodiments, the probe and the barcode comprise sequences of different lengths. In some embodiments, the probe binding site comprises a sequence that is longer than the sequence comprised by the barcode. In some embodiments, the probe binding site comprises a sequence that is shorter than the sequence comprised by the barcode.

[0165] In some embodiments, the method comprises the detection of a first probe binding site and/or a second probe binding site with a first complementary probe and/or a second complementary probe. In some embodiments, the barcode is randomly generated, and the probe binding site comprises the nucleic acid sequence of SEQ ID NO:31 (GAAACTNNNNNNNNAGACGGAG), wherein NNNNNNNN is the barcode and wherein N represents a nucleotide selected from the group consisting of A, T, C, and G. For example, in some embodiments, the method comprises detection of a barcode using a probe that binds to the probe binding site comprising the nucleotide sequence set forth in any one of SEQ ID NOs: 18-23 or a variant thereof comprising about 1, 2, 3, 4, or 5 nucleotide substitutions, insertions, or deletions.

[0166] In some embodiments, the probe binds directly to the barcode. In some embodiments, the probe comprises a sequence that is complementarity to the barcode. In some embodiments, the probe and the barcode share between about 70% and 100% complementarity. In some embodiments, the probe and the barcode sequence share at least about any of 70%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99%, 99%, or 100% complementarity.

[0167] In some embodiments, the probe sequence partially or completely overlaps with a barcode. In some embodiments, the probe sequence and the barcode partially overlap within the 5' region and/or 3' region of the barcode. In some embodiments, the probe sequence and the barcode partially overlap within the 5' region of the barcode. In some embodiments, the probe sequence and the barcode partially overlap within the 3' region of the barcode. In some embodiments, the probe sequence and the barcode partially overlap within the 5' region and the 3' region of the barcode.

[0168] In some embodiments, one or more probes is incubated with a sample comprising one or more cells, wherein the cells comprise one or more vectors encoding one or more transgenes and identifying regions comprising one or more barcodes for about any of 5, 10, 15, 20, 35, 30, 45, 60, 90, or 120 minutes. In some embodiments, the cells are intact. In some embodiments, the cells are fixed. In some embodiments, the cells are lysed. In some embodiments, one or more probes is incubated with the sample for about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 hours. In some embodiments, probes incubated with the sample are then detected by a method known in the art that is appropriate for the detection marker, as described herein. ii. Detection via Sequencing

[0169] In some embodiments, the first barcode and/or the second barcode are detected using sequencing and/or probe binding, wherein sequencing comprises one or more of Sanger sequencing, next-generation sequencing (NGS), shotgun sequencing, single molecule real time (SMRT) sequencing, nanopore DNA sequencing, massively parallel signature sequencing (MPSS), polony sequencing, 454 pyrosequencing, Illumina (Solexa) sequencing, combinatorial probe anchor synthesis (cPAS), SOLiD sequencing, Ion Torrent semiconductor sequencing, DNA nanoball sequencing, helioscope single molecule sequencing, and microfluidic systems. [0170] Next-generation sequencing methods are known in the art, and are described in, e.g., Metzker, M. (2010) Nature Biotechnology Reviews 11:31-46, which is incorporated herein by reference. Other examples of sequencing methods suitable for use when implementing the methods and systems disclosed herein are described in, e.g., International Patent Application Publication No. WO 2012/092426. In some instances, the sequencing may comprise, for example, targeted sequencing or direct sequencing. In some instances, sequencing may be performed using, e.g., Sanger sequencing. In some instances, the sequencing may comprise whole genome sequencing or whole exome sequence wherein detection of, e.g., genomic rearrangements, repetitive sequence elements, gene fusions, and novel transcripts within a cell is also desired.

[0171] The disclosed methods may be implemented using sequencing platforms such as the Roche 454, Illumina Solexa, ABI-SOLiD, ION Torrent, Complete Genomics, Pacific Bioscience, Helicos, and/or the Polonator platform. In some instances, sequencing may comprise Illumina MiSeq sequencing. In some instances, sequencing may comprise Illumina HiSeq sequencing. In some instances, sequencing may comprise Illumina NovaSeq sequencing. Optimized methods for sequencing a large number of target loci in nucleic acids extracted from a sample, such as a population of cells as described herein, are described in more detail in, e.g., International Patent Application Publication No. WO 2020/236941, the entire content of which is incorporated herein by reference.

[0172] Quantitative polymerase chain reaction (qPCR) or real-time PCR (RT-PCR) monitors the amplification of a targeted DNA molecule during the PCR reaction. Detection of PCR products in real-time may use non-specific fluorescent dyes that intercalate with any double-stranded DNA or sequence- specific DNA probes consisting of oligonucleotides that are labeled with a fluorescent reporter. This technology permits detection only after hybridization of the probe with its complementary nucleic acid sequence. DNA-binding dyes known to one of ordinary skill in the art include, but is not limited to, SYBR Green, DAPI, propidium iodide, Hoechst, ethidium bromide, mithramycin, chromomycin A3, etc. Oligonucleotides labeled with fluorescent reporters known to one of ordinary skill in the art include, but are not limited to, hydrolysis probes, molecular beacons, dual hybridization probes, eclipse probes, UniPrimer, Scorpions primers, LUX primers, and QZyme primers. [0173] Digital droplet PCR (ddPCR) is a digital PCR method utilizing a water-oil emulsion droplet system to precisely measure absolute quantities of nucleic acid molecules. ddPCR reactions are prepared with reporter fluorophores, such as FAM and HEX, or and intercalating dye. Droplets are formed in a water-oil emulsion to form massive partitions that separate the template DNA molecules. ddPCR fractionates a sample into 20,000 nanoliter-sized droplets, and PCR amplification of the template molecules occurs in each individual droplet using a thermal cycler. Partitioning enables the measurement of thousands of independent amplification events within a single sample. The droplets are streamed in a single file on a reader, which counts the fluorescent positive and negative droplets to calculate the DNA concentration. Fluorescence in two channels is measured for the individual droplets. Positive droplets containing at least one copy of the target DNA molecule will exhibit increased fluorescence, compared to negative droplets that do not contain the target DNA molecule. ddPCR data is shown as a plot as fluorescence intensity versus droplet number. Positive droplets are scored as positive, assigned a value of 1, and appear above a threshold line in the graph while negative droplets are scored as negative, assigned a value of 0, and appear below the threshold line. ddPCR is known to one of ordinary skill in the art. See, e.g., Hindson et al. (2011), Anal Chem. 83(22):8604-10; and Pinheiro et al. (2012), Anal Chem. 84(2):1003-l l.

[0174] In some embodiments, the method further comprises contacting the sample with i) a first probe, ii) a second probe, or iii) a first probe and a second probe, wherein the first probe has a different sequence from the second probe; and detecting binding of i) the first probe to a first barcode in the first vector, ii) the second probe to a second barcode in the second vector, or iii) the first probe to a first barcode in a first vector and the second probe to a second barcode in a second vector. In some embodiments, the first probe and the second probe comprise the same sequence. In some embodiments, the first probe and the second probe comprise different sequences. In some embodiments, the method further comprises contacting the sample with a third probe, wherein the third probe has a different sequence from the first probe or the second probe; and detecting the binding of the third probe to the third barcode in a third vector.

[0175] In some embodiments, the sample comprising one or more cells is screened and/or monitored using the methods provided herein. In some embodiments, the sample is screened and/or monitored for the presence of a transgene, e.g., by the detection of an associated barcode. In some embodiments, the sample is screened and/or monitored for the presence of two or more transgenes. In some embodiments, detection of the first barcode and the second barcode indicates the presence of i) a first vector encoding a first transgene and a second vector encoding a second transgene or ii) a first vector encoding a first transgene and a second transgene. In some embodiments, the detection of two or more barcodes is used to screen a sample comprising one or more cells (or one or more populations of cells), such as any cell or population of cells described in the present disclosure, for the presence of two or more barcodes. In some embodiments, the detection of two or more unique barcodes is used to screen a sample comprising one or more cells for the presence of two or more transgenes associated with the expression of the two or more barcodes.

2. Barcodes

[0176] The present disclosure provides vectors comprising a barcode and methods of selecting a cell or population of cells (e.g., T cells, B cells, NK cells, islet cells such as beta cells, or hypoimmunogenic cells thereof), or therapy (e.g., cell therapy) for the presence of a transgene encoded by a vector, wherein the vector comprises an identifying region comprising a barcode. Barcodes generally refer to short, unique DNA sequences used to identify a target. Nucleic acid barcodes with unique, identifiable sequences can include (e.g.) molecular barcodes that can be used to tag, mark, delineate, identify, etc. a specific sequence, e.g., a transgene such that confirmation of the presence of the barcode is indicative of the presence of the associated transgene. Barcodes may be non-naturally occurring sequences. Barcodes may be used to identify cells or cell populations wherein the cells or cell populations express or contain a vector encoding a transgene sequence and the barcode, for example to differentiate engineered cells or populations of cells from wild-type cells. Barcodes are associated with specific transgenes such that each barcode is associated with one or more specific transgene, and identifying the presence of one or more barcodes within a cell or population of cells is indicative of the presence of the associated one or more transgenes. The diversity of barcodes allows for the pooling and simultaneous sequencing of cells, population of cells, and/or samples obtained from a patient using a high throughput, multiplex system, for example to screen the cells, population of cells, and/or samples obtained from a patient for the presence of a transgene by detecting the presence of the barcode. Accordingly, the barcode(s) of the present disclosure can be used to detect the presence of a transgene. [0177] In some embodiments, the barcode is located on a vector. In some embodiments, one or more barcodes are located on the same vector. In some embodiments, one or more barcodes are located on different vectors. In some embodiments, the barcode is flanked by primer binding sites. In some embodiments, the barcode is flanked by primer binding sites comprising a forward primer and a reverse primer.

[0178] In some embodiments, the first barcode and/or the second barcode is located outside of the first transgene and/or second transgene. In some embodiments, the first barcode and/or the second barcode is located within the first transgene and/or the second transgene. In some embodiments, a portion of the first barcode and/or the second barcode is located within the first transgene and/or the second transgene and a portion of the first barcode and/or the second barcode is located outside of the first transgene and/or second transgene.

[0179] In some embodiments, the first barcode and/or the second barcode comprises a diverged nucleotide sequence within the transgene. A barcode can be a diverged sequence, for example wherein the diverged nucleotide sequence within the transgene encodes the same amino acid sequence as a non-diverged (e.g., wild-type) nucleotide sequence but wherein the diverged nucleotide sequence comprises a different nucleotide sequence from the non-diverged (e.g., wildtype) nucleotide sequence, for example silent mutations. A diverged sequence can include, for example, one or more types of mutations, such as substitutions, when combined, generate a unique nucleotide sequence that translates into the wild-type amino acid sequence. A diverged sequence can be a recombinant sequence. A diverged sequence can be an engineered sequence.

In some embodiments, the diverged nucleotide sequence is located at the junction of one or more transgene domains.

[0180] A barcode can encode the same amino acid sequence as a non-diverged nucleotide sequence, e.g., a reference wild-type amino acid sequence. A barcode can have at least about any of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a wild-type nucleotide sequence. In some embodiments, a barcode is a probe binding site. A barcode can be of any suitable length, for example, in some embodiments, the barcode is an oligonucleotide between about 6 to about 30 nucleotides, such as about any of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some embodiments, the barcode can be between about 6 to about 10 nucleotides in length, between about 10 to about 20 nucleotides in length, between about 15 to about 25 nucleotides in length, or between about 20 to about 30 nucleotides in length. The barcode in the present disclosure is distinct from a sequence present in nature, for example present in a subject who may or may not comprise mutations from the reference wild-type sequence (z.e., alternative alleles), such that the barcode can accurately be used to detect the presence of a transgene. The barcode may be a naturally occurring amino acid sequence provided the nucleotide sequence present in the subject is different from the nucleotide sequence of the barcode that is comprising a naturally occurring amino acid sequence. In some embodiments, the first barcode and the second barcode are about the same nucleotide length.

[0181] In some embodiments, the barcode is randomly generated. For example, in some embodiments, the randomly generated barcode comprises the nucleotide sequence of NNNNNNNN wherein N refers to any of the nucleic acid bases A, T, C, and G, such as a nucleotide sequence selected from the group consisting of CATCGGAA, GGACAATT, TGTCAACT, TTACAGTT, ATTCAAGG, and TACAGTTA, or a nucleotide sequence comprising 1, 2, 3, 4, or 5 nucleotide substitutions, insertions, or deletions from a nucleotide sequence selected from the group consisting of CATCGGAA, GGACAATT, TGTCAACT, TTACAGTT, ATTCAAGG, and TACAGTTA. In some embodiments, the barcode comprises a nucleotide sequence set forth in any one of CATCGGAA, GGACAATT, TGTCAACT, TTACAGTT, ATTCAAGG, and/or TACAGTTA. In some embodiments, the barcode comprises a nucleotide sequence set forth in any one of CATCGGAA, GGACAATT, TGTCAACT, TTACAGTT, ATTCAAGG, and/or TACAGTTA, or comprises a nucleotide sequence comprising 1, 2, 3, 4, or 5 nucleotide substitutions, insertions, or deletions from a nucleotide sequence set forth in any one of CATCGGAA, GGACAATT, TGTCAACT, TTACAGTT, ATTCAAGG, and/or TACAGTTA, and the presence of the barcode indicates the presence of a transgene encoded by the vector comprising the barcode. In some embodiments, the barcode comprising a nucleotide sequence selected from the group consisting of CATCGGAA, GGACAATT, TGTCAACT, TTACAGTT, ATTCAAGG, and TACAGTTA indicates the presence of a transgene encoded by the vector comprising the barcode. Exemplary barcode nucleic acid sequence #1

CATCGGAA

Exemplary barcode nucleic acid sequence #2 GGACAATT

Exemplary barcode nucleic acid sequence #3 TGTCAACT

Exemplary barcode nucleic acid sequence #4 TTACAGTT

Exemplary barcode nucleic acid sequence #5 ATTCAAGG

Exemplary barcode nucleic acid sequence #6 TACAGTTA 3. Primer Binding Sites

[0182] In some embodiments, the first barcode and/or the second barcode is located within a first and/or second identifying region, wherein the first and/or second identifying region comprises primer binding sites that flank the first barcode and/or the second barcode. Primers are shorts, single-stranded pieces of DNA that are complementary to target sequences. Primer binding sites are a region of nucleotide sequence where an RNA or DNA single- stranded primer binds to start (z.e., initiate) replication. A forward primer binding site is a stretch of the antisense strand of DNA that runs in 3' to 5' direction and is complementary to a forward primer. A reverse primer binding site is a stretch of the sense strand of DNA that runs in the 5' to 3' direction and is complementary to a reverse primer. Primer binding sites generally include both a forward primer binding site and a reverse primer binding site, wherein the region between and inclusive of the primer binding sites are replicated, for example for amplification. In some instances, a forward primer binding site or a reverse primer binding site may be utilized alone, for example for sequencing.

[0183] A primer binding site can be of any suitable length, for example, in some embodiments, the primer binding site is a stretch of nucleotides between about 10 to about 30 nucleotides. In some embodiments, the primer binding site comprises about any of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, or 30 nucleotides. In some embodiments, the primer binding site comprises about 10 to about 15, about 12 to about 17, about 15 to about 20, about 17 to about 22, about 20 to about 25, about 22 to about 27, or about 25 to about 30 nucleotides.

[0184] Primer binding sites may comprise natural or non-natural sequences. Primer binding sites allow for the amplification a specific segment of DNA by, for example, polymerase chain reaction or for determining the nucleotide sequence of DNA by, for example, sequencing techniques such as Sanger sequencing. The primer binding sites of the present disclosure provide one way by which a barcode can be identified and/or detected to screen for the presence or absence of a transgene.

[0185] Primer binding sites may be complementary to universal primers. A universal primer is able to bind to a sequence found in many commonly used plasmid cloning vectors, many of which are derived from pUC vectors. Examples of universal primers include, but are not limited to M13 Reverse (-27), M13 Forward (-41), M13 Forward (-20), M13 Forward (-21), M13 Reverse (-48), SP6, T3, T7, T7 EEV, T7 Reverse, T7 Term, pBluescript KS, pBluescript SK, 3'pGEX, 5'pGEX, GST-Tag, pTrcHis-Forward, pTrcHis-Reverse, CMV-Forward, CMV- Reverse, EGFP-C, EGFP-N, BGH-Reverse, pQEproseq, pQErevseq, Intein Forward, 5'-pBabe- Seq, 3'-pBabe-Seq, -96 gill Sequencing Primer, GALI Forward, pBAD Forward, pBAD Reverse, pTRE 3', pTRE 5', pYESTrp Forward, pYESTrp Reverse, RVprimer3, Rvprimer4, GLprimer 1, GLprimer 2, SeqL-A (ATTL1), SeqL-B (ATTL2), SV40-pArev, SV40-Promoter, U6 Primer, Xpress Forward, EBV-Rev primer, hU6-01, hU6-02, 16S rRNA For, 16S rRNA Rev, 3' RACE PCR, Anchored Oligo dT (20), Anchored Oligo dT (22), BGH Reverse, cDNA Cloning Primer, GAPDH For, GAPDH Rev, Neomycin For, Neomycin Rev, Oligo dT 15mer, Oligo dT 16mer, Oligo dT 18mer, Oligo dT 20mer, Oligo dT 20mer w/ 5' Phos, PCMV Forward, pGEX 3', pGEX 5', Random Hexamer, Random Hexamer w/ Biotin, and SP6 Upstream. In some embodiments, the universal primers are used to amplify the barcode sequence. In some embodiments, the universal primers are used to detect the barcode. In some embodiments, the detection of the barcode indicates the presence of a transgene encoded by a vector, wherein the vector comprises an identifying region comprising a barcode.

[0186] In some embodiments, the forward primer binding sites of the first barcode and the second barcode comprise the same sequence. In some embodiments, the forward primer binding site of the first barcode and/or the second barcode comprise different sequences. In some embodiments, the first and second forward primer binding sites are complementary to universal primers. In some embodiments, the first forward primer binding site and the second forward primer binding site is each between about 10 to about 30 nucleotides in length.

[0187] In some embodiments, the reverse primer binding sites of the first barcode and the second barcode comprise the same sequence. In some embodiments, the reverse primer binding site of the first barcode and/or the second barcode comprise different sequences. In some embodiments, the first and second reverse primer binding sites are complementary to universal primers. In some embodiments, the first reverse primer binding site and the second reverse primer binding site is each between about 10 to about 30 nucleotides in length.

[0188] In some embodiments, the forward primer binding sites of the first barcode and the second barcode comprise the same sequence, and the reverse primer binding sites of the first barcode and the second barcode comprise the same sequence. In some embodiments, the forward primer binding site of the first barcode and/or the second barcode comprise different sequences, and the reverse primer binding site of the first barcode and/or the second barcode comprise different sequences. In some embodiments, the first and second forward primer binding sites and the first and second reverse primer binding sites are complementary to universal primers. In some embodiments, the first forward primer binding site and the second forward primer binding site and the first reverse primer binding site and the second reverse primer binding site is each between about 10 to about 30 nucleotides in length.

[0189] In some embodiments, the forward and the reverse primer binding sites have a certain percentage complementarity with the primers that bind to each primer binding site. For example, in some embodiments, the forward primer binding site and the reverse primer binding site each comprises at least about 70% complementarity to the primer that binds to each primer binding site. In some embodiments, the forward binding site and the reverse primer binding site each comprises at least about 70%, about 80%, about 85%, about 90%, about 92%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, or about 100% complementarity to the primer that binds to each primer binding site. In some embodiments, the forward binding site and the reverse primer binding site each comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide substitutions, insertions, or deletions with respect to the nucleotide sequence of the primer that binds to each primer binding site. In some embodiments, the forward binding site and the reverse primer binding site each comprises 100% complementarity to the primer that binds to each primer binding site. In some embodiments, the primers bind to the primer binding site for amplification of the barcode.

[0190] In some embodiments, the distance between the forward and reverse primer binding sites is at least about 50 nucleotides in length, at least about 60 nucleotides in length, at least about 70 nucleotides in length, at least about 80 nucleotides in length, at least about 90 nucleotides in length, at least about 100 nucleotides in length, or more. In some embodiments, the distance between the forward and reverse primer binding sites is about 50 nucleotides to about 350 nucleotides in length, such as about 50 nucleotides to about 150 nucleotides, about 100 nucleotides to about 200 nucleotides, about 150 nucleotides to about 250 nucleotides, about 200 nucleotides to about 300 nucleotides, or about 250 nucleotides to about 350 nucleotides. [0191] In some embodiments, the forward primer binding site and the reverse primer binding site are unique compared to nucleotide sequences found within the host genome. In some embodiments, the forward primer binding site and the reverse primer binding site are distinguishable from nucleotide sequences found within the host genome. In some embodiments, the primers that bind to the forward primer binding site and the reverse primer binding site do not substantially bind to nucleotide sequences found within the host genome. In some embodiments, the forward primer binding site comprises the nucleotide sequence of SEQ ID NO:33 or a variant thereof comprising about 1, 2, 3, 4, or 5 nucleotide substitutions, insertions, or deletions. In some embodiments, the reverse primer binding site comprises the nucleotide sequence of SEQ ID NO:34 or a variant thereof comprising about 1, 2, 3, 4, or 5 nucleotide substitutions, insertions, or deletions. In some embodiments, the forward primer binding site comprises the nucleotide sequence of SEQ ID NO:33, and/or the reverse primer binding site comprises the nucleotide sequence of SEQ ID NO:34.

4. Identifying Regions

[0192] In some embodiments, the nucleic acid(s), for example any of the vectors described herein or nucleic acids derived thereof that integrate into a cell genome, comprise an identifying region comprising a barcode . In some embodiments, the identifying region further comprises one or more primer binding sites, for example a forward primer binding site and/or a reverse primer binding site. In some embodiments, the barcode is located in between the forward primer binding site and the reverse primer binding site, z.e., the barcode is flanked by the forward primer binding site and the reverse primer binding site.

[0193] A vector may have one or more identifying regions. For example, a vector may comprise any of 1, 2, 3, 4, 5, 6, 7, 8 , 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more identifying regions. The one or more identifying regions may comprise the same or different sequences. For example, a vector comprising one or more identifying regions wherein the identifying regions comprise the same sequences may be indicative of the presence of two identical copies inserted into the vector. In another example, a vector comprising one or more identifying regions wherein the identifying regions comprise difference sequences may be indicative of the presence of two different transgenes inserted into the vector. The identifying regions of the present disclosure are used to detect the presence of one or more barcodes that indicate the presence of one or more transgenes within an engineered cell or population of cells (e.g., T cells, B cells, NK cells, islet cells such as beta cells, or hypoimmunogenic cells thereof). For example, the identifying region may comprise any of 1, 2, 3, 4, 5, 6, 7, 8 , 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more barcodes.

[0194] In some embodiments, the identifying region comprises one or more probe binding sites. In some embodiments, the probe is designed to bind to the barcode. Probe binding sites are stretches of nucleic acid that, for example, a fluorescein-labeled oligonucleotide probe can recognize and hybridize, as described in more detail in Section II.1 above. Detection of the probe is also detection of the barcode, which indicate the presence of a transgene in a vector, including within a cell that contains or expresses the vector.

[0195] In some embodiments, the first identifying region and the second identifying region each has at least one barcode. In some embodiments, the first identifying region and/or the second identifying region has one barcode. In some embodiments, the first identifying region and/or the second identifying region has more than one barcode. In some embodiments, the first identifying region and/or the second identifying region comprises a first barcode and/or a second barcode comprising a nucleotide sequence selected from the group consisting of CATCGGAA, GGACAATT, TGTCAACT, TTACAGTT, ATTCAAGG, and TACAGTTA, or a variant thereof having at least about 80% (such as at least about any of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to any one of CATCGGAA, GGACAATT, TGTCAACT, TTACAGTT, ATTCAAGG, and/or TACAGTTA. In some embodiments, the first identifying region and/or the second identifying region comprises a first barcode and/or a second barcode comprising a nucleotide sequence selected from the group consisting of CATCGGAA, GGACAATT, TGTCAACT, TTACAGTT, ATTCAAGG, and TACAGTTA, or a variant thereof comprising up to about 6 (such as about any of 1, 2, 3, 4, 5, or 6) nucleotide substitutions.

[0196] In some embodiments, the first identifying region and the second identifying region each comprises a probe binding site. In some embodiments, the first identifying region and/or the second identifying region comprises two or more probe binding sites. In some embodiments, the first identifying region and/or the second identifying region comprises a first probe binding site and/or a second probe binding site comprising a nucleotide sequence set forth in any one of SEQ ID NOs:18-23, or a variant thereof having at least about 80% (such as at least about any of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to any one of SEQ ID NOs: 18-23. In some embodiments, the first identifying region and/or the second identifying region comprises a first probe binding site and/or a second probe binding site comprising a nucleotide sequence of any one of SEQ ID NOs: 18-23, or a variant thereof comprising up to about 6 (such as about any of 1, 2, 3, 4, 5, or 6) nucleotide substitutions. SEQ ID NO: 18 (Exemplary probe binding sequence #1; barcode bolded and underlined)

GAAACTCATCGGAAAGACGGAG

SEQ ID NO: 19 (Exemplary probe binding sequence #2; barcode bolded and underlined) GAAACTGGACAATTAGACGGAG

SEQ ID NO: 20 (Exemplary probe binding sequence #3; barcode bolded and underlined) GAAACTTGTCAACTAGACGGAG

SEQ ID NO:21 (Exemplary probe binding sequence #4; barcode bolded and underlined) GAAACTTTACAGTTAGACGGAG

SEQ ID NO: 22 (Exemplary probe binding sequence #5; barcode bolded and underlined) GAAACTATTCAAGGAGACGGAG

SEQ ID NO:23 (Exemplary probe binding sequence #6; barcode bolded and underlined) GAAACTTACAGTTAAGACGGAG

[0197] In some embodiments, the first identifying region and the second identifying region are each between about 10 to about 100 nucleotides in length, such as about any of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides in length. In some embodiments, the first identifying region and the second identifying region are each between about 18 to about 30 nucleotides in length. In some embodiments, the first identifying region and the second identifying region are each at least about any of 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some embodiments, the first identifying region and the second identifying region are about the same length. In some embodiments, the first identifying region and the second identifying region are different lengths. In some embodiments, the first identifying region and/or the second identifying region comprises a nucleotide sequence set forth in any one of SEQ ID NOs:24-30, or a variant thereof having at least about 80% (such as at least about any of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to any one of SEQ ID NOs:24-30. In some embodiments, the first identifying region and/or the second identifying region comprises a nucleotide sequence of any one of SEQ ID NOs:24-30, or a variant thereof comprising up to about 6 (such as about any of 1, 2, 3, 4, 5, or 6) nucleotide substitutions. In some embodiments, the first identifying region and/or the second identifying region comprises an antisense nucleic acid sequence of SEQ ID NO:32, wherein NNNNNNNN is the barcode and wherein N represents a nucleotide selected from the group consisting of A, T, C, and G.

SEQ ID NO: 24 (Barcode insert/identifying region #1; exemplary barcode underlined and bolded) ATCGAGTGTAGCCTTATCGCAGCTAGACGAAACTCATCGGAAAGACGGAGAAGTAA CGCAGGAATGAAAGACCCCAC

SEQ ID NO: 25 (Barcode insert/identifying region #2; exemplary barcode underlined and bolded) ATCGAGTGTAGCCTTATCGCAGCTAGACGAAACTGGACAATTAGACGGAGAAGTAA CGCAGGAATGAAAGACCCCAC

SEQ ID NO: 26 (Barcode insert/identifying region #3; exemplary barcode underlined and bolded) ATCGAGTGTAGCCTTATCGCAGCTAGACGAAACTTGTCAACTAGACGGAGAAGTAA CGCAGGAATGAAAGACCCCAC

SEQ ID NO: 27 (Barcode insert/identifying region #4; exemplary barcode underlined and bolded) ATCGAGTGTAGCCTTATCGCAGCTAGACGAAACTTTACAGTTAGACGGAGAAGTAA CGCAGGAATGAAAGACCCCAC

SEQ ID NO:28 (Barcode insert/identifying region #5; exemplary barcode underlined and bolded) ATCGAGTGTAGCCTTATCGCAGCTAGACGAAACTATTCAAGGAGACGGAGAAGTAA CGCAGGAATGAAAGACCCCAC

SEQ ID NO: 29 (Barcode insert/identifying region #6; exemplary barcode underlined and bolded) ATCGAGTGTAGCCTTATCGCAGCTAGACGAAACTTACAGTTAAGACGGAGAAGTAA CGCAGGAATGAAAGACCCCAC

SEQ ID NO:30 (Barcode insert/identifying region, Universal Gibson forward primer; randomly generated barcode underlined and bolded)

ATCGAGTGTAGCCTTATCGCAGCTAGACGAAACTNNNNNNNNAGACGGAGAAGTAA CGCAGGAATGAAAGACCCCAC

SEQ ID NO:32 (Barcode insert/identifying region, Universal Gibson reverse primer and antisense to Universal Gibson forward primer; randomly generated barcode underlined and bolded)

GTGGGGTCTTTCATTCCTGCGTTACTTCTCCGTCTNNNNNNNNAGTTTCGTCTAGCTG CGATAAGGCTACACTCGAT

[0198] In some embodiments, the first identifying region is located in a non-coding region or a coding region of the vector comprising the first transgene. In some embodiments, the second identifying region is located in a second non-coding region or a second coding region of the vector comprising the second transgene. In some embodiments, the first identifying region and the second identifying region are located on the same vector. In some embodiments, the first identifying region and the second identifying region are located in the same region of the vector that comprises the first transgene and the second transgene. In some embodiments, the first identifying region and the second identifying region are located on different vectors.

[0199] In some embodiments, the first identifying region is located within the first transgene and/or the second identifying region is located within the second transgene. In some embodiments, the first identifying region is located outside of the first transgene and the second identifying region is located outside of the second transgene. In some embodiments, the first identifying region is located within the first transgene and the second identifying region is located outside of the second transgene. In some embodiments, the first identifying region is located within the first transgene and the second identifying region is located outside of the second transgene. In some embodiments, the first identifying region and the second identifying region are located on the same vector. In some embodiments, the first identifying region and the second identifying region are located in the same region of the vector that comprises the first transgene and the second transgene. In some embodiments, the first identifying region and the second identifying region are located on different vectors.

[0200] In some embodiments, the first identifying region is located 3' to the first transgene and/or the second identifying region is located 3' to the second transgene. In some embodiments, the first identifying region is located 3' to the first transgene within about 1 to about 200 base pairs and/or the second identifying region is located 3' to the second transgene within about 1 to about 200 base pairs. In some embodiments, the first identifying region is located 3' to the first transgene at least about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 191, 192, 193, 194, or 195 base pairs and/or the second identifying region is located 3' to the second transgene at least about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 191, 192, 193, 194, or 195 base pairs. In some embodiments, the first identifying region is located 3' to the first transgene within 1-200 base pairs and/or the second identifying region is located 3' to the transgene promoter within 1- 200 base pairs.

[0201] In some embodiments, the first identifying region and/or the second identifying region is upstream of one or more additional regulatory elements. In some embodiments, the first identifying region and/or second identifying region is downstream of one or more additional regulatory elements. Regulatory elements can include, but are not limited to, any one or combination of: promoter sequences, enhancer sequences, intron sequences, terminator sequences, translation initiation signal sequences, polyadenylation signal sequences, replication element sequences, RNA processing and export element sequences, transposon sequences, transposase sequences, insulator sequences, 5' UTR sequences, 3' UTR sequences, mRNA 3' end processing sequences, boundary element sequences, locus control region (LCR) sequences, matrix attachment region (MAR) sequences, ubiquitous chromatin opening elements, linker sequences, secretion signal sequences, anchoring peptide sequences, localization signal sequences, fusion tag sequences, affinity tag sequences, chaperonin sequences, protease sequences, and posttranscriptional regulatory element sequences.

[0202] In some embodiments, the first identifying region is upstream of a first promoter and/or the second identifying region is upstream of a second promoter. In some embodiments, the first identifying region is downstream of the first promoter and/or the second identifying region is downstream of the second promoter. In some embodiments, the first identifying region is upstream of the first promoter and the second identifying region is downstream of the second promoter. In some embodiments, the first identifying region is downstream of the first promoter and the second identifying region is upstream of the second promoter. In some embodiments, the promoter is selected from the group consisting of a CAG promoter, cytomegalovirus (CMV) promoter, EFla promoter, EFla short promoter, PGK promoter, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, tk promoter of HSV, mouse mammary tumor virus (MMTV) promoter, LTR promoter of HIV, Epstein Barr virus (EBV) promoter, Rous sarcoma virus (RSV) promoter, UBC promoter, MoMuLV promoter, an avian leukemia virus promoter, actin promoter, myosin promoter, hemoglobin promoter, creatine kinase promoter, hybrid CMV enhancer/chicken P-actin (CBA) promoter, and CBA hybrid intron (CBh) promoter. In some embodiments, the first promoter and/or the second promoter is operably linked to the first transgene and/or the second transgene.

[0203] In some embodiments, the first promoter is selected from the group consisting of: an EFla promoter, an EFla short promoter, a CAG promoter, a ubiquitin/S27a promoter, an SV40 early promoter, an adenovirus major late promoter, a mouse metallothionein-I promoter, an RSV promoter, an MMTV promoter, a Moloney murine leukemia virus Long Terminal repeat region, a CMV promoter, an actin promoter, an immunoglobulin promoter, a heat shock promoter, polyoma virus promoter, a fowlpox virus promoter, a bovine papilloma virus promoter, an avian sarcoma virus promoter, a retrovirus promoter, a hepatitis-B virus promoter, a PGK promoter, an adenovirus late promoter, a vaccinia virus 7.5K promoter, a SV40 promoter, a tk promoter of HSV, a mouse mammary tumor virus (MMTV) promoter, an LTR promoter of HIV, a promoter of moloney virus, an Epstein Barr virus (EBV) promoter, a Rous sarcoma virus (RSV) promoter, a U6 promoter, and an UBC promoter. In some embodiments, the second promoter is selected from the group consisting of: an EFla promoter, an EFla short promoter, a CAG promoter, a ubiquitin/S27a promoter, an SV40 early promoter, an adenovirus major late promoter, a mouse metallothionein-I promoter, an RSV promoter, an MMTV promoter, a Moloney murine leukemia virus Long Terminal repeat region, a CMV promoter, an actin promoter, an immunoglobulin promoter, a heat shock promoter, polyoma virus promoter, a fowlpox virus promoter, a bovine papilloma virus promoter, an avian sarcoma virus promoter, a retrovirus promoter, a hepatitis-B virus promoter, a PGK promoter, an adenovirus late promoter, a vaccinia virus 7.5K promoter, a SV40 promoter, a tk promoter of HSV, a mouse mammary tumor virus (MMTV) promoter, an LTR promoter of HIV, a promoter of moloney virus, an Epstein Barr virus (EBV) promoter, a Rous sarcoma virus (RSV) promoter, a U6 promoter, and an UBC promoter. In some embodiments, the first transgene is operably linked to a first promoter. In some embodiments, the second transgene is operably linked to a second promoter. In some embodiments, the first promoter and the second promoter comprise the same sequence. [0204] In some embodiments, the first identifying region is located 5' to the first promoter and/or the second identifying region is located 5' to the second promoter. In some embodiments, the first identifying region is located 5' to the first promoter within about 1 to about 200 base pairs and/or the second identifying region is located 5' to the second promoter within about 1 to about 200 base pairs. In some embodiments, the first identifying region is located 5' to the first promoter at least about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 191, 192, 193, 194, or 195 base pairs and/or the second identifying region is located 5' to the second promoter at least about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 191, 192, 193, 194, or 195 base pairs. In some embodiments, the first identifying region is located 5' to the first promoter within 1-200 base pairs and/or the second identifying region is located 5' to the second promoter within 1-200 base pairs.

[0205] In some embodiments, the first identifying region is located 3' of the first promoter and/or the second identifying region is located 3' to the second promoter. In some embodiments, the first identifying region is located 3' to the first promoter within about 1 to about 200 base pairs and/or the second identifying region is located 3' to the second promoter within about 1 to about 200 base pairs. In some embodiments, the first identifying region is located 3' to the first promoter at least about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 191, 192, 193, 194, or 195 base pairs and/or the second identifying region is located 3' to the second promoter at least about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 191, 192, 193, 194, or 195 base pairs. In some embodiments, the first identifying region is located 3' to the first promoter within 1-200 base pairs and/or the second identifying region is located 3' to the second promoter within 1-200 base pairs.

[0206] In some embodiments, the first identifying region is located 5' to the first promoter within about 1 to about 200 base pairs and/or the second identifying region is located 3' to the second promoter within about 1 to about 200 base pairs. In some embodiments, the first identifying region is located 3' to the first promoter within about Ito about 200 base pairs and/or the second identifying region is located 5' to the second promoter within about 1 to about 200 base pairs. In some embodiments, the first identifying region of the first vector is inserted 5' to the first promoter, 3' to the first transgene, and 5' or 3' to a Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE).

[0207] In some embodiments, a vector comprising a transgene and an identifying region comprising a barcode of the present disclosure comprises a linker sequence, such as any of an internal ribosome entry site (IRES) sequence, a cleavable peptide sequence, a 2A peptide sequence, a F2A peptide sequence, a E2A peptide sequence, a P2A peptide sequence, a T2A peptide sequence, or a tPT2A peptide sequence, or any other suitable linker sequence known in the art.

5. Transgenes

[0208] The present disclosure provides a transgene encoded by a vector, wherein the vector comprises an identifying region comprising a barcode. In some embodiments, the vector encodes one or more transgenes. In some embodiments, the transgene encodes a chimeric antigen receptor (CAR), an antibody or an antibody fragment, a chimeric autoantibody receptor (CAAR), a B-cell autoantibody receptor (BAR), a T cell receptor (TCR), or one or more tolerogenic factors known in the art or described herein. In some embodiments, the vector comprises a CAR. [0209] In some embodiments, the one or more transgenes, e.g., the first and/or second transgene, encodes a protein, such as any of enzymes, structural polypeptides, signaling polypeptides, regulatory polypeptides, transport polypeptides, sensory polypeptides, motor polypeptides, defense polypeptides, storage polypeptides, transcription factors, antibodies, cytokines, hormones, catabolic polypeptides, anabolic polypeptides, proteolytic polypeptides, metabolic polypeptides, kinases, transferases, hydrolases, lyases, isomerases, ligases, enzyme modulator polypeptides, protein binding polypeptides, lipid binding polypeptides, membrane fusion polypeptides, cell differentiation polypeptides, epigenetic polypeptides, cell death polypeptides, nuclear transport polypeptides, nucleic acid binding polypeptides, reprogramming polypeptides, DNA editing polypeptides, DNA repair polypeptides, DNA recombination polypeptides, transposase polypeptides, DNA integration polypeptides, targeted endonucleases, recombinases, transposases, DNA polymerases, RNA polymerases, or reverse transcriptase. [0210] In some embodiments, the one or more transgenes, e.g., the first and/or second transgene, encodes a recombinant protein. In some embodiments, the one or more transgenes, e.g., the first and/or second transgene, encodes a wild-type protein. In some embodiments, the one or more transgenes, e.g., the first and/or second transgene, encodes a mutant or variant protein.

[0211] In some embodiments, the one or more transgenes, e.g., the first and second transgene, comprise a multi-cistronic construct comprising two or more sequences encoding two or more transgenes. In some embodiments, the one or more transgenes, e.g., the first and second transgene, are operably linked to one or more regulatory elements, such as any of the regulatory elements described herein (e.g., any of one or more promoter sequences, enhancer sequences, intron sequences, terminator sequences, translation initiation signal sequences, polyadenylation signal sequences, replication element sequences, RNA processing and export element sequences, transposon sequences, transposase sequences, insulator sequences, 5’UTR sequences, 3’UTR sequences, mRNA 3’ end processing sequences, boundary element sequences, locus control region (LCR) sequences, matrix attachment region (MAR) sequences, ubiquitous chromatin opening elements, linker sequences, secretion signal sequences, anchoring peptide sequences, localization signal sequences, fusion tag sequences, affinity tag sequences, chaperonin sequences, protease sequences, posttranscriptional regulatory element sequences, or any combination thereof). i. Engineered Receptors

[0212] In some embodiments, the one or more transgenes comprise sequence(s) encoding an engineered receptor, such as a chimeric antigen receptor (CAR), a chimeric autoantibody receptor (CAAR), a B-cell autoantibody receptor (BAR), or a T cell receptor (TCR). a. Chimeric Antigen Receptors (CARs)

[0213] In some embodiments, the one or more transgenes, e.g., the first and/or the second transgene, comprises a chimeric antigen receptor (CAR). CARs (also known as chimeric immunoreceptors, chimeric T cell receptors, or artificial T cell receptors) are receptor proteins that have been engineered to give host cells (e.g., T cells) the new ability to target a specific protein. The receptors are chimeric because they combine both antigen-binding and T cell activating functions into a single receptor. For example, a CAR may comprise an extracellular binding domain that specifically binds a target antigen, a transmembrane domain, and an intracellular signaling domain. In certain embodiments, the CAR may further comprise one or more additional elements, including one or more signal peptides, one or more extracellular hinge domains, and/or one or more intracellular costimulatory domains. Domains may be directly adjacent to one another, or there may be one or more amino acids linking the domains. The nucleotide sequence encoding a CAR may be derived from a mammalian sequence, for example, a mouse sequence, a primate sequence, a human sequence, or combinations thereof. In the cases where the nucleotide sequence encoding a CAR is non-human, the sequence of the CAR may be humanized. The nucleotide sequence encoding a CAR may also be codon-optimized for expression in a mammalian cell, for example, a human cell.

[0214] In some embodiments, the CAR is or comprises a first- generation CAR comprising an antigen binding domain, a transmembrane domain, and signaling domain (e.g., one, two or three signaling domains). In some embodiments, the CAR is or comprises a second-generation CAR comprising an antigen binding domain, a transmembrane domain, and two signaling domains. In some embodiments, the CAR is or comprises a third generation CAR comprising an antigen binding domain, a transmembrane domain, and at least three signaling domains. In some embodiments, the CAR is or comprises a fourth generation CAR comprising an antigen binding domain, a transmembrane domain, three or four signaling domains, and a domain which upon successful signaling of the CAR induces expression of a cytokine gene. In some embodiments, the antigen binding domain is or comprises an scFv or Fab.

[0215] In some embodiments, the antigen binding domain binds to a cell surface antigen of a cell. In some embodiments, the antigen binding domain targets an antigen characteristic of a cell type, such as a neoplastic cell, a T cell, a B cell, or a senescent cell. In some embodiments, the antigen binding domain binds to a cell surface antigen of a cell. In some embodiments, a cell surface antigen is characteristic of one type of cell. In some embodiments, a cell surface antigen is characteristic of more than one type of cell.

[0216] In some embodiments, the antigen characteristic of a neoplastic cell is selected from a cell surface receptor, an ion channel-linked receptor, an enzyme-linked receptor, a G protein- coupled receptor, receptor tyrosine kinase, tyrosine kinase associated receptor, receptor-like tyrosine phosphatase, receptor serine/ threonine kinase, receptor guanylyl cyclase, histidine kinase associated receptor, Epidermal Growth Factor Receptors (EGFR) (including ErbBl/EGFR, ErbB2/HER2, ErbB3/HER3, and ErbB4/HER4), Fibroblast Growth Factor Receptors (FGFR) (including FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF18, and FGF21) Vascular Endothelial Growth Factor Receptors (VEGFR) (including VEGF-A, VEGF- B, VEGF-C, VEGF-D, and PIGF), RET Receptor and the Eph Receptor Family (including EphAl, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphA9, EphAlO, EphBl, EphB2. EphB3, EphB4, and EphB6), CXCR1, CXCR2, CXCR3, CXCR4, CXCR6, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR8, CFTR, CIC-1, CIC-2, CIC-4, CIC-5, CIC-7, CIC- Ka, CIC-Kb, Bestrophins, TMEM16A, GABA receptor, glycin receptor, ABC transporters, NAV1.1, NAV1.2, NAV1.3, NAV1.4, NAV1.5, NAV1.6, NAV1.7, NAV1.8, NAV1.9, sphingosin-1 -phosphate receptor (S1P1R), NMDA channel, transmembrane protein, multispan transmembrane protein, T-cell receptor motifs, T-cell receptor alpha chains, T-cell receptor P chains, T-cell receptor y chains, T-cell receptor 5 chains, CCR7, CD3, CD4, CD5, CD7, CD8, CDllb, CDl lc, CD16, CD19, CD20, CD21, CD22, CD25, CD28, CD34, CD35, CD40, CD45RA, CD45RO, CD52, CD56, CD62E, CD68, CD80, CD95, CD117, CD127, CD133, CD137 (4-1 BB), CD163, F4/80, IE-4Ra, Sca-1, CTEA-4, GITR, GARP, FAP, granzyme B, EFA-1, transferrin receptor, NKp46, perforin, CD4+, (e.g., CD4+ Thl, Th2, Thl7, Th40, Th22, Th9, Tfh, Canonical Treg, FoxP3+, Tri, Th3, Tregl7, or TREG cells) CDCP1, NT5E, EpCAM, CEA, gpA33, Mucins, TAG-72, Carbonic anhydrase IX, PSMA, Folate binding protein, Gangliosides (e.g., CD2, CD3, GM2), Lewis-y2, VEGF, VEGFR 1/2/3, aVp3, a5 i, ErbBl/EGFR, ErbBl/HER2, ErB3, c-MET, IGF1R, EphA3, TRAIL-R1, TRAIL-R2, RANKL, FAP, Tenascin, PDL-1, BAFF, HDAC, ABE, FET3, KIT, MET, RET, IL-ip, ALK, RANKL, mTOR, CTLA-4, IL-6, IL-6R, JAK3, BRAF, PTCH, Smoothened, PIGF, ANPEP, TIMP1, PLAUR, PTPRJ, LTBR, or ANTXR1, Folate receptor alpha (FRa), ERBB2 (Her2/neu), EphA2, IL-13Ra2, epidermal growth factor receptor (EGFR), Mesothelin, TSHR, CD19, CD123, CD22, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII , GD2, GD3, BCMA, MUC16 (CA125), L1CAM, LeY, MSLN, IL13Ral, Ll-CAM, Tn Ag, prostate specific membrane antigen (PSMA), R0R1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, interleukin- 11 receptor a (IL-l lRa), PSCA, PRSS21, VEGFR2, LewisY, CD24, platelet-derived growth factor receptor-beta (PDGFR-beta), SSEA-4, CD20, MUC1, NCAM, Prostase, PAP, ELF2M, Ephrin B2, IGF-1 receptor, CAIX, LMP2, gplOO, bcr-abl, tyrosinase, Fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, Folate receptor beta, TEM1/CD248, TEM7R, CLDN6, GPRC5D, CX0RF61, CD97, CD179a, ALK, Polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-la, MAGE-A1, legumain, HPV E6, E7, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Major histocompatibility complex class I-related gene protein (MR1), urokinasetype plasminogen activator receptor (uPAR), Fos-related antigen 1, p53, p53 mutant, prostein, survivin, telomerase, PCTA-l/Galectin 8, MelanA/MARTl, Ras mutant, hTERT, sarcoma translocation breakpoints, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA 17, PAX3, Androgen receptor, Cyclin Bl, MYCN, RhoC, TRP-2, CYPIB I, BORIS, SART3, PAX5, OY- TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxyl esterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, a neoantigen, CD133, CD15, CD184, CD24, CD56, CD26, CD29, CD44, HLA-A, HLA-B, HLA-C, (HLA-A,B,C) CD49f, CD 151 CD340, CD200, tkrA, trkB, or trkC, or an antigenic fragment or antigenic portion thereof.

[0217] In some embodiments, the antigen characteristic of a T cell is selected from a cell surface receptor, a membrane transport protein (e.g., an active or passive transport protein such as, for example, an ion channel protein, a pore-forming protein, etc.), a transmembrane receptor, a membrane enzyme, and/or a cell adhesion protein characteristic of a T cell. In some embodiments, an antigen characteristic of a T cell may be a G protein-coupled receptor, receptor tyrosine kinase, tyrosine kinase associated receptor, receptor-like tyrosine phosphatase, receptor serine/threonine kinase, receptor guanylyl cyclase, histidine kinase associated receptor, AKT1, AKT2, AKT3, ATF2, BCL10, CALM1, CD3D (CD35), CD3E (CD3s), CD3G (CD3y), CD4, CD8, CD28, CD45, CD80 (B7-1), CD86 (B7-2), CD247 (CD3Q, CTLA4 (CD152), ELK1, ERK1 (MAPK3), ERK2, FOS, FYN, GRAP2 (GADS), GRB2, HLA-DRA, HLA-DRB1, HLA- DRB3, HLA-DRB4, HLA-DRB5, HRAS, IKBKA (CHUK), IKBKB, IKBKE, IKBKG (NEMO), IL2, ITPR1, ITK, JUN, KRAS2, LAT, LCK, MAP2K1 (MEK1), MAP2K2 (MEK2), MAP2K3 (MKK3), MAP2K4 (MKK4), MAP2K6 (MKK6), MAP2K7 (MKK7), MAP3K1 (MEKK1), MAP3K3, MAP3K4, MAP3K5, MAP3K8, MAP3K14 (NIK), MAPK8 (JNK1), MAPK9 (JNK2), MAPK10 (JNK3), MAPK11 (p38 ), MAPK12 (p38y), MAPK13 (p385), MAPK14 (p38a), NCK, NFAT1, NFAT2, NFKB1, NFKB2, NFKBIA, NRAS, PAK1, PAK2, PAK3, PAK4, PIK3C2B, PIK3C3 (VPS34), PIK3CA, PIK3CB, PIK3CD, PIK3R1, PKCA, PKCB, PKCM, PKCQ, PLCY1, PRF1 (Perforin), PTEN, RAC1, RAFI, RELA, SDF1, SHP2, SLP76, SOS, SRC, TBK1, TCRA, TEC, TRAF6, VAV1, VAV2, or ZAP70.

[0218] In some embodiments, the antigen characteristic of senescent cells is, for example, urokinase-type plasminogen activator receptor (uPAR). In some embodiments, the antigen binding domain binds an antigen associated with a senescent cell. In some instances, the antigen is expressed by a senescent cell. In some embodiments, the CAR may be used for treatment or prophylaxis of disorders characterized by the aberrant accumulation of senescent cells, e.g., liver and lung fibrosis, atherosclerosis, diabetes and osteoarthritis.

[0219] In some embodiments, the antigen characteristic of a B cell is selected from the group consisting of IL-10, TGFp, IgD, CD1, CD5, CD21, CD24, TLR4, CD21, CD22, CD23, Notch2, CD27, CXCR3, CXCR4, CXCR5, CXCR6, IgA, IgG, IgE, CD20, CD40, CD80, PDL-2, CD138, IL-6, CD38, CD78, CD319, CD25, CD30, CD19, CD22, ROR1, CD45, CD47, CD33, Igkappa, Iglambda, CD79a, CD79b, and IgM. In some embodiments, a CAR antigen binding domain binds to a ligand expressed on B cells, plasma cells, or plasmablasts, such as CD 10, CD19, CD20, CD22, CD24, CD27, CD38, CD45R, CD138, CD319, BCMA, CD28, TNF, interferon receptors, GM-CSF, ZAP-70, LFA-1, CD3 gamma, CD5, or CD2. See US 2003/0077249; WO 2017/058753; WO 2017/058850, the contents of which are herein incorporated by reference.

[0220] In some embodiments, the antigen binding domain targets an antigen characteristic of a disease, disorder, injury, or condition.

[0221] In certain embodiments, the antigen binding domain targets an antigen that is exclusively or preferentially expressed on tumor cells. Exemplary target antigens include, but are not limited to, CD5, CD 19, CD20, CD22, CD23, CD30, CD70, Kappa, Lambda, B cell maturation agent (BCMA), G-protein coupled receptor family C group 5 member D (GPRC5D) (associated with leukemias); CS1/SLAMF7, CD38, CD138, GPRC5D, TACI, and BCMA (associated with myelomas); GD2, HER2, EGFR, EGFRvIII, B7H3, PSMA, PSCA, CAIX, CD171, CEA, CSPG4, EPHA2, FAP, FRa, IE-13Ra, Mesothelin, MUC1, MUC16, and R0R1 (associated with solid tumors).

[0222] In some embodiments, the antigen binding domain targets an antigen characteristic of an autoimmune or inflammatory disorder. In some embodiments, the CAR binds an antigen associated with an autoimmune or inflammatory disorder. In some instances, the antigen is expressed by a cell associated with an autoimmune or inflammatory disorder. In some embodiments, the autoimmune or inflammatory disorder is selected from the group consisting of: chronic graft-vs-host disease (GVHD), lupus, arthritis, immune complex glomerulonephritis, goodpasture, uveitis, hepatitis, systemic sclerosis or scleroderma, type I diabetes, multiple sclerosis, cold agglutinin disease, Pemphigus vulgaris, Grave's disease, autoimmune hemolytic anemia, Hemophilia A, Primary Sjogren's Syndrome, thrombotic thrombocytopenia purpura, neuromyelits optica, Evan's syndrome, IgM mediated neuropathy, cyroglobulinemia, dermatomyositis, idiopathic thrombocytopenia, ankylosing spondylitis, bullous pemphigoid, acquired angioedema, chronic urticarial, antiphospholipid demyelinating polyneuropathy, and autoimmune thrombocytopenia or neutropenia or pure red cell aplasias, while exemplary nonlimiting examples of alloimmune diseases include allosensitization (see, for example, Blazar et al., 2015, Am. J. Transplant, 15(4) :931 -41 ) or xenosensitization from hematopoietic or solid organ transplantation, blood transfusions, pregnancy with fetal allosensitization, neonatal alloimmune thrombocytopenia, hemolytic disease of the newborn, sensitization to foreign antigens such as can occur with replacement of inherited or acquired deficiency disorders treated with enzyme or protein replacement therapy, blood products, or gene therapy. In some embodiments, the antigen characteristic of an autoimmune or inflammatory disorder is selected from a cell surface receptor, an ion channel-linked receptor, an enzyme-linked receptor, a G protein-coupled receptor, receptor tyrosine kinase, tyrosine kinase associated receptor, receptorlike tyrosine phosphatase, receptor serine/ threonine kinase, receptor guanylyl cyclase, or histidine kinase associated receptor.

[0223] In some embodiments, the antigen binding domain targets an antigen characteristic of an infectious disease. In some embodiments, the CAR binds an antigen associated with an infectious disease. In some instances, the antigen is expressed by a cell affected by an infectious disease. In some embodiments, the infectious disease is selected from HIV, hepatitis B virus, hepatitis C virus, Human herpes virus, Human herpes virus 8 (HHV-8, Kaposi sarcoma- associated herpes virus (KSHV)), Human T-lymphotrophic virus-1 (HTLV-1), Merkel cell polyomavirus (MCV), Simian virus 40 (SV40), Epstein-Barr virus, CMV, or human papillomavirus. In some embodiments, the antigen characteristic of an infectious disease is selected from a cell surface receptor, an ion channel-linked receptor, an enzyme-linked receptor, a G protein-coupled receptor, receptor tyrosine kinase, tyrosine kinase associated receptor, receptor-like tyrosine phosphatase, receptor serine/ threonine kinase, receptor guanylyl cyclase, histidine kinase associated receptor, HIV Env, gpl20, or CD4-induced epitope on HIV-1 Env. [0224] In some embodiments, the CAR comprises an antigen binding domain specific for any of: CD5, CD19, CD20, CD22, CD23, CD30, CD33, CD38, CD70, CD123, CD138, BCMA, GPRC5D, CD123, LeY, NKG2D ligand, WT1, GD2, HER2, EGFR, EGFRvIII, B7H3, PSMA, PSCA, CAIX, CD171, CEA, CSPG4, EPHA2, FAP, FRa, IL-13Ra, Mesothelin, MUC1, MUC16, ROR1, C-Met, CD133, Ep-CAM, GPC3, HPV16-E6, IL13Ra2, MAGEA3, MAGEA4, MARTI, NY-ESO-1, VEGFR2, a-Folate receptor, CD24, CD44v7/8, EGP-2, EGP-40, erb-B2, erb-B 2,3,4, FBP, Fetal acethylcholine e receptor, GD2, GD3, HMW-MAA, IL-l lRa, KDR, Lewis Y, Ll-cell adhesion molecule, MAGE-A1, Oncofetal antigen (h5T4), TAG-72, or CD19 and CD22. In some embodiments, the CAR comprises an antigen binding domain specific for any of: CD19, CD22, CD20, BCMA, an EBV antigen, CD27, CD30, CD19 and CD20, CD19 and CD22, CD19 and CD27, EBNA1, EBNA3A, BRLF1, BALF4, EBNA3C, LMP1, LMP2, LMP2A, LMP2B, BZLF1, BMLF1, gp350, gH/gL, EBNA1 and LMP1, EBNA1 and LMP2A, EBNA1 and LMP1 and LMP2A, LMP and BARF1 and EBNA1, CD 19 and an EBV antigen, CD20 and an EBV antigen, or CD22 and an EBV antigen. In some embodiments, the CAR comprises an antigen binding domain specific for any of: CD19, CD20, CD22, CD38, CD123, CD138, BCMA, CD19 and CD22, CD19 and CD20, CD19 and BCMA, CD19 and BAFFR, CD33 and CD123, HER2 and B7H3, HER2 and EGFR, HER2 and IL13Ra, HER2 and ROR1, B7H3 and EGFR, B7H3 and IL13Ra, B7H3 and ROR1, EGFR and IL13Ra, and/or EGFR and ROR1.

[0225] In some embodiments, a cell comprises a CAR comprising an antigen binding domain specific for two or more target antigens. In some embodiments, a cell comprises a CAR comprising an antigen binding domain specific to two or more epitopes of the same target antigen. In some embodiments, a cell comprises two CARs each comprising a different antigen binding domain from each other. For example, in some embodiments, the two or more CARs each comprise an antigen binding domain specific for a target antigen such that the cell comprises two or more CARs targeting any combinations of CD19xCD20, CD19xBCMA, CD20xBCMA, CD19xCD22, CD19xBAFFR, CD33xCD123, HER2xB7H3, HER2xEGFR, HER2xIL13Ra, HER2xRORl, B7H3xEGFR, B7H3xIL13Ra, B7H3xRORl, EGFRxIL13Ra, EGFRxRORl, and Her2xB7H3xEGFRxIL13Ra2xRORl.

[0226] In some embodiments, the CAR comprises a transmembrane domain comprising at least a transmembrane region of the alpha, beta, or zeta chain of a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or functional variant thereof. In some embodiments, the transmembrane domain comprises at least a transmembrane region(s) of CD8a, CD8P, 4-1BB/CD137, CD28, CD34, CD4, FcsRIy, CD16, OX40/CD134, CD3^, CD3s, CD3y, CD35, TCRa, TCRp, TCR^, CD32, CD64, CD64, CD45, CD5, CD9, CD22, CD37, CD80, CD86, CD40, CD40L/CD154, VEGFR2, FAS, FGFR2B, or functional variant thereof.

[0227] In some embodiments, the CAR comprises at least one signaling domain selected from one or more of B7-1/CD80, B7-2/CD86, B7-H1/PD-L1, B7-H2, B7-H3, B7-H4, B7-H6, B7-H7, BTLA/CD272, CD28, CTLA-4, Gi24/VISTA/B7-H5, ICOS/CD278, PD-1, PD-L2/B7- DC, PDCD6, 4-1BB/TNFSF9/CD137, 4-1BB Ligand/TNFSF9, BAFF/BLyS/TNFSF13B, BAFF R/TNFRSF13C, CD27/TNFRSF7, CD27 Ligand/TNFSF7, CD30/TNFRSF8, CD30 Ligand/TNFSF8, CD40/TNFRSF5, CD40/TNFSF5, CD40 Ligand/TNFSF5, DR3/TNFRSF25, GITR/TNFRSF18, GITR Ligand/TNFSF18, HVEM/TNFRSF14, LIGHT/TNFSF14, Ly mpho toxin- alpha/TNF-beta, OX40/TNFRSF4, 0X40 Ligand/TNFSF4, RELT/TNFRSF19L, TACI/TNFRSF I3B, TL1A/TNFSF15, TNF-alpha, TNF RII/TNFRSFI B, 2B4/CD244/SLAMF4, BLAME/SLAMF8, CD2, CD2F-10/SLAMF9, CD48/SLAMF2, CD58/LFA-3, CD84/SLAMF5, CD229/SLAMF3, CRACC/SLAMF7, NTB-A/SLAMF6, SLAM/CD150, CD7, CD53, CD82/Kai-1, CD90/Thyl, CD96, CD160, CD200, CD300a/LMIRl, HLA Class I, HLA-DR, Ikaros, Integrin alpha 4/CD49d, Integrin alpha 4 beta 1, Integrin alpha 4 beta 7/LPAM-l, LAG- 3, TCL1A, TCL1B, CRTAM, DAP12, Dectin- 1/CLEC7 A, DPPIV/CD26, EphB6, TIM-l/KIM- 1/HAVCR, TIM-4, TSLP, TSLP R, lymphocyte function associated antigen-1 (LFA-1), NKG2C, a CD3 zeta domain, an immunoreceptor tyrosine-based activation motif (ITAM), CD27, 4- IBB, CD134/OX40, CD30, CD40, PD-1, ICOS, LIGHT, NKG2C, a ligand that specifically binds with CD83, or functional fragment thereof.

[0228] In some embodiments, the CAR comprises a CD3 zeta domain or an immunoreceptor tyrosine -based activation motif (ITAM), or functional variant thereof. In some embodiments, the CAR comprises (i) a CD3 zeta domain, or an immunoreceptor tyrosine-based activation motif (ITAM), or functional variant thereof; and (ii) a CD28 domain, or a 4- IBB domain, or functional variant thereof. In some embodiments, the CAR comprises a (i) a CD3 zeta domain, or an immunoreceptor tyrosine-based activation motif (ITAM), or functional variant thereof; (ii) a CD28 domain or functional variant thereof; and (iii) a 4- IBB domain, or a CD 134 domain, or functional variant thereof. In some embodiments, the CAR comprises (i) a CD3 zeta domain, or an immunoreceptor tyrosine-based activation motif (ITAM), or functional variant thereof; (ii) a CD28 domain, or a 4- IBB domain, or functional variant thereof, and/or (iii) a 4- IBB domain, or a CD 134 domain, or functional variant thereof. In some embodiments, the CAR comprises a (i) a CD3 zeta domain, or an immunoreceptor tyrosine-based activation motif (ITAM), or functional variant thereof; (ii) a CD28 domain or functional variant thereof; (iii) a 4- 1BB domain, or a CD 134 domain, or functional variant thereof; and (iv) a cytokine or costimulatory ligand transgene.

[0229] In some embodiments, the CAR further comprises one or more spacers, e.g., wherein the spacer is a first spacer between the antigen binding domain and the transmembrane domain. In some embodiments, the first spacer includes at least a portion of an immunoglobulin constant region or variant or modified version thereof. In some embodiments, the spacer is a second spacer between the transmembrane domain and a signaling domain. In some embodiments, the second spacer is an oligopeptide, e.g., wherein the oligopeptide comprises glycine-serine doublets.

[0230] In some embodiments, the CAR is a first-generation CAR comprising an antigen binding domain, a transmembrane domain, and signaling domain. In some embodiments the signaling domain mediates downstream signaling during T cell activation.

[0231] In some embodiments, the CAR is a second-generation CAR. In some embodiments a second-generation CAR comprises an antigen binding domain, a transmembrane domain, and two signaling domains. In some embodiments a signaling domain mediates downstream signaling during T cell activation. In some embodiments a signaling domain is a costimulatory domain. In some embodiments, a costimulatory domain enhances cytokine production, CAR T cell proliferation, and or CAR T cell persistence during T cell activation. [0232] In some embodiments, the CAR is a third generation CAR. In some embodiments, a third generation CAR comprises an antigen binding domain, a transmembrane domain, and at least three signaling domains. In some embodiments a signaling domain mediates downstream signaling during T cell activation. In some embodiments a signaling domain is a costimulatory domain. In some embodiments, a costimulatory domain enhances cytokine production, CAR T cell proliferation, and or CAR T cell persistence during T cell activation. In some embodiments, a third generation CAR comprises at least two costimulatory domains. In some embodiments, the at least two costimulatory domains are not the same.

[0233] In some embodiments, the CAR is a fourth generation CAR. In some embodiments a fourth generation CAR comprises an antigen binding domain, a transmembrane domain, and at least two, three, or four signaling domains. In some embodiments a signaling domain mediates downstream signaling during T cell activation. In some embodiments a signaling domain is a costimulatory domain. In some embodiments, a costimulatory domain enhances cytokine production, CAR T cell proliferation, and or CAR T cell persistence during T cell activation. [0234] In some embodiments, a first, second, third, or fourth generation CAR further comprises a domain which upon successful signaling of the CAR induces expression of a cytokine gene. In some embodiments, a cytokine gene is endogenous or exogenous to a target cell comprising a CAR which comprises a domain which upon successful signaling of the CAR induces expression of a cytokine gene. In some embodiments a cytokine gene encodes a pro- inflammatory cytokine. In some embodiments a cytokine gene encodes IL-1, IL-2, IL-9, IL- 12, IL- 18, TNF, IFN-gamma, or a functional fragment thereof. In some embodiments, a domain which upon successful signaling of the CAR induces expression of a cytokine gene is or comprises a transcription factor or functional domain or fragment thereof. In some embodiments, a domain which upon successful signaling of the CAR induces expression of a cytokine gene is or comprises a transcription factor or functional domain or fragment thereof. In some embodiments a transcription factor or functional domain or fragment thereof is or comprises a nuclear factor of activated T cells (NF AT), an NF-KB, or functional domain or fragment thereof. See, e.g., Zhang. C. et al., Engineering CAR-T cells. Biomarker Research. 5:22 (2017); WO 2016126608; Sha, H. et al., Chimaeric antigen receptor T-cell therapy for tumour immunotherapy. Bioscience Reports Jan 27, 2017, 37 (1).

[0235] In some embodiments, a CAR antigen binding domain is or comprises an antibody or antigen-binding portion thereof. In some embodiments, a CAR antigen binding domain is or comprises an scFv or Fab. In some embodiments, a CAR antigen binding domain comprises an scFv or Fab fragment of a T-cell receptor alpha chain antibody, T-cell receptor P chain antibody, T-cell receptor y chain antibody, T-cell receptor 5 chain antibody, CCR7 antibody, CD3 antibody, CD4 antibody, CD5 antibody, CD7 antibody, CD8 antibody, CD 11b antibody, CD 11c antibody, CD 16 antibody, CD 19 antibody, CD20 antibody, CD21 antibody, CD22 antibody, CD25 antibody, CD28 antibody, CD34 antibody, CD35 antibody, CD40 antibody, CD45RA antibody, CD45RO antibody, CD52 antibody, CD56 antibody, CD62L antibody, CD68 antibody, CD80 antibody, CD95 antibody, CD117 antibody, CD127 antibody, CD133 antibody, CD137 (4-1 BB) antibody, CD 163 antibody, F4/80 antibody, IL-4Ra antibody, Sca-1 antibody, CTLA-4 antibody, GITR antibody, GARP antibody, LAP antibody, granzyme B antibody, LFA-1 antibody, MR1 antibody, uPAR antibody, transferrin receptor antibody, or any combination thereof.

[0236] In some embodiments, a CAR antigen binding domain is or comprises an antibody or antigen-binding portion thereof. In some embodiments, a CAR antigen binding domain is or comprises an scFv or Fab. In some embodiments, the CAR encoded by the first transgene and/or the second transgene is selected from the group consisting of: a CD5- specific CAR, a CD 19- specific CAR, a CD20-specific CAR, a CD22-specific CAR, a CD23-specific CAR, a CD30- specific CAR, a CD33-specific CAR, CD38-specific CAR, a CD70-specific CAR, a CD 123- specific CAR, a CD 138- specific CAR, a Kappa, Lambda, B cell maturation agent (BCMA)- specific CAR, a G-protein coupled receptor family C group 5 member D (GPRC5D)-specific CAR, a CD 123 -specific CAR, a LeY-specific CAR, a NKG2D ligand- specific CAR, a WT1- specific CAR, a GD2-specific CAR, a HER2-specific CAR, a EGFR-specific CAR, a EGFRvIII- specific CAR, a B7H3-specific CAR, a PSMA-specific CAR, a PSCA-specific CAR, a CAIX- specific CAR, a CD 171 -specific CAR, a CEA- specific CAR, a CSPG4- specific CAR, a EPHA2- specific CAR, a FAP-specific CAR, a FRa-specific CAR, a IL-13Ra-specific CAR, a Mesothelin-specific CAR, a MUC1 -specific CAR, a MUC16-specific CAR, a ROR1 -specific CAR, a C-Met-specific CAR, a CD 133- specific CAR, a Ep-C AM- specific CAR, a GPC3- specific CAR, a HPV16-E6- specific CAR, a IL 13 Ra2- specific CAR, a MAGEA3- specific CAR, a MAGEA4- specific CAR, a MARTI -specific CAR, a NY-ESO-l-specific CAR, a VEGFR2- specific CAR, a a-Folate receptor- specific CAR, a CD24-specific CAR, a CD44v7/8- specific CAR, a EGP-2- specific CAR, a EGP-40-specific CAR, a erb-B2-specific CAR, a erb-B 2,3,4- specific CAR, a FBP- specific CAR, a Fetal acethylcholine e receptor- specific CAR, a GD2- specific CAR, a GD3-specific CAR, a HMW-MAA-specific CAR, a IL- HRa- specific CAR, a KDR-specific CAR, a Lewis Y-specific CAR, a Ll-cell adhesion molecule- specific CAR, a MAGE- Al-specific CAR, a Oncofetal antigen (h5T4)-specific CAR, a TAG-72- specific CAR, and a CD19/CD22-bispecific CAR.

[0237] In some embodiments, a CAR comprises a signaling domain which is a costimulatory domain. In some embodiments, a CAR comprises a second costimulatory domain. In some embodiments, a CAR comprises at least two costimulatory domains. In some embodiments, a CAR comprises at least three costimulatory domains. In some embodiments, a CAR comprises a costimulatory domain selected from the group consisting of CD27, CD28, 4- 1BB, CD134/OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, and any combination thereof. In some embodiments, if a CAR comprises two or more costimulatory domains, two costimulatory domains are different. In some embodiments, if a CAR comprises two or more costimulatory domains, two costimulatory domains are the same.

[0238] In some embodiments, the CAR comprises a CD3 zeta domain or an immunoreceptor tyrosine -based activation motif (ITAM), or functional variant thereof. In some embodiments, the CAR comprises (i) a CD3 zeta domain, or an immunoreceptor tyrosine-based activation motif (ITAM), or functional variant thereof; and (ii) a CD28 domain, or a 4- IBB domain, or functional variant thereof. In some embodiments, the CAR comprises a (i) a CD3 zeta domain, or an immunoreceptor tyrosine-based activation motif (ITAM), or functional variant thereof; (ii) a CD28 domain or functional variant thereof; and (iii) a 4- IBB domain, or a CD 134 domain, or functional variant thereof. In some embodiments, the CAR comprises (i) a CD3 zeta domain, or an immunoreceptor tyrosine-based activation motif (ITAM), or functional variant thereof; (ii) a CD28 domain, or a 4- IBB domain, or functional variant thereof, and/or (iii) a 4- IBB domain, or a CD 134 domain, or functional variant thereof. In some embodiments, the CAR comprises a (i) a CD3 zeta domain, or an immunoreceptor tyrosine-based activation motif (ITAM), or functional variant thereof; (ii) a CD28 domain or functional variant thereof; (iii) a 4- 1BB domain, or a CD 134 domain, or functional variant thereof; and (iv) a cytokine or costimulatory ligand transgene.

[0239] In certain embodiments, the intracellular signaling domain comprises a CD28 transmembrane and signaling domain linked to a CD3 (e.g., CD3-zeta) intracellular domain. In some embodiments, the intracellular signaling domain comprises a chimeric CD28 and CD 137 (4- IBB, TNFRSF9) co- stimulatory domains, linked to a CD3-zeta intracellular domain.

[0240] In some embodiments, the CAR encompasses one or more, e.g., two or more, costimulatory domains and an activation domain, e.g., primary activation domain, in the cytoplasmic portion. Exemplary CARs include intracellular components of CD3-zeta, CD28, and 4-1BB.

[0241] In some embodiments the intracellular signaling domain includes intracellular components of a 4- IBB signaling domain and a CD3-zeta signaling domain. In some embodiments, the intracellular signaling domain includes intracellular components of a CD28 signaling domain and a CD3-zeta signaling domain.

[0242] In some embodiments, the CAR comprises an extracellular antigen binding domain (e.g., antibody or antibody fragment, such as an scFv or Fab) that binds to an antigen (e.g., tumor antigen), a spacer (e.g., containing a hinge domain, such as any as described herein), a transmembrane domain (e.g., any as described herein), and an intracellular signaling domain (e.g., any intracellular signaling domain, such as a primary signaling domain or costimulatory signaling domain as described herein). In some embodiments, the intracellular signaling domain is or includes a primary cytoplasmic signaling domain. In some embodiments, the intracellular signaling domain additionally includes an intracellular signaling domain of a costimulatory molecule (e.g., a costimulatory domain).

[0243] In some embodiments, the CAR contains one or more domains that combine an antigen- or ligand-binding domain (e.g., antibody or antibody fragment) that provides specificity for a desired antigen (e.g., tumor antigen) with intracellular signaling domains. In some embodiments, the intracellular signaling domain is a stimulating or an activating intracellular domain portion, such as a T cell stimulating or activating domain, providing a primary activation signal or a primary signal. In some embodiments, the intracellular signaling domain contains or additionally contains a costimulatory signaling domain to facilitate effector functions. In some embodiments, chimeric receptors when genetically engineered into immune cells can modulate T cell activity, and, in some cases, can modulate T cell differentiation or homeostasis, thereby resulting in genetically engineered cells or populations of cells (e.g., T cells, B cells, NK cells, islet cells such as beta cells, or hypoimmunogenic cells thereof) with improved longevity, survival and/or persistence in vivo, such as for use in adoptive cell therapy methods.

[0244] Exemplary antigen receptors, including CARs, and methods for engineering and introducing such receptors into cells, include those described, for example, in W0200014257, WO2013126726, WO2012/129514, WO2014031687, WO2013/ 166321, W02013/071154, W02013/ 123061, U.S. patent app. Pub. Nos. US2002131960, US2013287748, US20130149337, U.S. Patent Nos. 6,451,995, 7,446,190, 8,252,592, 8,339,645, 8,398,282, 7,446,179, 6,410,319, 7,070,995, 7,265,209, 7,354,762, 7,446,191, 8,324,353, and 8,479,118, and European patent app. No. EP2537416, and/or those described by Sadelain et al., Cancer Discov. 2013 April; 3(4): 388- 398; Davila et al., (2013) PLoS ONE 8(4): e61338; Turtle et al., Curr. Opin. Immunol., 2012 October; 24(5): 633-39; Wu et al., Cancer, 2012 March 18(2): 160-75. In some aspects, the antigen receptors include a CAR as described in U.S. Patent No.: 7,446,190, and those described in WO/2014055668. Examples of the CARs include CARs as disclosed in any of the aforementioned publications, such as WO2014031687, US 8,339,645, US 7,446,179, US 2013/0149337, US 7,446,190, US 8,389,282, Kochenderfer et al., (2013) Nature Reviews Clinical Oncology, 10, 267-276; Wang et al., (2012) J. Immunother. 35(9): 689-701; and Brentjens et al., Sci Transl Med. 2013 5(177). See also WO2014031687, US 8,339,645, US 7,446,179, US 2013/0149337, US 7,446,190, and US 8,389,282. The recombinant receptors, such as CARs, generally include an extracellular antigen binding domain, such as a portion of an antibody molecule, generally a variable heavy (VH) chain region and/or variable light (VL) chain region of the antibody, e.g., an scFv antibody fragment. In some embodiments, the antigen binding domain of the CAR molecule comprises an antibody, an antibody fragment, an scFv, a Fv, a Fab, a (Fab')2, a single domain antibody (SdAb), a VH or VL domain, or a camelid VHH domain.

[0245] In addition to the CARs described herein, various chimeric antigen receptors and nucleotide sequences encoding the same are known in the art and would be suitable for use according to the present disclosure. See, e.g., W02013040557; W02012079000; W02016030414; Smith T, et al., Nature Nanotechnology. 2017. DOI: 10.1038/NNAN0.2017.57, the disclosures of which are herein incorporated by reference. [0246] In some embodiments, the antigen targeted by the CAR is a polypeptide. In some embodiments, it is a carbohydrate or other molecule. In some embodiments, the antigen is selectively expressed or overexpressed on cells of a disease or condition, e.g., the tumor or pathogenic cells, as compared to normal or non-targeted cells or tissues. In other embodiments, the antigen is expressed on normal cells and/or is expressed on the engineered cells or populations of cells (e.g., T cells, B cells, NK cells, islet cells such as beta cells, or hypoimmunogenic cells thereof).

[0247] In some embodiments, the antigen targeted by the receptor includes antigens associated with a B cell malignancy, such as any of a number of known B cell markers. In some embodiments, the antigen targeted by the receptor is CD20, CD19, CD22, ROR1, CD45, CD47, CD21, CD5, CD33, Igkappa, Iglambda, CD79a, CD79b, or CD30.

[0248] In some embodiments, a cell comprises a CAR comprising an antigen binding domain specific for two or more target antigens. In some embodiments, a cell comprises a CAR comprising an antigen binding domain specific to two or more epitopes of the same target antigen. In some embodiments, a cell comprises two or more CARs each comprising a different antigen binding domain from each other. In some embodiments, the CAR binds to CD19. In some embodiments, the CAR binds to CD22. In some embodiments, the CAR binds to CD19 and CD22. In some embodiments, the CAR binds to CD20. In some embodiments, the CAR binds to CD19 and CD20. In some embodiments, the CAR binds to BCMA. In some embodiments, the CAR binds to CD19 and BCMA. In some embodiments, the CAR binds to BAFFR. In some embodiments, the CAR binds to CD19 and BAFFR. In some embodiments, the CAR binds to CD33. In some embodiments, the CAR binds to CD123. In some embodiments, the CAR binds to CD33 and CD123. In some embodiments, the CAR binds to HER2. In some embodiments, the CAR binds to B7H3. In some embodiments, the CAR binds to HER2 and B7H3. In some embodiments, the CAR binds to EGFR. In some embodiments, the CAR binds to HER2 and EGFR. In some embodiments, the CAR binds to IL13Ral and/or IL13Ra2 (i.e., IL13Ra). In some embodiments, the CAR binds to HER2 and IL13Ra. In some embodiments, the CAR binds to ROR1. In some embodiments, the CAR binds to HER2 and ROR1. In some embodiments, the CAR binds to B7H3 and EGFR. In some embodiments, the CAR binds to B7H3 and IL13Ra. In some embodiments, the CAR binds to B7H3 and R0R1. In some embodiments, the CAR binds to EGFR and IL13Ra. In some embodiments, the CAR binds to EGFR and R0R1. For example, in some embodiments, the two or more CARs each comprise an antigen binding domain specific for a target antigen such that the cell comprises two or more CARs targeting any combinations of CD19xCD20, CD19xBCMA, CD20xBCMA, CD19xCD22, CD19xBAFFR, CD33xCD123, HER2xB7H3, HER2xEGFR, HER2xIL13Ra, HER2xRORl, B7H3xEGFR, B7H3xIL13Ra, B7H3xRORl, EGFRxIL13Ra, EGFRxRORl, and Her2xB7H3xEGFRxIL13Ra2xRORl. In some embodiments, the CAR(s) targets an autoimmune disease. In some embodiments, the CAR(s) targets leukemia or lymphoma. In some embodiments, the CAR(s) targets acute myeloid leukemia. In some embodiments, the CAR(s) targets a solid tumor malignancy. In some embodiments, the CAR is selected from the group consisting of a first-generation CAR, a second generation CAR, a third generation CAR, and a fourth generation CAR. In some embodiments, the CAR includes a single binding domain that binds to a single target antigen. In some embodiments, the CAR includes a single binding domain that binds to more than one target antigen, e.g., 2, 3, or more target antigens. In some embodiments, the CAR includes two binding domains such that each binding domain binds to a different target antigen. In some embodiments, the CAR includes two binding domains such that each binding domain binds to the same target antigen. For example, detailed descriptions of exemplary CARs including CD19-specific, CD22-specific and CD19/CD22-bispecific CARs can be found in WO2012/079000, WO2016/149578, and W02020/014482, the disclosures including the sequence listings and figures are incorporated herein by reference in their entirety.

[0249] In some embodiments, the antigen targeted by the antigen-binding domain is CD 19. In some aspects, the antigen-binding domain of the recombinant receptor, e.g., CAR, and the antigen-binding domain binds, such as specifically binds or specifically recognizes, a CD19, such as a human CD19. In some embodiments, the scFv contains a VH and a VL derived from an antibody or an antibody fragment specific to CD 19. In some embodiments, the antibody or antibody fragment that binds CD 19 is a mouse derived antibody such as FMC63 and SJ25C1. In some embodiments, the antibody or antibody fragment is a human antibody, e.g., as described in U.S. Patent Publication No. US 2016/0152723. In some embodiments, the scFv is derived from FMC63. FMC63 generally refers to a mouse monoclonal IgGl antibody raised against Naim-1 and -16 cells expressing CD19 of human origin (Fing, N. R., et al., (1987). Leucocyte typing III. 302).

[0250] In some embodiments, the chimeric antigen receptor includes an extracellular portion containing an antibody or antibody fragment, e.g., as described above. In some embodiments, the chimeric antigen receptor includes an extracellular portion containing the antibody or fragment and an intracellular signaling domain.

[0251] In some embodiments of any of the CARs described herein, the antibody or fragment includes an scFv or a single-domain antibody fragment, for example, a VHH. In certain embodiments, the scFv may comprise a heavy chain variable region (VH) and a light chain variable region (VL) of an antibody connected by a linker. The VH and the VL may be connected in either order, i.e., VH-linker-VL or VL- linker- VH. Non-limiting examples of linkers include Whitlow linker, (G4S)n (n can be a positive integer, e.g., 1, 2, 3, 4, 5, 6, etc.) linker, and variants thereof.

[0252] In some embodiments, the antibody portion of a recombinant receptor, e.g., CAR, further includes spacer between the transmembrane domain and extracellular antigen binding domain. In some embodiments, the spacer includes at least a portion of an immunoglobulin constant region, such as a hinge region, e.g., an IgG4 hinge region, and/or a CH1/CL and/or Fc region. In some embodiments, the constant region or portion is of a human IgG, such as IgG4 or IgGl. In some aspects, the portion of the constant region serves as a spacer region between the antigen-recognition component, e.g., scFv, and transmembrane domain. The spacer can be of a length that provides for increased responsiveness of the cell following antigen binding, as compared to in the absence of the spacer. Exemplary spacers include, but are not limited to, those described in Hudecek et al., (2013) Clin. Cancer Res., 19:3153, WO2014031687, U.S. Patent No. 8,822,647 or published app. No. US 2014/0271635. In some embodiments, the constant region or portion is of a human IgG, such as IgG4 or IgGl.

[0253] In some embodiments, the antigen receptor comprises an intracellular domain linked directly or indirectly to the extracellular domain. In some embodiments, the chimeric antigen receptor includes a transmembrane domain linking the extracellular domain and the intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises an IT AM. For example, in some aspects, the antigen recognition domain e.g., extracellular domain) generally is linked to one or more intracellular signaling components, such as signaling components that mimic activation through an antigen receptor complex, such as a TCR complex, in the case of a CAR, and/or signal via another cell surface receptor. In some embodiments, the chimeric receptor comprises a transmembrane domain linked or fused between the extracellular domain (e.g., scFv) and intracellular signaling domain. Thus, in some embodiments, the antigen-binding component (e.g., antibody) is linked to one or more transmembrane and intracellular signaling domains.

[0254] In one embodiment, a transmembrane domain that naturally is associated with one of the domains in the receptor, e.g., CAR, is used. In some instances, the transmembrane domain is selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.

[0255] The transmembrane domain in some embodiments is derived either from a natural or from a synthetic source. Where the source is natural, the domain in some aspects is derived from any membrane-bound or transmembrane protein. Transmembrane regions include those derived from (z.e., comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD 134, CD 137, CD 154. Alternatively, the transmembrane domain in some embodiments is synthetic. In some aspects, the synthetic transmembrane domain comprises predominantly hydrophobic residues such as leucine and valine. In some aspects, a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain. In some embodiments, the linkage is by linkers, spacers, and/or transmembrane domain(s). In some aspects, the transmembrane domain contains a transmembrane portion of CD28.

[0256] In some embodiments, the extracellular domain and transmembrane domain can be linked directly or indirectly. In some embodiments, the extracellular domain and transmembrane are linked by a spacer, such as any described herein. In some embodiments, the receptor contains extracellular portion of the molecule from which the transmembrane domain is derived, such as a CD28 extracellular portion.

[0257] Among the intracellular signaling domains are those that mimic or approximate a signal through a natural antigen receptor, a signal through such a receptor in combination with a costimulatory receptor, and/or a signal through a costimulatory receptor alone. In some embodiments, a short oligo- or polypeptide linker, for example, a linker of between 2 and 10 amino acids in length, such as one containing glycines and serines, e.g., glycine-serine doublet, is present and forms a linkage between the transmembrane domain and the cytoplasmic signaling domain of the CAR.

[0258] T cell activation is in some aspects described as being mediated by two classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation through the TCR (primary cytoplasmic signaling sequences), and those that act in an antigenindependent manner to provide a secondary or co- stimulatory signal (secondary cytoplasmic signaling sequences). In some aspects, the CAR includes one or both of such signaling components.

[0259] The receptor, e.g., the CAR, generally includes at least one intracellular signaling component or components. In some aspects, the CAR includes a primary cytoplasmic signaling sequence that regulates primary activation of the TCR complex. Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine -based activation motifs or IT AMs. Examples of IT AMs containing primary cytoplasmic signaling sequences include those derived from CD3-zeta chain, FcR gamma, CD3 gamma, CD3 delta, and CD3 epsilon. In some embodiments, cytoplasmic signaling molecule(s) in the CAR contain(s) a cytoplasmic signaling domain, portion thereof, or sequence derived from CD3-zeta.

[0260] In some embodiments, the receptor includes an intracellular component of a TCR complex, such as a TCR CD3 chain that mediates T-cell activation and cytotoxicity, e.g., CD3- zeta chain. Thus, in some aspects, the antigen-binding portion is linked to one or more cell signaling modules. In some embodiments, cell signaling modules include CD3 transmembrane domain, CD3 intracellular signaling domains, and/or other CD transmembrane domains. In some embodiments, the intracellular component is or includes a CD3-zeta intracellular signaling domain. In some embodiments, the intracellular component is or includes a signaling domain from Fc receptor gamma chain. In some embodiments, the receptor, e.g., CAR, includes the intracellular signaling domain and further includes a portion, such as a transmembrane domain and/or hinge portion, of one or more additional molecules such as CD8, CD4, CD25, or CD16. For example, in some aspects, the CAR or other chimeric receptor is a chimeric molecule of CD3-zeta (CD3-z) or Fc receptor and a portion of one of CD8, CD4, CD25, or CD16. [0261] In some embodiments, upon ligation of the CAR or other chimeric receptor, the cytoplasmic domain or intracellular signaling domain of the receptor activates at least one of the normal effector functions or responses of the immune cell, e.g., T cell engineered to express the CAR. For example, in some contexts, the CAR induces a function of a T cell such as cytolytic activity or T-helper activity, such as secretion of cytokines or other factors. In some embodiments, a truncated portion of an intracellular signaling domain of an antigen receptor component or costimulatory molecule is used in place of an intact immunostimulatory chain, for example, if it transduces the effector function signal. In some embodiments, the intracellular signaling domain or domains include the cytoplasmic sequences of the T cell receptor (TCR), and in some aspects also those of co-receptors that in the natural context act in concert with such receptors to initiate signal transduction following antigen receptor engagement.

[0262] In the context of a natural TCR, full activation generally requires not only signaling through the TCR, but also a costimulatory signal. Thus, in some embodiments, to promote full activation, a component for generating secondary or co-stimulatory signal is also included in the CAR. In other embodiments, the CAR does not include a component for generating a costimulatory signal. In some aspects, an additional CAR is expressed in the same cell and provides the component for generating the secondary or costimulatory signal.

[0263] In some embodiments, the chimeric antigen receptor contains an intracellular domain of a T cell costimulatory molecule. In some embodiments, the CAR includes a signaling domain and/or transmembrane portion of a costimulatory receptor, such as CD28, 4- IBB, 0X40, DAP10, and ICOS. In some aspects, the same CAR includes both the activating and costimulatory components. In some embodiments, the chimeric antigen receptor contains an intracellular domain derived from a T cell costimulatory molecule or a functional variant thereof, such as between the transmembrane domain and intracellular signaling domain. In some aspects, the T cell costimulatory molecule is CD28 or 4- IBB. In some aspects, the T cell costimulatory molecule is 4- IBB.

[0264] In some embodiments, the activating domain is included within one CAR, whereas the costimulatory component is provided by another CAR recognizing another antigen. In some embodiments, the CARs include activating or stimulatory CARs, costimulatory CARs, both expressed on the same cell (see WO2014/055668). In some aspects, the cells include one or more stimulatory or activating CAR and/or a costimulatory CAR. In some embodiments, the cells further include inhibitory CARs (iCARs, see Fedorov et al., Sei. Transl. Medicine, 5(215) (December, 2013), such as a CAR recognizing an antigen other than the one associated with and/or specific for the disease or condition whereby an activating signal delivered through the disease-targeting CAR is diminished or inhibited by binding of the inhibitory CAR to its ligand, e.g., to reduce off-target effects.

[0265] In certain embodiments, the intracellular signaling domain comprises a CD28 transmembrane and signaling domain linked to a CD3 (e.g., CD3-zeta) intracellular domain. In some embodiments, the intracellular signaling domain comprises a chimeric CD28 and CD 137 (4- IBB, TNFRSF9) co- stimulatory domains, linked to a CD3-zeta intracellular domain.

[0266] In some embodiments, the CAR encompasses one or more, e.g., two or more, costimulatory domains and an activation domain, e.g., primary activation domain, in the cytoplasmic portion. Exemplary CARs include intracellular components of CD3-zeta, CD28, and 4-1BB.

[0267] In some embodiments the intracellular signaling domain includes intracellular components of a 4- IBB signaling domain and a CD3-zeta signaling domain. In some embodiments, the intracellular signaling domain includes intracellular components of a CD28 signaling domain and a CD3-zeta signaling domain.

[0268] In some embodiments, a CD 19 specific CAR includes an anti-CD19 single-chain antibody fragment (scFv), a transmembrane domain such as one derived from human CD8a, a 4- 1BB (CD 137) co- stimulatory signaling domain, and a CD3(^ signaling domain. In some embodiments, a CD22 specific CAR includes an anti-CD22 scFv, a transmembrane domain such as one derived from human CD8a, a 4- IBB (CD 137) co-stimulatory signaling domain, and a CD3(^ signaling domain. In some embodiments, a CD19/CD22-bispecific CAR includes an antiCD 19 scFv, an anti-CD22 scFv, a transmembrane domain such as one derived from human CD8a, a 4-1BB (CD137) co-stimulatory signaling domain, and a CD3(^ signaling domain.

[0269] In some embodiments, the CAR comprises a commercial CAR construct carried by a T cell. Non-limiting examples of commercial CAR-T cell based therapies include brexucabtagene autoleucel (TECARTUS®), axicabtagene ciloleucel (YESCARTA®), idecabtagene vicleucel (ABECMA®), lisocabtagene maraleucel (BREYANZI®), tisagenlecleucel (KYMRIAH®), Descartes-08 and Descartes- 11 from Cartesian Therapeutics, CTL110 from Novartis, P-BMCA-101 from Poseida Therapeutics, AUTO4 from Autolus Limited, UCARTCS from Cellectis, PBCAR19B and PBCAR269A from Precision Biosciences, FT819 from Fate Therapeutics, and CYAD-211 from Clyad Oncology.

[0270] In some embodiments, the CAR comprises an extracellular antigen binding domain (e.g., antibody or antibody fragment, such as an scFv) that binds to an antigen (e.g., tumor antigen), a spacer (e.g., containing a hinge domain, such as any as described herein), a transmembrane domain (e.g., any as described herein), and an intracellular signaling domain (e.g., any intracellular signaling domain, such as a primary signaling domain or costimulatory signaling domain as described herein). In some embodiments, the intracellular signaling domain is or includes a primary cytoplasmic signaling domain. In some embodiments, the intracellular signaling domain additionally includes an intracellular signaling domain of a costimulatory molecule (e.g., a costimulatory domain).

[0271] In some embodiments, the forward primer binding site for an exemplary CAR comprises the nucleotide sequence of SEQ ID NO:35 or a variant thereof comprising about 1, 2, 3, 4, or 5 nucleotide substitutions, insertions, or deletions. In some embodiments, the reverse primer binding site for an exemplary CAR comprises the nucleotide sequence of SEQ ID NO:36 or a variant thereof comprising about 1, 2, 3, 4, or 5 nucleotide substitutions, insertions, or deletions. In some embodiments, the forward primer binding site for an exemplary CAR comprises the nucleotide sequence of SEQ ID NO:35, and/or the reverse primer binding site for an exemplary CAR comprises the nucleotide sequence of SEQ ID NO:36. In some embodiments, the first CAR probe comprises the nucleotide sequence of SEQ ID NO:37 or a variant thereof comprising about 1, 2, 3, 4, or 5 nucleotide substitutions, insertions, or deletions.

[0272] In some embodiments, the second CAR probe comprises the nucleotide sequence of SEQ ID NO:38 or a variant thereof comprising about 1, 2, 3, 4, or 5 nucleotide substitutions, insertions, or deletions. In some embodiments, the first CAR probe comprises the nucleotide sequence of SEQ ID NO: 37, and/or the second CAR probe comprises the nucleotide sequence of SEQ ID NO:38. In some embodiments, the first CAR probe and/or the second CAR probe is attached to a fluorophore selected from the group consisting of Brilliant Ultra Violet 395, Alexa Fluor 350, Brilliant Ultra Violet 496, Qdot 525 Probe, Brilliant Ultra Violet 563, Qdot 565 Probe, Qdot 605 Probe, Brilliant Ultra Violet 615, Qdot 655 Probe, Brilliant Ultra Violet 661, Qdot 705 Probe, Brilliant Ultra Violet 737, Qdot 800 Probe, Brilliant Ultra Violet 805, Alexa Fluor 405, Brilliant Violet 421, Super Bright 436, eFluor 450, Pacific Blue, Coumarin and Coumarin Derivatives, Brilliant Violet 480, Cyan Fluorescent Protein (CFP), eFluor 506, Pacific Green, Pacific Orange, Super Bright 600, Super Bright 645, Brilliant Violet 650, Super Bright 702, Brilliant Violet 711, Super Bright 780, Brilliant Violet 786, Green Fluorescent Protein (GFP), BODIPY FL, NovaFluor Blue 510, Fluorescein (FITC), Alexa Fluor 488, Oregon Green 488, NovaFluor Blue 530, NovaFluor Blue 555, NovaFluor Blue 585, NovaFluor Blue 610-30S, NovaFluor Blue 610-70S, NovaFluor Blue 660-40S, NovaFluor Blue 660-120S, PerCP- Cyanine5.5, PerCP-eFluor 710, Alexa Fluor 532, Cy3, NovaFluor Yellow 570, Alexa Fluor 555, Alexa Fluor 561, Alexa Fluor 546, R-phycoerythrin (R-PE), Tetramethylrhodamine (TRITC), Red Fluorescent Protein (RFP), NovaFluor Yellow 590, Alexa Fluor 568, PE-eFluor 610, Texas Red (and Texas Red-X), NovaFluor Yellow 610, Alexa Fluor 594, NovaFluor Yellow 660, NovaFluor Yellow 690, NovaFluor Yellow 700, NovaFluor Yellow 730, NovaFluor Yellow 755, PE-Cyanine7, NovaFluor Red 660, Allophycocyanin (APC), Cy5, eFluor 660, Alexa Fluor 647, NovaFluor Red 685, NovaFluor Blue 690, Alexa Fluor 660, NovaFluor Red 700, Alexa Fluor 680, NovaFluor Red 710, Alexa Fluor 700, NovaFluor Red 725, NovaFluor Red 755, Alexa Fluor 750, APC-eFluor 780, FAM, HEX, Rhodamine Red-X, YY, Atto 550, Atto 590, Atto 700, Tamara, Rox, TruRed, Cy7, Red 613, Cy3.5 581, Cy5.5, DAPI, Hoechst, SYTOX blue, SYTOX green, SYTOX orange, YOYO-1, TOTO-1, TO-PRO-1, chromomycin A3, mithramycin, propidium iodide, ethidium bromide, SYBR Green, any KIRA VIA dyes (e.g., KIRVIA Blue 520), PE-Dazzle 594, PE-Fire 640, PE-Cy5, PE-Fire 700, PE-FIRE 810, PerCP, APC-Cyanine 7, APC-Fire 750, APC-Fire 810, Spark UV 387, Spark Violet 423, Spark Violet 500, Spark Violet 538, Spark Blue 550, Spark Blue 574, Spark YG 570, Spark YG 581, Spark YG 593, Spark NIR 685, Spark Red 718, Brilliant Violet 510, Brilliant Violet 570, Brilliant Violet 605, Brilliant Violet 750, 7-AAD, APC-H7, Apotracker Green, APC-R700, Brilliant Blue 515, Brilliant Blue 700, Calcein-AM, Calcein Red- AM, Calcein Violet-AM, CF 570, CytoPhase Violet, DRAQ5, DRAQ7, Helix NP Blue, Helix NP Green, Helix NP NIR, MitoSpy Green, MitoSpy NIR, MitoSpy Orange, MitoSpy Red, Tag-it Violet, VioBright FITC, Zombie Aqua, Zombie Green, Zombie NIR, Zombie Red, Zombie UV, Zombie Violet, and Zombie Yellow. In some embodiments, the first CAR probe is attached to HEX and the second CAR probe is attached to FAM. In some embodiments, the first CAR probe is attached to FAM and the second CAR probe is attached to HEX. Exemplary CAR Components

[0273] Exemplary components of CARs that may be used in the present disclosure are described below.

[0274] In some embodiments, the sequences of each component in a CAR can include any combination listed in Table 1.

Table 1. CAR components and Exemplary Sequences.

[0275] Signal peptides: In certain embodiments, the CAR may comprise a signal peptide at the N-terminus. Non-limiting examples of signal peptides include CD8a signal peptide, IgK signal peptide, and granulocyte-macrophage colony- stimulating factor receptor subunit alpha (GMCSFR-a, also known as colony stimulating factor 2 receptor subunit alpha (CSF2RA)) signal peptide, and variants thereof, the amino acid sequences of which are provided in Table 2 below.

Table 2. Exemplary sequences of signal peptides.

[0276] Hinge or spacer domains: In certain embodiments, the CAR may comprise a hinge domain, also referred to as a spacer. The terms “hinge” and “spacer” may be used interchangeably in the present disclosure. Non-limiting examples of hinge domains include CD8a hinge domain, CD28 hinge domain, IgG4 hinge domain, IgG4 hinge-CH2-CH3 domain, and variants thereof, the amino acid sequences of which are provided in Table 3 below.

Table 3. Exemplary sequences of hinge domains.

[0277] Transmembrane domains: In certain embodiments, the transmembrane domain of the CAR may comprise a transmembrane region of the alpha, beta, or zeta chain of a T cell receptor, CD28, CD3s, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or a functional variant thereof, including the human versions of each of these sequences. In other embodiments, the transmembrane domain may comprise a transmembrane region of CD8a, CD8P, 4-1BB/CD137, CD28, CD34, CD4, FcsRIy, CD16, OX40/CD134, CD3^, CD3s, CD3y, CD35, TCRa, TCRp, TCR^, CD32, CD64, CD64, CD45, CD5, CD9, CD22, CD37, CD80, CD86, CD40, CD40L/CD154, VEGFR2, FAS, FGFR2B, or a functional variant thereof, including the human versions of each of these sequences. In some embodiments, the transmembrane domain is selected from the group consisting of: alpha, beta, or zeta chain of a T cell receptor, CD28, CD3s, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CD8a, CD8P, 4-1BB/CD137, CD28, CD34, CD4, FcsRIy, CD16, OX40/CD134, CD3^, CD3s, CD3y, CD35, TCRa, TCRp, TCR^, CD32, CD64, CD64, CD45, CD5, CD9, CD22, CD37, CD80, CD86, CD40, CD40L/CD154, VEGFR2, FAS, FGFR2B, and any functional variant thereof. Table 4 provides the amino acid sequences of a few exemplary transmembrane domains.

Table 4. Exemplary sequences of transmembrane domains.

[0278] Intracellular domains: In certain embodiments, the intracellular signaling domain and/or intracellular costimulatory domain of the CAR may comprise one or more signaling domains selected from the group consisting of: B7-1/CD80, B7-2/CD86, B7-H1/PD-L1, B7-H2, B7-H3, B7-H4, B7-H6, B7-H7, BTLA/CD272, CD28, CTLA-4, Gi24/VISTA/B7-H5, ICOS/CD278, PD-1, PD-L2/B7-DC, PDCD6, 4-1BB/TNFSF9/CD137, 4-1BB Ligand/TNFSF9, BAFF/BLyS/TNFSF13B, BAFF R/TNFRSF13C, CD27/TNFRSF7, CD27 Ligand/TNFSF7, CD30/TNFRSF8, CD30 Ligand/TNFSF8, CD40/TNFRSF5, CD40/TNFSF5, CD40 Ligand/TNFSF5, DR3/TNFRSF25, GITR/TNFRSF18, GITR Ligand/TNFSF18, HVEM/TNFRSF14, LIGHT/TNFSF14, Lymphotoxin-alpha/TNFp, OX40/TNFRSF4, 0X40 Ligand/TNFSF4, RELT/TNFRSF19L, TACI/TNFRSF I 3B, TL1A/TNFSF15, TNFa, TNF RII/TNFRSF I B, 2B4/CD244/SLAMF4, BLAME/SLAMF8, CD2, CD2F-10/SLAMF9, CD48/SLAMF2, CD58/LFA-3, CD84/SLAMF5, CD229/SLAMF3, CRACC/SLAMF7, NTB- A/SLAMF6, SLAM/CD150, CD2, CD7, CD53, CD82/Kai-1, CD90/Thyl, CD96, CD160, CD200, CD300a/LMIRl, HLA Class I, HLA-DR, Ikaros, Integrin alpha 4/CD49d, Integrin alpha 4 beta 1, Integrin alpha 4 beta 7/LPAM-l, LAG-3, TCL1A, TCL1B, CRTAM, DAP12, Dectin- 1/CLEC7A, DPPIV/CD26, EphB6, TIM- 1 /KIM- 1 /HA VCR, TIM-4, TSLP, TSLP R, lymphocyte function associated antigen- 1 (LFA-1), NKG2C, CD3^, an immunoreceptor tyrosine-based activation motif (ITAM), CD27, 4-1BB, CD134/OX40, CD30, CD40, PD-1, ICOS, LIGHT, NKG2C, a ligand that specifically binds with CD83, and a functional variant thereof, including the human versions of each of these sequences. In some embodiments, the intracellular signaling domain and/or intracellular costimulatory domain comprises one or more signaling domains selected from a CD3^ domain, an ITAM, a CD28 domain, 4- IBB domain, or a functional variant thereof. Table 5 provides the amino acid sequences of a few exemplary intracellular costimulatory and/or signaling domains. In certain embodiments, the CD3^ signaling domain may have a mutation, e.g., a glutamine (Q) to lysine (K) mutation, at amino acid position 14. Table 5. Exemplary sequences of intracellular co stimulatory and/or signaling domains.

and/or cells expressing the CAR or other antigen receptor further includes a surrogate marker, such as a cell surface marker, which may be used to confirm transduction or engineering of the cell to express the receptor. In some embodiments, the marker includes all or part (e.g., truncated form) of CD34, a NGFR, or epidermal growth factor receptor, such as truncated version of such a cell surface receptor (e.g., tEGFR). In some embodiments, the nucleic acid encoding the marker is operably linked to a polynucleotide encoding for a linker sequence, such as a cleavable linker sequence, e.g., T2A. For example, a marker, and optionally a linker sequence, can be any as disclosed in published patent application No. WO2014031687. For example, the marker can be a truncated EGFR (tEGFR) that is, optionally, linked to a linker sequence, such as a T2A cleavable linker sequence. In some embodiments, the marker is a molecule, e.g., cell surface protein, not naturally found on T cells or not naturally found on the surface of T cells, or a portion thereof. In some embodiments, the molecule is a non- self molecule, e.g., non- self protein, i.e., one that is not recognized as “self’ by the immune system of the host into which the cells will be adoptively transferred. In some embodiments, the marker serves no therapeutic function and/or produces no effect other than to be used as a marker for genetic engineering, e.g., for selecting cells successfully engineered. In other embodiments, the marker may be a therapeutic molecule or molecule otherwise exerting some desired effect, such as a ligand for a cell to be encountered in vivo, such as a co stimulatory or immune checkpoint molecule to enhance and/or dampen responses of the cells upon adoptive transfer and encounter with ligand.

Exemplary CD 19 CARs

[0280] In some embodiments, the CAR is a CD 19 CAR (“CD 19-CAR”). In some embodiments, the CD 19 CAR may comprise an extracellular binding domain that specifically binds CD 19 and one or more of: any of the signal peptides described herein, any of the hinge domains described herein, any of the transmembrane domains described herein, any of the intracellular costimulatory domains described herein, and/or any of the intracellular signaling domains described herein, e.g., in tandem.

[0281] In some embodiments, the signal peptide of the CD19 CAR comprises a CD8a signal peptide. In some embodiments, the signal peptide comprises an IgK signal peptide. In some embodiments, the signal peptide comprises a GMCSFR-a or CSF2RA signal peptide. In some embodiments, the extracellular binding domain of the CD 19 CAR is specific to CD 19, for example, human CD 19. The extracellular binding domain of the CD 19 CAR can be codon- optimized for expression in a host cell or to have variant sequences to increase functions of the extracellular binding domain. In some embodiments, the extracellular binding domain comprises an immunogenically active portion of an immunoglobulin molecule, for example, an scFv. In some embodiments, the extracellular binding domain of the CD 19 CAR comprises an scFv derived from the FMC63 monoclonal antibody (FMC63), which comprises the heavy chain variable region (VH) and the light chain variable region (VL) of FMC63 connected by a linker. FMC63 and the derived scFv have been described in Nicholson et al., Mol. Immun. 34(16- 17): 1157- 1165 (1997) and PCT Application Publication No. WO2018/213337, the entire contents of each of which are incorporated by reference herein. In some embodiments, the amino acid sequences of the entire FMC63-derived scFv (also referred to as FMC63 scFv) and its different portions are provided in Table 6 below. In some embodiments, the CD19-specific scFv may comprise one or more CDRs having the CDR amino acid sequences set forth in Table 6. In some embodiments, the CD19-specific scFv may comprise a light chain with one or more CDRs having the light chain CDR amino acid sequences set forth in Table 6. In some embodiments, the CD19-specific scFv may comprise a heavy chain with one or more CDRs having the heavy chain CDR amino acid sequences set forth in Table 6. In any of these embodiments, the CD19-specific scFv may comprise one or more CDRs comprising one or more amino acid substitutions or comprising a sequence that is at least about 80% identical (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical), to any of the sequences identified in Table 6. In some embodiments, the extracellular binding domain of the CD 19 CAR comprises or consists of the one or more CDRs as described herein. In some embodiments, the linker linking the VH and the VL portions of the scFv is a Whitlow linker having an amino acid sequence set forth in Table 6. In some embodiments, the Whitlow linker may be replaced by a different linker, for example, a 3xG4S linker having an amino acid sequence set forth in Table 6.

Table 6. Exemplary sequences of anti-CD19 scFv and components.

[0282] In some embodiments, the extracellular binding domain of the CD 19 CAR is derived from an antibody specific to CD 19, including, for example, SJ25C1 (Bejcek et al., Cancer Res. 55:2346-2351 (1995)), HD37 (Pezutto et al., J. Immunol. 138(9):2793-2799 (1987)), 4G7 (Meeker et al., Hybridoma 3:305-320 (1984)), B43 (Bejcek (1995)), BLY3 (Bejcek (1995)), B4 (Freedman et al., 70:418-427 (1987)), B4 HB12b (Kansas & Tedder, J. Immunol. 147:4094-4102 (1991); Yazawa et al., Proc. Natl. Acad. Sci. USA 102:15178-15183 (2005); Herbst et al., J. Pharmacol. Exp. Ther. 335:213-222 (2010)), BU12 (Callard et al., J. Immunology, 148(10): 2983-2987 (1992)), and CLB-CD19 (De Rie Cell. Immunol. 118:368- 381(1989)). In any of these embodiments, the extracellular binding domain of the CD19 CAR can comprise or consist of the Vn, the VL, and/or one or more CDRs of any of the antibodies.

[0283] In some embodiments, the hinge domain of the CD19 CAR comprises a CD8a hinge domain, for example, a human CD8a hinge domain. In some embodiments, the hinge domain comprises a CD28 hinge domain, for example, a human CD28 hinge domain. In some embodiments, the hinge domain comprises an IgG4 hinge domain, for example, a human IgG4 hinge domain. In some embodiments, the hinge domain comprises a IgG4 hinge-Ch2-Ch3 domain, for example, a human IgG4 hinge-Ch2-Ch3 domain. In some embodiments, the transmembrane domain of the CD 19 CAR comprises a CD8a transmembrane domain, for example, a human CD8a transmembrane domain. In some embodiments, the transmembrane domain comprises a CD28 transmembrane domain, for example, a human CD28 transmembrane domain.

[0284] In some embodiments, the intracellular costimulatory domain of the CD19 CAR comprises a 4-1BB costimulatory domain. 4-1BB, also known as CD137, transmits a potent costimulatory signal to T cells, promoting differentiation and enhancing long-term survival of T lymphocytes. In some embodiments, the 4- IBB costimulatory domain is human. In some embodiments, the intracellular costimulatory domain comprises a CD28 costimulatory domain. CD28 is another co-stimulatory molecule on T cells. In some embodiments, the CD28 costimulatory domain is human. In some embodiments, the intracellular costimulatory domain of the CD19 CAR comprises a 4-1BB costimulatory domain and a CD28 costimulatory domain as described. In some embodiments, the intracellular signaling domain of the CD 19 CAR comprises a CD3-zeta (Q signaling domain. CD3-zeta associates with T cell receptors (TCRs) to produce a signal and contains immunoreceptor tyrosine-based activation motifs (IT AMs). The CD3-zeta signaling domain refers to amino acid residues from the cytoplasmic domain of the zeta chain that are sufficient to functionally transmit an initial signal necessary for T cell activation. In some embodiments, the CD3-zeta signaling domain is human.

[0285] In some embodiments, the CD19 CAR comprises a CD19-specific scFv , a CD8a hinge domain, a CD8a transmembrane domain, a 4- IBB costimulatory domain, a CD3^ signaling domain, and/or variants (z.e., having a sequence that is at least about 80% identical, for example, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity) thereof. In any of these embodiments, the CD19 CAR may additionally comprise a signal peptide (e.g., a CD8a signal peptide) as described above.

[0286] In some embodiments, the CD19 CAR comprises a CD19-specific scFv, an IgG4 hinge domain, a CD28 transmembrane domain, a 4- IBB costimulatory domain, a CD3^ signaling domain, and/or variants (z.e., having a sequence that is at least about 80% identical, for example, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity) thereof. In any of these embodiments, the CD19 CAR may additionally comprise a signal peptide (e.g., a CD8a signal peptide) as described above. [0287] In some embodiments, the CD19 CAR comprises a CD19-specific scFv, a CD28 hinge domain, a CD28 transmembrane domain, a CD28 costimulatory domain, a CD3^ signaling domain, and/or variants (z.e., having a sequence that is at least about 80% identical, for example, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity) thereof. In any of these embodiments, the CD 19 CAR may additionally comprise a signal peptide (e.g., a CD8a signal peptide) as described above.

[0288] In some embodiments, the CD 19 CAR is encoded by the sequence set forth in SEQ ID NO: 285 or a sequence at least about 80% identical (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical) to the nucleotide sequence set forth in SEQ ID NO: 285 (see Table 7). The encoded CD 19 CAR has a corresponding amino acid sequence set forth in SEQ ID NO: 286 or is at least about 80% identical (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical) to the amino acid sequence set forth in of SEQ ID NO: 286, with the following components: CD8a signal peptide, FMC63 scFv ( Vi.-Whitlow linker-Vn), CD8a hinge domain, CD8a transmembrane domain, 4- 1BB costimulatory domain, and CD3^ signaling domain.

[0289] In some embodiments, the CD 19 CAR is a commercially available embodiment of a CD 19 CAR. Non-limiting examples of commercially available embodiments of CD 19 CARs include tisagenlecleucel, lisocabtagene maraleucel, axicabtagene ciloleucel, and brexucabtagene autoleucel.

[0290] In some embodiments, the CAR is tisagenlecleucel or portions thereof. Tisagenlecleucel comprises a CD 19 CAR with the following components: CD8a signal peptide, FMC63 scFv (VL-3XG4S linker-Vn), CD8a hinge domain, CD8a transmembrane domain, 4- IBB costimulatory domain, and CD3^ signaling domain. The nucleotide and amino acid sequence of the CD19 CAR in tisagenlecleucel are provided in Table 7, with annotations of the sequences provided in Table 8.

[0291] In some embodiments, the CAR is lisocabtagene maraleucel or portions thereof. Lisocabtagene maraleucel comprises a CD 19 CAR with the following components: GMCSFR-a or CSF2RA signal peptide, FMC63 scFv (VL- Whitlow linker-Vn), IgG4 hinge domain, CD28 transmembrane domain, 4- IBB costimulatory domain, and CD3^ signaling domain. The nucleotide and amino acid sequence of the CD 19 CAR in lisocabtagene maraleucel are provided in Table 7, with annotations of the sequences provided in Table 9.

[0292] In some embodiments, the CAR is axicabtagene ciloleucel or portions thereof. Axicabtagene ciloleucel comprises a CD 19 CAR with the following components: GMCSFR-a or CSF2RA signal peptide, FMC63 scFv ( Vi.-Whitlow linker-Vn), CD28 hinge domain, CD28 transmembrane domain, CD28 costimulatory domain, and CD3^ signaling domain. The nucleotide and amino acid sequence of the CD 19 CAR in axicabtagene ciloleucel are provided in Table 7, with annotations of the sequences provided in Table 10.

[0293] In some embodiments, the CAR is brexucabtagene autoleucel or portions thereof. Brexucabtagene autoleucel comprises a CD 19 CAR with the following components: GMCSFR- a signal peptide, FMC63 scFv, CD28 hinge domain, CD28 transmembrane domain, CD28 costimulatory domain, and CD3^ signaling domain.

[0294] In some embodiments, the CAR is encoded by the sequence set forth in SEQ ID NO: 287, 289, or 291, or a sequence at least about 80% identical (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical) thereto. The encoded CD19 CAR has a corresponding amino acid sequence set forth in SEQ ID NO: 288, 290, or 292, respectively, or is at least about 80% identical (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical) thereto.

Table 7. Exemplary sequences of CD 19 CARs

Table 8. Annotation of tisagenlecleucel CD 19 CAR sequences

[0295] In some embodiments, the first barcode and/or the second barcode is located at the junction of the CD8a signal peptide and the FMC63 scFv. In some embodiments, the first barcode and/or the second barcode is located around about nucleotide positions 30-130, for example about 50 to about 100 nucleotide position, about 40 to about 90 nucleotide position, about 60 to about 110 nucleotide position, about 30 to about 80 nucleotide position, about 70 to about 120 nucleotide position, or about 80 to about 130 nucleotide position. In some embodiments, the first barcode and/or the second barcode is located at the junction of the FMC63 scFv and the CD8a hinge domain. In some embodiments, the first barcode and/or the second barcode is located around about nucleotide positions 740-840, for example about 750 to about 800 nucleotide position, about 740 to about 790 nucleotide position, about 760 to about 810 nucleotide position, about 770 to about 820 nucleotide position, about 780 to about 830 nucleotide position, or about 790 to about 840 nucleotide position. In some embodiments, the first barcode and/or the second barcode is located at the junction of the CD8a hinge domain and the CD8a transmembrane domain. In some embodiments, the first barcode and/or the second barcode is located around about nucleotide positions 875-975, for example about 875 to about 925 nucleotide position, about 880 to about 930 nucleotide position, about 890 to about 940 nucleotide position, about 900 to about 950 nucleotide position, about 910 to about 960 nucleotide position, or about 920 to about 975 nucleotide position. In some embodiments, the first barcode and/or the second barcode is located at the junction of the CD8a transmembrane domain and the 4- IBB costimulatory domain. In some embodiments, the first barcode and/or the second barcode is located around about nucleotide positions 950-1050, for example about 950 to about 1000 nucleotide position, about 960 to about 1100 nucleotide position, about 970 to about 1200 nucleotide position, about 980 to about 1030 nucleotide position, about 990 to about 1040 nucleotide position, or about 995 to about 1050 nucleotide position. In some embodiments, the first barcode and/or the second barcode is located at the junction of the 4- IBB costimulatory domain and the CD3(^ signaling domain. In some embodiments, the first barcode and/or the second barcode is located around about nucleotide positions 1075-1175, for example about 1075 to about 1125 nucleotide position, about 1080 to about 1130 nucleotide position, about 1090 to about 1140 nucleotide position, about 1100 to about 1150 nucleotide position, about 1110 to about 1160 nucleotide position, or about 1120 to about 1175 nucleotide position.

Table 9. Annotation of lisocabtagene maraleucel CD 19 CAR sequences

[0296] In some embodiments, the first barcode and/or the second barcode is located at the junction of the GMCSFR-a signal peptide and the FMC63 scFv. In some embodiments, the first barcode and/or the second barcode is located around about nucleotide positions 20-120, for example about 20 to about 70 nucleotide position, about 50 to about 100 nucleotide position, about 40 to about 90 nucleotide position, about 60 to about 110 nucleotide position, about 30 to about 80 nucleotide position, about 65 to about 120 nucleotide position, or about 80 to about 130 nucleotide position. In some embodiments, the first barcode and/or the second barcode is located at the junction of the FMC63 scFv and the IgG4 hinge domain. In some embodiments, the first barcode and/or the second barcode is located around about nucleotide positions 750-850, for example about 750 to about 805 nucleotide position, about 760 to about 810 nucleotide position, about 770 to about 820 nucleotide position, about 780 to about 830 nucleotide position, about 790 to about 840 nucleotide position, or about 800 to about 850 nucleotide position. In some embodiments, the first barcode and/or the second barcode is located at the junction of the IgG4 hinge domain and the CD28 transmembrane domain. In some embodiments, the first barcode and/or the second barcode is located around about nucleotide positions 790-890, for example about 790 to about 840 nucleotide position, about 800 to about 850 nucleotide position, about 810 to about 860 nucleotide position, about 820 to about 870 nucleotide position, about 830 to about 880 nucleotide position, or about 835 to about 890 nucleotide position. In some embodiments, the first barcode and/or the second barcode is located at the junction of the CD28 transmembrane domain and the 4- IBB costimulatory domain. In some embodiments, the first barcode and/or the second barcode is located around about nucleotide positions 870-970, for example about 870 to about 925 nucleotide position, about 880 to about 930 nucleotide position, about 890 to about 940 nucleotide position, about 900 to about 950 nucleotide position, about 910 to about 960 nucleotide position, or about 920 to about 970 nucleotide position. In some embodiments, the first barcode and/or the second barcode is located at the junction of the 4- IBB costimulatory domain and the CD3(^ signaling domain. In some embodiments, the first barcode and/or the second barcode is located around about nucleotide positions 1000-1100, for example about 1000 to about 1050 nucleotide position, about 1010 to about 1060 nucleotide position, about 1020 to about 1070 nucleotide position, about 1030 to about 1080 nucleotide position, about 1040 to about 1090 nucleotide position, or about 1045 to about 1100 nucleotide position. Table 10. Annotation of axicabtagene ciloleucel CD 19 CAR sequences

[0297] In some embodiments, the first barcode and/or the second barcode is located at the junction of the CSF2RA signal peptide and the FMC63 scFv. In some embodiments, the first barcode and/or the second barcode is located around about nucleotide positions 20-120, for example about 20 to about 70 nucleotide position, about 50 to about 100 nucleotide position, about 40 to about 90 nucleotide position, about 60 to about 110 nucleotide position, about 30 to about 80 nucleotide position, about 65 to about 120 nucleotide position, or about 80 to about 130 nucleotide position. In some embodiments, the first barcode and/or the second barcode is located at the junction of the FMC63 scFv and the CD28 hinge domain. In some embodiments, the first barcode and/or the second barcode is located around about nucleotide positions 750-850, for example about 750 to about 805 nucleotide position, about 760 to about 810 nucleotide position, about 770 to about 820 nucleotide position, about 780 to about 830 nucleotide position, about 790 to about 840 nucleotide position, or about 800 to about 850 nucleotide position. In some embodiments, the first barcode and/or the second barcode is located at the junction of the CD28 hinge domain and the CD28 transmembrane domain. In some embodiments, the first barcode and/or the second barcode is located around about nucleotide positions 880-980, for example about 880 to about 930 nucleotide position, about 890 to about 940 nucleotide position, about 900 to about 950 nucleotide position, about 910 to about 960 nucleotide position, about 920 to about 970 nucleotide position, or about 925 to about 980 nucleotide position. In some embodiments, the first barcode and/or the second barcode is located at the junction of the CD28 transmembrane domain and the CD28 costimulatory domain. In some embodiments, the first barcode and/or the second barcode is located around about nucleotide positions 960-1060, for example about 960 to about 1010 nucleotide position, about 970 to about 1020 nucleotide position, about 980 to about 1030 nucleotide position, about 990 to about 1040 nucleotide position, about 1000 to about 1050 nucleotide position, or about 1005 to about 1060 nucleotide position. In some embodiments, the first barcode and/or the second barcode is located at the junction of the CD28 costimulatory domain and the CD3(^ signaling domain. In some embodiments, the first barcode and/or the second barcode is located around about nucleotide positions 1080-1180, for example about 1080 to about 1135 nucleotide position, about 1090 to about 1140 nucleotide position, about 1100 to about 1150 nucleotide position, about 1110 to about 1160 nucleotide position, about 1120 to about 1170 nucleotide position, or about 1130 to about 1180 nucleotide position.

Exemplary CD20 CARs

[0298] In some embodiments, the CAR is a CD20 CAR (“CD20-CAR”). CD20 is an antigen found on the surface of B cells as early at the pro-B phase and progressively at increasing levels until B cell maturity, as well as on the cells of most B-cell neoplasms. CD20 positive cells are also sometimes found in cases of Hodgkin’s disease, myeloma, and thymoma. In some embodiments, the CD20 CAR may comprise an extracellular binding domain that specifically binds CD20 and one or more of: any of the signal peptides described herein, any of the hinge domains described herein, any of the transmembrane domains described herein, any of the intracellular costimulatory domains described herein, and/or any of the intracellular signaling domains described herein, e.g., in tandem.

[0299] In some embodiments, the signal peptide of the CD20 CAR comprises a CD8a signal peptide. In some embodiments, the signal peptide comprises an IgK signal peptide. In some embodiments, the signal peptide comprises a GMCSFR-a or CSF2RA signal peptide. [0300] In some embodiments, the extracellular binding domain of the CD20 CAR is specific to CD20, for example, human CD20. The extracellular binding domain of the CD20 CAR can be codon-optimized for expression in a host cell or to have variant sequences to increase functions of the extracellular binding domain. In some embodiments, the extracellular binding domain comprises an immunogenically active portion of an immunoglobulin molecule, for example, an scFv.

[0301] In some embodiments, the extracellular binding domain of the CD20 CAR is derived from an antibody specific to CD20, including, for example, Leul6, IF5, 1.5.3, rituximab, obinutuzumab, ibritumomab, ofatumumab, tositumumab, odronextamab, veltuzumab, ublituximab, and ocrelizumab. In any of these embodiments, the extracellular binding domain of the CD20 CAR can comprise or consist of the Vn, the VL, and/or one or more CDRs of any of the antibodies.

[0302] In some embodiments, the extracellular binding domain of the CD20 CAR comprises an scFv derived from the Leu 16 monoclonal antibody, which comprises the heavy chain variable region (VH) and the light chain variable region (VL) of Leul6 connected by a linker. See Wu el al., Protein Engineering. 14(12): 1025-1033 (2001). In some embodiments, the linker is a 3xG4S linker. In other embodiments, the linker is a Whitlow linker as described herein. In some embodiments, the amino acid sequences of different portions of the entire Leul6-derived scFv (also referred to as Leu 16 scFv) and its different portions are provided in Table 11 below. In some embodiments, the CD20-specific scFv may comprise one or more CDRs having the CDR amino acid sequences set forth in Table 11. In some embodiments, the CD20- specific scFv may comprise a light chain with one or more CDRs having light chain CDR amino acid sequences set forth in Table 11. In some embodiments, the CD20-specific scFv may comprise a heavy chain with one or more CDRs having heavy chain CDR amino acid sequences set forth in Table 11. In any of these embodiments, the CD20-specific scFv may comprise one or more CDRs comprising one or more amino acid substitutions, or comprising a sequence that is at least about 80% identical (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identity), to any of the sequences identified. In some embodiments, the extracellular binding domain of the CD20 CAR comprises or consists of the one or more CDRs as described herein.

Table 11. Exemplary sequences of anti-CD20 scFv and components.

[0303] In some embodiments, the hinge domain of the CD20 CAR comprises a CD8a hinge domain, for example, a human CD8a hinge domain. In some embodiments, the hinge domain comprises a CD28 hinge domain, for example, a human CD28 hinge domain. In some embodiments, the hinge domain comprises an IgG4 hinge domain, for example, a human IgG4 hinge domain. In some embodiments, the hinge domain comprises a IgG4 hinge-Ch2-Ch3 domain, for example, a human IgG4 hinge-Ch2-Ch3 domain.

[0304] In some embodiments, the transmembrane domain of the CD20 CAR comprises a CD8a transmembrane domain, for example, a human CD8a transmembrane domain. In some embodiments, the transmembrane domain comprises a CD28 transmembrane domain, for example, a human CD28 transmembrane domain.

[0305] In some embodiments, the intracellular costimulatory domain of the CD20 CAR comprises a 4- IBB costimulatory domain, for example, a human 4- IBB costimulatory domain. In some embodiments, the intracellular costimulatory domain comprises a CD28 costimulatory domain, for example, a human CD28 costimulatory domain.

[0306] In some embodiments, the intracellular signaling domain of the CD20 CAR comprises a CD3 zeta (Q signaling domain, for example, a human CD3(^ signaling domain.

[0307] In some embodiments, the CAR is a CD20 CAR, including, for example, a CD20 CAR comprising a CD20-specific scFv, a CD8a hinge domain, a CD8a transmembrane domain, a 4- IBB costimulatory domain, a CD3(^ signaling domain, and/or variants (z.e., having a sequence that is at least about 80% identical, for example, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity) thereof. In some embodiments, the first barcode and/or the second barcode is located at the junction of the CD20-specific scFv and the CD8a hinge domain. In some embodiments, the first barcode and/or the second barcode is located at the junction of the CD8a hinge domain and the CD28 transmembrane domain. In some embodiments, the first barcode and/or the second barcode is located at the junction of the CD28 transmembrane domain and the 4- IBB costimulatory domain. In some embodiments, the first barcode and/or the second barcode is located at the junction of the 4- IBB costimulatory domain and the CD3(^ signaling domain.

[0308] In some embodiments, the CAR is a CD20 CAR, including, for example, a CD20 CAR comprising a CD20-specific scFv, a CD28 hinge domain, a CD8a transmembrane domain, a 4- IBB costimulatory domain, a CD3(^ signaling domain, and/or variants (z.e., having a sequence that is at least about 80% identical, for example, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity) thereof. In some embodiments, the first barcode and/or the second barcode is located at the junction of the CD20-specific scFv and the CD28 hinge domain. In some embodiments, the first barcode and/or the second barcode is located at the junction of the CD28 hinge domain and the CD8a transmembrane domain. In some embodiments, the first barcode and/or the second barcode is located at the junction of the CD8a transmembrane domain and the 4- IBB costimulatory domain. In some embodiments, the first barcode and/or the second barcode is located at the junction of the 4- IBB costimulatory domain and the CD3(^ signaling domain.

[0309] In some embodiments, the CAR is a CD20 CAR, including, for example, a CD20 CAR comprising a CD20-specific scFv, an IgG4 hinge domain, a CD8a transmembrane domain, a 4- IBB costimulatory domain, a CD3(^ signaling domain, and/or variants (z.e., having a sequence that is at least about 80% identical, for example, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity) thereof. In some embodiments, the first barcode and/or the second barcode is located at the junction of the CD20-specific scFv and the IgG4 hinge domain. In some embodiments, the first barcode and/or the second barcode is located at the junction of the IgG4 hinge domain and the CD8a transmembrane domain. In some embodiments, the first barcode and/or the second barcode is located at the junction of the CD8a transmembrane domain and the 4- IBB costimulatory domain. In some embodiments, the first barcode and/or the second barcode is located at the junction of the 4- IBB costimulatory domain and the CD3(^ signaling domain.

[0310] In some embodiments, the CAR is a CD20 CAR, including, for example, a CD20 CAR comprising a CD20-specific scFv2, a CD8a hinge domain, a CD28 transmembrane domain, a 4- IBB costimulatory domain, a CD3(^ signaling domain, and/or variants (z.e., having a sequence that is at least about 80% identical, for example, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity) thereof. In some embodiments, the first barcode and/or the second barcode is located at the junction of the CD20-specific scFv2 and the CD8a hinge domain. In some embodiments, the first barcode and/or the second barcode is located at the junction of the CD8a hinge domain and the CD28 transmembrane domain. In some embodiments, the first barcode and/or the second barcode is located at the junction of the CD28 transmembrane domain and the 4- IBB costimulatory domain. In some embodiments, the first barcode and/or the second barcode is located at the junction of the 4- IBB costimulatory domain and the CD3(^ signaling domain.

[0311] In some embodiments, the CAR is a CD20 CAR, including, for example, a CD20 CAR comprising a CD20-specific scFv, a CD28 hinge domain, a CD28 transmembrane domain, a 4- IBB costimulatory domain, a CD3(^ signaling domain, and/or variants (z.e., having a sequence that is at least about 80% identical, for example, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity) thereof. In some embodiments, the first barcode and/or the second barcode is located at the junction of the CD20-specific scFv and the CD28 hinge domain. In some embodiments, the first barcode and/or the second barcode is located at the junction of the CD28 hinge domain and the CD28 transmembrane domain. In some embodiments, the first barcode and/or the second barcode is located at the junction of the CD28 transmembrane domain and the 4- IBB costimulatory domain. In some embodiments, the first barcode and/or the second barcode is located at the junction of the 4- IBB costimulatory domain and the CD3(^ signaling domain. [0312] In some embodiments, the CAR is a CD20 CAR, including, for example, a CD20 CAR comprising a CD20-specific scFv, an IgG4 hinge domain, a CD28 transmembrane domain, a 4- IBB costimulatory domain, a CD3^ signaling domain, and/or variants (z.e., having a sequence that is at least about 80% identical, for example, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity) thereof. In some embodiments, the first barcode and/or the second barcode is located at the junction of the CD20-specific scFv and the IgG4 hinge domain. In some embodiments, the first barcode and/or the second barcode is located at the junction of the IgG4 hinge domain and the CD28 transmembrane domain. In some embodiments, the first barcode and/or the second barcode is located at the junction of the CD28 transmembrane domain and the 4- IBB costimulatory domain. In some embodiments, the first barcode and/or the second barcode is located at the junction of the 4- IBB costimulatory domain and the CD3^ signaling domain.

Exemplary CD22 CARs

[0313] In some embodiments, the CAR is a CD22 CAR (“CD22-CAR”). CD22 is a transmembrane protein found mostly on the surface of mature B cells that functions as an inhibitory receptor for B cell receptor (BCR) signaling. CD22 is expressed in 60-70% of B cell lymphomas and leukemias (e.g., B-chronic lymphocytic leukemia, hairy cell leukemia, acute lymphocytic leukemia (ALL), and Burkitt's lymphoma) and is not present on the cell surface in early stages of B cell development or on stem cells. In some embodiments, the CD22 CAR may comprise an extracellular binding domain that specifically binds CD22 and one or more of: any of the signal peptides described herein, any of the hinge domains described herein, any of the transmembrane domains described herein, any of the intracellular costimulatory domains described herein, and/or any of the intracellular signaling domains described herein, e.g., in tandem.

[0314] In some embodiments, the signal peptide of the CD22 CAR comprises a CD8a signal peptide. In some embodiments, the signal peptide comprises an IgK signal peptide. In some embodiments, the signal peptide comprises a GMCSFR-a or CSF2RA signal peptide. [0315] In some embodiments, the extracellular binding domain of the CD22 CAR is specific to CD22, for example, human CD22. The extracellular binding domain of the CD22 CAR can be codon-optimized for expression in a host cell or to have variant sequences to increase functions of the extracellular binding domain. In some embodiments, the extracellular binding domain comprises an immunogenically active portion of an immunoglobulin molecule, for example, an scFv.

[0316] In some embodiments, the extracellular binding domain of the CD22 CAR is derived from an antibody specific to CD22, including, for example, SM03, inotuzumab, epratuzumab, moxetumomab, or pinatuzumab. In any of these embodiments, the extracellular binding domain of the CD22 CAR can comprise or consist of the VH, the VL, and/or one or more CDRs of any of the antibodies.

[0317] In some embodiments, the extracellular binding domain of the CD22 CAR comprises an scFv derived from the m971 monoclonal antibody (m971), which comprises the heavy chain variable region (VH) and the light chain variable region (VL) of m971 connected by a linker. In some embodiments, the linker is a 3xG4S linker. In other embodiments, the Whitlow linker may be used instead. In some embodiments, the amino acid sequences of the entire m971- derived scFv (also referred to as m971 scFv) and its different portions are provided in Table 12 below. In some embodiments, the extracellular binding domain of the CD22 CAR comprises an scFv derived from m971-L7, which is an affinity matured variant of m971 with significantly improved CD22 binding affinity compared to the parental antibody m971 (improved from about 2 nM to less than 50 pM). In some embodiments, the scFv derived from m971-L7 comprises the VH and the VL of m971-L7 connected by a 3xG4S linker. In other embodiments, the Whitlow linker may be used instead. In some embodiments, the amino acid sequences of the entire m971- L7-derived scFv (also referred to as m971-L7 scFv) and its different portions are provided in Table 12 below. In some embodiments, the CD22-specific scFv may comprise one or more CDRs having CDR amino acid sequences set forth in Table 12. In some embodiments, the CD22- specific scFv may comprise a heavy chain with one or more CDRs having heavy chain CDR amino acid sequences set forth in Table 12. In some embodiments, the CD22-specific scFv may comprise a light chain with one or more CDRs having light chain CDR amino acid sequences set forth in Table 12. In any of these embodiments, the CD22-specific scFv may comprise one or more CDRs comprising one or more amino acid substitutions, or comprising a sequence that is at least about 80% identical (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical), to any of the sequences identified. In some embodiments, the extracellular binding domain of the CD22 CAR comprises or consists of the one or more CDRs as described herein.

Table 12. Exemplary sequences of anti-CD22 scFv and components

[0318] In some embodiments, the extracellular binding domain of the CD22 CAR comprises immunotoxins HA22 or BL22. Immunotoxins BL22 and HA22 are therapeutic agents that comprise an scFv specific for CD22 fused to a bacterial toxin, and thus can bind to the surface of the cancer cells that express CD22 and kill the cancer cells. BL22 comprises a dsFv of an anti-CD22 antibody, RFB4, fused to a 38-kDa truncated form of Pseudomonas exotoxin A (Bang et al., Clin. Cancer Res., 11:1545-50 (2005)). HA22 (CAT8015, moxetumomab pasudotox) is a mutated, higher affinity version of BL22 (Ho et al., J. Biol. Chem., 280(1): 607- 17 (2005)). Suitable sequences of antigen binding domains of HA22 and BL22 specific to CD22 are disclosed in, for example, U.S. Patent Nos. 7,541,034; 7,355,012; and 7,982,011, which are hereby incorporated by reference in their entirety.

[0319] In some embodiments, the hinge domain of the CD22 CAR comprises a CD8a hinge domain, for example, a human CD8a hinge domain. In some embodiments, the hinge domain comprises a CD28 hinge domain, for example, a human CD28 hinge domain. In some embodiments, the hinge domain comprises an IgG4 hinge domain, for example, a human IgG4 hinge domain. In some embodiments, the hinge domain comprises a IgG4 hinge-Ch2-Ch3 domain, for example, a human IgG4 hinge-Ch2-Ch3 domain.

[0320] In some embodiments, the transmembrane domain of the CD22 CAR comprises a CD8a transmembrane domain, for example, a human CD8a transmembrane domain. In some embodiments, the transmembrane domain comprises a CD28 transmembrane domain, for example, a human CD28 transmembrane domain.

[0321] In some embodiments, the intracellular costimulatory domain of the CD22 CAR comprises a 4- IBB costimulatory domain, for example, a human 4- IBB costimulatory domain. In some embodiments, the intracellular costimulatory domain comprises a CD28 costimulatory domain, for example, a human CD28 costimulatory domain.

[0322] In some embodiments, the intracellular signaling domain of the CD22 CAR comprises a CD3-zeta (Q signaling domain, for example, a human CD3(^ signaling domain. [0323] In some embodiments, the CAR is a CD22 CAR, including, for example, a CD22 CAR comprising a CD22-specific scFv, a CD8a hinge domain, a CD8a transmembrane domain a 4- IBB costimulatory domain, a CD3(^ signaling domain, and/or variants (z.e., having a sequence that is at least about 80% identical, for example, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity) thereof. In some embodiments, the first barcode and/or the second barcode is located at the junction of the CD22-specific scFv and the CD8a hinge domain. In some embodiments, the first barcode and/or the second barcode is located at the junction of the CD8a hinge domain and the CD8a transmembrane domain. In some embodiments, the first barcode and/or the second barcode is located at the junction of the CD8a transmembrane domain and the 4- IBB costimulatory domain. In some embodiments, the first barcode and/or the second barcode is located at the junction of the 4- IBB costimulatory domain and the CD3(^ signaling domain. [0324] In some embodiments, the CAR is a CD22 CAR, including, for example, a CD22 CAR comprising a CD22-specific scFv, a CD28 hinge domain, a CD8a transmembrane domain, a 4- IBB costimulatory domain, a CD3(^ signaling domain, and/or variants (z.e., having a sequence that is at least about 80% identical, for example, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity) thereof. In some embodiments, the first barcode and/or the second barcode is located at the junction of the CD22-specific scFv and the CD28 hinge domain. In some embodiments, the first barcode and/or the second barcode is located at the junction of the CD28 hinge domain and the CD8a transmembrane domain. In some embodiments, the first barcode and/or the second barcode is located at the junction of the CD8a transmembrane domain and the 4- IBB costimulatory domain. In some embodiments, the first barcode and/or the second barcode is located at the junction of the 4- IBB costimulatory domain and the CD3(^ signaling domain.

[0325] In some embodiments, the CAR is a CD22 CAR, including, for example, a CD22 CAR comprising a CD22-specific scFv, an IgG4 hinge domain, a CD8a transmembrane domain, a 4- IBB costimulatory domain, a CD3(^ signaling domain, and/or variants (z.e., having a sequence that is at least about 80% identical, for example, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity) thereof. In some embodiments, the first barcode and/or the second barcode is located at the junction of the CD22-specific scFv and the IgG4 hinge domain. In some embodiments, the first barcode and/or the second barcode is located at the junction of the IgG4 hinge domain and the CD8a transmembrane domain. In some embodiments, the first barcode and/or the second barcode is located at the junction of the CD8a transmembrane domain and the 4- IBB costimulatory domain. In some embodiments, the first barcode and/or the second barcode is located at the junction of the 4- IBB costimulatory domain and the CD3(^ signaling domain.

[0326] In some embodiments, the CAR is a CD22 CAR, including, for example, a CD22 CAR comprising a CD22-specific scFv, a CD8a hinge domain, a CD28 transmembrane domain, a 4- IBB costimulatory domain, a CD3(^ signaling domain, and/or variants (z.e., having a sequence that is at least about 80% identical, for example, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity) thereof. In some embodiments, the first barcode and/or the second barcode is located at the junction of the CD22-specific scFv and the CD8a hinge domain. In some embodiments, the first barcode and/or the second barcode is located at the junction of the CD8a hinge domain and the CD28 transmembrane domain. In some embodiments, the first barcode and/or the second barcode is located at the junction of the CD28 transmembrane domain and the 4- IBB costimulatory domain. In some embodiments, the first barcode and/or the second barcode is located at the junction of the 4- IBB costimulatory domain and the CD3(^ signaling domain.

[0327] In some embodiments, the CAR is a CD22 CAR, including, for example, a CD22 CAR comprising a CD22-specific scFv, a CD28 hinge domain, a CD28 transmembrane domain, a 4- IBB costimulatory domain, a CD3(^ signaling domain, and/or variants (z.e., having a sequence that is at least about 80% identical, for example, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity) thereof. In some embodiments, the first barcode and/or the second barcode is located at the junction of the CD22-specific scFv and the CD28 hinge domain. In some embodiments, the first barcode and/or the second barcode is located at the junction of the CD28 hinge domain and the CD28 transmembrane domain. In some embodiments, the first barcode and/or the second barcode is located at the junction of the CD28 transmembrane domain and the 4- IBB costimulatory domain. In some embodiments, the first barcode and/or the second barcode is located at the junction of the 4- IBB costimulatory domain and the CD3(^ signaling domain.

[0328] In some embodiments, the CAR is a CD22 CAR, including, for example, a CD22 CAR comprising a CD22-specific scFv, an IgG4 hinge domain, a CD28 transmembrane domain, a 4- IBB costimulatory domain, a CD3(^ signaling domain, and/or variants (z.e., having a sequence that is at least about 80% identical, for example, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity) thereof. In some embodiments, the first barcode and/or the second barcode is located at the junction of the CD22-specific scFv and the IgG4 hinge domain. In some embodiments, the first barcode and/or the second barcode is located at the junction of the IgG4 hinge domain and the CD28 transmembrane domain. In some embodiments, the first barcode and/or the second barcode is located at the junction of the CD28 transmembrane domain and the 4- IBB costimulatory domain. In some embodiments, the first barcode and/or the second barcode is located at the junction of the 4- IBB costimulatory domain and the CD3^ signaling domain.

Exemplary BCMA CARs

[0329] In some embodiments, the CAR is a BCMA CAR (“BCMA-CAR”). BCMA is a tumor necrosis family receptor (TNFR) member expressed on cells of the B cell lineage, with the highest expression on terminally differentiated B cells or mature B lymphocytes. BCMA is involved in mediating the survival of plasma cells for maintaining long-term humoral immunity. The expression of BCMA has been recently linked to a number of cancers, such as multiple myeloma, Hodgkin's and non-Hodgkin's lymphoma, various leukemias, and glioblastoma. In some embodiments, the BCMA CAR may comprise an extracellular binding domain that specifically binds BCMA and one or more of: any of the signal peptides described herein, any of the hinge domains described herein, any of the transmembrane domains described herein, any of the intracellular costimulatory domains described herein, and/or any of the intracellular signaling domains described herein, e.g., in tandem.

[0330] In some embodiments, the signal peptide of the BCMA CAR comprises a CD8a signal peptide. In some embodiments, the signal peptide comprises an IgK signal peptide. In some embodiments, the signal peptide comprises a GMCSFR-a or CSF2RA signal peptide. [0331] In some embodiments, the extracellular binding domain of the BCMA CAR is specific to BCMA, for example, human BCMA. The extracellular binding domain of the BCMA CAR can be codon-optimized for expression in a host cell or to have variant sequences to increase functions of the extracellular binding domain.

[0332] In some embodiments, the extracellular binding domain comprises an immunogenically active portion of an immunoglobulin molecule, for example, an scFv. In some embodiments, the extracellular binding domain of the BCMA CAR is derived from an antibody specific to BCMA, including, for example, belantamab, erlanatamab, teclistamab, LCAR-B38M, or ciltacabtagene. In any of these embodiments, the extracellular binding domain of the BCMA CAR can comprise or consist of the Vn, the VL, and/or one or more CDRs of any of the antibodies.

[0333] In some embodiments, the extracellular binding domain of the BCMA CAR comprises an scFv derived from Cl 1D5.3, a murine monoclonal antibody as described in Carpenter et al., Clin. Cancer Res. 19(8):2048-2060 (2013). See also PCT Application Publication No. WO2010/104949. The Cl lD5.3-derived scFv may comprise the heavy chain variable region (VH) and the light chain variable region (VL) of Cl 1D5.3 connected by the Whitlow linker, the amino acid sequence of which is provided in Table 13 below. In some embodiments, the extracellular binding domain of the BCMA CAR comprises an scFv derived from another murine monoclonal antibody, C12A3.2, as described in Carpenter et al., Clin. Cancer Res. 19(8):2048-2060 (2013) and PCT Application Publication No. WO2010/104949, the amino acid sequence of which is also provided in Table 13 below. In some embodiments, the extracellular binding domain of the BCMA CAR comprises a fully human heavy-chain variable domain (FHVH) as described in Lam et al., Nat. Commun. 11 ( 1 ):283 (2020), also referred to as FHVH33. See also, PCT Application Publication No. WO2019/006072, and Table 13. In some embodiments, the extracellular binding domain of the BCMA CAR comprises an scFv derived from CT103A (or CAR0085) as described in U.S. Patent No. 11,026,975 B2, the amino acid sequence of which is provided in Table 13 below. In some embodiments, the BCMA-specific extracellular binding domain may comprise one or more CDRs having CDR amino acid sequences set forth in Table 13. In some embodiments, the BCMA-specific extracellular binding domain may comprise a light chain with one or more CDRs having light chain CDR amino acid sequences set forth in Table 13. In some embodiments, the BCMA-specific extracellular binding domain may comprise a heavy chain with one or more CDRs having heavy chain CDR amino acid sequences set forth in Table 13. In any of these embodiments, the BCMA-specific scFv may comprise one or more CDRs comprising one or more amino acid substitutions, or comprising a sequence that is at least about 80% identical (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical), to any of the sequences identified. In some embodiments, the extracellular binding domain of the BCMA CAR comprises or consists of the one or more CDRs as described herein.

[0334] In some embodiments, the extracellular binding domain of the BCMA CAR comprises a murine monoclonal antibody with high specificity to human BCMA, referred to as BB2121 in Friedman et al., Hum. Gene Ther. 29(5):585-601 (2018)). See also, PCT Application Publication No. WO2012163805. In some embodiments, the extracellular binding domain of the BCMA CAR comprises single variable fragments of two heavy chains (VHH) that can bind to two epitopes of BCMA as described in Zhao et al., J. Hematol. Oncol. 11(1): 141 (2018), also referred to as LCAR-B38M. See also, PCT Application Publication No. WO2018/028647. [0335] Additionally, CARs and binders directed to BCMA have been described in U.S. Application Publication Nos. 2020/0246381 Al and 2020/0339699 Al, the entire contents of each of which are incorporated by reference herein.

Table 13. Exemplary sequences of anti-BCMA binder and components.

[0336] In some embodiments, the hinge domain of the BCMA CAR comprises a CD8a hinge domain, for example, a human CD8a hinge domain. In some embodiments, the hinge domain comprises a CD28 hinge domain, for example, a human CD28 hinge domain. In some embodiments, the hinge domain comprises an IgG4 hinge domain, for example, a human IgG4 hinge domain. In some embodiments, the hinge domain comprises a IgG4 hinge-Ch2-Ch3 domain, for example, a human IgG4 hinge-Ch2-Ch3 domain.

[0337] In some embodiments, the transmembrane domain of the BCMA CAR comprises a CD8a transmembrane domain, for example, a human CD8a transmembrane domain. In some embodiments, the transmembrane domain comprises a CD28 transmembrane domain, for example, a human CD28 transmembrane domain.

[0338] In some embodiments, the intracellular costimulatory domain of the BCMA CAR comprises a 4- IBB costimulatory domain, for example, a human 4- IBB costimulatory domain. In some embodiments, the intracellular costimulatory domain comprises a CD28 costimulatory domain, for example, a human CD28 costimulatory domain.

[0339] In some embodiments, the intracellular signaling domain of the BCMA CAR comprises a CD3 zeta (Q signaling domain, for example, a human CD3(^ signaling domain.

[0340] In some embodiments, the CAR is a BCMA CAR, including, for example, a BCMA CAR comprising any of the BCMA-specific extracellular binding domains as described, a CD8a hinge domain, a CD8a transmembrane domain, a 4- IBB costimulatory domain, a CD3(^ signaling domain, and/or variants (z.e., having a sequence that is at least about 80% identical, for example, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity) thereof. In any of these embodiments, the BCMA CAR may additionally comprise a signal peptide (e.g., a CD8a signal peptide) as described above. In some embodiments, the first barcode and/or the second barcode is located at the junction of the BCMA-specific extracellular binding domain and the CD8a hinge domain. In some embodiments, the first barcode and/or the second barcode is located at the junction of the CD8a hinge domain and the CD8a transmembrane domain. In some embodiments, the first barcode and/or the second barcode is located at the junction of the CD8a transmembrane domain and the 4- IBB costimulatory domain. In some embodiments, the first barcode and/or the second barcode is located at the junction of the 4- IBB costimulatory domain and the CD3^ signaling domain.

[0341] In some embodiments, the CAR is a BCMA CAR, including, for example, a BCMA CAR comprising any of the BCMA-specific extracellular binding domains as described, a CD8a hinge domain, a CD8a transmembrane domain, a CD28 costimulatory domain, a CD3(^ signaling domain, and/or variants (z.e., having a sequence that is at least about 80% identical, for example, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity) thereof. In any of these embodiments, the BCMA CAR may additionally comprise a signal peptide as described above. In some embodiments, the first barcode and/or the second barcode is located at the junction of the BCMA-specific extracellular binding domain and the CD8a hinge domain. In some embodiments, the first barcode and/or the second barcode is located at the junction of the CD8a hinge domain and the CD8a transmembrane domain. In some embodiments, the first barcode and/or the second barcode is located at the junction of the CD8a transmembrane domain and the CD28 costimulatory domain. In some embodiments, the first barcode and/or the second barcode is located at the junction of the CD28 costimulatory domain and the CD3^ signaling domain.

[0342] In some embodiments, the CAR is a BCMA CAR as set forth in SEQ ID NO:346 or is at least about 80% identical (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical) thereto (see Table 13). The encoded BCMA CAR has a corresponding amino acid sequence set forth in SEQ ID NO:347 or is at least about 80% identical (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical) thereto, with the following components: CD8a signal peptide, CT103A scFv ( Vi.-Whitlow linker-Vn), CD8a hinge domain, CD8a transmembrane domain, 4- IBB costimulatory domain, and CD3^ signaling domain. In some embodiments, the first barcode and/or the second barcode is located at the junction of the CT103A scFv and the CD8a hinge domain. In some embodiments, the first barcode and/or the second barcode is located at the junction of the CD8a hinge domain and the CD8a transmembrane domain. In some embodiments, the first barcode and/or the second barcode is located at the junction of the CD8a transmembrane domain and the 4- IBB costimulatory domain. In some embodiments, the first barcode and/or the second barcode is located at the junction of the 4- IBB costimulatory domain and the CD3^ signaling domain.

[0343] In some embodiments, the CAR is a commercially available embodiment of BCMA CAR, including, for example, idecabtagene vicleucel (ide-cel, also called bb2121). In some embodiments, the CAR is idecabtagene vicleucel or portions thereof. Idecabtagene vicleucel comprises a BCMA CAR with the following components: the BB2121 binder, CD8a hinge domain, CD8a transmembrane domain, 4- IBB costimulatory domain, and CD3^ signaling domain.

[0344] In some cases, CARs are referred to as first, second, and/or third generation CARs. In some aspects, a first generation CAR is one that solely provides a CD3-chain induced signal upon antigen binding; in some aspects, a second-generation CARs is one that provides such a signal and costimulatory signal, such as one including an intracellular signaling domain from a costimulatory receptor such as CD28 or CD137; in some aspects, a third generation CAR is one that includes multiple costimulatory domains of different costimulatory receptors.

[0345] For example, in some embodiments, any of the CARs herein contain an antibody, e.g., an antibody fragment, a transmembrane domain that is or contains a transmembrane portion of CD28 or a functional variant thereof, and an intracellular signaling domain containing a signaling portion of CD28 or functional variant thereof and a signaling portion of CD3-zeta or functional variant thereof. In some embodiments, the CAR contains an antibody, e.g., antibody fragment, a transmembrane domain that is or contains a transmembrane portion of CD28 or a functional variant thereof, and an intracellular signaling domain containing a signaling portion of a 4- IBB or functional variant thereof and a signaling portion of CD3-zeta or functional variant thereof. In some such embodiments, the receptor further includes a spacer containing a portion of an Ig molecule, such as a human Ig molecule, such as an Ig hinge, e.g., an IgG4 hinge, such as a hinge-only spacer. [0346] In some aspects, the spacer contains only a hinge region of an IgG, such as only a hinge of IgG4 or IgGl. In other embodiments, the spacer is or contains an Ig hinge, e.g., an IgG4- derived hinge, optionally linked to a CH2 and/or CH3 domains. In some embodiments, the spacer is an Ig hinge, e.g., an IgG4 hinge, linked to CH2 and CH3 domains. In some embodiments, the spacer is an Ig hinge, e.g., an IgG4 hinge, linked to a CH3 domain only. In some embodiments, the spacer is or comprises a glycine-serine rich sequence or other flexible linker such as known flexible linkers.

[0347] For example, in some embodiments, any of the CARs herein include an antibody such as an antibody fragment, including scFvs, a spacer, such as a spacer containing a portion of an immunoglobulin molecule, such as a hinge region and/or one or more constant regions of a heavy chain molecule, such as an Ig-hinge containing spacer, a transmembrane domain containing all or a portion of a CD28-derived transmembrane domain, a CD28-derived intracellular signaling domain, and a CD3-zeta signaling domain. In some embodiments, the CAR includes an antibody or fragment, such as scFv, a spacer such as any of the Ig-hinge containing spacers, a CD28-derived transmembrane domain, a 4-lBB-derived intracellular signaling domain, and a CD3-zeta-derived signaling domain.

[0348] The recombinant receptors, such as any of the CARs of the disclosure, expressed by cells administered to a subject generally recognize or specifically bind to a molecule that is expressed in, associated with, and/or specific for the disease or condition or cells thereof being treated. Upon specific binding to the molecule, e.g., antigen, the receptor generally delivers an immuno stimulatory signal, such as an ITAM-transduced signal, into the cell, thereby promoting an immune response targeted to the disease or condition. For example, in some embodiments, the cells express a CAR that specifically binds to an antigen expressed by a cell or tissue of the disease or condition or associated with the disease or condition. In any of these embodiments, the extracellular binding domain of the CAR can be codon-optimized for expression in a host cell or have variant sequences to increase functions of the extracellular binding domain. b. T Cell Receptors

[0349] In some embodiments, the one or more transgenes comprise a T cell receptor (TCR). In some embodiments, the transgene encodes a T cell receptor (TCR) or antigen-binding portion thereof that recognizes a peptide epitope or T cell epitope of a target polypeptide, such as an antigen of a tumor, viral or autoimmune protein. [0350] In some embodiments, a T cell receptor or TCR is a molecule that contains a variable a and b chain (also known as TCRalpha and TCRbeta, respectively) or a variable g and d chains (also known as TCRalpha and TCRbeta, respectively), or antigen-binding portions thereof, and which is capable of specifically binding to a peptide bound to an MHC molecule. In some embodiments, the TCR is in the alpha-beta (a-P) form. Typically, TCRs that exist in alphabeta and gamma-delta forms are generally structurally similar, but T cells expressing them may have distinct anatomical locations or functions. A TCR can be found on the surface of a cell or in soluble form. Generally, a TCR is found on the surface of T cells (or T lymphocytes) where it is generally responsible for recognizing antigens bound to major histocompatibility complex (MHC) molecules.

[0351] Unless otherwise stated, the term “TCR” should be understood to encompass full TCRs as well as antigen-binding portions or antigen-binding fragments thereof. In some embodiments, the TCR is an intact or full-length TCR, including TCRs in the a-P form or y-5 form. In some embodiments, the TCR is an antigen-binding portion that is less than a full-length TCR but that binds to a specific peptide bound in an MHC molecule, such as binds to an MHC - peptide complex. In some cases, an antigen-binding portion or fragment of a TCR can contain only a portion of the structural domains of a full-length or intact TCR, but yet is able to bind the peptide epitope, such as MHC -peptide complex, to which the full TCR binds. In some cases, an antigen-binding portion contains the variable domains of a TCR, such as variable a chain and variable P chain of a TCR, sufficient to form a binding site for binding to a specific MHC- peptide complex. Generally, the variable chains of a TCR contain complementarity determining regions involved in recognition of the peptide, MHC and/or MHC -peptide complex. c. Chimeric Auto-Antibody Receptors (CAAR) and B-cell Autoantibody Receptors (BAR) [0352] In some embodiments, the one or more transgenes comprise a chimeric autoantibody receptor (CAAR).

[0353] In some embodiments, the CAAR binds, e.g., specifically binds, or recognizes, an autoantibody. In some embodiments, a cell expressing the CAAR, such as a T cell engineered to express a CAAR, can be used to bind to and kill autoantibody-expressing cells, but not normal antibody expressing cells. In some embodiments, CAAR-expressing cells can be used to treat an autoimmune disease associated with expression of self-antigens, such as autoimmune diseases. In some embodiments, CAAR-expressing cells can target B cells that ultimately produce the autoantibodies and display the autoantibodies on their cell surfaces, mark these B cells as disease-specific targets for therapeutic intervention. In some embodiments, CAAR-expressing cells can be used for efficiently targeting and killing the pathogenic B cells in autoimmune diseases by targeting the disease-causing B cells using an antigen- specific chimeric autoantibody receptor. In some embodiments, the recombinant receptor is a CAAR, such as any described in U.S. Patent Application Pub. No. US 2017/0051035.

[0354] In some embodiments, the CAAR comprises an antigen selected from a pancreatic P-cell antigen, synovial joint antigen, myelin basic protein, proteolipid protein, myelin oligodendritic glycoprotein, MuSK, keratinocyte adhesion protein desmoglein 3 (Dsg3), Ro- RNP complex, La antigen, myeloperoxidase, proteinase 3, cardiolipin, citrullinated proteins, carbamylated proteins, a3 chain of basement membrane collagen, or any combination thereof. [0355] In some embodiments, the CAAR comprises an autoantibody binding domain, a transmembrane domain, and one or more intracellular signaling regions or domains (also interchangeably called a cytoplasmic signaling domain or region). In some embodiments, the intracellular signaling region comprises an intracellular signaling domain. In some embodiments, the intracellular signaling domain is or comprises a primary signaling domain, a signaling domain that is capable of stimulating and/or inducing a primary activation signal in a T cell, a signaling domain of a T cell receptor (TCR) component (e.g., an intracellular signaling domain or region of a CD3-zeta) chain or a functional variant or signaling portion thereof), and/or a signaling domain comprising an immunoreceptor tyrosine- based activation motif (IT AM).

[0356] In some embodiments, the one or more transgenes comprise a B-cell autoantibody receptor (BAR). In some embodiments, the BAR comprises an FVIII antigen.

[0357] In some embodiments, the autoantibody binding domain comprises an autoantigen or a fragment thereof. The choice of autoantigen can depend upon the type of autoantibody being targeted. For example, the autoantigen may be chosen because it recognizes an autoantibody on a target cell, such as a B cell, associated with a particular disease state, e.g., an autoimmune disease, such as an autoantibody-mediated autoimmune disease. In some embodiments, the autoimmune disease includes pemphigus vulgaris (PV). Exemplary autoantigens include desmoglein 1 (Dsgl) and Dsg3.

[0358] In some embodiments, the first transgene and/or the second transgene encodes an antibody or an antibody fragment, a chimeric antigen receptor (CAR), a chimeric autoantibody receptor (CAAR), a B-cell autoantibody receptor (BAR), a T cell receptor (TCR), or one or more tolerogenic factors. In some embodiments, the first transgene and/or the second transgene encodes a CAR.

[0359] In some embodiments, the CAR encoded by the first transgene and/or the second transgene comprises a hinge domain, a transmembrane domain, and one or more signaling domains. In some embodiments, the hinge domain is a hinge domain of a naturally occurring protein. Hinge domains of any protein known in the art to comprise a hinge domain are compatible for use in the CARs as described herein. In some embodiments, the hinge domain is at least a portion of a hinge domain of a naturally occurring protein and confers flexibility to the CAR as described herein. In some embodiments, the hinge domain is a variant of a hinge domain of a naturally occurring protein (z.e., having a sequence that is at least about 80% identical, for example, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity).

[0360] In some embodiments, the hinge domain is selected from the group consisting of: CD8a hinge domain, CD28 hinge domain, IgG4 hinge domain, IgG4 hinge-CH2-CH3 domain, and any functional variant thereof. In some embodiments, the hinge domain is derived from CD8a. In some embodiments, the hinge domain is a portion of the hinge domain of CD8a, or any functional variant thereof. In some embodiments, the hinge domain is derived from CD28. In some embodiments, the hinge domain is a portion of the hinge domain of CD28, or any functional variant thereof. In some embodiments, the hinge domain is derived from IgG4. In some embodiments, the hinge domain is a portion of the hinge domain of IgG4, or any functional variant thereof. In some embodiments, the hinge domain is derived from IgG4 hinge-CH2-CH3. In some embodiments, the hinge domain is a portion of the hinge domain of IgG4 hinge-CH2- CH3, or any functional variant thereof.

[0361] In some embodiments, the transmembrane domain is selected from the group consisting of: alpha, beta, or zeta chain of a T cell receptor, CD28, CD3s, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CD8a, CD8P, 4-1BB/CD137, CD28, CD34, CD4, FcsRIy, CD16, OX40/CD134, CD3^, CD3s, CD3y, CD35, TCRa, TCRp, TCR^, CD32, CD64, CD64, CD45, CD5, CD9, CD22, CD37, CD80, CD86, CD40, CD40L/CD154, VEGFR2, FAS, FGFR2B, and any functional variant thereof. In some embodiments, the signaling domain is selected from the group consisting of: B7-1/CD80, B7-2/CD86, B7-H1/PD-L1, B7-H2, B7-H3, B7-H4, B7-H6, B7-H7, BTLA/CD272, CD28, CTLA-4, G124/VISTA/B7-H5, ICOS/CD278, PD-1, PD-L2/B7-DC, PDCD6, 4- 1BB/TNFSF9/CD137, 4-1BB Ligand/TNFSF9, BAFF/BLyS/TNFSF13B, BAFF R/TNFRSF13C, CD27/TNFRSF7, CD27 Ligand/TNFSF7, CD30/TNFRSF8, CD30 Ligand/TNFSF8, CD40/TNFRSF5, CD40/TNFSF5, CD40 Ligand/TNFSF5, DR3/TNFRSF25, GITR/TNFRSF18, GITR Ligand/TNFSF18, HVEM/TNFRSF14, LIGHT/TNFSF14, Lymphotoxin-alpha/TNFp, OX40/TNFRSF4, 0X40 Ligand/TNFSF4, RELT/TNFRSF19L, TACI/TNFRSFI 3B, TL1A/TNFSF15, TNFa, TNF RII/TNFRSF 1 B, 2B4/CD244/SLAMF4, BLAME/SLAMF8, CD2, CD2F-10/SLAMF9, CD48/SLAMF2, CD58/LFA-3, CD84/SLAMF5, CD229/SLAMF3, CRACC/SLAMF7, NTB-A/SLAMF6, SLAM/CD150, CD7, CD53, CD82/Kai-1, CD90/Thyl, CD96, CD160, CD200, CD300a/LMIRl, HLA Class I, HLA-DR, Ikaros, Integrin alpha 4/CD49d, Integrin alpha 4 beta 1, Integrin alpha 4 beta 7/LPAM-l, LAG- 3, TCL1A, TCL1B, CRTAM, DAP12, Dectin- 1/CLEC7 A, DPPIV/CD26, EphB6, TIM-l/KIM- 1/HAVCR, TIM-4, TSLP, TSLP R, lymphocyte function associated antigen-1 (LFA-1), NKG2C, CD3^, an immunoreceptor tyrosine-based activation motif (IT AM), CD27, 4- IBB,

CD 134/0X40, CD30, CD40, PD-1, ICOS, LIGHT, NKG2C, a ligand that specifically binds with CD83, any functional variant thereof, and any combination thereof.

[0362] In some embodiments, the CAR encoded by the first transgene and/or the second transgene comprises a CD8a hinge domain, a CD8a transmembrane domain, a 4- IBB domain, and a CD3zeta domain. In some embodiments, the CAR encoded by the first transgene and/or the second transgene is selected from the group consisting of: a CD5- specific CAR, a CD 19- specific CAR, a CD20-specific CAR, a CD22-specific CAR, a CD23-specific CAR, a CD30- specific CAR, a CD33-specific CAR, CD38-specific CAR, a CD70-specific CAR, a CD 123- specific CAR, a CD 138- specific CAR, a Kappa, Lambda, B cell maturation agent (BCMA)- specific CAR, a G-protein coupled receptor family C group 5 member D (GPRC5D)-specific CAR, a CD 123 -specific CAR, a LeY-specific CAR, a NKG2D ligand- specific CAR, a WT1- specific CAR, a GD2-specific CAR, a HER2-specific CAR, a EGFR-specific CAR, a EGFRvIII- specific CAR, a B7H3-specific CAR, a PSMA-specific CAR, a PSCA-specific CAR, a CAIX- specific CAR, a CD 171 -specific CAR, a CEA- specific CAR, a CSPG4- specific CAR, a EPHA2- specific CAR, a FAP-specific CAR, a FRa-specific CAR, a IL-13Ra-specific CAR, a Mesothelin-specific CAR, a MUC1 -specific CAR, a MUC16-specific CAR, a ROR1 -specific CAR, a C-Met-specific CAR, a CD 133- specific CAR, a Ep-C AM- specific CAR, a GPC3- specific CAR, a HPV16-E6- specific CAR, a IL 13 Ra2- specific CAR, a MAGEA3- specific CAR, a MAGEA4- specific CAR, a MARTI -specific CAR, a NY-ESO-l-specific CAR, a VEGFR2- specific CAR, a a-Folate receptor- specific CAR, a CD24-specific CAR, a CD44v7/8- specific CAR, a EGP-2- specific CAR, a EGP-40-specific CAR, a erb-B2-specific CAR, a erb-B 2,3,4- specific CAR, a FBP- specific CAR, a Fetal acethylcholine e receptor- specific CAR, a GD2- specific CAR, a GD3-specific CAR, a HMW-MAA-specific CAR, a IL- HRa- specific CAR, a KDR-specific CAR, a Lewis Y-specific CAR, a Ll-cell adhesion molecule- specific CAR, a MAGE- Al-specific CAR, a Oncofetal antigen (h5T4)-specific CAR, a TAG-72- specific CAR, and a CD19/CD22-bispecific CAR. In some embodiments, the CAR encoded by the first transgene and/or the second transgene is a CD19 CAR or a CD22 CAR. In some embodiments, the CAR encoded by the first transgene and/or the second transgene further comprises one or more co-stimulatory domain(s).

[0363] In some embodiments, the diverged nucleotide sequence within the transgene is located at the junction of: (i) the signaling domain and the co-stimulatory domain; or (ii) the hinge domain and the transmembrane domain. In some embodiments, the diverged nucleotide sequence within the transgene is located at the junction of the 4- IBB and CD3-zeta domains. ii. Tolerogenic Factors

[0364] In some embodiments, the one or more transgenes, e.g., the first and/or the second transgene, comprises a tolerogenic factor. Tolerogenic factors include any factors that promote or contribute to promoting or inducing tolerance to an engineered cell or population of cells (e.g., a hypoimmunogenic islet cell such as a hypoimmunogenic beta cell) of the disclosure by the immune system (e.g., innate or adaptive immune system).

[0365] In some embodiments, the tolerogenic factor comprises one or more of CD16, CD24, CD35, CD39, CD46, CD47, CD52, CD55, CD59, CD64, CD200, CCL22, CTLA4-Ig, Cl inhibitor, FASL, IDO1, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, IL- 10, IL-35, PD-L1, SERPINB9, CCL21, MFGE8, DUX4, B2M-HLA-E, CD27, IL-39, CD16 Fc Receptor, IL15-RF, H2-M3 (HLA-G), A20/TNFAIP3, CR1, HLA-F, DUX4, MANF, or any combinations, functional fragments, or variants thereof. In some embodiments, the tolerogenic factor is CD47, PD-L1, HLA-E or HLA-G, CCL21, FasL, Serpinb9, CD200, Mfge8, or any combinations, functional fragments, or variants thereof. In some embodiments, expression of the tolerogenic (e.g., immune) factor affects immune recognition and tolerance of an engineered cell or population of cells (e.g., T cells, B cells, NK cells, islet cells such as beta cells, or hypoimmunogenic cells thereof) of the disclosure in a recipient. In some embodiments, the engineered cell or population of cells contains an exogenous sequence that encodes the one or more tolerogenic factors. In some embodiments, the tolerogenic factor is overexpressed in the cell. In some embodiments, the expression of the tolerogenic factor is overexpressed or increased in the engineered cell or population of cells, e.g., compared to a similar cell of the same cell type that has not been engineered with the modification, such as a reference or nonengineered cell, e.g., a cell not engineered with a transgene encoding the tolerogenic factor. [0366] In some embodiments, the tolerogenic factor is CD47, or a functional fragment or variant thereof. CD47 is a leukocyte surface antigen and has a role in cell adhesion and modulation of integrins. It is normally expressed on the surface of a cell and signals to circulating macrophages not to eat the cell. In some embodiments, the engineered cell or population of cells (e.g., T cells, B cells, NK cells, islet cells such as beta cells, or hypoimmunogenic cells thereof) contains a transgene sequence that encodes CD47, such as human CD47, or a functional fragment or variant thereof. In some embodiments, CD47, or a functional fragment or variant thereof, is overexpressed in the cell. In some embodiments, the expression of CD47, or a functional fragment or variant thereof, is overexpressed or increased in the engineered cell or population of cells compared to a similar cell of the same cell type that has not been engineered, such as a reference or non-engineered cell, e.g., a cell not engineered with a transgene encoding CD47. In some embodiments, the engineered cell or population of cells contains an overexpressed transgene that encodes CD47, or a functional fragment or variant thereof, such as human CD47. Useful genomic, polynucleotide and polypeptide information about human CD47 are provided in, for example, the NP_001768.1, NP_942088.1, NM_001777.3 and NM_198793.2.

[0367] In some embodiments, the cell outlined herein comprises a transgene sequence encoding a CD47 polypeptide that has at least 95% sequence identity (e.g., 95%, 96%, 97%, 98%, 99%, or more) to an amino acid sequence as set forth in NCBI Ref. Sequence Nos. NP_001768.1 and NP_942088.1, or to fragments thereof. In some embodiments, the cell outlined herein comprises a transgene sequence encoding a CD47 polypeptide having an amino acid sequence as set forth in NCBI Ref. Sequence Nos. NP_001768.1 and NP_942088.1, or a functional fragment or variant thereof. In some embodiments, the cell comprises a transgene sequence encoding a CD47 polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) to the sequence set forth in NCBI Ref. Nos. NM_001777.3 and NM_198793.2, or to fragments thereof. In some embodiments, the cell comprises a transgene sequence encoding a CD47 polynucleotide as set forth in NCBI Ref. Sequence Nos. NM_001777.3 and NM_198793.2, or fragments thereof.

[0368] In some embodiments, the cell outlined herein comprises a transgene sequence encoding a CD47 polypeptide has at least 95% sequence identity (e.g., 95%, 96%, 97%, 98%, 99%, or more) to an amino acid sequence as set forth in NCBI Ref. Sequence Nos. NP_001768.1 and NP_942088.1, or to fragments thereof. In some embodiments, the cell outlined herein comprises a transgene sequence encoding a CD47 polypeptide having an amino acid sequence as set forth in NCBI Ref. Sequence Nos. NP_001768.1 and NP_942088.1, or a functional fragment or variant thereof. In some embodiments, the cell comprises a transgene sequence encoding a CD47 polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) to the sequence set forth in NCBI Ref. Nos. NM_001777.3 and NM_198793.2, or to a functional fragment thereof. In some embodiments, the cell comprises a transgene sequence encoding a CD47 polynucleotide as set forth in NCBI Ref. Sequence Nos. NM_001777.3 and NM_198793.2, or fragments thereof.

[0369] In some embodiments, the cell comprises a CD47 polypeptide having at least 95% sequence identity (e.g., 95%, 96%, 97%, 98%, 99%, or more) to an amino acid sequence as set forth in NCBI Ref. Sequence Nos. NP_001768.1 and NP_942088.1, or a functional fragment thereof. In some embodiments, the cell outlined herein comprises a CD47 polypeptide having an amino acid sequence as set forth in NCBI Ref. Sequence Nos. NP_001768.1 and NP_942088.1, or a functional fragment or variant thereof.

[0370] In some embodiments, the cell comprises a CD47 polypeptide having at least 95% sequence identity (e.g., 95%, 96%, 97%, 98%, 99%, or more) to an amino acid sequence selected from the group consisting of SEQ ID NOs:l-5, or a functional fragment thereof. In some embodiments, the cell outlined herein comprises a CD47 polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs:l-5, or a functional fragment or variant thereof, as laid out in Table 14 below. Table 14. CD47 polypeptide sequences.

[0371] In some embodiments, the cell comprises a CD47 polypeptide encoded by a nucleic acid sequence having at least 95% sequence identity (e.g., 95%, 96%, 97%, 98%, 99%, or more) to a nucleic acid sequence selected from the group consisting of SEQ ID NOs:6-l 1, or a functional fragment thereof. In some embodiments, the cell outlined herein comprises a CD47 polypeptide encoded by a nucleic acid sequence having a nucleic acid sequence selected from the group consisting of SEQ ID NOs:6-l 1, or a functional fragment or variant thereof, as laid out in Table 15 below.

Table 15. CD47 nucleic acid sequences.

[0372] In some embodiments, all or a functional portion of CD47 can be linked to other components such as a signal peptide, a leader sequence, a secretory signal, a label (e.g., a reporter gene), or any combination thereof. In some embodiments, the nucleic acid sequence encoding a signal peptide of CD47 is replaced with a nucleic acid sequence encoding a signal peptide from a heterologous protein. The heterologous protein can be, for example, CD8a, CD28, tissue plasminogen activator (tPA), growth hormone, granulocyte-macrophage colony stimulating factor (GM-CSF), GM-CSF receptor (GM-CSFRa), or an immunoglobulin e.g., IgE or IgK). In some embodiments, the signal peptide is a signal peptide from an immunoglobulin (such as IgG heavy chain or IgG-kappa light chain), a cytokine (such as interleukin-2 (IL-2), or CD33), a serum albumin protein (e.g., HSA or albumin), a human azurocidin preprotein signal sequence, a luciferase, a trypsinogen (e.g., chymotrypsinogen or trypsinogen) or other signal peptide able to efficiently express a protein by or on a cell.

[0373] In some embodiments, the engineered cell or population of cells (e.g., T cells, B cells, NK cells, islet cells such as beta cells, or hypoimmunogenic cells thereof) contains a transgene that encodes CD200, such as human CD200, or a functional fragment or variant thereof. In some embodiments, CD200 is overexpressed in the cell. In some embodiments, the expression of CD200 is increased in the engineered cell or population of cells compared to a similar reference or non-engineered cell (including with any other modifications) except that the reference or non-engineered cell does not include the transgene encoding CD200. Useful genomic, polynucleotide and polypeptide information about human CD200 are provided in, for example, the GeneCard Identifier GC03P112332, HGNC No. 7203, NCBI Gene ID 4345, Uniprot No. P41217, and NCBI RefSeq Nos. NP_001004196.2, NM_001004196.3, NP_001305757.1, NM_001318828.1, NP_005935.4, NM_005944.6, XP_005247539.1, and XM_005247482.2. In certain embodiments, the transgene encoding CD200, or a functional fragment or variant thereof, is operably linked to a promoter.

[0374] In some embodiments, the engineered cell or population of cells (e.g., T cells, B cells, NK cells, islet cells such as beta cells, or hypoimmunogenic cells thereof) contains a transgene that encodes HLA-E, such as human HLA-E, or a functional fragment or variant thereof. In some embodiments, HLA-E is overexpressed in the cell. In some embodiments, the expression of HLA-E is increased in the engineered cell or population of cells compared to a similar reference or non-engineered cell (including with any other modifications) except that the reference or non-engineered cell does not include the transgene encoding HLA-E. Useful genomic, polynucleotide, and polypeptide information about human HLA-E are provided in, for example, the GeneCard Identifier GC06P047281, HGNC No. 4962, NCBI Gene ID 3133, Uniprot No. P13747, and NCBI RefSeq Nos. NP_005507.3 and NM_005516.5. In certain embodiments, the transgene encoding HLA-E, or a functional fragment or variant thereof, is operably linked to a promoter.

[0375] In some embodiments, the engineered cell or population of cells (e.g., T cells, B cells, NK cells, islet cells such as beta cells, or hypoimmunogenic cells thereof) contains a transgene that encodes HLA-G, such as human HLA-G, or a functional fragment or variant thereof. In some embodiments, HLA-G is overexpressed in the cell. In some embodiments, the expression of HLA-G is increased in the engineered cell or population of cells compared to a similar reference or non-engineered cell (including with any other modifications) except that the reference or non-engineered cell does not include the transgene encoding HLA-G. Useful genomic, polynucleotide and polypeptide information about human HLA-G are provided in, for example, the GeneCard Identifier GC06P047256, HGNC No. 4964, NCBI Gene ID 3135, Uniprot No. P17693, and NCBI RefSeq Nos. NP_002118.1 and NM_002127.5. In certain embodiments, the transgene encoding HLA-G, or a functional fragment or variant thereof, is operably linked to a promoter.

[0376] In some embodiments, the engineered cell or population of cells (e.g., T cells, B cells, NK cells, islet cells such as beta cells, or hypoimmunogenic cells thereof) contains a transgene that encodes PD-L1, such as human PD-L1, or a functional fragment or variant thereof. In some embodiments, PD-L1 is overexpressed in the cell. In some embodiments, the expression of PD-L1 is increased in the engineered cell or population of cells compared to a similar reference or non-engineered cell (including with any other modifications) except that the reference or non-engineered cell does not include the transgene encoding PD-L1. Useful genomic, polynucleotide and polypeptide information about human PD-L1 or CD274 are provided in, for example, the GeneCard Identifier GC09P005450, HGNC No. 17635, NCBI Gene ID 29126, Uniprot No. Q9NZQ7, and NCBI RefSeq Nos. NP_001254635.1, NM_001267706.1, NP_054862.1, and NM_014143.3. In certain embodiments, the polynucleotide encoding PD-L1, or a functional fragment or variant thereof, is operably linked to a promoter.

[0377] In some embodiments, the engineered cell or population of cells (e.g., T cells, B cells, NK cells, islet cells such as beta cells, or hypoimmunogenic cells thereof) contains a transgene that encodes FasL, such as human FasL, or a functional fragment or variant thereof. In some embodiments, FasL is overexpressed in the cell. In some embodiments, the expression of FasL is increased in the engineered cell or population of cells compared to a similar reference or non-engineered cell (including with any other modifications) except that the reference or nonengineered cell does not include the transgene encoding FasL. Useful genomic, polynucleotide and polypeptide information about human Fas ligand (which is known as FasL, FASLG, CD178, TNFSF6, and the like) are provided in, for example, the GeneCard Identifier GC01P172628, HGNC No. 11936, NCBI Gene ID 356, Uniprot No. P48023, and NCBI RefSeq Nos.

NP_000630.1, NM_000639.2, NP_001289675.1, and NM_001302746.1. In certain embodiments, the transgene encoding Fas-L is operably linked to a promoter.

[0378] In some embodiments, the engineered cell or population of cells (e.g., T cells, B cells, NK cells, islet cells such as beta cells, or hypoimmunogenic cells thereof) contains a transgene that encodes CCL21, such as human CCL21, or a functional fragment or variant thereof. In some embodiments, CCL21 is overexpressed in the cell. In some embodiments, the expression of CCL21 is increased in the engineered cell or population of cells compared to a similar reference or non-engineered cell (including with any other modifications) except that the reference or non-engineered cell does not include the transgene encoding CCL21. Useful genomic, polynucleotide and polypeptide information about human CCL21 are provided in, for example, the GeneCard Identifier GC09M034709, HGNC No. 10620, NCBI Gene ID 6366, Uniprot No. 000585, and NCBI RefSeq Nos. NP_002980.1 and NM_002989.3. In certain embodiments, the transgene encoding CCL21, or a functional fragment or variant thereof, is operably linked to a promoter.

[0379] In some embodiments, the engineered cell or population of cells (e.g., T cells, B cells, NK cells, islet cells such as beta cells, or hypoimmunogenic cells thereof) contains a transgene that encodes CCL22, such as human CCL22, or a functional fragment or variant thereof. In some embodiments, CCL22 is overexpressed in the cell. In some embodiments, the expression of CCL22 is increased in the engineered cell or population of cells compared to a similar reference or non-engineered cell (including with any other modifications) except that the reference or non-engineered cell does not include the transgene encoding CCL22. Useful genomic, polynucleotide and polypeptide information about human CCL22 are provided in, for example, the GeneCard Identifier GC16P057359, HGNC No. 10621, NCBI Gene ID 6367, Uniprot No. 000626, and NCBI RefSeq Nos. NP_002981.2, NM_002990.4, XP_016879020.1, and XM_017023531.1. In certain embodiments, the transgene encoding CCL22, or a functional fragment or variant thereof, is operably linked to a promoter.

[0380] In some embodiments, the engineered cell or population of cells (e.g., T cells, B cells, NK cells, islet cells such as beta cells, or hypoimmunogenic cells thereof) contains a transgene that encodes Mfge8, such as human Mfge8, or a functional fragment or variant thereof. In some embodiments, Mfge8 is overexpressed in the cell. In some embodiments, the expression of Mfge8 is increased in the engineered cell or population of cells compared to a similar reference or non-engineered cell or population of cells (including with any other modifications) except that the reference or non-engineered cell or population of cells does not include the transgene encoding Mfge8. Useful genomic, polynucleotide and polypeptide information about human Mfge8 are provided in, for example, the GeneCard Identifier GC15M088898, HGNC No. 7036, NCBI Gene ID 4240, Uniprot No. Q08431, and NCBI RefSeq Nos. NP_001108086.1, NM_001114614.2, NP_001297248.1, NM_001310319.1, NP_001297249.1, NM_001310320.1, NP_001297250.1, NM_001310321.1, NP_005919.2, and NM_005928.3. In certain embodiments, the transgene encoding Mfge8, or a functional fragment or variant thereof , is operably linked to a promoter.

[0381] In some embodiments, the engineered cell or population of cells (e.g., T cells, B cells, NK cells, islet cells such as beta cells, or hypoimmunogenic cells thereof) contains a transgene that encodes SerpinB9, such as human SerpinB9, or a functional fragment or variant thereof. In some embodiments, SerpinB9 is overexpressed in the cell. In some embodiments, the expression of SerpinB9 is increased in the engineered cell or population of cells compared to a similar reference or non-engineered cell or population of cells (including with any other modifications) except that the reference or non-engineered cell or population of cells does not include the transgene encoding SerpinB9. Useful genomic, polynucleotide and polypeptide information about human SerpinB9 are provided in, for example, the GeneCard Identifier GC06M002887, HGNC No. 8955, NCBI Gene ID 5272, Uniprot No. P50453, and NCBI RefSeq Nos. NP_004146.1, NM_004155.5, XP_005249241.1, and XM_005249184.4. In certain embodiments, the transgene encoding SerpinB9, or a functional fragment or variant thereof, is operably linked to a promoter.

Hi. Antibodies

[0382] In some embodiments, the one or more transgenes, e.g., the first and/or the second transgene, encode an antibody.

[0383] In some embodiments, the encoded antibody is a therapeutic antibody, such as an antibody that binds to any of CD47, Sirpa, CD52, amyloid beta, angiopoietin-like protein 3 (ANGPTL3), B cell activating factor (BAFF), A proliferation-inducing ligand (APRIL), B cell maturation antigen (BCMA), B. anthracis protective antigen (B. anthracis PA), calcitonin gene- related peptide (CGRP), calcitonin gene-related peptide receptor (CGRP-R), C-C chemokine receptor type 4 (CCR4), CD147, CD19, CD3, CD2, CD20, CD22, CD25, epithelial cell adhesion molecule (EpCAM), Glycoprotein 100 (GP100), CD30, CD33, CD38, CD4, CD52, CD6, CD79b, CD80, CD86, Clostridium difficile Toxin B, coagulation factor IX (Factor IX), coagulation factor X (Factor X), complement Cis (Cis), complement C5 (C5), cytotoxic T- lymphocyte antigen 4 (CTLA4), dabigatran, DNA/Histone Hl, Ebola virus Glycoprotein, epidermal growth factor receptor (EGFR), C-Met, epithelial cell adhesion molecule (EpCAM), Factor IX, Factor VIII, fibroblast growth factor 23 (FGF23), ganglioside GM3 (GM3), GD2 ganglioside (GD2), human epidermal growth factor receptor 2 (HER2), immunoglobulin E (IgE), insulin-like growth factor 1 receptor (IGF-1R), integrin alpha lib beta 3 (Integrin allbp3), integrin alpha4 beta7 (Integrin a4p7), integrin subunit alpha 4 (ITGA4), integrin subunit alpha L (ITGAL), interferon alpha receptor 1 (IFNAR-1), interferon gamma (IFN-y), interleukin 1 alpha (IL-la), interleukin 1 beta (IL-1 P), interleukin 12, (IL-12), interleukin 23 (IL- 23), interleukin 13 (IL-13), interleukin 17 receptor alpha (IL-17 RA), interleukin 17A (IL-17A), interleukin 17F (IL-17F), interleukin 23 pl9 (IL-23pl9), interleukin 31 receptor subunit alpha (IL-31RA), interleukin 4 receptor alpha (IL-4RA), interleukin 5 (IL-5), interleukin 5 receptor subunit alpha (IL-5RA), interleukin 6 (IL-6), interleukin 6 receptor (IL-6R), interleukin 8 (IL-8), Kallikrein, Lipid A region of endotoxin, lymphocyte activation gene 3 (LAG-3), programmed cell death 1 (PD-1), nectin cell adhesion molecule 4 (Nectin 4), neonatal Fc Receptor (FcRn), platelet- derived growth factor receptor alpha (PDGFRA), programmed cell death 1 ligand 1 (PD-L1), proprotein convertase subtilisin/kexin Type 9 (PCSK9), P-selectin, rabies virus glycoprotein (Rabies virus GP), receptor activator of nuclear factor kappaB ligand (RANKL), respiratory syncytial virus F protein (RSV-F), SARS-Cov-2 spike protein (SARS-Cov-2 S protein), sclerostin, SLAM family member 7 (SLAMF7), thrombopoietin receptor (TPOR), thymic stromal lymphopoietin (TSLP), tissue factor (TF), transferrin receptor protein 1 (TFR1), transforming growth factor-beta (TGF-P), tumor necrosis factor-alpha (TNF-a), tumor-associated calcium signal transducer 2 (TACSTD-2), vascular endothelial growth factor (VEGF), vascular endothelial growth factor A (VEGF-A), angiopoietin 2 (ANG-2), vascular endothelial growth factor receptor 2 (VEGFR-2), von Willebrand Factor (vWF), or any combination thereof. iv. Fusogens and Viral Vectors

[0384] In some embodiments, the one or more transgenes, e.g., the first and/or the second transgene, comprise a sequence encoding one or more fusogens.

[0385] Fusogens can promote mixing between lipids in a particle, such as a virus particle or virus-like particle (VLP), and lipids in a target cell. Thus, fusogens may facilitate the fusion of the particle, such as a virus particle or a VLP, to a membrane of the target cell. In some embodiments, the particle, such as a virus particle or a VLP, integrates into the membrane of the target cell following fusion, e.g., facilitated by a fusogen. In some embodiments, the membrane is the plasma membrane of the target cell. In some embodiments, fusogens promote formation of one or more pores between the interior of the particle, e.g., the viral particle or VLP, and the cytosol of the target cell.

[0386] In some embodiments, a fusogen of the disclosure contains a mammalian protein, such as any suitable mammalian protein known in the art or described herein. In some embodiments, the fusogen contains a viral protein, such as any suitable viral protein known in the art or described herein. In some embodiments, the one or more fusogens are selected from NiV-F, NiV-G, Gag-Pol, and Rev.

[0387] In some embodiments, the one or more transgenes comprise sequence(s) encoding viral or virus-like vectors or particles, such as a retroviral vector or particle (e.g., a lentiviral vector or particle), an AAV vector or particle, or a virus-like particle. In some embodiments, the one or more transgenes comprise both sequence(s) encoding viral or virus-like vectors or particles and a fusogen.

[0388] In some embodiments, a fusogen of the disclosure is or contains a mammalian protein. Exemplary mammalian fusogens include a SNARE family protein such as vSNAREs or tSNAREs, a syncytin protein such as Syncytin-1 (DOI: 10.1128/JVI.76.13.6442-6452.2002) and Syncytin-2, myomaker (biorxiv.org/content/early/2017/04/02/123158, doi.org/10.1101/123158, doi: 10.1096/fj.201600945R, doi:10.1038/naturel2343), myomixer (www.nature.com/nature/joumal/v499/n7458/full/naturel2343.html, doi: 10.1038/naturel2343), myomerger (science. sciencemag.org/content/early/2017/04/05/science.aam9361 , DOI: 10.1126/science.aam9361), FGFRL1 (fibroblast growth factor receptor-like 1), Minion (doi.org/10.1101/122697), an isoform of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (e.g., as disclosed in US 6,099,857A), a gap junction protein such as connexin 43, connexin 40, connexin 45, connexin 32, or connexin 37 (e.g., as disclosed in US 2007/0224176), Hap2, any protein capable of inducing syncytium formation between heterologous cells, any protein with fusogenic properties, a homologue thereof, a fragment thereof, a variant thereof, and a protein fusion containing one or more proteins or fragments thereof, such as one or more of any of the foregoing. In some embodiments, the fusogen is encoded by a human endogenous retroviral element (hERV) found in the human genome. Additional exemplary fusogens are disclosed in US 6,099,857A and US 2007/0224176, the entire contents of each of which are hereby incorporated by reference.

[0389] In some embodiments, a fusogen of the disclosure is or contains a non-mammalian protein. In some embodiments, the fusogen is or contains a viral protein. In some embodiments, the fusogen is a viral fusion protein. In some embodiments, the fusogen is a viral envelope protein. In some embodiments, a viral fusogen is a Class I viral membrane fusion protein, a Class II viral membrane protein, a Class III viral membrane fusion protein, a viral membrane glycoprotein, or other viral fusion proteins, or a homologue thereof, a fragment thereof, a variant thereof, or a protein fusion containing one or more proteins or fragments thereof, such as one or more of any of the foregoing.

[0390] In some embodiments, Class I viral membrane fusion proteins include Baculovirus F protein, e.g., F proteins of the nucleopolyhedrovirus (NPV) genera, e.g., Spodoptera exigua MNPV (SeMNPV) F protein and Fymantria dispar MNPV (FdMNPV), and paramyxovirus F proteins. In some embodiments, Class II viral membrane proteins include tick bone encephalitis E (TBEV E) and Semliki Forest Virus E1/E2. In some embodiments, Class III viral membrane fusion proteins include rhabdovirus G (e.g., fusogenic protein G of the Vesicular Stomatatis Virus (VSV-G), Cocal virus G protein), herpesvirus glycoprotein B (e.g., Herpes Simplex virus 1 (HSV-1) gB), Epstein Barr Virus glycoprotein B (EBV gB), thogotovirus G, baculovirus gp64 (e.g., Autographa California multiple NPV (AcMNPV) gp64), and Boma disease virus (BDV) glycoprotein (BDV G).

[0391] Examples of other viral proteins that may find use in the fusogens of the present disclosure include other membrane glycoproteins and viral fusion proteins; viral syncytia proteins such as influenza hemagglutinin (HA) or mutants, or fusion proteins thereof; human immunodeficiency virus type 1 envelope protein (HIV-1 ENV), gpl20 from HIV binding LFA-1 to form lymphocyte syncytium, HIV gp41, HIV gp!60, or HIV Trans-Activator of Transcription (TAT); viral glycoprotein VSV-G, viral glycoprotein from vesicular stomatitis virus of the Rhabdoviridae family; glycoproteins gB and gH-gL of the varicella-zoster virus (VZV); murine leukaemia virus (MLV)-lOAl; Gibbon Ape Leukemia Virus glycoprotein (GaLV); type G glycoproteins in Rabies, Mokola, vesicular stomatitis virus and Togaviruses; murine hepatitis virus JHM surface projection protein; porcine respiratory coronavirus spike- and membrane glycoproteins; avian infectious bronchitis spike glycoprotein and its precursor; bovine enteric coronavirus spike protein; the F and H, HN or G genes of a Morbillivirus (e.g., measles virus (MeV), canine distemper virus, Cetacean morbillivirus, Peste-des-petits-ruminants virus, Phocine distemper virus, Rinderpest virus), Newcastle disease virus, human parainfluenza virus 3, simian virus 41, Sendai virus and human respiratory syncytial virus; gH of human herpesvirus 1 and simian varicella virus, with the chaperone protein gL; human, bovine and cercopithicine herpesvirus gB; envelope glycoproteins of Friend murine leukaemia virus and Mason Pfizer monkey virus; mumps virus hemagglutinin neuraminidase, and glycoproteins Fl and F2; membrane glycoproteins from Venezuelan equine encephalomyelitis; paramyxovirus F protein; SIV gpl60 protein; Ebola virus G protein; or Sendai virus fusion protein, or a homologue thereof, a fragment thereof, a variant thereof, and a protein fusion containing one or more proteins or fragments thereof, such as one or more of any of the foregoing. Viral fusogens can also be categorized as class I fusogens, class II fusogens, class III fusogens, and class IV fusogens. In some embodiments, class I fusogens such as human immunodeficiency virus (HIV) gp41 have a characteristic post- fusion conformation with a signature trimer of a-helical hairpins with a central coiled-coil structure. Class I viral fusion proteins include proteins having a central post-fusion six-helix bundle. Class I viral fusion proteins include influenza HA, parainfluenza F, HIV Env, Ebola GP, hemagglutinins from orthomyxoviruses, F proteins from paramyxoviruses (e.g., Measles, (Katoh et al. BMC Biotechnology 2010, 10:37)), ENV proteins from retroviruses, and fusogens of filoviruses and coronaviruses. In some embodiments, class II viral fusogens such as dengue E glycoprotein, have a structural signature of P- sheets forming an elongated ectodomain that refolds to result in a trimer of hairpins. In some embodiments, the class II viral fusogen lacks the central coiled coil. Class II viral fusogen can be found in alphaviruses (e.g., El protein) and flaviviruses (e.g., E glycoproteins). Class II viral fusogens include fusogens from Semliki Forest virus, Sinbis, rubella virus, and dengue virus. In some embodiments, class III viral fusogens such as the vesicular stomatitis virus G glycoprotein, combine structural signatures found in classes I and II. In some embodiments, a class III viral fusogen comprises a helices (e.g., forming a six-helix bundle to fold back the protein as with class I viral fusogens), and P sheets with an amphiphilic fusion peptide at its end, reminiscent of class II viral fusogens. Class III viral fusogens can be found in rhabdoviruses and herpesviruses. In some embodiments, class IV viral fusogens are fusion-associated small transmembrane (FAST) proteins (doi:10.1038/sj.emboj.7600767, Nesbitt, Rae L., "Targeted Intracellular Therapeutic Delivery Using Liposomes Formulated with Multifunctional FAST proteins" (2012). Electronic Thesis and Dissertation Repository. Paper 388), which are encoded by nonenveloped reoviruses. In some embodiments, the class IV viral fusogens are sufficiently small that they do not form hairpins (doi: 10.1146/annurev-cellbio-101512-122422, doi:10.1016/j.devcel.2007.12.008). [0392] In some some embodiments, the fusogen is a Baboon Endogenous Retrovirus (BaEV) envelope glycoprotein. Exemplary BaEV envelope glycoproteins and variants thereof are described in PCT/US2022/031459; US9249426; Aguila et al., Journal of Virology 2003 77(2):1281-1291; Bernadin et al., Blood Advances 2019 3(3):461-475; Colamartino et al., Frontiers in Immunology 2019 10:2873; Girard-Gagnepain et al., Blood 2014 124(8): 1221- 1231; and Levy et al., Journal of Thrombosis and Haemostasis 2016 14:2478-2492. Wild-type BaEV envelope glycoproteins are retroviral envelope proteins containing a C-terminal cytoplasmic tail, a transmembrane domain, and an extracellular domain. Maturation of the precursor protein in the Golgi results in two subunits, the surface unit protein or gp70, and the transmembrane protein p20E. The surface unit protein or gp70 and the transmembrane protein p20E remain associated in a labile interaction that may include a disulfide bond. In wild-type BaEV envelope glycoproteins, fusogenicity is controlled by a short, fusion inhibitory R peptide, which is localized on the C- terminal of the cytoplasmic tail domain. The fusion inhibitory R peptide harbors the tyrosine endocytosis signal YXXL, and its cleavage by the viral protease is thought to potentiate fusogenic activation through molecular rearrangements in the membrane-spanning domain and the extracellular region of the envelope glycoprotein (S alamango et al., (2015) Journal of virology 89(24): 12492-12500). In wild-type BaEV envelope glycoproteins, the gp70 mediates receptor binding to the ASCT-2 and ASCT-1 receptors on host cells. In some embodiments, the glycoprotein 70 (g70) subunit or a biologically active portion thereof binds the ASCT-2 and ASCT-1 receptors. In wild-type BaEV envelope glycoproteins, the p20E acts as a class I viral fusion protein. The interaction of the gp70 subunit with a host cell membrane triggers refolding of the p20E and is believed to activate the fusogenic potential by unmasking the fusion peptide. In some embodiments, the fusogen is a truncated BaEV envelope glycoprotein. Exemplary BaEV envelope glycoproteins and truncates thereof are described in PCT/US2022/031459, which is incorporated herein by reference in its entirety. In some embodiments, the fusogen is a modified BaEV envelope glycoprotein. In some embodiments, the cytoplasmic tail domain of the BaEV envelope glycoprotein is devoid of the fusion inhibitory R peptide. The fusion inhibitory R peptide of the BaEV envelope glycoprotein is typically located between amino acids 547 and 564 of the wild-type BaEV envelope glycoprotein. In some embodiments, the fusogen is a mutant of a BaEV envelope glycoprotein which is at least 80%, preferably at least 85%, still preferably at least 90%, more preferably at least 95%, still more preferably at least 99% identical to said wild-type BaEV envelope glycoprotein, provided that said mutant glycoprotein retains the fusogenic potential of the wild-type envelope glycoprotein. In some embodiments, the cytoplasmic tail domain of the BaEV envelope glycoprotein is replaced by the cytoplasmic tail domain of a murine leukemia virus (MLV) envelope glycoprotein. The Murine Leukemia Virus envelope glycoprotein is notably described in Ott et al., (1990) J. Virol. 64:757-766. In some embodiments, the Murine Leukemia Virus envelope glycoprotein is that of strain 4070A. The term “MLV envelope glycoprotein” refers to the wild-type form of the MLV envelope glycoprotein or to a mutant of said wild-type MLV envelope glycoprotein which is at least 80%, preferably at least 85%, still preferably at least 90%, more preferably at least 95%, still more preferably at least 99% identical to said wild-type MLV envelope glycoprotein, provided that said mutant glycoprotein retains the capacity of the wild-type envelope glycoprotein of interacting with viral core proteins, in particular with lentiviral core proteins. Typically, the cytoplasmic tail domain of the MLV envelope glycoprotein is located between amino acids 622 and 654 of the wild-type MLV envelope glycoprotein.

[0393] In some embodiments, the fusogen is or contains a G or H protein. In some embodiments, the G or H protein is a Paramyxovirus (e.g., Morbillivirus or Henipavirus) G or H protein or a biologically active portion thereof. In some embodiments, the Henipavirus G protein is a Hendra (HeV) virus G protein, a Nipah (NiV) virus G-protein (NiV-G), a Cedar (CedPV) virus G-protein, a Mojiang virus G-protein, a bat Paramyxovirus G-protein, a Kumasi virus G- protein, a Langya virus G-protein, or a biologically active portion thereof. The Henipavirus attachment G proteins are type II transmembrane glycoproteins containing an N-terminal cytoplasmic tail, a transmembrane domain, and an extracellular domain containing an extracellular stalk, and a globular head. The N-terminal cytoplasmic domain is within the inner lumen of the lipid bilayer and the C-terminal portion is the extracellular domain that is exposed on the outside of the lipid bilayer. Regions of the stalk in the C-terminal region have been shown to be involved in interactions with F protein and triggering of F protein fusion (Liu et al., 2015 J of Virology 89:1838). In wild-type NiV-G protein, the globular head mediates receptor binding to henipavirus entry receptors ephrin B2 and ephrin B3 but is dispensable for membrane fusion (Brandel-Tretheway et al., Journal of Virology. 2019. 93(13)e00577-19). In particular embodiments herein, tropism of the G protein is modified. Binding of the G protein to a binding partner can trigger fusion mediated by a compatible F protein or biologically active portion thereof. G proteins are expressed as sequences including an N-terminal methionine required for start of translation. As such N-terminal methionines are commonly cleaved co- or post- translationally, the mature protein G proteins in some cases lack the N-terminal methionine. G glycoproteins are highly conserved between henipavirus species. For example, the G protein of NiV and HeV viruses share 79% amino acid identity. Studies have shown a high degree of compatibility among G proteins with F proteins of different species as demonstrated by heterotypic fusion activation (Brandel-Tretheway et al., Journal of Virology, 2019). As described herein, fusogens can contain heterologous proteins from different species. In some embodiments, the fusogen comprises a mutant of a wild-type G protein (e.g., any of Hendra (HeV) virus G protein, a Nipah (NiV) virus G-protein (NiV-G), a Cedar (CedPV) virus G-protein, a Mojiang virus G-protein, a bat Paramyxovirus G-protein, a Kumasi virus G-protein, or a Langya virus G- protein, or a biologically active portion thereof) having at least at or about 80%, at least at or about 81%, at least at or about 82%, at least at or about 83%, at least at or about 84%, at least at or about 85%, at least at or about 86%, at least at or about 87%, at least at or about 88%, at least at or about 89%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at least at or about 96%, at least at or about 97%, at least at or about 98%, or at least at or about 99% identity to the wildtype G protein, so long as the mutant G protein retains the fusogenic activity of the wild type G protein. In some embodiments, the mutant G protein is a functionally active variant or biologically active portion containing one or more amino acid mutations, such as one or more amino acid insertions, deletions, substitutions, or truncations. In some embodiments, the mutations described herein relate to amino acid insertions, deletions, substitutions, or truncations of amino acids compared to a reference G protein sequence. In some embodiments, the reference G protein sequence is the wild-type sequence of a G protein or a biologically active portion thereof. In some embodiments, the functionally active variant or the biologically active portion thereof is a mutant of a wild-type Hendra (HeV) virus G protein, a wild-type Nipah (NiV) virus G-protein (NiV-G), a wild-type Cedar (CedPV) virus G-protein, a wild-type Mojiang virus G- protein, a wild-type bat Paramyxovirus G-protein, a Kumasi virus G-protein, a Langya virus G- protein, or biologically active portion thereof. In some embodiments, the G protein is a mutant G protein that is a biologically active portion that is an N-terminally and/or C-terminally truncated fragment of a wild-type Hendra (HeV) virus G protein, a wild-type Nipah (NiV) virus G-protein (NiV-G), a wild-type Cedar (CedPV) virus G-protein, a wild-type Mojiang virus G-protein, a wild-type bat Paramyxovirus G-protein, a Kumasi virus G-protein, or a Langya virus G-protein. In particular embodiments, the truncation is an N-terminal truncation of all or a portion of the cytoplasmic domain. In some embodiments, the mutant G protein is a biologically active portion that is truncated and lacks up to 49 contiguous amino acid residues at or near the N-terminus of the wild-type G protein. In some embodiments, the mutant F protein is truncated and lacks up to 49 contiguous amino acids, such as up to 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 30, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 contiguous amino acids at the N-terminus of the wild-type G protein. In particular embodiments, the G protein or functionally active mutant or biologically active portion is a protein that retains fusogenic activity, e.g., in conjunction with a Henipavirus F protein, e.g., NiV-F or HeV-F. Fusogenic activity includes the activity of the G protein in conjunction with a Henipavirus F protein to promote or facilitate fusion of two membrane lumens, such as the lumen of a lipid particle, e.g., a viral particle or VLP, having embedded in its lipid bilayer a henipavirus F and G protein, and a cytoplasm of a target cell, e.g., a cell that contains a surface receptor or molecule that is recognized or bound by the targeted envelope protein. In some embodiments, the F protein and G protein are from the same Henipavirus species (e.g., NiV-G and NiV-F). In some embodiments, the F protein and G protein are from different Henipavirus species e.g., NiV-G and HeV-F). In some embodiments, retaining fusogenic activity includes activity (in conjunction with a Henipavirus F protein) that is between at or about 10% and at or about 150% or more of the level or degree of binding of the corresponding wild-type G protein, such as at or at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or 120% of the level or degree of fusogenic activity of the corresponding wild-type G protein.

[0394] In some specific embodiments, the G protein is a wild-type Nipah virus G (NiV-G) protein or a Hendra virus G protein or is a functionally active variant or biologically active portion thereof. In some embodiments, the G protein is a mutant NiV-G protein that is a biologically active portion of a wild-type NiV-G. In some embodiments, the biologically active portion is an N-terminally truncated fragment. In some embodiments, the mutant NiV-G protein is truncated and lacks up to 5, up to 6, up to 7, up to 8, up to 9, up to 10, up to 11, up to 12, up to 13, up to 14, up to 15, up to 16, up to 17, up to 18, up to 19, up to 20, up to 21, up to 22, up to

23, up to 24, up to 25, up to 26, up to 27, up to 28, up to 29, up to 30, up to 31, up to 32, up to

33, up to 34, up to 35, up to 36, up to 37, up to 38, up to 39, up to 40, up to 41, up to 42, up to

43, up to 44, up to 45, up to 46, up to 47, up to 48, up to 49, or up to 50, contiguous amino acid residues at or near the N-terminus of the wild-type NiV-G protein. In some embodiments, the NiV-G protein is a biologically active portion that does not contain a cytoplasmic domain. [0395] In some specific embodiments, the G protein is a wild type HeV-G protein or a mutant HeV-G protein that is a biologically active portion of a wild-type HeV-G. In some embodiments, the biologically active portion is an N-terminally truncated fragment. In some embodiments, the biologically active portion is an N-terminally truncated fragment. In some embodiments, the mutant HeV-G protein is truncated and lacks up to 5, up to 6, up to 7, up to 8, up to 9, up to 10, up to 11, up to 12, up to 13, up to 14, up to 15, up to 16, up to 17, up to 18, up to 19, up to 20, up to 21, up to 22, up to 23, up to 24, up to 25, up to 26, up to 27, up to 28, up to 29, up to 30, up to 31, up to 32, up to 33, up to 34, up to 35, up to 36, up to 37, up to 38, up to 39, up to 40, up to 41, up to 42, up to 43, up to 44, or up to 45 contiguous amino acid residues at or near the N-terminus of the wild-type HeV-G protein. In some embodiments, the HeV-G protein is a biologically active portion that does not contain a cytoplasmic domain. In some embodiments, the mutant HeV-G protein lacks the N-terminal cytoplasmic domain of the wildtype HeV-G protein.

[0396] In some embodiments, the G protein or the functionally active variant or biologically active portion thereof binds to Ephrin B2 or Ephrin B3. In some embodiments, the functionally active variant or biologically active portion thereof retains binding to Ephrin B2 or B3, e.g., that is at least or at least about any of: 5% of the level or degree of binding of the corresponding wild-type G protein, 10% of the level or degree of binding of the corresponding wild-type G protein, 15% of the level or degree of binding of the corresponding wild-type G protein, 20% of the level or degree of binding of the corresponding wild-type G protein, 25% of the level or degree of binding of the corresponding wild-type G protein, 30% of the level or degree of binding of the corresponding wild-type G protein, 35% of the level or degree of binding of the corresponding wild-type G protein, 40% of the level or degree of binding of the corresponding wild-type G protein, 45% of the level or degree of binding of the corresponding wild-type G protein, 50% of the level or degree of binding of the corresponding wild-type G protein, 55% of the level or degree of binding of the corresponding wild-type G protein, 60% of the level or degree of binding of the corresponding wild- type G protein, 65% of the level or degree of binding of the corresponding wild-type G protein, 70% of the level or degree of binding of the corresponding wild- type G protein, 75% of the level or degree of binding of the corresponding wild-type G protein, 80% of the level or degree of binding of the corresponding wild-type G protein, 85% of the level or degree of binding of the corresponding wild-type G protein, 90% of the level or degree of binding of the corresponding wild-type G protein, or 95% of the level or degree of binding of the corresponding wild-type protein or a functionally active variant or biologically active portion thereof. In some embodiments, the G protein is NiV-G or a functionally active variant or biologically active portion thereof and binds to Ephrin B2 or Ephrin B3.

[0397] In some embodiments, the G protein or functionally active variant or biologically active portion thereof exhibits reduced binding for the native binding partner of a wild-type G protein. In some embodiments, the mutant G protein or the biologically active portion thereof is a mutant of wild-type Niv-G and exhibits reduced binding to one or both of the native binding partners Ephrin B2 or Ephrin B3. In some embodiments, the mutant G-protein or the biologically active portion, such as a mutant NiV-G protein, exhibits reduced binding to the native binding partner. In some embodiments, the reduced binding to Ephrin B2 or Ephrin B3 is reduced by greater than at or about 5%, at or about 10%, at or about 15%, at or about 20%, at or about 25%, at or about 30%, at or about 40%, at or about 50%, at or about 60%, at or about 70%, at or about 80%, at or about 90%, or at or about 100%. In some embodiments, the G protein contains one or more amino acid substitutions in a residue that is involved in the interaction with one or both of Ephrin B2 and Ephrin B3. In some embodiments, the amino acid substitutions correspond to mutations E501A, W504A, Q530A and E533A.

[0398] In some embodiments, the G protein mutations described herein can improve transduction efficiency. In some embodiments, the mutations described herein allow for specific targeting of other desired cell types that are not Ephrin B2 or Ephrin B3. In some embodiments, the mutations described herein result in at least the partial inability to bind at least one natural receptor, such as reduce the binding to at least one of Ephrin B2 or Ephrin B3. In some embodiments, the mutations described herein interfere with natural receptor recognition.

[0399] In some embodiments, the fusogen contains a protein with a hydrophobic fusion peptide domain. In some embodiments, the fusogen is or contains an F protein. In some embodiments, the fusogen contains a henipavirus F protein molecule or biologically active portion thereof. In some embodiments, the Henipavirus F protein is a Hendra (Hev) virus F protein, a Nipah (NiV) virus F-protein, a Cedar (CedPV) virus F protein, a Mojiang virus F protein, a bat Paramyxovirus F protein, a Kumasi virus F protein, a Eangya virus F protein, or a biologically active portion thereof. In some embodiments, the N-terminal hydrophobic fusion peptide domain of the F protein molecule or biologically active portion thereof is exposed on the outside of a lipid bilayer. F proteins of henipaviruses are encoded as F0 precursors containing a signal peptide. Following cleavage of the signal peptide, the mature F0 is transported to the cell surface, then endocytosed and cleaved by cathepsin E into the mature fusogenic subunits Fl and F2. The Fl and F2 subunits are associated by a disulfide bond and recycled back to the cell surface. The Fl subunit contains the fusion peptide domain located at the N terminus of the Fl subunit, where it is able to insert into a cell membrane to drive fusion. In some aspects, fusion is blocked by association of the F protein with G protein, until the G protein engages with a target molecule resulting in its disassociation from F and exposure of the fusion peptide to mediate membrane fusion. Among different henipavirus species, the sequence and activity of the F protein is highly conserved. For example, the F protein of NiV and HeV viruses share 89% amino acid sequence identity. Further, in some cases, the henipavirus F proteins exhibit compatibility with G proteins from other species to trigger fusion (Brandel-Tretheway et al., Journal of Virology. 2019. 93(13):e00577-19). In some aspects, the F protein is heterologous to the G protein, i.e., the F and G protein or biologically active portions are from different henipavirus species. For example, the F protein is from Hendra virus and the G protein is from Nipah virus. In other aspects, the F protein can be a chimeric F protein containing regions of F proteins from different species of Henipavirus. In some embodiments, switching a region of amino acid residues of the F protein from one species of Henipavirus to another can result in fusion to the G protein of the species comprising the amino acid insertion. (Brandel-Tretheway et al., Journal of Virology. 2019. 93(13):e00577-19). In some cases, the chimeric F protein contains an extracellular domain from one henipavirus species and a transmembrane and/or cytoplasmic domain from a different henipavirus species. For example, the F protein contains an extracellular domain of Hendra virus and a transmembrane/cytoplasmic domain of Nipah virus. F proteins are expressed as sequences including an N-terminal signal sequence. As such N- terminal signal sequences are commonly cleaved co- or post-translationally, the mature F protein can lack the N-terminal signal sequence.

[0400] In some embodiments, the fusogen comprises a mutant of a wild- type F protein (e.g., any Henipavirus F protein such as a Hendra (Hev) virus F protein, a Nipah (NiV) virus F- protein, a Cedar (CedPV) virus F protein, a Mojiang virus F protein, a bat Paramyxovirus F protein, a Kumasi virus F protein, or a Langya virus F protein) having at least at or about 80%, at least at or about 81%, at least at or about 82%, at least at or about 83%, at least at or about 84%, at least at or about 85%, at least at or about 86%, at least at or about 87%, at least at or about 88%, at least at or about 89%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at least at or about 96%, at least at or about 97%, at least at or about 98%, or at least at or about 99% identity to the wild-type F protein, so long as the mutant F protein retains the fusogenic activity of the wild type F protein. In particular embodiments, the F protein or the functionally active variant or biologically active portion thereof retains fusogenic activity in conjunction with a Henipavirus G protein, such as a G protein set forth above (e.g., NiV-G or HeV-G). Fusogenic activity includes the activity of the F protein in conjunction with a G protein to promote or facilitate fusion of two membrane lumens, such as the lumen of a lipid particle, e.g., a VLP, having embedded in its lipid bilayer a henipavirus F and G protein, and a cytoplasm of a target cell, e.g., a cell that contains a surface receptor or molecule that is recognized or bound by the targeted envelope protein. In some embodiments, the F protein and G protein are from the same Henipavirus species (e.g., NiV-G and NiV-F). In some embodiments, the F protein and G protein are from different Henipavirus species (e.g., NiV-G and HeV-F). In particular embodiments, the F protein or the functionally active variant or biologically active portion retains the cleavage site cleaved by cathepsin L. Reference to retaining fusogenic activity includes activity (in conjunction with a Henipavirus G protein) that is between at or about 10% and at or about 150% or more of the level or degree of binding of the corresponding wild-type F protein, such as at or at least about 10% , 15% , 20% , 25% , 30% , 35% , 40% , 45% , 50% , 55% , 60% , 65% , 70% , 75% , 80% , 85% , 90% , 95%, 100%, or 120% of the level or degree of fusogenic activity of the corresponding wild-type F protein. In some embodiments, the F protein is a mutant F protein that is a functionally active fragment or a biologically active portion containing one or more amino acid mutations, such as one or more amino acid insertions, deletions, substitutions, or truncations. In some embodiments, the mutations described herein relate to amino acid insertions, deletions, substitutions, or truncations of amino acids compared to a reference F protein sequence. In some embodiments, the reference F protein sequence is the wild-type sequence of an F protein or a biologically active portion thereof. In some embodiments, the mutant F protein or the biologically active portion thereof is a mutant of a wild-type Hendra (Hev) virus F protein, a Nipah (NiV) virus F-protein, a Cedar (CedPV) virus F protein, a Mojiang virus F protein, a bat Paramyxovirus F protein, a Kumasi virus F protein, or a Langya virus F protein. In some embodiments, the mutant F protein is a biologically active portion of a wild-type F protein that is an N-terminally and/or C-terminally truncated fragment. In some embodiments, the mutant F protein or the biologically active portion of a wild-type F protein thereof comprises one or more amino acid substitutions. In some embodiments, the mutations described herein can improve transduction efficiency. In some embodiments, the mutations described herein can increase fusogenic capacity. Exemplary mutations include any as described, see, e.g., Khetawat and Broder 2010 Virology Journal 7:312; Witting et al., 2013 Gene Therapy 20:997-1005; published international; patent application No. WO/2013/148327. In some embodiments, the mutant F protein is a biologically active portion that is truncated and lacks up to 20 contiguous amino acid residues at or near the C-terminus of the wild-type F protein. In some embodiments, the mutant F protein is truncated and lacks up to 20 contiguous amino acids, such as up to 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 contiguous amino acids at the C-terminus of the wild-type F protein. In some embodiments, the mutant F protein is truncated and lacks up to 19 contiguous amino acids, such as up to 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 contiguous amino acids at the C-terminus of the wild-type F protein. In some embodiments, the F protein or the functionally active variant or biologically active portion thereof comprises an Fl subunit or a fusogenic portion thereof. In some embodiments, the Fl subunit is a proteolytically cleaved portion of the FO precursor. In some embodiments, the FO precursor is inactive. In some embodiments, the cleavage of the FO precursor forms a disulfide-linked F1+F2 heterodimer. In some embodiments, the cleavage exposes the fusion peptide and produces a mature F protein. In some embodiments, the cleavage occurs at or around a single basic residue. In some embodiments, the cleavage occurs at Arginine 109 of NiV-F protein. In some embodiments, cleavage occurs at Lysine 109 of the Hendra virus F protein.

[0401] In some specific embodiments, the F protein is a wild- type Nipah virus F (NiV-F) protein, or is a functionally active variant or biologically active portion thereof having at least at or about 80%, at least at or about 81%, at least at or about 82%, at least at or about 83%, at least at or about 84%, at least at or about 85%, at or about 86%, at least at or about 87%, at least at or about 88%, or at least at or about 89%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at or about 96%, at least at or about 97%, at least at or about 98%, or at least at or about 99% sequence identity to wild type NiV-F. In some examples, the F protein is cleaved into an Fl subunit c and an F2 subunit. In particular embodiments, the F protein or the functionally active variant or biologically active portion thereof retains the cleavage site cleaved by cathepsin L. In some embodiments, the F protein is a mutant NiV-F protein that is a biologically active portion thereof that is truncated and lacks up to 20 contiguous amino acid residues at or near the C- terminus of the wild-type NiV-F protein. In some embodiments, the F protein is a mutant NiV-F protein that is a biologically active portion thereof that comprises a 20 amino acid truncation at or near the C-terminus of the wild-type NiV-F protein; and a point mutation on an N-linked glycosylation site. In some embodiments, the F protein is a mutant NiV-F protein that is a biologically active portion thereof that comprises a 25 amino acid truncation at or near the C- terminus of the wild-type NiV-F protein. In some embodiments, the F protein is a mutant NiV-F protein that is a biologically active portion thereof that comprises a 22 amino acid truncation at or near the C-terminus of the wild-type NiV-F protein.

[0402] In some embodiments, a particle, e.g., a viral particle or VLP, is pseudotyped with a fusogen. In some embodiments, the fusogen is exposed on the surface of the particle, e.g., the viral particle or VLP. In some embodiments, the fusogen is exposed on the surface of the lipid bilayer of the particle, e.g., the viral particle or VLP. In some embodiments, a portion of the fusogen is embedded in the lipid bilayer of the particle, e.g., the viral particle or VLP. In some embodiments, the particle is a viral particle or a virus-like particle. In some embodiments, the fusogen is exposed on the surface of the viral envelope of the particle, e.g., the viral particle or VLP. In some embodiments, a portion of the fusogen is embedded in the viral envelope of the particle, e.g., the viral particle or VLP.

[0403] In some embodiments, a fusogen according to the present disclosure may be present at a copy number of at least, or no more than, 10, 50, 100, 500, 1,000, 2,000, 5,000, 10,000, 20,000, 50,000, 100,000, 200,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000, 100,000,000, 500,000,000, or 1,000,000,000 copies. In some embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the fusogen is disposed in the cell membrane. In some embodiments, the cell comprises the fusogen internally, e.g., in the cytoplasm or an organelle. In some embodiments, the fusogen comprises (or is identified as comprising) about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 5%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, or more, or about 1-30%, 5- 20%, 10-15%, 12- 15%, 13-14%, or 13.6% of the total protein in the cell or in a particle, e.g., a viral particle or VLP, e.g., as determined by a mass spectrometry assay. In embodiments, the fusogen comprises (or is identified as comprising) about 13.6% of the total protein in the cell. In some embodiments, the fusogen is (or is identified as being) more or less abundant than one or more additional proteins of interest. In an embodiment, the fusogen has (or is identified as having) a ratio to EGFP of about 140, 145, 150, 151, 152, 153, 154, 155, 156, 157 (e.g., 156.9), 158, 159, 160, 165, or 170. In another embodiment, the fusogen has (or is identified as having) a ratio to CD63 of about 2700, 2800, 2900, 2910 (e.g., 2912), 2920, 2930, 2940, 2950, 2960, 2970, 2980, 2990, or 3000, or about 1000-5000, 2000-4000, 2500-3500, 2900-2930, 2910-2915, or 2912.0, e.g., by a mass spectrometry assay. In an embodiment, the fusogen has (or is identified as having) a ratio to ARRDC1 of about 600, 610, 620, 630, 640, 650, 660 (e.g., 664.9), 670, 680, 690, or 700. In another embodiment, the fusogen has (or is identified as having) a ratio to GAPDH of about 50, 55, 60, 65, 70 (e.g., 69), 75, 80, or 85, or about 1-30%, 5-20%, 10-15%, 12-15%, 13-14%, or 13.6%. In another embodiment, the fusogen has (or is identified as having) a ratio to CNX of about 500, 510, 520, 530, 540, 550, 560 (e.g., 558.4), 570, 580, 590, or 600, or about 300-800, 400-700, 500-600, 520-590, 530-580, 540-570, 550-560, or 558.4, e.g., by a mass spectrometry assay.

[0404] In some embodiments, a fusogen according to the present disclosure is a protein fusogen. In some embodiments, the fusogen is a native protein or a derivative of a native protein. In some embodiments, the fusogen is a synthetic protein. In some embodiments, the fusogen is a mammalian protein or a homologue of a mammalian protein e.g., having 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity to the mammalian protein), such as any described herein. In some embodiments, the fusogen is a non-mammalian protein. In some embodiments, the fusogen is a viral protein or a homologue of a viral protein (e.g., having 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity to the viral protein), such as any described herein. In some embodiments, the fusogen is a fragment of any of the foregoing. In some embodiments, the fusogen is a variant of any of the foregoing. In some embodiments, the fusogen is a protein fusion containing one or more proteins or fragments thereof. In some embodiments, the fusogen is a protein fusion containing one or more of any of the foregoing.

[0405] In some embodiments, a fusogen according to the present disclosure is mutated to reduce binding for the native binding partner of the fusogen. In some embodiments the fusogen is randomly mutated. In some embodiments the fusogen is rationally mutated. In some embodiments the fusogen is subjected to directed evolution. In some embodiments the fusogen is truncated and only a subset of the peptide is inserted into the engineered cell or population of cells (e.g., T cells, B cells, NK cells, islet cells such as beta cells, or hypoimmunogenic cells thereof) described herein, such as inserted into a safe harbor genomic locus described herein. v. Safety Switches

[0406] In some embodiments, the one or more transgenes, e.g., the first and/or the second transgene, comprises a safety switch, also known as a “suicide gene” or “suicide switch.” A suicide gene or suicide switch can be incorporated to function as a “safety switch” that can cause the death of the cell, such as after the engineered cell or population of cells (e.g., T cells, B cells, NK cells, islet cells such as beta cells, or hypoimmunogenic cells thereof) is administered to a subject and if the cells should grow and divide in an undesired manner. The “suicide gene” ablation approach includes a suicide gene in a gene transfer vector encoding a protein that results in cell killing only when activated by a specific compound. A suicide gene may encode an enzyme that selectively converts a nontoxic compound into highly toxic metabolites. The result is specifically eliminating cells expressing the enzyme. Inclusion of a safety switch or suicide gene allows for controlled killing of the cells in the event of cytotoxicity or other negative consequences to the recipient, thus increasing the safety of cell-based therapies, including those using tolerogenic factors.

[0407] In some embodiments, a safety switch can be incorporated into, such as introduced, into an engineered cell or population of cells (e.g., T cells, B cells, NK cells, islet cells such as beta cells, or hypoimmunogenic cells thereof) provided herein to provide the ability to induce death or apoptosis of the engineered cell or population of cells containing the safety switch, for example if the cells grow and divide in an undesired manner or cause excessive toxicity to the host. Thus, the use of safety switches enables one to conditionally eliminate aberrant cells in vivo and can be a critical step for the application of cell therapies in the clinic. Safety switches and their uses thereof are described in, for example, Duzgune§, Origins of Suicide Gene Therapy (2019); Duzgune§ (eds), Suicide Gene Therapy. Methods in Molecular Biology, vol. 1895 (Humana Press, New York, NY) (for HSV-tk, cytosine deaminase, nitroreductase, purine nucleoside phosphorylase, and horseradish peroxidase); Zhou and Brenner, Exp Hematol 44(11): 1013-1019 (2016) (for iCaspase9); Wang et al., Blood 18(5): 1255-1263 (2001) (for huEGFR); U.S. Patent Application Publication No. 20180002397 (for HER1); and Philip et al., Bloodl24(8): 1277- 1287 (2014) (for RQR8).

[0408] In some embodiments, the safety switch can cause cell death in a controlled manner, for example, in the presence of a drug or prodrug or upon activation by a selective exogenous compound. In some embodiments, the safety switch is selected from the group consisting of herpes simplex virus thymidine kinase (HSV-tk), cytosine deaminase (CyD), nitroreductase (NTR), purine nucleoside phosphorylase (PNP), horseradish peroxidase, inducible caspase 9 (iCasp9), rapamycin-activated caspase 9 (rapaCasp9), CCR4, CD 16, CD 19, CD20, CD30, EGFR, GD2, HER1, HER2, MUC1, PSMA, and RQR8.

[0409] In some embodiments, the safety switch may be a transgene encoding a product with cell killing capabilities when activated by a drug or prodrug, for example, by turning a nontoxic prodrug to a toxic metabolite inside the cell. In these embodiments, cell killing is activated by contacting an engineered cell or population of cells (e.g., T cells, B cells, NK cells, islet cells such as beta cells, or hypoimmunogenic cells thereof) with the drug or prodrug. In some cases, the safety switch is HSV-tk, which converts ganciclovir (GCV) to GCV-triphosphate, thereby interfering with DNA synthesis and killing dividing cells. In some cases, the safety switch is CyD or a variant thereof, which converts the antifungal drug 5-fluorocytosine (5-FC) to cytotoxic 5-fluorouracil (5-FU) by catalyzing the hydrolytic deamination of cytosine into uracil. 5-FU is further converted to potent anti-metabolites (5- FdUMP, 5-FdUTP, 5-FUTP) by cellular enzymes. These compounds inhibit thymidylate synthase and the production of RNA and DNA, resulting in cell death. In some cases, the safety switch is NTR or a variant thereof, which can act on the prodrug CB 1954 via reduction of the nitro groups to reactive N-hydroxylamine intermediates that are toxic in proliferating and nonproliferating cells. In some cases, the safety switch is PNP or a variant thereof, which can turn prodrug 6-methylpurine deoxyriboside or fludarabine into toxic metabolites to both proliferating and nonproliferating cells. In some cases, the safety switch is horseradish peroxidase or a variant thereof, which can catalyze indole-3- acetic acid (IAA) to a potent cytotoxin and thus achieve cell killing. In other embodiments, the suicide gene is a cytosine deaminase (e.g., the Escherichia coli cytosine deaminase (EC-CD)) gene, and the trigger is 5-fluorocytosine (5-FC) (Barese et al., Mol. Therap. 20(10): 1932-1943 (2012) and Xu et al., Cell Res. 8:73-8 (1998), both incorporated herein by reference in their entirety). In some embodiments, the suicide gene or suicide switch and genes associated with the suicide gene or the safety switch are expressed from a bicistronic cassette. In some embodiments, the suicide gene or suicide switch and one or more tolerogenic factors are expressed from a bicistronic cassette.

[0410] In some embodiments, the safety switch may be an iCasp9. Caspase 9 is a component of the intrinsic mitochondrial apoptotic pathway which, under physiological conditions, is activated by the release of cytochrome C from damaged mitochondria. Activated caspase 9 then activates caspase 3, which triggers terminal effector molecules leading to apoptosis. The iCasp9 may be generated by fusing a truncated caspase 9 (without its physiological dimerization domain or caspase activation domain) to a FK506 binding protein (FKBP), FKBP12-F36V, via a peptide linker. The iCasp9 has low dimer-independent basal activity and can be stably expressed in host cells (e.g., human T cells) without impairing their phenotype, function, or antigen specificity. However, in the presence of chemical inducer of dimerization (CID), such as rimiducid (AP1903), AP20187, and rapamycin, iCasp9 can undergo inducible dimerization and activate the downstream caspase molecules, resulting in apoptosis of cells expressing the iCasp9. See, e.g., PCT Application Publication No. WO2011/146862; Stasi et al., N. Engl. J. Med. 365; 18 (2011); Tey et al., Biol. Blood Marrow Transplant 13:913-924 (2007). In particular, the rapamycin-inducible caspase 9 variant is called rapaCasp9. See Stavrou et al., Mai. Ther. 26(5): 1266- 1276 (2018). Thus, iCasp9 can be used as a safety switch to achieve controlled killing of the host cells.

[0411] In some embodiments, the safety switch may be a membrane-expressed protein, which allows for cell depletion after administration of a specific antibody to that protein. Safety switches of this category may include, for example, one or more transgene encoding CCR4, CD16, CD19, CD20, CD30, EGFR, GD2, HER1, HER2, MUC1, PSMA, or RQR8 for surface expression thereof. These proteins may have surface epitopes that can be targeted by specific antibodies. In some embodiments, the safety switch comprises CCR4, which can be recognized by an anti-CCR4 antibody. Non-limiting examples of suitable anti-CCR4 antibodies include mogamulizumab and biosimilars thereof. In some embodiments, the safety switch comprises CD 16 or CD30, which can be recognized by an anti-CD16 or anti-CD30 antibody. Non-limiting examples of such antiCD 16 or anti-CD30 antibody include AFM13 and biosimilars thereof. In some embodiments, the safety switch comprises CD 19, which can be recognized by an antiCD 19 antibody. Non-limiting examples of such anti-CD19 antibody include MOR208 and biosimilars thereof. In some embodiments, the safety switch comprises CD20, which can be recognized by an anti-CD20 antibody. Non-limiting examples of such anti-CD20 antibody include obinutuzumab, ublituximab, ocaratuzumab, rituximab, rituximab-Rllb, and biosimilars thereof. Cells that express the safety switch are thus CD20-positive and can be targeted for killing through administration of an anti-CD20 antibody as described. In some embodiments, the safety switch comprises EGFR, which can be recognized by an anti-EGFR antibody. Nonlimiting examples of such anti-EGFR antibody include tomuzotuximab, RO5083945 (GA201), cetuximab, and biosimilars thereof. In some embodiments, the safety switch comprises GD2, which can be recognized by an anti-GD2 antibody. Non-limiting examples of such anti-GD2 antibody include Hul4.18K322A, Hul4.18-IL2, Hu3F8, dinituximab, c.60C3-Rllc, and bio similars thereof.

[0412] In some embodiments, the safety switch may be an exogenously administered agent that recognizes one or more tolerogenic factors on the surface of the engineered cell or population of cells (e.g., T cells, B cells, NK cells, islet cells such as beta cells, or hypoimmunogenic cells thereof). In some embodiments, the exogenously administered agent is an antibody directed against or specific to a tolerogenic agent, e.g., an anti-CD47 antibody. By recognizing and blocking a tolerogenic factor on the engineered cell or population of cells, an exogenously administered antibody may block the immune inhibitory functions of the tolerogenic factor thereby re- sensitizing the immune system to the engineered cells or populations of cells. For instance, for an engineered cell or population of cells that overexpresses CD47, an exogenously administered anti-CD47 antibody may be administered to the subject, resulting in masking of CD47 on the engineered cell or population of cells and triggering of an immune response to the engineered cell or population of cells.

[0413] In some embodiments, the safety switch is a system wherein, upon activation, cells downregulate expression of one or more tolerogenic factors and/or upregulate expression of one or more immune signaling molecules, thereby marking the cell for elimination by the host immune system. In some embodiments, the one or more tolerogenic factors may be any tolerogenic factor known in the art or described herein, such as any of CD16, CD24, CD35, CD39, CD46, CD47, CD52, CD55, CD59, CD64, CD200, CCL22, CTLA4-Ig, Cl inhibitor, FASL, IDO1, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, IL- 10, IL-35, PD-L1, SERPINB9, CCL21, MFGE8, DUX4, B2M-HLA-E, CD27, IL-39, CD16 Fc Receptor, IL15-RF, H2-M3 (HLA-G), A20/TNFAIP3, CR1, HLA-F, MANF, or any combination thereof. In some embodiments, the one or more immune signaling molecules may be any suitable immune signaling molecule known in the art or described herein, such as any of B2M, HLA-A, HLA-B, HLA-C, HLA-D, HLA-E, RFXANK, CIITA, CTLA-4, PD-1, RAET1E/ULBP4, RAET1G/ULBP5, RAET1H/ULBP2, RAET1/ULBP1, RAET1L/ULBP6, RAET1N/ULBP3, ligands of NKG2D, or any combination thereof.

[0414] In some embodiments, the safety switch can include any of the strategies as described in WO2021146627A1, which is incorporated by reference in its entirety.

[0415] In some embodiments, a safety switch may be introduced into an engineered cell or population of cells (e.g., T cells, B cells, NK cells, islet cells such as beta cells, or hypoimmunogenic cells thereof) of the disclosure as part of an expression vector comprising, e.g., an inducible safety switch (e.g., suicide switch). iv. Genome-Editing Complexes

[0416] In some embodiments, the one or more transgenes, e.g., the first transgene and/or the second transgene, encodes a genome editing complex.

[0417] In some specific embodiments, the genome editing complex comprises a genome targeting entity and/or a genome modifying entity. In some embodiments, the genome targeting entity is a nucleic acid-guided targeting entity, such as any of a sequence specific nuclease, a nucleic acid programmable DNA binding protein, an RNA guided nuclease, RNA-guided nuclease comprising a Cas nuclease and a guide RNA (CRISPR-Cas combination), a ribonucleoprotein (RNP) complex comprising a gRNA and a Cas nuclease, a homing endonuclease, a zinc finger nuclease (ZF) nucleic acid binding entity, a transcription activatorlike effector (TALE) nucleic acid binding entity, a meganuclease, a Cas nuclease, a core Cas protein, a homing endonuclease, an endonuclease-deficient-Cas protein, an enzymatically inactive Cas protein, a CRIS PR-associated transposase (CAST), a Type II or Type V Cas protein, or a functional portion thereof. In some embodiments, the genome targeting entity is selected from the group consisting of Casl, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8a, Cas8b, Cas8c, Cas9, CaslO, Casl2, Casl2a (Cpfl), Casl2b (C2cl), Casl2c (C2c3), Casl2d (CasY), Casl2e (CasX), Casl2f (C2cl0), Casl2g, Casl2h, Casl2i, Casl2k (C2c5), Casl3, Casl3a (C2c2), Casl3b, Casl3c, Casl3d, C2c4, C2c8, C2c9, Cmrl, Cmr2, Cmr3, Cmr4, Cmr5, Cmr6, Csdl, Csd2, Cas5d, Csel, Cse2, Cse3, Cse4, Cas5e, Csfl, Csml, Csm2, Csm3, Csm4, Csm5, Csnl, Csn2, Cstl, Cst2, Cas5t, Cshl, Csh2, Cas5h, Csal, Csa2, Csa3, Csa4, Csa5, Cas5a, CsxlO, Csxl l, Csyl, Csy2, Csy3, Csy4, Mad7, SpCas9, eSpCas9, SpCas9-HFl, HypaSpCas9, HeFSpCas9, and evoSpCas9 high-fidelity variants of SpCas9, SaCas9, NmeCas9, CjCas9, StCas9, TdCas9, LbCasl2a, AsCasl2a, AacCasl2b, BhCasl2b v4, TnpB, dCas (D10A), dCas (H840A), dCasl3a, dCasl3b, or a functional portion thereof. In some embodiments, the genome modifying entity comprises one or more of the following genome modifying activities: cleaving, deaminating, nicking, polymerizing, interrogating, integrating, cutting, unwinding, breaking, altering, methylating, demethylating, or otherwise destabilizing a target sequence. In some embodiments, the genome modifying entity comprises a recombinase, integrase, transposase, endonuclease, exonuclease, nickase, helicase, DNA polymerase, RNA polymerase, reverse transcriptase, deaminase, flippase, methylase, demethylase, acetylase, a nucleic acid modifying protein, an RNA modifying protein, a DNA modifying protein, an Argonaute protein, an epigenetic modifying protein, a histone modifying protein, or a functional portion thereof. In some embodiments, the genome modifying entity is selected from a sequence specific nuclease, a nucleic acid programmable DNA binding protein, an RNA guided nuclease, RNA-guided nuclease comprising a Cas nuclease and a guide RNA (CRISPR-Cas combination), a ribonucleoprotein (RNP) complex comprising the gRNA and the Cas nuclease, a homing endonuclease, a zinc finger nuclease (ZFN), a transcription activator- like effector nuclease (TALEN), a meganuclease, a Cas nuclease, a core Cas protein, a TnpB nuclease, an endonuclease-deficient-Cas protein, an enzymatically inactive Cas protein, a CRISPR-associated transposase (CAST), a Type II or Type V Cas protein, base editing, prime editing, a Programmable Addition via Site-specific Targeting Elements (PASTE), or a functional portion thereof. In certain specific embodiments, the genome modifying entity is selected from Casl, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8a, Cas8b, Cas8c, Cas9, CaslO, Casl2, Casl2a (Cpfl), Casl2b (C2cl), Casl2c (C2c3), Casl2d (CasY), Casl2e (CasX), Casl2f (C2cl0), Casl2g, Casl2h, Casl2i, Casl2k (C2c5), Casl3, Casl3a (C2c2), Casl3b, Casl3c, Casl3d, C2c4, C2c8, C2c9, Cmrl, Cmr2, Cmr3, Cmr4, Cmr5, Cmr6, Csdl, Csd2, Cas5d, Csel, Cse2, Cse3, Cse4, Cas5e, Csfl, Csml, Csm2, Csm3, Csm4, Csm5, Csnl, Csn2, Cstl, Cst2, Cas5t, Cshl, Csh2, Cas5h, Csal, Csa2, Csa3, Csa4, Csa5, Cas5a, CsxlO, Csxll, Csyl, Csy2, Csy3, Csy4, Mad7, SpCas9, eSpCas9, SpCas9-HFl, HypaSpCas9, HeFSpCas9, and evoSpCas9 high-fidelity variants of SpCas9, SaCas9, NmeCas9, CjCas9, StCas9, TdCas9, LbCasl2a, AsCasl2a, AacCasl2b, BhCasl2b v4, TnpB, FokI, dCas (D10A), dCas (H840A), dCasl3a, dCasl3b, a base editor, a prime editor, a target-primed reverse transcription (TPRT) editor, APOBEC1, cytidine deaminase, adenosine deaminase, uracil glycosylase inhibitor (UGI), adenine base editors (ABE), cytosine base editors (CBE), reverse transcriptase, serine integrase, recombinase, transposase, polymerase, adenine-to-thymine or “ATBE” (or thymine-to-adenine or “TABE”) transversion base editor, ten-eleven translocation methylcytosine dioxygenases (TETs), TET1, TET3, TET1CD, histone acetyltransferase p300, histone methyltransferase SMYD3, histone methyltransferase PRDM9, H3K79 methyltransferase D0T1L, transcriptional repressor, or a functional portion thereof. In certain embodiments, the genome modifying entity is a Cas protein such as Cas9, or Mad7. In some embodiments, the genome targeting entity and the genome modifying entity are (a) different domains of a single polypeptide; (b) two different polypeptides that are operably linked together; or (c) two different polypeptides that are not linked together. a. Genome editing complexes

[0418] In some embodiments, one or more transgene(s), such as any of the transgene(s) described herein, comprises a genome editing complex.

[0419] Any of a variety of agents associated with gene editing technologies can be included in a genome editing complex. The genome editing complex can be used for knock-in or integration of DNA sequences into a region of the genome. In some embodiments, the genome editing complex mediates single-strand breaks (SSB). In some embodiments, the genome editing complex mediates double-strand breaks (DSB), including in connection with non-homologous end-joining (NHEJ) or homology-directed repair (HDR). In some embodiments, the genome editing complex does not mediate SSB. In some embodiments, the genome editing complex does not mediate DSB. In some embodiments, the genome editing complex can be used for DNA base editing or prime-editing. In some embodiments, the genome editing complex can be used for Programmable Addition via Site-specific Targeting Elements (PASTE). In some embodiments, the genome editing complex can cleave, deaminate, nick, polymerize, interrogate, integrate, cut, unwind, break, alter, methylate, demethylate, or otherwise destabilize a target locus.

[0420] In some embodiments, the genome editing complex is or encodes one or more polypeptides having an activity selected from nuclease activity (e.g., programmable nuclease activity); nickase activity (e.g., programmable nickase activity); homing activity (e.g., programmable DNA binding activity); nucleic acid polymerase activity (e.g., DNA polymerase or RNA polymerase activity); integrase activity; recombinase activity; or base editing activity (e.g., cytidine deaminase or adenosine deaminase activity).

[0421] In some embodiments, the genome editing complex is one for use in target-primed reverse transcription (TPRT) or “prime editing”. In some embodiments, prime editing mediates targeted insertions, deletions, all 12 possible base-to-base conversions, and combinations thereof in human cells without requiring DSBs or donor DNA templates. Prime editing is a genome editing method that directly writes new genetic information into a specified DNA site using a nucleic acid programmable DNA binding protein (“napDNAbp”) working in association with a polymerase (z.e., in the form of a fusion protein or otherwise provided in trans with the napDNAbp), wherein the prime editing system is programmed with a prime editing (PE) guide RNA (“PEgRNA”) that both specifies the target site and templates the synthesis of the desired edit in the form of a replacement DNA strand by way of an extension (either DNA or RNA) engineered onto a guide RNA (e.g., at the 5' or 3' end, or at an internal portion of a guide RNA). The replacement strand containing the desired edit (e.g., a single nucleobase substitution) shares the same sequence as the endogenous strand of the target site to be edited (with the exception that it includes the desired edit). Through DNA repair and/or replication machinery, the endogenous strand of the target sequence is replaced by the newly synthesized replacement strand containing the desired edit. In some cases, prime editing may be thought of as a “search- and-replace” genome editing technology since the prime editors search and locate the desired target sequence to be edited, and encode a replacement strand containing a desired edit which is installed in place of the corresponding target sequence endogenous DNA strand at the same time. For example, prime editing can be adapted for conducting precision CRISPR/Cas-based genome editing in order to bypass double stranded breaks. In some embodiments, the genome editing complex is or encodes a primer editor that is a reverse transcriptase, or any DNA polymerase known in the art. Thus, in one aspect, the prime editor may comprise Cas9 (or an equivalent napDNAbp) which is programmed to target a DNA sequence by associating it with a specialized guide RNA (i.e., PEgRNA) containing a spacer sequence that anneals to a complementary protospacer in the target DNA. Such methods include any disclosed in Anzalone et al., (doi.org/10.1038/s41586-019-1711-4), or in PCT publication Nos. WO2020191248, WO202 1226558, or W02022067130, which are hereby incorporated in their entirety. In some embodiments, the genome editing complex is or encodes a Cas protein-reverse transcriptase fusion or related systems to target a specific DNA sequence with a guide RNA, generate a single strand nick at the target sequence, and use the nicked DNA as a primer for reverse transcription of an engineered reverse transcriptase template that is integrated with the guide RNA. In some embodiments, the prime editor protein is paired with two prime editing guide RNAs (pegRNAs) that template the synthesis of complementary DNA flaps on opposing strands of genomic DNA, resulting in the replacement of endogenous DNA sequence between the PE-induced nick sites with pegRNA-encoded sequences.

[0422] In some embodiments, the genome editing complex is or encodes a base editor (e.g., a nucleobase editor). Base editors (BEs) are typically fusions of a Cas (“CRISPR-associated”) domain and a nucleobase modification domain (e.g., a natural or evolved deaminase, such as a cytidine deaminase that include APOBEC1 (“apolipoprotein B mRNA editing enzyme, catalytic polypeptide 1”), CDA (“cytidine deaminase”), and AID (“activation-induced cytidine deaminase”)) domains. In some cases, base editors may also include proteins or domains that alter cellular DNA repair processes to increase the efficiency and/or stability of the resulting single-nucleotide change. Currently available base editors include cytidine base editors (e.g., BE4) that convert target C*G to T«A and adenine base editors (e.g., ABE7.10) that convert target A«T to G*C. Cas9-targeted deamination was first demonstrated in connection with a Base Editor (BE) system designed to induce base changes without introducing double-strand DNA breaks. Further Rat deaminase APOBEC1 (rAPOBECl) fused to deactivated Cas9 (dCas9) was used to successfully convert cytidines to thymidines upstream of the PAM of the sgRNA. This first BE system was optimized by changing the dCas9 to a “nickase” Cas9 D10A, which nicks the strand opposite the deaminated cytidine. Without being bound by theory, this is expected to initiate long-patch base excision repair (BER), where the deaminated strand is preferentially used to template the repair to produce a U:A base pair, which is then converted to T:A during DNA replication. In some embodiments, the genome editing complex is a nucleobase editor containing a first DNA binding protein domain that is catalytically inactive, a domain having base editing activity, and a second DNA binding protein domain having nickase activity, where the DNA binding protein domains are expressed on a single fusion protein or are expressed separately (e.g., on separate expression vectors). In some embodiments, the base editor is a fusion protein comprising a domain having base editing activity (e.g., cytidine deaminase or adenosine deaminase), and two nucleic acid programmable DNA binding protein domains (napDNAbp), a first comprising nickase activity and a second napDNAbp that is catalytically inactive, wherein at least the two napDNAbp are joined by a linker. In some embodiments, the base editor is a fusion protein that comprises a DNA domain of a CRISPR-Cas (e.g., Cas9) having nickase activity (nCas; nCas9), a catalytically inactive domain of a CRISPR-Cas protein (e.g., Cas9) having nucleic acid programmable DNA binding activity (dCas; e.g., dCas9), and a deaminase domain, wherein the dCas is joined to the nCas by a linker, and the dCas is immediately adjacent to the deaminase domain. In some embodiments, the base editor is an adenine-to-thymine or “ATBE” (or thymine-to-adenine or “TABE”) transversion base editor. Exemplary base editor and base editor systems include any as described in patent publication Nos. US20220127622, US20210079366, US20200248169, US20210093667, US20210071163, W02020181202, WO2021158921, WO2019126709, W02020181178, W02020181195, W02020214842, W02020181193, which are hereby incorporated in their entirety. [0423] In some embodiments, the genome editing complex is for use in Programmable Addition via Site-specific Targeting Elements (PASTE). In some aspects, PASTE is a platform in which genomic insertion is directed via a CRISPR-Cas9 nickase fused to both a reverse transcriptase and serine integrase. As described in loannidi et al.

(doi.org/10.1101/2021.11.01.466786), PASTE does not generate double stranded breaks but allows for integration of sequences as large as ~36 kb. In some embodiments, the serine integrase can be any known in the art. In some embodiments, the serine integrase has sufficient orthogonality such that PASTE can be used for multiplexed gene integration, simultaneously integrating at least two different genes in at least two genomic loci. In some embodiments, PASTE has editing efficiencies comparable to or better than those of homology directed repair or non-homologous end joining based integration, with activity in nondividing cells and fewer detectable off-target events.

[0424] In some embodiments, the genome editing complex comprises a genome targeting entity and/or a genome modifying entity.

[0425] In some embodiments, the genome targeting entity is a nucleic acid-guided targeting entity, such as any of a sequence specific nuclease, a nucleic acid programmable DNA binding protein, an RNA guided nuclease, RNA-guided nuclease comprising a Cas nuclease and a guide RNA (CRISPR-Cas combination), a ribonucleoprotein (RNP) complex comprising a gRNA and a Cas nuclease, a zinc finger nuclease (ZF) nucleic acid binding entity, a transcription activatorlike effector (TALE) nucleic acid binding entity, a meganuclease, a Cas nuclease, a core Cas protein, a homing endonuclease, an endonuclease-deficient-Cas protein, an enzymatically inactive Cas protein, a CRIS PR-associated transposase (CAST), a Type II or Type V Cas protein, or a functional portion thereof. In other embodiments, the genome targeting entity comprises any of Casl, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8a, Cas8b, Cas8c, Cas9, Cas 10, Casl2, Casl2a (Cpfl), Casl2b (C2cl), Casl2c (C2c3), Casl2d (CasY), Casl2e (CasX), Casl2f (C2cl0), Casl2g, Casl2h, Casl2i, Casl2k (C2c5), Casl3, Casl3a (C2c2), Casl3b, Casl3c, Casl3d, C2c4, C2c8, C2c9, Cmrl, Cmr2, Cmr3, Cmr4, Cmr5, Cmr6, Csdl, Csd2, Cas5d, Csel, Cse2, Cse3, Cse4, Cas5e, Csfl, Csml, Csm2, Csm3, Csm4, Csm5, Csnl, Csn2, Cstl, Cst2, Cas5t, Cshl, Csh2, Cas5h, Csal, Csa2, Csa3, Csa4, Csa5, Cas5a, CsxlO, Csxl l, Csyl, Csy2, Csy3, Csy4, Mad7, SpCas9, eSpCas9, SpCas9-HFl, HypaSpCas9, HeFSpCas9, and evoSpCas9 high-fidelity variants of SpCas9, SaCas9, NmeCas9, CjCas9, StCas9, TdCas9, LbCasl2a, AsCasl2a, AacCasl2b, BhCasl2b v4, TnpB, dCas (D10A), dCas (H840A), dCasl3a, dCasl3b, or a functional portion thereof.

[0426] In some embodiments, the genome modifying entity comprises one or more genome modifying activities, such as any of: cleaving, deaminating, nicking, polymerizing, interrogating, integrating, cutting, unwinding, breaking, altering, methylating, demethylating, or otherwise destabilizing a target locus, as well as any combination thereof. In some embodiments, the genome modifying entity is selected from Casl, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8a, Cas8b, Cas8c, Cas9, CaslO, Casl2, Casl2a (Cpfl), Casl2b (C2cl), Casl2c (C2c3), Casl2d (CasY), Casl2e (CasX), Casl2f (C2cl0), Casl2g, Casl2h, Casl2i, Casl2k (C2c5), Casl3, Casl3a (C2c2), Casl3b, Casl3c, Casl3d, C2c4, C2c8, C2c9, Cmrl, Cmr2, Cmr3, Cmr4, Cmr5, Cmr6, Csdl, Csd2, Cas5d, Csel, Cse2, Cse3, Cse4, Cas5e, Csfl, Csml, Csm2, Csm3, Csm4, Csm5, Csnl, Csn2, Cstl, Cst2, Cas5t, Cshl, Csh2, Cas5h, Csal, Csa2, Csa3, Csa4, Csa5, Cas5a, CsxlO, Csxl l, Csyl, Csy2, Csy3, Csy4, Mad7, SpCas9, eSpCas9, SpCas9-HFl, HypaSpCas9, HeFSpCas9, and evoSpCas9 high-fidelity variants of SpCas9, SaCas9, NmeCas9, CjCas9, StCas9, TdCas9, LbCasl2a, AsCasl2a, AacCasl2b, BhCasl2b v4, TnpB, FokI, dCas (D10A), dCas (H840A), dCasl3a, dCasl3b, a base editor, a prime editor, a target-primed reverse transcription (TPRT) editor, APOBEC1, cytidine deaminase, adenosine deaminase, uracil glycosylase inhibitor (UGI), adenine base editors (ABE), cytosine base editors (CBE), reverse transcriptase, serine integrase, recombinase, transposase, polymerase, adenine-to-thymine or “ATBE” (or thymine-to-adenine or “TABE”) transversion base editor, ten-eleven translocation methylcytosine dioxygenases (TETs), TET1, TET3, TET1CD, histone acetyltransferase p300, histone methyltransferase SMYD3, histone methyltransferase PRDM9, H3K79 methyltransferase DOT1L, a transcriptional repressor, or a functional portion thereof. In other embodiments, the genome modifying entity is selected from a sequence specific nuclease, a recombinant nuclease, a nucleic acid programmable DNA binding protein, an RNA guided nuclease, RNA-guided nuclease comprising a Cas nuclease and a guide RNA (CRISPR-Cas combination), a ribonucleoprotein (RNP) complex comprising the gRNA and the Cas nuclease, a homing endonuclease, a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a meganuclease, a Cas nuclease, a core Cas protein, a TnpB nuclease, an endonuclease-deficient-Cas protein, an enzymatically inactive Cas protein, a CRIS PR-associated transposase (CAST), a Type II or Type V Cas protein, base editing, prime editing, a Programmable Addition via Site-specific Targeting Elements (PASTE), or a functional portion thereof. In some specific embodiments, the genome modifying entity comprises a recombinase, integrase, transposase, endonuclease, exonuclease, nickase, helicase, DNA polymerase, RNA polymerase, reverse transcriptase, deaminase, flippase, methylase, demethylase, acetylase, a nucleic acid modifying protein, an RNA modifying protein, a DNA modifying protein, an Argonaute protein, an epigenetic modifying protein, a histone modifying protein, or a functional portion thereof. In some embodiments, the genome modifying entity is a Cas protein, a transcription activator-like effector nuclease (TALEN), or a zinc finger nuclease (ZFN). In some embodiments, the recombinant nuclease is a Cas nuclease. In some embodiments, the recombinant nuclease is a TALEN. In some embodiments, the recombinant nuclease is a ZFN. [0427] In some embodiments, the genome targeting entity and the genome modifying entity in a genome editing complex are different domains of a single polypeptide. In other embodiments, the genome targeting entity and genome modifying entity are two different polypeptides that are operably linked together. In certain embodiments, the genome targeting entity and genome modifying entity are two different polypeptides that are not linked together. [0428] In some specific embodiments, the genome editing complex comprises a guide nucleic acid having a targeting sequence that is complementary to at least one target locus, optionally wherein the guide nucleic acid is a guide RNA (gRNA). In some embodiments, the genome editing complex is an RNA-guided nuclease, such as a Cas nuclease (e.g., a Type II or Type V Cas protein) and a guide RNA (CRISPR-Cas combination). In some embodiments, the CRISPR-Cas combination is a ribonucleoprotein (RNP) complex comprising the gRNA and the Cas nuclease. Exemplary and non-limiting Cas nucleases that may be used in the genome editing complexes of the disclosure include Cas3, Cas4, Cas5, Cas8a, Cas8b, Cas8c, Cas9, Cas 10, Casl2, Casl2a (Cpfl), Casl2b (C2cl), Casl2c (C2c3), Casl2d (CasY), Casl2e (CasX), Casl2f (C2cl0), Casl2g, Casl2h, Casl2i, Casl2k (C2c5), Casl3, Casl3a (C2c2), Casl3b, Casl3c, Casl3d, C2c4, C2c8, C2c9, Cmr5, Csel, Cse2, Csfl, Csm2, Csn2, CsxlO, Csxl l, Csyl, Csy2, Csy3, and Mad7. In some embodiments, the Cas protein is selected from Cas3, Cas9, Cas 10, Casl2, Casl3, or Mad7. In some embodiments, the Cas protein is Mad7. In some embodiments, the Cas protein is a Cas 12a (also known as cpfl) from a Prevotella, Francisella novicida, Acidaminococcus sp., Lachnospiraceae bacterium, or Francisella bacteria. In some embodiments, the Cas protein is a Cas 12b from a Bacillus, optionally Bacillus hisashii. In some embodiments, the Cas protein is Cas9 from Streptococcus pyogenes (SpCas). In some embodiments, the Cas9 protein is from Staphylococcus aureus (SaCas9). In some embodiments, the Cas9 protein is from Neisseria meningitidis (NmeCas9). In some embodiments, the Cas9 protein is from Campylobacter jejuni (CjCas9). In some embodiments, the Cas9 protein is from Streptococcus thermophilis (StCas9). The Cas9 nuclease can, in some embodiments, be a Cas9 or functional fragment thereof from any bacterial species. See, e.g., Makarova et al. Nature Reviews, Microbiology, 9: 467-477 (2011), including supplemental information, hereby incorporated by reference in its entirety.

[0429] In some embodiments, the Cas is wild-type Cas9, which can site-specifically cleave double-stranded DNA, resulting in the activation of the double-strand break (DSB) repair machinery. DSBs can be repaired by the cellular Non-Homologous End Joining (NHEJ) pathway (Overballe-Petersen et al., 2013, Proc Natl Acad Sci USA, Vol. 110: 19860-19865), resulting in insertions and/or deletions (indels), which disrupt the targeted locus. Alternatively, if a donor template with homology to the targeted locus is supplied, the DSB may be repaired by the homology-directed repair (HDR) pathway allowing for precise replacement mutations to be made (Overballe- Petersen et al., 2013, Proc Natl Acad Sci USA, Vol. 110: 19860-19865; Gong et al., 2005, Nat. Struct Mol Biol, Vol. 12: 304-312). In some embodiments, the Cas is a mutant form known as Cas9 D10A, with only nickase activity. This means that Cas9D10A cleaves only one DNA strand and does not activate NHEJ. Instead, when provided with a homologous repair template, DNA repairs are conducted via the high-fidelity HDR pathway only, resulting in reduced indel mutations (Cong et al., 2013, Science, Vol. 339: 819-823; Jinek et al., 2012, Science, Vol.337: 816-821; Qi et al., 2013 Cell, Vol. 152: 1173-1183). Cas9D10A is even more appealing in terms of target specificity when loci are targeted by paired Cas9 complexes designed to generate adjacent DNA nicks (Ran et al., 2013, Cell, Vol. 154: 1380-1389). In some embodiments, the Cas is a nuclease-deficient Cas9 (Qi et al., 2013 Cell, Vol. 152: 1173-1183). For instance, mutations H840A in the HNH domain and D10A in the RuvC domain inactivate cleavage activity, but do not prevent DNA binding. Therefore, this variant can be used to target in a sequence-specific manner any region of the genome without cleavage. Instead, by fusing with various effector domains, dCas9 can be used either as a gene silencing or activation tool. Furthermore, it can be used as a visualization tool by coupling the guide RNA or the Cas9 protein to a fluorophore or a fluorescent protein. [0430] In some embodiments, the Cas protein comprises one or more mutations such that the Cas protein is converted into a nickase that is able to cleave only one strand of a double stranded DNA molecule (e.g., an SSB). For example, Cas9, which is normally capable of inducing a double strand break, can be converted into a Cas9 nickase, which is capable of inducing a single strand break, by mutating one of two Cas9 catalytic domains: the RuvC domain, which comprises the RuvC I, RuvC II, and RuvC III motifs, or the NHN domain. In some embodiments, the Cas protein comprises one or more mutations in the RuvC catalytic domain or the HNH catalytic domain. In some embodiments, the genome modifying entity is a recombinant nuclease that has been modified to have nickase activity. In some embodiments, the recombinant nuclease cleaves the strand to which the guide RNA, e.g., sgRNA, hybridizes, but does not cleave the strand that is complementary to the strand to which the guide RNA, e.g., sgRNA, hybridizes. In some embodiments, the recombinant nuclease does not cleave the strand to which the guide RNA, e.g., sgRNA, hybridizes, but does cleave the strand that is complementary to the strand to which the guide RNA, e.g., sgRNA, hybridizes.

[0431] In some embodiments, the genome editing complex is capable of inducing a DSB and comprises a nuclease or a functional fragment thereof, and a first guide RNA, e.g., a first sgRNA, and a second guide RNA, e.g., a second sgRNA. The guide RNA, e.g., the first guide RNA or the second guide RNA, in some embodiments, binds to the nuclease and targets the nuclease to a specific location within the target gene such as at a location within the sense strand or the antisense strand of the target gene that is or includes the cleavage site. In some embodiments, the recombinant nuclease is a Cas protein from any bacterial species or is a functional fragment thereof. In some embodiments, the Cas protein is Cas9 nuclease. Cas9 can, in some embodiments, be a Cas9 or functional fragment thereof from any bacterial species. See, e.g., Makarova et al., Nature Reviews, Microbiology, 9: 467-477 (2011), including supplemental information, hereby incorporated by reference in its entirety.

[0432] In some embodiments, the Cas9 is from Streptococcus pyogenes (SpCas9) and comprises one or more mutations in the RuvC catalytic domain or the HNH catalytic domain. In some embodiments, the one or more mutations in the RuvC catalytic domain or the HNH catalytic domain inactivates the catalytic activity of the domain. In some embodiments, the recombinant nuclease has RuvC activity but does not have HNH activity. In some embodiments, the recombinant nuclease does not have RuvC activity but does have HNH activity. In some embodiments, the Cas9 is from Streptococcus pyogenes (SpCas9) and comprises one or more mutations selected from the group consisting of D10A, H840A, H854A, and H863A. In some embodiments, the Cas9 is from Streptococcus pyogenes (SpCas9) and comprises one or more mutations in the RuvC I, RuvC II, or RuvC III motifs. In some embodiments, the Cas9 is from Streptococcus pyogenes (SpCas9) and comprises a mutation in the RuvC I motif. In some embodiments, the Cas9 is from Streptococcus pyogenes (SpCas9) and comprises a D10A mutation in the RuvC I motif. In some embodiments, the Cas9 is from Streptococcus pyogenes (SpCas9) and comprises one or more mutations in the HNH catalytic domain. In some embodiments, the one or more mutations in the HNH catalytic domain is selected from the group consisting of H840A, H854A, and H863A. In some embodiments, the Cas9 is from Streptococcus pyogenes (SpCas9) and comprises a H840A mutation in the HNH catalytic domain. In some embodiments, the Cas9 is from Streptococcus pyogenes (SpCas9) and comprises a H840A mutation. In some embodiments, the Cas9 is from Streptococcus pyogenes (SpCas9) and comprises a D10A mutation. In some embodiments, the Cas9 is from Streptococcus pyogenes (SpCas9) and comprises one or more mutations selected from the group consisting of N497A, R661A, Q695A, and Q926A. In some embodiments, the Cas9 is from Streptococcus pyogenes (SpCas9) and comprises one or more mutations selected from the group consisting of R780A, K810A, K855A, H982A, K1003A, R1060A, and K848A. In some embodiments, the Cas9 is from Streptococcus pyogenes (SpCas9) and comprises one or more mutations selected from the group consisting of N692A, M694A, Q695A, and H698A. In some embodiments, the Cas9 is from Streptococcus pyogenes (SpCas9) and comprises one or more mutations selected from the group consisting of M495V, Y515N, K526E, and R661Q. In some embodiments, the Cas9 is from Streptococcus pyogenes (SpCas9) and comprises one or more mutations selected from the group consisting of F539S, M763I, and K890N. In some embodiments, the Cas9 is from Streptococcus pyogenes (SpCas9) and comprises one or more mutations selected from the group consisting of E480K, E543D, E1219V, A262T, S409I, M694I, E108G, S217A.

[0433] In some embodiments, the Cas9 is from Streptococcus pyogenes (SaCas9). In some embodiments, the SaCas9 is wild-type SaCas9. In some embodiments, the SaCas9 comprises one or more mutations in REC3 domain. In some embodiments, the SaCas9 comprises one or more mutations in RECI domain. In some embodiments, the SaCas9 comprises one or more mutations selected from the group consisting of N260D, N260Q, N260E, Q414A, Q414L. In some embodiments, the SaCas9 comprises one or more mutations in the recognition lobe. In some embodiments, the SaCas9 comprises one or more mutations selected from the group consisting of R245A, N413A, N419A. In some embodiments, the SaCas9 comprises one or more mutations in the RuvC-III domain. In some embodiments, the SaCas9 comprises a R654A mutation.

[0434] In some embodiments, the Cas protein is Casl2. In some embodiments, the Cas protein is Cas 12a (z.e., cpfl). In some embodiments, the Cas 12a is from the group consisting of Francisella novicida U112 (FnCasl2a), Acidaminococcus sp. BV3L6 (AsCasl2a), Moraxella bovoculi AAXl l_00205 (Mb3Casl2a), Lachnospiraceae bacterium ND2006 (LbCasl2a), Thiomicro spir a sp. Xs5 (TsCasl2a), Moraxella bovoculi AAX08_00205 (Mb2Casl2a), and Butyrivibrio sp. NC3005 (BsCasl2a). In some embodiments, the Cas 12a recognizes a T-rich 5’ protospacer adjacent motif (PAM). In some embodiments, the Casl2a processes its own crRNA without requiring a transactivating crRNA (tracrRNA). In some embodiments, the Cas 12a processes both RNase and DNase activity. In some embodiments, the Cas 12a is a split Cas 12a platform, consisting of N-terminal and C-terminal fragments of Cas 12a. In some embodiments, the split Cas 12a platform is from Lachnospiraceae bacterium.

[0435] In some embodiments, the Cas protein is a Mad7 protein. Mad7 is an engineered class 2 type V-A CRISPR-Cas (Casl2a/Cpfl) system isolated from Eubacterium rectale. Mad7 is an engineered Casl2a variant with 76% homology to wild-type Casl2a. Mad7 is highly proficient in generating genomic insertions and/or deletions (indels) indels, small DNA insertions (e.g., 23 bases), and larger integrations ranging from about 1 to 14 kb in size (see, e.g., Liu, Z. et al., CRISPR J, 3(2):97- 108, 2020). Mad7 only requires a crRNA for gene editing and allows for specific targeting of AT rich regions of the genome.

[0436] In some embodiments, the Cas is a TnpB protein. In some embodiments, TnpB proteins may comprise a Ruv-C-like domain. The RuvC domain may be a split RuvC domain comprising RuvC-I, RuvC-II, and RuvC-III subdomains. In some embodiments, the TnpB may further comprise one or more of a HTH domain, a bridge helix domain, and a zinc finger domain. TnpB proteins do not comprise an HNH domain. In some embodiments, a TnpB protein comprises, starting at the N-terminus: a HTH domain, a RuvC-I subdomain, a bridge helix domain, a RuvC-II sub-domain, a zinger finger domain, and a RuvC-III sub-domain. In some embodiments, a RuvC-III sub-domain forms the C-terminus of a TnpB protein. In some embodiments, the TnpB protein is from Epsilonproteobacteria bacterium, Actinoplanes lobatus strain DSM 43150, Actinomadura celluolosilytica strain DSM 45823, Actinomadura namibiensis strain DSM 44197, Alicyclobacillus macro sprangiidus strain DSM 17980, Lipingzhangella halophila strain DSM 102030, or Ktedonobacter recemifer. In some embodiments, the TnpB protein is from Ktedonobacter racemifer, or comprises a conserved RNA region with similarity to the 5’ ITR of K. racemifer TnpB loci. In some embodiments, the TnpB may comprise a Fanzor protein, a TnpB homolog found in eukaryotic genomes.

[0437] In some embodiments, the genome editing complex comprises, or is used in combination with, a guide RNA, e.g., single guide RNA (sgRNA), for inducing a DSB at the cleavage site. In some embodiments, the genome editing complex comprises, or is used in combination with, more than one guide RNA, e.g., a first sgRNA and a second sgRNA, for inducing a DSB at the cleavage site through an SSB on each strand. In some embodiments, the genome editing complex can be used in combination with a donor template, e.g., a singlestranded DNA oligonucleotide (ssODN), for HD R- mediated integration of the donor template into the target gene, such as at the targeting sequence. In some embodiments, the genome editing complex can be used in combination with a donor template, e.g., an ssODN, and a guide RNA, e.g., a sgRNA, for HDR-mediated integration of the donor template into the target gene, such as at the targeting sequence. In some embodiments, the genome editing complex can be used in combination with a donor template, e.g., an ssODN, and a first guide RNA, e.g., a first sgRNA, and a second guide RNA, e.g., a second sgRNA, for HDR-mediated integration of the donor template into the target gene, such as at the targeting sequence.

[0438] In particular embodiments, the genome-modifying agent is a Cas protein, such as Cas9. In some embodiments, delivery of the CRISPR/Cas can be used to introduce single point mutations (deletions or insertions) in a particular target gene, via a single gRNA. Using a pair of gRNA-directed Cas9 nucleases instead, it is also possible to induce large deletions or genomic rearrangements, such as inversions or translocations.

[0439] In some embodiments, the genome-modifying agent is targeted to the cleavage site by interacting with a guide RNA, e.g., sgRNA, that hybridizes to a DNA sequence that immediately precedes a Protospacer Adjacent Motif (PAM) sequence or a target adjacent motif or transposon- associated motif (TAM) sequence. In general, a guide RNA, e.g., sgRNA, is any nucleotide sequence comprising a sequence, e.g., a crRNA sequence, that has sufficient complementarity with a target gene sequence to hybridize with the target gene sequence at the cleavage site and direct sequence- specific binding of the recombinant nuclease to a portion of the target gene that includes the cleavage site. Full complementarity (100%) is not necessarily required, as long as there is sufficient complementarity to cause hybridization and promote formation of a complex, e.g., CRISPR complex, that includes the recombinant nuclease and the guide RNA, e.g., sgRNA. In some embodiments, the cleavage site is situated at a site within the target gene that is homologous to the sequence of the guide RNA, e.g., sgRNA. In some embodiments, the cleavage site is situated approximately 3 nucleotides upstream of the PAM or TAM sequence. In some embodiments, the cleavage site is situated approximately 3 nucleotides upstream of the juncture between the guide RNA and the PAM or TAM sequence. In some embodiments, the cleavage site is situated 3 nucleotides upstream of the PAM or TAM sequence. In some embodiments, the cleavage site is situated 4 nucleotides upstream of the PAM or TAM sequence. In some embodiments, the cleavage site is situated approximately 25 nucleotides upstream from the PAM or TAM sequence. In some embodiments, the cleavage site is situated approximately 23 nucleotides upstream from the PAM or TAM sequence. In some embodiments, the cleavage site is situated approximately 19 nucleotides upstream from the PAM or TAM sequence. In some embodiments, the cleavage site is situated approximately 18 nucleotides upstream from the PAM or TAM sequence. In some embodiments, the cleavage site is situated approximately 12 nucleotides upstream from the PAM or TAM sequence. In some embodiments, the cleavage site is situated approximately 8 nucleotides upstream from the PAM or TAM sequence.

[0440] In some embodiments, the genome editing complex capable of inducing a DSB comprises a fusion protein comprising a DNA binding domain and a DNA cleavage domain. In some embodiments, the DNA cleavage domain is or comprises a recombinant nuclease. In some embodiments, the fusion protein is a TALEN comprising a DNA binding domain and a DNA cleavage domain. In some embodiments, the DNA binding domain is a transcription activatorlike (TAL) effector DNA binding domain. In some embodiments, the TAL effector DNA binding domain is from Xanthomonas bacteria. In some embodiments, the DNA cleavage domain is a Fokl nuclease domain. In some embodiments, the TAL effector DNA binding domain is engineered to target a specific target sequence, e.g., a portion of a target gene that includes a cleavage site. [0441] In some embodiments, the fusion protein is a zinc finger nuclease (ZFN) comprising a zinc finger DNA binding domain and a DNA cleavage domain. In some embodiments, the DNA cleavage domain is a Fokl nuclease domain. In some embodiments, the zinc finger DNA binding domain is engineered to target a specific target sequence, e.g., a portion of a target gene, that includes a cleavage site, such as the targeting sequence.

[0442] In some embodiments, genome editing can be accomplished by introducing into a cell a genome editing complex of the disclosure. For example, introducing into a cell a genome editing complex capable of inducing a SSB at a cleavage site within the sense strand and an SSB at a cleavage site within the antisense strand of an endogenous target gene in the cell.

[0443] In some embodiments, the cleavage site in the sense strand is less than 400, less than 350, less than 300, less than 250, less than 200, less than 175, less than 150, less than 125, less than 100, less than 90, less than 80, less than 75, less than 70, less than 65, less than 60, less than 55, less than 50, less than 45, less than 40, or less than 35 nucleotides from the nucleotide that is complementary to the cleavage site in the antisense strand. In some embodiments, the cleavage site in the antisense strand is less than 400, less than 350, less than 300, less than 250, less than 200, less than 175, less than 150, less than 125, less than 100, less than 90, less than 80, less than 75, less than 70, less than 65, less than 60, less than 55, less than 50, less than 45, less than 40, or less than 35 nucleotides from the nucleotide that is complementary to the cleavage site in the sense strand. In some embodiments, the cleavage site in the sense strand is between 20 and 400, 20 and 350, 20 and 300, 20 and 250, 20 and 200, 20 and 150, 20 and 125, 20 and 100, 20 and 90, 20 and 80, 20 and 70, 30 and 400, 30 and 350, 30 and 300, 30 and 250, 30 and 200, 30 and 150, 30 and 125, 30 and 100, 30 and 90, 30 and 80, 30 and 70, 40 and 400, 40 and 350, 40 and 300, 40 and 250, 40 and 200, 40 and 150, 40 and 125, 40 and 100, 40 and 90, 40 and 80, or 40 and 70 nucleotides from the nucleotide that is complementary to the cleavage site in the antisense strand. In some embodiments, the cleavage site in the antisense strand is between 20 and 400, 20 and 350, 20 and 300, 20 and 250, 20 and 200, 20 and 150, 20 and 125, 20 and 100, 20 and 90, 20 and 80, 20 and 70, 30 and 400, 30 and 350, 30 and 300, 30 and 250, 30 and 200, 30 and 150, 30 and 125, 30 and 100, 30 and 90, 30 and 80, 30 and 70, 40 and 400, 40 and 350, 40 and 300, 40 and 250, 40 and 200, 40 and 150, 40 and 125, 40 and 100, 40 and 90, 40 and 80, or 40 and 70 nucleotides from the nucleotide that is complementary to the cleavage site in the sense strand. [0444] In some embodiments, the genome editing complex capable of inducing an SSB at a cleavage site within the sense strand and an SSB at a cleavage site within the antisense strand comprise a recombinant nuclease. In some embodiments, the recombinant nuclease includes a recombinant nuclease that induces the SSB in the sense strand, and a recombinant nuclease that induced the SSB in the antisense strand, and both of which recombinant nucleases are referred to as the recombinant nuclease. Accordingly, in some embodiments, the method involves introducing into a cell the genome editing complex comprising a recombinant nuclease for inducing an SSB at a cleavage site in the sense strand and an SSB at a cleavage site in the antisense strand within an endogenous target gene in the cell. Although, in some embodiments, it is described that “a” “the” recombinant nuclease induces an SSB in the antisense strand an SSB in the sense strand, it is to be understood that this includes situations where two of the same recombinant nucleases is used, such that one of the recombinant nucleases induces the SSB in the sense strand and the other recombinant nuclease induces the SSB in the antisense strand. In some embodiments, the recombinant nuclease that induces the SSB lacks the ability to induce a DSB by cleaving both strands of double stranded DNA.

[0445] In some embodiments, the genome editing complex capable of inducing an SSB comprises a recombinant nuclease and a first guide RNA, e.g., a first sgRNA, and a second guide RNA, e.g., a second sgRNA.

[0446] In some embodiments, the genome editing complex capable of inducing an SSB at a cleavage site within the sense strand and an SSB at a cleavage site within the antisense strand comprise a fusion protein comprising a DNA binding domain and a DNA cleavage domain. In some embodiments, the DNA cleavage domain is or comprises a recombinant nuclease. In some embodiments, the fusion protein is a TALEN comprising a DNA binding domain and a DNA cleavage domain. In some embodiments, the DNA binding domain is a transcription activatorlike (TAL) effector DNA binding domain. In some embodiments, the TAL effector DNA binding domain is from Xanthomonas bacteria. In some embodiments, the DNA cleavage domain is a Fokl nuclease domain. In some embodiments, the TAL effector DNA binding domain is engineered to target a specific target sequence, e.g., a portion of a target gene that includes a cleavage site. In some embodiments, the fusion protein is a zinc finger nuclease (ZFN) comprising a zinc finger DNA binding domain and a DNA cleavage domain. In some embodiments, the DNA cleavage domain is a Fokl nuclease domain. In some embodiments, the zinc finger DNA binding domain is engineered to target a specific target sequence, e.g., a portion of a target gene that includes a cleavage site, such as the targeting sequence.

[0447] In some embodiments, the genome editing complex capable of inducing an SSB at a cleavage site within the sense strand and an SSB at a cleavage site within the antisense strand involve use of the CRISPR/Cas gene editing system. In some embodiments the genome editing complex comprises a recombinant nuclease, such as Mad7 or Cas9.

[0448] In some embodiments, the genome modifying entity is targeted to the cleavage site by interacting with a guide RNA, e.g., a first guide RNA, such as a first sgRNA, or a second guide RNA, such as a second sgRNA, that hybridizes to a DNA sequence on the sense strand or the antisense strand that immediately precedes a PAM or TAM sequence. In some embodiments, the genome modifying entity is targeted to the cleavage site on the sense strand by interacting with a first guide RNA, e.g., first sgRNA, that hybridizes to a sequence on the sense strand that immediately precedes a PAM sequence. In some embodiments, the genome modifying entity is targeted to the cleavage site on the antisense strand by interacting with a second guide RNA, e.g., second sgRNA, that hybridizes to a sequence on the antisense strand that immediately precedes a PAM sequence. In some embodiments, the first guide RNA, e.g., first sgNA, that is specific to the sense strand of a target gene of interest is used to target a genome modifying entity to induce an SSB at a cleavage site within the sense strand of the target gene. In some embodiments, the first guide RNA, e.g., first sgNA, that is specific to the antisense strand of a target gene of interest is used to target the genome modifying entity to induce an SSB at a cleavage site within the antisense strand of the target gene. In some embodiments, the second guide RNA, e.g., second sgNA, that is specific to the sense strand of a target gene of interest used to target the genome modifying entity to induce an SSB at a cleavage site within the sense strand of the target gene. In some embodiments, the second guide RNA, e.g., second sgNA, that is specific to the antisense strand of a target gene of interest is used to target the genome modifying entity to induce an SSB at a cleavage site within the antisense strand of the target gene. In some embodiments, the first guide RNA, e.g., first sgNA, that is specific to the sense strand of a target gene of interest is used to target the genome modifying entity to induce an SSB at a cleavage site within the sense strand of the target gene; and the second guide RNA, e.g., second sgNA, that is specific to the antisense strand of a target gene of interest is used to target the genome modifying entity to induce an SSB at a cleavage site within the antisense strand of the target gene. In some embodiments, the genome modifying entity is, e.g., Mad7 or Cas9. In some embodiments, the first guide RNA, e.g., first sgNA, that is specific to the antisense strand of a target gene of interest is used to target the genome modifying entity to induce an SSB at a cleavage site within the antisense strand of the target gene; and the second guide RNA, e.g., second sgNA, that is specific to the sense strand of a target gene of interest is used to target the genome modifying entity to induce an SSB at a cleavage site within the sense strand of the target gene.

[0449] In some embodiments, the cleavage site is situated at a site within the target gene that is homologous to a sequence comprised within the guide RNA, e.g., sgRNA. In some embodiments, the cleavage site of the sense strand is situated at a site within the sense strand of the target genomic locus that is homologous to a sequence comprised within the first guide RNA, e.g., the first sgRNA. In some embodiments, the cleavage site of the antisense strand is situated at a site within the antisense strand of the target genomic locus that is homologous to a sequence comprised within the first guide RNA, e.g., the first sgRNA. In some embodiments, the cleavage site of the sense strand is situated at a site within the sense strand of the target genomic locus that is homologous to a sequence comprised within the second guide RNA, e.g., the second sgRNA. In some embodiments, the cleavage site of the antisense strand is situated at a site within the antisense strand of the target genomic locus that is homologous to a sequence comprised within the second guide RNA, e.g., the second sgRNA. In some embodiments, the cleavage site of the sense strand is situated at a site within the sense strand of the target genomic locus that is homologous to a sequence comprised within the first guide RNA, e.g., the first sgRNA; and the cleavage site of the antisense strand is situated at a site within the antisense strand of the target genomic locus that is homologous to a sequence comprised within the second guide RNA, e.g., the second sgRNA. In some embodiments, the cleavage site of the antisense strand is situated at a site within the antisense strand of the target genomic locus that is homologous to a sequence comprised within the first guide RNA, e.g., the first sgRNA; and the cleavage site of the sense strand is situated at a site within the sense strand of the target genomic locus that is homologous to a sequence comprised within the second guide RNA, e.g., the second sgRNA. In some embodiments, the cleavage site of the antisense strand is situated at a site within the antisense strand of the target genomic locus that is homologous to a sequence comprised within the second guide RNA, e.g., the second sgRNA; and the cleavage site of the sense strand is situated at a site within the sense strand of the target genomic locus that is homologous to a sequence comprised within the first guide RNA, e.g., the first sgRNA.

[0450] In some embodiments, the sense strand comprises the targeting sequence, and the targeting sequence includes a protospacer adjacent motif (PAM) or a target adjacent motif or transposon-associated motif (TAM) sequence. In some embodiments, the sense strand comprises the targeting sequence, and the targeting sequence includes a PAM or TAM sequence; and the antisense strand comprises a sequence that is complementary to the targeting sequence and includes a PAM or TAM sequence. In some embodiments, the antisense strand comprises the targeting sequence, and the targeting sequence includes a PAM or TAM sequence. In some embodiments, the antisense strand comprises the targeting sequence, and the targeting sequence includes a PAM or TAM sequence; and the sense strand comprises a sequence that is complementary to the targeting sequence and includes a PAM or TAM sequence.

[0451] In some embodiments, the cleavage site on the sense strand and/or the antisense strand is situated approximately 3 nucleotides upstream of the PAM or TAM sequence. In some embodiments, the cleavage site on the sense strand and/or the antisense strand is situated approximately 3 nucleotides upstream of the juncture between the guide RNA and the PAM or TAM sequence. In some embodiments, the cleavage site on the sense strand and/or the antisense strand is situated 3 nucleotides upstream of the PAM or TAM sequence. In some embodiments, the cleavage site on the sense strand and/or the antisense strand is situated 4 nucleotides upstream of the PAM or TAM sequence.

[0452] In some embodiments, the PAM or TAM sequence that is recognized by a recombinant nuclease is in the sense strand. In some embodiments, the PAM or TAM sequence that is recognized by a recombinant nuclease is in the antisense strand. In some embodiments, the PAM or TAM sequence that is recognized by a recombinant nuclease is in the sense strand and is in the antisense strand. In some embodiments, the PAM or TAM sequence on the sense strand and the PAM or TAM sequence on the antisense strand are outwardly facing. In some embodiments, the PAM or TAM sequence on the sense strand and the PAM or TAM sequence on the antisense strand comprise the same nucleic acid sequence, which can be any PAM or TAM sequence disclosed herein or known in the art. In some embodiments, the PAM or TAM sequence on the sense strand and the PAM or TAM sequence on the antisense strand each comprise a different nucleic acid sequence, each of which can be any of the PAM or TAM sequences disclosed herein or known in the art.

[0453] As is known in the art, the PAM or TAM sequence that is recognized by a recombinant nuclease, such as Cas9 or Mad7, differs depending on the particular recombinant nuclease and the organism species it is from. b. Guide RNAs and Compositions Thereof

[0454] In some embodiments, one or more transgene sequences, such as any of the transgene sequences described herein, comprises a nucleic acid-guided targeting entity, such as any of a sequence-specific nuclease, a nucleic acid programmable DNA binding protein, an RNA guided nuclease, RNA-guided nuclease comprising a Cas nuclease and a guide RNA (CRISPR-Cas combination), a ribonucleoprotein (RNP) complex comprising a gRNA and a Cas nuclease, a zinc finger nuclease (ZF) nucleic acid binding entity, a transcription activator-like effector (TALE) nucleic acid binding entity, a meganuclease, a Cas nuclease, a core Cas protein, a homing endonuclease, an endonuclease-deficient-Cas protein, an enzymatically inactive Cas protein, a CRISPR-associated transposase (CAST), a Type II or Type V Cas protein, or a functional portion thereof.

[0455] In some embodiments, the transgene sequence comprises a nucleic acid-guided nuclease, such as a guide RNA-guided nuclease, e.g., a Cas protein or Mad7, as described herein. In such cases, the nucleic acid-guided nuclease is targeted to a cleavage site by interacting with a guide RNA, e.g., sgRNA, that hybridizes to a DNA sequence that immediately precedes a Protospacer Adjacent Motif (PAM) or target adjacent motif or transposon-associated motif (TAM) sequence. The guide RNA can bind to the nucleic acid-guided nuclease and target the nucleic acid-guided nuclease to a specific location within the target sequence, such as at a location within the sense strand or the antisense strand of a target gene, e.g., within the host cell’s genome, that is or includes the cleavage site.

[0456] In general, a guide RNA, e.g., sgRNA, is any nucleotide sequence comprising a sequence, e.g., a crRNA sequence, that has sufficient complementarity with a target gene sequence to hybridize with the target gene sequence at the cleavage site and direct sequencespecific binding of the nucleic acid-guided nuclease to a portion of the target gene that includes the cleavage site. Full complementarity (100%) is not necessarily required, so long as there is sufficient complementarity to cause hybridization and promote formation of a complex, e.g., CRISPR complex, that includes the nucleic acid-guided nuclease and the guide RNA, e.g., sgRNA.

[0457] Methods for designing guide RNAs, e.g., sgRNAs, and their exemplary targeting sequences, e.g., crRNA sequences, can include those described in, e.g., International PCT Pub. Nos. WO2015/161276, W02017/193107, and WO2017/093969. Exemplary guide RNA structures, including particular domains, are described in WO2015/161276, e.g., in FIGS. 1A-1G therein. Since a guide RNA is an RNA molecule, it will comprise the base uracil (U), while any DNA encoding the guide RNA molecule will comprise the base thymine (T). In some embodiments, the guide RNA, e.g., sgRNA, comprises a CRISPR targeting RNA sequence (crRNA) and a trans-activating crRNA sequence (tracrRNA). In some embodiments, the guide RNA, e.g., sgRNA, is an RNA comprising, from 5' to 3': a crRNA sequence and a tracrRNA sequence. In some embodiments, the crRNA and tracrRNA do not naturally occur together in the same sequence.

[0458] In some embodiments, the crRNA comprises a nucleotide sequence that is homologous, e.g., is at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous, or is about 100% homologous, to a portion of the target genomic locus that includes the cleavage site. In some embodiments, the crRNA comprises a nucleotide sequence that is 100% homologous to a portion of the target sequence, e.g., genomic locus, that includes the cleavage site. In some embodiments, the portion of the target sequence that includes the cleavage site is a portion of the sense strand of the target sequence that includes the cleavage site. In some embodiments, the portion of the target sequence that includes the cleavage site is a portion of the antisense strand of the target sequence that includes the cleavage site.

[0459] In some embodiments, one or more transgenes may comprise more than one guide RNA e.g., sgRNA), such as a first and a second guide RNA. In some embodiments, the first guide RNA, e.g., the first sgRNA, and the second guide RNA, e.g., the second sgRNA, each comprise a crRNA and a tracrRNA. In some embodiments, each of the first guide RNA, e.g., first sgRNA, and the second guide RNA, e.g., second sgRNA, is an RNA comprising, from 5' to 3': a crRNA sequence and a tracrRNA sequence. In some embodiments, the crRNA and tracrRNA do not naturally occur together in the same sequence. In some embodiments, the sgRNA comprises a crRNA sequence that is homologous to a sequence in the target sequence that includes the cleavage site. In some embodiments, the first sgRNA comprises a crRNA sequence that is homologous to a sequence in the antisense strand of the target sequence that includes the cleavage site; and/or the second sgRNA comprises a crRNA sequence that is homologous to a sequence in the sense strand of the target sequence that includes the cleavage site.

[0460] In some embodiments, the crRNA sequence has 100% sequence identity to a sequence in the target sequence that includes the cleavage site. In some embodiments, the crRNA sequence of the first sgRNA has 100% sequence identity to a sequence in the sense strand of the target sequence that includes the cleavage site; and/or the crRNA sequence of the second sgRNA has 100% sequence identity to a sequence in the antisense strand of the target sequence that includes the cleavage site. In some embodiments, the crRNA sequence of the first sgRNA has 100% sequence identity to a sequence in the antisense strand of the target sequence that includes the cleavage site; and/or the crRNA sequence of the second sgRNA has 100% sequence identity to a sequence in the sense strand of the target sequence that includes the cleavage site.

[0461] Guidance on the selection of crRNA sequences can be found, e.g., in Fu Y et al., Nat Biotechnol 2014 (doi: 10.1038/nbt.2808) and Sternberg SH et al., Nature 2014 (doi: 10.1038/naturel3011). Examples of the placement of crRNA sequences within the guide RNA, e.g., sgRNA, structure include those described in WO2015/161276, e.g., in FIGS. 1A-1G therein.

[0462] In some embodiments, the crRNA is 15-27 nucleotides in length, i.e., the crRNA is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 nucleotides in length. In some embodiments, the crRNA is 18-22 nucleotides in length. In some embodiments, the crRNA is 19-21 nucleotides in length. In some embodiments, the crRNA is 20 nucleotides in length. In some embodiments, the crRNA is 21 nucleotides in length.

[0463] In some embodiments, the crRNA is homologous to a portion of a target sequence that includes the cleavage site. In some embodiments, the crRNA is homologous to a portion of the sense strand of the target sequence that includes the cleavage site. In some embodiments, the crRNA is homologous to a portion of the antisense strand of the target sequence that includes the cleavage site. In some embodiments, the crRNA of the first sgRNA is homologous to a portion of the sense strand of the target sequence that includes the cleavage site; and the crRNA of the second sgRNA is homologous to a portion of the antisense strand of the target sequence that includes the cleavage site. [0464] In some embodiments, the crRNA is homologous to a portion of the antisense strand of a target sequence that includes the cleavage site. In some embodiments, the crRNA is homologous to a portion of the sense strand of the target sequence that includes the cleavage site. In some embodiments, the crRNA of the first sgRNA is homologous to a portion of the antisense strand of the target sequence that includes the cleavage site; and the crRNA of the second sgRNA is homologous to a portion of the sense strand of the target sequence that includes the cleavage site.

[0465] In some embodiments, the crRNA is homologous to a portion of a target genomic locus that includes the cleavage site, and is 15-27 nucleotides in length, i.e., the crRNA is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 nucleotides in length. In some embodiments, the portion of the target sequence that includes the cleavage site is on the sense strand. In some embodiments, the portion of the target sequence that includes the cleavage site is on the antisense strand.

[0466] In some embodiments, the crRNA is homologous to a portion, i.e., sequence, in the sense strand or the antisense strand of the target sequence that includes the cleavage site and is immediately upstream of a PAM or TAM sequence.

[0467] In some embodiments, the tracrRNA sequence may be or comprise any sequence for tracrRNA that is used in any CRISPR/Cas system known in the art. Exemplary CRISPR/Cas systems, sgRNA, crRNA, and tracrRNA, and their manufacturing process and use include those described in, e.g., International PCT Pub. Nos. WO2015/161276, W02017/193107 and WO2017/093969, and those described in, e.g., U.S. Patent Application Publication Nos. 20150232882, 20150203872, 20150184139, 20150079681, 20150073041, 20150056705, 20150031134, 20150020223, 20140357530, 20140335620, 20140310830, 20140273234, 20140273232, 20140273231, 20140256046, 20140248702, 20140242700, 20140242699, 20140242664, 20140234972, 20140227787, 20140189896, 20140186958, 20140186919, 20140186843, 20140179770, 20140179006, 20140170753, 20140093913, and 20140080216.

[0468] In some embodiments, the genome editing complex comprises a guide nucleic acid having a targeting domain that is complementary to at least one target sequence, optionally wherein the guide nucleic acid is a guide RNA (gRNA). In some cases, the genome editing complex is an RNA-guided nuclease, for example, a Cas nuclease and a guide RNA (CRISPR- Cas combination). In some embodiments, the CRISPR-Cas combination is a ribonucleoprotein (RNP) complex comprising the gRNA and the Cas nuclease. In some embodiments, the Cas nuclease is a Type II or Type V Cas protein. In some embodiments, the Cas nuclease is selected from Cas3, Cas4, Cas5, Cas8a, Cas8b, Cas8c, Cas9, Cas 10, Cas 12, Cas 12a (Cpfl), Cas 12b (C2cl), Casl2c (C2c3), Casl2d (CasY), Casl2e (CasX), Casl2f (C2cl0), Casl2g, Casl2h, Casl2i, Casl2k (C2c5), Casl3, Casl3a (C2c2), Casl3b, Casl3c, Casl3d, C2c4, C2c8, C2c9, Cmr5, Csel, Cse2, Csfl, Csm2, Csn2, CsxlO, Csxll, Csyl, Csy2, Csy3, or Mad7. vii. Transcription Factors

[0469] In some embodiments, the one or more transgenes, e.g., the first and/or the second transgene, can encode proteins that include, but are not limited to, DNA-binding proteins, transcription factors, chromatin remodeling factors, methylated DNA binding proteins, polymerases, methylases, demethylases, acetylases, deacetylases, kinases, phosphatases, integrases, recombinases, ligases, topoisomerases, gyrases, and helicases. In some embodiments, the one or more transgenes, e.g., the first and/or the second transgene, encode one or more transcription factors.

[0470] Transcription factors are proteins involved in the process of transcribing DNA into RNA. Transcription factors include many proteins, excluding RNA polymerase, that initiate and regulate the transcription of genes. Transcription factors can be activators, which increase transcription, or repressors, which inhibit transcription. A distinct feature of transcription factors is the DNA-binding domains that are responsible for binding to specific sequences of DNA called enhancer or promoter sequences. Some transcription factors bind to a DNA promoter sequence near the transcription start site and help form the transcription initiation complex. Other transcription factors bind to regulatory sequences, such as enhancer sequences, and can either stimulate or repress transcription of the related gene. These regulatory sequences can be thousands of base pairs upstream or downstream from the gene being transcribed. Regulation of transcription is the most common form of gene control. The action of transcription factors allows for unique expression of each gene in different cell types and during development, in homeostasis, and during ageing.

[0471] In some embodiments, the one or more transcription factors comprise one or more of OCT4, SOX2, NANOG, KLF4, LIN28, C-MYC, ECAT1, UTF1, ESRRB, SV40LT, HESRG, CDH1, TDGF1, DPPA4, DNMT3B, ZIC3, p53DD, and L1TD1, or functional domains or fragments thereof. In other embodiments, the transcription factor is or comprises a nuclear factor of activated T cells (NFAT) transcription factor, an NF-KB transcription factor, or functional domain or fragment thereof.

[0472] The transcription factors, OCT4, SOX2, NANOG, and KLF4 are triggers for the induction of somatic cells to pluripotent stem cells and are essential master regulators for stem cell biology. OCT4, SOX2, KLF4, and NANOG are expressed at similar levels in iPSCs and embryonic stem cells, and expression of all four transcription factors decreases after differentiation (see, e.g., Ma, Y. et al., Cy to technology, 66(6): 967-978, 2014, hereby incorporated by reference in its entirety). OCT4 is expressed in all pluripotent cells during mouse embryogenesis and in undifferentiated embryonic stem (ES) cells. OCT4 is downregulated during trophoblast differentiation, and mutant embryos lacking OCT4 exclusively develop into trophoblast-like cells, suggesting that OCT4 is required to either establish or maintain pluripotency in the embryo. Unlike transcription factors which act in a binary on-off mode, OCT4 may regulate cell fate in a quantitative manner and cooperates with the other proteins in a network of transcription factors (see, e.g., Pan, G. et al., FASEB J Off Publ Fed Am Soc Exp Biol., 20:1730-1732, 2006, hereby incorporated by reference in its entirety). NANOG plays a critical role in the regulation of cell fate during embryonic development by maintaining the pluripotent epiblast and preventing differentiation (see, e.g., Chambers et al., Cell, 113:643-655, 2003, hereby incorporated by reference in its entirety). SOX2 is required in induced pluripotent stem cells and may regulate OCT4 expression. SOX2 and OCT4 can perpetuate their own expression when expressed concurrently (see, e.g., Masui et al., Nat Cell Biol., 9:625-635, 2007, hereby incorporated by reference in its entirety). SOX2 was found to determine the differentiation lineage of human mesenchymal stem cells. KLF4 may be the central position for the OCT4, SOX2, KLF4, and NANOG transcriptional network. In embryonic stem cells, KLF4 shares multiple target genes with both OCT4 and SOX2. The KLF4 C-terminus contains three tandem zinc fingers and is the domain that interacts with OCT4 and activates NANOG gene transcription.

[0473] The RNA-binding protein Lin28 (Lin28a) is a pluripotency factor that directly binds to many cytoplasmic mRNAs and to the microRNA let-7. LIN28 is expressed early during development and in undifferentiated tissues, and it is downregulated as development and cellular differentiation proceed. LIN28 is highly expressed in embryonic stem cells (ESCs) and is downregulated in response to differentiation, and thus is one of several factors leveraged to reprogram mammalian somatic cells to pluripotent cells (see, e.g., Tsialikas, J. and Romer- Seibert, J., Development, 142(14):2397-2404, 2015, hereby incorporated by reference in its entirety).

[0474] The transcription factor, cellular-MYC (z.e., C-MYC) is thought to regulate approximately 15% of all genes through its binding to enhancer box (E-box) sequences and is itself tightly regulated. The N-terminal transactivation domain (NTD) contains the transcription activation domain (TAD) and two MYC boxes, MBI and MB II, which are highly conserved sequence elements involved in transcription regulation and protein stability. The central portion of C-MYC contains a nuclear localization signal and two further conserved sequence elements, MBIII and MBIV. The C-terminal domain contains the bHLHZip motif, which remains partially unstructured until it dimerizes with another bHLHZip protein, MAX. It then forms an ordered alpha-helix structure, which is subject to multiple post-translational modifications and protein interactions that regulate the function of C-MYC. C-MYC has been implicated in multiple cellular processes, including proliferation, differentiation, apoptosis, and metabolism. Gene targets of C-MYC are involved in chromatin modification, DNA replication, and ribosome and mitochondrial biogenesis.

[0475] The transcription factor Embryonic Stem Cell-Associated Transcript 1 (ECAT1, also known as KHDC3L) may function as a regulator of genomic imprinting in the oocyte. ECAT1 is specifically expressed in oocytes and preimplantation embryos, and it is absent in differentiated cells. ECAT1 belongs to the KHDC1 family, members of which contain an atypical KH domain that may not bind RNA, whereas canonical KH domains may bind RNA. ECAT1 has been found to localize to the cytoskeleton and is predominantly at the cortical region in growing oocytes. After the first cellular division, ECAT1 becomes asymmetrically confined to the outer cortical region and excluded from the cell-to-cell contact region until the blastocyst stage where it is homogeneously redistributed to the nucleus (see, e.g., Akoury, E. et al., Hum Reprod., 30( 1): 159- 169, 2015, hereby incorporated by reference in its entirety).

[0476] Undifferentiated embryonic cell transcription factor 1 (UTF1), is expressed in ESCs and primordial germ cells. UTF1 is controlled by OCT4 and SOX2. It is involved in chromatin organization, acts as an epigenetic factor controlling H3K27me3 deposition at bivalent genes, and promotes cell differentiation during exit from pluripotency (See, e.g., Bao, Q. et al., Sci Rep., 7:14612, 2017, hereby incorporated by reference in its entirety). Estrogen-related-receptor beta (z.e., ESRRB) is a member of the nuclear orphan receptor family. ESRRB activates 0CT4 and sustains self-renewal. ESRRB then interacts with 0CT4 to positively regulate NANOG gene expression. (See, e.g., Zhang, X. et al., J Biol Chem., 283(51):35825-35833, 2008, hereby incorporated by reference in its entirety).

[0477] Simian Virus 40 Large T antigen (z.e., SV40LT) is responsible for both viral and cellular transcriptional regulation, virion assembly, viral DNA replication, and alteration of the cell cycle. Embryonic stem cell-related gene (HESRG or ESRG) is predominantly expressed in undifferentiated human embryonic stem cells but not in differentiated cells, and it is activated in an early stage of somatic cell reprogramming following the forced expression of OCT3/4, SOX2, and KLF4. CDH1 is a tumor suppressor gene. Teratocarcinoma-derived growth factor 1 (TDGF1) is an extracellular, membrane-bound signaling protein that plays an essential role in embryonic development and tumor growth. Developmental pluripotency-associated protein 4 (DPPA4) is a nuclear factor that is involved in the maintenance of pluripotency in stem cells and essential for embryogenesis. DNA methyltransferase 3 beta (DNMT3B) is DNA methyltransferase which is thought to function in de novo methylation, rather than maintenance methylation; the protein localizes primarily to the nucleus, and its expression is developmentally regulated. ZIC3 is a nuclear protein that may function as a transcription factor in early stages of left-right body axis formation. p53DD is a dominant negative truncated p53 mutant, which impairs the wild-type p53 transactivation function. LINE-1 type transposase domain containing 1 (L1TD1) is an RNA-binding protein that is involved with self-renewal of undifferentiated human embryonic stem cells and cancer cell proliferation.

6. Vectors

[0478] Vectors are nucleic acid (e.g., DNA) molecules that serve as vehicles to transfer foreign nucleic acid (e.g., DNA or a fragment thereof) into a host cell, in which the genetic material can be replicated and expressed. Vectors can be, for example, plasmids, viral vectors, cosmids, and artificial chromosomes. In the present disclosure, the vector may be an expression vector carrying a transgene and a unique barcode sequence used to identify the transgene in an engineered cell or population of cells (e.g., T cells, B cells, NK cells, islet cells such as beta cells, or hypoimmunogenic cells thereof). In some embodiments, a vector as provided herein (such as the first and/or the second vector) is a plasmid or a lentiviral vector. [0479] Vectors may include additional components that are necessary for expression of a gene-of-interest (e.g., a transgene) in cells (e.g., multiple cloning site, promoter, regulatory elements, etc.). For example, a multiple cloning site acts as an insertion site for heterologous genes, reporters, additional gene regulatory elements, selectable markers, etc. Vectors may have one or more multiple cloning sites containing restriction sites. Furthermore, a promoter is a nucleic acid region that is upstream, or 5', of a target gene, wherein transcription of the target gene is initiated at the promoter by a cell’s transcription machinery. Promoters may be prokaryotic or eukaryotic, and furthermore can be expressed ubiquitously, expressed within specific cell types or subtypes, inducible, repressed, or otherwise cycled on/off in a controlled fashion or in response to cellular signaling pathway activity. Gene regulatory elements control the level and isoform type during gene expression. Regulatory elements may work at the transcriptional and/or the post-transcriptional stages. Gene regulatory elements may include promoters, enhancers, silencers, and insulators.

[0480] While not necessary for expression of a transgene encoded by a vector, a selectable marker can help identify and/or select for cells expressing the vector. Selectable markers may include antibiotic resistance genes, fluorescent proteins, toxins, etc. Selectable markers can permit positive selection (z.e., cells comprising the selectable marker are not killed) or negative selection (z.e., cells comprising the selectable marker are killed). Non-limiting examples of antibiotic resistance selectable markers known to those skill in the art include, for example, ampicillin, blasticidin, carbenicillin, chloramphenicol, hygromycin B, kanamycin, puromycin, spectinomycin, tetracycline, and zeocin.

[0481] The present disclosure provides vectors in which an identifying region comprising a barcode sequence and primer binding sites can have a transgene inserted by (e.g.) the Gibson Assembly method into a specific location within or section of the vector nucleotide sequence, which is known in the art. The Gibson Assembly method is a cloning method that typically does not require restriction enzyme digestion. Briefly, the 5' end of one or more blunt end, doublestranded DNA is digested, cleaved, or otherwise pared back using a T5 exonuclease. A Phusion DNA polymerase and Taq DNA ligase are used to fill in the gaps in the plasmid and seal the nicks in the DNA backbone, respectively. See, Gibson et al. (2009) Nat Methods. 6(5):343-5, which is incorporated by reference. [0482] Additional methods of inserting a transgene into a vector provided herein include recombineering, homologous recombination, standard restriction enzyme cloning, CRISPR systems, zinc-finger nuclease (ZFN) systems, TALENS, etc., as is known by one skilled in the art.

[0483] In one aspect is provided a vector comprising an identifying region and a transgene sequence, wherein the identifying region comprises a barcode that identifies the vector, and wherein the identifying region is located within about 300 bp upstream of the transgene sequence, within about 300 bp downstream of the transgene sequence, or within the transgene sequence. In some embodiments, the vector further comprises a promoter operably linked to a transgene (e.g., a first transgene and/or a second transgene), including any of the transgenes described herein (see Section II.5: “Transgenes”). In some embodiments, the vector comprises a first barcode sequence and/or a second barcode sequence, including any of the barcodes or barcode sequences as described in Section II.2 (“Barcodes”). In some embodiments, the first barcode sequence and/or the second barcode sequence of the first identifying region and/or the second identifying region comprises a diverged nucleotide sequence. In some embodiments, the diverged nucleotide sequence within the first transgene and/or the second transgene encodes the same amino acid sequence as a non-diverged nucleotide sequence. In some embodiments, the diverged nucleotide sequence is located at a junction between one or more transgene domains. In some embodiments, the vector comprises a plasmid, a phagemid, a viral vector, a cosmid, or a transposon.

[0484] In some embodiments, a barcode sequence is a probe binding site. In some embodiments, the first identifying region and the second identifying region each has a probe binding site, such as any of the probe binding sites described herein (see Section II.4: “Identifying Regions”). In some embodiments, the first identifying region and/or the second identifying region has more than one probe binding site. In some embodiments, the first identifying region and/or the second identifying region has two or more probe binding sites. [0485] In some embodiments, the first barcode sequence and/or the second barcode sequence comprises primer binding sites, wherein the forward primer binding site is 5' of the barcode sequence, and wherein the reverse primer binding site is 3' of the barcode sequence. In some embodiments, the primer binding sites of the first barcode sequence and of the second barcode sequence comprise any of the primer binding sequences described herein, including any primer binding sites that are complementary to universal primers, such as any of the universal primers described in Section II.3 (“Primer Binding Sites”).

[0486] The vector(s) of the present disclosure comprises a first identifying region and/or a second identifying region, including any identifying region described herein (see Section II.4: “Identifying Regions”). In some embodiments, the first identifying region and the second identifying region each has at least one barcode sequence. In some embodiments, the first identifying region and/or the second identifying region has one barcode sequence. In some embodiments, the first identifying region and/or the second identifying region has more than one barcode sequence. In some embodiments, the first barcode sequence and/or the second barcode sequence is located outside of the first transgene sequence and/or the second transgene sequence. In some embodiments, the first barcode sequence and/or the second barcode sequence is located within the first transgene sequence and/or the second transgene sequence. In some embodiments, a portion of the first barcode sequence and/or the second barcode sequence is located with the first and/or the second transgene sequence, and a portion of the first barcode sequence and/or the second barcode sequence is located outside of the first and/or second transgene sequence. In some embodiments, the first identifying region is located within the first transgene sequence and/or a second identifying region that is located within the second transgene sequence. In some embodiments, the first identifying region is located outside of the first transgene sequence and the second identifying region is located outside of the second transgene sequence. In some embodiments, the first identifying region is located within the first transgene sequence and the second identifying region is located outside of the second transgene sequence. In some embodiments, the first identifying region is located within the first transgene sequence and the second identifying region is located outside of the second transgene sequence.

[0487] In some embodiments, the first identifying region is located in a non-coding region or a coding region of the vector comprising the first transgene. In some embodiments, the second identifying region is located in a second non-coding region or a second coding region of the vector comprising the second transgene. In some embodiments, the first identifying region and the second identifying region are located on the same vector. In some embodiments, the first identifying region and the second identifying region are located in the same region of the vector comprising the first transgene and the second transgene. In some embodiments, the first identifying region and the second identifying region are located on different vectors. In some embodiments, the first identifying region is upstream of the first promoter and/or the second identifying region is upstream of the second promoter. In some embodiments, the first and/or the second identifying region is located about 1 to about 200 base pairs (for example, at least about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, or 200 base pairs) 5' of the first and/or the second promoter. In some embodiments, the first and/or the second identifying region is located about 1 to about 200 base pairs (for example, at least about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, or 200 base pairs) 3' of the first and/or the second promoter. In some embodiments, the first identifying region is downstream of the first promoter and/or the second identifying region is downstream of the second promoter. In some embodiments, the first identifying region is upstream of the first promoter and the second identifying region is downstream of the second promoter. In some embodiments, the first identifying region is downstream of the first promoter and the second identifying region is upstream of the second promoter. In some embodiments, the first identifying region and/or the second identifying region is located about 1 to about 200 base pairs (e.g., at least about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, or 200 base pairs) 3' of the first transgene.

[0488] In some embodiments, the first identifying region and/or the second identifying region is upstream of one or more additional regulatory elements. In some embodiments, the first identifying region and/or second identifying region is downstream of one or more additional regulatory elements. Regulatory elements can include, but are not limited to, any one or combination of: promoter sequences, enhancer sequences, intron sequences, terminator sequences, translation initiation signal sequences, polyadenylation signal sequences, replication element sequences, RNA processing and export element sequences, transposon sequences, transposase sequences, insulator sequences, 5' UTR sequences, 3' UTR sequences, mRNA 3' end processing sequences, boundary element sequences, locus control region (LCR) sequences, matrix attachment region (MAR) sequences, ubiquitous chromatin opening elements, linker sequences, secretion signal sequences, anchoring peptide sequences, localization signal sequences, fusion tag sequences, affinity tag sequences, chaperonin sequences, protease sequences, and posttranscriptional regulatory element sequences.

[0489] The vector(s) of the present disclosure further comprise a first promoter and/or a second promoter, such as any of the promoters described herein (see Section II.4: “Identifying Regions”), wherein the first transgene is operably linked to the first promoter and/or the second transgene is operably linked to the second promoter. In some embodiments, the first identifying region is upstream of a first promoter and/or a second identifying region is upstream of a second promoter. In some embodiments, the first identifying region is downstream of the first promoter and/or the second identifying region is downstream of the second promoter. In some embodiments, the first identifying region is upstream of the first promoter and the second identifying region is downstream of the second promoter. In some embodiments, the first promoter and the second promoter comprise the same sequence. In some embodiments, the first promoter and the second promoter comprise different sequences.

[0490] In some embodiments, the vector comprises a plasmid, a phagemid, a viral vector, a cosmid, or a transposon. In some embodiments, the vector (such as a first vector and/or a second vector) encodes a transgene (such as a first transgene and/or a second transgene), wherein the transgene encodes an antibody or an antibody fragment, a chimeric antigen receptor (CAR), a T cell receptor (TCR), or one or more tolerogenic factors, such as any of the transgenes described herein (see Section II.5: “Transgenes”). In some embodiments, the first transgene and/or the second transgene encodes a CAR, including any of the CARs described in the present disclosure. In some embodiments, the CAR encoded by the first transgene and/or the second transgene comprises a hinge domain, a transmembrane domain, and one or more signaling domains. In some embodiments, the hinge domain is selected from the group consisting of: CD8a hinge domain, CD28 hinge domain, IgG4 hinge domain, IgG4 hinge-CH2-CH3 domain, and any functional variant thereof. In some embodiments, the transmembrane domain is selected from the group consisting of: alpha, beta, or zeta chain of a T cell receptor, CD28, CD3s, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CD8a, CD8P, 4-1BB/CD137, CD28, CD34, CD4, FcsRIy, CD16, OX40/CD134, CD3^, CD3s, CD3y, CD35, TCRa, TCRp, TCR^, CD32, CD64, CD64, CD45, CD5, CD9, CD22, CD37, CD80, CD86, CD40, CD40L/CD154, VEGFR2, FAS, FGFR2B, and any functional variant thereof. In some embodiments, the signaling domain is selected from the group consisting of: B7-1/CD80, B7-2/CD86, B7-H1/PD-L1, B7-H2, B7-H3, B7-H4, B7-H6, B7-H7, BTLA/CD272, CD28, CTLA-4, Gi24/VISTA/B7-H5, ICOS/CD278, PD-1, PD-L2/B7-DC, PDCD6, 4- 1BB/TNFSF9/CD137, 4-1BB Ligand/TNFSF9, BAFF/BLyS/TNFSF13B, BAFF R/TNFRSF13C, CD27/TNFRSF7, CD27 Ligand/TNFSF7, CD30/TNFRSF8, CD30 Ligand/TNFSF8, CD40/TNFRSF5, CD40/TNFSF5, CD40 Ligand/TNFSF5, DR3/TNFRSF25, GITR/TNFRSF18, GITR Ligand/TNFSF18, HVEM/TNFRSF14, LIGHT/TNFSF14, Lymphotoxin-alpha/TNFp, OX40/TNFRSF4, 0X40 Ligand/TNFSF4, RELT/TNFRSF19L, TACI/TNFRSFI 3B, TL1A/TNFSF15, TNFa, TNF RII/TNFRSF 1 B, 2B4/CD244/SLAMF4, BLAME/SLAMF8, CD2, CD2F-10/SLAMF9, CD48/SLAMF2, CD58/LFA-3, CD84/SLAMF5, CD229/SLAMF3, CRACC/SLAMF7, NTB-A/SLAMF6, SLAM/CD150, CD2, CD7, CD53, CD82/Kai-1, CD90/Thyl, CD96, CD160, CD200, CD300a/LMIRl, HLA Class I, HLA-DR, Ikaros, Integrin alpha 4/CD49d, Integrin alpha 4 beta 1, Integrin alpha 4 beta 7/LPAM-l, LAG- 3, TCL1A, TCL1B, CRTAM, DAP12, Dectin- 1/CLEC7 A, DPPIV/CD26, EphB6, TIM-l/KIM- 1/HAVCR, TIM-4, TSLP, TSLP R, lymphocyte function associated antigen-1 (LFA-1), NKG2C, CD3(^, an immunoreceptor tyrosine-based activation motif (IT AM), CD27, CD28, 4- IBB, CD134/OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, any functional variant thereof, and any combination thereof. In some embodiments, the CAR encoded by the transgene comprises a CD8a hinge domain, a CD8a transmembrane domain, a 4- IBB domain, and a CD3-zeta domain. In some embodiments, the CAR encoded by the first transgene and/or the second transgene is selected from the group consisting of: a CD5- specific CAR, a CD 19- specific CAR, a CD20-specific CAR, a CD22-specific CAR, a CD23-specific CAR, a CD30- specific CAR, a CD33-specific CAR, CD38-specific CAR, a CD70-specific CAR, a CD 123- specific CAR, a CD 138- specific CAR, a Kappa, Lambda, B cell maturation agent (BCMA)- specific CAR, a G-protein coupled receptor family C group 5 member D (GPRC5D)-specific CAR, a CD 123 -specific CAR, a LeY-specific CAR, a NKG2D ligand- specific CAR, a WT1- specific CAR, a GD2-specific CAR, a HER2-specific CAR, a EGER-specific CAR, a EGFRvIII- specific CAR, a B7H3-specific CAR, a PSMA-specific CAR, a PSCA-specific CAR, a CAIX- specific CAR, a CD 171 -specific CAR, a CEA- specific CAR, a CSPG4- specific CAR, a EPHA2- specific CAR, a FAP-specific CAR, a FRa-specific CAR, a IL-13Ra-specific CAR, a Mesothelin-specific CAR, a MUC1 -specific CAR, a MUC16-specific CAR, a R0R1 -specific CAR, a C-Met-specific CAR, a CD 133- specific CAR, a Ep-C AM- specific CAR, a GPC3- specific CAR, a HPV16-E6- specific CAR, a IL 13 Ra2- specific CAR, a MAGEA3- specific CAR, a MAGEA4- specific CAR, a MARTI -specific CAR, a NY-ESO-l-specific CAR, a VEGFR2- specific CAR, a a-Folate receptor- specific CAR, a CD24-specific CAR, a CD44v7/8- specific CAR, a EGP-2- specific CAR, a EGP-40-specific CAR, a erb-B2-specific CAR, a erb-B 2,3,4- specific CAR, a FBP- specific CAR, a Fetal acethylcholine e receptor- specific CAR, a GD2- specific CAR, a GD3-specific CAR, a HMW-MAA-specific CAR, a IL- HRa- specific CAR, a KDR-specific CAR, a Lewis Y-specific CAR, a Ll-cell adhesion molecule- specific CAR, a MAGE- Al-specific CAR, a Oncofetal antigen (h5T4)-specific CAR, a TAG-72- specific CAR, and a CD19/CD22-bispecific CAR. In some embodiments, the CAR encoded by the transgene (e.g., the first transgene and/or the second transgene) is a CD 19 CAR or a CD22 CAR. In some embodiments, the CAR encoded by the first transgene and/or the second transgene comprises from the N-terminus to the C-terminus: a CD19 antibody-binding domain, a CD8a hinge domain, a CD8a transmembrane domain, a 4- IBB domain, and a CD3zeta domain. In some embodiments, the CAR encoded by the first transgene and/or the second transgene comprises from the N-terminus to the C-terminus: a CD22 antibody-binding domain, a CD8a hinge domain, a CD8a transmembrane domain, a 4- IBB domain, and a CD3zeta domain.

[0491] In some embodiments, the CAR encoded by the first transgene and/or the second transgene further comprises one or more co-stimulatory domain(s). In some embodiments, the diverged nucleotide sequence (e.g., the diverged barcode sequence) within the transgene is located at the junction of: i) the signaling domain and the co-stimulatory domain; or ii) the hinge domain and the transmembrane domain. In some embodiments, the diverged nucleotide sequence within the transgene is located at the junction of the 4- IBB and CD3-zeta domains.

[0492] In some embodiments, the elements of the vector from 5' to 3' comprises an identifying region comprising a forward primer binding site, a barcode, and a reverse primer binding site; a promoter; and a transgene. In some embodiments, the elements of the vector from 5' to 3' comprises a first identifying region comprising a forward primer binding site, a barcode, and a reverse primer binding site; a first promoter; a first transgene; a second identifying region comprising a forward primer binding site, a barcode, and a reverse primer binding site; a second promoter; and a second transgene. In some embodiments, the elements of the vector from 5' to 3' comprises a promoter; an identifying region comprising a forward primer binding site, a barcode, and a reverse primer binding site; and a transgene. In some embodiments, the elements of the vector from 5' to 3' comprises a first promoter; a first identifying region comprising a forward primer binding site, a barcode, and a reverse primer binding site; a first transgene; a second promoter; a second identifying region comprising a forward primer binding site, a barcode, and a reverse primer binding site; and a second transgene. In some embodiments, the elements of the vector from 5' to 3' comprises a first promoter; a first identifying region comprising a forward primer binding site, a barcode, and a reverse primer binding site; a first transgene; a second identifying region comprising a forward primer binding site, a barcode, and a reverse primer binding site; a second promoter; and a second transgene. In some embodiments, the elements of the vector from 5' to 3' comprises a first identifying region comprising a forward primer binding site, a barcode, and a reverse primer binding site; a first promoter; a first transgene; a second promoter; a second identifying region comprising a forward primer binding site, a barcode, and a reverse primer binding site; and a second transgene. z. Elements for Insertion of Transgenes

[0493] The vector(s) of the present disclosure further may encode an enzyme recognition sequence, such as a recombinase, integrase, or meganuclease recognition sequence. In some embodiments, the vector comprises one or more recombination and/or cassette exchange sequences. These sites located within the vector of the present disclosure can then be leveraged to insert desired sequences, including large DNA payloads (e.g., > 100 bp) using a recombinase, integrase, meganuclease, or HDR-mediated insertion or substitution through recognition of the enzyme recognition sequence.

[0494] In some embodiments, a vector of the present disclosure comprises one or more recombination sequences, such as any of Cre recombination sequences, Bxbl recombination sequences, Flp recombination sequences, Al 18 recombination sequences, (pC31 recombination sequences, R recombinase recombination sequences, Lambda recombination sequences, HK101 recombination sequences, pSAM2 recombination sequences, Beta-siz recombination sequences, CipH recombination sequences, ParA recombination sequences, Gamma-delta recombination sequences, TP901 recombination sequences, or any combination thereof. In some embodiments, the recombination sequences flank the region of insertion of or the region encoding a first transgene and/or a second transgene encoded by a first vector and/or a second vector. In some embodiments, the recombination sequences flank to region of insertion of or the region encoding a first transgene and a first identifying region comprising a first barcode and/or a second transgene and a second identifying region comprising a second barcode encoded by a first vector and/or a second vector. In some embodiments, the recombination sequences are used in a method of making any of the vectors described herein, wherein the method of making a vector comprises homologous recombination and/or recombineering of a transgene or a transgene and an identifying region comprising a barcode into a vector backbone, such as any vector backbone known in the art or described herein.

[0495] In some embodiments, a vector of the disclosure includes enzyme recognition sequences for the bacteriophage Pl Cre/lox system, e.g., Cre recombination sequences. The Cre protein is a 343 amino acid protein that has two domains: the larger carboxyl (catalytic) domain and the smaller amino domain. When expressed in a cell containing the target lox sites, these lox sites are recombined by the Cre enzyme. LoxP locus ofX-over Pl) is a specific sequence of the bacteriophage Pl that is 34 bp and is targeted by the Cre enzyme. The site includes an asymmetric 8 bp sequence in between two sets of symmetric 13 bp sequences. The loxP sequence and various mutants are given in Table 16 below. The 13 bp sequences are palindromic but the 8 bp spacer sequence is not palindromic, thus giving the loxP sequence its directionality. In particular, loxP sites are directional such that inverted loxP sites on the same chromosome arm will cause an inversion of the DNA sequence located between the two loxP sites, whereas a direct repeat of loxP sites will cause a deletion event. LoxP sites being located on different chromosomes can cause translocation events to be catalysed upon Cre recombinase induction. When loxP sites are located in the original sequence and on a donor plasmid sequence, the donor sequence can be swapped with the original sequence in a process called recombinase- mediated cassette exchange (RMCE). In some embodiments, the Cre recombination sequences can include any of the loxP or variant sequences listed in Table 16 below. In some embodiments, the Cre recombination sequences comprise one or more of loxP, lox511, loxN, and lox2272, as well as combinations thereof. The Cre recombinase enzyme is not found naturally within mammalian cells, such as any of the mammalian cells described herein. Thus, Cre recombinase can be provided to cells to induce recombination as a polypeptide or as a nucleic acid encoding the Cre recombinase. In cases where a nucleic acid encoding the Cre recombinase is provided, expression of Cre recombinase may be controlled by an inducible promoter, including but not limited to, for example, the pL, pBAD, Tet-on/Tet-Off, Lac switch, ecdysone, cumate, or tamoxifen inducible promoters.

Table 16. Exemplary enzyme recognition sequences.

[0496] In some embodiments, a vector of the present disclosure includes enzyme recognition sequences for the mycobacteriophage large serine recombinase Bxbl, e.g., Bxbl recombination sequences. Bxbl recombinase catalyzes site- specific recombination between its corresponding attP and attB recognition sites. Depending on the relative orientation of the attP and attB sites, the reaction can result in excision, inversion, or integration of sequences between the recognition sites to yield the product sites known as attL and attR. The attP and attB sites are each 39 bp and 34 bp in length, respectively. The catalyzed reaction is unidirectional and irreversible unless an excisionase is present. In some embodiments, the Bxbl recombination sequences in a vector of the present disclosure comprise an attP or attB sequence. In some embodiments, the Bxbl recombination sequences are selected from attPO, attP15, attBO, attB 15, attP6, attP13, attB6, or attB 13, as well as combinations thereof. In some embodiments, the Bxbl recombination sequences comprise a minimal attP or attB sequence, such as any of SEQ ID NOs: 244, 277, 309, 367, and 370-374.

[0497] In some embodiments, a vector of the present disclosure includes enzyme recognition sequences for the .S'. cerevisiae recombinase, flippase (Flp), e.g., Flp recombination sequences. Flp recombines the sequences between the short flippase recognition target (FRT) sites, which are 34 bp long. The wild-type FRT F site includes an asymmetric 8 bp sequence in between two sets of 13 bp flanking arm sequences (5' GAAGTTCCTATTCTCTAGAAAGTATAGGAACTTC 3'; see Table 16, SEQ ID NO: 245). In some embodiments, the Flp recombination sequences are selected from F, F3, F5, F10, Fl l, F12, F13, F14, F15, and F16, as well as combinations thereof.

[0498] In some embodiments, a vector of the present disclosure further comprises one or more regulatory element sequences. In some embodiments, the one or more regulatory element sequences comprise one or more promoter sequences, enhancer sequences, intron sequences, terminator sequences, translation initiation signal sequences, poly adenylation signal sequences, replication element sequences, RNA processing and export element sequences, transposon sequences, transposase sequences, insulator sequences, 5’UTR sequences, 3’UTR sequences, mRNA 3’ end processing sequences, boundary element sequences, locus control region (ECR) sequences, matrix attachment region (MAR) sequences, ubiquitous chromatin opening elements, linker sequences, secretion signal sequences, anchoring peptide sequences, localization signal sequences, fusion tag sequences, affinity tag sequences, chaperonin sequences, protease sequences, or posttranscriptional regulatory element sequences. [0499] In some embodiments, a vector of the present disclosure comprises a promoter, such as any of a CAG promoter, cytomegalovirus (CMV) promoter, EFla promoter, EFla short promoter, PGK promoter, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, tk promoter of HSV, mouse mammary tumor virus (MMTV) promoter, LTR promoter of HIV, Epstein Barr virus (EBV) promoter, Rous sarcoma virus (RSV) promoter, UBC promoter, MoMuLV promoter, an avian leukemia virus promoter, actin promoter, myosin promoter, hemoglobin promoter, creatine kinase promoter, hybrid CMV enhancer/chicken P- actin (CBA) promoter, or CBA hybrid intron (CBh) promoter, or any other suitable promoter known in the art.

[0500] In some embodiments, a vector of the present disclosure comprises a polyadenylation signal, such as any of Rb B-globin polyA, Rb a-globin polyA, Human Growth Hormone polyA, Human B-globin polyA, Bovine Growth Hormone polyA, or SV40 late polyA, or any other suitable polyadenylation signal known in the art.

[0501] In some embodiments, a vector of the present disclosure comprises a linker sequence, such as any of an internal ribosome entry site (IRES) sequence, a cleavable peptide sequence, a 2A peptide sequence, a F2A peptide sequence, a E2A peptide sequence, a P2A peptide sequence, a T2A peptide sequence, or a tPT2A peptide sequence, or any other suitable linker sequence known in the art.

[0502] In some embodiments, a vector of the present disclosure comprises one or more sequences encoding one or more selection markers. In some embodiments, the one or more selection markers comprise a fluorescent protein. In some embodiments, the fluorescent protein is selected from green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (EYFP), blue fluorescent protein (BFP), enhanced blue fluorescent protein (EBFP), cyan fluorescent protein (CFP), enhanced cyan fluorescent protein (ECFP), superfolder GFP, superfolder YFP, orange fluorescent protein, red fluorescent protein, small ultrared fluorescent protein, FMN-binding fluorescent protein, dsRed, qFP611, Dronpa, TagRFP, KFP, EosFP, IrisFP, Dendra, Kaede, KikGrl, emerald fluorescent protein, Azami Green, mWasabi, TagGFP, TurboGFP, AcGFP, ZsGreen, or T-Sapphire, or any suitable fluorescent protein known in the art. In some embodiments, fluorescent protein selection markers are screened using flow cytometry to select for engineered cells or population of cells (e.g., T cells, B cells, NK cells, islet cells such as beta cells, or hypoimmunogenic cells thereof). In other embodiments, fluorescent protein selection markers are screened using spectral imaging to select for engineered cells or populations of cells. Selection for engineered cells or populations of cells can include, for example, selecting and/or sorting for engineered cells or a population of engineered cells to expand and culture in vitro, such as prior to therapeutic administration as described further herein. In some instances, selection for engineered cells can include, for example, selecting and/or sorting for engineered cells or a population of engineered cells to administer to an individual (such as a patient, for example a human patient) in need thereof.

[0503] In some embodiments, a vector of the present disclosure comprises one or more sequences encoding one or more selection proteins. In some embodiments, the one or more selection proteins are selected from a membrane-bound selection molecule, a blasticidin selection protein, a hygromycin selection protein, a puromycin selection protein, a zeocin selection protein, a neomycin selection protein, a ganciclovir selection protein, a 5’- fluorocytosine selection protein, a G418 (geneticin) selection protein, or a cytosine deaminase selection protein, or any other suitable selection protein known in the art. In some embodiments, antibiotic resistance screening is used to select for engineered cells or population of cells. In some embodiments, membrane-bound selection molecules can include, for example, membranebound biotin mimetic peptides (BMPs) or biotin acceptor peptides (BAPs) (see, e.g., W02012085911, hereby incorporated by reference in its entirety, for a list of BMPs and BAPs). In some embodiments, the membrane -bound selection molecule can include membrane peptides with mutated posttranslational modifications, for example glycosylation, acetylation, phosphorylation, nitrosylation, methylation, lipidation, etc. In some embodiments, membranebound selection molecules may include, for example, extracellular domain peptides of exogenous receptors, extracellular and transmembrane domain peptides of exogenous receptors, and/or full-length peptides of exogenous receptors that are not otherwise produced by the modified cell as described herein or population of such cells. In some embodiments, membranebound selection molecules are screened using flow cytometry to select for engineered cells or populations of cells. In some embodiments, membrane-bound selection molecules are screened using cytometry by time of flight (i.e., CyTOF) to select for engineered cells or populations of cells. In some embodiments, membrane-bound selection molecules are screened using spectral imaging to select for engineered cells or populations of cells. In some embodiments, the engineered cell or population of cells as described herein is sorted or selected by flow cytometry, CyTOF, or spectral imaging using antibodies that bind to membrane-bound selection molecules.

Hi. Insertion of Transgene Sequences into Vectors

[0504] In some embodiments, transgenes, such as any of the transgenes described herein, may be inserted at a vector, such as any vector known in the art or described herein. For example, in some embodiments, the transgene (e.g., the first transgene and/or the second transgene) may be inserted into a vector sequence as described herein. Insertion of transgenes into a vector may be accomplished by various methods and agents, for example, using recombination-based methods, zinc finger nucleases, restriction enzymes, transcription activatorlike effector nucleases (TALENs), Programmable Addition via Site-specific Targeting Elements (PASTE), nucleic acid-guided nuclease editing, and the like. In some embodiments, one or more transgenes may be inserted into a vector of the disclosure by one or more genetic rearrangements. In some embodiments, such genetic rearrangements are mediated by a sitespecific recombinase. Thus, in some cases, a vector of the disclosure comprises, or is otherwise engineered to comprise a site-specific recombinase. In some embodiments, the site-specific recombinase comprises one or more of Cre, Bxbl, Flp, Al 18, (pC31, R recombinase, Lambda, HK101, pSAM2, Beta-siz, CipH, ParA, Gamma-delta, or TP901.

[0505] In some embodiments, the site-specific recombinase is capable of targeting one or more recombination or cassette exchange sequences in the vector. In further embodiments, the site-specific recombinase is capable of inserting one or more transgenes into the vector. In some embodiments, the site-specific recombinase and the recombination or cassette exchange sequences are located on different vectors. In some embodiments, the site-specific recombinase and the recombination or cassette exchange sequences are located on the same vector.

[0506] In some embodiments, the vector comprises Cre recombination sequences, such as loxP, lox511, loxN, and lox2272, and the site-specific recombinase is Cre recombinase. In some embodiments, the vector comprises Bxbl recombination sequences, such as attP and an attB sequence (e.g., any of attPO, attP15, attBO, attB 15, attP6, attP13, attB6, and attB 13, including minimal attP or attB sequences, such as any of SEQ ID NOs: 244, 277, 309, 367, and 370-374), and the site-specific recombinase is Bxbl recombinase. In some embodiments, the vector comprises Flp recombination sequences, such as FRT, F, F3, F5, F10, Fl l, F12, F13, F14, F15, and Fl 6, and the site-specific recombinase is Flp recombinase. In some embodiments, the vector comprises Al 18 recombination sequences, and the site-specific recombinase is Al 18. In some embodiments, the vector comprises (pC31 recombination sequences, and the site-specific recombinase is (pC31 recombinase.

[0507] In some embodiments, the first transgene and/or the second transgene to be inserted into a vector are encoded by a nucleic acid. In some embodiments, the nucleic acid encoding first transgene and/or the second transgene and the vector have matching recombination or cassette exchange sequences, such that the corresponding recombinase can insert the nucleic acid into a region of the vector by recombination. In some embodiments, the vector and the nucleic acid encoding the first transgene and/or the second transgene comprise Cre recombination sequences, such as loxP, lox511, loxN, and lox2272, and the site-specific recombinase is Cre recombinase. In some embodiments, the vector and the nucleic acid encoding the first transgene and/or the second transgene comprise Bxbl recombination sequences, such as attP and an attB sequence (e.g., any of attPO, attP15, attBO, attB15, attP6, attP13, attB6, and attB13, including minimal attP or attB sequences, such as any of SEQ ID NOs: 244, 277, 309, 367, and 370-374), and the sitespecific recombinase is Bxbl recombinase. In some embodiments, the vector and the nucleic acid encoding the first transgene and/or the second transgene comprise Flp recombination sequences, such as FRT, F, F3, F5, F10, Fl l, F12, F13, F14, F15, and F16, and the site-specific recombinase is Flp recombinase. In some embodiments, the vector and the nucleic acid encoding the first transgene and/or the second transgene comprise Al 18 recombination sequences, and the site-specific recombinase is Al 18 recombinase. In some embodiments, the vector and the nucleic acid encoding the first transgene and/or the second transgene comprise (pC31 recombination sequences, and the site-specific recombinase is (pC31 recombinase.

[0508] Any of the vectors described herein can be inserted into a cell or a population of cells, such as any cell or population of cells described herein. Methods for the insertion or introduction of a first vector and/or a second vector of the present disclosure into cells are known to those of skill in the art and include, but are not limited to, lipid-mediated transfer (e.g., liposomes, including neutral and cationic lipids), electroporation, direct injection, cell fusion, particle bombardment, calcium phosphate co-precipitation, DEAE-dextran-mediated transfer and viral vector-mediated transfer. 7. Selecting as Suitable

[0509] In one aspect is provided a method of selecting a cell, population of cells, or therapy (e.g., cell therapy) comprising, detecting the presence or absence of a first barcode and/or a second barcode in the cell or population of cells, wherein the presence of the first barcode indicates the presence of a first transgene and the presence of the second barcode indicates the presence of a second transgene, and selecting the cell or population of cells as being suitable for: a) administration to a subject; b) manufacturing a drug product; c) further gene editing or genome editing; d) creating a cell bank; e) differentiation into a drug product; f) packaging for distribution; and/or g) cryopreservation, based on the presence or absence of the first barcode and/or the second barcode. In some embodiments, the therapy is a cell therapy. [0510] In another aspect is provided a method of manufacturing a therapy wherein the method comprises detecting the presence or absence of a first barcode and/or a second barcode in a cell or population of cells wherein the presence of the first barcode indicates the presence of a first transgene and the presence of the second barcode indicates the presence of a second transgene, and wherein the therapy is determined to be suitable for any one or more uses for: a) further gene editing or genome editing; b) creating a cell bank; c) differentiation into a drug product; d) packaging for distribution; and/or e) cryopreservation, based on the presence or absence of the first barcode and/or the second barcode. In some embodiments, the therapy is a cell therapy.

[0511] In some embodiments, the cell or population of cells is selected as being suitable for: a) administration to a subject; b) manufacturing a drug product; c) further gene editing or genome editing; d) creating a cell bank; e) differentiation into a drug product; f) packaging for distribution; and/or g) cryopreservation based on the presence or absence of the first barcode and/or second barcode. In some embodiments, the cell or population of cells is selected as being suitable for: a) administration to a subject; b) manufacturing a drug product; c) further gene editing or genome editing; d) creating a cell bank; e) differentiation into a drug product; f) packaging for distribution; and/or g) cryopreservation based on the presence or absence of a third (or more) barcode(s). In some embodiments, the therapy is a cell therapy. In some embodiments, the method further comprises administering to a subject in need thereof an effective dose of a cell, population of cells, or cell therapy to treat a disease in the subject. Administration to a Subject

[0512] In some embodiments, a cell, population of cells, or therapy (e.g., cell therapy) wherein the presence of the first and/or second transgene or the first, second, and/or third (or more) transgenes is detected is selected as suitable for administration to a subject. In some embodiments, a cell, population of cells, or therapy (e.g., cell therapy) wherein the absence of the first and/or second transgene or the first, second, and/or third (or more) transgenes is detected is selected as suitable for administration to a subject.

[0513] In some embodiments, detecting the presence or absence of a first and/or second transgene in a cell, population of cells, or therapy (e.g., cell therapy) and selecting the cell, population of cells, or therapy (e.g., cell therapy) as suitable for administration to a subject further comprises administering to a subject in need thereof an effective dose of the cell, population of cells, or cell therapy to treat a disease in the subject. In some embodiments, the cell, population of cells, or therapy (e.g., cell therapy) is selected as suitable for administration of a first dose. In some embodiments, the cell, population of cells, or therapy (e.g., cell therapy) is selected as suitable for administration of a second or more dose(s).

[0514] In some embodiments, the cell therapy comprises administering one or more doses of engineered immune cells expressing the first transgene and/or the second transgene. In some embodiments, the first transgene and/or the second transgene is a CAR, such as CD 19 CAR and/or CD22 CAR, such that detecting the presence or absence of the first barcode and/or the second barcode in the cell is indicative of the presence or absence of the first transgene and/or the second transgene, for example a CAR such as CD 19 CAR and/or CD22 CAR. Therefore, in some embodiments, detecting the presence of the first barcode and/or the second barcode as indicative of the presence of a CAR, such as CD 19 CAR and/or CD22 CAR, is further indicative of suitability for administration of the cell therapy to an individual in need thereof. In some embodiments, the cell therapy further comprises one or more additional administrations of the cell, population of cells, or cell therapy to the subject comprising: i) the same or different dose as the initial dose of the cell, population of cells, or cell therapy administered to the subject; and/or ii) a cell, population of cells, or cell therapy comprising the same or different transgene encoded by a vector as the initial cell, population of cells, or cell therapy administered to the subject.

[0515] Administration to a subject is described in more detail in Section VI, Subsection 1 below: “Therapeutic uses and products: Uses”. Manufacturing Drug Product(s)/Therapy

[0516] In some embodiments, a cell, population of cells, or therapy (e.g., cell therapy) wherein the presence of the first and/or second transgene or the first, second, and/or third (or more) transgenes is detected is selected as suitable for manufacturing a drug product. In some embodiments, a cell, population of cells, or therapy (e.g., cell therapy) wherein the absence of the first and/or second transgene or the first, second, and/or third (or more) transgenes is detected is selected as suitable for manufacturing a drug product. In some embodiments, the drug product is a cell therapy. In some embodiments, the cell or population of cells is a primary cell or induced pluripotent stem cell as described in Section III.A and Section III.C below. In some embodiments, the cell or population of cells is used generate a drug product, for example a peptide, propeptide, polypeptide, antibody, cytokine, chemokine, etc. In some embodiments, the cell or population of cells is a producer cell, for example packaging cell line, as described in Section III.B below.

[0517] In some embodiments, a cell or population of cells is selected as suitable for use in the differentiation of the cell or population of cells into a therapeutic product, wherein the presence of the first barcode and/or the second barcode or the first, second, and/or third (or more) barcode(s) is detected. In some embodiments, a cell or population of cells is selected as suitable for use in the differentiation of the cell or population of cells into a therapeutic produce, wherein the absence of the first barcode and/or the second barcode or the first, second, and/or third (or more) barcode(s) is detected. In some embodiments, the cell, population of cells, or cell therapy (e.g., cell therapy derived from the cell or population of cells) comprises stem cells, induced pluripotent stem cells, hematopoietic stem cells, or primary cells that are differentiated or derived into another cell type for any of the therapeutic uses described herein. For example, in some embodiments, induced pluripotent stem cells that comprise the first transgene and/or the second transgene as indicated by the presence of the first barcode and/or the second barcode can be differentiated into an islet cell, such as a beta cell, for use as a cell therapy in a patient in need thereof (e.g., a diabetes patient). In some embodiments, the selected cell, population of cells, or cell therapy are enriched for the presence of the first barcode and/or the second barcode. In some embodiments, the selected cell, population of cells, or cell therapy are further enriched for markers of the differentiated cell or therapeutic product to be administered (e.g., markers of islet cell phenotype, for example beta cell phenotype). In some embodiments, the selected cell, population of cells, or cell therapy are purified from a population of cells that comprises a mixture of cells wherein the first barcode and/or the second barcode are detected as being absent in one subset of cells and as being present in a second subset of cells, such that one subset is purified and the other subset is removed or otherwise not selected.

Gene/Genome Editing

[0518] In some embodiments, a cell, population of cells, or therapy (e.g., cell therapy) wherein the presence of the first and/or second transgene or the first, second, and/or third (or more) transgenes is detected is selected as suitable for further gene editing or genome editing. In some embodiments, a cell, population of cells, or therapy (e.g., cell therapy) wherein the absence of the first and/or second transgene or the first, second, and/or third (or more) transgenes is detected is selected as suitable for further gene editing or genome editing. In some embodiments, the cell, population of cells, or therapy (e.g., cell therapy) is selected based on detection of the presence of a first transgene, wherein the cell, population of cells, or therapy (e.g., cell therapy) undergoes further gene editing to express a second (or more) transgene(s). In some embodiments, the cell, population of cells, or therapy (e.g., cell therapy) is selected based on detection of the absence of a first transgene, wherein the cell, population of cells, or therapy (e.g., cell therapy) undergoes further gene editing to express a second (or more) transgene(s). In some embodiments, further gene editing or genome editing can comprise disruption of endogenous genes or genomic loci. In some embodiments, further gene editing or genome editing can comprise insertion and expression of a third or more transgene(s), such as any of the transgenes described herein.

[0519] In some embodiments, further gene editing or genome editing comprises introducing a modification that inactivates or disrupts one or more alleles of a gene selected from the group consisting of OITA, B2M, MHC-I, MHC-II, HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, and HLA-DR.

[0520] In some embodiments, further gene editing or genome editing comprises introducing a modification that increases expression of a transgene selected from the group consisting of CD16, CD24, CD35, CD39, CD46, CD47, CD52, CD55, CD59, CD64, CD200, CCL22, CTLA4-Ig, Cl inhibitor, FASL, IDO1, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, IL-10, IL-35, PD-L1, SERPINB9, CCL21, MFGE8, DUX4, B2M-HLA-E, CD27, IL-39, CD16 Fc Receptor, IL15-RF, H2-M3 (HLA-G), A20/TNFAIP3, CR1, HLA-F, and MANF. [0521] In some embodiments, further gene editing or genome editing comprises introducing a safety switch, such as any safety switch selected from the group consisting of cytosine deaminase (CyD), herpesvirus thymidine kinase (HSV-Tk), an inducible caspase 9 (iCaspase9), and rapamycin-activated caspase 9 (rapaCasp9).

[0522] In some embodiments, further gene editing or genome editing comprises introducing one or more transgene as described herein.

[0523] Methods of gene editing and genome editing can use any suitable methods known in the art, including, but not limited to, CRISPR/Cas, zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALENs), base editing, prime editing, programmable addition via site-specific targeting elements (PASTE), restriction enzymes, etc. Methods of delivery of gene editing and/or genome editing components to the cell, population of cells, or therapy (e.g., cell therapy) include, but are not limited to, DEAE-dextran mediated delivery, calcium phosphate precipitate method, cationic lipids mediated delivery, liposome mediated transfection, electroporation, microprojectile bombardment, receptor-mediated gene delivery, delivery mediated by poly lysine, histone, chitosan, and peptides. Standard methods for transfection and transformation of a cell, population of cells, or therapy (e.g., cell therapy) are well known in the art.

Cell Bank

[0524] In some embodiments, a cell, population of cells, or therapy (e.g., cell therapy) wherein the presence of the first and/or second transgene or the first, second, and/or third (or more) transgenes is detected is selected as suitable for creating a cell bank. In some embodiments, a cell, population of cells, or therapy (e.g., cell therapy) wherein the absence of the first and/or second transgene or the first, second, and/or third (or more) transgenes is detected is selected as suitable for creating a cell bank.

[0525] In some embodiments, the cell, population of cells, or therapy (e.g., cell therapy) are maintained in a cell bank. In some embodiments, the cell, population of cells, or therapy (e.g., cell therapy) are cryopreserved and deposited in a biorepository cell bank. In some embodiments, the cell bank is a working cell bank or a master cell bank. In some embodiments, the cell, population of cells, or therapy (e.g., cell therapy) are maintained as a collection of cryopreserved aliquots of specific cell number within a cell bank. For example, cells can be aliquoted and cryopreserved at about any of IxlO⁶, 1.5xl0⁶, 2xl0⁶, 2.5xl0⁶, 3xl0⁶, 3.5xl0⁶, 4xl0⁶, 4.5xl0⁶, 5xl0⁶, 5.5xl0⁶, 6xl0⁶, 6.5xl0⁶, 7xl0⁶, 7.5xl0⁶, 8xl0⁶, 8.5xlO⁶, 9xl0⁶, 9.5xl0⁶, or IxlO⁷ cells per vial. In some embodiments, cells are aliquoted and cryopreserved as cell pellets. In some embodiments, cells are aliquoted and cryopreserved in a suspension in freezing medium.

[0526] In some embodiments, the cell bank is on-site at the manufacturing location. In some embodiments, the cell bank is on-site at the location of cell therapy administration to the subject. In some embodiments, the cell bank is off-site. In some embodiments, the cell, population of cells, or therapy (e.g., cell therapy) are cryopreserved and deposited in the cell bank at different locations. In some embodiments, the cell, population of cells, or therapy (e.g., cell therapy) are shipped to the cell bank for long-term storage. In some embodiments, the cell, population of cells, or therapy (e.g., cell therapy) are cryopreserved and deposited in the cell bank at the same location. In some embodiments, the cell bank is a private cell bank. In some embodiments, the cell bank is a commercial cell bank. In some embodiments, the cell bank is for research purposes. In some embodiments, the cell bank is for therapeutic or medical purposes. In some embodiments, cell, population of cells, or therapy (e.g., cell therapy) deposited in the cell bank can be used for research purposes, quality control purposes, and/or therapeutic or medical purposes.

Packaging for Distribution

[0527] In some embodiments, a cell, population of cells, or therapy (e.g., cell therapy) wherein the presence of the first and/or second transgene or the first, second, and/or third (or more) transgenes is detected is selected as suitable for packaging for distribution. In some embodiments, a cell, population of cells, or therapy (e.g., cell therapy) wherein the absence of the first and/or second transgene or the first, second, and/or third (or more) transgenes is detected is selected as suitable for packaging for distribution.

[0528] In some embodiments, the cell, population of cells, or therapy (e.g., cell therapy) can be packaged in a cell flask with culture medium. In some embodiments, the cell, population of cells, or therapy (e.g., cell therapy) are cryopreserved as described below. In some embodiments, the cryopreserved cell, population of cells, or therapy (e.g., cell therapy) is frozen as a cell pellet or a suspension in freezing medium, e.g., within a cryovial. In some embodiments, the cell, population of cells, or therapy (e.g., cell therapy) is embedded in alginate cross-linked beads. [0529] In some embodiments, the cell, population of cells, or therapy (e.g., cell therapy) is packaged, e.g., in primary, secondary, and/or tertiary packaging, that meets Current Good Manufacturing Practice (CGMP) guidelines. In some embodiments, the primary, secondary, and/or tertiary packaging maintains product stability and closure integrity. In some embodiments, the primary, secondary, and/or tertiary packaging display chain of identity information. In some embodiments, the primary, secondary, and/or tertiary packaging is sterile and prevents contamination of microorganisms. In some embodiments, the primary, secondary, and/or tertiary packaging prevents contamination of gasses such as CO2, and liquid phase nitrogen and/or vapor phase nitrogen.

Cryopreservation

[0530] In some embodiments, a cell, population of cells, or therapy (e.g., cell therapy) wherein the presence of the first and/or second transgene or the first, second, and/or third (or more) transgenes is detected is selected as suitable for cryopreservation. In some embodiments, a cell, population of cells, or therapy (e.g., cell therapy) wherein the absence of the first and/or second transgene or the first, second, and/or third (or more) transgenes is detected is selected as suitable for cryopreservation. Cryopreservation preserves the cell, population of cells, or therapy for an extended period of time by freezing at low temperatures. For example, the cell, population of cells, or therapy (e.g., cell therapy) can be frozen at about any of -50°C, -55°C, -60°C, -65°C, - 70°C, -75°C, -76°C, -77°C, -78°C, -79°C, -80°C or lower temperature. In some embodiments, the cell, population of cells, or therapy (e.g., cell therapy) can be frozen at about any of -90°C, - 95°C, -100°C, -105°C, -110°C, -115°C, -120°C, -125°C, -130°C, -135°C, -140°C, -145°C, -150°C, -155°C, -160°C, -165°C, -170°C, -175°C, -180°C, -185°C, -190°C, -191°C, -192°C, -193°C, - 194°C, -195°C, -196°C, or lower temperature. In some embodiments, cryopreservation is achieved using liquid nitrogen. In some embodiments, cryopreservation is achieved using dry ice. In some embodiments, cryopreservation is achieved using a supercooling freezer. In some embodiments, cryopreservation is achieved using vitrification. In some embodiments, cryopreservation is achieved using slow freezing.

[0531] Depending on the cell types or given cells among different mammalian species, there is great diversity in cryobiological response and cryosurvival during the freezing and thawing cycle. Cryoprotectants can minimize damage to the cell viability or biology during freezing and thawing cycles. In some embodiments, the cell, population of cells, or therapy (e.g., cell therapy) is cryopreserved with a cryoprotectant. Cryoprotectants affect the rates of water transport, nucleation, and ice crystal growth to preserve the fine structure of the cell, population of cells, or therapy (e.g., cell therapy) and are typically added to the cells prior to freezing. Cryoprotectants can be cell membrane-permeating or nonmembrane -permeating. Examples of cryoprotectants include, but are not limited to, dimethyl sulfoxide (DMSO), ethylene glycol (EG), glycerol, 1,2-propanediol, 2-methyl-2,4-pentanediol, polyvinyl pyrrolidone, hydroxyethyl starch, trehalose, alginates, polyvinyl alcohol, chitosan, CELLBANKER® freezing media, sericin, various sugars, etc.

[0532] In some embodiments, cryopreservation is achieved using snap freezing. In some embodiments, cryopreservation is achieved using liquid phase nitrogen (also called “liquid nitrogen” or “LN2”). In some embodiments, cryopreservation is achieved using vapor phase nitrogen. In some embodiments, cryopreservation is achieved using dry ice.

[0533] In some embodiments, cryopreservation is achieved using slow freezing. In some embodiments, cells are cooled at a rate of about -1°C to about -2°C per minute, of about -1°C to about -3°C per minute, of about -1°C to about -4°C per minute, of about -1°C to about -5°C per minute, of about -2°C to about -3 °C per minute, of about -2°C to about -4°C per minute, of about -2°C to about -5°C per minute, of about -2°C to about -6°C per minute, of about -3°C to about - 4°C per minute, of about -3°C to about -5°C per minute, of about -3°C to about -6°C per minute, or of about -3°C to about -7°C per minute. In some embodiments, slow freezing can be achieved or aided by use of CoolCell™ Freezer Container, Mr. Frosty™ Freezing Container, or other similar devices.

[0534] In some embodiments, the cell, population of cells, or therapy (e.g., cell therapy) are cryopreserved prior to transportation (e.g., transportation to a lab for testing or transportation to a medical facility for administration to a subjection). In some embodiments, any cell, population of cells, or therapy (e.g., cell therapy) not administered to a subject is cryopreserved. In some embodiments, the cell, population of cells, or therapy (e.g., cell therapy) are cryopreserved for long-term storage. In some embodiments, the cell, population of cells, or therapy (e.g., cell therapy) are cryopreserved prior to deposit in a cell bank. In some embodiments, the cell, population of cells, or therapy (e.g., cell therapy) are cryopreserved prior to differentiation into a drug product. In some embodiments, the cell, population of cells, or therapy (e.g., cell therapy) are cryopreserved after differentiation into a drug product. In some embodiments, the cell, population of cells, or therapy (e.g., cell therapy) are cryopreserved prior to further gene editing or genome editing. In some embodiments, the cell, population of cells, or therapy (e.g., cell therapy) are cryopreserved after further gene editing or genome editing. In some embodiments, the cell, population of cells, or therapy (e.g., cell therapy) are cryopreserved prior to packaging for distribution. In some embodiments, the cell, population of cells, or therapy (e.g., cell therapy) are cryopreserved after packaging for distribution. In some embodiments, the cell, population of cells, or therapy (e.g., cell therapy) that is cryopreserved is administered to a subject, for example for a first, second, third, fourth, or more round of administration. In some embodiments, the cell, population of cells, or therapy (e.g., cell therapy) that is cryopreserved retains its biological properties. In some embodiments, the cell, population of cells, or therapy (e.g., cell therapy) that is cryopreserved has high viability upon thawing.

Additional Assays

[0535] Secondary assays may be further used to select a cell, population of cells, or therapy (e.g., cell therapy) for any of the uses described above in combination with the selection and detection methods provided herein. These secondary assays can be used to further identify the cell(s), population(s) of cells, or therapy (e.g., cell therapy) that will have an acceptable safety profile or activity profile and/or that will meet regulatory standards for administration to a subject (e.g., a human patient). Accordingly, in some embodiments, the method of selecting further comprises selecting the cell, population of cells, or cell therapy based on information obtained from one or more assays. For example, in some embodiments, the one or more assays are selected from the group consisting of a phenotypic assay, a genotypic assay, a viral sequence assay, a safety assay, an identity assay, a purity assay, and a cell count assay.

[0536] In some embodiments, the phenotypic assay is selected from the group consisting of a FACS assay, an ELISA assay, a T cell killing assay, an NK cell killing assay, a macrophage cell killing assay, a cell function assay, and any combination thereof. In some embodiments, the genotypic assay is selected from the group consisting of PCR, whole genome sequencing, whole exome sequencing, gene-targeted sequencing, qPCR, RT-qPCR, RNA sequencing, microarray analysis, in situ hybridization, serial analysis of gene expression, and any combination thereof. In some embodiments, the phenotypic and/or the genotypic assay(s) are combined with the identity assay described below to characterize the cell, population of cells, or therapy (e.g., cell therapy) produced as per the present disclosure. In some embodiments, the assay(s) may inform on the likelihood of therapeutic efficacy.

[0537] In some embodiments, the viral assay is selected from the group consisting of a Treponema pallidum antibody (Syphilis) test, a CMV antibody (Anti-CMV IgG and IgM) test, a Hepatitis B Core antibody (Anti-HBc) test, a Hepatitis B Surface Antigen (HBsAg) test, a Hepatitis C Virus antibody (Anti-HCV) test, a Human Immunodeficiency Virus antibody (HIVl/2 plus O) test, a human T-Lympho tropic Virus antibody (HTLV-I/II) test, a Trypanosoma cruzi antibody test, a human Herpes Virus 6 DNA test, a human Herpes Virus 7 DNA test, an Epstein-Barr Virus (EBV) DNA test, a Parvovirus B19 DNA test, a human Herpes Virus 8 DNA test, a Hepatitis A PCR test, a Hepatitis E Virus (HEV) Quantitative RT-PCR test, and any combination thereof.

[0538] In some embodiments, the safety assay is selected from the group consisting of mycoplasma testing, sterility testing, endotoxin testing, karyotyping, replication-competent lentivirus testing, vector copy number testing, virus screening, cytokine independent outgrowth testing, balanced translocation testing, and any combination thereof. These assays, in conjunction with the purity assays identified below, identify the cell, population of cells, or therapy (e.g., cell therapy) that is safe for administration to a subject, for example a human patient, and is in line with federal regulatory requirements for safety, sterility, and purity as per the FDA’s Good Manufacturing Practices guidelines.

[0539] In some embodiments, the purity assay is selected from the group consisting of cell viability, mycoplasma testing, sterility testing, endotoxin testing, presence/absence of residual activation beads, presence/absence of residual TCRa/p, presence/absence of a chimeric antigen receptor (CAR), presence/absence of B2M expression, presence/absence of OITA expression, presence/absence of HLA-A/B/C expression, presence/absence of HLA-DP/DQ/DR expression, presence/absence of tolerogenic factor expression, presence/absence of safety switch, and any combination thereof. These assays confirm no contamination by microorganisms that may otherwise be harmful to the subject or may reduce efficacy of the administered therapy.

[0540] In some embodiments, the identity assay is flow cytometry for the polypeptide(s) encoded by a first transgene and/or a second transgene. The identity assay confirms that the cell, population of cells, or therapy (e.g., cell therapy) demonstrate the expected identity (e.g., any cells differentiated from a stem cell, induced pluripotent stem cell, etc. is confirmed to have differentiated into the expected cell type as described in more detail in Section III below).

Assays for Hypoimmuno genic Phenotypes

[0541] In some embodiments, the cell, population of cells, or cell therapy display a hypoimmunogenic phenotype upon administration to a subject in need thereof, e.g., such that the cell, population of cells, or cell therapy (e.g., engineered islet cells as described herein) do no induce or induce a reduced immune response upon administration to the subject. As such, the cell, population of cells, or cell therapy are less likely to be, unlikely to be, or will not be rejected by the subject’s native immune system upon administration to the subject. For example, in some embodiments, the cell, population of cells, or cell therapy (e.g., engineered islet cells), such as beta cells, are modified such that they are able to evade immune recognition and responses when administered to the subject (e.g., patient, such as recipient subject). The cell, population of cells, or cell therapy (e.g., engineered islets) can evade being killed by immune cells in vitro and in vivo. In some embodiments, the cell, population of cells, or cell therapy (e.g., engineered islet cells) evade being killed by the host macrophages, T cells, and/or NK cells. In some embodiments, the cell, population of cells, or cell therapy (e.g., engineered islet cells) are ignored by immune cells or a subject’s immune system. In other words, the cell, population of cells, or cell therapy (e.g., engineered islet cells) administered in accordance with the methods described herein are not detectable by immune cells of the immune system. In some embodiments, the cell, population of cells, or cell therapy (e.g., engineered islet cells) are cloaked and therefore avoid immune rejection. [0542] Methods of determining whether a cell, population of cells, or cell therapy (for example, engineered islets) provided herein evades immune recognition include, but are not limited to, IFN-y Elispot assays, microglia killing assays, cell engraftment animal models, cytokine release assays, ELISAs, killing assays using bioluminescence imaging or chromium release assay or Xcelligence analysis, mixed-lymphocyte reactions, immunofluorescence analysis, etc.

[0543] In some embodiments, the immunogenicity of the cell, population of cells, or cell therapy (e.g., engineered islets) is evaluated in a complement-dependent cytotoxicity (CDC) assay. CDC can be assayed in vitro by incubating cells with IgG or IgM antibodies targeting an HLA- independent antigen expressed on the cell surface in the presence of serum containing complement and analyzing cell killing. In some embodiments, CDC can be assayed by incubating cells with ABO blood type incompatible serum, wherein the cells comprise A antigens or B antigens, and the serum comprises antibodies against the A antigens and/or B antigens of the cells.

[0544] In some embodiments, once the cell, population of cells, or cell therapy (e.g., engineered islets) have been modified or generated as described herein, they may be assayed for their hypoimmunogenicity. Any of a variety of assays can be used to assess if the cells are hypoimmunogenic or can evade the immune system. Exemplary assays include any as is described in WO2016183041 and WO2018132783.

[0545] In some embodiments, the cell, population of cells, or cell therapy (e.g., engineered islets) described herein survive in a host without stimulating the host immune response for one week or more (e.g., one week, two weeks, one month, two months, three months, 6 months, one year, two years, three years, four years, five years or more, e.g. for the life of the cell and/or its progeny). The cell, population of cells, or cell therapy (e.g., engineered islets) maintain expression of the transgenes and/or are deleted or reduced in expression of target genes for as long as they survive in the host. In some aspects, if the transgenes are no longer expressed and/or if target genes are expressed, the cell, population of cells, or cell therapy (e.g., engineered islets) may be removed by the host's immune system. In some embodiments, the persistence or survival of the cell, population of cells, or cell therapy (e.g., engineered islets) may be monitored after their administration to a recipient by further expressing a transgene encoding a protein that allows the cells to be detected in vivo (e.g. a fluorescent protein, such as GFP, a truncated receptor or other surrogate marker or other detectable marker).

[0546] The hypoimmunogenic cells, population of cells, or cell therapy are administered in a manner that permits them to engraft to the intended tissue site and reconstitute or regenerate the functionally deficient area. In some embodiments, the hypoimmunogenic cells, population of cells, or cell therapy are assayed for engraftment (e.g., successful engraftment). In some embodiments, the engraftment of the hypoimmunogenic cells, population of cells, or cell therapy is evaluated after a pre-selected amount of time. In some embodiments, the engrafted cells, population of cells, or cell therapy are monitored for cell survival. For example, the cell survival may be monitored via bioluminescence imaging (BEI), wherein the cells, population of cells, or cell therapy are transduced with a luciferase expression construct for monitoring cell survival. In some embodiments, the engrafted cells, population of cells, or cell therapy are visualized by immuno staining and imaging methods known in the art. In some embodiments, the engrafted cells, population of cells, or cell therapy express known biomarkers that may be detected to determine successful engraftment. For example, flow cytometry may be used to determine the surface expression of particular biomarkers. In some embodiments, the hypoimmunogenic cells, population of cells, or cell therapy are engrafted to the intended tissue site as expected (e.g. successful engraftment of the hypoimmunogenic cells). In some embodiments, the hypoimmunogenic cells, population of cells, or cell therapy are engrafted to the intended tissue site as needed, such as at a site of cellular deficiency. In some embodiments, the hypoimmunogenic cells, population of cells, or cell therapy are engrafted to the intended tissue site in the same manner as a cell, population of cells, or cell therapy of the same type not comprising the modifications. [0547] In some embodiments, the hypoimmunogenic cells, population of cells, or cell therapy are assayed for function. In some embodiments, the hypoimmunogenic cells, population of cells, or cell therapy are assayed for function prior to their engraftment to the intended tissue site. In some embodiments, the hypoimmunogenic cells, population of cells, or cell therapy are assayed for function following engraftment to the intended tissue site. In some embodiments, the function of the hypoimmunogenic cells, population of cells, or cell therapy is evaluated after a pre-selected amount. In some embodiments, the function of the engrafted cells, population of cells, or cell therapy is evaluated by the ability of the cells to produce a detectable phenotype. For example, engrafted beta islet cells function may be evaluated based on the restoration of lost glucose control due to diabetes. In some embodiments, the function of the hypoimmunogenic cells, population of cells, or cell therapy is as expected (e.g., successful function of the hypoimmunogenic cells while avoiding antibody-mediated rejection). In some embodiments, the function of the hypoimmunogenic cells, population of cells, or cell therapy is as needed, such as sufficient function at a site of cellular deficiency while avoiding antibody-mediated rejection. For example, in some embodiments, the engineered islets function in the same manner as a non-modified cell of the same type. z. Complement dependent cytotoxicity

[0548] In some embodiments, the cell, population of cells, or cell therapy (e.g., engineered islets) provided herein evade complement dependent cytotoxicity (CDC). In some embodiments, the CDC is secondary to a thrombotic reaction of IB MIR. In some embodiments, the CDC occurs independently of IBMIR. [0549] In some embodiments, susceptibility of cells to CDC can be analyzed in vitro according to standard protocols understood by one of ordinary skill in the art. In some embodiments, CDC can be analyzed in vitro by mixing serum comprising the components of the complement system (e.g., human serum), with target cells bound by an antibody (e.g., an IgG or IgM antibody), and then to determine cell death. In some embodiments, susceptibility of cells to CDC can be analyzed in vitro by incubating cells in the presence of ABO-incompatible or Rh factor incompatible serum, comprising the components of the complement system and antibodies against ABO type A, ABO type B, and/or Rh factor antigens of the cells.

[0550] A common CDC assay determines cell death via pre-loading the target cells with a radioactive compound. As cells die, the radioactive compound is released from them. Hence, the efficacy of the antibody to mediate cell death is determined by the radioactivity level. Unlike radioactive CDC assays, non-radioactive CDC assays often determine the release of abundant cell components, such as GAPDH, with fluorescent or luminescent determination. In some embodiments, cell killing by CDC can be analyzed using a label-free platform such as xCELLigence™ (Agilent).

Assays to Assess Hypoimmunogenic islets/SC-[> cell function

[0551] In some embodiments, the cell, population of cells, or cell therapy, such as engineered hypoimmunogenic islet cells, exhibits one or more functions of a wild-type or control cell, for example beta islet cell, optionally wherein the one or more functions of the beta islet cell is selected from the group consisting of in vitro glucose-stimulated insulin secretion (GSIS), glucose metabolism, maintaining fasting blood glucose levels, secreting insulin in response to glucose injections in vivo, and clearing glucose after a glucose injection in vivo.

[0552] A key functional feature of a P cell is its ability to repeatedly perform glucose stimulated insulin secretion (GSIS). In certain embodiments, assays can be performed to determine the physiological function in vitro of secreting insulin in response to glucose. In some embodiments, the engineered hypoimmunogenic islet cell (e.g., beta (P) cell) is capable of glucose-stimulated insulin secretion (GSIS), optionally wherein the insulin secretion is in a perfusion GSIS assay. In certain embodiments, the GSIS assay may be a perfusion GSIS (dynamic GSIS) assay (for example as in Velazco-Cruz, Stem Cell Reports, 2019). In some embodiments, the GSIS is dynamic GSIS comprising first and second phase dynamic insulin secretion. In some embodiments, the GSIS is static GSIS, optionally wherein the static incubation index is greater than at or about 1, greater than at or about 2, greater than at or about 5, greater than at or about 10 or greater than at or about 20.

[0553] In some embodiments, the level of insulin secretion by the engineered hypoimmunogenic islets is at least 20% of that observed for primary islets, optionally cadaveric islets. In some embodiments, the level of insulin secretion by the engineered hypoimmunogenic islets is at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70% of that observed for primary islets, optionally cadaveric islets. In some embodiments, the total insulin content of the engineered hypoimmunogenic islets is greater than at or about 500 pIU Insulin per 5000 cells, greater than at or about 1000 pIU Insulin per 5000 cells, greater than at or about 2000 pIU Insulin per 5000 cells, greater than at or about 3000 pIU Insulin per 5000 cells or greater than at or about 4000 pIU Insulin per 5000 cells. In some embodiments, the proinsulin to insulin ratio of the beta cell is between at or about 0.02 and at or about 0.1, optionally at or about 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09 and any value between any of the foregoing.

[0554] In some embodiments, the cell, population of cells, or cell therapy (e.g., engineered hypoimmunogenic islet cell) exhibits functionality for more than 2 weeks following transplantation into a subject. In some embodiments, the cell, population of cells, or cell therapy (e.g., engineered hypoimmunogenic islets) exhibits functionality for more than 3 weeks, for more than 4 weeks, for more than 8 weeks, for more than 3 months, for more than 6 months or for more than 12 months following transplantation into a subject. For example, in some embodiments, the functionality of the hypoimmunogenic islet cell is selected from the group consisting of maintaining fasting blood glucose levels, secreting insulin in response to glucose injections in vivo, and clearing glucose after a glucose injection in vivo.

[0555] In certain embodiments, other assays can be performed to examine the expression of specific genes, pathways, and transcription factors in the cell, population of cells, or cell therapy, e.g., hypoimmunogenic islet cell. Such assays include those detecting the presence of Yap (Rosado-Olivieri et al., 2019), the ROCKII pathway (Ghazizadeh et al., 2017), the transforming growth factor b (TGF-b) pathway (Velazco-Cruz, et al., 2019), the cytoskeleton (Hogrebe et al., 2020), and the expression of SIX2 (Velazco-Cruz et al., Cell Reports, 2020). In certain embodiments, other transcription factors important for the SC-P cell phenotype include PDX1, NKX6-1, NKX2-2, and NEURODI (Hogrebe et al., 2020). [0556] In certain embodiments, an assay measuring changes in intracellular Ca²⁺ may be performed as described in Pagliuca et al. (Cell, 2014). Beta cells (i.e., P cells) sense changing glucose levels through calcium signaling; increasing glucose levels leads to membrane depolarization causing an influx of calcium ions which triggers insulin exocytosis (Mohammed et al., 2009). In certain embodiments, the functional SC-P cells exhibit calcium flux similarly to primary human islet cells.

[0557] In certain embodiments, assays can also be performed to assess in vivo functionality of the SC-P cells. An example of such an assay can be found in Pagliuca et al. (Cell, 2014). Briefly, to test their capacity to function in vivo, SC-P cells are transplanted under the kidney capsule of immunocompromised mice and the ability of the cells to produce insulin is analyzed. III. CELLS

[0558] The present disclosure provides cells, for example a population of cells, that have been engineered (hereafter interchangeably “cell” or “engineered cell”), e.g., to comprise one or more vectors that encode one or more transgene(s) and accompanying barcode(s), as well as compositions, methods, uses, and kits related thereto, as described herein. A cell according to the present disclosure may be any suitable cell known in the art, such as bacterial, mammalian, insect, fungal, or plant cells. The cells may be cell lines, primary cells, stem cells, differentiated cells derived or produced from such stem cells, hematopoietic stem cells, induced pluripotent stem cells, and the like. In some embodiments, the cell is a human cell. In some embodiments, the cell is a murine cell.

A. Primary and Induced Pluripotent Stem Cell-Derived Cells

[0559] In some embodiments, the present disclosure provides a cell (e.g., stem cell, induced pluripotent stem cell, differentiated cell derived or produced from such stem cell, hematopoietic stem cell, or primary cell), or population thereof, wherein the cell or population thereof comprises any of the vectors described herein. In some embodiments, the host cell is a mammalian cell. In some embodiments, the mammalian cell is a human cell. In some embodiments, the cell is in an immune cell. In some embodiments, the cell is a primary human cell.

[0560] In some embodiments, the cells (i.e., engineered cells or populations of cells) as provided herein are pluripotent stems cells or are cells differentiated from pluripotent stem cells. In some embodiments, the cells are primary cells. [0561] The cell may be a vertebrate cell, for example, a mammalian cell, such as a human cell or a mouse cell. The cell may also be a vertebrate stem cell, for example, a mammalian stem cell, such as a human stem cell or a mouse stem cell. Preferably, the cell or stem cell is amenable to modification. Preferably, the cell or stem cell, or a cell derived from such a stem cell, has or is believed to have therapeutic value, such that the cell or stem cell or a cell derived or differentiated from such stem cell may be used to treat a disease, disorder, defect or injury in a subject in need of treatment for same.

[0562] In some embodiments, the cell is a stem cell or progenitor cell (e.g., iPSC, embryonic stem cell, hematopoietic stem cell, mesenchymal stem cell, endothelial stem cell, epithelial stem cell, adipose stem or progenitor cells, germline stem cells, lung stem or progenitor cells, mammary stem cells, olfactory adult stem cells, hair follicle stem cells, multipotent stem cells, amniotic stem cells, cord blood stem cells, or neural stem or progenitor cells). In some embodiments, the stem cells are adult stem cells (e.g., somatic stem cells or tissue specific stem cells). In some embodiments, the stem or progenitor cell is capable of being differentiated (e.g., the stem cell is totipotent, pluripotent, or multipotent). In some embodiments, the cell is isolated from embryonic or neonatal tissue. In some embodiments, the cell is a fibroblast, monocytic precursor, B cell, exocrine cell, pancreatic progenitor, endocrine progenitor, hepatoblast, myoblast, preadipocyte, progenitor cell, hepatocyte, chondrocyte, smooth muscle cell, K562 human erythroid leukemia cell line, bone cell, synovial cell, tendon cell, ligament cell, meniscus cell, adipose cell, dendritic cells, or natural killer cell. In some embodiments, the cell is manipulated (e.g., converted or differentiated) into a muscle cell, erythroid-megakaryocytic cell, eosinophil, iPSC, macrophage, T cell, islet beta-cell, neuron, cardiomyocyte, blood cell, endocrine progenitor, exocrine progenitor, ductal cell, acinar cell, alpha cell, beta cell, delta cell, PP cell, hepatocyte, cholangiocyte, or brown adipocyte. In some embodiments, the cell is a muscle cell (e.g., skeletal, smooth, or cardiac muscle cell), erythroid- megakaryocytic cell, eosinophil, iPSC, macrophage, T cell, islet beta-cell, neuron, cardiomyocyte, blood cell (e.g., red blood cell, white blood cell, or platelet), endocrine progenitor, exocrine progenitor, ductal cell, acinar cell, alpha cell, beta cell, delta cell, PP cell, hepatocyte, cholangiocyte, or white or brown adipocyte. In some embodiments, the cell is a hormone- secreting cell (e.g., a cell that secretes insulin, oxytocin, endorphin, vasopressin, serotonin, somatostatin, gastrin, secretin, glucagon, thyroid hormone, bombesin, cholecystokinin, testosterone, estrogen, or progesterone, renin, ghrelin, amylin, or pancreatic polypeptide), an epidermal keratinocyte, an epithelial cell (e.g., an exocrine secretory epithelial cell, a thyroid epithelial cell, a keratinizing epithelial cell, a gall bladder epithelial cell, or a surface epithelial cell of the cornea, tongue, oral cavity, esophagus, anal canal, distal urethra, or vagina), a kidney cell, a germ cell, a skeletal joint synovium cell, a periosteum cell, a bone cell (e.g., osteoclast, osteocyte, or osteoblast), a perichondrium cell (e.g., a chondroblast or chondrocyte), a cartilage cell (e.g., chondrocyte), a fibroblast, an endothelial cell, a pericardium cell, a meningeal cell, a keratinocyte precursor cell, a keratinocyte stem cell, a pericyte, a glial cell, an ependymal cell, a cell isolated from an amniotic or placental membrane, or a serosal cell (e.g., a serosal cell lining body cavities).

[0563] In some embodiments, the cell is a somatic cell. In some embodiments, the cells are derived from skin or other organs, e.g., heart, brain or spinal cord, liver, lung, kidney, pancreas, bladder, bone marrow, spleen, intestine, or stomach. The cells can be from humans or other mammals (e.g., rodent, non-human primate, bovine, or porcine cells).

[0564] In some embodiments, the cell is a mammalian cell, for example a primary cell or a cell line, selected from the group consisting of: islet cells, beta islet cells, pancreatic islet cells, immune cells, B cells, T cells, natural killer (NK) cells, natural killer T (NKT) cells, macrophage cells, endothelial cells, muscle cells, cardiac muscle cells, smooth muscle cells, skeletal muscle cells, dopaminergic neurons, retinal pigmented epithelium cells, optic cells, hepatocytes, thyroid cells, skin cells, glial progenitor cells, neural cells, cardiac cells, stem cells, hematopoietic stem cells, induced pluripotent stem cells (iPSCs), mesenchymal stem cells (MSCs), embryonic stem cells (ESCs), pluripotent stem cell (PSCs), blood cells, endothelial stem cells, epithelial stem cells, adipose stem or progenitor cells, germline stem cells, lung stem or progenitor cells, mammary stem cells, olfactory adult stem cells, hair follicle stem cells, multipotent stem cells, amniotic stem cells, cord blood stem cells, neural stem or progenitor cells, CHO cells, BHK cells, MDCK cells, C3H 10T1/2 cells, FLY cells, Psi-2 cells, BOSC 23 cells, PA317 cells, WEHI cells, COS cells, BSC 1 cells, BSC 40 cells, BMT 10 cells, VERO cells, W138 cells, MRC5 cells, A549 cells, HT1080 cells, 293 cells, 293T cells, B-50 cells, 3T3 cells, NIH3T3 cells, HepG2 cells, Saos-2 cells, Huh7 cells, HeLa cells, W163 cells, 211 cells, NSO, PerC6, Sp2/0, BHK, C127, and 211 A cells. [0565] In some embodiments, the cell is an immune cell, such as, for example, an immune cell selected from the group consisting of: a natural killer (NK) cell, a natural killer T (NKT) cell, a T cell (e.g., CTL), CAR-T cell, a CD 14+ cell, a dendritic cell, a PBMC cell, and any combination thereof. In some embodiments, the cell is an NK cell, a T cell (e.g., CTL), or a PBMC.

Primary cells

[0566] In some embodiments, the cell or population of cells as provided herein comprise cells derived from primary cells obtained or isolated from one or more individual subjects or donors. In some embodiments, the cells are derived from a pool of isolated primary cells obtained from one or more (e.g., two or more, three or more, four or more, five or more, ten or more, twenty or more, fifty or more, or one hundred or more) different donor subjects. In some embodiments, the primary cells isolated or obtained from the plurality of different donor subjects (e.g., two or more, three or more, four or more, five or more, ten or more, twenty or more, fifty or more, or one hundred or more) are pooled together in a batch and are engineered in accord with the provided methods.

[0567] In some embodiments, the primary cells are from a pool of primary cells from one or more donor subjects that are different than the recipient subject (e.g., the patient administered the cells). The primary cells can be obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100 or more donor subjects and pooled together. The primary cells can be obtained from 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10, or more 20 or more, 50 or more, or 100 or more donor subjects and pooled together. In some embodiments, the primary cells are harvested from one or a plurality of individuals, and in some instances, the primary cells or the pool of primary T cells are cultured in vitro. In some embodiments, the primary cells or the pool of primary T cells are engineered in accord with the methods provided herein.

[0568] In some embodiments, the methods include obtaining or isolating a desired type of primary cell (e.g., T cells, NK cells, NKT cells, endothelial cell, islet cell, beta islet cell, hepatocyte or other primary cells as described herein) from individual donor subjects, pooling the cells to obtain a batch of the primary cell type, and engineering the cells by the methods provided herein. In some embodiments, the methods include obtaining or isolating a desired type of primary cell (e.g., T cells, NK cells, endothelial cell, beta islet cell, hepatocyte or other primary cells as described herein), engineering cells of each of the individual donors by the methods provided herein, and pooling engineered cells or populations of cells of at least two individual samples to obtain a batch of engineered cells or populations of cells of the primary cell type.

[0569] In some embodiments, the primary cells are isolated or obtained from an individual or from a pool of primary cells isolated or obtained from more than one individual donor. The primary cells may be any type of primary cell described herein, including any described in herein. In some embodiments, the primary cells are selected from T cells, NK cells, beta islet cells, endothelial cells, epithelial cells such as RPE, thyroid, skin, or hepatocytes. In some embodiments, the primary cells from an individual donor or a pool of individual donors are engineered to contain and express any of the vectors described herein.

[0570] In some embodiments, the engineered cell or population of cells is a muscle cell (e.g., skeletal, smooth, or cardiac muscle cell), a skin cell, optic cells, immune cells, B cells, natural killer T cells, macrophages, erythroid-megakaryocytic cell, eosinophil, iPSC, macrophage, T cell, islet cluster, islet cell, beta-cell, neuron, cardiomyocyte, blood cell (e.g., red blood cell, white blood cell, or platelet), endocrine progenitor, exocrine progenitor, ductal cell, acinar cell, alpha cell, beta islet cell, delta cell, PP cell, hepatocyte, cholangiocyte, or white or brown adipocyte. In some embodiments, the cell is a hormone- secreting cell (e.g., a cell that secretes insulin, oxytocin, endorphin, vasopressin, serotonin, somatostatin, gastrin, secretin, glucagon, thyroid hormone, bombesin, cholecystokinin, testosterone, estrogen, or progesterone, renin, ghrelin, amylin, or pancreatic polypeptide), an epidermal keratinocyte, an epithelial cell (e.g., an exocrine secretory epithelial cell, a thyroid epithelial cell, a keratinizing epithelial cell, a gall bladder epithelial cell, or a surface epithelial cell of the cornea, tongue, oral cavity, esophagus, anal canal, distal urethra, or vagina), a kidney cell, a germ cell, a skeletal joint synovium cell, a periosteum cell, a bone cell (e.g., osteoclast, osteocyte, or osteoblast), a perichondrium cell (e.g., a chondroblast or chondrocyte), a cartilage cell (e.g., chondrocyte), a fibroblast, an endothelial cell, a pericardium cell, a meningeal cell, a keratinocyte precursor cell, a keratinocyte stem cell, a pericyte, a glial cell, an ependymal cell, a cell isolated from an amniotic or placental membrane, or a serosal cell (e.g., a serosal cell lining body cavities). Induced pluripotent stem cells

[0571] In some embodiments, the cell or population thereof as provided herein are induced pluripotent stem cells or are engineered cells or populations of cells that are derived from or differentiated from induced pluripotent stem cells. The generation of mouse and human pluripotent stem cells (generally referred to as iPSCs; miPSCs for murine cells or hiPSCs for human cells) is generally known in the art. As will be appreciated by those skilled in the art, there are a variety of different methods for the generation of iPCSs. The original induction was done from mouse embryonic or adult fibroblasts using the viral introduction of four transcription factors, Oct3/4, Sox2, c-Myc and Klf4; see Takahashi and Yamanaka Cell 126:663-676 (2006), hereby incorporated by reference in its entirety and specifically for the techniques outlined therein. Since then, a number of methods have been developed; see Seki et al., World J. Stem Cells 7(1): 116-125 (2015) for a review, and Lakshmipathy and Vermuri, editors, Methods in Molecular Biology: Pluripotent Stem Cells, Methods and Protocols, Springer 2013, both of which are hereby expressly incorporated by reference in their entirety, and in particular for the methods for generating hiPSCs (see, for example, Chapter 3 of the latter reference).

[0572] Generally, iPSCs are generated by the transient expression of one or more “reprogramming factors” in the host cell, usually introduced using episomal vectors. Under these conditions, small amounts of the cells are induced to become iPSCs (in general, the efficiency of this step is low, as no selection markers are used). Once the cells are "reprogrammed" and become pluripotent, they lose the episomal vector(s) and produce the factors using the endogenous genes.

[0573] As is also appreciated by those of skill in the art, the number of reprogramming factors that can be used or are used can vary. Commonly, when fewer reprogramming factors are used, the efficiency of the transformation of the cells to a pluripotent state goes down, as well as the "pluripotency", e.g., fewer reprogramming factors may result in cells that are not fully pluripotent but may only be able to differentiate into fewer cell types.

[0574] In some embodiments, a single reprogramming factor, OCT4, is used. In other embodiments, two reprogramming factors, OCT4 and KLF4, are used. In other embodiments, three reprogramming factors, OCT4, KLF4, and SOX2, are used. In other embodiments, four reprogramming factors, OCT4, KLF4, SOX2, and c-Myc, are used. In other embodiments, 5, 6, or 7 reprogramming factors can be selected from the group including, but not limited to: SOKMNLT, SOX2, OCT4 (POU5F1), KLF4, MYC, NANOG, LIN28, and SV40L T antigen. In general, these reprogramming factor genes are provided on episomal vectors such as are known in the art and commercially available.

[0575] In some embodiments, the hosts cells used for transfecting the one or more reprogramming factors are non-pluripotent stem cells. In general, as is known in the art, iPSCs are made from non-pluripotent cells such as, but not limited to, blood cells, fibroblasts, etc., by transiently expressing the reprogramming factors as described herein. In some embodiments, the non-pluripotent cells, such as fibroblasts, are obtained or isolated from one or more individual subjects or donors prior to reprogramming the cells. In some embodiments, iPSCs are made from a pool of isolated non-pluripotent stems cells, e.g., fibroblasts, obtained from one or more (e.g., two or more, three or more, four or more, five or more, ten or more, twenty or more, fifty or more, or one hundred or more) different donor subjects. In some embodiments, the non- pluripotent cells, such as fibroblasts, are isolated or obtained from a plurality of different donor subjects (e.g., two or more, three or more, four or more, five or more, ten or more, twenty or more, fifty or more, or one hundred or more), pooled together in a batch, reprogrammed as iPSCs, and engineered in accord with the provided methods.

[0576] In some embodiments, the iPSCs are derived from, such as by transiently transfecting one or more reprogramming factors into cells from a pool of non-pluripotent cells (e.g., fibroblasts) from one or more donor subjects that are different than the recipient subject (e.g., the patient administered the cells). The non-pluripotent cells (e.g., fibroblasts) to be induced to iPSCs can be obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100 or more donor subjects and pooled together. The non-pluripotent cells (e.g., fibroblasts) can be obtained from 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10, or more, 20 or more, 50 or more, or 100 or more donor subjects and pooled together. In some embodiments, the non-pluripotent cells (e.g., fibroblasts) are harvested from one or a plurality of individuals, and in some instances, the non-pluripotent cells (e.g., fibroblasts) or the pool of non-pluripotent cells (e.g., fibroblasts) are cultured in vitro and transfected with one or more reprogramming factors to induce generation of iPSCs. In some embodiments, the non- pluripotent cells (e.g., fibroblasts) or the pool of non-pluripotent cells (e.g., fibroblasts) are engineered or modified in accord with the methods provided herein. In some embodiments, the engineered iPSCs or a pool of engineered iPSCs are then subjected to a differentiation process for differentiation into any cells of an organism and tissue.

[0577] Once the engineered iPSCs cells have been generated, they may be assayed for their hypoimmunogenicity and/or retention of pluripotency as is described in W02016183041 and WO2018132783. In some embodiments, hypoimmunogenicity is assayed using a number of techniques as exemplified in Figure 13 and Figure 15 of WO2018132783. These techniques include transplantation into allogeneic hosts and monitoring for hypoimmunogenic pluripotent cell growth (e.g., teratomas) that escape the host immune system. In some instances, hypoimmunogenic pluripotent cell derivatives are transduced to express luciferase and can then be followed using bioluminescence imaging. Similarly, the T cell and/or B cell response of the host animal to such cells are tested to confirm that the cells do not cause an immune reaction in the host animal. T cell responses can be assessed by Elispot, ELISA, FACS, PCR, or mass cytometry (CYTOF). B cell responses or antibody responses are assessed using FACS or Luminex. Additionally, or alternatively, the cells may be assayed for their ability to avoid innate immune responses, e.g., NK cell killing, as is generally shown in Figures 14 and 15 of WO2018132783.

[0578] In some embodiments, the immunogenicity of the cells is evaluated using T cell immunoassays such as T cell proliferation assays, T cell activation assays, and T cell killing assays recognized by those skilled in the art. In some cases, the T cell proliferation assay includes pretreating the cells with interferon-gamma and coculturing the cells with labelled T cells and assaying the presence of the T cell population (or the proliferating T cell population) after a preselected amount of time. In some cases, the T cell activation assay includes coculturing T cells with the cells outlined herein and determining the expression levels of T cell activation markers in the T cells.

[0579] In vivo assays can be performed to assess the immunogenicity of the cells outlined herein. In some embodiments, the survival and immunogenicity of engineered or modified iPSCs is determined using an allogeneic humanized immunodeficient mouse model. In some instances, the engineered or modified iPSCs are transplanted into an allogeneic humanized NSG- SGM3 mouse and assayed for cell rejection, cell survival, and teratoma formation. In some instances, grafted engineered iPSCs or differentiated cells thereof display long-term survival in the mouse model. [0580] Additional techniques for determining immunogenicity including hypoimmunogenicity of the cells are described in, for example, Deuse et al., Nature Biotechnology, 2019, 37, 252-258 and Han et al., Proc Natl Acad Sci USA, 2019, 116(21), 10441-10446, the disclosures including the figures, figure legends, and description of methods are incorporated herein by reference in their entirety.

[0581] Similarly, the retention of pluripotency is tested in a number of ways. In one embodiment, pluripotency is assayed by the expression of certain pluripotency-specific factors as generally described herein and shown in Figure 29 of WO2018132783. Additionally, or alternatively, the pluripotent cells are differentiated into one or more cell types as an indication of pluripotency.

[0582] Once the engineered pluripotent stem cells (engineered iPSCs) have been generated, they can be maintained in an undifferentiated state as is known for maintaining iPSCs. For example, the cells can be cultured on Matrigel using culture media that prevents differentiation and maintains pluripotency. In addition, they can be in culture medium under conditions to maintain pluripotency.

[0583] Any of the pluripotent stem cells described herein can be differentiated into any cells of an organism and tissue. In an aspect, provided herein are engineered cells or populations of cells that are differentiated into different cell types from iPSCs for subsequent transplantation into recipient subjects. Differentiation can be assayed as is known in the art, generally by evaluating the presence of cell-specific markers. As will be appreciated by those in the art, the differentiated engineered (e.g., hypoimmunogenic) pluripotent cell derivatives can be transplanted using techniques known in the art that depends on both the cell type and the ultimate use of these cells. Exemplary types of differentiated cells and methods for producing the same are described below. In some embodiments, the iPSCs may be differentiated to any type of cell described herein. In some embodiments, the iPSCs are differentiated into cell types selected from T cells, NK cells, beta islet cells, endothelial cells, epithelial cells such as RPE, thyroid, skin, or hepatocytes. In some embodiments, host cells such as non-pluripotent cells (e.g., fibroblasts) from an individual donor or a pool of individual donors are isolated or obtained, generated into iPSCs in which the iPSCs are then engineered to contain modifications (e.g., genetic modifications) described herein and then differentiated into a desired cell type. Islet cells

[0584] In some embodiments, the engineered cells or populations of cells that are provided herein are islet cells, such as beta cells, primary beta cells, pancreatic islet cells or pancreatic beta cells. In some embodiments, the islet cells are isolated or obtained from one or more individual donor subjects, such as one or more individual healthy donor (e.g., a subject that is not known or suspected of, e.g., not exhibiting clinical signs of, a disease or infection). As will be appreciated by those skilled in the art, methods of isolating or obtaining islet cells from an individual can be achieved using known techniques.

[0585] In some embodiments, islet cells, such as beta cells, primary beta cells, pancreatic islet cells or pancreatic beta cells, are obtained (e.g., harvested, extracted, removed, or taken) from a subject or an individual. In some embodiments, islet cells are produced from a pool of islet cells such that the islet cells are from one or more subjects (e.g., one or more human including one or more healthy humans). In some embodiments, the pool of islet cells is from 1- 100, 1-50, 1-20, 1-10, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, or 100 or more subjects.

[0586] In some embodiments, the engineered cells or populations of cells as provided herein are islet cells derived from engineered iPSCs that contain any of the vectors described herein and that are differentiated into beta islet cells. As will be appreciated by those skilled in the art, the methods for differentiation depend on the desired cell type using known techniques. In some embodiments, islet cells are derived from the engineered pluripotent cells described herein. Useful methods for differentiating pluripotent stem cells into islet cells are described, for example, in U.S. Patent No. 9,683,215; U.S. Patent No. 9,157,062; U.S. Patent No. 8,927,280; U.S. Patent Pub. No. 2021/0207099; Hogrebe et al., Nat. Biotechnol., 2020, 38:460-470; and Hogrebe et al., Nat. Protoc., 2021, the contents of which are herein incorporated by reference in their entirety.

[0587] In some embodiments, the engineered pluripotent cells described herein are differentiated into beta-like cells or islet organoids. Pagliuca et al. (Cell, 2014, 159(2) :428-39) and Vegas et al. (Nat Med, 2016, 22(3):306-l 1) report on the successful differentiation of P-cells from hiPSCs, the contents of which are incorporated herein by reference in their entirety.

[0588] In some embodiments, the method of producing engineered islet cells from engineered pluripotent cells by in vitro differentiation comprises: (a) culturing the engineered iPSCs in a first culture medium comprising one or more factors selected from the group consisting insulin-like growth factor, transforming growth factor, FGF, EGF, HGF, SHH, VEGF, transforming growth factor-P superfamily, BMP2, BMP7, a GSK inhibitor, an ALK inhibitor, a BMP type 1 receptor inhibitor, and retinoic acid to produce a population of immature pancreatic islet cells; and (b) culturing the immature islet cells in a second culture medium that is different than the first culture medium to produce engineered islet cells. In some embodiments, the GSK inhibitor is CHIR-99021, a derivative thereof, or a variant thereof. In some instances, the GSK inhibitor is at a concentration ranging from about 2 mM to about 10 mM. In some embodiments, the ALK inhibitor is SB-431542, a derivative thereof, or a variant thereof. In some instances, the ALK inhibitor is at a concentration ranging from about 1 pM to about 10 pM. In some embodiments, the first culture medium and/or second culture medium are absent of animal serum.

[0589] Differentiation is assayed as is known in the art, e.g., by evaluating the presence of beta cell- associated or specific markers, including but not limited to, insulin. Differentiation can also be measured functionally, such as measuring glucose metabolism, see generally Muraro et al., Cell Syst. 2016 Oct 26; 3(4): 385-394.e3, hereby incorporated by reference in its entirety. [0590] Additional descriptions of islet cells including for use in the present technology are found in W02020/018615, the disclosure of which is herein incorporated by reference in its entirety.

[0591] In some embodiments, the engineered islet cells, such as primary beta islet cells isolated from one or more individual donors (e.g., healthy donors) or endothelial cells differentiated from iPSCs derived from one or more individual donors (e.g., healthy donors), are maintained in culture and in some cases expanded and/or cryopreserved. In certain embodiments, the engineered islet cells are cryopreserved.

[0592] Exemplary islet cell types include, but are not limited to, pancreatic islet progenitor cell, immature pancreatic islet cell, mature pancreatic islet cell, and the like.

[0593] In some embodiments, the islet cells engineered as disclosed herein, such as primary beta islet cells isolated from one or more individual donors (e.g., healthy donors) or beta islet cells differentiated from iPSCs derived from one or more individual donors (e.g., healthy donors), secrete insulin. In some embodiments, a pancreatic islet cell exhibits at least two characteristics of an endogenous pancreatic islet cell, for example, but not limited to, secretion of insulin in response to glucose, and expression of beta cell markers.

[0594] Exemplary beta cell markers or beta cell progenitor markers include, but are not limited to, c-peptide, Pdxl, glucose transporter 2 (Glut2), HNF6, VEGF, glucokinase (GCK), prohormone convertase (PC 1/3), Cdcpl, NeuroD, Ngn3, Nkx2.2, Nkx6.1, Nkx6.2, Pax4, Pax6, Ptfla, Isll, Sox9, Soxl7, and FoxA2.

[0595] In some embodiments, the islet cells, such as primary beta islet cells isolated from one or more individual donors (e.g., healthy donors) or beta islet cells differentiated from iPSCs derived from one or more individual donors e.g., healthy donors), produce insulin in response to an increase in glucose. In various embodiments, the pancreatic islet cells secrete insulin in response to an increase in glucose. In some embodiments, the cells have a distinct morphology such as a cobblestone cell morphology and/or a diameter of about 17 pm to about 25 pm.

[0596] In some embodiments, the present disclosure is directed to engineered beta islet cells, such as primary beta islet cells isolated from one or more individual donors (e.g., healthy donors) or beta islet cells differentiated from iPSCs derived from one or more individual donors (e.g., healthy donors).

[0597] In some embodiments, the provided engineered islet cells evade immune recognition. In some embodiments, the engineered islet cells described herein, such as primary beta islet cells isolated from one or more individual donors (e.g., healthy donors) or beta islet cells differentiated from iPSCs derived from one or more individual donors (e.g., healthy donors), do not activate an immune response. z. Engineered Islets

[0598] In some embodiments, the provided engineered islets, also includes a modification to modulate (e.g., increase) expression of one or more tolerogenic factor. In some embodiments, the modulation of expression of the tolerogenic factor (e.g., increased expression), and the modulation of expression of the one or more MHC class I molecules and/or one or more MHC class II molecules (e.g., reduced or eliminated expression) is relative to the amount of expression of said molecule(s) in a cell that does not comprise the modification(s), such as a control cell. In some embodiments, the modulation of expression is relative to the amount of expression of said molecule(s) in a wild-type cell. In some embodiments, the control or wild-type cell is an islet cell that has not been engineered with the modifications. In some embodiments, modulation of expression of the tolerogenic factor (e.g., increased expression), and the modulation of expression of the one or more MHC class I molecules and/or one or more MHC class II molecules (e.g., reduced or eliminated expression) is relative to the amount of expression of said molecule(s) in a control or wild-type cell of the same cell type that does comprise not the modification(s). In some embodiments, the control or wild-type cell does not express the one or more tolerogenic factor, the one or more MHC class I molecules, and/or the one or more MHC class II molecules. In some embodiments, it is understood that where the control or wild-type cell does not express the tolerogenic factor, the provided engineered islet cell includes a modification to overexpress the one or more tolerogenic factor or increase the expression of the one or more tolerogenic factor from 0%. It is understood that if the islet cell prior to the engineering does not express a detectable amount of the tolerogenic factor, then a modification that results in any detectable amount of an expression of the tolerogenic factor is an increase in the expression compared to the similar beta cell that does not contain the modifications.

[0599] In some embodiments, the provided engineered islets includes a modification to increase expression of one or more tolerogenic factors. In some embodiments, the tolerogenic factor is one or more of DUX4, B2M-HLA-E, CD35, CD52, CD16, CD52, CD47, CD46, CD55, CD59, CD27, CD200, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, PD-L1, IDO1, CTLA4-Ig, Cl-Inhibitor, IL-10, IL-35, FASL, CCL21, MFGE8, SERPINB9, CD35, IL-39, CD16 Fc Receptor, IL15-RF, and H2-M3 (including any combination thereof). In some embodiments, the tolerogenic factor is one or more of CD47, PD-L1, HLA-E or HLA-G, CCL21, FasL, Serpinb9, CD200, and Mfge8 (including any combination thereof). In some embodiments, the modification to increase expression of one or more tolerogenic factors is or includes increased expression of CD47. In some embodiments, the modification to increase expression of one or more tolerogenic factors is or includes increased expression of PD-L1. In some embodiments, the modification to increase expression of one or more tolerogenic factors is or includes increased expression of HLA-E. In some embodiments, the modification to increase expression of one or more tolerogenic factors is or includes increased expression of HLA-G. In some embodiments, the modification to increase expression of one or more tolerogenic factors is or includes increased expression of CCL21, PD-L1, FasL, Serpinb9, H2-M3 (HLA-G), CD47, CD200, and Mfge8.

[0600] In some embodiments, the engineered islets includes one or more modifications, such as genomic modifications, that reduce expression of one or more MHC class I molecules and a modification that increases expression of CD47. In other words, the engineered islets comprises exogenous CD47 proteins and exhibit reduced or silenced surface expression of one or more one or more MHC class I molecules. In some embodiments, the engineered islets includes one or more genomic modifications that reduce expression of one or more MHC class II molecules and a modification that increases expression of CD47. In some instances, the engineered islets comprises exogenous CD47 nucleic acids and proteins and exhibit reduced or silenced surface expression of one or more MHC class I molecules. In some embodiments, the engineered islets includes one or more genomic modifications that reduce or eliminate expression of one or more MHC class II molecules, one or more genomic modifications that reduce or eliminate expression of one or more MHC class II molecules, and a modification that increases expression of CD47. In some embodiments, the engineered islets comprises exogenous CD47 proteins, exhibit reduced or silenced surface expression of one or more MHC class I molecules and exhibit reduced or lack surface expression of one or more MHC class II molecules. In many embodiments, the engineered islets is a B2M^indel/indel , ciITA^indel/indel, CD47tg cell.

[0601] In some embodiments, the engineered islets elicits a reduced level of immune activation or no immune activation upon administration to a recipient subject. In some embodiments, the engineered islets elicits a reduced level of systemic TH1 activation or no systemic TH1 activation in a recipient subject. In some embodiments, the engineered islets elicits a reduced level of immune activation of peripheral blood mononuclear cells (PBMCs) or no immune activation of PBMCs in a recipient subject. In some embodiments, the engineered islets elicits a reduced level of donor- specific IgG antibodies or no donor specific IgG antibodies against the cells upon administration to a recipient subject. In some embodiments, the engineered islets elicits a reduced level of IgM and IgG antibody production or no IgM and IgG antibody production against the cells in a recipient subject. In some embodiments, the engineered islets elicits a reduced level of cytotoxic T cell killing of the cells upon administration to a recipient subject.

[0602] In some embodiments, the engineered islets provided herein comprises a “suicide gene” or “suicide switch”. A suicide gene or suicide switch can be incorporated to function as a “safety switch” that can cause the death of the engineered islets, such as after the engineered islets is administered to a subject and if the engineered islets should grow and divide in an undesired manner. The “suicide gene” ablation approach includes a suicide gene in a gene transfer vector encoding a protein that results in cell killing only when activated by a specific compound. A suicide gene may encode an enzyme that selectively converts a nontoxic compound into highly toxic metabolites. The result is specifically eliminating cells expressing the enzyme. In some embodiments, the suicide gene is the herpesvirus thymidine kinase (HSV- tk) gene and the trigger is ganciclovir. In other embodiments, the suicide gene is the Escherichia coli cytosine deaminase (EC-CD) gene and the trigger is 5-fluorocytosine (5-FC) (Barese et al, Mol. Therap. 20(10): 1932-1943 (2012), Xu et al, Cell Res. 8:73-8 (1998), both incorporated herein by reference in their entirety).

[0603] In other embodiments, the suicide gene is an inducible Caspase protein. An inducible Caspase protein comprises at least a portion of a Caspase protein capable of inducing apoptosis. In preferred embodiments, the inducible Caspase protein is iCasp9. It comprises the sequence of the human FK506-binding protein, FKBP12, with an F36V mutation, connected through a series of amino acids to the gene encoding human caspase 9. FKBP12-F36V binds with high affinity to a small-molecule dimerizing agent, API 903. Thus, the suicide function of iCasp9 in the instant invention is triggered by the administration of a chemical inducer of dimerization (CID). I n some embodiments, the CID is the small molecule drug API 903. Dimerization causes the rapid induction of apoptosis. (See WO2011146862; Stasi et al, N. Engl. J. Med 365; 18 (2011); Tey et al, Biol. Blood Marrow Transplant. 13:913-924 (2007), each of which are incorporated by reference herein in their entirety.)

[0604] Inclusion of a safety switch or suicide gene allows for controlled killing of the cells in the event of cytotoxicity or other negative consequences to the recipient, thus increasing the safety of cell-based therapies, including those using tolerogenic factors.

[0605] In some embodiments, a safety switch can be incorporated into, such as introduced, into the engineered islets provided herein to provide the ability to induce death or apoptosis of the engineered islets containing the safety switch, for example if the cells grow and divide in an undesired manner or cause excessive toxicity to the host. Thus, the use of safety switches enables one to conditionally eliminate aberrant cells in vivo and can be a critical step for the application of cell therapies in the clinic. Safety switches and their uses thereof are described in, for example, Duzgune§, Origins of Suicide Gene Therapy (2019); Duzgune§ (eds), Suicide Gene Therapy. Methods in Molecular Biology, vol. 1895 (Humana Press, New York, NY) (for HSV- tk, cytosine deaminase, nitroreductase, purine nucleoside phosphorylase, and horseradish peroxidase); Zhou and Brenner, Exp Hematol 44(11): 1013-1019 (2016) (for iCaspase9); Wang et al., Blood 18(5): 1255- 1263 (2001) (for huEGFR); U.S. Patent Application Publication No. 20180002397 (for HER1); and Philip et al., Bloodl24(8): 1277- 1287 (2014) (for RQR8).

[0606] In some embodiments, the safety switch can cause cell death in a controlled manner, for example, in the presence of a drug or prodrug or upon activation by a selective exogenous compound. In some embodiments, the safety switch is selected from the group consisting of herpes simplex virus thymidine kinase (HSV-tk), cytosine deaminase (CyD), nitroreductase (NTR), purine nucleoside phosphorylase (PNP), horseradish peroxidase, inducible caspase 9 (iCasp9), rapamycin-activated caspase 9 (rapaCasp9), CCR4, CD16, CD19, CD20, CD30, EGFR, GD2, HER1, HER2, MUC1, PSMA, and RQR8.

[0607] In some embodiments, the safety switch may be a transgene encoding a product with cell killing capabilities when activated by a drug or prodrug, for example, by turning a non-toxic prodrug to a toxic metabolite inside the cell. In these embodiments, cell killing is activated by contacting a engineered islets with the drug or prodrug. In some cases, the safety switch is HSV- tk, which converts ganciclovir (GCV) to GCV-triphosphate, thereby interfering with DNA synthesis and killing dividing cells. In some cases, the safety switch is CyD or a variant thereof, which converts the antifungal drug 5-fluorocytosine (5-FC) to cytotoxic 5-fluorouracil (5-FU) by catalyzing the hydrolytic deamination of cytosine into uracil. 5-FU is further converted to potent anti-metabolites (5- FdUMP, 5-FdUTP, 5-FUTP) by cellular enzymes. These compounds inhibit thymidylate synthase and the production of RNA and DNA, resulting in cell death. In some cases, the safety switch is NTR or a variant thereof, which can act on the prodrug CB 1954 via reduction of the nitro groups to reactive N-hydroxylamine intermediates that are toxic in proliferating and nonproliferating cells. In some cases, the safety switch is PNP or a variant thereof, which can turn prodrug 6-methylpurine deoxyriboside or fludarabine into toxic metabolites to both proliferating and nonproliferating cells. In some cases, the safety switch is horseradish peroxidase or a variant thereof, which can catalyze indole-3-acetic acid (IAA) to a potent cytotoxin and thus achieve cell killing.

[0608] In some embodiments, the safety switch may be an iCasp9. Caspase 9 is a component of the intrinsic mitochondrial apoptotic pathway which, under physiological conditions, is activated by the release of cytochrome C from damaged mitochondria. Activated caspase 9 then activates caspase 3, which triggers terminal effector molecules leading to apoptosis. The iCasp9 may be generated by fusing a truncated caspase 9 (without its physiological dimerization domain or caspase activation domain) to a FK506 binding protein (FKBP), FKBP12-F36V, via a peptide linker. The iCasp9 has low dimer-independent basal activity and can be stably expressed in host cells (e.g., human T cells) without impairing their phenotype, function, or antigen specificity. However, in the presence of chemical inducer of dimerization (CID), such as rimiducid (AP1903), AP20187, and rapamycin, iCasp9 can undergo inducible dimerization and activate the downstream caspase molecules, resulting in apoptosis of cells expressing the iCasp9. See, e.g., PCT Application Publication No. WO2011/146862; Stasi et al., N. Engl. J. Med. 365; 18 (2011); Tey et al., Biol. Blood Marrow Transplant 13:913-924 (2007). In particular, the rapamycininducible caspase 9 variant is called rapaCasp9. See Stavrou et al., Mai. Ther.

26(5): 1266- 1276 (2018). Thus, iCasp9 can be used as a safety switch to achieve controlled killing of the host cells.

[0609] In some embodiments, the safety switch may be a membrane-expressed protein which allows for cell depletion after administration of a specific antibody to that protein. Safety switches of this category may include, for example, one or more transgene encoding CCR4, CD16, CD19, CD20, CD30, EGFR, GD2, HER1, HER2, MUC1, PSMA, or RQR8 for surface expression thereof. These proteins may have surface epitopes that can be targeted by specific antibodies. In some embodiments, the safety switch comprises CCR4, which can be recognized by an anti-CCR4 antibody. Non-limiting examples of suitable anti-CCR4 antibodies include mogamulizumab and biosimilars thereof. In some embodiments, the safety switch comprises CD 16 or CD30, which can be recognized by an anti-CD16 or anti-CD30 antibody. Non-limiting examples of such anti-CD16 or anti-CD30 antibody include AFM13 and biosimilars thereof. In some embodiments, the safety switch comprises CD 19, which can be recognized by an antiCD 19 antibody. Non-limiting examples of such anti-CD19 antibody include MOR208 and biosimilars thereof. In some embodiments, the safety switch comprises CD20, which can be recognized by an anti-CD20 antibody. Non-limiting examples of such anti-CD20 antibody include obinutuzumab, ublituximab, ocaratuzumab, rituximab, rituximab-Rllb, and biosimilars thereof. Cells that express the safety switch are thus CD20-positive and can be targeted for killing through administration of an anti-CD20 antibody as described. In some embodiments, the safety switch comprises EGFR, which can be recognized by an anti-EGFR antibody. Nonlimiting examples of such anti-EGFR antibody include tomuzotuximab, RO5083945 (GA201), cetuximab, and biosimilars thereof. In some embodiments, the safety switch comprises GD2, which can be recognized by an anti-GD2 antibody. Non-limiting examples of such anti-GD2 antibody include Hul4.18K322A, Hul4.18-IL2, Hu3F8, dinituximab, c.60C3-Rllc, and bio similars thereof.

[0610] In some embodiments, the safety switch may be an exogenously administered agent that recognizes one or more tolerogenic factor on the surface of the engineered islets. In some embodiments, the exogenously administered agent is an antibody directed against or specific to a tolerogenic agent, e.g., an anti-CD47 antibody. By recognizing and blocking a tolerogenic factor on engineered islets, an exogenously administered antibody may block the immune inhibitory functions of the tolerogenic factor thereby re- sensitizing the immune system to the engineered islets. For instance, for a engineered islets that overexpresses CD47 an exogenously administered anti-CD47 antibody may be administered to the subject, resulting in masking of CD47 on the engineered islets and triggering of an immune response to the engineered islets.

[0611] In some embodiments, the method further comprises introducing an expression vector comprising an inducible safety switch (e.g., suicide switch) into the cell.

[0612] In some embodiments, the tolerogenic factor is CD47 and the cell includes an exogenous polynucleotide encoding a CD47 protein. In some embodiments, the cell expresses an exogenous CD47 polypeptide.

[0613] In some embodiments, a method disclosed herein comprises administering to a subject in need thereof a CD47-SIRPa blockade agent, wherein the subject was previously administered a engineered islets engineered to express an exogenous CD47 polypeptide. In some embodiments, the CD47-SIRPa blockade agent comprises a CD47 -binding domain. In some embodiments, the CD47-binding domain comprises signal regulatory protein alpha (SIRPa) or a fragment thereof. In some embodiments, the CD47-SIRPa blockade agent comprises an immunoglobulin G (IgG) Fc domain. In some embodiments, the IgG Fc domain comprises an IgGl Fc domain. In some embodiments, the IgGl Fc domain comprises a fragment of a human antibody. In some embodiments, the CD47-SIRPa blockade agent is selected from the group consisting of TTI-621, TTI-622, and ALX148. In some embodiments, the CD47- SIRPa blockade agent is TTI-621, TTI-622, and ALX148. In some embodiments, the CD47- SIRPa blockade agent is TTI-622. In some embodiments, the CD47-SIRPa blockade agent is ALX148. In some embodiments, the IgG Fc domain comprises an IgG4 Fc domain. In some embodiments, the CD47-SIRPa blockade agent is an antibody. In some embodiments, the antibody is selected from the group consisting of MIAP410, B6H12, and Magrolimab. In some embodiments, the antibody is MIAP410. In some embodiments, the antibody is B6H12. In some embodiments, the antibody is Magrolimab. In some embodiments, the antibody is selected from the group consisting of AO-176, IBI188 (letaplimab), STI-6643, and ZL-1201. In some embodiments, the antibody is AO-176 (Arch). In some embodiments, the antibody is IBI188 (letaplimab) (Innovent). In some embodiments, the antibody is STI-6643 (Sorrento). In some embodiments, the antibody is ZL-1201 (Zai).

[0614] In some embodiments, useful antibodies or fragments thereof that bind CD47 can be selected from a group that includes magrolimab ((Hu5F9-G4)) (Forty Seven, Inc.; Gilead Sciences, Inc.), urabrelimab, CC-90002 (Celgene; Bristol-Myers Squibb), IBL188 (Innovent Biologies), IBL322 (Innovent Biologies), TG-1801 (TG Therapeutics; also known as NI-1701, Novimmune SA), ALX148 (ALX Oncology), TJ011133 (also known as TJC4, LMab Biopharma), FA3M3, ZL-1201 (Zai Lab Co., Ltd), AK117 (Akesbio Australia Pty, Ltd.), AO- 176 (Arch Oncology), SRF231 (Surface Oncology), GenSci-059 (GeneScience), C47B157 (Janssen Research and Development), C47B161 (Janssen Research and Development), C47B167 (Janssen Research and Development), C47B222 (Janssen Research and Development), C47B227 (Janssen Research and Development), Vx-1004 (Corvus Pharmaceuticals), HMBD004 (Hummingbird Bioscience Pte Ltd), SHR-1603 (Hengrui), AMMS4-G4 (Beijing Institute of Biotechnology), RTX-CD47 (University of Groningen), and IMC-002. (Samsung Biologies; ImmuneOncia Therapeutics). In some embodiments, the antibody or fragment thereof does not compete for CD47 binding with an antibody selected from a group that includes magrolimab, urabrelimab, CC-90002, IBL188, IBL322, TG-1801 (NI-1701), ALX148, TJ011133, FA3M3, ZL1201, AK117, AO-176, SRF231, GenSci-059, C47B157, C47B161, C47B167, C47B222, C47B227, Vx-1004, HMBD004, SHR-1603, AMMS4-G4, RTX-CD47, and IMC-002. In some embodiments, the antibody or fragment thereof competes for CD47 binding with an antibody selected from magrolimab, urabrelimab, CC-90002, IBL188, IBL322, TG-1801 (NI-1701), ALX148, TJ011133, FA3M3, ZL1201, AK117, AO-176, SRF231, GenSci-059, C47B157, C47B161, C47B167, C47B222, C47B227, Vx-1004, HMBD004, SHR-1603, AMMS4-G4, RTX-CD47, and IMC-002. In some embodiments, the antibody or fragment thereof that binds CD47 is selected from a group that includes a single-chain Fv fragment (scFv) against CD47, a Fab against CD47, a VHH nanobody against CD47, a DARPin against CD47, and variants thereof. In some embodiments, the scFv against CD47, a Fab against CD47, and variants thereof are based on the antigen binding domains of any of the antibodies selected from a group that includes magrolimab, urabrelimab, CC-90002, IBI-188, IBI-322, TG-1801 (NI-1701), ALX148, TJ011133, FA3M3, ZL1201, AK117, AO-176, SRF231, GenSci-059, C47B157, C47B161, C47B167, C47B222, C47B227, Vx-1004, HMBD004, SHR-1603, AMMS4-G4, RTX-CD47, and IMC-002.

[0615] In some embodiments, the CD47 antagonist provides CD47 blockade. Methods and agents for CD47 blockade are described in PCT/US2021/054326, which is incorporated by reference in its entirety.

[0616] In some embodiments, the engineered islets is derived from a source cell already comprising one or more of the desired modifications. In some embodiments, in view of the teachings provided herein one of ordinary skill in the art will readily appreciate how to assess what modifications are required to arrive at the desired final form of a engineered islets and that not all reduced or increased levels of target components are achieved via active engineering. In some embodiments, the modifications of the engineered islets may be in any order, and not necessarily the order listed in the descriptive language provided herein.

[0617] Once altered, the presence of expression of any of the molecule described herein can be assayed using known techniques, such as Western blots, ELISA assays, FACS assays, flow cytometry, and the like. ii. Exemplary Embodiments of Engineered islets

[0618] In some embodiments, the engineered hypoimmunogenic islets comprise any of the vectors described herein (e.g., in section II.6 above), wherein the vector generates germline modifications that: (a) inactivate or disrupt one or more alleles of: (i) one or more major histocompatibility complex (MHC) class I molecules or one or more molecules that regulate expression of the one or more MHC class I molecules, and/or (ii) one or more MHC class II molecules or one or more molecules that regulate expression of the one or more MHC class II molecules; and/or (b) increase expression of one or more tolerogenic factors, wherein the increased expression is relative to a control or wild- type islet that does not comprise the modifications. In some embodiments, the one or more molecules that regulate expression of the one or more MHC class I molecules is B2M. In some embodiments, the one or more molecules that regulate expression of the one or more MHC class II molecules is OITA. In some embodiments, expression of HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, and HLA-DR are reduced in the engineered hypoimmunogenic islets. In some embodiments, the engineered hypoimmunogenic islet cell further comprises a modification to increase expression of an exogenous safety switch.

[0619] In some embodiments, the modifications comprise a modification that regulates the expression of the one or more MHC class I molecules, and the modification inactivates or disrupts one or more alleles of B2M. In some embodiments, the modification that inactivates or disrupts one or more alleles of B2M reduces mRNA expression of the B2M gene. In some embodiments, the modification that inactivates or disrupts one or more alleles of B2M reduces protein expression of B2M. In some embodiments, the modification that inactivates or disrupts one or more alleles of B2M comprises: i) inactivation or disruption of one allele of the B2M gene; ii) inactivation or disruption of both alleles of the B2M gene; or iii) inactivation or disruption of all B2M coding alleles in the cell. In some embodiments, the inactivation or disruption comprises an indel in the B2M gene. In some embodiments, the inactivation or disruption comprises a frameshift mutation or a deletion of a contiguous stretch of genomic DNA of the B2M gene.

[0620] In some embodiments, the one or more modifications comprise a modification that regulates cell surface protein expression of the one or more MHC class I molecules and the modification inactivates or disrupts one or more alleles of B2M. In some embodiments, the modification that regulates cell surface protein expression reduces cell surface trafficking of the one or more MHC class I molecules. In some embodiments, the one or more modifications reduce a function of the one or more MHC class I molecules, optionally wherein the function is antigen presentation.

[0621] In some embodiments, the modification is a modification that regulates expression of the one or more MHC class II molecules, and the modification inactivates or disrupts one or more alleles of CIITA. In some embodiments, the modification that inactivates or disrupts one or more alleles of CIITA reduces protein expression of CIITA. In some embodiments, the modification that inactivates or disrupts one or more alleles of CIITA comprises: i) inactivation or disruption of one allele of the CIITA gene; ii) inactivation or disruption of both alleles of the CIITA gene; or iii) inactivation or disruption of all CIITA coding alleles in the cell. In some embodiments, the inactivation or disruption comprises an indel in the CIITA gene. In some embodiments, the inactivation or disruption is a frameshift mutation or a deletion of a contiguous stretch of genomic DNA of the CIITA gene.

[0622] In some embodiments, the vector comprises one or more transgenes comprising one or more tolerogenic factors selected from the group consisting of CD16, CD24, CD35, CD39, CD46, CD47, CD52, CD55, CD59, CD64, CD200, CCL22, CTLA4-Ig, Cl inhibitor, FASL, IDO1, HLA- C, HLA-E, HLA-E heavy chain, HLA-G, IL-10, IL-35, PD-L1, SERPINB9, CCL21, MFGE8, DUX4, B2M-HLA-E, CD27, IL-39, CD16 Fc Receptor, IL15-RF, H2-M3 (HLA-G), A20/TNFAIP3, CR1, HLA-F, and MANF. In some embodiments, at least one of the one or more tolerogenic factors is CD47. In some embodiments, at least one of the one or more tolerogenic factors is PD-L1. In some embodiments, at least one of the one or more tolerogenic factors is HLA- E. In some embodiments, at least one of the one or more tolerogenic factors is HLA-G.

[0623] In some embodiments, the engineered hypoimmunogenic islet cells further comprises a modification to increase expression of an exogenous safety switch. In some embodiments, the safety switch is a system wherein upon activation, cells downregulate expression of the one or more tolerogenic factors and/or upregulate expression of one or more immune signaling molecules, thereby marking the cell for elimination by the host immune system. In some embodiments, the one or more immune signaling molecules is selected from the group consisting of B2M, HLA-A, HLA-B, HLA-C, HLA-D, HLA-E, RFXANK, CIITA, CTLA-4, PD-1, RAET1E/ULBP4, RAET1G/ULBP5, RAET1H/ULBP2, RAET1/ULBP1, RAET1L/ULBP6, RAET1N/ULBP3, and other ligands of NKG2D. In some embodiments, the safety switch is a suicide gene. In some embodiments, the suicide gene is selected from the group consisting of cytosine deaminase (CyD), herpesvirus thymidine kinase (HSV-Tk), an inducible caspase 9 (iCaspase9), and rapamycin-activated caspase 9 (rapaCasp9). In some embodiments, the engineered hypoimmunogenic islet cells have the genotype B2Af"^de//mde/; cjjTA'^l’ ''ⁱ'^,"’ ''ⁱ ; CD47tg; safety switch transgene.

[0624] In some embodiments, the one or more tolerogenic factors comprises CD47 and the engineered hypoimmunogenic islet cells expresses CD47 at a first level that is greater than at or about 5-fold over a second level expressed by the control or wild-type islet cells, optionally wherein CD47 is expressed at a first level that is greater than at or about 10-fold, greater than at or about 20-fold, greater than at or about 30-fold, greater than at or about 40-fold, greater than at or about 50-fold, greater than at or about 60-fold, or greater than at or about 70-fold over a second level expressed by the control or wild-type islet cells. In some embodiments, the one or more tolerogenic factors comprises CD47 and CD47 is expressed by the engineered hypoimmunogenic islet cells at greater than at or about 20,000 molecules per cell, optionally, wherein CD47 is expressed by the engineered hypoimmunogenic islet cells at greater than at or about 30,000 molecules per cell, greater than at or about 50,000 molecules per cell, greater than at or about 100,000 molecules per cell, greater than at or about 200,000 molecules per cell, greater than at or about 300,000 molecules per cell, greater than at or about 400,000 molecules per cell, greater than at or about 500,000 molecules per cell, or greater than at or about 600,000 molecules per cell.

[0625] In some embodiments, the engineered islets exhibit increased expression of CD47 and reduced expression of one or more molecules of the MHC class I complex. In some embodiments, the engineered islets exhibit increased expression of CD47 and reduced expression of one or more molecules of the MHC class I and MHC class II complexes. In some embodiments, the engineered islets express one or more exogenous complement inhibitor polypeptides selected from CD46, CD59, CD55, and any combinations thereof.

[0626] In some embodiments, engineered islets exhibit increased expression of CD47 and reduced expression of B2M. In some embodiments, engineered islets exhibit increased expression of CD47 and reduced expression of OITA. In some embodiments, the engineered islets exhibit increased expression of CD47 and reduced expression of NLRC5. In some embodiments, the engineered islets exhibit increased expression of CD47 and reduced expression of one or more molecules of B2M and OITA. In some embodiments, the engineered islets exhibit increased expression of CD47 and reduced expression of one or more molecules of B2M and NLRC5. In some embodiments, the engineered islets exhibit increased expression of CD47 and reduced expression of one or more molecules of B2M, OITA and NLRC5. Any of the engineered islets described herein can also exhibit increased expression of one or more factors selected from the group including, but not limited to, DUX4, CD24, CD27, CD200, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, PD-L1, IDO1, CTLA4-Ig, Cl-Inhibitor, IL-10, IL-35, IL-39, EasL, CCL21, CCL22, Mfge8, and Serpinb9. In some embodiments, the engineered islets express one or more exogenous complement inhibitor polypeptides selected from CD46, CD59, CD55, and any combinations thereof.

[0627] In some embodiments, the engineered islets exhibit increased expression of CD47 and at least one complement inhibitor selected from the group consisting of CD46, CD59, CD55, and any combination thereof, and reduced expression of one or more molecules of the MHC class I complex. In some embodiments, the engineered islets exhibit increased expression of CD47 and at least one complement inhibitor selected from the group consisting of CD46, CD59, CD55, and any combination thereof, and reduced expression of one or more molecules of the MHC class II complex. In some embodiments, the engineered islets exhibit increased expression of CD47 and at least one complement inhibitor selected from the group consisting of CD46, CD59, CD55, and any combination thereof, and reduced expression of one or more molecules of the MHC class II and MHC class II complexes. In some embodiments, the engineered islets and populations thereof exhibit increased expression of CD47 and at least one complement inhibitor selected from the group consisting of CD46, CD59, CD55, and any combination thereof, and reduced expression of B2M. In some embodiments, the engineered islets and populations thereof exhibit increased expression of CD47 and at least one complement inhibitor selected from the group consisting of CD46, CD59, CD55, and any combination thereof, and reduced expression of CIITA. In some embodiments, the engineered islets and populations thereof exhibit increased expression of CD47 and at least one complement inhibitor selected from the group consisting of CD46, CD59, CD55, and any combination thereof, and reduced expression of NLRC5. In some embodiments, the engineered islets and populations thereof exhibit increased expression of CD47 and at least one complement inhibitor selected from the group consisting of CD46, CD59, CD55, and any combination thereof, and reduced expression of one or more molecules of B2M and CIITA. In some embodiments, the engineered islets and populations thereof exhibit increased expression of CD47 and at least one complement inhibitor selected from the group consisting of CD46, CD59, CD55, and any combination thereof, and reduced expression of one or more molecules of B2M and NLRC5. In some embodiments, the engineered islets and populations thereof exhibit increased expression of CD47 and at least one complement inhibitor selected from the group consisting of CD46, CD59, CD55, and any combination thereof, and reduced expression of one or more molecules of CIITA and NLRC5. In some embodiments, the engineered islets and populations thereof exhibit increased expression of CD47 at least one complement inhibitor selected from the group consisting of CD46, CD59, CD55, and any combination thereof, and reduced expression of one or more molecules of B2M, CIITA and NLRC5.

[0628] In some embodiments, a engineered islets exhibits increased or decreased expression of the one more target molecules (e.g. MHC class I or class II, or CD47) in which the increase or decrease in expression is retained or similar (e.g. 75% to 100% of the level) compared to the unmodified or wild-type cell. Also provided herein is a population of engineered islets that include a plurality of cells that exhibit increased or decreased expression of the one more target molecules (e.g. MHC class I or class II, or CD47).

[0629] In some embodiments, at least at or about 50%, at least at or about 60%, at least at or about 70%, at least at or about 80%, or at least at or about 90% of the cells in the population are eliminated for expression of MHC class I or for B2M. In some embodiments, at least at or about 50%, at least at or about 60%, at least at or about 70%, at least at or about 80%, or at least at or about 90% of the cells in the population are eliminated for expression of MHC class II or for CIITA.

[0630] In some embodiments, least at or about 50%, at least at or about 60%, at least at or about 70%, at least at or about 80%, or at least at or about 90% of the cells in the population level have increased expression of the tolerogenic factor (CD47) that is greater than at or about 5-fold, greater than at or about 10-fold, greater than at or about 20-fold, greater than at or about 30-fold, greater than at or about 40-fold, greater than at or about 50-fold, greater than at or about 60-fold, or greater than at or about 70-fold over a wild-type primary beta cell or an unmodified pluripotent stem cell or an unmodified SC-beta differentiated from the unmodified pluripotent stem cell. In some embodiments, at least at or about 50%, at least at or about 60%, at least at or about 70%, at least at or about 80%, or at least at or about 90% of the cells in the population expresses the tolerogenic factor (e.g. CD47) at greater than at or about 20,000 molecules per cell, at greater than at or about 30,000 molecules per cell, greater than at or about 50,000 molecules per cell, greater than at or about 100,000 molecules per cell, greater than at or about 200,000 molecules per cell, greater than at or about 300,000 molecules per cell, greater than at or about 400,000 molecules per cell, greater than at or about 500,000 molecules per cell, or greater than at or about 600,000 molecules per cell.

[0631] One skilled in the art will appreciate that levels of expression such as increased or reduced expression of a gene, protein or molecule can be referenced or compared to a comparable cell. In some embodiments, a modified cell (e.g., an engineered islet, such as a modified beta islet cell) having increased expression of a protein (e.g., CD46, CD59. CD55, CD47, or any other tolerogenic factor) refers to a modified cell having a higher level of the protein compared to an unmodified cell. In some embodiments, an engineered islets having increased expression of a protein (e.g., CD46, CD59. CD55, CD47, or any other tolerogenic factor) is a cell comprising modifications, wherein the cell comprising modifications has a higher level of the protein compared to a cell without said modifications (e.g., beta cell without the modifications may comprise other modifications). In some embodiments, an engineered islet having reduced expression of a protein (e.g., B2M, or CIITA) is a cell comprising modifications, wherein the cell comprising modifications has a lower level of the protein or RNA compared to a cell without said modifications (e.g., beta cell without the modifications may comprise other modifications). In some embodiments, the engineered islets express one or more exogenous complement inhibitor polypeptides selected from CD46, CD59, CD55, and any combinations thereof.

[0632] In one embodiment, provided herein are engineered islets expressing exogenous CD47 polypeptides and having reduced expression of either one or more MHC class I complex proteins, one or more MHC class II complex proteins, or any combination of MHC class I and class II complex proteins. In another embodiment, the engineered islets express exogenous CD47 polypeptides and express reduced levels of B2M and CIITA polypeptides. In some embodiments, the engineered islets express exogenous CD47 polypeptides and possess modifications of the B2M and CIITA genes. In some instances, the modifications inactivate the B2M and CIITA genes. In some embodiments, the modified cells express one or more exogenous complement inhibitor polypeptides selected from CD46, CD59, CD55, and any combinations thereof. ii. Exemplary Features of SC-[> cells

[0633] The provided modified SC-beta cells, including those obtained by in vitro differentiation from pluripotent stem cells such as modified pluripotent stem cells, are highly functional SC-P cells. The modified SC-beta cell or population exhibits a GSIS response both in vitro and in vivo. The isolated SC-beta cell or population also exhibits at least one characteristic feature of a mature endogenous beta cell. In some aspects, a modified SC-beta cell or population thereof exhibits a stimulation index of between about 1.4 and about 2.4. In some aspects, a modified SC-beta cell or population thereof produces insulin at between approximately 300 uIU to about 4000 uIU per 30 minute per 106 total cells incubation at a high glucose concentration. [0634] In certain embodiments, static insulin secretion is greater than about 1 uIU/ 103 cells/hour at high glucose (e.g., 20 mM). In certain embodiments, the static insulin secretion is greater than about 1.5 uIU/103 cells/hour at high glucose. In certain embodiments, the static insulin secretion is greater than about 2.0 uIU/103 cells/hour at high glucose. In certain embodiments, the static insulin secretion is greater than about 2.5 uIU/103 cells/hour at high glucose. In certain embodiments, the static insulin secretion is greater than about 3.0 uIU/103 cells/hour at high glucose. In certain embodiments, the static insulin secretion is greater than about 3.5 uIU/103 cells/hour at high glucose. In certain embodiments, the static insulin secretion is greater than about 4.0 uIU/103 cells/hour at high glucose. In certain embodiments, the static stimulation index is defined as a ratio of 20 mM glucose to 2 mM glucose, incubated for 1 hr. [0635] Assays to assess functional activity of the SC-b cells include static and dynamic glucose stimulated insulin secretion (GSIS) assays to measure glucose responsiveness, insulin content and proinsulin-to-insulin ratio to determine intracellular insulin levels and processing, flow cytometry to measure the percentages of the different hormone-producing cells, and qRT- PCR and immuno staining to confirm the presence of P-cell-specific markers. In particular, SC-P cells can be identified by their coexpression of C-peptide and NKX6-1, while chromogranin A (CHGA) marks the general endocrine population. After aggregation, >80% of the cells in these clusters should express CHGA, with -20-60% of these cells being C-peptide+/NKX6-l+.

[0636] In certain embodiments, the SC-P cells achieve both first and second-phase dynamic insulin secretion. In some embodiments, the SC-P cells achieve equivalent functional capabilities of human islets. In certain embodiments, the SC-P cells retain functionality for 1 or more days. In certain embodiments, the SC-pp cells retain functionality for more than 1 week. In certain embodiments, the SC-P cells may be used to treat or reverse severe preexisting diabetes at a rate similar to primary human islets, outperforming cells generated with a suspension-based protocol. In certain embodiments, the SC-P cells, when transplanted, are capable of maintaining normoglycemia indefinitely.

Hepatocyte cells

[0637] In some embodiments, the engineered cells or populations of cells that are provided herein are hepatocytes, such as primary hepatocytes or hepatocytes differentiated from pluripotent cells, stem cells or iPSCs. In some embodiments, primary hepatocytes are isolated or obtained from one or more individual donor subjects, such as one or more individual healthy donor (e.g., a subject that is not known or suspected of, e.g., not exhibiting clinical signs of, a disease or infection). As will be appreciated by those in the art, methods of isolating or obtaining hepatocytes from an individual can be achieved using known techniques. [0638] In some embodiments, primary hepatocytes are obtained (e.g., harvested, extracted, removed, or taken) from a subject or an individual. In some embodiments, primary hepatocytes are produced from a pool of hepatocytes such that the hepatocytes are from one or more subjects (e.g., one or more human including one or more healthy humans). In some embodiments, the pool of primary hepatocytes is from 1-100, 1-50, 1-20, 1-10, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, or 100 or more subjects.

[0639] In some embodiments, the cells as provided herein are engineered hepatocytes differentiated from engineered iPSCs that contain any of the vectors described herein and that are differentiated into hepatocytes. As will be appreciated by those skilled in the art, the methods for differentiation depend on the desired cell type using known techniques.

[0640] In some embodiments, engineered pluripotent cells containing any of the vectors described herein are differentiated into hepatocytes. There are a number of techniques that can be used to differentiate engineered pluripotent cells into hepatocytes; see for example, Pettinato et al., doi: 10.1038/spre32888; Snykers et al., Methods Mol Biol, 2011 698:305-314; Si-Tayeb et al., Hepatology, 2010, 51:297-305; and Asgari et al., Stem Cell Rev, 2013, 9(4):493- 504, all of which are incorporated herein by reference in their entirety. Differentiation can be assayed as is known in the art, generally by evaluating the presence of hepatocyte-associated and/or specific markers, including, but not limited to, albumin, alpha fetoprotein, and fibrinogen. Differentiation can also be measured functionally, such as the metabolization of ammonia, LDL storage and uptake, ICG uptake and release, and glycogen storage.

[0641] In some embodiments, the engineered hepatocytes, such as primary hepatocytes isolated from one or more individual donors (e.g., healthy donors) or hepatocytes differentiated from iPSCs derived from one or more individual donors (e.g., healthy donors), are maintained in culture and in some cases expanded and/or cryopreserved. In certain embodiments, the population of hepatocytes are cryopreserved.

[0642] In some embodiments, the present disclosure is directed to engineered hepatocytes, such as primary hepatocytes isolated from one or more individual donors (e.g., healthy donors) or hepatocytes differentiated from iPSCs derived from one or more individual donors (e.g., healthy donors). [0643] In some embodiments, the provided engineered hepatocytes evade immune recognition. In some embodiments, the engineered hepatocytes described herein, such as primary hepatocytes isolated from one or more individual donors (e.g., healthy donors) or hepatocytes differentiated from iPSCs derived from one or more individual donors (e.g., healthy donors), do not activate an immune response.

T cells

[0644] In some embodiments, the engineered cells or populations of cells provided herein are T lymphocytes (also called T cells), such as primary T lymphocytes or T cells. In some embodiments, primary T lymphocytes are isolated or obtained from one or more individual donor subjects, such as one or more individual healthy donor (e.g., a subject that is not known or suspected of, e.g., not exhibiting clinical signs of, a disease or infection). In some instances, the T cells are populations or subpopulations of primary T cells from one or more individuals. As will be appreciated by those skilled in the art, methods of isolating or obtaining T lymphocytes from an individual can be achieved using known techniques.

[0645] In some embodiments, primary T cells are obtained (e.g., harvested, extracted, removed, or taken) from a subject or an individual. In some embodiments, primary T cells are produced from a pool of T cells such that the T cells are from one or more subjects (e.g., one or more human including one or more healthy humans). In some embodiments, the pool of primary T cells is from 1-100, 1-50, 1-20, 1-10, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, or 100 or more subjects.

[0646] In some embodiments, the engineered cells or populations of cells as provided herein are T lymphocytes differentiated from engineered pluripotent cells that contain any of the vectors described herein and that are differentiated into T lymphocytes. As will be appreciated by those skilled in the art, the methods for differentiation depend on the desired cell type using known techniques.

[0647] Methods for generating T cells from pluripotent stem cells (e.g., iPSC) are described, for example, in Iriguchi et al., Nature Communications 12, 430 (2021); Themeli et al., Nature Biotechnology 31:928-933 (2013).

[0648] Non-limiting examples of T cells, such as primary T cells, include CD3+ T cells, CD4+ T cells, CD8+ T cells, naive T cells, regulatory T (Treg) cells, non-regulatory T cells, Thl cells, Th2 cells, Th9 cells, Thl7 cells, T-follicular helper (Tfh) cells, cytotoxic T lymphocytes (CTL), effector T (Teff) cells, central memory T (Tcm) cells, effector memory T (Tern) cells, effector memory T cells express CD45RA (TEMRA cells), tissue-resident memory (Trm) cells, virtual memory T cells, innate memory T cells, memory stem cell (Tsc), y5 T cells, and any other subtype of T cells. In some embodiments, the primary T cells are selected from the group consisting of: cytotoxic T cells, helper T cells, memory T cells, regulatory T cells, tumor infiltrating lymphocytes, and any combination thereof.

[0649] Exemplary T cells that may be used according to the present disclosure include, without limitation, cytotoxic T cells, helper T cells, memory T cells, central memory T cells, effector memory T cells, effector memory RA T cells, regulatory T cells, tissue infiltrating lymphocytes, and combinations thereof. In many embodiments, the T cells express CCR7, CD27, CD28, and CD45RA. In some embodiments, the central T cells express CCR7, CD27, CD28, and CD45RO. In other embodiments, the effector memory T cells express PD-1, CD27, CD28, and CD45RO. In other embodiments, the effector memory RA T cells express PD-1, CD57, and CD45RA.

[0650] In some embodiments, the engineered T cells described herein, such as primary T cells isolated from one or more individual donors (e.g., healthy donors) or T cells differentiated from iPSCs derived from one or more individual donors (e.g., healthy donors), comprise T cells engineered to express a chimeric antigen receptor including but not limited to an engineered receptor (e.g., a CAR, CAAR, BAR, or TCR) as described herein or known in the art. Any suitable engineered receptor (e.g., a CAR, CAAR, BAR, or TCR) can be included in the T cells, including any of the engineered receptors described herein. In some embodiments, the engineered T cells express at least an engineered receptor that specifically binds to an antigen or epitope of interest expressed on the surface of at least one of a damaged cell, a dysplastic cell, an infected cell, an immunogenic cell, an inflamed cell, a malignant cell, a metaplastic cell, a mutant cell, or any combination thereof.

[0651] In some embodiments, the T cell includes a transgene encoding an engineered receptor (e.g., a CAR, CAAR, BAR, or TCR), wherein the transgene is inserted in a vector, such as any of the vectors described herein. Any suitable method can be used to insert the engineered receptor into the T cell, including for example lentiviral based transduction methods. In some embodiments, the transgene is inserted into a vector, such as any of the vectors described herein. [0652] In some embodiments, the present disclosure is directed to engineered T cells, such as primary T cells isolated from one or more individual donors (e.g., healthy donors) or T cells differentiated from iPSCs derived from one or more individual donors (e.g., healthy donors).

[0653] In some embodiments, the provided engineered T cells evade immune recognition. In some embodiments, the engineered T cells described herein, such as primary T cells isolated from one or more individual donors (e.g., healthy donors) or T cells differentiated from iPSCs derived from one or more individual donors (e.g., healthy donors), do not activate an immune response.

Natural killer cells

[0654] In some embodiments, the engineered cells or populations of cells that are provided herein are Natural Killer (NK) cells. In some embodiments, the NK cells are isolated or obtained from one or more individual donor subjects, such as one or more individual healthy donor (e.g., a subject that is not known or suspected of, e.g., not exhibiting clinical signs of, a disease or infection). In some instances, the NK cells are populations or subpopulations of NK cells from one or more individuals. As will be appreciated by those skilled in the art, methods of isolating or obtaining NK cells from an individual can be achieved using known techniques.

[0655] In some embodiments, the engineered cells or populations of cells as provided herein are NK cells differentiated from engineered pluripotent cells that contain any of the vectors described herein and that are differentiated into NK cells. As will be appreciated by those skilled in the art, the methods for differentiation depend on the desired cell type using known techniques.

[0656] Methods for generating NK cells from pluripotent stem cells (e.g., iPSC) are described, for example, in U.S. Patent No. 10626373; Shankar et al., Stem Cell Res Ther. 2020; 11: 234; Euchner et al., Frontiers in Immunology, 2021; 12, Article 640672, doi=10.3389/fimmu.2021.640672.

[0657] In some embodiments, NK cells are obtained (e.g., harvested, extracted, removed, or taken) from a subject or an individual. In some embodiments, NK cells are produced from a pool of NK cells such that the NK cells are from one or more subjects (e.g., one or more human including one or more healthy humans). In some embodiments, the pool of primary NK cells is from 1-100, 1-50, 1-20, 1-10, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, or 100 or more subjects. [0658] In some embodiments, NK cells, including primary NK cells isolated from one or more individual donors (e.g., healthy donors) or NK cells differentiated from iPSCs derived from one or more individual donors (e.g., healthy donors) express CD56 (e.g., CD56dim or CD56bright) and lack CD3 (e.g., CD3neg). In some embodiments, NK cells as described herein may also express the low-affinity Fey receptor CD 16, which mediate ADCC. In some embodiments, the NK cells also express one or more natural killer cell receptors NKG2A and NKG2D or one or more natural cytotoxicity receptors NKp46, NKp44, NKp30. For example, for the case of primary NK cells, in specific cases, the primary cells may be isolated from a starting source of NK cells, such as a sample containing peripheral blood mononuclear cells (PBMCs), by depletion of cells positive for CD3, CD14, and/or CD19. For instance, the cells may be subject to depletion using immunomagnetic beads having attached thereto antibodies to CD3, CD14, and/or CD19, respectively, thereby producing an enriched population of NK cells. In other cases, primary NK cells may be isolated from a starting source that is a mixed population (e.g., PBMCs) by selecting cells for the presence of one or more markers on the NK cells, such as CD56, CD 16, NKp46, and/or NKG2D.

[0659] In some embodiments, prior to the engineering as described herein, the NK cells, such as isolated primary NK cells, may be subject to one or more expansion or activation step. In some embodiments, expansion may be achieved by culturing of the NK cells with feeder cells, such as antigen presenting cells that may or may not be irradiated. The ratio of NK cells to antigen presenting cells (APCs) in the expansion step may be of a certain number, such as 1:1, 1:1.5, 1:2, or 1:3, for example. In certain aspects, the APCs are engineered to express membranebound IL-21 (mblL- 21). In particular aspects, the APCs are alternatively or additionally engineered to express IL-21, IL-15, and/or IL-2. In particular embodiments, the media in which the expansion step(s) occurs comprises one or more agents to facilitate expansion, such as one or more recombinant cytokines. In specific embodiments, the media comprises one or more recombinant cytokines from IL-2, IL-15, IL-18, and/or IL-21. In some embodiments, the steps for engineering the NK cells by introducing the vectors as described herein is carried out 2-12 days after initiation of the expansion, such as on or about day 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. [0660] In some embodiments, the engineered NK cells described herein, such as primary NK cells isolated from one or more individual donors (e.g., healthy donors), comprise NK cells engineered to express an engineered receptor (e.g., a CAR, CAAR, BAR, or TCR), e.g., as described herein. Any suitable engineered receptor can be included in the engineered NK cells, including an engineered receptor (e.g., a CAR, CAAR, BAR, or TCR) described herein. In some embodiments, the engineered NK cells express at least one engineered receptor that specifically binds to an antigen or epitope of interest expressed on the surface of at least one of a damaged cell, a dysplastic cell, an infected cell, an immunogenic cell, an inflamed cell, a malignant cell, a metaplastic cell, a mutant cell, or any combination thereof.

[0661] In some embodiments, the engineered NK cell includes a transgene encoding an engineered receptor (e.g., a CAR, CAAR, BAR, or TCR), wherein the transgene is inserted in any of the vectors described herein. Any suitable method can be used to insert the engineered receptor into the vector(s) of the engineered NK cell, including, for example, lentiviral based transduction methods. In some embodiments, the transgene is inserted into any of the vectors described herein.

[0662] In some embodiments, the present technology is directed to engineered NK cells, such as primary NK cells isolated from one or more individual donors (e.g., healthy donors) or NK cells differentiated from iPSCs derived from one or more individual donors (e.g., healthy donors).

[0663] In some embodiments, the provided engineered NK cells evade immune recognition. In some embodiments, the engineered NK cells described herein, such as primary NK cells isolated from one or more individual donors (e.g., healthy donors), do not activate an immune response.

Endothelial cells

[0664] In some embodiments, the engineered cells or populations of cells that are provided herein are endothelial cells, such as primary endothelial cells. In some embodiments, primary endothelial cells are isolated or obtained from one or more individual donor subjects, such as one or more individual healthy donor (e.g., a subject that is not known or suspected of, e.g., not exhibiting clinical signs of, a disease or infection). As will be appreciated by those skilled in the art, methods of isolating or obtaining endothelial cells from an individual can be achieved using known techniques.

[0665] In some embodiments, primary endothelial cells are obtained (e.g., harvested, extracted, removed, or taken) from a subject or an individual. In some embodiments, primary endothelial cells are produced from a pool of endothelial cells such that the endothelial cells are 16 from one or more subjects e.g., one or more human including one or more healthy humans). In some embodiments, the pool of primary endothelial cells is from 1-100, 1-50, 1-20, 1-10, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, or 100 or more subjects.

[0666] In some embodiments, the engineered cells or populations of cells as provided herein are engineered endothelial cells differentiated from engineered iPSCs that contain any of the vectors described herein and that are differentiated into an endothelial cell type. As will be appreciated by those skilled in the art, the methods for differentiation depend on the desired cell type using known techniques.

[0667] In some embodiments, the engineered pluripotent cells described herein are differentiated into endothelial colony forming cells (ECFCs) to form new blood vessels. Techniques to differentiate endothelial cells are known. See, e.g., Prasain et al., doi: 10.1038/nbt.3048, incorporated herein by reference in its entirety. Differentiation can be assayed as is known in the art, generally by evaluating the presence of endothelial cell associated or specific markers or by measuring functionality.

[0668] In some embodiments, the method of producing engineered endothelial cells from engineered pluripotent cells by in vitro differentiation comprises: (a) culturing a population of engineered iPSCs in a first culture medium comprising a GSK inhibitor; (b) culturing the population of engineered iPSCs cells in a second culture medium comprising VEGF and bFGF to produce a population of pre-endothelial cells; and (c) culturing the population of pre- endothelial cells in a third culture medium comprising a ROCK inhibitor and an ALK inhibitor to produce a population of differentiated endothelial cells that are engineered to contain the one or more exogenous sequences as described herein.

[0669] In some embodiments, the GSK inhibitor is CHIR-99021, a derivative thereof, or a variant thereof. In some instances, the GSK inhibitor is at a concentration ranging from about 1 mM to about 10 mM. In some embodiments, the ROCK inhibitor is Y-27632, a derivative thereof, or a variant thereof. In some instances, the ROCK inhibitor is at a concentration ranging from about 1 pM to about 20 pM. In some embodiments, the ALK inhibitor is SB-431542, a derivative thereof, or a variant thereof. In some instances, the ALK inhibitor is at a concentration ranging from about 0.5 pM to about 10 pM. [0670] In some embodiments, the first culture medium comprises from 2 pM to about 10 pM of CHIR-99021. In some embodiments, the second culture medium comprises 50 ng/ml VEGF and 10 ng/ml bFGF. In other embodiments, the second culture medium further comprises Y-27632 and SB-431542. In various embodiments, the third culture medium comprises 10 pM Y-27632 and 1 pM SB-431542. In certain embodiments, the third culture medium further comprises VEGF and bFGF. In particular instances, the first culture medium and/or the second medium is absent of insulin.

[0671] The engineered cells or populations of cells provided herein can be cultured on a surface, such as a synthetic surface to support and/or promote differentiation of pluripotent cells into engineered endothelial cells. In some embodiments, the surface comprises a polymer material including, but not limited to, a homopolymer or copolymer of selected one or more acrylate monomers. Non-limiting examples of acrylate monomers and methacrylate monomers include tetra(ethylene glycol) diacrylate, glycerol dimethacrylate, 1,4-butanediol dimethacrylate, poly(ethylene glycol) diacrylate, di(ethylene glycol) dimethacrylate, tetra(ethyiene glycol) dimethacrylate, 1,6-hexanediol propoxylate diacrylate, neopentyl glycol diacrylate, trimethylolpropane benzoate diacrylate, trimethylolpropane eihoxylate (1 EO/QH) methyl, tricyclo[5.2.1.02,6] decane dimethanol diacrylate, neopentyl glycol exhoxylate diacrylate, and trimethylolpropane triacrylate. Acrylate synthesized as known in the art or obtained from a commercial vendor, such as Polysciences, Inc., Sigma Aldrich, Inc. and Sartomer, Inc.

[0672] In some embodiments, the endothelial cells may be seeded onto a polymer matrix. In some cases, the polymer matrix is biodegradable. Suitable biodegradable matrices are well known in the art and include collagen-GAG, collagen, fibrin, PLA, PGA, and PLA/PGA copolymers. Additional biodegradable materials include poly(anhydrides), poly(hydroxy acids), poly(ortho esters), poly(propylfumerates), poly(caprolactones), polyamides, polyamino acids, polyacetals, biodegradable polycy anoacrylates, biodegradable polyurethanes and polysaccharides.

[0673] Non-biodegradable polymers may also be used as well. Other non-biodegradable, yet biocompatible polymers include polypyrrole, polyanibnes, polythiophene, polystyrene, polyesters, non-biodegradable polyurethanes, polyureas, poly(ethylene vinyl acetate), polypropylene, polymethacrylate, polyethylene, polycarbonates, and poly (ethylene oxide). The polymer matrix may be formed in any shape, for example, as particles, a sponge, a tube, a sphere, a strand, a coiled strand, a capillary network, a film, a fiber, a mesh, or a sheet. The polymer matrix can be modified to include natural or synthetic extracellular matrix materials and factors.

[0674] The polymeric material can be dispersed on the surface of a support material. Useful support materials suitable for culturing cells include a ceramic substance, a glass, a plastic, a polymer or co-polymer, any combinations thereof, or a coating of one material on another. In some instances, a glass includes soda-lime glass, pyrex glass, vycor glass, quartz glass, silicon, or derivatives of these or the like.

[0675] In some instances, plastics or polymers including dendritic polymers include poly(vinyl chloride), poly(vinyl alcohol), poly(methyl methacrylate), poly(vinyl acetate- maleic anhydride), poly (dimethylsiloxane) monomethacrylate, cyclic olefin polymers, fluorocarbon polymers, polystyrenes, polypropylene, polyethyleneimine or derivatives of these or the like. In some instances, copolymers include poly(vinyl acetate-co-maleic anhydride), poly(styrene-co- maleic anhydride), poly(ethylene-co-acrylic acid) or derivatives of these or the like.

[0676] Additional descriptions of endothelial cells and their differentiation for use in the methods provided herein are found in W02020/018615, the disclosure of which is herein incorporated by reference in its entirety.

[0677] In some embodiments, the engineered endothelial cells, such as primary endothelial cells isolated from one or more individual donors (e.g., healthy donors) or endothelial cells differentiated from iPSCs derived from one or more individual donors (e.g., healthy donors), are maintained in culture and in some cases expanded and/or cryopreserved. In certain embodiments, the engineered endothelial cells are cryopreserved.

[0678] In some embodiments, the present disclosure is directed to engineered endothelial cells, such as primary endothelial cells isolated from one or more individual donors (e.g., healthy donors) or endothelial cells differentiated from iPSCs derived from one or more individual donors (e.g., healthy donors).

[0679] In some embodiments, the provided engineered endothelial cells evade immune recognition. In some embodiments, the engineered endothelial cells described herein, such as primary endothelial cells isolated from one or more individual donors (e.g., healthy donors) or endothelial cells differentiated from iPSCs derived from one or more individual donors (e.g., healthy donors), do not activate an immune response. [0680] Exemplary endothelial cell types include, but are not limited to, a capillary endothelial cell, vascular endothelial cell, aortic endothelial cell, arterial endothelial cell, venous endothelial cell, renal endothelial cell, brain endothelial cell, liver endothelial cell, and the like. [0681] The endothelial cells outlined herein, such as isolated primary endothelial cells or differentiated endothelial cells, can express one or more endothelial cell markers. Non-limiting examples of such markers include VE-cadherin (CD 144), ACE (angiotensin-converting enzyme) (CD 143), BNH9/BNF13, CD31, CD34, CD54 (ICAM-1), CD62E (E-Selectin), CD105 (Endoglin), CD 146, Endocan (ESM-1), Endoglyx-1, Endomucin, Eotaxin-3, EPAS1 (Endothelial PAS domain protein 1), Factor VIII related antigen, FLI-1, Flk-1 (KDR, VEGFR-2), FLT-1 (VEGFR-1), GATA2, GBP-1 (guanylate- binding protein-1), GRO-alpha, HEX, ICAM-2 (intercellular adhesion molecule 2), LM02, LYVE-1, MRB (magic roundabout), Nucleolin, PAL- E (pathologische anatomic Leiden- endothelium), RTKs, sVCAM-1, TALI, TEM1 (Tumor endothelial marker 1), TEM5 (Tumor endothelial marker 5), TEM7 (Tumor endothelial marker 7), thrombomodulin (TM, CD141), VCAM-1 (vascular cell adhesion molecule- 1) (CD106), VEGF, vWF (von Willebrand factor), ZO-1, endothelial cell-selective adhesion molecule (ESAM), CD 102, CD93, CD 184, CD304, and DLL4.

Epithelial cells

[0682] In some embodiments, the engineered cells or populations of cells that are provided herein are retinal pigmented epithelium (RPE) cells, such as primary RPE cells. In some embodiments, the primary RPE cells are isolated or obtained from one or more individual donor subjects, such as one or more individual healthy donor (e.g., a subject that is not known or suspected of, e.g., not exhibiting clinical signs of, a disease or infection). As will be appreciated by those skilled in the art, methods of isolating or obtaining RPE cells from an individual can be achieved using known techniques.

[0683] In some embodiments, primary RPE cells are obtained (e.g., harvested, extracted, removed, or taken) from a subject or an individual. In some embodiments, primary RPE cells are produced from a pool of RPE cells such that the RPE cells are from one or more subjects (e.g., one or more human including one or more healthy humans). In some embodiments, the pool of primary RPE cells is from 1-100, 1-50, 1-20, 1-10, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, or 100 or more subjects. [0684] In some embodiments, the cells as provided herein are engineered RPE cells differentiated from engineered iPSCs that contain any of the vectors described herein and that are differentiated into an engineered RPE cell. As will be appreciated by those skilled in the art, the methods for differentiation depend on the desired cell type using known techniques.

[0685] Useful methods for differentiating pluripotent stem cells into RPE cells are described in, for example, US9,458,428 and US9,850,463, the disclosures of which are herein incorporated by reference in their entirety. Additional methods for producing RPE cells from human induced pluripotent stem cells can be found in, for example, Lamba et al., PNAS, 2006, 103(34): 12769-12774; Mellough et al., Stem Cells, 2012, 30(4):673-686; Idelson et al., Cell Stem Cell, 2009, 5(4): 396-408; Rowland et al., Journal of Cellular Physiology, 2012, 227(2):457-466; Buchholz et al., Stem Cells Trans Med, 2013, 2(5): 384-393; and da Cruz et al., Nat Biotech, 2018, 36:328-337.

[0686] Human pluripotent stem cells have been differentiated into RPE cells using the techniques outlined in Kamao et al., Stem Cell Reports 2014:2:205-18, hereby incorporated by reference in its entirety; see also Mandai et al., N Engl J Med, 2017, 376:1038-1046, the contents herein incorporated in its entirety. Differentiation can be assayed as is known in the art, generally by evaluating the presence of RPE-associated and/or specific markers or by measuring functionality.

[0687] In some embodiments, the method of producing engineered retinal pigmented epithelium (RPE) cells from engineered pluripotent cells by in vitro differentiation comprises: (a) culturing the population of engineered pluripotent cells in a first culture medium comprising any one of the factors selected from activin A, bFGF, BMP4/7, DKK1, IGF1, noggin, a BMP inhibitor, an ALK inhibitor, a ROCK inhibitor, and a VEGFR inhibitor to produce a population of pre-RPE cells; and (b) culturing the pre-RPE cells in a second culture medium that is different than the first culture medium to produce engineered RPE cells. In some embodiments, the ALK inhibitor is SB-431542, a derivative thereof, or a variant thereof. In some instances, the ALK inhibitor is at a concentration ranging from about 2 mM to about 10 pM. In some embodiments, the ROCK inhibitor is Y-27632, a derivative thereof, or a variant thereof. In some instances, the ROCK inhibitor is at a concentration ranging from about 1 pM to about 10 pM. In some embodiments, the first culture medium and/or second culture medium are absent of animal serum. [0688] Differentiation can be assayed as is known in the art, generally by evaluating the presence of RPE associated and/or specific markers or by measuring functionality. See, for example, Kamao et al., Stem Cell Reports, 2014, 2(2):205-18, the contents of which are herein incorporated by reference in its entirety.

[0689] Additional descriptions of RPE cells, including methods for their differentiation and for use in the present technology, are found in W02020/018615, the disclosure of which is herein incorporated by reference in its entirety.

[0690] In some embodiments, the engineered RPE cells, such as primary RPE cells isolated from one or more individual donors (e.g., healthy donors) or RPE cells differentiated from iPSCs derived from one or more individual donors (e.g., healthy donors), are maintained in culture and in some cases expanded and/or cryopreserved. In certain embodiments, the population of RPE cells are cryopreserved.

[0691] Exemplary RPE cell types include, but are not limited to, retinal pigmented epithelium (RPE) cell, RPE progenitor cell, immature RPE cell, mature RPE cell, functional RPE cell, and the like.

[0692] In some embodiments, the RPE cells, such as primary RPE cells isolated from one or more individual donors (e.g., healthy donors) or RPE cells differentiated from iPSCs derived from one or more individual donors (e.g., healthy donors), have a genetic expression profile similar or substantially similar to that of native RPE cells. Such RPE cells may possess the polygonal, planar sheet morphology of native RPE cells when grown to confluence on a planar substrate.

[0693] In some embodiments, the present technology is directed to engineered RPE cells, such as primary RPE cells isolated from one or more individual donors (e.g., healthy donors) or RPE cells differentiated from iPSCs derived from one or more individual donors (e.g., healthy donors).

[0694] In some embodiments, the provided engineered RPE cells evade immune recognition. In some embodiments, the engineered RPE cells described herein, such as primary RPE cells isolated from one or more individual donors (e.g., healthy donors) or RPE cells differentiated from iPSCs derived from one or more individual donors (e.g., healthy donors), do not activate an immune response. [0695] In some embodiments, the engineered cells or populations of cells provided herein are thyroid cells, such as primary thyroid cells. In some embodiments, the primary thyroid cells are isolated or obtained from one or more individual donor subjects, such as one or more individual healthy donor (e.g., a subject that is not known or suspected of, e.g., not exhibiting clinical signs of, a disease or infection). As will be appreciated by those skilled in the art, methods of isolating or obtaining thyroid cells from an individual can be achieved using known techniques.

[0696] In some embodiments, primary thyroid cells are obtained (e.g., harvested, extracted, removed, or taken) from a subject or an individual. In some embodiments, primary thyroid cells are produced from a pool of thyroid cells such that the thyroid cells are from one or more subjects (e.g., one or more human including one or more healthy humans). In some embodiments, the pool of primary thyroid cells is from 1-100, 1-50, 1-20, 1-10, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, or 100 or more subjects.

[0697] In some embodiments, the cells as provided herein are thyroid cells differentiated from engineered iPSCs that contain modifications (e.g., genetic modifications) described herein and that are differentiated into a thyroid cell. As will be appreciated by those skilled in the art, the methods for differentiation depend on the desired cell type using known techniques.

[0698] In some embodiments, engineered pluripotent cells containing any of the vectors described herein are differentiated into thyroid progenitor cells and thyroid follicular organoids that can secrete thyroid hormones. Techniques to differentiate thyroid cells are known the art. See, e.g., Kurmann et al., Cell Stem Cell, 2015 Nov 5;17(5):527-42, incorporated herein by reference in its entirety and specifically for the methods and reagents for the generation of thyroid cells from human pluripotent stem cells. Differentiation can be assayed as is known in the art, generally by evaluating the presence of thyroid cell associated or specific markers or by measuring functionality.

[0699] In some embodiments, the population of engineered thyroid cells, such as primary thyroid cells isolated from one or more individual donors (e.g., healthy donors) or thyroid cells differentiated from iPSCs derived from one or more individual donors (e.g., healthy donors), are maintained in culture and in some cases expanded and/or cryopreserved. In certain embodiments, the thyroid cells are cryopreserved. [0700] In some embodiments, the present technology is directed to engineered thyroid cells, such as primary thyroid cells isolated from one or more individual donors (e.g., healthy donors) or thyroid cells differentiated from iPSCs derived from one or more individual donors (e.g., healthy donors).

[0701] In some embodiments, the provided engineered thyroid cells evade immune recognition. In some embodiments, the engineered thyroid cells described herein, such as primary thyroid cells isolated from one or more individual donors (e.g., healthy donors) or thyroid cells differentiated from iPSCs derived from one or more individual donors (e.g., healthy donors), do not activate an immune response.

Cardiac cells

[0702] In some embodiments, the engineered cells or populations of cells provided herein are cardiac cells. In some embodiments, the engineered cardiac cells comprise cells differentiated from HIP cells comprising any of the vectors as described herein. As will be appreciated by those skilled in the art, the methods for differentiation depend on the desired cell type using known techniques. Exemplary cardiac cell types include, but are not limited to, a cardiomyocyte, nodal cardiomyocyte, conducting cardiomyocyte, working cardiomyocyte, cardiomyocyte precursor cell, cardiomyocyte progenitor cell, cardiac stem cell, cardiac muscle cell, atrial cardiac stem cell, ventricular cardiac stem cell, epicardial cell, hematopoietic cell, vascular endothelial cell, endocardial endothelial cell, cardiac valve interstitial cell, cardiac pacemaker cell, and the like.

[0703] In some embodiments, cardiomyocyte precursor cells include a cell that is capable of giving rise to progeny that include mature (end-stage) cardiomyocytes. Cardiomyocyte precursor cells can often be identified using one or more markers selected from GATA-4, Nkx2.5, and the MEF-2 family of transcription factors. In some instances, cardiomyocytes refer to immature cardiomyocytes or mature cardiomyocytes that express one or more markers (sometimes at least 2, 3, 4, or 5 markers) from the following list: cardiac troponin I (cTnl), cardiac troponin T (cTnT), sarcomeric myosin heavy chain (MHC), GATA-4, Nkx2.5, N- cadherin, p2-adrenoceptor, ANF, the MEF-2 family of transcription factors, creatine kinase MB (CK-MB), myoglobin, and atrial natriuretic factor (ANF). In some embodiments, the cardiac cells of the disclosure demonstrate spontaneous periodic contractile activity. In some cases, when those cardiac cells are cultured in a suitable tissue culture environment with an appropriate Ca²⁺ concentration and electrolyte balance, the cells can be observed to contract in a periodic fashion across one axis of the cell, and then release from contraction, without having to add any additional components to the culture medium. In some embodiments, the cardiac cells of the disclosure are hypoimmunogenic cardiac cells.

[0704] In some embodiments, the method of producing hypoimmunogenic cardiac cells from hypoimmunogenic pluripotent (HIP) cells by in vitro differentiation comprises: (a) culturing HIP cells in a culture medium comprising a GSK inhibitor; (b) culturing the HIP cells in a culture medium comprising a WNT antagonist to produce a population of pre-cardiac cells; and (c) culturing the pre-cardiac cells in a culture medium comprising insulin to produce hypoimmune cardiac cells. In some embodiments, the GSK inhibitor is CHIR-99021, a derivative thereof, or a variant thereof. In some instances, the GSK inhibitor is at a concentration ranging from about 2 mM to about 10 mM. In some embodiments, the WNT antagonist is IWR1, a derivative thereof, or a variant thereof. In some instances, the WNT antagonist is at a concentration ranging from about 2 mM to about 10 mM.

[0705] In some embodiments, the hypoimmunogenic cardiac cells is isolated from noncardiac cells. In some embodiments, the isolated hypoimmunogenic cardiac cells are expanded. In certain embodiments, the isolated hypoimmunogenic cardiac cells are expanded and cryopreserved.

[0706] Other useful methods for differentiating induced pluripotent stem cells or pluripotent stem cells into cardiac cells are described, for example, in US2017/0152485; US2017/0058263; US2017/0002325; US2016/0362661; US2016/0068814; US9,062,289; US7,897,389; and US7,452,718. Additional methods for producing cardiac cells from induced pluripotent stem cells or pluripotent stem cells are described in, for example, Xu et al., Stem Cells and Development, 2006, 15(5): 631-9, Burridge et al., Cell Stem Cell, 2012, 10: 16-28, and Chen et al., Stem Cell Res, 2015, 15(2):365-375.

[0707] In various embodiments, hypoimmunogenic cardiac cells can be cultured in medium comprising a BMP pathway inhibitor, a WNT signaling activator, a WNT signaling inhibitor, a WNT agonist, a WNT antagonist, a Src inhibitor, a EGFR inhibitor, a PCK activator, a cytokine, a growth factor, a cardiotropic agent, a compound, and/or the like.

[0708] The WNT signaling activator includes, but is not limited to, CHIR99021. The PCK activator includes, but is not limited to, PMA. The WNT signaling inhibitor includes, but is not limited to, a compound selected from KY02111, SO3031 (KY01-I), SO2031 (KY02-I), and SO3042 (KY03-I), and XAV939. The Src inhibitor includes, but is not limited to, A419259. The EGFR inhibitor includes, but is not limited to, AG1478.

[0709] Non-limiting examples of an agent for generating a cardiac cell from an iPSC include activin A, BMP4, Wnt3a, VEGF, soluble frizzled protein, cyclosporin A, angiotensin II, phenylephrine, ascorbic acid, dimethylsulfoxide, 5-aza-2'-deoxycytidine, and the like.

[0710] The cells provided herein can be cultured on a surface, such as a synthetic surface to support and/or promote differentiation of hypoimmunogenic pluripotent cells into cardiac cells. In some embodiments, the surface comprises a polymer material including, but not limited to, a homopolymer or copolymer of selected one or more acrylate monomers. Non-limiting examples of acrylate monomers and methacrylate monomers include tetra(ethylene glycol) diacrylate, glycerol dimethacrylate, 1,4-butanediol dimethacrylate, poly(ethylene glycol) diacrylate, di(ethylene glycol) dimethacrylate, tetra(ethyiene glycol) dimethacrylate, 1,6- hexanediol propoxylate diacrylate, neopentyl glycol diacrylate, trimethylolpropane benzoate diacrylate, trimethylolpropane eihoxylate (1 EO/QH) methyl, tricyclo[5.2.1.02,6] decane dimethanol diacrylate, neopentyl glycol exhoxylate diacrylate, and trimethylolpropane triacrylate. Acrylate synthesized is known in the art or obtained from a commercial vendor, such as Polysciences, Inc., Sigma Aldrich, Inc. and Sartomer, Inc.

[0711] The polymeric material can be dispersed on the surface of a support material. Useful support materials suitable for culturing cells include a ceramic substance, a glass, a plastic, a polymer or co-polymer, any combinations thereof, or a coating of one material on another. In some instances, a glass includes soda-lime glass, pyrex glass, vycor glass, quartz glass, silicon, derivatives of these, or the like.

[0712] In some instances, plastics or polymers including dendritic polymers include poly(vinyl chloride), poly(vinyl alcohol), poly(methyl methacrylate), poly(vinyl acetate- maleic anhydride), poly (dimethylsiloxane) monomethacrylate, cyclic olefin polymers, fluorocarbon polymers, polystyrenes, polypropylene, polyethyleneimine, derivatives of these, or the like. In some instances, copolymers include poly(vinyl acetate-co-maleic anhydride), poly(styrene-co- maleic anhydride), poly(ethylene-co-acrylic acid), derivatives of these, or the like.

[0713] The efficacy of cardiac cells prepared as described herein can be assessed in animal models for cardiac cryoinjury, which causes 55% of the left ventricular wall tissue to become scar tissue without treatment (Li et al., Ann. Thorac. Surg. 62:654, 1996; Sakai et al., Ann. Thorac. Surg. 8:2074, 1999, Sakai et al., Thorac. Cardiovasc. Surg. 118:715, 1999).

[0714] In some embodiments, the engineered cardiac cells, such cardiac cells differentiated from iPSCs derived from one or more individual donors (e.g., healthy donors), are maintained in culture and in some cases expanded and/or cryopreserved. In certain embodiments, the cardiac cells are cryopreserved.

[0715] In some embodiments, the present technology is directed to engineered cardiac cells, such as cardiac cells differentiated from iPSCs derived from one or more individual donors (e.g., healthy donors).

[0716] In some embodiments, the provided engineered cardiac cells evade immune recognition. In some embodiments, the engineered cardiac cells described herein, such as cardiac cells differentiated from iPSCs derived from one or more individual donors (e.g., healthy donors), do not activate an immune response.

Neural cells

[0717] In some embodiments, the engineered cells or populations of cells provided herein are neural cells, including neural cell types differentiated from engineered pluripotent cells (e.g., iPSCs) as described herein. As will be appreciated by those skilled in the art, the methods for differentiation depend on the desired cell type using known techniques. Exemplary neural cell types include, but are not limited to, cerebral endothelial cells, neurons (e.g., dopaminergic neurons), glial cells, and the like.

[0718] In some embodiments, differentiation of induced pluripotent stem cells is performed by exposing or contacting cells to specific factors which are known to produce a specific cell lineage(s), so as to target their differentiation to a specific, desired lineage and/or cell type of interest. In some embodiments, terminally differentiated cells display specialized phenotypic characteristics or features. In certain embodiments, the stem cells described herein are differentiated into a neuroectodermal, neuronal, neuroendocrine, dopaminergic, cholinergic, serotonergic (5-HT), glutamatergic, GABAergic, adrenergic, noradrenergic, sympathetic neuronal, parasympathetic neuronal, sympathetic peripheral neuronal, or glial cell population. In some instances, the glial cell population includes a microglial (e.g., amoeboid, ramified, activated phagocytic, and activated non-phagocytic) cell population or a macroglial (e.g., central nervous system cell: astrocyte, oligodendrocyte, ependymal cell, and radial glia; and peripheral nervous system cell: Schwann cell and satellite cell) cell population, or the precursors and progenitors of any of the preceding cells.

[0719] Protocols for generating different types of neural cells are described in PCT Application No. WO2010144696, US Patent Nos. 9,057,053; 9,376,664; and 10,233,422. Additional descriptions of methods for differentiating hypoimmunogenic pluripotent cells can be found, for example, in Deuse et al., Nature Biotechnology, 2019, 37, 252-258 and Han et al., Proc Natl Acad Sci USA, 2019, 116(21), 10441-10446.

[0720] In some embodiments, the engineered neural cells, such as neural cells differentiated from iPSCs derived from one or more individual donors (e.g., healthy donors), are maintained in culture and in some cases expanded and/or cryopreserved. In certain embodiments, the neural cells are cryopreserved.

[0721] In some embodiments, the present disclosure is directed to engineered neural cells, such as neural cells differentiated from iPSCs derived from one or more individual donors (e.g., healthy donors), that is engineered to express any of the vectors described herein.

[0722] In some embodiments, the provided engineered neural cells evade immune recognition. In some embodiments, the engineered neural cells described herein, such as neural cells differentiated from iPSCs derived from one or more individual donors (e.g., healthy donors), do not activate an immune response.

[0723] In some embodiments, the engineered cells or populations of cells provided herein are cerebral endothelial cells (ECs), precursors, and/or progenitors thereof, e.g., differentiated from pluripotent stem cells (e.g., induced pluripotent stem cells). In some embodiments, the cerebral endothelial cells (ECs), precursors, and/or progenitors thereof are differentiated from pluripotent stem cells on a surface by culturing the cells in a medium comprising one or more factors that promote the generation of cerebral ECs or neural cell. In some instances, the medium includes one or more of the following: CHIR-99021, VEGF, basic FGF (bFGF), or Y-27632. In some embodiments, the medium includes a supplement designed to promote survival and functionality for neural cells. In some embodiments, cerebral endothelial cells (ECs), precursors, and progenitors thereof are differentiated from pluripotent stem cells on a surface by culturing the cells in an unconditioned or conditioned medium. In some instances, the medium comprises factors or small molecules that promote or facilitate differentiation. In some embodiments, the medium comprises one or more factors or small molecules selected from the group consisting of VEGR, FGF, SDF-1, CHIR-99021, Y-27632, SB 431542, and any combination thereof. In some embodiments, the surface for differentiation comprises one or more extracellular matrix proteins. The surface can be coated with the one or more extracellular matrix proteins. The cells can be differentiated in suspension and then put into a gel matrix form, such as matrigel, gelatin, or fibrin/thrombin forms to facilitate cell survival. In some cases, differentiation is assayed as is known in the art, generally by evaluating the presence of cell-specific markers. In some embodiments, the cerebral endothelial cells express or secrete a factor selected from CD31, VE cadherin, and any combinations thereof. In certain embodiments, the cerebral endothelial cells express or secrete one or more of the factors selected from CD31, CD34, CD45, CD117 (c-kit), CD 146, CXCR4, VEGF, SDF-1, PDGF, GLUT-1, PECAM-1, eNOS, claudin-5, occludin, ZO-1, p-glycoprotein, von Willebrand factor, VE-cadherin, low density lipoprotein receptor EDER, low density lipoprotein receptor-related protein 1 LRP1, insulin receptor INSR, leptin receptor LEPR, basal cell adhesion molecule BCAM, transferrin receptor TFRC, advanced glycation end product- specific receptor AGER, receptor for retinol uptake STRA6, large neutral amino acids transporter small subunit 1 SLC7A5, excitatory amino acid transporter 3 SLC1A1, sodium- coupled neutral amino acid transporter 5 SLC38A5, solute carrier family 16 member 1 SLC16A1, ATP-dependent translocase ABCB1, ATP-ABCC2-binding cassette transporter ABCG2, multidrug resistance-associated protein 1 ABCC1, canalicular multispecific organic anion transporter 1 ABCC2, multidrug resistance-associated protein 4 ABCC4, multidrug resistance-associated protein 5 ABCC5, or any combination thereof. In some embodiments, the cerebral ECs are characterized with one or more of the features selected from high expression of tight junctions, high electrical resistance, low fenestration, small perivascular space, high prevalence of insulin and transferrin receptors, high number of mitochondria, or any combination thereof. In some embodiments, cerebral ECs are selected or purified using a positive selection strategy. In some instances, the cerebral ECs are sorted against an endothelial cell marker such as, but not limited to, CD31. In other words, CD31 positive cerebral ECs are isolated. In some embodiments, cerebral ECs are selected or purified using a negative selection strategy. In some embodiments, undifferentiated or pluripotent stem cells are removed by selecting for cells that express a pluripotency marker including, but not limited to, TRA-1-60 and SSEA-1.

[0724] In some embodiments, the engineered cells or population of cells provided herein are dopaminergic neurons, including neuronal stem cells, neuronal progenitor cells, immature dopaminergic neurons, and mature dopaminergic neurons. In some embodiments, the dopaminergic neurons are differentiated from HIP cells. In some cases, dopaminergic neurons include neuronal cells which express tyrosine hydroxylase (TH), the rate-limiting enzyme for dopamine synthesis. In some embodiments, dopaminergic neurons secrete the neurotransmitter dopamine, and have little or no expression of dopamine hydroxylase. A dopaminergic (DA) neuron can express one or more of the following markers: neuron- specific enolase (NSE), 1- aromatic amino acid decarboxylase, vesicular monoamine transporter 2, dopamine transporter, Nurr-1, or dopamine-2 receptor (D2 receptor). In certain cases, neural stem cells include a population of pluripotent cells that have partially differentiated along a neural cell pathway and express one or more neural markers including, for example, nestin. Neural stem cells may differentiate into neurons or glial cells (e.g., astrocytes and oligodendrocytes). Neural progenitor cells can include cultured cells which express FOXA2 and low levels of P-tubulin, but not tyrosine hydroxylase. Such neural progenitor cells have the capacity to differentiate into a variety of neuronal subtypes; particularly a variety of dopaminergic neuronal subtypes, upon culturing the appropriate factors, such as those described herein. In some embodiments, DA neurons, precursors, and progenitors thereof are differentiated from pluripotent stem cells by culturing the stem cells in medium comprising one or more factors or additives. Useful factors and additives that promote differentiation, growth, expansion, maintenance, and/or maturation of DA neurons include, but are not limited to, Wntl, FGF2, FGF8, FGF8a, sonic hedgehog (SHH), brain derived neurotrophic factor (BDNF), transforming growth factor a (TGF-a), TGF-P, interleukin 1 beta, glial cell line-derived neurotrophic factor (GDNF), a GSK-3 inhibitor (e.g., CHIR-99021), a TGF-P inhibitor (e.g., SB-431542), B-27 supplement, dorsomorphin, purmorphamine, noggin, retinoic acid, cAMP, ascorbic acid, neurturin, knockout serum replacement, N-acetyl cysteine, c- kit ligand, modified forms thereof, mimics thereof, analogs thereof, or variants thereof. In some embodiments, the DA neurons are differentiated in the presence of one or more factors that activate or inhibit the WNT pathway, NOTCH pathway, SHH pathway, BMP pathway, FGF pathway, and the like. Differentiation protocols and detailed descriptions thereof are provided in, e.g., US9,968,637, US7,674,620, Kim et al., Nature, 2002, 418,50-56; Bjorklund et al., PNAS, 2002, 99(4), 2344-2349; Grow et al., Stem Cells Transl Med. 2016, 5(9): 1133-44, and Cho et al., PNAS, 2008, 105:3392-3397, the disclosures in their entirety including the detailed description of the examples, methods, figures, and results are herein incorporated by reference. In some embodiments, hypoimmunogenic dopaminergic neurons can be isolated from nonneuronal cells. In some embodiments, isolated hypoimmunogenic dopaminergic neurons can be expanded. In certain embodiments, the isolated hypoimmunogenic dopaminergic neurons are expanded and cryopreserved. To characterize and monitor DA differentiation and assess the DA phenotype, expression of any number of molecular and genetic markers can be evaluated. For example, the presence of genetic markers can be determined by various methods known to those skilled in the art. Expression of molecular markers can be determined by quantifying methods such as, but not limited to, qPCR-based assays, immunoassays, immunocytochemistry assays, immunoblotting assays, or the like. Exemplary markers for DA neurons include, but are not limited to, TH, b-tubulin, paired box protein (Pax6), insulin gene enhancer protein (Isll), nestin, diaminobenzidine (DAB), G protein-activated inward rectifier potassium channel 2 (GIRK2), microtubule-associated protein 2 (MAP-2), NURR1, dopamine transporter (DAT), forkhead box protein A2 (F0XA2), FOX3, doublecortin, LIM homeobox transcription factor 1-beta (LMX1B), or the like. In some embodiments, the DA neurons express one or more of the markers selected from corin, F0XA2, TuJl, NURR1, or any combination thereof. In some embodiments, DA neurons are assessed according to cell electrophysiological activity. The electrophysiology of the cells can be evaluated by using assays known to those skilled in the art. For instance, whole-cell and perforated patch clamp, assays for detecting electrophysiological activity of cells, assays for measuring the magnitude and duration of action potential of cells, and functional assays for detecting dopamine production of DA cells. In some embodiments, DA neuron differentiation is characterized by spontaneous rhythmic action potentials, and high- frequency action potentials with spike frequency adaption upon injection of depolarizing current. In other embodiments, DA differentiation is characterized by the production of dopamine. The level of dopamine produced is calculated by measuring the width of an action potential at the point at which it has reached half of its maximum amplitude (spike half-maximal width). Useful descriptions of neurons derived from stem cells and methods of making thereof can be found, for example, in Kirkeby et al., Cell Rep, 2012, 1:703-714; Kriks et al., Nature, 2011, 480:547-551; Wang et al., Stem Cell Reports, 2018, 11(1): 171- 182; Lorenz Studer, “Chapter 8 - Strategies for Bringing Stem Cell-Derived Dopamine Neurons to the clinic-The NYSTEM Trial” in Progress in Brain Research, 2017, volume 230, pg. 191-212; Liu et al., Nat Protoc, 2013, 8:1670-1679; Upadhya et al., Curr Protoc Stem Cell Biol, 38, 2D.7.1-2D.7.47; US Publication Appl. No. 20160115448, and US8,252,586; US8,273,570; US9,487,752 and US 10,093,897, the contents are incorporated herein by reference in their entirety. In addition to DA neurons, other neuronal cells, precursors, and progenitors thereof can be differentiated from the HIP cells outlined herein by culturing the cells in medium comprising one or more factors or additives. Non-limiting examples of factors and additives include GDNF, BDNF, GM-CSF, B27, basic FGF, basic EGF, NGF, CNTF, SMAD inhibitor, Wnt antagonist, SHH signaling activator, or any combination thereof. In some embodiments, the SMAD inhibitor is selected from the group consisting of SB431542, LDN-193189, Noggin PD169316, SB203580, LY364947, A77-01, A-83-01, BMP4, GW788388, GW6604, SB-505124, lerdelimumab, metelimumab, GC-I008, AP-12009, AP- 11014, LY550410, LY580276, LY364947, LY2109761, SB-505124, E-616452 (RepSox ALK inhibitor), SD-208, SMI6, NPC-30345, K 26894, SB-203580, SD-093, activin-M108A, P144, soluble TBR2-Fc, DMH-1, dorsomorphin dihydrochloride, or derivatives thereof. In some embodiments, the Wnt antagonist is selected from the group consisting of XAV939, DKK1, DKK-2, DKK-3, DKK-4, SFRP-1, SFRP-2, SFRP-3, SFRP-4, SFRP-5, WIF-1, Soggy, IWP-2, IWR1, ICG-001, KY0211, Wnt-059, LGK974, IWP-L6, and derivatives thereof. In some embodiments, the SHH signaling activator is selected from the group consisting of Smoothened agonist (SAG), SAG analog, SHH, C25-SHH, C24-SHH, purmorphamine, Hg-Ag, and derivatives thereof. In some embodiments, the neurons express one or more of the markers selected from the group consisting of glutamate ionotropic receptor NMDA type subunit 1 GRIN1, glutamate decarboxylase 1 GAD1, gamma-aminobutyric acid GABA, tyrosine hydroxylase TH, LIM homeobox transcription factor 1-alpha LMX1A, Forkhead box protein 01 FOXO1, Forkhead box protein A2 FOXA2, Forkhead box protein 04 FOXO4, FOXG1, 2', 3'- cyclic-nucleotide 3'-phosphodiesterase CNP, myelin basic protein MBP, tubulin beta chain 3 TUB3, tubulin beta chain 3 NEUN, solute carrier family 1 member 6 SLC1A6, SST, PV, calbindin, RAX, LHX6, LHX8, DLX1, DLX2, DLX5, DLX6, SOX6, MAFB, NPAS1, ASCL1, SIX6, OLIG2, NKX2.1, NKX2.2, NKX6.2, VGLUT1, MAP2, CTIP2, SATB2, TBR1, DLX2, ASCL1, ChAT, NGFI-B, c-fos, CRF, RAX, POMC, hypocretin, NADPH, NGF, Ach, VAChT, PAX6, EMX2p75, CORIN, TUJ1, NURR1, and any combination thereof.

[0725] In some embodiments, the engineered cells or population of cells provided herein are glial cells such as, but not limited to, microglia, astrocytes, oligodendrocytes, ependymal cells and Schwann cells, glial precursors, and glial progenitors thereof that are produced by differentiating pluripotent stem cells into therapeutically effective glial cells, and the like. Differentiation of hypoimmunogenic pluripotent stem cells produces hypoimmunogenic neural cells, such as hypoimmunogenic glial cells. In some embodiments, glial cells, precursors, and progenitors thereof can be generated by culturing pluripotent stem cells in medium comprising one or more agents selected from the group consisting of retinoic acid, IL-34, M-CSF, FLT3 ligand, GM-CSF, CCL2, a TGFbeta inhibitor, a BMP signaling inhibitor, a SHH signaling activator, FGF, platelet derived growth factor PDGF, PDGFR-alpha, HGF, IGF1, noggin, SHH, dorsomorphin, noggin, and any combination thereof. In certain instances, the BMP signaling inhibitor is LDN193189, SB431542, or a combination thereof. In some embodiments, the glial cells express NKX2.2, PAX6, SOXIO, brain derived neurotrophic factor BDNF, neutrotrophin-3 NT-3, NT-4, EGF, ciliary neurotrophic factor CNTF, nerve growth factor NGF, FGF8, EGFR, OLIG1, OLIG2, myelin basic protein MBP, GAP-43, LNGFR, nestin, GFAP, CDl lb, CDl lc, CX3CR1, P2RY12, IBA-1, TMEM119, CD45, or any combination thereof. Exemplary differentiation medium can include any specific factors and/or small molecules that may facilitate or enable the generation of a glial cell type as recognized by those skilled in the art. To determine if the cells generated according to the in vitro differentiation protocol display glial cell characteristics and features, the cells can be transplanted into an animal model. In some embodiments, the glial cells are injected into an immunocompromised mouse, e.g., an immunocompromised shiverer mouse. The glial cells are administered to the brain of the mouse and after a pre-selected amount of time, the engrafted cells are evaluated. In some instances, the engrafted cells in the brain are visualized by using immunostaining and imaging methods. In some embodiments, it is determined that the glial cells express known glial cell biomarkers.

Useful methods for generating glial cells, precursors, and progenitors thereof from stem cells are found, for example, in US7,579,188; US7,595,194; US8,263,402; US8,206,699; US8,252,586; US9,193,951; US9,862,925; US8,227,247; US9,709,553; US2018/0187148; US2017/0198255; US2017/0183627; US2017/0182097; US2017/253856; US2018/0236004; WO2017/172976; and WO2018/093681. Methods for differentiating pluripotent stem cells are described in, e.g., Kikuchi et al., Nature, 2017, 548, 592-596; Kriks et al., Nature, 2011, 547-551; Doi et al., Stem Cell Reports, 2014, 2, 337-50; Perrier et al., Proc Natl Acad Sci USA, 2004, 101, 12543-12548; Chambers et al., Nat Biotechnol, 2009, 27, 275-280; and Kirkeby et al., Cell Reports, 2012, 1, 703-714. Additional descriptions of neural cells including dopaminergic neurons for use in the present technology are found in W02020/018615, the disclosure of which is herein incorporated by reference in its entirety.

Hematopoietic stem cells

[0726] In some embodiments, the engineered cells or populations of cells provided herein are hematopoietic stem cells. In some cases, the hematopoietic stem cell is an immature cell that can develop into all types of blood cells, including white blood cells, red blood cells, and platelets. Hematopoietic stem cells (HSC) are found in the peripheral blood and the bone marrow. In some cases, the hematopoietic stem cell is isolated from the peripheral blood or bone marrow.

B. Producer Cells

[0727] An engineered cell or population of cells of the present disclosure may find use in the production of molecules of interest, such as antibodies, fusogens, viral or virus-like particles, and engineered receptors (e.g., CARs, CAARs, TCRs and BARs). In some cases, the engineered cells or populations of cells may be used for the production of one or more proteins encoded by a transgene described herein, e.g., wherein the sequences encoding the transgene(s) are accompanied by a barcode sequence as described herein. Non-limiting examples of cells that may be used include CHO cells, BHK cells, MDCK cells, C3H 10T1/2 cells, FLY cells, Psi-2 cells, BOSC 23 cells, PA317 cells, WEHI cells, COS cells, BSC 1 cells, BSC 40 cells, BMT 10 cells, VERO cells, W138 cells, MRC5 cells, A549 cells, HT1080 cells, 293 cells, 293T cells, B- 50 cells, 3T3 cells, NIH3T3 cells, HepG2 cells, Saos-2 cells, Huh7 cells, HeLa cells, W163 cells, 211 cells, NS0, PerC6, Sp2/0, BHK, C127, and 211 A cells. In some embodiments, the engineered cell or population of cells of the disclosure is a HEK293 cell, e.g., a HEK293T cell. In some embodiments, the engineered cell or population of cells of the disclosure is a HeLa cell. In some embodiments, the engineered cell or population of cells of the disclosure is a NS0 cell. In some embodiments, the engineered cell or population of cells of the disclosure is a PerC6 cell. [0728] In some embodiments, engineered cells or population of cells of the present disclosure may be used for the production of virus or virus-like particles. In such cases, multiple cell culture systems including bacteria, mammalian cell lines, insect cell lines, yeast, and plant cells can be used. In some embodiments, elements for the production of a viral or virus-like particle, e.g., a recombinant viral particle such as a replication incompetent lentiviral vector, are included in a packaging cell line or are present on a packaging vector. In some embodiments, viral particles can include packaging elements, rev, gag, and pol, delivered to the packaging cell line via one or more packaging vectors. In some embodiments, the packaging vector is an expression vector or viral vector that lacks a packaging signal and comprises a polynucleotide encoding one, two, three, four, or more viral structural and/or accessory genes. Typically, the packaging vectors are included in a packaging cell, and are introduced into the cell via transfection, transduction, or infection. A retroviral, e.g., lentiviral, transfer vector can be introduced into a packaging cell line, via transfection, transduction or infection, to generate a source cell or cell line. The packaging vectors can be introduced into human cells or cell lines by standard methods including, e.g., calcium phosphate transfection, lipofection or electroporation. In some embodiments, the packaging vectors are introduced into the cells together with a dominant selectable marker, such as neomycin, hygromycin, puromycin, blastocidin, zeocin, thymidine kinase, DHFR, Gin synthetase, or ADA, followed by selection in the presence of the appropriate drug and isolation of clones. A selectable marker gene can be linked physically to genes encoded by the packaging vector, e.g., by IRES or self-cleaving viral peptides. In some embodiments, the packaging vector is a packaging plasmid.

[0729] In some embodiments, engineered cells or population of cells of the disclosure comprise cells from producer (or packaging) cell lines. In some embodiments, the producer cell line further stably expresses one or more transgene sequences encoded by any of the vectors described herein. Suitable cell lines that can be used include, for example, CHO cells, BHK cells, MDCK cells, C3H 10T1/2 cells, FLY cells, Psi-2 cells, BOSC 23 cells, PA317 cells, WEHI cells, COS cells, BSC 1 cells, BSC 40 cells, BMT 10 cells, VERO cells, W138 cells, MRC5 cells, A549 cells, HT1080 cells, HEK293 cells, HEK293T cells, B-50 cells, 3T3 cells, NIH3T3 cells, HepG2 cells, Saos-2 cells, Huh7 cells, HeLa cells, W163 cells, PerC6 cells, NSO cells, 211 cells, and 211 A cells. In embodiments, the packaging cells are HEK293 cells, HEK293T cells, HeLa cells, PerC6 cells, or NSO cells. In some embodiments, producer cells e.g., a source cell line) include a cell line that is capable of producing recombinant retroviral particles, comprising a packaging cell line and a transfer vector construct comprising a packaging signal. Methods of preparing viral stock solutions are illustrated by, e.g., Y. Soneoka et al. (1995) Nucl. Acids Res. 23:628-633, and N. R. Landau et al., (1992) J. Virol. 66:5110- 5113, which are incorporated herein by reference. Infectious virus particles may be collected from the packaging cells, e.g., by cell lysis, or collection of the supernatant of the cell culture. Optionally, the collected virus particles may be enriched or purified. In some embodiments, the source cell comprises one or more plasmids coding for viral structural proteins and replication enzymes (e.g., gag, pol, and env) that can package viral particles (z.e., a packaging plasmid). In some embodiments, the sequences coding for at least two of the gag, pol, and env precursors are on the same plasmid. In some embodiments, the sequences coding for the gag, pol, and env precursors are on different plasmids. In some embodiments, the sequences coding for the gag, pol, and env precursors have the same expression signal, e.g., promoter. In some embodiments, the sequences coding for the gag, pol, and env precursors have a different expression signal, e.g., different promoters. In some embodiments, expression of the gag, pol, and env precursors is inducible. In some embodiments, the plasmids coding for viral structural proteins and replication enzymes are transfected at the same time or at different times. In some embodiments, the plasmids coding for viral structural proteins and replication enzymes are transfected at the same time or at a different time from the packaging vector. In some embodiments, the source cell line comprises one or more stably integrated viral structural genes. In some embodiments expression of the stably integrated viral structural genes is inducible.

[0730] In some embodiments, expression of the viral structural genes is regulated at the transcriptional level. In some embodiments, expression of the viral structural genes is regulated at the translational level. In some embodiments, expression of the viral structural genes is regulated at the post-translational level. In some embodiments, expression of the viral structural genes is regulated by a tetracycline (Tet)-dependent system, in which a Tet-regulated transcriptional repressor (Tet-R) binds to DNA sequences included in a promoter and represses transcription by steric hindrance (Yao et al., 1998; Jones et al., 2005). Upon addition of doxycycline (dox), Tet-R is released, allowing transcription. Multiple other suitable transcriptional regulatory promoters, transcription factors, and small molecule inducers are suitable to regulate transcription of viral structural genes.

[0731] In some embodiments, engineered cells or populations of cells (e.g., T cells, B cells, NK cells, islet cells such as beta cells, or hypoimmunogenic cells thereof) of the present disclosure may be used for the production of retroviruses such as lentiviruses.

[0732] In some embodiments, third-generation lentivirus components, human immunodeficiency virus type 1 (HIV) Rev, Gag/Pol, and an envelope under the control of Tet- regulated promoters and coupled with antibiotic resistance cassettes are separately integrated into the source cell genome. In some embodiments the source cell only has one copy of each of Rev, Gag/Pol, and an envelope protein integrated into the genome.

[0733] In some embodiments, a nucleic acid encoding the exogenous agent (e.g., a retroviral nucleic acid encoding the vector comprising one or more transgene) is also integrated into the source cell genome. In some embodiments a nucleic acid encoding the vector comprising one or more transgene is maintained episomally. In some embodiments a nucleic acid encoding the vector comprising one or more transgene is transfected into the source cell that has stably integrated Rev, Gag/Pol, and an envelope protein in the genome. See, e.g., Milani et al., EMBO Molecular Medicine, 2017, which is herein incorporated by reference in its entirety.

[0734] In some embodiments, a retroviral nucleic acid described herein is unable to undergo reverse transcription. Such a nucleic acid, in some embodiments, is able to transiently express a vector comprising one or more transgene. The retrovirus may comprise a disabled reverse transcriptase protein, or may not comprise a reverse transcriptase protein. In some embodiments, the retroviral nucleic acid comprises a disabled primer binding site (PBS) and/or att site. In some embodiments, one or more viral accessory genes, including rev, tat, vif, nef, vpr, vpu, vpx, S2, or functional equivalents thereof, are disabled or absent from the retroviral nucleic acid. In embodiments, one or more accessory genes selected from S2, rev, and tat are disabled or absent from the retroviral nucleic acid.

[0735] Typically, modem retroviral vector systems include viral genomes bearing cisacting vector sequences for transcription, reverse-transcription, integration, translation, and packaging of viral RNA into the viral particles, and producer cells lines which express the transacting retroviral gene sequences e.g., gag, pol, and env) needed for production of virus particles. By separating the cis-and trans-acting vector sequences completely, the virus is unable to maintain replication for more than one cycle of infection. Generation of live vims can be avoided by a number of strategies, e.g., by minimizing the overlap between the cis-and trans-acting sequences to avoid recombination.

[0736] In some embodiments, engineered cells or populations of cells (e.g., T cells, B cells, NK cells, islet cells such as beta cells, or hypoimmunogenic cells thereof) of the present disclosure may be used for the production of vims or vims-like particles (VLPs). A virus-like particle (VLP) which comprises a sequence that is devoid of or lacking viral genetic material may be the result of removing or eliminating the viral RNA from the sequence. Similar to viral particles, VLPs contain a viral outer envelope made from the host cell (z.e., producer cell or source cell) lipid-bi layer as well as at least one viral structural protein. In some embodiments, a viral structural protein refers to any viral protein or fragment thereof which contributes to the structure of the viral core or capsid. Generally, for viral particles, expression of the gag precursor protein alone mediates vector assembly and release. In some aspects, gag proteins or fragments thereof have been demonstrated to assemble into structures analogous to viral cores. In one embodiment this may be achieved by using an endogenous packaging signal binding site on gag. Alternatively, the endogenous packaging signal binding site is on pol. In this embodiment, the RNA which is to be delivered will contain a cognate packaging signal. In another embodiment, a heterologous binding domain (which is heterologous to gag) located on the RNA to be delivered, and a cognate binding site located on gag or pol, can be used to ensure packaging of the RNA to be delivered. The heterologous sequence could be non-viral or it could be viral, in which case it may be derived from a different virus. The VLP could be used to deliver therapeutic RNA, in which case functional integrase and/or reverse transcriptase is not required. These VLPs could also be used to deliver a therapeutic gene of interest, in which case pol is typically included. [0737] In an embodiment, gag-pol are altered, and the packaging signal is replaced with a corresponding packaging signal. In this embodiment, the particle can package the RNA with the new packaging signal. The advantage of this approach is that it is possible to package an RNA sequence that is devoid of viral sequence for example, RNAi.

[0738] An alternative approach is to rely on over-expression of the RNA to be packaged. In one embodiment the RNA to be packaged is over-expressed in the absence of any RNA containing a packaging signal. This may result in a significant level of therapeutic RNA being packaged, and that this amount is sufficient to transduce a cell and have a biological effect. [0739] In some embodiments, a polynucleotide, such as a transgene, comprises a nucleotide sequence encoding a viral gag protein or retroviral gag and pol proteins, wherein the gag protein or pol protein comprises a heterologous RNA binding domain capable of recognizing a corresponding sequence in an RNA sequence to facilitate packaging of the RNA sequence into a viral vector particle. In some embodiments, the heterologous RNA binding domain comprises an RNA binding domain derived from a bacteriophage coat protein, a Rev protein, a protein of the U1 small nuclear ribonucleoprotein particle, a Nova protein, a TF111 A protein, a TIS 11 protein, a trp RNA-binding attenuation protein (TRAP), or a pseudouridine synthase. [0740] In some embodiments, the assembly of a viral based vector vehicle particle (z.e., a VLP) is initiated by binding of the core protein to a unique encapsidation sequence within the viral genome (e.g., UTR with stem-loop structure). In some embodiments, the interaction of the core with the encapsidation sequence facilitates oligomerization.

[0741] In some embodiments, the source cell for VLP production comprises one or more plasmids coding for viral structural proteins (e.g., gag, pol) which can package viral particles (z.e., a packaging plasmid). In some embodiments, the sequences coding for at least two of the gag and pol precursors are on the same plasmid. In some embodiments, the sequences coding for the gag and pol precursors are on different plasmids. In some embodiments, the sequences coding for the gag and pol precursors have the same expression signal, e.g., promoter. In some embodiments, the sequences coding for the gag and pol precursors have a different expression signal, e.g., different promoters. In some embodiments, expression of the gag and pol precursors is inducible.

[0742] In some embodiments, formation of VLPs or any viral-based particle can be detected by any suitable technique known in the art. Examples of such techniques include, e.g., electron microscopy, dynamic light scattering, selective chromatographic separation, and/or density gradient centrifugation.

[0743] In some embodiments, the cell is a cell line, such as CHO cells, BHK cells, MDCK cells, C3H 10T1/2 cells, FLY cells, Psi-2 cells, BOSC 23 cells, PA317 cells, WEHI cells, COS cells, BSC 1 cells, BSC 40 cells, BMT 10 cells, VERO cells, W138 cells, MRC5 cells, A549 cells, HT1080 cells, 293 cells, 293T cells, B-50 cells, 3T3 cells, NIH3T3 cells, HepG2 cells, Saos-2 cells, Huh7 cells, HeLa cells, W163 cells, 211 cells, NS0, PerC6, Sp2/0, BHK, C127 and 211 A cells.

C. Population of Cells

[0744] In some embodiments, the invention provides a population of cells comprising any of the vectors described herein. The population of cells can include a plurality of cells selected from any of the cells described herein.

[0745] In some embodiments, the population of cells comprises one or more subpopulation of cells. In some embodiments, the population of cells comprises a first subpopulation of cells and a second subpopulation of cells. [0746] In some embodiments, the one or more subpopulation of cells comprises one or more vectors, wherein the one or more vectors comprises one or more transgene sequences accompanied by a unique barcode sequence. The transgene sequences can be selected from any of the transgenes described herein, for example, sequences of antibodies, fusogens and viral vectors, engineered receptors, tolerogenic factors, safety switches, genome editing complexes, transcription factors, etc. In some embodiments, the transgene sequences encode CARs.

[0747] In some embodiments, the first subpopulation of cells and/or the second subpopulation of cells comprises a CAR. In some embodiments, the first subpopulation of cells and the second subpopulation of cells comprise the same CAR. In some embodiments, the first subpopulation of cells and the second subpopulation of cells comprise different CARs. In some embodiments, the first subpopulation of cells and the second subpopulation of cells comprise two or more CARs. In some embodiments, the first subpopulation of cells and the second subpopulation of cells comprise two or more CARs located on one vector. In some embodiments, the first subpopulation of cells and the second subpopulation of cells comprise two or more CARs located on different vectors. In some embodiments, the first subpopulation of cells and the second subpopulation of cells comprise one CAR that is the same CAR and one CAR that is a different CAR. The CAR can be selected from any of the CARs described herein.

[0748] In some embodiments, the CAR is a CD 19 CAR. In some embodiments, the CAR is a CD22 CAR. In some embodiments, the first subpopulation of cells comprises a CD 19 CAR and the second subpopulation of cells comprises a CD22 CAR. In some embodiments, the first subpopulation of cells comprises a CD22 CAR and the second subpopulation of cells comprises a CD 19 CAR.

[0749] In some embodiments, the population of cells further comprises a third subpopulation of cells. In some embodiments, the third subpopulation of cells comprises a vector comprising a first barcode and a first transgene encoding a CD 19 CAR and a second barcode and a second transgene encoding a CD22 CAR. In some embodiments, the third subpopulation of cells comprises a first vector comprising a first barcode and a first transgene encoding a CD 19 CAR and a second vector comprising a second barcode and a second transgene encoding a CD22 CAR. In some embodiments, the third subpopulation of cells comprises a vector comprising a barcode and a transgene encoding a CD19/CD22-bispecific CAR. In some embodiments, the first subpopulation of cells comprises a CD 19 CAR, the second subpopulation of cells comprises a CD22 CAR, and the third subpopulation of cells comprises a CD 19 CAR and a CD22 CAR or a CD19/CD22 bivalent CAR.

[0750] In some embodiments, the population of cells further comprises a fourth subpopulation of cells. In some embodiments, the fourth subpopulation of cells does not comprise a CAR. In some embodiments, the fourth subpopulation of cells comprises any of the CARs described herein. In some embodiments, the fourth subpopulation of cells comprises a CD 19 CAR. In some embodiments, the fourth subpopulation of cells comprises a CD22 CAR. In some embodiments, the fourth subpopulation of cells comprises a CD 19 and a CD22 CAR. In some embodiments, the fourth subpopulation of cells comprises a CD19/CD22-bispecific CAR. In some embodiments, the first subpopulation of cells comprises a CD 19 CAR, the second subpopulation of cells comprises a CD22 CAR, the third subpopulation of cells comprises a CD 19 CAR and a CD22 CAR or a CD19/CD22 bivalent CAR, and the fourth subpopulation of cells does not comprise a CAR.

[0751] In some embodiments, the first subpopulation of cells and the second subpopulation of cells comprise cells of the same cell type. In some embodiments, the first subpopulation of cells and the second subpopulation of cells comprise cells of different cell types. Examples of cells include, without limitation, islet cells, beta islet cells, pancreatic islet cells, immune cells, B cells, T cells, natural killer (NK) cells, natural killer T (NKT) cells, macrophage cells, endothelial cells, muscle cells, cardiac muscle cells, smooth muscle cells, skeletal muscle cells, dopaminergic neurons, retinal pigmented epithelium cells, optic cells, hepatocytes, thyroid cells, skin cells, glial progenitor cells, neural cells, cardiac cells, stem cells, hematopoietic stem cells, induced pluripotent stem cells (iPSCs), mesenchymal stem cells (MSCs), embryonic stem cells (ESCs), pluripotent stem cell (PSCs), blood cells, endothelial stem cells, epithelial stem cells, adipose stem or progenitor cells, germline stem cells, lung stem or progenitor cells, mammary stem cells, olfactory adult stem cells, hair follicle stem cells, multipotent stem cells, amniotic stem cells, cord blood stem cells, neural stem or progenitor cells, CHO cells, BHK cells, MDCK cells, C3H 10T1/2 cells, FLY cells, Psi-2 cells, BOSC 23 cells, PA317 cells, WEHI cells, COS cells, BSC 1 cells, BSC 40 cells, BMT 10 cells, VERO cells, W138 cells, MRC5 cells, A549 cells, HT1080 cells, 293 cells, 293T cells, B-50 cells, 3T3 cells, NIH3T3 cells, HepG2 cells, Saos-2 cells, Huh7 cells, HeLa cells, W163 cells, 211 cells, NS0, PerC6, Sp2/0, BHK, C127 and 211 A cells. [0752] In some embodiments, the population of cells comprises one or more subpopulations of cells. For example, in some embodiments, the population of cells comprises 1, 2, 3, 4, 5, or more subpopulations of cells. In some embodiments, each subpopulation of cells comprises one or more vectors, wherein each vector comprises a unique barcode sequence associated with a sequence encoding a transgene. In some embodiments, each unique barcode sequence is used to identify a cell or a population of cells, e.g., a subpopulation of cells, comprising the transgene. In some embodiments, the population of cells comprises three or more subpopulations of cells, wherein each subpopulation of cells comprises a vector comprising a unique barcode sequence associated with a sequence encoding a transgene, wherein each unique barcode sequence is used to identify a population of cells comprising the transgene.

[0753] In some embodiments, the population of cells comprises a first vector encoding a first transgene and a second vector encoding a second transgene. In some embodiments, the first vector comprising the first transgene encodes a CD 19 CAR. In some embodiments, the second vector comprising the second transgene encodes a CD22 CAR. In some embodiments, the population of cells comprises one vector encoding a first transgene and a second transgene, wherein the first transgene encodes a CD 19 CAR and wherein the second transgene encodes a CD22 CAR.

[0754] In some embodiments, the invention further provides a composition comprising any of the cells or population of cells, e.g., subpopulation of cells, described herein.

D. Samples

[0755] In some embodiments, the sample is a biological sample. In some embodiments, the sample is from a human. In some embodiments, the sample comprises an engineered cell or population of cells (e.g., T cells, B cells, NK cells, islet cells such as beta cells, or hypoimmunogenic cells thereof). In some embodiments, the comprises a population of cells. In some embodiments, the sample comprises any cell described herein. In some embodiments, the sample comprises any population of cells described herein. In some embodiments, the sample is a tissue sample, and may include, for example, tissue sample, biopsy, resection, smear, aspirate, PBMC, cell lines, hair follicles, or any combination thereof.

[0756] The disclosed method may be used with any of a variety of samples comprising cells that are collected from a subject (e.g., a patient). Examples of a sample include, but are not limited to, a liquid biopsy sample, a blood sample (e.g., a peripheral whole blood sample), a blood plasma sample, a blood serum sample, a lymph sample, a circulating tumor cell (CTC) sample, a cerebrospinal fluid (CSF) sample, a pericardial fluid sample, a pleural fluid sample, an ascites (peritoneal fluid) sample, an interstitial fluid sample, a feces (or stool) sample, or other body fluid, secretion, and/or excretion sample that can comprise a cell of interest (or sample derived therefrom), as well as, e.g., a tissue sample, biopsy, resection, smear, or aspirate. A tissue sample may be harvested from any tissue in the body, e.g., lung, liver, pancreas, bone, gynecological, cardiac, muscle, adipose, brain, gastrointestinal, breast, eye, cartilage, tumor, etc. [0757] In some embodiments, the sample may be collected by surgical resection, needle biopsy, fine needle aspiration, collection cup or tube, oral swab, nasal swab, vaginal swab or a cytology smear, etc. In some embodiments, the sample comprises a mixture of single cells. In some embodiments, the sample is further processed, e.g., digested, dissected, or otherwise broken down into a single cell mixture. In some embodiments, the sample is further processed to select for cells of a specific lineage or phenotype, for example a T cell, B cell, NK cell, macrophage, monocyte, dendritic cell, pancreatic islet cell e.g., beta cell), keratinocyte, hepatocyte, stellate cell, cardiac cell, etc. wherein the cell presents specific lineage markers and can be selected or sorted, for example by magnetic-activated cell selection (MACS) or fluorescent-activated cell sorting (FACS).

[0758] In some embodiments, the sample cell population is at least about 60% pure, e.g., 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%, at least about 99%, or 100% pure cell population, wherein a “pure” cell population comprises a population of cells of one cell type or one cell lineage, for example a population of: CD8+ cytotoxic T cells; regulatory T cells (Tregs); CD4+ THI cells; CD4+ TH2 cells; hepatocytes; stellate cells; keratinocytes; natural killer (NK) cells; beta cells; etc.

[0759] In some embodiments, the sample comprises a mixed cell population. For example, in some embodiments, the sample comprises a mixed cell population comprising one, two, three, four, five, or more cell types or cell lineages within the sample. In some embodiments, the sample can comprise one, two, three, four, five, or more populations of cells. IV. CELL COMPOSITIONS

[0760] Certain aspects of the present disclosure relate to compositions comprising any of the cells (e.g., engineered cells or populations of cells (e.g., T cells, B cells, NK cells, islet cells such as beta cells, or hypoimmunogenic cells thereof)) described herein, or populations of such cells (e.g., engineered cells). In some embodiments, the compositions are pharmaceutical compositions.

[0761] In some embodiments, the compositions provided herein further include a pharmaceutically acceptable excipient or carrier. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn- protein complexes); and/or non-ionic surfactants such as polysorbates (TWEEN™), poloxamers (PLURONICS™) or polyethylene glycol (PEG). In some embodiments, the pharmaceutical composition includes a pharmaceutically acceptable buffer (e.g., neutral buffer saline or phosphate buffered saline). In some embodiments, the pharmaceutical composition can contain one or more excipients for modifying, maintaining or preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption, or penetration of the composition. In some aspects, a skilled artisan understands that a pharmaceutical composition containing cells may differ from a pharmaceutical composition containing a protein.

[0762] The pharmaceutical composition in some embodiments contains cells (e.g., engineered cells or populations of cells (e.g., T cells, B cells, NK cells, islet cells such as beta cells, or hypoimmunogenic cells thereof)) as described herein in amounts effective to treat or prevent a disease or condition, such as a therapeutically effective or prophylactically effective amount. Therapeutic or prophylactic efficacy in some embodiments is monitored by periodic assessment of treated subjects. For repeated administrations over several days or longer, depending on the condition, the treatment is repeated until a desired suppression of disease symptoms occurs. However, other dosage regimens may be useful and can be determined. The desired dosage can be delivered by a single bolus administration of the composition, by multiple bolus administrations of the composition, or by continuous infusion administration of the composition. In some embodiments, the cells can be grafted to the subject (e.g., patient), for example by bone graft, skin graft (e.g., cell suspension autograft), or the like. In some embodiments, the cells can be transplanted to the subject (e.g., the patient), for example beta cell transplantation, islet cell transplantation, hepatocyte transplantation, stem cell transplantation (e.g., hematopoietic cell transplantation), or the like.

[0763] In some embodiments, pharmaceutical compositions comprising one or more cell types is administered to the subject (e.g., the patient). In some embodiments, the pharmaceutical compositions comprising one or more cell types is administered by the same route. In some embodiments, the pharmaceutical compositions comprising one or more cell types is administered by different routes.

[0764] In some embodiments, compositions comprising cells (e.g., engineered cells or populations of cells (e.g., T cells, B cells, NK cells, islet cells such as beta cells, or hypoimmunogenic cells thereof)) as described herein are administered using standard administration techniques, formulations, and/or devices. Also provided are formulations and devices, such as syringes and vials, for storage and administration of the compositions. Compositions of the disclosure cells can be administered via localized injection, including catheter administration, systemic injection, localized injection, intravenous injection, or parenteral administration. When administering a therapeutic composition (e.g., a pharmaceutical composition containing an engineered cell or population of cells), it will generally be formulated in a unit dosage injectable form (solution, suspension, emulsion).

[0765] Formulations include those for intravenous, intraperitoneal, or subcutaneous, administration. In some embodiments, the cells of the disclosure are administered parenterally. The term “parenteral,” as used herein, includes intravenous, intramuscular, intravitreal, subcutaneous, rectal, vaginal, and intraperitoneal administration. In some embodiments, the cells of the disclosure are administered to a subject using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection.

[0766] Compositions in some embodiments are provided as sterile liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions, or dispersions, which may in some aspects be buffered to a selected pH. Liquid compositions are somewhat more convenient to administer, especially by injection. Liquid compositions can comprise carriers, which can be a solvent or dispersing medium containing, for example, water, saline, phosphate buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol) and suitable mixtures thereof. Sterile injectable solutions can be prepared by incorporating the cells in a solvent, such as in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, or the like.

[0767] In some embodiments, a pharmaceutically acceptable carrier can include all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration (Gennaro, 2000, Remington: The science and practice of pharmacy, Lippincott, Williams & Wilkins, Philadelphia, PA). Examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solutions, dextrose solution, and 5% human serum albumin. Liposomes and nonaqueous vehicles such as fixed oils may also be used. Supplementary active compounds can also be incorporated into the compositions. The pharmaceutical carrier should be one that is suitable for the cells (e.g., engineered cells or population of cells (e.g., T cells, B cells, NK cells, islet cells such as beta cells, or hypoimmunogenic cells thereof)), such as a saline solution, a dextrose solution or a solution comprising human serum albumin. In some embodiments, the pharmaceutically acceptable carrier or vehicle for such compositions is any non-toxic aqueous solution in which the cells (e.g., engineered cells or populations of cells) can be maintained, or remain viable, for a time sufficient to allow administration of live cells. For example, the pharmaceutically acceptable carrier or vehicle can be a saline solution or buffered saline solution.

[0768] In some embodiments, the composition, including pharmaceutical composition, is sterile. In some embodiments, isolation, enrichment, or culturing of the cells is carried out in a closed or sterile environment, for example and for instance in a sterile culture bag, to minimize error, user handling and/or contamination. In some embodiments, sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes. In some embodiments, culturing is carried out using a gas permeable culture vessel. In some embodiments, culturing is carried out using a bioreactor.

[0769] Also provided herein are compositions that are suitable for cryopreserving the provided cells (e.g., engineered cells or population of cells (e.g., T cells, B cells, NK cells, islet cells such as beta cells, or hypoimmunogenic cells thereof)). In some embodiments, the provided cells (e.g., engineered cells or populations of cells) are cryopreserved in a cryopreservation medium. In some embodiments, the cryopreservation medium is a serum free cryopreservation medium. In some embodiments, the composition comprises a cryoprotectant. In some embodiments, the cryoprotectant is or comprises DMSO and/or glycerol. In some embodiments, the cryopreservation medium is between at or about 5% and at or about 10% DMSO (v/v). In some embodiments, the cryopreservation medium is at or about 5% DMSO (v/v). In some embodiments, the cryopreservation medium is at or about 6% DMSO (v/v). In some embodiments, the cryopreservation medium is at or about 7% DMSO (v/v). In some embodiments, the cryopreservation medium is at or about 7.5% DMSO (v/v). In some embodiments, the cryopreservation medium is at or about 8% DMSO (v/v). In some embodiments, the cryopreservation medium is at or about 9% DMSO (v/v). In some embodiments, the cryopreservation medium is at or about 10% DMSO (v/v). In some embodiments, the cryopreservation medium contains a commercially available cryopreservation solution (CryoStor™ CS10). CryoStor™ CS10 is a cryopreservation medium containing 10% dimethyl sulfoxide (DMSO). In some embodiments, compositions formulated for cry opreservation can be stored at low temperatures, such as ultra- low temperatures, for example, storage with temperature ranges from -40 °C to -150 °C, such as or about 80 °C ± 6.0 ° C.

[0770] In some embodiments, the pharmaceutical composition comprises cells (e.g., engineered cells or population of cells (e.g., T cells, B cells, NK cells, islet cells such as beta cells, or hypoimmunogenic cells thereof)) described herein and a pharmaceutically acceptable carrier comprising 31.25 % (v/v) Plasma-Lyte A, 31.25 % (v/v) of 5% dextro se/0.45% sodium chloride, 10% dextran 40 (LMD)/5% dextrose, 20% (v/v) of 25% human serum albumin (HSA), and 7.5% (v/v) dimethylsulfoxide (DMSO).

[0771] In some embodiments, the cryopreserved cells (e.g., engineered cells or population of cells (e.g., T cells, B cells, NK cells, islet cells such as beta cells, or hypoimmunogenic cells thereof)) are prepared for administration by thawing. In some cases, the cells can be administered to a subject immediately after thawing. In such an embodiment, the composition is ready-to-use without any further processing. In other cases, the cells are further processed after thawing, such as by resuspension with a pharmaceutically acceptable carrier, incubation with an activating or stimulating agent, or are activated washed and resuspended in a pharmaceutically acceptable buffer prior to administration to a subject.

V. METHODS OF MONITORING A THERAPY

[0772] Monitoring a therapy as described herein may assist in evaluating the therapeutic efficacy of cells or one or more population(s) of cells intended for treatment of a subject in need thereof. For example, a cell or population of cells administered to a subject (e.g., a human patient) for cell therapy that comprises a vector expressing a CAR may be monitored for suppression or loss of CAR expression over time. Monitoring a therapy as described herein can confer greater, increased, enhanced, etc. efficacy of the cell or population of cells administered to the subject for cell therapy by providing key data to a medical professional who is monitoring the subject to make treatment decisions, for example changes to dosage level or dosage frequency, the administration of additional therapeutic agents, etc. Thus, provided herein is a method of monitoring a therapy for enhanced efficacy compared to the efficacy of a therapy administered to an individual wherein the therapy is not monitored. Exemplary ways in which a therapy may be monitored are described below.

[0773] The present disclosure provides methods of monitoring a sample obtained from a subject receiving cell therapy for the presence or absence of a first barcode and/or a second barcode in the cell, population of cells, or cell therapy wherein the method comprises: (i) detecting the first barcode and/or the second barcode in the sample obtained from the subject receiving the cell, population of cells, or cell therapy, wherein detection of the first barcode indicates the presence of the first transgene, and/or detection of the second barcode indicates the presence of the second transgene in the sample; and (ii) determining the percentage, ratio, relative, or absolute number of cells expressing the first transgene and/or the second transgene. In some embodiments, the method further comprises determining the percentage, ratio, relative, or absolute number of cells expressing the first transgene and/or the second transgene at a second time point. [0774] In some embodiments, determining the percentage, ratio, relative, or absolute number of cells expressing the first transgene and/or the second transgene in a sample at a first time point and a second time point is used to monitor: i) cell therapy persistence; ii) uncontrolled expansion of the cells expressing the first transgene and/or the second transgene; or iii) changes in a subject’s health or disease profile, in a subject receiving treatment. In some embodiments, the method further comprises one or more additional administrations of the cell, population of cells, or cell therapy to the subject comprising: i) the same or different dose as the initial dose of the cell, population of cells, or cell therapy administered to the subject; and/or ii) a cell, population of cells, or cell therapy comprising the same or different transgene encoded by a vector as the initial cell, population of cells, or cell therapy administered to the subject.

[0775] Also provided is a method of monitoring a cell therapy administered to a subject, comprising: (i) obtaining a sample from the subject who was administered the cell therapy, (ii) detecting the presence or absence of a first barcode and/or a second barcode in the sample, wherein the presence of the first barcode indicates the presence of a first transgene and the presence of the second barcode indicates the presence of a second transgene, and (iii) determining the percentage, ratio, relative, or absolute number of cells expressing the first transgene and/or the second transgene based on the presence or absence of the first barcode and/or second barcode.

[0776] There are multiple instances and/or reasons for using the methods of monitoring a sample as described herein. For example, by monitoring the sample administered to a subject (e.g., a human patient) over time, a prescribing physician can monitor the therapeutic efficacy; how or if the administered cells change, lose, or suppress vector or transgene expression over time; how long the cells can persist in specific target tissues of the body (e.g., immune cells engrafting into the bone marrow, lymph nodes, spleen, blood or lymph fluid, or organs of interest); observe cell expansion over time (e.g., T cell engraftment and proliferation in an otherwise immunocompromised patient); or confirm the safety of the administered cells (e.g., monitor the cells for any cellular transform into precancerous or cancerous cells over time). [0777] Accordingly, in some embodiments, the methods of monitoring a sample as described herein can be performed after at least about any one or more of: about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 13 weeks, about 14 weeks, about 15 weeks, about 16 weeks, about 17 weeks, about 18 weeks, about 19 weeks, about 20 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 13 months, about 14 months, about 15 months, about 16 months, about 17 months, about 18 months, about 19 months, about 20 months, about 21 months, about 22 months, about 23 months, about 24 months, about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years, about 11 years, about 12 years, about 13 years, about 14 years, about 15 years or more, or until the administered cells are removed from the subject (e.g., the patient) or are no longer identifiable by the methods of monitoring described herein, for example due to the cell death of the administered cells over time or via a rapid and targeted response (e.g., Graft versus Host Disease, GvHD).

[0778] In some embodiments, the method comprises monitoring responsiveness to cell therapy in a subject. Measures of responsiveness are dependent upon the disease to be treated. For example, responsiveness to cell therapy in a subject to be treated for a cancer may include (i) reduction in tumor burden, (2) increase in tumor-infiltrating lymphocytes, (3) reduction in immune checkpoint blockade markers, (4) increased overall survival, (5) complete response or complete cure, (6) going into remission, (7) no indication or signs of cancer recurrence, (8) reduction or elimination of cancer cells in the subject, etc. Responsiveness to cell therapy in a subject to be treated for autoimmune disorder may include (e.g.) (1) reduction in serum levels of pro-inflammatory cytokines, (2) reduction in tissue levels of pro-inflammatory cytokines, (3) reduction or elimination of pain or discomfort associated with the autoimmune disorder, (4) going into remission of the autoimmune disorder, (5) increased duration of time between autoimmune flare-ups or outbreaks, (6) decreased markers of cytotoxic T cell activity and/or B cell activity, (7) increased presence of regulatory T cells (Tregs) in the affected tissue(s), (8) decreased levels of immune hyperresponsivity, etc. In some embodiments, if the monitoring indicates that the subject is responsive to the cell therapy, then there is no change in treatment. In some embodiments, if the monitoring indicates that the subject is not responsive to the cell therapy, then one or more actions are taken from the group consisting of rescreening or retesting of the sample, collecting and screening another sample from the subject, administering one or more additional therapeutic doses to the subject, changing the treatment of the subject, administering one or more additional therapeutic agents to the subject, and/or ceasing treatment of the subject. In some embodiments, the additional therapeutic agent is selected from the group consisting of a small molecule inhibitor, a chemotherapeutic agent, a cancer immunotherapy, an antibody, a cellular therapy, a nucleic acid, a surgery, a radiotherapy, an anti-angiogenic therapy, an anti-DNA repair therapy, an anti-inflammatory therapy, an anti-neoplastic agent, a cell growth factor, a growth inhibitory agent, a cytotoxic agent, a hormonal therapy, a metabolic agent, a protein, a peptide, a pro-peptide, and any combination thereof. For example, in some embodiments, the additional therapeutic agents are selected from the group consisting of insulin, amylinomimetic(s), dopamine-2 agonist(s), DPP-4 inhibitor(s), metformin, alpha-glucosidase inhibitor(s), SGLT2 inhibitor(s), statins, GLP-1 receptor agonist(s), incretin, meglitinide(s), sulfonylureas, thiazolidinediones, nonsteroidal anti-inflammatory drugs (NSAIDs), antimalarial drugs, corticosteroids, azathioprine, mycophenolate, methotrexate, cyclosporine, voclosporin, leflunomide, belimumab, anifrolumab, abatacept, rituximab, vitamin D supplementation, dehydroepiandrosterone (DHEA), and any combination thereof. In some embodiments, cessation of the therapy comprises triggering a safety switch in the cell, population of cells, or cell therapy administered to the subject.

[0779] In some embodiments, the cell therapy is monitored for safety, for example for toxicity or onset of any one or more adverse events (AEs), such as adverse events of special interest (AESIs). Adverse events depend upon the therapy administered, for example CAR-T cell therapies have been associated with the following adverse events: cytokine release syndrome (CRS), neurological events (i.e., CAR T-cell-related encephalopathy syndrome (CRES) or immune effector cell-associated neurotoxicity syndrome (ICANS)), cytopenias, infection, off- target cytotoxic activity and accompanying tissue damage, organ failure, graft- versus-host disease (GvHD), etc. In some embodiments, one or more safety assay(s) is conducted, wherein the safety assay is selected from the group consisting of mycoplasma testing, sterility testing, endotoxin testing, karyotyping, replication-competent lentivirus testing, vector copy number testing, virus screening, cytokine independent outgrowth testing, balanced translocation testing, and any combination thereof. In some embodiments, if the monitoring indicates that the cell therapy is safe for administration to the subject, then there is no change in treatment. In some embodiments, if the monitoring indicates that the cell therapy is not safe for administration to the subject, then one or more actions are taken selected from the group consisting of rescreening or retesting of the sample, collecting and screening another sample from the subject, changing the treatment of the subject, administering one or more additional therapeutic agents to the subject, and ceasing treatment of the subject. In some embodiments, the additional therapeutic agent is selected from the group consisting of a small molecule inhibitor, a chemotherapeutic agent, a cancer immunotherapy, an antibody, a cellular therapy, a nucleic acid, a surgery, a radiotherapy, an anti- angiogenic therapy, an anti-DNA repair therapy, an anti-inflammatory therapy, an anti- neoplastic agent, a cell growth factor, a growth inhibitory agent, a cytotoxic agent, a hormonal therapy, a metabolic agent, a protein, a peptide, a pro-peptide, and any combination thereof. For example, in some embodiments, the additional therapeutic agents are selected from the group consisting of insulin, amylinomimetic(s), dopamine-2 agonist(s), DPP-4 inhibitor(s), metformin, alpha-glucosidase inhibitor(s), SGLT2 inhibitor(s), statins, GLP-1 receptor agonist(s), incretin, meglitinide(s), sulfonylureas, thiazolidinediones, nonsteroidal anti-inflammatory drugs (NSAIDs), antimalarial drugs, corticosteroids, azathioprine, mycophenolate, methotrexate, cyclosporine, voclosporin, leflunomide, belimumab, anifrolumab, abatacept, rituximab, vitamin D supplementation, dehydroepiandrosterone (DHEA), and any combination thereof. In some embodiments, cessation of the therapy comprises triggering a safety switch in the cell, population of cells, or cell therapy administered to the subject.

[0780] In some embodiments, the cell therapy is monitored for cell persistence, cell activity, and/or cell expansion in vivo. Methods of monitoring cell persistence and/or cell expansion may include (e.g.) obtaining PBMC samples from the subject and detecting the ratio of cells expressing one or more barcode(s) to cells expressing no barcode for example using single cell sequencing, or detecting the ratio of cells expressing one or more transgene(s) to cells expressing no transgene for example using FACS analysis or immunohistochemistry staining. Methods of monitoring cell activity can include, but is not limited to, increased gene expression or protein production of cytotoxic cytokines (e.g., IFN-y, granzyme B, TNF-a) or decreased expression of senescence or exhaustion markers (e.g., PD-1, CTLA-4, TIM-3, LAG-3, KLRG1, CD57), which may be tested immediately upon harvest from the subject or culturing in vitro and re-activating with antigen or anti-CD3 and anti-CD28 antibodies and testing after 12-24 hrs. In some embodiments, if the monitoring confirms cell persistence, cell activity, and/or cell expansion in vivo upon administration to the subject, then there is no change in treatment. In some embodiments, if the monitoring does not confirm cell persistence, cell activity, and/or cell expansion in vivo upon administration to the subject, then one or more actions are taken selected from the group consisting of rescreening or retesting of the sample, collecting and screening another sample from the subject, administering one or more additional therapeutic doses to the subject, changing the treatment of the subject, administering one or more additional therapeutic agents to the subject, and ceasing treatment of the subject. In some embodiments, the additional therapeutic agent is selected from the group consisting of a small molecule inhibitor, a chemotherapeutic agent, a cancer immunotherapy, an antibody, a cellular therapy, a nucleic acid, a surgery, a radiotherapy, an anti- angiogenic therapy, an anti-DNA repair therapy, an antiinflammatory therapy, an anti-neoplastic agent, a cell growth factor, a growth inhibitory agent, a cytotoxic agent, a hormonal therapy, a metabolic agent, a protein, a peptide, a pro-peptide, and any combination thereof. For example, in some embodiments, the additional therapeutic agents are selected from the group consisting of insulin, amylinomimetic(s), dopamine-2 agonist(s), DPP-4 inhibitor(s), metformin, alpha-glucosidase inhibitor(s), SGLT2 inhibitor(s), statins, GLP- 1 receptor agonist(s), incretin, meglitinide(s), sulfonylureas, thiazolidinediones, nonsteroidal anti-inflammatory drugs (NSAIDs), antimalarial drugs, corticosteroids, azathioprine, mycophenolate, methotrexate, cyclosporine, voclosporin, leflunomide, belimumab, anifrolumab, abatacept, rituximab, vitamin D supplementation, dehydroepiandrosterone (DHEA), and any combination thereof. In some embodiments, cessation of the therapy comprises triggering a safety switch in the cell, population of cells, or cell therapy administered to the subject.

[0781] In some embodiments, the cell therapy is monitored for reduced or eliminated expression of the first transgene and/or the second transgene in the cell or population of cells. In some embodiments, reduced or eliminated expression of the first transgene and/or the second transgene may be indicative of a reduction or loss in therapeutic efficacy or may occur prior to a reduction or clearance of the cell therapy from the subject to which the cell therapy was administered, e.g., via apoptosis. In some embodiments, if the monitoring confirms that there is no reduction or elimination of expression of the first transgene and/or the second transgene in the cell or population of cells, then there is no change in treatment. In some embodiments, if the monitoring confirms reduced or eliminated expression of the first transgene and/or the second transgene in the cell or population of cells, then one or more actions are taken selected from the group consisting of rescreening or retesting of the sample, collecting and screening another sample from the subject, changing the treatment of the subject, administering one or more additional therapeutic agents to the subject, and ceasing treatment of the subject. In some embodiments, the additional therapeutic agent is selected from the group consisting of a small molecule inhibitor, a chemotherapeutic agent, a cancer immunotherapy, an antibody, a cellular therapy, a nucleic acid, a surgery, a radiotherapy, an anti- angiogenic therapy, an anti-DNA repair therapy, an anti-inflammatory therapy, an anti-neoplastic agent, a cell growth factor, a growth inhibitory agent, a cytotoxic agent, a hormonal therapy, a metabolic agent, a protein, a peptide, a pro-peptide, and any combination thereof. In some embodiments, cessation of the therapy comprises triggering a safety switch in the cell, population of cells, or cell therapy administered to the subject. For example, in some embodiments, the additional therapeutic agents are selected from the group consisting of insulin, amylinomimetic(s), dopamine-2 agonist(s), DPP-4 inhibitor(s), metformin, alpha-glucosidase inhibitor(s), SGLT2 inhibitor(s), statins, GLP-1 receptor agonist(s), incretin, meglitinide(s), sulfonylureas, thiazolidinediones, nonsteroidal antiinflammatory drugs (NSAIDs), antimalarial drugs, corticosteroids, azathioprine, mycophenolate, methotrexate, cyclosporine, voclosporin, leflunomide, belimumab, anifrolumab, abatacept, rituximab, vitamin D supplementation, dehydroepiandrosterone (DHEA), and any combination thereof.

[0782] In some embodiments, the cell therapy is monitored for its therapeutic kinetics, for example pharmacokinetic and/or pharmacodynamic properties. These may include, for example, but are not limited to, antigen binding affinity, target (re)binding, rate of dissociation (e.g., rate of drug dissociation or rate of antigen dissociation), time-dependent activity, half-life, bioactivity, cell-based washout, etc. In some embodiments, if the monitoring confirms acceptable therapeutic kinetics, then there is no change in treatment. In some embodiments, if the monitoring does not confirm acceptable therapeutic kinetics, then one or more actions are taken selected from the group consisting of rescreening or retesting of the sample, collecting and screening another sample from the subject, administering one or more additional therapeutic doses to the subject, changing the treatment of the subject, administering one or more additional therapeutic agents to the subject, and ceasing treatment of the subject. In some embodiments, the additional therapeutic agent is selected from the group consisting of a small molecule inhibitor, a chemotherapeutic agent, a cancer immunotherapy, an antibody, a cellular therapy, a nucleic acid, a surgery, a radiotherapy, an anti- angiogenic therapy, an anti-DNA repair therapy, an anti- inflammatory therapy, an anti-neoplastic agent, a cell growth factor, a growth inhibitory agent, a cytotoxic agent, a hormonal therapy, a metabolic agent, a protein, a peptide, a pro-peptide, and any combination thereof. For example, in some embodiments, the additional therapeutic agents are selected from the group consisting of insulin, amylinomimetic(s), dopamine-2 agonist(s), DPP-4 inhibitor(s), metformin, alpha-glucosidase inhibitor(s), SGLT2 inhibitor(s), statins, GLP- 1 receptor agonist(s), incretin, meglitinide(s), sulfonylureas, thiazolidinediones, nonsteroidal anti-inflammatory drugs (NSAIDs), antimalarial drugs, corticosteroids, azathioprine, mycophenolate, methotrexate, cyclosporine, voclosporin, leflunomide, belimumab, anifrolumab, abatacept, rituximab, vitamin D supplementation, dehydroepiandrosterone (DHEA), and any combination thereof. In some embodiments, cessation of the therapy comprises triggering a safety switch in the cell, population of cells, or cell therapy administered to the subject.

[0783] In some embodiments, the cell therapy is monitored for controlling tumor growth, tumor expansion, and/or tumor recurrence. In some embodiments, the cell therapy is monitored for preventing antigen escape of a tumor. In some instances, controlling tumor growth, tumor expansion, and/or tumor recurrence and/or preventing antigen escape of a tumor correlates to or is directly responsible for therapeutic efficacy of the cell therapy. In some embodiments, if the monitoring indicates that the cell therapy controls tumor growth, tumor expansion, and/or tumor recurrence, and/or prevents antigen escape of a tumor, then there is no change in treatment. In some embodiments, if the monitoring indicates that the cell therapy does not control tumor growth, tumor expansion, and/or tumor recurrence, and/or does not prevent antigen escape of a tumor, then one or more actions are taken selected from the group consisting of rescreening or retesting of the sample, collecting and screening another sample from the subject, changing the treatment of the subject, administering one or more additional therapeutic agents to the subject, and ceasing treatment of the subject. In some embodiments, the additional therapeutic agent is selected from the group consisting of a small molecule inhibitor, a chemotherapeutic agent, a cancer immunotherapy, an antibody, a cellular therapy, a nucleic acid, a surgery, a radiotherapy, an anti- angiogenic therapy, an anti-DNA repair therapy, an anti-inflammatory therapy, an anti- neoplastic agent, a cell growth factor, a growth inhibitory agent, a cytotoxic agent, a hormonal therapy, a metabolic agent, a protein, a peptide, a pro-peptide, and any combination thereof. In some embodiments, cessation of the therapy comprises triggering a safety switch in the cell, population of cells, or cell therapy administered to the subject. [0784] In some embodiments, the cell therapy is monitored for the presence or absence of a therapeutic viral vector. In some embodiments, the cell therapy is monitored for vector copy number. In some embodiments, the presence of a therapeutic viral vector is indicative of increased therapeutic efficacy of the cell therapy administered to the subject. In some embodiments, if the monitoring confirms the presence of a therapeutic viral vector, then there is no change in treatment. In some embodiments, if the monitoring confirms the absence of a therapeutic viral vector, then one or more actions are taken selected from the group consisting of rescreening or retesting of the sample, collecting and screening another sample from the subject, administering one or more additional therapeutic doses to the subject, changing the treatment of the subject, administering one or more additional therapeutic agents to the subject, and ceasing treatment of the subject. In some embodiments, the additional therapeutic agent is selected from the group consisting of a small molecule inhibitor, a chemotherapeutic agent, a cancer immunotherapy, an antibody, a cellular therapy, a nucleic acid, a surgery, a radiotherapy, an anti- angiogenic therapy, an anti-DNA repair therapy, an anti-inflammatory therapy, an anti-neoplastic agent, a cell growth factor, a growth inhibitory agent, a cytotoxic agent, a hormonal therapy, a metabolic agent, a protein, a peptide, a pro-peptide, and any combination thereof. In some embodiments, cessation of the therapy comprises triggering a safety switch in the cell, population of cells, or cell therapy administered to the subject.

[0785] In some embodiments, the population of cells administered to the subject (e.g., the patient) comprises two or more subpopulations of cells. In some embodiments, each subpopulation of cells is monitored as described herein. In some embodiments, the percentage, ratio, relative, or absolute number of cells from each subpopulation are determined at one or more time points after administration to the subject (e.g., the patient). In some embodiments, the percentage, ratio, relative, or absolute number of cells from each subpopulation are indicative of therapeutic success, e.g., the diminishment, amelioration, or elimination of the disease or disorder being treated and/or the prevention of recurrence(s). In some embodiments, the percentage, ratio, relative, or absolute number of cells from each subpopulation indicate that the treatment comprising the administration of the cells or population of cells should be modified to a higher or lower dose or re-administered at the same dose for one or more additional rounds of cell (or composition thereof) administration. [0786] In some embodiments, the method further comprises one or more additional administrations of cell therapy to a subject comprising: i) the same or different dose as the initial cell therapy administered to the subject; or ii) cell therapy comprising the same or different transgene encoded by a vector as the initial cell therapy administered to the subject.

[0787] In some embodiments, the method further comprises determining the percentage, ratio, relative, or absolute number of cells expressing the first transgene and/or the second transgene at a second time point. In some embodiments, the determining the percentage, ratio, relative, or absolute number of cells expressing the first transgene and/or the second transgene in a sample at a first time point and a second time point is used to monitor: i) cell therapy persistence; ii) uncontrolled expansion of the cells expressing the first transgene and/or the second transgene; or changes in a subject’s health or disease profile, in a subject receiving treatment.

Number of cells with one or more transgenes

[0788] In some embodiments, the cell therapy is monitored for the number of cells with a first transgene, the number of cells with a second transgene, and/or the number of cells with a first transgene and a second transgene. In some embodiments, the number of cells with a first transgene is about any of about l.OxlO⁵ to about 3.0xl0⁵, about 2.0xl0⁵ to about 4.0xl0⁵, about 3.0xl0⁵ to about 5.0xl0⁵, about 4.0xl0⁵ to about 6.0xl0⁵, about 5.0xl0⁵ to about 7.0xl0⁵, about

8.0xl0⁵ to about l.OxlO⁶, about 9.0xl0⁵ to about 2.0xl0⁶, about l.OxlO⁶ to about 3.0xl0⁶, about

2.0xl0⁶ to about 4.0xl0⁶, about 3.0xl0⁶ to about 5.0xl0⁶, about 4.0xl0⁶ to about 6.0xl0⁶, about

5.0xl0⁶ to about 7.0xl0⁶, about 8.0xl0⁶ to about l.OxlO⁷, about 9.0xl0⁶ to about 2.0xl0⁷, about l.OxlO⁷ to about 3.0xl0⁷, about 2.0xl0⁷ to about 4.0xl0⁷, about 3.0xl0⁷ to about 5.0xl0⁷, about

4.0xl0⁷ to about 6.0xl0⁷, about 5.0xl0⁷ to about 7.0xl0⁷, about 8.0xl0⁷ to about l.OxlO⁸, about

9.0xl0⁷ to about 2.0xl0⁸, about l.OxlO⁸ to about 3.0xl0⁸, or more. In some embodiments, the number of cells with a second transgene is about any of about l.OxlO⁵ to about 3.0xl0⁵, about 2.0xl0⁵ to about 4.0xl0⁵, about 3.0xl0⁵ to about 5.0xl0⁵, about 4.0xl0⁵ to about 6.0xl0⁵, about

5.0xl0⁵ to about 7.0xl0⁵, about 8.0xl0⁵ to about l.OxlO⁶, about 9.0xl0⁵ to about 2.0xl0⁶, about l.OxlO⁶ to about 3.0xl0⁶, about 2.0xl0⁶ to about 4.0xl0⁶, about 3.0xl0⁶ to about 5.0xl0⁶, about

4.0xl0⁶ to about 6.0xl0⁶, about 5.0xl0⁶ to about 7.0xl0⁶, about 8.0xl0⁶ to about l.OxlO⁷, about

9.0xl0⁶ to about 2.0xl0⁷, about l.OxlO⁷ to about 3.0xl0⁷, about 2.0xl0⁷ to about 4.0xl0⁷, about

3.0xl0⁷ to about 5.0xl0⁷, about 4.0xl0⁷ to about 6.0xl0⁷, about 5.0xl0⁷ to about 7.0xl0⁷, about 8.0xl0⁷ to about l.OxlO⁸, about 9.0xl0⁷ to about 2.0xl0⁸, about l.OxlO⁸ to about 3.0xl0⁸, or more. In some embodiments, the number of cells with a first transgene and a second transgene is about any of about l.OxlO⁵ to about 3.0xl0⁵, about 2.0xl0⁵ to about 4.0xl0⁵, about 3.0xl0⁵ to about 5.0xl0⁵, about 4.0xl0⁵ to about 6.0xl0⁵, about 5.0xl0⁵ to about 7.0xl0⁵, about 8.0xl0⁵ to about l.OxlO⁶, about 9.0xl0⁵ to about 2.0xl0⁶, about l.OxlO⁶ to about 3.0xl0⁶, about 2.0xl0⁶ to about 4.0xl0⁶, about 3.0xl0⁶ to about 5.0xl0⁶, about 4.0xl0⁶ to about 6.0xl0⁶, about 5.0xl0⁶ to about 7.0xl0⁶, about 8.0xl0⁶ to about l.OxlO⁷, about 9.0xl0⁶ to about 2.0xl0⁷, about l.OxlO⁷ to about 3.0xl0⁷, about 2.0xl0⁷ to about 4.0xl0⁷, about 3.0xl0⁷ to about 5.0xl0⁷, about 4.0xl0⁷ to about 6.0xl0⁷, about 5.0xl0⁷ to about 7.0xl0⁷, about 8.0xl0⁷ to about l.OxlO⁸, about 9.0xl0⁷ to about 2.0xl0⁸, about l.OxlO⁸ to about 3.0xl0⁸, or more.

Percentage of cells with one or more transgenes

[0789] In some embodiments, the cell therapy is monitored for the percentage of cells with a first transgene, the percentage of cells with a second transgene, and/or the percentage of cells with a first transgene and a second transgene. In some embodiments, a composition of cells or population of cells for cell therapy is further selected based upon the percentage of cells that have a first transgene, the percentage of cells that have a second transgene, and/or the percentage of cells that have a first transgene and a second transgene. In some embodiments, the composition is selected when the percentage of cells that have a first transgene is at least 25%, for example at least about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more. In some embodiments, the composition is selected when the percentage of cells that have a second transgene is at least 25%, for example at least about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more. In some embodiments, the composition is selected when the percentage of cells that have a first transgene and a second transgene is at least 25%, for example at least about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more. In some embodiments, the composition is selected when the percentage of cells that have a first transgene is at least about 25%, the percentage of cells that have a second transgene is at least about 25%, and/or the percentage of cells that have a first transgene and a second transgene is at least about 25%. In some embodiments, the composition is selected when the percentage of cells that have at least one or more transgenes is at least about 25%, for example at least about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more. In some embodiments, the composition is selected when the total percentage of a combination of cells comprising the first transgene, the second transgene, or the first and second transgene is at least about 80%, such as at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%. In some embodiments, the composition is selected when the percentage of cells that have no transgene is less than about 10%, for example less than about any of 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1.5%, 1%, 0.5%, or less.

Ratio of cells with one or more transgenes

[0790] In some embodiments, the cell therapy is monitored for the ratio of cells with a second transgene to cells with a first transgene. In some embodiments, the ratio of cells with a second transgene to cells with a first transgene is about 90:1, about 80:1, about 70:1, about 60:1, about 50:1, about 40:1, about 30:1, about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, about 1:25, about 1:30, about 1:40, about 1:50, about 1:60, about 1:70, about 1:80, or about 1:90. In some embodiments, the cell therapy is monitored for the ratio of cells with a first transgene to cells with a first transgene and a second transgene. In some embodiments, the ratio of cells with a first transgene to cells with a first transgene and a second transgene is about 90:1, about 80:1, about 70:1, about 60:1, about 50:1, about 40:1, about 30:1, about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, about 1:25, about 1:30, about 1:40, about 1:50, about 1:60, about 1:70, about 1:80, or about 1:90. In some embodiments, the cell therapy is monitored for the ratio of cells with a second transgene to cells with a first transgene and a second transgene. In some embodiments, the ratio of cells with a second transgene to cells with a first transgene and a second transgene is about 90:1, about 80:1, about 70:1, about 60:1, about 50:1, about 40:1, about 30:1, about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, about 1:25, about 1:30, about 1:40, about 1:50, about 1:60, about 1:70, about 1:80, or about 1:90. In some embodiments, the cell therapy is monitored for the ratio of cells with a first transgene to cells with a second transgene to cells with a first transgene and a second transgene. In some embodiments, the ratio of cells with a first transgene to cells with a second transgene to cells with a first transgene and a second transgene is about 10:1:1, about 10:2:1, about 10:3:1, about 10:4:1, about 10:5:1, about 10:6:1, about 10:7:1, about 10:8:1, about 10:9:1, about 10:10:1, about 9:1:1, about 9:2:1, about 9:3:1, about 9:4:1, about 9:5:1, about 9:6:1, about 9:7:1, about 9:8:1, about 9:9:1, about 8:1:1, about 8:2:1, about 8:3:1, about 8:4:1, about 8:5:1, about 8:6:1, about 8:7:1, about 8:8:1, about 7:1:1, about 7:2:1, about 7:3:1, about

7:4:1, about 7:5:1, about 7:6:1, about 7:7:1, about 6:1:1, about 6:2:1, about 6:3:1, about 6:4:1, about 6:5:1, about 6:6:1, about 5:1:1, about 5:2:1, about 5:3:1, about 5:4:1, about 5:5:1, about

4:1:1, about 4:2:1, about 4:3:1, about 4:4:1, about 3:1:1, about 3:2:1, about 3:3:1, about 2:1:1, about 2:2:1, about 1:1:1, about 1:2:1, about 1:2:2, about 1:3:1, about 1:3:2, about 1:3:3, about

1:4:1, about 1:4:2, about 1:4:3, about 1:4:4, about 1:5:1, about 1:5:2, about 1:5:3, about 1:5:4, about 1:5:5, about 1:6:1, about 1:6:2, about 1:6:3, about 1:6:4, about 1:6:5, about 1:6:6, about

1:7:1, about 1:7:2, about 1:7:3, about 1:7:4, about 1:7:5, about 1:7:6, about 1:7:7, about 1:8:1, about 1:8:2, about 1:8:3, about 1:8:4, about 1:8:5, about 1:8:6, about 1:8:7, about 1:8:8, about

1:9:1, about 1:9:2, about 1:9:3, about 1:9:4, about 1:9:5, about 1:9:6, about 1:9:7, about 1:9:8, about 1:9:9, about 1:10:1, about 1:10:2, about 1:10:3, about 1:10:4, about 1:10:5, about 1:10:6, about 1:10:7, about 1:10:8, about 1:10:9, or about 1:10:10.

[0791] In some embodiments, the cell therapy is monitored for the ratio of cells with a second transgene to cells with a first transgene to cells with no transgene. In some embodiments, the ratio of cells with a second transgene to cells with a first transgene to cells with no transgene is about 10:1:1, about 10:2:1, about 10:3:1, about 10:4:1, about 10:5:1, about 10:6:1, about 10:7:1, about 10:8:1, about 10:9:1, about 10:10:1, about 9:1:1, about 9:2:1, about 9:3:1, about 9:4:1, about 9:5:1, about 9:6:1, about 9:7:1, about 9:8:1, about 9:9:1, about 8:1:1, about 8:2:1, about 8:3:1, about 8:4:1, about 8:5:1, about 8:6:1, about 8:7:1, about 8:8:1, about 7:1:1, about 7:2:1, about 7:3:1, about 7:4:1, about 7:5:1, about 7:6:1, about 7:7:1, about 6:1:1, about 6:2:1, about 6:3:1, about 6:4:1, about 6:5:1, about 6:6:1, about 5:1:1, about 5:2:1, about 5:3:1, about 5:4:1, about 5:5:1, about 4:1:1, about 4:2:1, about 4:3:1, about 4:4:1, about 3:1:1, about 3:2:1, about 3:3:1, about 2:1:1, about 2:2:1, about 1:1:1, about 1:2:1, about 1:3:1, about 1:4:1, about 1:5:1, about 1:6:1, about 1:7:1, about 1:8:1, about 1:9:1, about 1:10:1, 10:1:0, about 10:2:0, about 10:3:0, about 10:4:0, about 10:5:0, about 10:6:0, about 10:7:0, about 10:8:0, about 10:9:0, about 10:10:0, about 9:1:0, about 9:2:0, about 9:3:0, about 9:4:0, about 9:5:0, about 9:6:0, about 9:7:0, about 9:8:0, about 9:9:0, about 8:1:0, about 8:2:0, about 8:3:0, about 8:4:0, about 8:5:0, about 8:6:0, about 8:7:0, about 8:8:0, about 7:1:0, about 7:2:0, about 7:3:0, about 7:4:0, about 7:5:0, about 7:6:0, about 7:7:0, about 6:1:0, about 6:2:0, about 6:3:0, about 6:4:0, about 6:5:0, about 6:6:0, about 5:1:0, about 5:2:0, about 5:3:0, about 5:4:0, about 5:5:0, about 4:1:0, about 4:2:0, about 4:3:0, about 4:4:0, about 3:1:0, about 3:2:0, about 3:3:0, about 2:1:0, about 2:2:0, about 1:1:0, about 1:2:0, about 1:3:0, about 1:4:0, about 1:5:0, about 1:6:0, about 1:7:0, about 1:8:0, about 1:9:0, or about 1:10:0. In some embodiments, the cell therapy is monitored for the ratio of cells with a first transgene to cells with a first transgene and a second transgene to cells with no transgene. In some embodiments, the ratio of cells with a first transgene to cells with a first transgene and a second transgene to cells with no transgene is about 10:1:1, about 10:2:1, about 10:3:1, about 10:4:1, about 10:5:1, about 10:6:1, about 10:7:1, about 10:8:1, about 10:9:1, about 10:10:1, about 9:1:1, about 9:2:1, about 9:3:1, about 9:4:1, about 9:5:1, about 9:6:1, about 9:7:1, about 9:8:1, about 9:9:1, about 8:1:1, about 8:2:1, about 8:3:1, about 8:4:1, about 8:5:1, about 8:6:1, about 8:7:1, about 8:8:1, about 7:1:1, about 7:2:1, about 7:3:1, about 7:4:1, about 7:5:1, about 7:6:1, about 7:7:1, about 6:1:1, about 6:2:1, about 6:3:1, about 6:4:1, about 6:5:1, about 6:6:1, about 5:1:1, about 5:2:1, about 5:3:1, about 5:4:1, about 5:5:1, about 4:1:1, about 4:2:1, about 4:3:1, about 4:4:1, about 3:1:1, about 3:2:1, about 3:3:1, about 2:1:1, about 2:2:1, about 1:1:1, about 1:2:1, about 1:3:1, about 1:4:1, about 1:5:1, about 1:6:1, about 1:7:1, about 1:8:1, about 1:9:1, about 1:10:1, 10:1:0, about 10:2:0, about 10:3:0, about 10:4:0, about 10:5:0, about 10:6:0, about 10:7:0, about 10:8:0, about 10:9:0, about 10:10:0, about 9:1:0, about 9:2:0, about 9:3:0, about 9:4:0, about 9:5:0, about 9:6:0, about 9:7:0, about 9:8:0, about 9:9:0, about 8:1:0, about 8:2:0, about 8:3:0, about 8:4:0, about 8:5:0, about 8:6:0, about 8:7:0, about 8:8:0, about 7:1:0, about 7:2:0, about 7:3:0, about 7:4:0, about 7:5:0, about 7:6:0, about 7:7:0, about 6:1:0, about 6:2:0, about 6:3:0, about 6:4:0, about 6:5:0, about 6:6:0, about 5:1:0, about 5:2:0, about 5:3:0, about 5:4:0, about 5:5:0, about 4:1:0, about 4:2:0, about 4:3:0, about 4:4:0, about 3:1:0, about 3:2:0, about 3:3:0, about 2: 1:0, about 2:2:0, about 1:1:0, about 1:2:0, about 1:3:0, about 1:4:0, about 1:5:0, about 1:6:0, about 1:7:0, about 1:8:0, about 1:9:0, or about 1:10:0. In some embodiments, the cell therapy is monitored for the ratio of cells with a second transgene to cells with a first transgene and a second transgene to cells with no transgene. In some embodiments, the ratio of cells with a second transgene to cells with a first transgene and a second transgene to cells with no transgene is about 10:1:1, about 10:2:1, about 10:3:1, about 10:4:1, about 10:5:1, about 10:6:1, about 10:7:1, about 10:8:1, about 10:9:1, about 10:10:1, about 9:1:1, about 9:2:1, about 9:3:1, about 9:4:1, about 9:5:1, about 9:6:1, about 9:7:1, about 9:8:1, about 9:9:1, about 8:1:1, about 8:2:1, about 8:3:1, about 8:4:1, about 8:5:1, about 8:6:1, about 8:7:1, about 8:8:1, about 7:1:1, about 7:2:1, about 7:3:1, about 7:4:1, about 7:5:1, about 7:6:1, about 7:7:1, about 6:1:1, about 6:2:1, about 6:3:1, about 6:4:1, about 6:5:1, about 6:6:1, about 5:1:1, about 5:2:1, about 5:3:1, about 5:4:1, about 5:5:1, about 4:1:1, about 4:2:1, about 4:3:1, about 4:4:1, about 3:1:1, about 3:2:1, about 3:3:1, about 2:1:1, about 2:2:1, about 1:1:1, about 1:2:1, about 1:3:1, about 1:4:1, about 1:5:1, about 1:6:1, about 1:7:1, about 1:8:1, about 1:9:1, about 1:10:1, 10:1:0, about 10:2:0, about 10:3:0, about 10:4:0, about 10:5:0, about 10:6:0, about 10:7:0, about 10:8:0, about 10:9:0, about 10:10:0, about 9:1:0, about 9:2:0, about 9:3:0, about 9:4:0, about 9:5:0, about 9:6:0, about 9:7:0, about 9:8:0, about 9:9:0, about 8:1:0, about 8:2:0, about 8:3:0, about 8:4:0, about 8:5:0, about 8:6:0, about 8:7:0, about 8:8:0, about 7:1:0, about 7:2:0, about 7:3:0, about 7:4:0, about 7:5:0, about 7:6:0, about 7:7:0, about 6:1:0, about 6:2:0, about 6:3:0, about 6:4:0, about 6:5:0, about 6:6:0, about 5:1:0, about 5:2:0, about 5:3:0, about 5:4:0, about 5:5:0, about 4:1:0, about 4:2:0, about 4:3:0, about 4:4:0, about 3:1:0, about 3:2:0, about 3:3:0, about 2:1:0, about 2:2:0, about 1:1:0, about 1:2:0, about 1:3:0, about 1:4:0, about 1:5:0, about 1:6:0, about 1:7:0, about 1:8:0, about 1:9:0, or about 1:10:0.

[0792] In some embodiments, the cell therapy is monitored for the ratio of cells with a first transgene to cells with a second transgene to cells with a first transgene and a second transgene to cells with no transgene. In some embodiments, the ratio of cells with a first transgene to cells with a second transgene to cells with a first transgene and a second transgene to cells with no transgene is about 10:1:1:1, about 10:2:1:1, about 10:3:1:1, about 10:4:1:1, about 10:5:1:1, about 10:6:1:1, about 10:7:1:1, about 10:8:1:1, about 10:9:1:1, about 10:10:1:1, 10:1:2:1, about 10:2:2:1, about 10:3:2:1, about 10:4:2:1, about 10:5:2:1, about 10:6:2:1, about 10:7:2:1, about 10:8:2:1, about 10:9:2:1, about 10:10:2:1, 10:1:3:1, about 10:2:3:1, about 10:3:3:1, about 10:4:3:1, about 10:5:3:1, about 10:6:3:1, about 10:7:3:1, about 10:8:3:1, about 10:9:3:1, about 10:10:3:1, 10:1:4:1, about 10:2:4:1, about 10:3:4:1, about 10:4:4:1, about 10:5:4:1, about 10:6:4:1, about 10:7:4:1, about 10:8:4:1, about 10:9:4:1, about 10:10:4:1, 10:1:5:1, about 10:2:5:1, about 10:3:5:1, about 10:4:5:1, about 10:5:5:1, about 10:6:5:1, about 10:7:5:1, about 10:8:5:1, about 10:9:5:1, about 10:10:5:1, 10:1:6:1, about 10:2:6:1, about 10:3:6:1, about 10:4:6:1, about 10:5:6:1, about 10:6:6:1, about 10:7:6:1, about 10:8:6:1, about 10:9:6:1, about 10:10:6:1, 10:1:7:1, about 10:2:7:1, about 10:3:7:1, about 10:4:7:1, about 10:5:7:1, about 10:6:7:1, about 10:7:7:1, about 10:8:7:1, about 10:9:7:1, about 10:10:7:1, 10:1:8:1, about 10:2:8:1, about 10:3:8:1, about 10:4:8:1, about 10:5:8:1, about 10:6:8:1, about 10:7:8:1, about 10:8:8:1, about 10:9:8:1, about 10:10:8:1, 10:1:9:1, about 10:2:9:1, about 10:3:9:1, about 10:4:9:1, about 10:5:9:1, about 10:6:9:1, about 10:7:9:1, about 10:8:9:1, about 10:9:9:1, about 10:10:9:1, 10:1:10:1, about 10:2:10:1, about 10:3:10:1, about 10:4:10:1, about 10:5:10:1, about 10:6:10:1, about 10:7:10:1, about 10:8:10:1, about 10:9:10:1, about 10:10:10:1, about 9: 1: 1 : 1, about 9:2: 1:1, about 9:3: 1:1, about 9:4: 1:1, about 9:5: 1:1, about 9:6: 1:1, about 9:7: 1:1, about 9:8: 1 : 1, about 9:9: 1 : 1, about 9: 1:2: 1, about 9:2:2:1, about 9:3:2:1, about 9:4:2:1, about 9:5:2:1, about 9:6:2:1, about 9:7:2:1, about 9:8:2:1, about 9:9:2:1, about 9: 1:3: 1, about 9:2:3:1, about 9:3:3: 1, about 9:4:3:1, about 9:5:3: 1, about 9:6:3: 1, about 9:7:3:1, about 9:8:3: 1, about 9:9:3: 1, about 9: 1:4: 1, about 9:2:4:1, about 9:3:4:1, about 9:4:4:1, about 9:5:4:1, about 9:6:4:1, about 9:7:4:1, about 9:8:4:1, about 9:9:4:1, about 9: 1:5: 1, about 9:2:5:1, about 9:3:5: 1, about 9:4:5:1, about 9:5:5: 1, about 9:6:5: 1, about 9:7:5: 1, about 9:8:5: 1, about 9:9:5: 1, about 9: 1:6:1, about 9:2:6:1, about 9:3:6: 1, about 9:4:6:1, about 9:5:6: 1, about 9:6:6: 1, about 9:7:6:1, about 9:8:6: 1, about 9:9:6: 1, about 9: 1:7:1, about 9:2:7: 1, about 9:3:7: 1, about 9:4:7: 1, about 9:5:7: 1, about 9:6:7:1, about 9:7:7:1, about 9:8:7: 1, about 9:9:7:1, about 9: 1:8: 1, about 9:2:8:1, about 9:3:8: 1, about 9:4:8:1, about 9:5:8: 1, about 9:6:8: 1, about 9:7:8: 1, about 9:8:8: 1, about 9:9:8: 1, about 9: 1:9: 1, about 9:2:9:1, about 9:3:9: 1, about 9:4:9:1, about 9:5:9: 1, about 9:6:9: 1, about 9:7:9:1, about 9:8:9: 1, about 9:9:9: 1, about 8: 1:1:1, about 8:2: 1:1, about 8:3: 1:1, about 8:4: 1:1, about 8:5: 1: 1, about 8:6: 1: 1, about 8:7: 1: 1, about 8:8: 1: 1, about 8: 1 :2: 1, about 8:2:2:1, about 8:3:2: 1, about 8:4:2:1, about 8:5:2: 1, about 8:6:2:1, about 8:7:2:1, about 8:8:2: 1, about 8: 1 :3: 1, about 8:2:3: 1, about 8:3:3: 1, about 8:4:3: 1, about 8:5:3: 1, about 8:6:3: 1, about 8:7:3: 1, about 8:8:3: 1, about 8: 1 :4: 1, about 8:2:4:1, about 8:3:4: 1, about 8:4:4:1, about 8:5:4: 1, about 8:6:4:1, about 8:7:4:1, about 8:8:4: 1, about 8: 1 :5: 1, about 8:2:5: 1, about 8:3:5: 1, about 8:4:5: 1, about 8:5:5: 1, about 8:6:5: 1, about 8:7:5: 1, about 8:8:5: 1, about 8: 1 :6: 1, about 8:2:6:1, about 8:3:6: 1, about 8:4:6:1, about 8:5:6: 1, about 8:6:6: 1, about 8:7:6: 1, about 8:8:6: 1, about 8: 1 :7: 1, about 8:2:7:1, about 8:3:7: 1, about 8:4:7:1, about 8:5:7: 1, about 8:6:7: 1, about 8:7:7:1, about 8:8:7: 1, about 8: 1 :8: 1, about 8:2:8: 1, about 8:3:8: 1, about 8:4:8: 1, about 8:5:8: 1, about 8:6:8: 1, about 8:7:8: 1, about 8:8:8: 1 , about 7: 1 : 1 : 1, about 7:2: 1 : 1, about 7:3: 1 : 1, about 7:4: 1 : 1, about 7:5: 1 : 1, about 7:6: 1 : 1, about 7:7:1:1, about 7: 1 :2: 1, about 7:2:2:1, about 7:3:2:1, about 7:4:2:1, about 7:5:2:1, about 7:6:2:1, about 7:7:2:1, about 7: 1 :3: 1, about 7:2:3:1, about 7:3:3:1, about 7:4:3:1, about 7:5:3: 1, about 7:6:3:1, about 7:7:3:1, about 7: 1 :4: 1, about 7:2:4:1, about 7:3:4:1, about 7:4:4:1, about 7:5:4:1, about 7:6:4:1, about 7:7:4:1, about 7: 1 :5: 1, about 7:2:5:1, about 7:3:5: 1, about 7:4:5:1, about 7:5:5: 1, about 7:6:5:1, about 7:7:5:1, about 7: 1 :6: 1, about 7:2:6:1, about 7:3:6:1, about 7:4:6: 1, about 7:5:6: 1, about 7:6:6: 1, about 7:7:6: 1, about 7: 1:7:1, about 7:2:7: 1, about 7:3:7:1, about 7:4:7:1, about 7:5:7:1, about 7:6:7:1, about 7:7:7:1, about 6: 1 : 1 : 1, about 6:2: 1 : 1, about 6:3: 1:1, about 6:4: 1:1, about 6:5: 1:1, about 6:6: 1:1, about 6: 1:2:1, about 6:2:2: 1, about 6:3:2:1, about 6:4:2:1, about 6:5:2:1, about 6:6:2:1, about 6: 1:3: 1, about 6:2:3:1, about 6:3:3: 1, about 6:4:3:1, about 6:5:3: 1, about 6:6:3: 1, about 6: 1:4: 1, about 6:2:4:1, about 6:3:4:1, about 6:4:4:1, about 6:5:4:1, about 6:6:4:1, about 6: 1:5: 1, about 6:2:5:1, about 6:3:5: 1, about 6:4:5:1, about 6:5:5: 1, about 6:6:5: 1, about 6: 1:6: 1, about 6:2:6:1, about 6:3:6: 1, about 6:4:6:1, about 6:5:6: 1, about 6:6:6: 1, about 5: 1:1:1, about 5:2: 1:1, about 5:3: 1:1, about 5:4: 1:1, about 5:5: 1:1, about 5: 1:2:1, about 5:2:2: 1, about 5:3:2: 1, about 5:4:2: 1, about 5:5:2: 1, about 5: 1:3:1, about 5:2:3:1, about 5:3:3: 1, about 5:4:3:1, about 5:5:3: 1, about 5: 1:4: 1, about 5:2:4:1, about 5:3:4:1, about 5:4:4: 1, about 5:5:4: 1, about 5: 1:5:1, about 5:2:5: 1, about 5:3:5: 1, about 5:4:5: 1, about 5:5:5: 1 , about 4: 1 : 1 : 1, about 4:2: 1 : 1, about 4:3: 1 : 1, about 4:4: 1 : 1, about 4: 1 :2: 1, about 4:2:2:1, about 4:3:2:1, about 4:4:2:1, about 4:1:3:1, about 4:2:3:1, about 4:3:3:1, about 4:4:3:1, about 4: 1 :4: 1, about 4:2:4:1, about 4:3:4:1, about 4:4:4:1, about 3: 1 : 1 : 1, about 3:2: 1 : 1, about 3:3: 1 : 1, about 3: 1:2: 1, about 3:2:2:1, about 3:3:2:1, about 3: 1:3: 1, about 3:2:3:1, about 3:3:3: 1, about 2: 1 : 1 : 1, about 2:2: 1 : 1, about 2: 1 :2: 1, about 2:2:2:1, about 1 : 1 : 1 : 1, about 1 :2: 1 : 1, about 1 :3: 1 : 1, about 1:4: 1: 1, about 1:5:1: 1, about 1:6: 1: 1, about 1:7: 1: 1, about 1:8: 1: 1, about 1:9: 1: 1, about 1:10:1:1, about 1: 1:2: 1, about 1: 1:3: 1, about 1: 1:4:1, about 1: 1:5: 1, about 1: 1:6: 1, about 1: 1:7: 1, about 1: 1:8:1, about 1: 1:9:1, or about 1:1:10:1.

VI. THERAPEUTIC USES AND PRODUCTS

[0793] In some aspects, provided herein are methods of treating a disease or disorder in a subject, comprising administering to the subject an effective amount of a cell therapy and detecting the presence or absence of the engineered cells or populations of cells, as described in the present application. In other aspects, provided herein are uses of a cell therapy comprising detecting the presence or absence of the engineered cells or populations of cells of the present disclosure for treating a disease or disorder in a subject, or for the manufacture of medicaments for treating a disease or disorder in a subject.

1. Uses

[0794] Such methods and uses include, for example, administration of engineered cells or population of said cells (e.g., T cells, B cells, NK cells, islet cells such as beta cells, or hypoimmunogenic cells thereof), or compositions containing the same, to a subject having a disease, condition, or disorder. It is within the level of a skilled artisan to choose the appropriate engineered cell or population of cells as provided herein for a particular disease, condition, or disorder. In some embodiments, the engineered cells or populations of cells or compositions thereof are administered in an effective amount to effect treatment of the disease or disorder, e.g., a therapeutically effective amount. Uses include uses of the engineered cells or populations of cells or compositions thereof in such methods and treatments, and in the preparation of a medicament in order to carry out such therapeutic methods. In some embodiments, the methods thereby treat the disease or condition or disorder in the subject.

[0795] In some embodiments, the cell therapy comprises administering one or more doses of engineered immune cells expressing the first transgene and/or the second transgene. In some embodiments, the cell therapy comprises administering one or more doses of engineered T cells expressing a first CAR and/or a second CAR, such as any of the CARs described herein. In some embodiments, the engineered cell or population of cells comprises a CAR, wherein the CAR comprises an antigen binding domain, and wherein the antigen binding domain recognizes an antigen characteristic of a T cell, e.g., as described above.

[0796] In some embodiments, the engineered cell or population of cells comprises a CAR, wherein the CAR comprises an antigen binding domain, and wherein the antigen binding domain recognizes an antigen characteristic of a B cell, e.g., as described above. In some embodiments, the engineered cell or population of cells comprises a CAR, wherein the CAR comprises an antigen binding domain, and wherein the antigen binding domain recognizes an antigen characteristic of an autoimmune or inflammatory disorder, e.g., as described above. In some embodiments, the engineered cell or population of cells comprises a CAR, wherein the CAR comprises an antigen binding domain, and wherein the antigen binding domain recognizes an antigen characteristic of a senescent cell, e.g., as described above. In some embodiments, the engineered cell or population of cells comprises a CAR, wherein the CAR comprises an antigen binding domain, and wherein the antigen binding domain recognizes an antigen characteristic of an infectious disease, e.g., as described above.

[0797] In some embodiments, the engineered cell or population of cells comprises a CAR comprising an antigen binding domain selected from the group consisting of: CD5, CD19, CD20, CD22, CD23, CD30, CD33, CD38, CD70, CD123, CD138, BCMA, GPRC5D, CD123, LeY, NKG2D ligand, WT1, GD2, HER2, EGFR, EGFRvIII, B7H3, PSMA, PSCA, CAIX, CD171, CEA, CSPG4, EPHA2, FAP, FRa, IE-13Ra, Mesothelin, MUC1, MUC16, ROR1, C- Met, CD133, Ep-CAM, GPC3, HPV16-E6, IE13Ra2, MAGEA3, MAGEA4, MARTI, NY-ESO- 1, VEGFR2, a-Folate receptor, CD24, CD44v7/8, EGP-2, EGP-40, erb-B2, erb-B 2,3,4, FBP, Fetal acethylcholine e receptor, GD2, GD3, HMW-MAA, IL-l lRa, KDR, Lewis Y, Ll-cell adhesion molecule, MAGE-A1, Oncofetal antigen (h5T4), TAG-72, CD19, and CD22, e.g., as described above. In some embodiments, the engineered cell or population of cells comprises a CAR comprising an antigen binding domain selected from the group consisting of: CD 19, CD22, CD20, BCMA, an EBV antigen, CD27, CD30, CD19 and CD20, CD19 and CD22, CD19 and CD27, EBNA1, EBNA3A, BRLF1, BALF4, EBNA3C, LMP1, LMP2, LMP2A, LMP2B, BZLF1, BMLF1, gp350, gH/gL, EBNA1 and LMP1, EBNA1 and LMP2A, EBNA1 and LMP1 and LMP2A, LMP and BARF1 and EBNA1, CD 19 and an EBV antigen, CD20 and an EBV antigen, or CD22 and an EBV antigen. In some embodiments, the engineered cell or population of cells comprises a CAR comprising an antigen binding domain selected from the group consisting of: CD19, CD20, CD22, CD38, CD123, CD138, BCMA, or CD19 and CD22, e.g., as described above.

[0798] In other embodiments, the engineered cell or population of cells comprises a chimeric autoantibody receptor (CAAR), such as any of the CAARs described herein. In other embodiments, the CAAR comprises an antigen selected from the group consisting of a pancreatic P-cell antigen, synovial joint antigen, myelin basic protein, proteolipid protein, myelin oligodendritic glycoprotein, MuSK, keratinocyte adhesion protein desmoglein 3 (Dsg3), Ro- RNP complex, La antigen, myeloperoxidase, proteinase 3, cardiolipin, citrullinated proteins, carbamylated proteins, and a3 chain of basement membrane collagen.

[0799] In some embodiments, the engineered cell or population of cells (e.g., B cells) comprises a B-cell autoantibody receptor (BAR), such as any of the BARs described herein. In some embodiments, the BAR comprises an FVIII antigen. [0800] In some embodiments, the engineered cell or population of cells (e.g., T cells) comprises a T cell receptor (TCR), as described in more detail herein. In some embodiments, the engineered cell or population of cells comprises a safety switch, such as any of the safety switches described herein. In some embodiments, the engineered cell or population of cells comprises a transcription factor, as described in more detail herein. In some embodiments, the engineered cell or population of cells comprises a tolerogenic factor, such as any of the tolerogenic factors described herein. In some embodiments, the tolerogenic factor is selected from the group consisting of: CD16, CD24, CD35, CD39, CD46, CD47, CD52, CD55, CD59, CD64, CD200, CCL22, CTLA4-Ig, Cl inhibitor, FASL, IDO1, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, IL-10, IL-35, PD-L1, SERPINB9, CCL21, MFGE8, DUX4, B2M-HLA-E, CD27, IL-39, CD16 Fc Receptor, IL15-RF, H2-M3 (HLA-G), A20/TNFAIP3, CR1, HLA-F, and MANF.

[0801] In some embodiments, the disease or disorder to be treated is selected from autoimmune or inflammatory disorders, including but not limited to, any of the disorders selected from the group consisting of: arthritis, rheumatoid arthritis, acute arthritis, chronic rheumatoid arthritis, gout or gouty arthritis, acute gouty arthritis, acute immunological arthritis, chronic inflammatory arthritis, degenerative arthritis, type II collagen-induced arthritis, infectious arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis, Still's disease, vertebral arthritis, juvenile-onset rheumatoid arthritis, osteoarthritis, arthritis chronica progrediente, arthritis deformans, polyarthritis chronica primaria, reactive arthritis, ankylosing spondylitis, inflammatory hyperproliferative skin diseases, psoriasis, plaque psoriasis, gutatte psoriasis, pustular psoriasis, psoriasis of the nails, atopy, atopic diseases, hay fever, Job's syndrome, dermatitis, contact dermatitis, chronic contact dermatitis, exfoliative dermatitis, exfoliative psoriatic dermatitis, allergic dermatitis, allergic contact dermatitis, dermatitis herpetiformis, nummular dermatitis, seborrheic dermatitis, non-specific dermatitis, primary irritant contact dermatitis, atopic dermatitis, x-linked hyper IgM syndrome, allergic intraocular inflammatory diseases, urticaria, chronic allergic urticaria, chronic idiopathic urticaria, chronic autoimmune urticaria, myositis, polymyositis/dermatomyositis, juvenile dermatomyositis, toxic epidermal necrolysis, scleroderma, systemic scleroderma, sclerosis, systemic sclerosis, multiple sclerosis (MS), MS associated with EBV infection, spino-optical MS, primary progressive MS (PPMS), relap sing-remitting MS (RRMS), progressive relapsing MS, secondary progressive MS (SPMS), progressive systemic sclerosis, atherosclerosis, arteriosclerosis, sclerosis disseminata, ataxic sclerosis, neuromyelitis optica spectrum disorder, inflammatory bowel disease (IBD), Crohn's disease, autoimmune-mediated gastrointestinal diseases, colitis, ulcerative colitis, colitis ulcerosa, microscopic colitis, collagenous colitis, colitis polyposa, necrotizing enterocolitis, transmural colitis, autoimmune inflammatory bowel disease, bowel inflammation, pyoderma gangrenosum, erythema nodosum, primary sclerosing cholangitis, respiratory distress syndrome, adult or acute respiratory distress syndrome (ARDS), meningitis, inflammation of all or part of the uvea, iritis, choroiditis, an autoimmune hematological disorder, rheumatoid spondylitis, rheumatoid synovitis, hereditary angioedema, cranial nerve damage, meningitis, herpes gestationis, pemphigoid gestationis, pruritis scroti, autoimmune premature ovarian failure, sudden hearing loss due to an autoimmune condition, IgE-mediated diseases, anaphylaxis, allergic and atopic rhinitis, encephalitis, Rasmussen's encephalitis, limbic and/or brainstem encephalitis, uveitis, anterior uveitis, acute anterior uveitis, granulomatous uveitis, nongranulomatous uveitis, phacoantigenic uveitis, posterior uveitis, autoimmune uveitis, glomerulonephritis (GN) with or without nephrotic syndrome, chronic or acute glomerulonephritis, primary GN, immune-mediated GN, membranous GN (membranous nephropathy), idiopathic membranous GN or idiopathic membranous nephropathy, membrano- or membranous proliferative GN (MPGN), Type I or Type II GN, rapidly progressive GN, proliferative nephritis, autoimmune polyglandular endocrine failure, balanitis including balanitis circumscripta plasmacellularis, balanoposthitis, erythema annulare centrifugum, erythema dyschromicum perstans, erythema multiform, granuloma annulare, lichen nitidus, lichen sclerosus et atrophicus, lichen simplex chronicus, lichen spinulosus, lichen planus, lamellar ichthyosis, epidermolytic hyperkeratosis, premalignant keratosis, pyoderma gangrenosum, allergic conditions and responses, allergic reaction, eczema, allergic or atopic eczema, asteatotic eczema, dyshidrotic eczema, vesicular palmoplantar eczema, asthma, asthma bronchiale, bronchial asthma, auto-immune asthma, conditions involving infiltration of T cells or chronic inflammatory responses, immune reactions against foreign antigens, immune reactions against fetal A-B-0 blood groups during pregnancy, chronic pulmonary inflammatory disease, autoimmune myocarditis, leukocyte adhesion deficiency, lupus, lupus nephritis, lupus cerebritis, pediatric lupus, non-renal lupus, extra-renal lupus, discoid lupus, discoid lupus erythematosus, alopecia lupus, systemic lupus erythematosus (SLE), cutaneous SLE, subacute cutaneous SLE, neonatal lupus syndrome (NLE), lupus erythematosus disseminatus, Type I diabetes, Type II diabetes, latent autoimmune diabetes in adults, Type 1.5 diabetes, juvenile onset (Type I) diabetes mellitus, pediatric insulin-dependent diabetes mellitus (IDDM), adult onset diabetes mellitus (Type II diabetes), idiopathic diabetes, insipidus, diabetic retinopathy, diabetic nephropathy, diabetic large-artery disorder, immune responses associated with acute or delayed hypersensitivity mediated by cytokines or T-lymphocytes, tuberculosis, sarcoidosis, granulomatosis, lymphomatoid granulomatosis, Wegener's granulomatosis, agranulocytosis, vasculitides, vasculitis, large- vessel vasculitis, polymyalgia rheumatica, giant cell (Takayasu's) arteritis, medium-vessel vasculitis, Kawasaki's disease, polyarteritis nodosa/periarteritis nodosa, microscopic polyarteritis, immunovasculitis, CNS vasculitis, cutaneous vasculitis, hypersensitivity vasculitis, necrotizing vasculitis, systemic necrotizing vasculitis, ANCA- associated vasculitis, Churg-Strauss vasculitis, syndrome (CSS), ANCA-associated small-vessel vasculiti, temporal arteritis, aplastic anemia, autoimmune aplastic anemia, Coombs positive anemia, Diamond Blackfan anemia, hemolytic anemia, immune hemolytic anemia, autoimmune hemolytic anemia (AIHA), pernicious anemia (anemia pemiciosa), Addison's disease, pure red cell anemia, aplasia (PRCA), Factor VIII deficiency, hemophilia A, autoimmune neutropenia, pancytopenia, leukopenia, diseases involving leukocyte diapedesis, CNS inflammatory disorders, Alzheimer's disease, Parkinson's disease, multiple organ injury syndrome, multiple organ injury syndrome secondary to septicemia, trauma, or hemorrhage, antigen-antibody complex-mediated diseases, anti-glomerular basement membrane disease, anti-phospholipid antibody syndrome, anti-phospholipid syndrome, allergic neuritis, Behcet's disease/syndrome, Castleman's syndrome, Goodpasture's syndrome, Reynaud's syndrome, Sjogren's syndrome, Stevens-Johnson syndrome, pemphigoid, pemphigoid bullous, skin pemphigoid, pemphigus, pemphigus vulgaris, pemphigus foliaceus, pemphigus mucus-membrane pemphigoid, pemphigus erythematosus, autoimmune polyendocrinopathies, Reiter's disease or syndrome, thermal injury, preeclampsia, an immune complex disorder, immune complex nephritis, antibody-mediated nephritis, polyneuropathies, chronic neuropathy, IgM polyneuropathies, IgM-mediated neuropathy, thrombocytopenia, thrombotic thrombocytopenic purpura (TTP), post-transfusion purpura (PTP), heparin-induced thrombocytopenia, autoimmune or immune-mediated thrombocytopenia, idiopathic thrombocytopenic purpura (ITP), chronic or acute ITP, acquired thrombocytopenic purpura, scleritis, idiopathic cerato-scleritis, episcleritis, autoimmune disease of the testis or ovary, autoimmune orchitis or oophoritis, primary hypothyroidism, hypoparathyroidism, autoimmune endocrine diseases, thyroiditis, autoimmune thyroiditis, Hashimoto's disease, chronic thyroiditis, Hashimoto's thyroiditis, subacute thyroiditis, autoimmune thyroid disease, idiopathic hypothyroidism, Grave's disease, polyglandular syndromes, autoimmune polyglandular syndromes, polyglandular endocrinopathy syndromes, paraneoplastic syndromes, neurologic paraneoplastic syndromes, Lambert-Eaton myasthenic syndrome, Eaton-Lambert syndrome, stiff-man or stiff-person syndrome, encephalomyelitis, allergic encephalomyelitis, encephalomyelitis allergica, experimental allergic encephalomyelitis (EAE), myasthenia gravis, thymoma-associated myasthenia gravis, cerebellar degeneration, neuromyotonia, opsoclonus, opsoclonus myoclonus syndrome (OMS), sensory neuropathy, multifocal motor neuropathy, Sheehan's syndrome, hepatitis, autoimmune hepatitis, chronic hepatitis, lupoid hepatitis, giant cell hepatitis, chronic active hepatitis, autoimmune chronic active hepatitis, lymphoid interstitial pneumonitis (LIP), bronchiolitis obliterans, Guillain-Barre syndrome, Berger's disease (IgA nephropathy), idiopathic IgA nephropathy, linear IgA dermatosis, acute febrile neutrophilic dermatosis, subcorneal pustular dermatosis, transient acantholytic dermatosis, cirrhosis, primary biliary cirrhosis, pneumonocirrhosis, autoimmune enteropathy syndrome, Celiac or Coeliac disease, celiac sprue (gluten enteropathy), refractory sprue, idiopathic sprue, cryoglobulinemia, amyotrophic lateral sclerosis (ALS; Lou Gehrig's disease), coronary artery disease, autoimmune ear disease, autoimmune inner ear disease (AIED), autoimmune hearing loss, polychondritis, refractory or relapsed or relapsing polychondritis, pulmonary alveolar proteinosis, Cogan's syndrome/nonsyphilitic interstitial keratitis, Bell's palsy, Sweet's disease/syndrome, rosacea autoimmune, zoster-associated pain, amyloidosis, a non-cancerous lymphocytosis, a primary lymphocytosis, monoclonal B cell lymphocytosis, benign monoclonal gammopathy or monoclonal gammopathy of undetermined significance, peripheral neuropathy, paraneoplastic syndrome, channelopathies, epilepsy, migraine, arrhythmia, muscular disorders, deafness, blindness, periodic paralysis, channelopathies of the CNS, autism, inflammatory myopathy, focal or segmental or focal segmental glomerulosclerosis (ESGS), endocrine ophthalmopathy, uveoretinitis, chorioretinitis, autoimmune hepatological disorder, fibromyalgia, multiple endocrine failure, Schmidt's syndrome, adrenalitis, gastric atrophy, presenile dementia, demyelinating diseases, autoimmune demyelinating diseases, chronic inflammatory demyelinating polyneuropathy, Dressier's syndrome, alopecia areata, alopecia totalis, CREST syndrome, calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, telangiectasia, male or female autoimmune infertility, anti-spermatozoan antibodies, mixed connective tissue disease, Chagas' disease, rheumatic fever, recurrent abortion, farmer's lung, erythema multiforme, post-cardiotomy syndrome, post myocardial infarction cardiotomy syndrome, Cushing's syndrome, bird-fancier's lung, allergic granulomatous angiitis, benign lymphocytic angiitis, Alport's syndrome, alveolitis, allergic alveolitis, fibrosing alveolitis, interstitial lung disease, transfusion reaction, leprosy, malaria, parasitic diseases, leishmaniasis, kypanosomiasis, schistosomiasis, ascariasis, aspergillosis, Samter's syndrome, Caplan's syndrome, dengue, endocarditis, endomyocardial fibrosis, diffuse interstitial pulmonary fibrosis, interstitial lung fibrosis, pulmonary fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis, endophthalmitis, erythema elevatum et diutinum, erythroblastosis fetalis, eosinophilic faciitis, Shulman's syndrome, Felty's syndrome, flariasis, cyclitis, chronic cyclitis, heterochronic cyclitis, iridocyclitis (acute or chronic), Fuch's cyclitis, Henoch-Schonlein purpura, human immunodeficiency virus (HIV) infection, SCID, acquired immune deficiency syndrome (AIDS), echovirus infection, sepsis, endotoxemia, pancreatitis, thyroxicosis, parvovirus infection, rubella virus infection, post-vaccination syndromes, congenital rubella infection, Epstein-Barr virus infection, mumps, Evan's syndrome, autoimmune gonadal failure, Sydenham's chorea, poststreptococcal nephritis, thromboangitis ubiterans, thyrotoxicosis, tabes dorsalis, chorioiditis, giant cell polymyalgia, chronic hypersensitivity pneumonitis, keratoconjunctivitis sicca, epidemic keratoconjunctivitis, idiopathic nephritic syndrome, minimal change nephropathy, benign familial and ischemia-reperfusion injury, transplant organ reperfusion, retinal autoimmunity, joint inflammation, bronchitis, chronic obstructive airway /pulmonary disease, silicosis, aphthae, aphthous stomatitis, arteriosclerotic disorders, aspermiogenese, autoimmune hemolysis, Boeck's disease, cryoglobulinemia, Dupuytren's contracture, endophthalmia phacoanaphylactica, enteritis allergica, erythema nodosum leprosum, idiopathic facial paralysis, chronic fatigue syndrome, febris rheumatica, Hamman-Rich's disease, sensoneural hearing loss, haemoglobinuria paroxysmatica, hypogonadism, ileitis regionalis, leucopenia, mononucleosis infectiosa, traverse myelitis, primary idiopathic myxedema, nephrosis, ophthalmia symphatica, orchitis granulomatosa, pancreatitis, polyradiculitis acuta, pyoderma gangrenosum, Quervain's thyreoiditis, acquired splenic atrophy, non-malignant thymoma, vitiligo, toxic-shock syndrome, food poisoning, conditions involving infiltration of T cells, leukocyte-adhesion deficiency, immune responses associated with acute or delayed hypersensitivity mediated by cytokines or T- lymphocytes, diseases involving leukocyte diapedesis, multiple organ injury syndrome, antigenantibody complex-mediated diseases, antiglomerular basement membrane disease, allergic neuritis, autoimmune polyendocrinopathies, oophoritis, primary myxedema, autoimmune atrophic gastritis, sympathetic ophthalmia, rheumatic diseases, mixed connective tissue disease, nephrotic syndrome, insulitis, polyendocrine failure, autoimmune polyglandular syndrome type I, adult-onset idiopathic hypoparathyroidism (AOIH), cardiomyopathy, dilated cardiomyopathy, epidermolisis bullosa acquisita (EBA), hemochromatosis, myocarditis, nephrotic syndrome, primary sclerosing cholangitis, purulent or nonpurulent sinusitis, acute or chronic sinusitis, ethmoid, frontal, maxillary, sphenoid sinusitis, an eosinophil-related disorder, eosinophilia, pulmonary infiltration eosinophilia, eosinophilia-myalgia syndrome, Loffler's syndrome, chronic eosinophilic pneumonia, tropical pulmonary eosinophilia, bronchopneumonic aspergillosis, aspergilloma, granulomas containing eosinophils, anaphylaxis, seronegative spondyloarthritides, polyendocrine autoimmune disease, sclerosing cholangitis, sclera, episclera, chronic mucocutaneous candidiasis, Bruton's syndrome, transient hypogammaglobulinemia of infancy, Wiskott-Aldrich syndrome, ataxia telangiectasia syndrome, angiectasis, autoimmune disorders associated with collagen disease, rheumatism, neurological disease, lymphadenitis, reduction in blood pressure response, vascular dysfunction, tissue injury, cardiovascular ischemia, hyperalgesia, renal ischemia, cerebral ischemia, disease accompanying vascularization, allergic hypersensitivity disorders, glomerulonephritides, reperfusion injury, ischemic re-perfusion disorder, reperfusion injury of myocardial or other tissues, lymphomatous tracheobronchitis, inflammatory dermatoses, dermatoses with acute inflammatory components, multiple organ failure, bullous diseases, renal cortical necrosis, acute purulent meningitis, central nervous system inflammatory disorders, ocular or orbital inflammatory disorders, granulocyte transfusion-associated syndromes, cytokine-induced toxicity, narcolepsy, acute serious inflammation, chronic intractable inflammation, pyelitis, endarterial hyperplasia, peptic ulcer, valvulitis, emphysema, alopecia areata, adipose tissue inflammation/diabetes type II, obesity- associated adipose tissue inflammation/insulin resistance, endometriosis, and pulmonary hemosiderosis.

[0802] In some embodiments, the disease or disorder is selected from the group consisting of: diabetes, cancer, vascularization disorders, ocular disease, thyroid disease, skin diseases, liver diseases, a condition or disease associated with a vascular condition or disease, a vascular condition or disease, a condition or disease associated with autoimmune thyroiditis, autoimmune thyroiditis, a condition or disease associated with a liver disease, liver disease, cirrhosis of the liver, a condition or disease associated with a corneal disease, corneal disease, Fuchs dystrophy or congenital hereditary endothelial dystrophy, a condition or disease associated with a kidney disease, kidney disease, a disease associated with cancer, cancer, B cell acute lymphoblastic leukemia (B-ALL), diffuse large B-cell lymphoma, liver cancer, pancreatic cancer, breast cancer, ovarian cancer, colorectal cancer, lung cancer, non- small cell lung cancer, acute myeloid lymphoid leukemia, multiple myeloma, gastric cancer, gastric adenocarcinoma, pancreatic adenocarcinoma, glioblastoma, neuroblastoma, lung squamous cell carcinoma, hepatocellular carcinoma, bladder cancer, a condition or disease associated with a hematopoietic disease or disorder, a hematopoietic disease or disorder, myelodysplasia, aplastic anemia, Fanconi anemia, paroxysmal nocturnal hemoglobinuria, Sickle cell disease, Diamond Blackfan anemia, Schachman Diamond disorder, Kostmann's syndrome, chronic granulomatous disease, adrenoleukodystrophy, leukocyte adhesion deficiency, hemophilia, thalassemia, betathalassemia, leukaemia, acute lymphocytic leukemia (ALL), acute myelogenous (myeloid) leukemia (AML), adult lymphoblastic leukaemia, chronic lymphocytic leukemia (CLL), B-cell chronic lymphocytic leukemia (B-CLL), chronic myeloid leukemia (CML), juvenile chronic myelogenous leukemia (CML), juvenile myelomonocytic leukemia (JMML), severe combined immunodeficiency disease (SCID), X-linked severe combined immunodeficiency, Wiskott- Aldrich syndrome (WAS), adenosine-deaminase (ADA) deficiency, chronic granulomatous disease, Chediak-Higashi syndrome, Hodgkin's lymphoma, non-Hodgkin's lymphoma (NHL), AIDS, a condition or disease associated with leukemia or myeloma, leukemia, myeloma, a condition or disease associated with an autoimmune disease or condition, an autoimmune disease or condition, acute disseminated encephalomyelitis, acute hemorrhagic leukoencephalitis, Addison's disease, Agammaglobulinemia, alopecia areata, amyotrophic lateral sclerosis, ankylosing spondylitis, antiphospholipid syndrome, antisynthetase syndrome, atopic allergy, autoimmune aplastic anemia, autoimmune cardiomyopathy, autoimmune enteropathy, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, autoimmune lymphoproliferative syndrome, autoimmune peripheral neuropathy, autoimmune pancreatitis, autoimmune polyendocrine syndrome, autoimmune progesterone dermatitis, autoimmune thrombocytopenic purpura, autoimmune urticaria, autoimmune uveitis, Balo disease, Balo concentric sclerosis, Bechets syndrome, Berger's disease, Bickerstaffs encephalitis, Blau syndrome, bullous pemphigoid, cancer, Castleman's disease, celiac disease, chronic inflammatory demyelinating polyneuropathy, chronic recurrent multifocal osteomyelitis, Churg- Strauss syndrome, cicatricial pemphigoid, Cogan syndrome, cold agglutinin disease, complement component 2 deficiency, cranial arteritis, CREST syndrome, Crohn's disease, Cushing's syndrome, cutaneous leukocytoclastic angiitis, Dego's disease, Dercum's disease, dermatitis herpetiformis, dermatomyositis, diabetes mellitus type 1, diffuse cutaneous systemic sclerosis, Dressier's syndrome, discoid lupus erythematosus, eczema, enthesitis-related arthritis, eosinophilic fasciitis, eosinophilic gastroenteritis, epidermolysis bullosa acquisita, erythema nodosum, essential mixed cryoglobulinemia, Evan's syndrome, fibrodysplasia ossificans progressiva, fibrosing alveolitis, gastritis, gastrointestinal pemphigoid, giant cell arteritis, glomerulonephritis, goodpasture's syndrome, Grave's disease, Guillain-Barre syndrome (GBS), Hashimoto's encephalitis, Hashimoto's thyroiditis, hemolytic anaemia, Henoch-Schonlein purpura, herpes gestationis, hypogammaglobulinemia, idiopathic inflammatory demyelinating disease, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura, IgA nephropathy, inclusion body myositis, inflammatory demyelinating polyneuropathy, interstitial cystitis, juvenile idiopathic arthritis, juvenile rheumatoid arthritis, Kawasaki's disease, Lambert-Eaton myasthenic syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, linear IgA disease (LAD), Lou Gehrig's disease, lupoid hepatitis, lupus erythematosus, Majeed syndrome, Meniere's disease, microscopic polyangiitis, Miller-Fisher syndrome, mixed connective tissue disease, morphea, Mucha-Habermann disease, multiple sclerosis, myasthenia gravis, myositis, neuropyelitis optica, neuromyotonia, ocular cicatricial pemphigoid, opsoclonus myoclonus syndrome, ord thyroiditis, palindromic rheumatism, paraneoplastic cerebellar degeneration, paroxysmal nocturnal hemoglobinuria (PNH), Parry Romberg syndrome, Parsonnage-Turner syndrome, pars planitis, pemphigus, pemphigus vulgaris, pernicious anemia, perivenous encephalomyelitis, POEMS syndrome, polyarteritis nodosa, polymyalgia rheumatica, polymyositis, primary biliary cirrhosis, primary sclerosing cholangitis, progressive inflammatory neuropathy, psoriasis, psoriatic arthritis, pyoderma gangrenosum, pure red cell aplasia, Rasmussen's encephalitis, Raynaud phenomenon, relapsing polychondritis, Reiter's syndrome, restless leg syndrome, retroperitoneal fibrosis, rheumatoid arthritis, rheumatoid fever, sarcoidosis, Schmidt syndrome, Schnitzler syndrome, scleritis, scleroderma, Sjogren's syndrome, spondylarthropathy, Still's disease, stiff person syndrome, subacute bacterial endocarditis, Susac's syndrome, Sweet's syndrome, Sydenham chorea, sympathetic ophthalmia, Takayasu's arteritis, temporal arteritis, Tolosa-Hunt syndrome, transverse myelitis, ulcerative colitis, undifferentiated connective tissue disease, undifferentiated spondylarthropathy, vasculitis, vitiligo, Wegener's granulomatosis, a condition or disease associated with Parkinson’s disease, Huntington disease, multiple sclerosis, a neurodegenerative disease or condition, attention deficit hyperactivity disorder (ADHD), Tourette Syndrome (TS), schizophrenia, psychosis, depression, a neuropsychiatric disorder stroke, amyotrophic lateral sclerosis (ALS), Parkinson’s disease, Huntington disease, multiple sclerosis, a neurodegenerative disease or condition, schizophrenia, psychosis, depression, or a neuropsychiatric disorder stroke.

[0803] In some embodiments, the engineered cells (e.g., T cells, B cells, NK cells, islet cells such as beta cells, or hypoimmunogenic cells thereof) provided herein, populations of said engineered cells, or compositions thereof can be used in cell therapy. Therapeutic cells outlined herein may be useful to treat a disorder such as, but not limited to, a cancer, a genetic disorder, a chronic infectious disease, an autoimmune disorder, a neurological disorder, or the like.

[0804] In some embodiments, the engineered cells (e.g., T cells, B cells, NK cells, islet cells such as beta cells, or hypoimmunogenic cells thereof) provided herein, populations of said engineered cells, or compositions thereof are administered prior to providing a tissue, organ, or partial organ transplant to a patient in need thereof. In some embodiments, the patient exhibits a reduced immune response to the engineered cells or populations of cells. In particular embodiments, the patient does not exhibit an immune response to the engineered cells or populations of cells. In certain embodiments, the engineered cells or populations of cells are administered to the patient for the treatment of a cellular deficiency in a particular tissue or organ, and the patient subsequently receives a tissue or organ transplant for the same particular tissue or organ. In such embodiments, the engineered cell treatment functions as a bridge therapy to the eventual tissue or organ replacement. For example, in some embodiments, the patient has a liver disorder and receives an engineered hepatocyte treatment as provided herein, prior to receiving a liver transplant. In certain embodiments, the engineered cells or populations of cells are administered to the patient for the treatment of a cellular deficiency in a particular tissue or organ and the patient subsequently receives a tissue or organ transplant for a different tissue or organ. For example, in some embodiments, the patient is a diabetes patient who is treated with engineered pancreatic beta cells as provided herein prior to receiving a pancreas transplant. In some embodiments, the method is for the treatment of a cellular deficiency. In exemplary embodiments, the tissue or organ transplant is a heart transplant, a lung transplant, a kidney transplant, a liver transplant, a pancreas transplant, an intestine transplant, a stomach transplant, a cornea transplant, a bone marrow transplant, a blood vessel transplant, a heart valve transplant, or a bone transplant.

[0805] The engineered cells (e.g., T cells, B cells, NK cells, islet cells such as beta cells, or hypoimmunogenic cells thereof) provided herein, populations of said engineered cells, or compositions thereof can be administered to any suitable patients including, for example, a candidate for a cellular therapy for the treatment of a disease or disorder, such as any of the diseases or disorders described herein. Candidates for cellular therapy include any patient having a disease or condition that may potentially benefit from the therapeutic effects of the engineered cells or population of cells described herein. In some embodiments, the patient is an allogeneic recipient of the administered cells. In some embodiments, the provided engineered cells or populations of cells are effective for use in allogeneic cell therapy. A candidate who benefits from the therapeutic effects of the engineered cells or populations of cells provided herein may exhibit an elimination, reduction, or amelioration of the disease or condition.

[0806] The specific amount and dosage regimen of engineered cells (e.g., T cells, B cells, NK cells, islet cells such as beta cells, or hypoimmunogenic cells thereof) provided herein, populations of said engineered cells, or compositions thereof will vary depending on the weight, gender, age, and health of the individual; the formulation; the biochemical nature; bioactivity; bioavailability and side effects of the cells; and the number and identity of the cells in the complete therapeutic regimen.

[0807] The engineered cells (e.g., T cells, B cells, NK cells, islet cells such as beta cells, or hypoimmunogenic cells thereof) provided herein, populations of said engineered cells, or compositions thereof can be administered by any suitable route, such as by intravenous, intraperitoneal, or subcutaneous administration, as described in Section IV (“Cell Compositions") above. In some embodiments, the engineered cells provided herein, populations of said engineered cells, or compositions thereof are administered parenterally. In some embodiments, the engineered cells provided herein, populations of said engineered cells, and compositions thereof are administered to a subject intravenously.

[0808] Any therapeutically effective amount of engineered cells (e.g., T cells, B cells, NK cells, islet cells such as beta cells, or hypoimmunogenic cells thereof) provided herein, and populations of said engineered cells, can be included in compositions for therapeutic treatments or uses, depending on the indication being treated. In some embodiments, the pharmaceutical composition includes at least about 1 x 10², 5 x 10², 1 x 10³, 5 x 10³, 1 x 10⁴, 5 x 10⁴, 1 x 10⁵, 5 x

10⁵, 1 x 10⁶, 5 x 10⁶, 1 x 10⁷, 5 x 10⁷, 1 x 10⁸, 5 x 10⁸, 1 x 10⁹, 5 x 10⁹, 1 x IO¹⁰, or 5 x IO¹⁰ cells.

In some embodiments, the pharmaceutical composition includes up to about 1 x 10², 5 x 10², 1 x 10³, 5 x 10³, 1 x 10⁴, 5 x 10⁴, 1 x 10⁵, 5 x 10⁵, 1 x 10⁶, 5 x 10⁶, 1 x 10⁷, 5 x 10⁷, 1 x 10⁸, 5 x 10⁸,

1 x 10⁹, 5 x 10⁹, 1 x IO¹⁰, or 5 x IO¹⁰ cells. In some embodiments, the pharmaceutical composition includes up to about 6.0 x 10⁸ cells. In some embodiments, the pharmaceutical composition includes up to about 8.0 x 10⁸ cells. In some embodiments, the pharmaceutical composition includes at least about 1 x 10² - 5 x 10²; 5 x 10² - l x 10³; 1 x 10³ - 5 x 10³; 5 x 10³ - 1 x 10⁴; 1 x 10⁴ - 5 x 10⁴; 5 x 10⁴ - 1 x 10⁵; 1 x 10⁵ - 5 x 10⁵; 5 x 10⁵ - 1 x 10⁶; 1 x 10⁶ - 5 x 10⁶; 5 x 10⁶ - 1 x 10⁷; 1 x 10⁷ - 5 x 10⁷; 5 x 10⁷ - 1 x 10⁸; 1 x 10⁸ - 5 x 10⁸; 5 x 10⁸ - 1 x 10⁹; 1 x 10⁹ - 5 x 10⁹; 5 x 10⁹ - 1 x IO¹⁰; or 1 x IO¹⁰ - 5 x IO¹⁰ cells. In exemplary embodiments, the pharmaceutical composition includes from about 1.0 x 10⁶ to about 2.5 x 10⁸ cells. In some embodiments, the pharmaceutical composition comprises engineered beta cells, wherein the dose is from about 1 x 10⁷ cells to about 3 x 10⁸ cells, or from about 6,500 islet equivalents (IEQ) to about 600,000 IEQ (e.g., from about 6,500 IEQ to about 7,500 IEQ, from about 7,000 IEQ to about 8,000 IEQ, from about 7,500 IEQ to about 10,000 IEQ, from about 10,000 IEQ to about 20,000 IEQ, from about 15,000 IEQ to about 25,000 IEQ, from about 20,000 IEQ to about 50,000 IEQ, from about 25,000 IEQ to about 75,000 IEQ, from about 50,000 IEQ to about 150,000 IEQ, from about 100,000 IEQ to about 200,000 IEQ, from about 150,000 IEQ to about 250,000 IEQ, from about 200,000 IEQ to about 400,000 IEQ, from about 300,000 IEQ to about 500,000 IEQ, or from about 400,000 IEQ to about 600,000 IEQ). In some embodiments, the pharmaceutical composition has a volume of at least about any of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, or 500 mL. In exemplary embodiments, the pharmaceutical composition has a volume of up to about any of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, or 500 mL. In exemplary embodiments, the pharmaceutical composition has a volume of about any of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, or 500 mL. In some embodiments, the pharmaceutical composition has a volume of from about 1-50 mL, 50-100 mL, 100-150 mL, 150-200 mL, 200-250 mL, 250-300 mL, 300-350 mL, 350-400 mL, 400-450 mL, or 450-500 mL. In some embodiments, the pharmaceutical composition has a volume of from about 1-50 mL, 50-100 mL, 100-150 mL, 150-200 mL, 200-250 mL, 250-300 mL, 300-350 mL, 350-400 mL, 400-450 mL, or 450-500 mL. In some embodiments, the pharmaceutical composition has a volume of from about 1-10 mL, 10-20 mL, 20-30 mL, 30-40 mL, 40-50 mL, 50-60 mL, 60-70 mL, 70-80 mL, 70-80 mL, 80-90 mL, or 90-100 mL. In some embodiments, the pharmaceutical composition has a volume that ranges from about 5 mL to about 80 mL. In exemplary embodiments, the pharmaceutical composition has a volume that ranges from about 10 mL to about 70 mL. In many embodiments, the pharmaceutical composition has a volume that ranges from about 10 mL to about 50 mL. In some embodiments, a dose of the pharmaceutical composition includes about 1.0 x 10⁵ to about 2.5 x 10⁸ cells at a volume of about 10 mL to 50 mL, and the pharmaceutical composition is administered as a single dose.

[0809] In some embodiments, the pharmaceutical composition is administered as a single dose of from about 1.0 x 10⁵ to about 1.0 x 10⁷ cells per kg body weight for subjects 50 kg or less. In some embodiments, the pharmaceutical composition is administered as a single dose of from about 0.5 x 10⁵ to about 1.0 x 10⁷, about 1.0 x 10⁵ to about 1.0 x 10⁷, about 1.0 x 10⁵ to about 1.0 x 10⁷, about 5.0 x 10⁵ to about 1 x 10⁷, about 1.0 x 10⁶ to about 1 x 10⁷, about 5.0 x 10⁶ to about 1.0 x 10⁷, about 1.0 x 10⁵ to about 5.0 x 10⁶, about 1.0 x 10⁵ to about 1.0 x 10⁶, about 1.0 x 10⁵ to about 5.0 x 10⁵, about 1.0 x 10⁵ to about 5.0 x 10⁶, about 2.0 x 10⁵ to about 5.0 x 10⁶, about 3.0 x 10⁵ to about 5.0 x 10⁶, about 4.0 x 10⁵ to about 5.0 x 10⁶, about 5.0 x 10⁵ to about 5.0 x 10⁶, about 6.0 x 10⁵ to about 5.0 x 10⁶, about 7.0 x 10⁵ to about 5.0 x 10⁶, about 8.0 x 10⁵ to about 5.0 x 10⁶, or about 9.0 x 10⁵ to about 5.0 x 10⁶ cells per kg body weight for subjects 50 kg or less. In some embodiments, the dose is from about 0.2 x 10⁶ to about 5.0 x 10⁶ cells per kg body weight for subjects 50 kg or less. In many embodiments, the dose is at a range that is lower than from about 0.2 x 10⁶ to about 5.0 x 10⁶ cells per kg body weight for subjects 50 kg or less.

In many embodiments, the dose is at a range that is higher than from about 0.2 x 10⁶ to about 5.0 x 10⁶ cells per kg body weight for subjects 50 kg or less. In exemplary embodiments, the single dose is at a volume of about 10 mL to 50 mL. In some embodiments, the dose is administered intravenously. In exemplary embodiments, the cells are administered in a single dose of from about 1.0 x 10⁶ to about 5.0 x 10⁸ cells for subjects above 50 kg. In some embodiments, the pharmaceutical composition is administered as a single dose of from about 0.5 x 10⁶ to about 1.0 x 10⁹, about 1.0 x 10⁶ to about 1.0 x 10⁹, about 1.0 x 10⁶ to about 1.0 x 10⁹, about 5.0 x 10⁶ to about 1.0 x 10⁹, about 1.0 x 10⁷ to about 1.0 x 10⁹, about 5.0 x 10⁷ to about 1.0 x 10⁹, about 1.0 x 10⁶ to about 5.0 x 10⁷, about 1.0 x 10⁶ to about 1.0 x 10⁷, about 1.0 x 10⁶ to about 5.0 x 10⁷, about 1.0 x 10⁷ to about 5.0 x 10⁸, about 2.0 x 10⁷ to about 5.0 x 10⁸, about 3.0 x 10⁷ to about 5.0 x 10⁸, about 4.0 x 10⁷ to about 5.0 x 10⁸, about 5.0 x 10⁷ to about 5.0 x 10⁸, about 6.0 x 10⁷ to about 5.0 x 10⁸, about 7.0 x 10⁷ to about 5.0 x 10⁸, about 8.0 x 10⁷ to about 5.0 x 10⁸, or about 9.0 x 10⁷ to about 5.0 x 10⁸ cells per kg body weight for subjects 50 kg or less. In certain embodiments, the cells are administered in a single dose of about 1.0 x 10⁷ to about 2.5 x 10⁸ cells for subjects above 50 kg. In some embodiments, the cells are administered in a single dose of a range that is less than about 1.0 x 10⁷ to about 2.5 x 10⁸ cells for subjects above 50 kg. In some embodiments, the cells are administered in a single dose of a range that is higher than about 1.0 x 10⁷ to about 2.5 x 10⁸ cells for subjects above 50 kg. In some embodiments, the pharmaceutical composition comprises engineered beta cells, wherein the dose is from about 1.25 x 10⁵ cells/kg to about 1.2 x 10⁷ cells/kg, or from about 80 lEQ/kg to about 24,000 lEQ/kg (e.g., from about 80 lEQ/kg to about 800 lEQ/kg, from about 500 lEQ/kg to about 1500 lEQ/kg, from about 1000 lEQ/kg to about 2000 lEQ/kg, from about 1500 lEQ/kg to about 3000 lEQ/kg, from about 2000 lEQ/kg to about 4000 lEQ/kg, from about 3000 lEQ/kg to about 5000 lEQ/kg from about 4000 lEQ/kg to about 6000 lEQ/kg, from about 5000 lEQ/kg to about 10,000 lEQ/kg, from about 7500 lEQ/kg to about 15,000 lEQ/kg, from about 15,000 lEQ/kg to about 20,000 lEQ/kg, or from about 20,000 lEQ/kg to about 24,000 lEQ/kg). In some embodiments, the dose is administered intravenously. In exemplary embodiments, the single dose is at a volume of about 10 mL to 50 mL. In some embodiments, the dose is administered intravenously.

[0810] In some embodiments, the dose is administered intravenously at a rate of about 1 to 50 mL per minute, 1 to 40 mL per minute, 1 to 30 mL per minute, 1 to 20 mL per minute, 10 to 20 mL per minute, 10 to 30 mL per minute, 10 to 40 mL per minute, 10 to 50 mL per minute, 20 to 50 mL per minute, 30 to 50 mL per minute, or 40 to 50 mL per minute. In numerous embodiments, the pharmaceutical composition is stored in one or more infusion bags for intravenous administration. In some embodiments, the dose is administered completely at no more than 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, 70 minutes, 80 minutes, 90 minutes, 120 minutes, 150 minutes, 180 minutes, 240 minutes, or 300 minutes.

[0811] In some embodiments, a single dose of the pharmaceutical composition is present in a single infusion bag. In other embodiments, a single dose of the pharmaceutical composition is divided into 2, 3, 4, or 5 separate infusion bags.

[0812] In some embodiments, the cells described herein are administered in a plurality of doses such as 2, 3, 4, 5, 6, or more doses. In some embodiments, each dose of the plurality of doses is administered to the subject ranging from 1 to 24 hours apart. In some instances, a subsequent dose is administered from about 1 hour to about 24 hours (e.g., about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours) after an initial or preceding dose. In some embodiments, each dose of the plurality of doses is administered to the subject ranging from about 1 day to about 28 days apart. In some instances, a subsequent dose is administered from about 1 day to about 28 days (e.g., about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 days) after an initial or preceding dose. In certain embodiments, each dose of the plurality of doses is administered to the subject ranging from about 1 week to about 6 weeks apart. In certain instances, a subsequent dose is administered from about 1 week to about 6 weeks (e.g., about any of 1, 2, 3, 4, 5, or 6 weeks) after an initial or preceding dose. In several embodiments, each dose of the plurality of doses is administered to the subject ranging from about 1 month to about 12 months apart. In several instances, a subsequent dose is administered from about 1 month to about 12 months (e.g., about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months) after an initial or preceding dose. [0813] In some embodiments, a subject is administered a first dosage regimen at a first timepoint, and then subsequently administered a second dosage regimen at a second timepoint. In some embodiments, the first dosage regimen is the same as the second dosage regimen. In other embodiments, the first dosage regimen is different than the second dosage regimen. In some instances, the number of cells in the first dosage regimen and the second dosage regimen are the same. In some instances, the number of cells in the first dosage regimen and the second dosage regimen are different. In some cases, the number of doses of the first dosage regimen and the second dosage regimen are the same. In some cases, the number of doses of the first dosage regimen and the second dosage regimen are different.

[0814] In some embodiments, a subject, individual, or patient to be treated according to the methods or therapeutic uses described herein is a mammal. In some embodiments, the subject, individual, or patient to be treated according to the methods or therapeutic uses described herein is a human.

[0815] In some embodiments, the engineered cells or populations of cells provided herein (e.g., engineered iPSCs, stem cells, pluripotent cells, or the like) are differentiated into different cell types for subsequent transplantation into recipient subjects. Differentiation can be assayed as is known in the art, generally by evaluating the presence of cell-specific markers. As will be appreciated by those in the art, the differentiated engineered cell derivatives can be transplanted using techniques known in the art that depends on both the cell type and the ultimate use of these cells. Exemplary types of differentiated cells and methods for producing the same are described below. In some embodiments, the engineered cells or populations of cells provided herein (e.g., engineered iPSCs, stem cells, pluripotent cells, or the like) may be differentiated to any type of cell described herein, including any described herein. In some embodiments, the differentiated engineered cells or populations of cells are transplanted or administered to a patient, e.g., as described above. In some embodiments, the patient is a human individual. In some embodiments, the patient is the same as the donor. In some embodiments, the patient is a different individual than the donor. In some embodiments, the engineered cells or populations of cells provided herein (e.g., engineered iPSCs, stem cells, pluripotent cells, or the like) are differentiated into cell types selected from T cells, NK cells, beta islet cells, endothelial cells, epithelial cells such as RPE, thyroid, skin, or hepatocytes. In some embodiments, host cells such as non-pluripotent cells (e.g., fibroblasts) from an individual donor or a pool of individual donors are isolated or obtained, generated into iPSCs in which the iPSCs are then engineered (e.g., genetic modifications) as described herein, differentiated into a desired cell type, and administered and/or transplanted into the individual patient. In some embodiments, the population of engineered cells isolated from one or more individual donors (e.g., healthy donors) or engineered cells differentiated from engineered iPSCs, stem cells, pluripotent cells, or the like derived from one or more individual donors (e.g., healthy donors), are maintained in culture, in some cases expanded, prior to administration. In certain embodiments, the population of engineered cells are cryopreserved prior to administration.

[0816] In some embodiments, the engineered cells or populations of cells described herein, such as cells isolated from one or more individual donors (e.g., healthy donors) or engineered cells differentiated from engineered iPSCs, stem cells, pluripotent cells, or the like derived from one or more individual donors (e.g., healthy donors), do not activate an immune response in the patient (e.g., in the recipient upon administration). In some embodiments, the number of cells administration is at a lower dosage than would be required for immunogenic cells (e.g., a population of cells of the same or similar cell type or phenotype but that do not contain the vectors comprising the one or more transgenes (e.g., a first and a second or more transgene(s)) described herein, such as one or more CARs or tolerogenic factors). In some embodiments, the number of cells administration is at a lower dosage than would be required to reduce immune rejection of immunogenic cells (e.g., a population of cells of the same or similar cell type or phenotype but that do not contain the vectors comprising the one or more transgenes (e.g., a first and a second or more transgene(s)) described herein, such as one or more CARs or tolerogenic factors).

[0817] In some embodiments, the engineered cells or populations of cells for use as described herein are pancreatic cells, such as islet cells, e.g., beta islet cells, including primary cells, which may be transplanted (either as a cell suspension or within a gel matrix as discussed herein) into the portal vein/liver, the omentum, the gastrointestinal mucosa, the bone marrow, a muscle, or subcutaneous pouches. In some embodiments, pancreatic cells described herein are administered to a subject to treat diabetes or other diseases, disorders or conditions associated with pancreatic dysfunction.

[0818] In some embodiments, the engineered cells or populations of cells for use as described herein are hepatocytes, including primary hepatocytes, which can be administered as a cell therapy to address loss of hepatocyte function, diseases of the liver, liver dysfunction, or cirrhosis of the liver.

[0819] In some embodiments, the engineered cells or populations of cells for use as described herein are immune cells (e.g., T cells, B cells, NK cells, macrophages, neutrophils, dendritic cells, MDSCs, or the like). Immune cells such as those provided herein may be useful for the treatment of suitable cancers including, but not limited to, B cell acute lymphoblastic leukemia (B-ALL), diffuse large B-cell lymphoma, liver cancer, pancreatic cancer, breast cancer, ovarian cancer, colorectal cancer, lung cancer, non- small cell lung cancer, acute myeloid lymphoid leukemia, multiple myeloma, gastric cancer, gastric adenocarcinoma, pancreatic adenocarcinoma, glioblastoma, neuroblastoma, lung squamous cell carcinoma, hepatocellular carcinoma, or bladder cancer.

[0820] In some embodiments, the engineered cells or populations of cells for use as described herein are endothelial cells, including primary endothelial cells. In some embodiments, the endothelial cells are endothelial colony forming cells (ECFCs) that may be used to form new blood vessels to address peripheral arterial disease. The engineered endothelial cells provided herein may be useful in the treatment of cardiovascular disease, vascular disease, peripheral vascular disease, ischemic disease, myocardial infarction, congestive heart failure, peripheral vascular obstructive disease, stroke, reperfusion injury, limb ischemia, neuropathy (e.g., peripheral neuropathy or diabetic neuropathy), organ failure (e.g., liver failure, kidney failure, or the like), diabetes, rheumatoid arthritis, osteoporosis, vascular injury, tissue injury, hypertension, angina pectoris and myocardial infarction due to coronary artery disease, renal vascular hypertension, renal failure due to renal artery stenosis, claudication of the lower extremities, or the like. In certain embodiments, the patient has suffered from or is suffering from a transient ischemic attack or stroke, which in some cases, may be due to cerebrovascular disease. In some embodiments, the engineered endothelial cells are administered to treat tissue ischemia, e.g., as occurs in atherosclerosis, myocardial infarction, or limb ischemia and to repair injured blood vessels. In some instances, the cells are used in bioengineering grafts. For instance, the engineered endothelial cells can be used in cell therapy for the repair of ischemic tissues, formation of blood vessels and heart valves, engineering of artificial vessels, repair of damaged vessels, or inducing the formation of blood vessels in engineered tissues (e.g., prior to transplantation). Additionally, the endothelial cells can be engineered to deliver agents to target and treat tumors. In some embodiments, the engineered endothelial cells of the disclosure may be useful for repair or replacement of tissue in need of vascular cells or vascularization. Such repair or replacement may involve administering to a human patient in need of such treatment, a composition containing the engineered endothelial cells, such as engineered isolated primary endothelial cells or differentiated endothelial cells, to promote vascularization in such tissue. The tissue in need of vascular cells or vascularization can be (e.g.) a cardiac tissue, liver tissue, pancreatic tissue, renal tissue, muscle tissue, neural tissue, or bone tissue, among others, which can be a tissue damaged and/or characterized by excess cell death, a tissue at risk for damage, or an artificially engineered tissue. In some embodiments, vascular diseases, which may be associated with cardiac diseases or disorders, can be treated by administering the engineered endothelial cells of the disclosure, such as but not limited to, definitive vascular endothelial cells, or endocardial endothelial cells derived as described herein. Such vascular diseases include, but are not limited to, coronary artery disease, cerebrovascular disease, aortic stenosis, aortic aneurysm, peripheral artery disease, atherosclerosis, varicose veins, angiopathy, infarcted area of heart lacking coronary perfusion, non-healing wounds, diabetic or non-diabetic ulcers, or any other disease or disorder in which it is desirable to induce formation of blood vessels. In certain embodiments, the engineered endothelial cells may be used for improving prosthetic implants (e.g., vessels made of synthetic materials such as Dacron and Gortex) which are used in vascular reconstructive surgery. For example, prosthetic arterial grafts are often used to replace diseased arteries which perfuse vital organs or limbs. In other embodiments, the engineered endothelial cells are used to cover the surface of prosthetic heart valves to decrease the risk of the formation of emboli by making the valve surface less thrombogenic. The engineered endothelial cells as outlined can be transplanted into the patient using well known surgical techniques for grafting tissue and/or isolated cells into a vessel. In some embodiments, the cells are introduced into the patient’s heart tissue by injection (e.g., intramyocardial injection, intracoronary injection, trans- endocardial injection, trans-epicardial injection, percutaneous injection), infusion, grafting, or implantation. In certain embodiments, the engineered endothelial cells can be engineered in a way that improves their performance in the context of an implanted graft. Non-limiting illustrative examples include secretion or expression of a thrombolytic agent to prevent intraluminal clot formation, secretion of an inhibitor of smooth muscle proliferation to prevent luminal stenosis due to smooth muscle hypertrophy, and expression and/or secretion of an endothelial cell mitogen or autocrine factor to stimulate endothelial cell proliferation and improve the extent or duration of the endothelial cell lining of the graft lumen. In some embodiments, the engineered endothelial cells are utilized for delivery of therapeutic levels of a secreted product to a specific organ or limb. For example, a vascular implant lined with endothelial cells engineered (transduced) in vitro can be grafted into a specific organ or limb. The secreted product of the transduced endothelial cells will be delivered in high concentrations to the perfused tissue, thereby achieving a desired effect to a targeted anatomical location. In other embodiments, the engineered endothelial cells comprise an exogenous sequence that encodes a gene that disrupts or inhibits angiogenesis when expressed by endothelial cells in a vascularizing tumor. In some embodiments, endothelial cells described herein, such as isolated primary endothelial cells or differentiated endothelial cells, are administered to a recipient subject to treat a vascular disorder selected from vascular injury, cardiovascular disease, vascular disease, peripheral vascular disease, ischemic disease, myocardial infarction, congestive heart failure, peripheral vascular obstructive disease, hypertension, ischemic tissue injury, reperfusion injury, limb ischemia, stroke, neuropathy (e.g., peripheral neuropathy or diabetic neuropathy), organ failure (e.g., liver failure, kidney failure, or the like), diabetes, rheumatoid arthritis, osteoporosis, cerebrovascular disease, hypertension, angina pectoris and myocardial infarction due to coronary artery disease, renal vascular hypertension, renal failure due to renal artery stenosis, claudication of the lower extremities, or other vascular conditions or diseases.

[0821] In some embodiments, the engineered cells or populations of cells for use as described herein are cardiac cells, including primary cardiac cells, which may be useful for treating any of the diseases or disorders selected from the group consisting of: pediatric cardiomyopathy, age-related cardiomyopathy, dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, chronic ischemic cardiomyopathy, peripartum cardiomyopathy, inflammatory cardiomyopathy, idiopathic cardiomyopathy, other cardiomyopathy, myocardial ischemic reperfusion injury, ventricular dysfunction, heart failure, congestive heart failure, coronary artery disease, end-stage heart disease, atherosclerosis, ischemia, hypertension, restenosis, angina pectoris, rheumatic heart, arterial inflammation, cardiovascular disease, myocardial infarction, myocardial ischemia, congestive heart failure, myocardial infarction, cardiac ischemia, cardiac injury, myocardial ischemia, vascular disease, acquired heart disease, congenital heart disease, congenital heart defect, atherosclerosis, coronary artery disease, dysfunctional conduction systems, dysfunctional coronary arteries, pulmonary hypertension, cardiac arrhythmias, muscular dystrophy, muscle mass abnormality, muscle degeneration, myocarditis, infective myocarditis, drug- or toxin-induced muscle abnormalities, hypersensitivity myocarditis, autoimmune endocarditis, heart valve disease or dysfunction, endocarditis, rheumatic fever, mitral valve prolapse, infective endocarditis, cardiomegaly, and/or mitral insufficiency, among others. [0822] In some embodiments, the engineered cells or populations of cells for use as described herein are neural cells, including primary neural cells, such as cerebral endothelial cells, dopaminergic neurons, or glial cells. The engineered neural cells of the disclosure may be useful for treating, e.g., Parkinson’s disease, Alzheimer’s disease, Huntington disease, multiple sclerosis, other neurodegenerative disease or condition, attention deficit hyperactivity disorder (ADHD), stroke, amyotrophic lateral sclerosis (ALS), Tourette Syndrome (TS), schizophrenia, psychosis, depression, cerebral hemorrhage, epileptic seizures, or other neuropsychiatric disorders or conditions. In some embodiments, neural cells described herein are administered to a subject to treat or ameliorate stroke. In some embodiments, engineered neurons and glial cells are administered to a subject with amyotrophic lateral sclerosis (ALS). In some embodiments, engineered cerebral endothelial cells are administered to alleviate the symptoms or effects of cerebral hemorrhage. In some embodiments, engineered dopaminergic neurons are administered to a patient with Parkinson’s disease. In some embodiments, engineered noradrenergic neurons and/or GABAergic interneurons are administered to a patient who has experienced an epileptic seizure. In some embodiments, engineered motor neurons, interneurons, Schwann cells, oligodendrocytes, and/or microglia are administered to a patient who has experienced a spinal cord injury. In some embodiments, engineered dopaminergic (DA) neurons derived from HIP cells are administered to a patient, e.g., human patient, to treat a neurodegenerative disease or condition, such as Parkinson’s disease, Huntington disease, or multiple sclerosis. In other embodiments, the DA neurons are used to treat or ameliorate one or more symptoms of a neuropsychiatric disorder, such as attention deficit hyperactivity disorder (ADHD), Tourette Syndrome (TS), schizophrenia, psychosis, or depression. In yet other embodiments, the DA neurons are used to treat a patient with impaired DA neurons.

[0823] In some embodiments, the engineered cells or populations of cells for use as described herein are hematopoietic stem cells, including primary hematopoietic stem cells. In some embodiments, the engineered hematopoietic stem cells may be useful for treating hematopoietic diseases or disorders, such as any selected from the group consisting of: myelodysplasia, aplastic anemia, Fanconi anemia, paroxysmal nocturnal hemoglobinuria, Sickle cell disease, Diamond Blackfan anemia, Schachman Diamond disorder, Kostmann's syndrome, chronic granulomatous disease, adrenoleukodystrophy, leukocyte adhesion deficiency, hemophilia, thalassemia, beta- thalassemia, leukaemia such as acute lymphocytic leukemia (ALL), acute myelogenous (myeloid) leukemia (AML), adult lymphoblastic leukaemia, chronic lymphocytic leukemia (CLL), B-cell chronic lymphocytic leukemia (B-CLL), chronic myeloid leukemia (CML), juvenile chronic myelogenous leukemia (CML), juvenile myelomonocytic leukemia (JMML), severe combined immunodeficiency disease (SCID), X-linked severe combined immunodeficiency, Wiskott-Aldrich syndrome (WAS), adenosine-deaminase (ADA) deficiency, chronic granulomatous disease, Chediak-Higashi syndrome, Hodgkin's lymphoma, non-Hodgkin's lymphoma (NHL), or AIDS. In some embodiments, the engineered hematopoietic stem cells may be useful for treating a cellular deficiency associated with leukemia or myeloma, or to treat leukemia or myeloma. In some embodiments, the engineered hematopoietic stem cells may be useful for treating a cellular deficiency associated with an autoimmune disease or condition or to treat an autoimmune disease or condition. In some embodiments, the autoimmune disease or condition is selected from the group consisting of: acute disseminated encephalomyelitis, acute hemorrhagic leukoencephalitis, Addison's disease, Agammaglobulinemia, alopecia areata, amyotrophic lateral sclerosis, ankylosing spondylitis, antiphospholipid syndrome, antisynthetase syndrome, atopic allergy, autoimmune aplastic anemia, autoimmune cardiomyopathy, autoimmune enteropathy, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, autoimmune lymphoproliferative syndrome, autoimmune peripheral neuropathy, autoimmune pancreatitis, autoimmune polyendocrine syndrome, autoimmune progesterone dermatitis, autoimmune thrombocytopenic purpura, autoimmune urticaria, autoimmune uveitis, Balo disease, Balo concentric sclerosis, Bechets syndrome, Berger's disease, Bickerstaffs encephalitis, Blau syndrome, bullous pemphigoid, cancer, Castleman's disease, celiac disease, chronic inflammatory demyelinating polyneuropathy, chronic recurrent multifocal osteomyelitis, Churg-Strauss syndrome, cicatricial pemphigoid, Cogan syndrome, cold agglutinin disease, complement component 2 deficiency, cranial arteritis, CREST syndrome, Crohn's disease, Cushing's syndrome, cutaneous leukocytoclastic angiitis, Dego's disease, Dercum's disease, dermatitis herpetiformis, dermatomyositis, diabetes mellitus type 1 , diffuse cutaneous systemic sclerosis, Dressier's syndrome, discoid lupus erythematosus, eczema, enthesitis-related arthritis, eosinophilic fasciitis, eosinophilic gastroenteritis, epidermolysis bullosa acquisita, erythema nodosum, essential mixed cryoglobulinemia, Evan's syndrome, firodysplasia ossificans progressiva, fibrosing aveolitis, gastritis, gastrointestinal pemphigoid, giant cell arteritis, glomerulonephritis, goodpasture's syndrome, Grave's disease, Guillain-Barre syndrome (GBS), Hashimoto's encephalitis, Hashimoto's thyroiditis, hemolytic anaemia, Henoch-Schonlein purpura, herpes gestationis, hypogammaglobulinemia, idiopathic inflammatory demyelinating disease, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura, IgA nephropathy, inclusion body myositis, inflammatory demyelinating polyneuropathy, interstitial cystitis, juvenile idiopathic arthritis, juvenile rheumatoid arthritis, Kawasaki's disease, Lambert-Eaton myasthenic syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, linear IgA disease (LAD), Lou Gehrig's disease, lupoid hepatitis, lupus erythematosus, Majeed syndrome, Meniere's disease, microscopic polyangiitis, Miller-Eisher syndrome, mixed connective tissue disease, morphea, Mucha-Habermann disease, multiple sclerosis, myasthenia gravis, myositis, neuropyelitis optica, neuromyotonia, ocular cicatricial pemphigoid, opsoclonus myoclonus syndrome, ord thyroiditis, palindromic rheumatism, paraneoplastic cerebellar degeneration, paroxysmal nocturnal hemoglobinuria (PNH), Parry Romberg syndrome, Parsonnage-Turner syndrome, pars planitis, pemphigus, pemphigus vulgaris, permicious anemia, perivenous encephalomyelitis, POEMS syndrome, polyarteritis nodosa, polymyalgia rheumatica, polymyositis, primary biliary cirrhosis, primary sclerosing cholangitis, progressive inflammatory neuropathy, psoriasis, psoriatic arthritis, pyoderma gangrenosum, pure red cell aplasia, Rasmussen's encephalitis, Raynaud phenomenon, relapsing polychondritis, Reiter's syndrome, restless leg syndrome, retroperitoneal fibrosis, rheumatoid arthritis, rheumatoid fever, sarcoidosis, Schmidt syndrome, Schnitzler syndrome, scleritis, scleroderma, Sjogren's syndrome, spondylarthropathy, Still's disease, stiff person syndrome, subacute bacterial endocarditis, Susac's syndrome, Sweet's syndrome, Sydenham chorea, sympathetic ophthalmia, Takayasu's arteritis, temporal arteritis, Tolosa-Hunt syndrome, transverse myelitis, ulcerative colitis, undifferentiated connective tissue disease, undifferentiated spondylarthropathy, vasculitis, vitiligo, or Wegener's granulomatosis.

[0824] In some embodiments, the donor of the therapeutic engineered cells or populations of cells provided herein is different from the patient (e.g., the recipient that is administered the therapeutic cells). In some embodiments, the pool of engineered cells or populations of cells does not include cells from the patient. In some embodiments, one or more of the donor subjects from which the pool of engineered cells or populations of cells is obtained are different from the patient. In some embodiments, the engineered cells or populations of cells differentiated into cell types as described herein may be used for subsequent transplantation or engraftment into subjects (e.g., recipients). In some embodiments, the population of engineered cells isolated from one or more individual donors (e.g., healthy donors) or engineered cells differentiated from iPSCs derived from one or more individual donors (e.g., healthy donors), are maintained in culture, in some cases expanded, prior to administration. In certain embodiments, the population of engineered cells is cryopreserved prior to administration.

[0825] In some embodiments, the engineered cells or populations of cells for use as described herein are T cells, and the pharmaceutical composition includes from about 2.0 x 10⁶ to about 2.0 x 10⁸ cells, such as but not limited to, primary T cells or T cells differentiated from engineered induced pluripotent stem cells. In some cases, the dose includes about 1.0 x 10⁵ to about 2.5 x 10⁸ primary T cells described herein at a volume of about 10 mL to 50 mL. In several cases, the dose includes about 1.0 x 10⁵ to about 2.5 x 10⁸ primary T cells that have been described above at a volume of about 10 mL to 50 mL. In various cases, the dose includes about 1.0 x 10⁵ to about 2.5 x 10⁸ T cells differentiated from engineered induced pluripotent stem cells described herein at a volume of about 10 mL to 50 mL. In other cases, the dose is at a range that is lower than about 1.0 x 10⁵ to about 2.5 x 10⁸ T cells, including primary T cells or T cells differentiated from engineered induced pluripotent stem cells. In yet other cases, the dose is at a range that is higher than about 1.0 x 10⁵ to about 2.5 x 10⁸ T cells, including primary T cells and T cells differentiated from engineered induced pluripotent stem cells. In some embodiments, the cells are engineered T cells (e.g., primary T cells or T cells differentiated from engineered induced pluripotent stem cells) and the first dosage regimen includes engineered T cells expressing a first CAR and the second dosage regimen includes engineered T cells expressing a second CAR such that the first CAR and the second CAR are different. For instance, the first CAR and second CAR bind different target antigens. In some cases, the first CAR includes an scFv that binds an antigen, and the second CAR includes an scFv that binds a different antigen. In some embodiments, the first CAR and second CAR bind different target antigen epitopes but the same target antigen. In some cases, the first CAR includes an scFv that binds a first epitope, and the second CAR includes an scFv that binds a different epitope of the same antigen. In some embodiments, the first dosage regimen includes engineered T cells expressing a first CAR, and the second dosage regimen includes engineered T cells or primary T cells expressing a second CAR such that the first CAR and the second CAR are the same. The first dosage regimen can be administered to the subject at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1-3 months, 1-6 months, 4-6 months, 3-9 months, 3-12 months, or more months apart from the second dosage regimen. In some embodiments, a subject is administered a plurality of dosage regimens during the course of a disease (e.g., cancer) and at least two of the dosage regimens comprise the same type of engineered T cells described herein. In other embodiments, at least two of the plurality of dosage regimens comprise different types of engineered T cells described herein.

[0826] In some embodiments, the therapeutic uses and methods provided herein further comprise administering one or more additional therapeutic agents to the subject. In some embodiments, the one or more additional therapeutics are selected from the group consisting of a small molecule inhibitor, a chemotherapeutic agent, a cancer immunotherapy, an antibody, a cellular therapy, a nucleic acid, a surgery, a radiotherapy, an anti- angiogenic therapy, an anti- DNA repair therapy, an anti-inflammatory therapy, an anti-neoplastic agent, a cell growth factor, a growth inhibitory agent, a cytotoxic agent, a hormonal therapy, a metabolic agent, a protein, a peptide, a pro-peptide, and any combination thereof. In some embodiments, the patient has been previously administered one or more therapeutic agents, such as any of a small molecule inhibitor, a chemotherapeutic agent, a cancer immunotherapy, an antibody, a cellular therapy, a nucleic acid, a surgery, a radiotherapy, an anti- angiogenic therapy, an anti-DNA repair therapy, an anti-inflammatory therapy, an anti-neoplastic agent, a cell growth factor, a growth inhibitory agent, a cytotoxic agent, a hormonal therapy, a metabolic agent, a protein, a peptide, a propeptide, or any combination thereof. For example, in some embodiments, the one or more additional therapeutic agents are selected from the group consisting of insulin, amylinomimetic(s), dopamine-2 agonist(s), DPP-4 inhibitor(s), metformin, alpha-glucosidase inhibitor(s), SGLT2 inhibitor(s), statins, GLP-1 receptor agonist(s), incretin, meglitinide(s), sulfonylureas, thiazolidinediones, nonsteroidal anti-inflammatory drugs (NSAIDs), antimalarial drugs, corticosteroids, azathioprine, mycophenolate, methotrexate, cyclosporine, voclosporin, leflunomide, belimumab, anifrolumab, abatacept, rituximab, vitamin D supplementation, dehydroepiandrosterone (DHEA), and any combination thereof. In some embodiments, the therapeutic uses and methods provided herein further comprise administering one or more immunosuppressive agents to the patient. In some embodiments, the therapeutic uses and methods provided herein further comprise administering an agent that triggers a safety switch to the patient. In some embodiments, the one or more additional therapeutic agents are administered via routes that include, but are not limited to, oral, intravenous, intracavitary, intratumoral, intraarterial, intravitreal, intramuscular, subcutaneous, parenteral, transmucosal, transdermal, ocular, topical, intraperitoneal, intracranial, intrapleural, and epidermal routes. In some embodiments, the engineered cells provided herein, populations of said engineered cells, or compositions thereof, and the one or more additional therapies are administered simultaneously or sequentially.

[0827] In some embodiments, the therapeutic uses and methods provided herein further comprise monitoring therapeutic efficacy, such as by monitoring whether the treated subject exhibits an elimination, reduction, or amelioration of the disease or condition, as described in Section V (“Methods of Monitoring a Sample”) above. In some embodiments, the therapeutic uses and methods provided herein further comprise monitoring the prophylactic efficacy of the method. Therapeutic or prophylactic efficacy in some embodiments is monitored by periodic assessment of treated subjects. In some embodiments, a subject is treated according to the therapeutic uses and methods provided herein until a desired suppression of one or more disease symptoms occurs.

2. Products

[0828] Further provided herein are methods of making therapeutic products, which, for example, may be used in any of the therapeutic uses and methods provided herein.

[0829] In one aspect, provided herein are methods for making a therapeutic cell product. In some embodiments, the methods comprise inserting a vector comprising a transgene (e.g., encoding a therapeutic protein) into a cell, such as any of the cells described herein. In some embodiments, the therapeutic protein is any protein suitable for the treatment of a disease or disorder. In some embodiments, the therapeutic protein is a CAR, CAAR, BAR, or TCR, for example, as described herein. In some embodiments, the methods further comprise culturing the cell under conditions that allow for the survival and proliferation of the engineered cell or population of cells, and recovering the engineered cell or population of cells, for example, by separating, enriching, and/or purifying the engineered cell or population of cells from a larger pool of cells. In some embodiments, the therapeutic protein is selected from the group consisting of: enzymes, structural polypeptides, signaling polypeptides, regulatory polypeptides, transport polypeptides, sensory polypeptides, motor polypeptides, defense polypeptides, storage polypeptides, transcription factors, antibodies, cytokines, hormones, catabolic polypeptides, anabolic polypeptides, proteolytic polypeptides, metabolic polypeptides, kinases, transferases, hydrolases, lyases, isomerases, ligases, enzyme modulator polypeptides, protein binding polypeptides, lipid binding polypeptides, membrane fusion polypeptides, cell differentiation polypeptides, epigenetic polypeptides, cell death polypeptides, nuclear transport polypeptides, nucleic acid binding polypeptides, reprogramming polypeptides, DNA editing polypeptides, DNA repair polypeptides, DNA recombination polypeptides, transposase polypeptides, DNA integration polypeptides, targeted endonucleases, recombinases, transposases, DNA polymerases, RNA polymerases, and reverse transcriptase. In some cases, the therapeutic protein is a therapeutic antibody, such as any of the antibodies described in Section II(6)(iii) herein. [0830] In other aspects, provided herein are methods for making viral or virus-like particles for use in engineering the cell or population of cells derived from the sample as described herein.

[0831] In some embodiments, provided herein are methods for making a lentivirus particle for use in engineering the cell or population of cells derived from the sample as described herein. In some embodiments, the methods may comprise inserting nucleic acid(s) or vector(s) encoding a lentivirus particle into a cell, such as any of the cells provided herein. In some embodiments, the methods further comprise culturing the cell under conditions that allow for production of the lentivirus particle, and optionally recovering the lentivirus particle, for example, by separating, enriching, and/or purifying the lentivirus particle. The lentivirus particles can then be used to engineer a cell or population of cells as described herein to express one or more vectors comprising one or more transgenes and accompanying barcodes.

[0832] In other embodiments, provided herein are methods for making a viral particle or virus-like particle comprising a fusogen for use in engineering the cell or population of cells derived from the sample as described herein. In some embodiments, the methods may comprise inserting nucleic acid(s) or vector(s) encoding a viral particle or virus-like particle and a fusogen into a cell, such as any of the cells provided herein. In some embodiments, the methods further comprise culturing the cell under conditions that allow for production of the viral particle or virus-like particle comprising the fusogen, and optionally recovering the viral particle or viruslike particle comprising the fusogen, for example, by separating, enriching, and/or purifying the viral particle or virus-like particle comprising the fusogen from the cell. The viral particles or virus-like particles can then be used to engineer a cell or population of cells as described herein to express one or more vectors comprising one or more transgenes and accompanying barcodes. [0833] In other embodiments, provided herein are methods for making an AAV particle for use in engineering the cell or population of cells derived from the sample as described herein. In some embodiments, the methods may comprise inserting nucleic acid(s) or vector(s) encoding an AAV particle into a cell, such as any of the cells provided herein. In some embodiments, the methods further comprise culturing the cell under conditions that allow for production of the AAV particle, and optionally recovering the AAV particle, for example, by separating, enriching, and/or purifying the AAV particle. The AAV particles can then be used to engineer a cell or population of cells as described herein to express one or more vectors comprising one or more transgenes and accompanying barcodes.

[0834] In some embodiments, the methods further comprise administering the therapeutic product, i.e., the cells, the population of cells, or the composition thereof to a patient.

3. Kits

[0835] In some aspects, provided herein are kits for detecting the presence or absence of one or more barcodes or articles of manufacture comprising any of the cells, populations of cells, or compositions thereof of the present disclosure. In some embodiments, the kits for detecting the presence or absence of one or more barcodes or articles of manufacture comprise instructions for use according to the methods and/or uses provided herein.

[0836] In some aspects, provided herein is a kit for detecting the presence or absence of one or more barcodes or article of manufacture comprising an engineered cell or population of cells provided herein, populations of cells, or compositions thereof. In some embodiments, the kit for detecting the presence or absence of one or more barcodes or article of manufacture further comprises a package insert comprising instructions for use of the engineered cells or populations of cells according to any of the methods or uses of the present disclosure, such as a therapeutic method or use described herein.

[0837] In some embodiments, the kits for detecting the presence or absence of one or more barcodes or articles of manufacture contain materials useful for clinical transplantation therapies, including cell therapies. In some embodiments, the kits for detecting the presence or absence of one or more barcodes or articles of manufacture contain a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. (e.g., glass or plastic containers). Generally, the container holds a composition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The label or package insert may indicate that the composition is used for treating a particular condition. The label or package insert may further comprise instructions for administering the composition to a patient, for example, instructions customarily included in commercial packages of therapeutic products such as information about the indications, usage, dosage, administration, contraindications and/or warnings concerning the use of such therapeutic products. In some embodiments, the kit for detecting the presence or absence of one or more barcodes or article of manufacture comprises a combination treatment as described herein.

[0838] In some embodiments, the kits or articles of manufacture contain materials useful for detecting the presence or absence of one or more barcodes associated with one or more transgenes encoded by one or more vectors inserted into the cell or population of cells as described herein. In some embodiments, the kits or articles of manufacture for detecting the presence or absence of one or more barcodes contain a container and a label or package insert on or associated with the container and, optionally, instructions for detecting the barcode(s) in a cell, population of cells, or composition thereof administered to a patient, for example as part of the method of monitoring a sample in a patient administered the cell, population of cells, or composition thereof. In some embodiments, the instructions further describe how to calculate the percentage, ratio, relative, and/or absolute number of cells (e.g., from one or more subpopulations of cells) administered to a patient.

EXAMPLES

[0839] The following examples are included for illustrative purposes only and are not intended to limit the scope of the present disclosure.

Example 1: Generation of CAR transfer plasmids containing a unique barcode sequence (barcode)

[0840] This example describes the generation of exemplary vectors containing a unique barcode sequence in one or more regions of the vector, for example, upstream of the promoter region or within the sequence encoding the CAR. These vectors can be used in collecting correlative data for assessing engineered and therapeutic cells.

[0841] The unique barcode sequences may be inserted into different locations on vectors encoding various CARs. In a first exemplary insertion strategy, as shown in FIG. 1, the unique barcode sequence, containing universal forward and reverse primers and unique FAM or HEX probe, was ligated immediately 5' of a promoter as a Gibson insert into a linearized vector. FIG. 1 further provides five exemplary barcode sequences. The inserted barcodes allow for the identification of vectors encoding different transgenes through detection of the presence of the barcode using universal primers or labeled probe. Alternatively, in a second exemplary insertion strategy, the unique barcode sequences were inserted into CAR-encoding regions. For example, the unique barcode sequence was located at the junction of the 4- IBB signaling domain and the CD3zeta signaling domain of in both a CD 19 CAR and a CD22 CAR (FIG. 2).

Example 2: Assessing vector detection with probes and droplet digital PCR (ddPCR) [0842] Digital droplet PCR (ddPCR) was used to assess the specificity of the unique barcodes for a CD 19 CAR and a CD22 CAR. As shown in FIG. 3A, a 96- well plate was set up in which columns 1-6 contained the CD22 CAR plasmid and columns 7-12 contained the CD 19 CAR plasmid. The amount of plasmid (pg) was serially diluted from the top of the plate to the bottom of the plate. The corresponding number of copies of plasmid, calculated according to the amount of DNA, is also shown. Three probes (CD22CARvl, CD22CARv2, and CD19CAR) were used to test the specificity of the detection. For wells containing CD22 CAR, the CD19CAR probe served as a negative control while the two CD22CAR probes (vl and v2) were expected to generate a strong signal. For wells containing the CD 19 CAR, the two CD22CAR probes (vl and v2) served as negative controls while the CD19CAR probe was expected to generate a strong signal.

[0843] As expected, both CD22CARvl and CD22CARv2 probes detected high fluorescence intensity in all wells containing CD22 CAR (FIG. 3B and FIG. 3C). The CD19CAR probe primarily was unable to detect any signal, as evidenced by the negative droplets below the threshold in FIG. 3D. The calculated number of copies of CD22 CAR aligned with the actual number of copies per reaction for both CD22CAR probes, compared with no amplification using the CD19CAR probe as expected (FIG. 3E). When the recovery efficiency for amplified plasmid was determined, at least 70% of the amplified plasmid for CD22CARvl and v2 was recovered (FIG. 3F). Taken together, the data showed the CD22CAR probes specifically and efficiently identified the unique barcode sequence associated with CD22 CAR. [0844] In the wells plated with CD 19 CAR, the CD19CAR probe was able to bind to the CD 19 CAR, as shown by high fluorescence intensity in FIG. 31. As expected, the CD22CARvl and CD22CARv2 probes failed to bind and fluorescence was below the threshold in all wells (FIG. 3G and FIG. 3H). The calculated number of copies of CD 19 CAR aligned with the actual number of copies per reaction when the CD19CAR probe was used (FIG. 3J). When the recovery efficiency for amplified plasmid was determined, at least 70% of the amplified plasmid for the CD19CAR probe was recovered (FIG. 3K). Taken together, the data showed the CD19CAR probe specifically and efficiently identified the unique barcode sequence associated with CD 19 CAR.

[0845] These results demonstrate that the CAR-specific probes used for ddPCR amplification are specific to their CAR plasmids. These data support the use of engineered vectors containing unique barcode sequences in one or more regions of the vector, which further permit for the collecting and assessing of correlative data for engineered and/or therapeutic cells as described in the present disclosure.

Example 3: Multiplex vector detection using probes and droplet digital PCR (ddPCR) [0846] In order to determine whether the probes are specific in a multiplex assay, a 96- well plate was set up in which a third of the plate contained CD22 CAR, another third contained CD 19 CAR, and the last third contained a mixture of CD22 CAR and CD 19 CAR (FIG. 4A). The amount of plasmid (pg) was serially diluted from the top of the plate to the bottom of the plate. The corresponding number of copies of plasmid, calculated according to the amount of DNA, is also shown. Three probes (CD22CARvl, CD22CARv2, and CD19CAR) were used to test the specificity of the detection. The CD22CAR probes were HEX probes and the CD19CAR probe was a FAM probe.

[0847] In wells containing only CD 19 CAR, binding of the CD19CAR probe led to high fluorescence signal intensity while signal from binding of the CD22CARvl probe was undetectable (FIG. 4B and FIG. 4C). Conversely, in wells containing only CD22 CAR, binding of the CD22CARvl probe led to high fluorescence signal intensity while signal from binding of the CD19CAR probe was undetectable (FIG. 4D and FIG. 4E). In the wells containing a mixture of CD22 CAR and CD 19 CAR, there was high FAM (FIG. 4F) and high HEX signal intensity (FIG. 4G). The calculated number of copies of CD 19 CAR aligned with the actual number of copies per reaction in the singleplex and multiplex detection of CD 19 CAR using the CD19CAR probe, while there was no amplification in the CD22 CAR-only wells, as was expected (FIG. 4H). The calculated number of copies of CD22 CAR aligned with the actual number of copies per reaction in the singleplex and multiplex detection of CD22 CAR using the CD22CARvl probe, while there was no amplification in the CD 19 CAR-only wells, as was expected (FIG. 41). When the second CD22CAR probe was tested in a similar fashion, positive droplets were detected using the CD19CAR probe in wells containing only CD 19 CAR (FIG. 4J), but the droplets were at the threshold when the CD22CARv2 probe was used on only CD 19 CAR, suggesting a lack of signal (FIG. 4K). Positive droplets were detected using the CD22CARv2 probe in wells containing only CD22 CAR (FIG. 4M). No significant signal was detected when the CD19CAR probe was used on only CD22 CAR (FIG. 4L). In the wells containing a mixture of CD22 CAR and CD 19 CAR, there was high FAM (FIG. 4N) and high HEX (FIG. 40) signal intensity. The calculated number of copies of CD19 CAR aligned with the actual number of copies per reaction in the singleplex and multiplex detection of CD 19 CAR using the CD19CAR probe, while there was no amplification in the CD22 CAR-only wells, as was expected (FIG. 4P). The calculated number of copies of CD22 CAR agreed aligned with the actual number of copies per reaction in the singleplex and multiplex detection of CD22 CAR using the CD22CARv2 probe, while there was no amplification in the CD 19 CAR-only wells, as expected (FIG. 4Q). Taken together, the data show the CD19CAR probe and CD22CARvl/v2 probes specifically recognized the unique barcode sequences associated with CD 19 CAR and CD22 CAR in a multiplex ddPCR assay.

[0848] These results demonstrated that the CD 19 CAR and CD22CAR probes can be used together in a multiplex assay format for rapid and reliable target detection and sample analyses. While both probes target the same region within the CAR construct, i.e., the junction between 4- 1BB and CD3zeta domains, the DNA sequences recognized by the CD 19 CAR and CD22CAR probes did not inhibit the assay efficiency. These data support the use of engineered vectors containing unique barcode sequences in one or more regions of the vector for multiplex target detection and sample analyses.

[0849] The present disclosure is not intended to be limited in scope to the particular disclosed embodiments, which are provided, for example, to illustrate various aspects of the present disclosure. Various modifications to the compositions and methods described will become apparent from the description and teachings herein. Such variations may be practiced without departing from the true scope and spirit of the disclosure and are intended to fall within the scope of the present disclosure.

Claims

CLAIMS What is claimed:

1. A method of selecting a cell, population of cells, or therapy comprising, detecting the presence or absence of a first barcode and/or a second barcode in the cell or population of cells, wherein the presence of the first barcode indicates the presence of a first transgene and the presence of the second barcode indicates the presence of a second transgene, and selecting the cell or population of cells as being suitable for a) administration to a subject; b) manufacturing a drug product; c) gene editing or further gene editing; d) viral transduction or further viral transduction; e) creating a cell bank; f) differentiation into a cellular intermediate or a fully differentiated drug product; g) packaging for distribution; and/or h) cryopreservation or formulation, based on the presence or absence of the first barcode and/or second barcode.

2. A method of manufacturing a therapy, the method comprising, detecting the presence or absence of a first barcode and/or a second barcode in a cell or population of cells wherein the presence of the first barcode indicates the presence of a first transgene and the presence of the second barcode indicates the presence of a second transgene, and wherein the therapy is determined to be suitable for any one or more uses for a) administration to a subject; b) gene editing or further gene editing; c) viral transduction or further viral transduction d) creating a cell bank; e) differentiation into a cellular intermediate or a fully differentiated drug product; f) packaging for distribution; and/or g) cryopreservation or formulation, based on the presence or absence of the first barcode and/or second barcode.

3. The method of claim 1 or claim 2, wherein the therapy is a cell therapy.

4. The method of claim 3, wherein the cell therapy is generated in vivo or ex vivo.

5. The method of claim 4, wherein the cell therapy is generated in vivo in a subject in need thereof to treat a disease in the subject, wherein a vector comprising the first transgene and/or the second transgene and the first barcode and/or the second barcode is administered to the subject.

6. The method of claim 5, wherein the vector is packaged in a fusosome for trafficking to the target cell or target population of cells in vivo within the subject.

7. The method of any one of claims 1-4, further comprising administering to a subject in need thereof an effective dose of a cell, population of cells, or cell therapy to treat a disease in the subject.

8. The method of claim 7, wherein the cell therapy comprises administering one or more doses of engineered immune cells expressing the first transgene and/or the second transgene.

9. The method of any one of claims 3-8, further comprising monitoring a sample obtained from the subject administered the therapy for the presence or absence of the first barcode and/or the second barcode in the cell, population of cells, or therapy, the method comprising:

(i) detecting the first barcode and/or the second barcode in the sample obtained from the subject receiving the cell, population of cells, or therapy, wherein detection of the first barcode indicates the presence of the first transgene, and/or detection of the second barcode indicates the presence of the second transgene in the sample; and

(ii) determining the percentage, ratio, relative, or absolute number of cells expressing the first transgene and/or the second transgene.

10. The method of claim 9, further comprising determining the percentage, ratio, relative, or absolute number of cells expressing the first transgene and/or the second transgene at a second time point.

11. The method of claim 10, wherein determining the percentage, ratio, relative, or absolute number of cells expressing the first transgene and/or the second transgene in a sample at a first time point and a second time point is used to monitor: i) cell therapy persistence; ii) cell therapy efficacy; iii) expansion of the cells expressing the first transgene and/or the second transgene; or iv) changes in a subject’s health or disease profile, in a subject receiving treatment.

12. The method of any one of claims 7-11, further comprising one or more additional administrations of the cell, population of cells, or therapy to the subject comprising: i) the same or different dose as the initial dose of the cell, population of cells, or therapy administered to the subject; and/or ii) a cell, population of cells, or therapy comprising the same or different transgene encoded by a vector as the initial cell, population of cells, or therapy administered to the subject.

13. A method of monitoring a therapy administered to a subject, comprising, obtaining a sample from the subject who was administered the therapy, detecting the presence or absence of a first barcode and/or a second barcode in the sample, wherein the presence of the first barcode indicates the presence of a first transgene, and the presence of the second barcode indicates the presence of a second transgene, and determining the percentage, ratio, relative, or absolute number of cells expressing the first transgene and/or the second transgene based on the presence or absence of the first barcode and/or second barcode.

14. The method of claim 13, wherein the method comprises monitoring responsiveness to cell therapy in the subject.

15. The method of claim 14, wherein if the monitoring indicates that the subject is responsive to the cell therapy, then there is no change in treatment.

16. The method of claim 14, wherein if the monitoring indicates that the subject is not responsive to the cell therapy, then one or more actions are taken from the group consisting of rescreening or retesting of the sample, collecting and screening another sample from the subject, administering one or more additional therapeutic doses to the subject, changing the treatment of the subject, administering one or more additional therapeutic agents to the subject, and/or ceasing treatment of the subject.

17. The method of claim 13, wherein the cell therapy is monitored for its efficacy, optionally wherein the cell therapy is monitored for enhanced efficacy compared to the efficacy of a cell therapy administered to an individual wherein the cell therapy is not monitored.

18. The method of claim 17, wherein if the monitoring indicates that the cell therapy is efficacious, then there is no change in treatment.

19. The method of claim 17, wherein if the monitoring indicates that the cell therapy is not efficacious, then one or more actions are taken from the group consisting of rescreening or retesting of the sample, collecting and screening another sample from the subject, administering one or more additional therapeutic doses to the subject, changing the treatment of the subject, administering one or more additional therapeutic agents to the subject, and/or ceasing treatment of the subject.

20. The method of claim 13, wherein the cell therapy is monitored for its safety.

21. The method of claim 20, wherein if the monitoring indicates that the cell therapy is safe for administration to the subject, then there is no change in treatment.

22. The method of claim 20, wherein if the monitoring indicates that the cell therapy is not safe for administration to the subject, then one or more actions are taken selected from the group consisting of rescreening or retesting of the sample, collecting and screening another sample from the subject, changing the treatment of the subject, administering one or more additional therapeutic agents to the subject, and ceasing treatment of the subject.

23. The method of claim 13, wherein the cell therapy is monitored for cell persistence, cell activity, and/or cell expansion.

24. The method of claim 23, wherein if the monitoring confirms cell persistence, cell activity, and/or cell expansion upon administration to the subject, then there is no change in treatment.

25. The method of claim 23, wherein if the monitoring does not confirm cell persistence, cell activity, and/or cell expansion upon administration to the subject, then one or more actions are taken selected from the group consisting of rescreening or retesting of the sample, collecting and screening another sample from the subject, administering one or more additional therapeutic doses to the subject, changing the treatment of the subject, administering one or more additional therapeutic agents to the subject, and ceasing treatment of the subject.

26. The method of claim 13, wherein the cell therapy is monitored for reduced or eliminated expression of the first transgene and/or the second transgene in the cell or population of cells.

27. The method of claim 13, wherein the cell therapy is monitored for increased expression of the first transgene and/or the second transgene in the cell or population of cells.

28. The method of claim 13, wherein the cell therapy is monitored for gene editing efficiency in the cell or population of cells.

29. The method of claim 26, wherein if the monitoring confirms that there is no reduction or elimination of expression of the first transgene and/or the second transgene in the cell or population of cells, then there is no change in treatment.

30. The method of claim 26, wherein if the monitoring confirms reduced or eliminated expression of the first transgene and/or the second transgene in the cell or population of cells, then one or more actions are taken selected from the group consisting of rescreening or retesting of the sample, collecting and screening another sample from the subject, changing the treatment of the subject, administering one or more additional therapeutic agents to the subject, and ceasing treatment of the subject.

31. The method of claim 26, wherein if the monitoring confirms that there is a reduction or elimination of expression of the first transgene and/or the second transgene in the cell or population of cells, then there is no change in treatment.

32. The method of claim 26, wherein if the monitoring confirms that there is no reduced or eliminated expression of the first transgene and/or the second transgene in the cell or population of cells, then one or more actions are taken selected from the group consisting of rescreening or retesting of the sample, collecting and screening another sample from the subject, changing the treatment of the subject, administering one or more additional therapeutic agents to the subject, and ceasing treatment of the subject.

33. The method of claim 27, wherein if the monitoring confirms that there is increased expression of the first transgene and/or the second transgene in the cell or population of cells, then there is no change in treatment.

34. The method of claim 27, wherein if the monitoring confirms no increase in expression of the first transgene and/or the second transgene in the cell or population of cells, then one or more actions are taken selected from the group consisting of rescreening or retesting of the sample, collecting and screening another sample from the subject, changing the treatment of the subject, administering one or more additional therapeutic agents to the subject, and ceasing treatment of the subject.

35. The method of claim 13, wherein the cell therapy is monitored for its therapeutic kinetics.

36. The method of claim 35, wherein if the monitoring confirms acceptable therapeutic kinetics, then there is no change in treatment.

37. The method of claim 35, wherein if the monitoring does not confirm acceptable therapeutic kinetics, then one or more actions are taken selected from the group consisting of rescreening or retesting of the sample, collecting and screening another sample from the subject, administering one or more additional therapeutic doses to the subject, changing the treatment of the subject, administering one or more additional therapeutic agents to the subject, and ceasing treatment of the subject.

38. The method of claim 13, wherein the cell therapy is monitored for controlling tumor growth, tumor expansion, and/or tumor recurrence.

39. The method of claim 13, wherein the cell therapy is monitored for preventing antigen escape of a tumor.

40. The method of claim 38 or claim 39, wherein if the monitoring indicates that the cell therapy controls tumor growth, tumor expansion, and/or tumor recurrence, and/or prevents antigen escape of a tumor, then there is no change in treatment.

41. The method of claim 38 or claim 39, wherein if the monitoring indicates that the cell therapy does not control tumor growth, tumor expansion, and/or tumor recurrence, and/or does not prevent antigen escape of a tumor, then one or more actions are taken selected from the group consisting of rescreening or retesting of the sample, collecting and screening another sample from the subject, changing the treatment of the subject, administering one or more additional therapeutic agents to the subject, and ceasing treatment of the subject.

42. The method of claim 13, wherein the cell therapy is monitored for the presence or absence of a therapeutic viral vector.

43. The method of claim 42, wherein if the monitoring confirms the presence of a therapeutic viral vector, then there is no change in treatment.

44. The method of claim 42, wherein if the monitoring confirms the absence of a therapeutic viral vector, then one or more actions are taken selected from the group consisting of rescreening or retesting of the sample, collecting and screening another sample from the subject, administering one or more additional therapeutic doses to the subject, changing the treatment of the subject, administering one or more additional therapeutic agents to the subject, and ceasing treatment of the subject.

45. The method of claim 42, wherein if the monitoring confirms the absence of a therapeutic viral vector, then there is no change in treatment.

46. The method of claim 42, wherein if the monitoring confirms the presence of a therapeutic viral vector, then one or more actions are taken selected from the group consisting of rescreening or retesting of the sample, collecting and screening another sample from the subject, administering one or more additional therapeutic doses to the subject, changing the treatment of the subject, administering one or more additional therapeutic agents to the subject, and ceasing treatment of the subject.

47. The method of any one of claims 16, 19, 22, 25, 30, 32, 34, 37, 41, and 44, wherein the additional therapeutic agent is selected from the group consisting of a small molecule inhibitor, a chemotherapeutic agent, a cancer immunotherapy, an antibody, a cellular therapy, a nucleic acid, a surgery, a radiotherapy, an anti- angiogenic therapy, an anti-DNA repair therapy, an antiinflammatory therapy, an anti-neoplastic agent, a growth inhibitory agent, a cytotoxic agent, a hormonal therapy, a metabolic agent, a protein, a peptide, a pro-peptide, and any combination thereof.

48. The method of any one of claims 16, 19, 22, 25, 30, 32, 34, 37, 41, and 44, wherein cessation of the therapy comprises triggering a safety switch in the cell, population of cells, or cell therapy administered to the subject.

49. The method of any one of claims 13-48, wherein the cell therapy is monitored for vector copy number.

50. The method of any one of claims 13-49, wherein the cell therapy is monitored for the number of cells with a first transgene, the number of cells with a second transgene, the number of cells with a first transgene and a second transgene, and/or the number of cells without a first transgene or a second transgene.

51. The method of any one of claims 13-50, wherein the cell therapy is monitored for the percentage of cells with a first transgene, the percentage of cells with a second transgene, the percentage of cells with a first transgene and a second transgene, and/or the percentage of cells without a first transgene or a second transgene.

52. The method of any one of claims 13-51, wherein the cell therapy is monitored for the ratio of cells with a first transgene to cells without a transgene.

53. The method of any one of claims 13-51, wherein the cell therapy is monitored for the ratio of cells with a second transgene to cells without a transgene.

54. The method of any one of claims 13-51, wherein the cell therapy is monitored for the ratio of cells with a second transgene to cells with a first transgene.

55. The method of any one of claims 13-51, wherein the cell therapy is monitored for the ratio of cells with a first transgene to cells with a first transgene and a second transgene.

56. The method of any one of claims 13-51, wherein the cell therapy is monitored for the ratio of cells with a first transgene to cells with a first transgene and a second transgene to cells without a first transgene or second transgene.

57. The method of any one of claims 13-56, wherein the cell therapy is monitored for the ratio of cells with a second transgene to cells with a first transgene and a second transgene.

58. The method of any one of claims 13-57, wherein the cell therapy is monitored for the ratio of cells with a second transgene to cells with a first transgene and a second transgene to cells without a first transgene or second transgene.

59. The method of any one of claims 52-57, wherein the cell therapy is monitored for the ratio of cells with a first transgene to cells with a second transgene to cells with a first transgene and a second transgene.

60. The method of any one of claims 54-57, wherein the cell therapy is monitored for the ratio of cells with a first transgene to cells with a second transgene to cells with a first transgene and a second transgene to cells without a first transgene or second transgene.

61. The method of any one of claims 1-60, further selecting a composition of cells or population of cells for cell therapy based upon the percentage of cells that have a first transgene, the percentage of cells that have a second transgene, the percentage of cells that have a first transgene and a second transgene, and/or the percentage of cells that do not have a first transgene or a second transgene.

62. The method of claim 61, wherein the composition is selected when the percentage of cells that have a first transgene is at least 15%.

63. The method of claim 61 or claim 62, wherein the composition is selected when the percentage of cells that have a second transgene is at least 15%.

64. The method of any one of claims 61-63, wherein the composition is selected when the percentage of cells that have a first transgene and a second transgene is at least 15%.

65. The method of any one of claims 1-64, further selecting a composition of cells or population of cells for cell therapy based upon: i. the ratio of cells that have a first transgene to cells that have a second transgene, ii. the ratio of cells that have a first transgene to cells that have a first transgene and a second transgene, iii. the ratio of cells that have a second transgene to cells that have a first transgene and a second transgene, iv. the ratio of cells that have a first transgene to cells that have a first transgene and a second transgene to cells that do not have a first transgene or second transgene, v. the ratio of cells that have a second transgene to cells that have a first transgene and a second transgene to cells that do not have a first transgene or second transgene, and/or vi. the ratio of cells that have a first transgene to cells that have a second transgene to cells that have a first transgene and a second transgene to cells that do not have a first transgene or second transgene.

66. The method of claim 65, wherein the composition is selected when the ratio of cells with the first transgene to cells with no transgene is at least 1:6.

67. The method of claim 65, wherein the composition is selected when the ratio of cells with the second transgene to cells with no transgene is at least 1:6.

68. The method of any one of claims 65-67, wherein the composition is selected when the ratio of cells with the first transgene and the second transgene to cells with no transgene is at least 1:6.

69. The method of any one of claims 65-68, wherein the composition is selected when the ratio of cells with the first transgene to cells with the second transgene is at least 1:1.

70. The method of any one of claims 1-69, wherein the cell, population of cells, or cell therapy is capable of immune evasion.

71. The method of any one of claims 1-69, wherein the cell, population of cells, or cell therapy is capable of evading a host adaptive immune response.

72. The method of any one of claims 1-71, wherein the cell, population of cells, or cell therapy is capable of evading a host innate immune response.

73. The method of any one of claims 1-72, wherein the cell, population of cells, or cell therapy is capable of evading a T cell response, an NK cell response, a macrophage cell response, a microglial cell response, or a combination thereof.

74. The method of any one of claims 1-73, wherein the cell, population of cells, or cell therapy is for use in treating a disease or disorder.

75. The method of claim 74, wherein the disease is a cancer or an autoimmune disease.

76. The method of claim 74 or claim 75, wherein the cancer is selected from the group consisting of ovarian cancer, gastric adenocarcinoma, pancreatic adenocarcinoma, glioblastoma, hepatocellular carcinoma, B-cell chronic lymphocytic leukemia (B-CLL), juvenile chronic myelogenous leukemia (CML), juvenile myelomonocytic leukemia (JMML), Acute Lymphoblastic Leukemia (ALL), Acute Myeloid Leukemia (AML), Adrenocortical Carcinoma, Kaposi Sarcoma (Soft Tissue Sarcoma), AIDS-Related Lymphoma (Lymphoma), Primary CNS Lymphoma (Lymphoma), Anal Cancer, Appendix Cancer, Astrocytomas, Atypical

Teratoid/Rhabdoid Tumor, Basal Cell Carcinoma of the Skin, Bile Duct Cancer, Bladder Cancer, Bone Cancer (includes Ewing Sarcoma and Osteosarcoma and Malignant Fibrous Histiocytoma), Brain Tumors, Breast Cancer, Bronchial Tumors, Burkitt Lymphoma, Carcinoid Tumor, Carcinoma, Cardiac Tumors, Atypical Teratoid/Rhabdoid Tumor, Medulloblastoma, Germ Cell Tumor, Primary CNS Lymphoma, Cervical Cancer, Cholangiocarcinoma, Chordoma, Chronic Lymphocytic Leukemia (CLL), Chronic Myelogenous Leukemia (CML), Chronic Myeloproliferative Neoplasms, Colorectal Cancer, Craniopharyngioma, Cutaneous T-Cell Lymphoma, Ductal Carcinoma In Situ (DCIS), Endometrial Cancer, Ependymoma, Esophageal Cancer, Esthesioneuroblastoma, Ewing Sarcoma, Extracranial Germ Cell Tumor, Extragonadal Germ Cell Tumor, Intraocular Melanoma, Retinoblastoma, Fallopian Tube Cancer, Fibrous Histiocytoma of Bone, Osteosarcoma, Gallbladder Cancer, Gastric Cancer, Gastrointestinal Carcinoid Tumor, Gastrointestinal Stromal Tumors (GIST) (Soft Tissue Sarcoma), Germ Cell Tumors, Central Nervous System Germ Cell Tumors, Extracranial Germ Cell Tumors, Extragonadal Germ Cell Tumors, Ovarian Germ Cell Tumors, Testicular Cancer, Gestational Trophoblastic Disease, Hairy Cell Leukemia, Head and Neck Cancer, Hepatocellular Cancer, Histiocytosis (Langerhans Cell), Hodgkin Lymphoma, Hypopharyngeal Cancer, Islet Cell Tumors, Pancreatic Neuroendocrine Tumors, Kaposi Sarcoma (Soft Tissue Sarcoma), Renal Cell Cancer, Langerhans Cell Histiocytosis, Laryngeal Cancer, Leukemia, Lip and Oral Cavity Cancer, Liver Cancer, Lung Cancer (Non-Small Cell, Small Cell, Pleuropulmonary Blastoma, and Tracheobronchial Tumor), Lung Squamous Cell Carcinoma, Lymphoma, Male Breast Cancer, Malignant Fibrous Histiocytoma of Bone and Osteosarcoma, Merkel Cell Carcinoma, Mesothelioma, Metastatic Cancer, Metastatic Squamous Neck Cancer with Occult Primary, Midline Tract Carcinoma With NUT Gene Changes, Oropharyngeal Cancer, Multiple Endocrine Neoplasia Syndromes, Multiple Myeloma/Plasma Cell Neoplasms, Mycosis Fungoides (Lymphoma), Myelodysplastic Syndromes, Myelodysplastic/Myeloproliferative Neoplasms, Chronic Myeloproliferative Neoplasms, acute B lymphoblastic leukemia (B-ALL), large B cell lymphoma (LBCL), diffuse large B cell lymphoma (DLBCL), high-grade B cell lymphoma (HGBCL), primary mediastinal B cell lymphoma (PMBCL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), or small lymphocytic lymphoma (SLL) Follicular Lymphoma, Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN), Systemic Mastocytosis, Nasal Cavity and Paranasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma, Non-Hodgkin Lymphoma, Pancreatic Cancer, Pancreatic Neuroendocrine Tumors (Islet Cell Tumors), Papillomatosis, Paraganglioma, Paranasal Sinus and Nasal Cavity Cancer, Parathyroid Cancer, Penile Cancer, Pharyngeal Cancer, Pheochromocytoma, Pituitary Tumor, Pleuropulmonary Blastoma, Primary Central Nervous System (CNS) Lymphoma, Primary Peritoneal Cancer, Prostate Cancer, Recurrent Cancer, Rhabdomyosarcoma, Salivary Gland Cancer, Vascular Tumors, Small Intestine Cancer, Soft Tissue Sarcoma, T-Cell Lymphoma, Thymoma and Thymic Carcinoma, Transitional Cell Cancer of the Renal Pelvis and Ureter, Vaginal Cancer, Vulvar Cancer, and Wilms Tumor.

77. The method of any one of claims 74-76, wherein the cancer is selected from the group consisting of Non-Hodgkin’s Lymphoma (NHL), Chronic Lymphocytic Leukemia (CLL), large B cell lymphoma (LBCL), diffuse LBCL (DLBCL), high-grade B cell lymphoma (HGBCL), primary mediastinal B cell lymphoma (PMBCL), mantle cell lymphoma (MCL), follicular lymphoma (FL), marginal zone lymphoma (MZL), and small lymphocytic lymphoma (SLL)

78. The method of any one of claims 74-77, further comprising administration of one or more additional therapeutic agents.

79. The method of claim 78, wherein the one or more additional therapeutic agents are selected from the group consisting of a chemotherapeutic agent, a gene therapy, a metabolite, a metabolic inhibitor, a cytokine, an immunotherapy, a radiotherapy, an oncolytic virus, an anticancer vaccine, a therapeutic antibody, a small molecule inhibitor, an additional cellular therapy, a nucleic acid, a surgery, an anti-angiogenic therapy, an anti-DNA repair therapy, an antiinflammatory therapy, an anti-neoplastic agent, a growth inhibitory agent, a cytotoxic agent, and any combination thereof.

80. The method of claim 74 or claim 75, wherein the autoimmune disease is selected from the group consisting of arthritis, rheumatoid arthritis, acute arthritis, chronic rheumatoid arthritis, gout or gouty arthritis, acute gouty arthritis, acute immunological arthritis, chronic inflammatory arthritis, degenerative arthritis, type II collagen-induced arthritis, infectious arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis, Still's disease, vertebral arthritis, juvenile-onset rheumatoid arthritis, osteoarthritis, arthritis chronica progrediente, arthritis deformans, polyarthritis chronica primaria, reactive arthritis, ankylosing spondylitis, inflammatory hyperproliferative skin diseases, psoriasis, plaque psoriasis, gutatte psoriasis, pustular psoriasis, psoriasis of the nails, atopy, atopic diseases, hay fever, Job's syndrome, dermatitis, contact dermatitis, chronic contact dermatitis, exfoliative dermatitis, exfoliative psoriatic dermatitis, allergic dermatitis, allergic contact dermatitis, dermatitis herpetiformis, nummular dermatitis, seborrheic dermatitis, non-specific dermatitis, primary irritant contact dermatitis, atopic dermatitis, x-linked hyper IgM syndrome, allergic intraocular inflammatory diseases, urticaria, chronic allergic urticaria, chronic idiopathic urticaria, chronic autoimmune urticaria, myositis, polymyositis/dermatomyositis, juvenile dermatomyositis, toxic epidermal necrolysis, scleroderma, systemic scleroderma, sclerosis, systemic sclerosis, multiple sclerosis (MS), MS associated with EBV infection, spino-optical MS, primary progressive MS (PPMS), relapsingremitting MS (RRMS), progressive relapsing MS, secondary progressive MS (SPMS), progressive systemic sclerosis, atherosclerosis, arteriosclerosis, sclerosis disseminata, ataxic sclerosis, neuromyelitis optica spectrum disorder, inflammatory bowel disease (IBD), Crohn's disease, autoimmune-mediated gastrointestinal diseases, colitis, ulcerative colitis, colitis ulcerosa, microscopic colitis, collagenous colitis, colitis polyposa, necrotizing enterocolitis, transmural colitis, autoimmune inflammatory bowel disease, bowel inflammation, pyoderma gangrenosum, erythema nodosum, primary sclerosing cholangitis, respiratory distress syndrome, adult or acute respiratory distress syndrome (ARDS), meningitis, inflammation of all or part of the uvea, iritis, choroiditis, an autoimmune hematological disorder, rheumatoid spondylitis, rheumatoid synovitis, hereditary angioedema, cranial nerve damage, meningitis, herpes gestationis, pemphigoid gestationis, pruritis scroti, autoimmune premature ovarian failure, sudden hearing loss due to an autoimmune condition, IgE-mediated diseases, anaphylaxis, allergic and atopic rhinitis, encephalitis, Rasmussen's encephalitis, limbic and/or brainstem encephalitis, uveitis, anterior uveitis, acute anterior uveitis, granulomatous uveitis, nongranulomatous uveitis, phacoantigenic uveitis, posterior uveitis, autoimmune uveitis, glomerulonephritis (GN) with or without nephrotic syndrome, chronic or acute glomerulonephritis, primary GN, immune-mediated GN, membranous GN (membranous nephropathy), idiopathic membranous GN or idiopathic membranous nephropathy, membrano- or membranous proliferative GN (MPGN), Type I or Type II GN, rapidly progressive GN, proliferative nephritis, autoimmune polyglandular endocrine failure, balanitis including balanitis circumscripta plasmacellularis, balanoposthitis, erythema annulare centrifugum, erythema dyschromicum perstans, erythema multiform, granuloma annulare, lichen nitidus, lichen sclerosus et atrophicus, lichen simplex chronicus, lichen spinulosus, lichen planus, lamellar ichthyosis, epidermolytic hyperkeratosis, premalignant keratosis, pyoderma gangrenosum, allergic conditions and responses, allergic reaction, eczema, allergic or atopic eczema, asteatotic eczema, dyshidrotic eczema, vesicular palmoplantar eczema, asthma, asthma bronchiale, bronchial asthma, auto-immune asthma, conditions involving infiltration of T cells or chronic inflammatory responses, immune reactions against foreign antigens, immune reactions against fetal A-B-0 blood groups during pregnancy, chronic pulmonary inflammatory disease, autoimmune myocarditis, leukocyte adhesion deficiency, lupus, lupus nephritis, lupus cerebritis, pediatric lupus, non-renal lupus, extra-renal lupus, discoid lupus, discoid lupus erythematosus, alopecia lupus, systemic lupus erythematosus (SLE), cutaneous SLE, subacute cutaneous SLE, neonatal lupus syndrome (NLE), lupus erythematosus disseminatus, CNS lupus, anti- neutrophilic cytoplasmic autoantibody (ANCA) associated vasculitis, granulomatous polyangiitis, microscopic polyangiitis, autoimmune blistering skin diseases, anti-NMDA receptor neuropathy, stiff persons disease, anti-NMDA receptor encephalitis, anti-synthetase autoimmune syndromes, rapidly progressive glomerulopathy, Type I diabetes, Type II diabetes, latent autoimmune diabetes in adults, Type 1.5 diabetes, juvenile onset (Type I) diabetes mellitus, pediatric insulin-dependent diabetes mellitus (IDDM), adult onset diabetes mellitus (Type II diabetes), idiopathic diabetes, insipidus, diabetic retinopathy, diabetic nephropathy, diabetic large-artery disorder, immune responses associated with acute or delayed hypersensitivity mediated by cytokines or T-lymphocytes, tuberculosis, sarcoidosis, granulomatosis, lymphomatoid granulomatosis, Wegener's granulomatosis, agranulocytosis, vasculitides, vasculitis, large- vessel vasculitis, polymyalgia rheumatica, giant cell (Takayasu's) arteritis, medium-vessel vasculitis, Kawasaki's disease, polyarteritis nodosa/periarteritis nodosa, microscopic polyarteritis, immunovasculitis, CNS vasculitis, cutaneous vasculitis, hypersensitivity vasculitis, necrotizing vasculitis, systemic necrotizing vasculitis, ANCA- associated vasculitis, Churg-Strauss vasculitis, syndrome (CSS), ANCA-associated small-vessel vasculiti, temporal arteritis, aplastic anemia, autoimmune aplastic anemia, Coombs positive anemia, Diamond Blackfan anemia, hemolytic anemia, immune hemolytic anemia, autoimmune hemolytic anemia (AIHA), pernicious anemia (anemia pemiciosa), Addison's disease, pure red cell anemia, aplasia (PRCA), Factor VIII deficiency, hemophilia A, autoimmune neutropenia, pancytopenia, leukopenia, diseases involving leukocyte diapedesis, CNS inflammatory disorders, Alzheimer's disease, Parkinson's disease, multiple organ injury syndrome, multiple organ injury syndrome secondary to septicemia, trauma, or hemorrhage, antigen-antibody complex-mediated diseases, anti-glomerular basement membrane disease, anti-phospholipid antibody syndrome, anti-phospholipid syndrome, allergic neuritis, Behcet's disease/syndrome, Castleman's syndrome, Goodpasture's syndrome, Reynaud's syndrome, Sjogren's syndrome, Stevens-Johnson syndrome, pemphigoid, pemphigoid bullous, skin pemphigoid, pemphigus, pemphigus vulgaris, pemphigus foliaceus, pemphigus mucus-membrane pemphigoid, pemphigus erythematosus, autoimmune polyendocrinopathies, Reiter's disease or syndrome, thermal injury, preeclampsia, an immune complex disorder, immune complex nephritis, antibody-mediated nephritis, polyneuropathies, chronic neuropathy, IgM polyneuropathies, IgM-mediated neuropathy, thrombocytopenia, thrombotic thrombocytopenic purpura (TTP), post-transfusion purpura (PTP), heparin-induced thrombocytopenia, autoimmune or immune-mediated thrombocytopenia, idiopathic thrombocytopenic purpura (ITP), chronic or acute ITP, acquired thrombocytopenic purpura, scleritis, idiopathic cerato-scleritis, episcleritis, autoimmune disease of the testis or ovary, autoimmune orchitis or oophoritis, primary hypothyroidism, hypoparathyroidism, autoimmune endocrine diseases, thyroiditis, autoimmune thyroiditis, Hashimoto's disease, chronic thyroiditis, Hashimoto's thyroiditis, subacute thyroiditis, autoimmune thyroid disease, idiopathic hypothyroidism, Grave's disease, polyglandular syndromes, autoimmune polyglandular syndromes, polyglandular endocrinopathy syndromes, paraneoplastic syndromes, neurologic paraneoplastic syndromes, Lambert-Eaton myasthenic syndrome, Eaton-Lambert syndrome, stiff-man or stiff-person syndrome, encephalomyelitis, allergic encephalomyelitis, encephalomyelitis allergica, experimental allergic encephalomyelitis (EAE), myasthenia gravis, thymoma-associated myasthenia gravis, cerebellar degeneration, neuromyotonia, opsoclonus, opsoclonus myoclonus syndrome (OMS), sensory neuropathy, multifocal motor neuropathy, Sheehan's syndrome, hepatitis, autoimmune hepatitis, chronic hepatitis, lupoid hepatitis, giant cell hepatitis, chronic active hepatitis, autoimmune chronic active hepatitis, lymphoid interstitial pneumonitis (LIP), bronchiolitis obliterans, Guillain-Barre syndrome, Berger's disease (IgA nephropathy), idiopathic IgA nephropathy, linear IgA dermatosis, acute febrile neutrophilic dermatosis, subcorneal pustular dermatosis, transient acantholytic dermatosis, cirrhosis, primary biliary cirrhosis, pneumonocirrhosis, autoimmune enteropathy syndrome, Celiac or Coeliac disease, celiac sprue (gluten enteropathy), refractory sprue, idiopathic sprue, cryoglobulinemia, amyotrophic lateral sclerosis (ALS; Lou Gehrig's disease), coronary artery disease, autoimmune ear disease, autoimmune inner ear disease (AIED), autoimmune hearing loss, polychondritis, refractory or relapsed or relapsing polychondritis, pulmonary alveolar proteinosis, Cogan's syndrome/nonsyphilitic interstitial keratitis, Bell's palsy, Sweet's disease/syndrome, rosacea autoimmune, zoster-associated pain, amyloidosis, a non-cancerous lymphocytosis, a primary lymphocytosis, monoclonal B cell lymphocytosis, benign monoclonal gammopathy or monoclonal gammopathy of undetermined significance, peripheral neuropathy, paraneoplastic syndrome, channelopathies, epilepsy, migraine, arrhythmia, muscular disorders, deafness, blindness, periodic paralysis, channelopathies of the CNS, autism, inflammatory myopathy, focal or segmental or focal segmental glomerulosclerosis (FSGS), endocrine ophthalmopathy, uveoretinitis, chorioretinitis, autoimmune hepatological disorder, fibromyalgia, multiple endocrine failure, Schmidt's syndrome, adrenalitis, gastric atrophy, presenile dementia, demyelinating diseases, autoimmune demyelinating diseases, chronic inflammatory demyelinating polyneuropathy, Dressier's syndrome, alopecia areata, alopecia totalis, CREST syndrome, calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, telangiectasia, male or female autoimmune infertility, anti-spermatozoan antibodies, mixed connective tissue disease, Chagas' disease, rheumatic fever, recurrent abortion, farmer's lung, erythema multiforme, post-cardiotomy syndrome, post myocardial infarction cardiotomy syndrome, Cushing's syndrome, bird-fancier's lung, allergic granulomatous angiitis, benign lymphocytic angiitis, Alport's syndrome, alveolitis, allergic alveolitis, fibrosing alveolitis, interstitial lung disease, transfusion reaction, leprosy, malaria, parasitic diseases, leishmaniasis, kypanosomiasis, schistosomiasis, ascariasis, aspergillosis, Samter's syndrome, Caplan's syndrome, dengue, endocarditis, endomyocardial fibrosis, diffuse interstitial pulmonary fibrosis, interstitial lung fibrosis, pulmonary fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis, endophthalmitis, erythema elevatum et diutinum, erythroblastosis fetalis, eosinophilic faciitis, Shulman's syndrome, Felty's syndrome, flariasis, cyclitis, chronic cyclitis, heterochronic cyclitis, iridocyclitis (acute or chronic), Fuch's cyclitis, Henoch-Schonlein purpura, human immunodeficiency virus (HIV) infection, SCID, acquired immune deficiency syndrome (AIDS), echovirus infection, sepsis, endotoxemia, pancreatitis, thyroxicosis, parvovirus infection, rubella virus infection, post-vaccination syndromes, congenital rubella infection, Epstein-Barr virus infection, mumps, Evan's syndrome, autoimmune gonadal failure, Sydenham's chorea, poststreptococcal nephritis, thromboangitis ubiterans, thyrotoxicosis, tabes dorsalis, chorioiditis, giant cell polymyalgia, chronic hypersensitivity pneumonitis, keratoconjunctivitis sicca, epidemic keratoconjunctivitis, idiopathic nephritic syndrome, minimal change nephropathy, benign familial and ischemia-reperfusion injury, transplant organ reperfusion, retinal autoimmunity, joint inflammation, bronchitis, chronic obstructive airway /pulmonary disease, silicosis, aphthae, aphthous stomatitis, arteriosclerotic disorders, aspermiogenese, autoimmune hemolysis, Boeck's disease, cryoglobulinemia, Dupuytren's contracture, endophthalmia phacoanaphylactica, enteritis allergica, erythema nodosum leprosum, idiopathic facial paralysis, chronic fatigue syndrome, febris rheumatica, Hamman-Rich's disease, sensoneural hearing loss, haemoglobinuria paroxysmatica, hypogonadism, ileitis regionalis, leucopenia, mononucleosis infectiosa, traverse myelitis, primary idiopathic myxedema, nephrosis, ophthalmia symphatica, orchitis granulomatosa, pancreatitis, polyradiculitis acuta, pyoderma gangrenosum, Quervain's thyreoiditis, acquired splenic atrophy, non-malignant thymoma, vitiligo, toxic-shock syndrome, food poisoning, conditions involving infiltration of T cells, leukocyte-adhesion deficiency, immune responses associated with acute or delayed hypersensitivity mediated by cytokines or T- lymphocytes, diseases involving leukocyte diapedesis, multiple organ injury syndrome, antigenantibody complex-mediated diseases, antiglomerular basement membrane disease, allergic neuritis, autoimmune polyendocrinopathies, oophoritis, primary myxedema, autoimmune atrophic gastritis, sympathetic ophthalmia, rheumatic diseases, mixed connective tissue disease, nephrotic syndrome, insulitis, polyendocrine failure, autoimmune polyglandular syndrome type I, adult-onset idiopathic hypoparathyroidism (AOIH), cardiomyopathy, dilated cardiomyopathy, epidermolisis bullosa acquisita (EBA), hemochromatosis, myocarditis, nephrotic syndrome, primary sclerosing cholangitis, purulent or nonpurulent sinusitis, acute or chronic sinusitis, ethmoid, frontal, maxillary, sphenoid sinusitis, an eosinophil-related disorder, eosinophilia, pulmonary infiltration eosinophilia, eosinophilia-myalgia syndrome, Loffler's syndrome, chronic eosinophilic pneumonia, tropical pulmonary eosinophilia, bronchopneumonic aspergillosis, aspergilloma, granulomas containing eosinophils, anaphylaxis, seronegative spondyloarthritides, polyendocrine autoimmune disease, sclerosing cholangitis, sclera, episclera, chronic mucocutaneous candidiasis, Bruton's syndrome, transient hypogammaglobulinemia of infancy, Wiskott-Aldrich syndrome, ataxia telangiectasia syndrome, angiectasis, autoimmune disorders associated with collagen disease, rheumatism, neurological disease, lymphadenitis, reduction in blood pressure response, vascular dysfunction, tissue injury, cardiovascular ischemia, hyperalgesia, renal ischemia, cerebral ischemia, disease accompanying vascularization, allergic hypersensitivity disorders, glomerulonephritides, reperfusion injury, ischemic re-perfusion disorder, reperfusion injury of myocardial or other tissues, lymphomatous tracheobronchitis, inflammatory dermatoses, dermatoses with acute inflammatory components, multiple organ failure, bullous diseases, renal cortical necrosis, acute purulent meningitis, central nervous system inflammatory disorders, ocular or orbital inflammatory disorders, granulocyte transfusion-associated syndromes, cytokine-induced toxicity, narcolepsy, acute serious inflammation, chronic intractable inflammation, pyelitis, endarterial hyperplasia, peptic ulcer, valvulitis, emphysema, alopecia areata, adipose tissue inflammation/diabetes type II, obesity- associated adipose tissue inflammation/insulin resistance, endometriosis, and pulmonary hemosiderosis.

81. The method of claim 80, wherein the autoimmune disease is selected from the group consisting of systemic lupus erythematosus (SLE), lupus nephritis, CNS lupus, anti-neutrophilic cytoplasmic autoantibody (ANCA) associated vasculitis, granulomatous polyangiitis, microscopic polyangiitis, multiple sclerosis, pemphigus vulgaris, autoimmune blistering skin diseases, membranous nephropathy, anti-NMDA receptor neuropathy, neuromyelitis optica, idiopathic thrombocytopenic purpura, autoimmune hepatitis, type 1 diabetes mellitus, rheumatoid arthritis, juvenile rheumatoid arthritis, chronic inflammatory demyelinating polyneuropathy, polymyositis/dermatomyositis, stiff persons disease, anti-NMDA receptor encephalitis, anti-synthetase autoimmune syndromes, anti-phospholipid antibody syndrome, Sjogren’s syndrome, cryoglobulinemia, focal segmental glomerulosclerosis, rapidly progressive glomerulopathy, and autoimmune hemolytic anemia.

82. The method of claim 80 or claim 81, wherein the autoimmune disease is diabetes.

83. The method of any one of claims 75-77 and 80-82, further comprising administration of one or more additional therapeutic agents.

84. The method of claim 83, wherein the one or more additional therapeutic agents are selected from the group consisting of insulin, amylinomimetic(s), dopamine-2 agonist(s), DPP-4 inhibitor(s), metformin, alpha-glucosidase inhibitor(s), SGLT2 inhibitor(s), statins, GLP-1 receptor agonist(s), incretin, meglitinide(s), sulfonylureas, thiazolidinediones, nonsteroidal antiinflammatory drugs (NSAIDs), antimalarial drugs, corticosteroids, azathioprine, mycophenolate, methotrexate, cyclosporine, voclosporin, leflunomide, belimumab, anifrolumab, abatacept, rituximab, vitamin D supplementation, dehydroepiandrosterone (DHEA), and any combination thereof.

85. The method of any one of claims 1-84, wherein the cell, population of cells, or cell therapy comprising the first barcode and/or the second barcode comprises improved traceability compared to a cell, population of cells, or cell therapy that does not comprise the first barcode and/or the second barcode.

86. The method of any one of claims 1-85, wherein the cell, population of cells, or cell therapy comprising the first barcode and/or the second barcode comprises enhanced safety compared to a cell, population of cells, or cell therapy that does not comprise the first barcode and/or the second barcode.

87. The method of any one of claims 1-86, further comprising selecting the cell, population of cells, or cell therapy based on information obtained from one or more assays.

88. The method of claim 87, wherein the one or more assays are selected from the group consisting of a phenotypic assay, a functional assay, a genotypic assay, a viral assay, a safety assay, an identity assay, a purity assay, and a cell count assay.

89. The method of claim 88, wherein the phenotypic assay is selected from the group consisting of a FACS assay, an ELISA assay, and any combination thereof.

90. The method of claim 88, wherein the functional assay is selected from the group consisting of a hormone secretion assay, a cell response assay, a cell killing assay, a T cell proliferation assay, a T cell activation assay, a T cell killing assay, an NK cell killing assay, a macrophage cell killing assay, a cell function assay, a mixed meal tolerance test, continuous blood glucose level monitoring, monitoring blood glucose levels after a period of fasting, glucose tolerance tests, glucose utilization and oxidation, insulin secretion by a U-PLEX® Meso Scale Discovery (MSD) assay, glucose- stimulated insulin secretion (GSIS) assays, insulin content and proinsulin-to-insulin ratio, flow cytometry to measure the percentages of the different hormone-producing cells, qRT-PCR and immuno staining for cell-specific markers. C- Peptide assays, IFN-y ELISpot assays, microglia killing assays, cell engraftment animal models, cytokine release assays, ELISAs, killing assays using bioluminescence imaging or chromium release assay or Xcelligence analysis, mixed-lymphocyte reactions, immunofluorescence analysis, complement-dependent cytotoxicity (CDC) assay, instant blood-mediated inflammatory reaction (IBMIR), and any combination thereof.

91. The method of claim 88, wherein the genotypic assay is selected from the group consisting of PCR, whole genome sequencing, whole exome sequencing, gene-targeted sequencing, hybrid capture sequencing, epigenetic sequencing, methylation sequencing, qPCR, RT-qPCR, RNA sequencing, microarray analysis, in situ hybridization, serial analysis of gene expression, and any combination thereof.

92. The method of claim 88, wherein the viral assay is selected from the group consisting of a Treponema pallidum antibody (Syphilis) test, a CMV antibody (Anti-CMV IgG and IgM) test, a Hepatitis B Core antibody (Anti-HBc) test, a Hepatitis B Surface Antigen (HBsAg) test, a Hepatitis C Virus antibody (Anti-HCV) test, a Human Immunodeficiency Virus antibody (HIVl/2 plus O) test, a human T-Lympho tropic Virus antibody (HTLV-I/II) test, a Trypanosoma cruzi antibody test, a human Herpes Virus 6 DNA test, a human Herpes Virus 7 DNA test, an Epstein-Barr Virus (EBV) DNA test, a Parvovirus B19 DNA test, a human Herpes Virus 8 DNA test, a Hepatitis A PCR test, a Hepatitis E Virus (HEV) Quantitative RT-PCR test, and any combination thereof.

93. The method of claim 88, wherein the safety assay is selected from the group consisting of mycoplasma testing, sterility testing, endotoxin testing, karyotyping, replication-competent lentivirus testing, vector copy number testing, virus screening, cytokine independent outgrowth testing, balanced translocation testing, and any combination thereof.

94. The method of claim 88, wherein the identity assay is flow cytometry for the polypeptide(s) encoded by a first transgene and/or a second transgene.

95. The method of claim 88, wherein the purity assay is selected from the group consisting of cell viability, mycoplasma testing, sterility testing, endotoxin testing, presence/absence of residual activation beads, presence/absence of residual TCRa/p, presence/absence of a chimeric antigen receptor (CAR), presence/absence of B2M expression, presence/absence of CIITA expression, presence/absence of HLA-A/B/C expression, presence/absence of HLA-DP/DQ/DR expression, presence/absence of tolerogenic factor expression, presence/absence of safety switch, and any combination thereof.

96. The method of any one of claims 1-95, wherein the first transgene and/or the second transgene are encoded by a vector.

97. The method of claim 96, wherein the first transgene and the second are encoded by the same vector.

98. The method of claim 96, wherein the first transgene and the second transgene are encoded by different vectors.

99. The method of any one of claims 96-98, wherein the vector comprising the first transgene and/or the second transgene further comprises an identifying region comprising the first barcode and/or the second barcode.

100. The method of any one of claims 1-99, wherein the detecting the presence or absence of a first barcode and/or a second barcode in the cell or population of cells comprises sequencing and/or probe binding.

101. The method of claim 100, wherein the sequencing comprises one or more of Sanger sequencing, NGS, PCR, qPCR, RT-PCR, or digital droplet PCR (ddPCR).

102. The method of claim 100, further comprising contacting the sample with i) a first probe, ii) a second probe, or iii) a first probe and a second probe, wherein the first probe has a different sequence from the second probe; and detecting binding of i) the first probe to the first barcode in a first vector, ii) the second probe to the second barcode in a second vector, or iii) the first probe to the first barcode sequence in the first vector and the second probe to the second barcode in a second vector.

103. The method of claim 102, wherein the first probe and the second probe are each conjugated to a detection marker selected from the group consisting of a fluorophore, biotin, an enzyme, a radioisotope, and a non-radio active heavy metal isotope.

104. The method of any one of claims 100-103, wherein the first probe is attached to a fluorophore selected from the group consisting of Brilliant Ultra Violet 395, Alexa Fluor 350, Brilliant Ultra Violet 496, Qdot 525 Probe, Brilliant Ultra Violet 563, Qdot 565 Probe, Qdot 605 Probe, Brilliant Ultra Violet 615, Qdot 655 Probe, Brilliant Ultra Violet 661, Qdot 705 Probe, Brilliant Ultra Violet 737, Qdot 800 Probe, Brilliant Ultra Violet 805, Alexa Fluor 405, Brilliant Violet 421, Super Bright 436, eFluor 450, Pacific Blue, Coumarin and Coumarin Derivatives, Brilliant Violet 480, Cyan Fluorescent Protein (CFP), eFluor 506, Pacific Green, Pacific Orange, Super Bright 600, Super Bright 645, Brilliant Violet 650, Super Bright 702, Brilliant Violet 711, Super Bright 780, Brilliant Violet 786, Green Fluorescent Protein (GFP), BODIPY FL, NovaFluor Blue 510, Fluorescein (FITC), Alexa Fluor 488, Oregon Green 488, NovaFluor Blue 530, NovaFluor Blue 555, NovaFluor Blue 585, NovaFluor Blue 610-30S, NovaFluor Blue 610- 70S, NovaFluor Blue 660-40S, NovaFluor Blue 660-120S, PerCP-Cyanine5.5, PerCP-eFluor 710, Alexa Fluor 532, Cy3, NovaFluor Yellow 570, Alexa Fluor 555, Alexa Fluor 561, Alexa Fluor 546, R-phycoerythrin (R-PE), Tetramethylrhodamine (TRITC), Red Fluorescent Protein (RFP), NovaFluor Yellow 590, Alexa Fluor 568, PE-eFluor 610, Texas Red (and Texas Red-X), NovaFluor Yellow 610, Alexa Fluor 594, NovaFluor Yellow 660, NovaFluor Yellow 690, NovaFluor Yellow 700, NovaFluor Yellow 730, NovaFluor Yellow 755, PE-Cyanine7, NovaFluor Red 660, Allophycocyanin (APC), Cy5, eFluor 660, Alexa Fluor 647, NovaFluor Red 685, NovaFluor Blue 690, Alexa Fluor 660, NovaFluor Red 700, Alexa Fluor 680, NovaFluor Red 710, Alexa Fluor 700, NovaFluor Red 725, NovaFluor Red 755, Alexa Fluor 750, APC- eFluor 780, FAM, HEX, Rhodamine Red-X, Tamara, YY, Atto 550, Atto 590, Atto 700, Rox, TruRed, Cy7, Red 613, Cy3.5 581, Cy5.5, DAPI, Hoechst, SYTOX blue, SYTOX green, SYTOX orange, YOYO-1, TOTO-1, TO-PRO-1, chromomycin A3, mithramycin, propidium iodide, ethidium bromide, SYBR Green, any KIRA VIA dyes (e.g., KIR VIA Blue 520), PE- Dazzle 594, PE-Fire 640, PE-Cy5, PE-Fire 700, PE-FIRE 810, PerCP, APC-Cyanine 7, APC- Fire 750, APC-Fire 810, Spark UV 387, Spark Violet 423, Spark Violet 500, Spark Violet 538, Spark Blue 550, Spark Blue 574, Spark YG 570, Spark YG 581, Spark YG 593, Spark NIR 685, Spark Red 718, Brilliant Violet 510, Brilliant Violet 570, Brilliant Violet 605, Brilliant Violet 750, 7-AAD, APC-H7, Apotracker Green, APC-R700, Brilliant Blue 515, Brilliant Blue 700, Calcein-AM, Calcein Red- AM, Calcein Violet- AM, CF 570, CytoPhase Violet, DRAQ5, DRAQ7, Helix NP Blue, Helix NP Green, Helix NP NIR, MitoSpy Green, MitoSpy NIR, MitoSpy Orange, MitoSpy Red, Tag-it Violet, VioBright FITC, Zombie Aqua, Zombie Green, Zombie NIR, Zombie Red, Zombie UV, Zombie Violet, and Zombie Yellow.

105. The method of any one of claims 100-104, wherein the second probe is attached to a fluorophore selected from the group consisting of Brilliant Ultra Violet 395, Alexa Fluor 350, Brilliant Ultra Violet 496, Qdot 525 Probe, Brilliant Ultra Violet 563, Qdot 565 Probe, Qdot 605 Probe, Brilliant Ultra Violet 615, Qdot 655 Probe, Brilliant Ultra Violet 661, Qdot 705 Probe, Brilliant Ultra Violet 737, Qdot 800 Probe, Brilliant Ultra Violet 805, Alexa Fluor 405, Brilliant Violet 421, Super Bright 436, eFluor 450, Pacific Blue, Coumarin and Coumarin Derivatives, Brilliant Violet 480, Cyan Fluorescent Protein (CFP), eFluor 506, Pacific Green, Pacific Orange, Super Bright 600, Super Bright 645, Brilliant Violet 650, Super Bright 702, Brilliant Violet 711, Super Bright 780, Brilliant Violet 786, Green Fluorescent Protein (GFP), BODIPY FL, NovaFluor Blue 510, Fluorescein (FITC), Alexa Fluor 488, Oregon Green 488, NovaFluor Blue 530, NovaFluor Blue 555, NovaFluor Blue 585, NovaFluor Blue 610-30S, NovaFluor Blue 610- 70S, NovaFluor Blue 660-40S, NovaFluor Blue 660-120S, PerCP-Cyanine5.5, PerCP-eFluor 710, Alexa Fluor 532, Cy3, NovaFluor Yellow 570, Alexa Fluor 555, Alexa Fluor 561, Alexa Fluor 546, R-phycoerythrin (R-PE), Tetramethylrhodamine (TRITC), Red Fluorescent Protein (RFP), NovaFluor Yellow 590, Alexa Fluor 568, PE-eFluor 610, Texas Red (and Texas Red-X), NovaFluor Yellow 610, Alexa Fluor 594, NovaFluor Yellow 660, NovaFluor Yellow 690, NovaFluor Yellow 700, NovaFluor Yellow 730, NovaFluor Yellow 755, PE-Cyanine7, NovaFluor Red 660, Allophycocyanin (APC), Cy5, eFluor 660, Alexa Fluor 647, NovaFluor Red 685, NovaFluor Blue 690, Alexa Fluor 660, NovaFluor Red 700, Alexa Fluor 680, NovaFluor Red 710, Alexa Fluor 700, NovaFluor Red 725, NovaFluor Red 755, Alexa Fluor 750, APC- eFluor 780, FAM, HEX, Rhodamine Red-X, YY, Atto 550, Atto 590, Atto 700, Tamara, Rox, TruRed, Cy7, Red 613, Cy3.5 581, Cy5.5, DAPI, Hoechst, SYTOX blue, SYTOX green, SYTOX orange, YOYO-1, TOTO-1, TO-PRO-1, chromomycin A3, mithramycin, propidium iodide, ethidium bromide, SYBR Green, any KIRA VIA dyes (e.g., KIR VIA Blue 520), PE- Dazzle 594, PE-Fire 640, PE-Cy5, PE-Fire 700, PE-FIRE 810, PerCP, APC-Cyanine 7, APC- Fire 750, APC-Fire 810, Spark UV 387, Spark Violet 423, Spark Violet 500, Spark Violet 538, Spark Blue 550, Spark Blue 574, Spark YG 570, Spark YG 581, Spark YG 593, Spark NIR 685, Spark Red 718, Brilliant Violet 510, Brilliant Violet 570, Brilliant Violet 605, Brilliant Violet 750, 7-AAD, APC-H7, Apotracker Green, APC-R700, Brilliant Blue 515, Brilliant Blue 700, Calcein-AM, Calcein Red- AM, Calcein Violet- AM, CF 570, CytoPhase Violet, DRAQ5, DRAQ7, Helix NP Blue, Helix NP Green, Helix NP NIR, MitoSpy Green, MitoSpy NIR, MitoSpy Orange, MitoSpy Red, Tag-it Violet, VioBright FITC, Zombie Aqua, Zombie Green, Zombie NIR, Zombie Red, Zombie UV, Zombie Violet, and Zombie Yellow.

106. The method of any one of claims 1-105, wherein the first barcode sequence and/or the second barcode sequence is located outside of the first and/or second transgene sequence.

107. The method of any one of claims 1-105, wherein the first barcode sequence and/or the second barcode sequence is located within the first and/or the second transgene sequence.

108. The method of any one of claims 1-105, wherein a portion of the first barcode sequence and/or the second barcode sequence is located within the first/and or the second transgene sequence and a portion of the first barcode sequence and/or the second barcode sequence is located outside of the first and/or second transgene sequence.

109. The method of any one of claims 1-105, wherein the first barcode sequence and/or the second barcode sequence comprises a diverged nucleotide sequence within the first transgene and/or the second transgene.

110. The method of claim 109, wherein the diverged nucleotide sequence within the first transgene and/or the second transgene encodes the same amino acid sequence as a non-diverged nucleotide sequence.

111. The method of claim 109 or 110, wherein the diverged nucleotide sequence is located at the junction of one or more transgene domains.

112. The method of any one of claims 1-111, wherein the first barcode sequence and the second barcode sequence are each between about 6 to about 30 nucleotides in length.

113. The method of any one of claims 1-112, wherein the first barcode sequence and the second barcode sequence are about the same nucleotide length.

114. The method of any one of claims 1-113, wherein the barcode sequence comprises a nucleic acid sequence selected from the group consisting of CATCGGAA, GGACAATT, TGTCAACT, TTACAGTT, ATTCAAGG, and TACAGTTA.

115. The method of any one of claims 1-114, wherein the first barcode sequence and/or the second barcode sequence is located within a first identifying region and/or a second identifying region, wherein the first identifying region and/or the second identifying region comprises primer binding sites that flank the first barcode sequence and/or the second barcode sequence.

116. The method of claim 115, wherein each primer binding site comprises: i) a forward primer binding site; and/or ii) a reverse primer binding site.

117. The method of claim 115 or 116, wherein the forward primer binding sites of the first barcode sequence and the second barcode sequence comprise the same sequence.

118. The method of claim 115 or 116, wherein the forward primer binding sites of the first barcode sequence and the second barcode sequence comprise different sequences.

119. The method of any one of claims 115-118, wherein the reverse primer binding sites of the first barcode sequence and the second barcode sequence comprise the same sequence.

120. The method of any one of claims 115-118, wherein the reverse primer binding sites of the first barcode sequence and the second barcode sequence comprise different sequences.

121. The method of any one of claims 1-120, wherein the first and second forward primer binding sites and the first and second reverse primer binding sites are complementary to universal primers.

122. The method of any one of claims 1-121, wherein the first forward primer binding site and the second forward primer binding site is each between about 10 to about 30 nucleotides in length.

123. The method of any one of claims 1-122, wherein the first reverse primer binding site and the second reverse primer binding site is each between about 10 to about 30 nucleotides in length.

124. The method of any one of claims 1-123, wherein the first identifying region is located in a non-coding region or a coding region of the vector comprising the first transgene.

125. The method of claim 1-124, wherein the second identifying region is located in a second non-coding region or a second coding region of the vector comprising the second transgene.

126. The method of any one of claims 97 or 100-125, wherein the first identifying region and the second identifying region are located in the same region of the same vector that comprises the first transgene and the second transgene.

127. The method of any one of claims 1-125, wherein the first identifying region is upstream of a first promoter and/or the second identifying region is upstream of a second promoter.

128. The method of any one of claims 1-125, wherein the first identifying region is downstream of the first promoter and/or the second identifying region is downstream of the second promoter.

129. The method of any one of claims 1-125, wherein the first identifying region is upstream of the first promoter and the second identifying region is downstream of the second promoter.

130. The method of any one of claims 1-125, wherein the first identifying region is downstream of the first promoter and the second identifying region is upstream of the second promoter.

131. The method of any one of claims 1-130, wherein the first identifying region and/or the second identifying region is upstream of one or more additional regulatory elements.

132. The method of any one of claims 1-130, wherein the first identifying region and/or second identifying region is downstream of one or more additional regulatory elements.

133. The method of claim 131 or 132, wherein the one or more additional regulatory elements is selected from the group consisting of: promoter sequences, enhancer sequences, intron sequences, terminator sequences, translation initiation signal sequences, polyadenylation signal sequences, replication element sequences, RNA processing and export element sequences, transposon sequences, transposase sequences, insulator sequences, 5' UTR sequences, 3' UTR sequences, mRNA 3' end processing sequences, boundary element sequences, locus control region (LCR) sequences, matrix attachment region (MAR) sequences, ubiquitous chromatin opening elements, linker sequences, secretion signal sequences, anchoring peptide sequences, localization signal sequences, fusion tag sequences, affinity tag sequences, chaperonin sequences, protease sequences, posttranscriptional regulatory element sequences, and any combination thereof.

134. The method of any one of claims 99-133, wherein the first identifying region is located within the first transgene sequence and/or the second identifying region is located within the second transgene sequence.

135. The method of any one of claims 99-133, wherein the first identifying region is located outside of the first transgene sequence and/or the second identifying region is located outside of the second transgene sequence.

136. The method of any one of claims 99-133, wherein the first identifying region is located within the first transgene sequence and the second identifying region is located outside of the second transgene sequence.

137. The method of any one of claims 99-133, wherein the first identifying region is located outside the first transgene sequence and the second identifying region is located within of the second transgene sequence.

138. The method of claim 127, wherein the first identifying region is located 5' to the first promoter within about 1 to about 200 base pairs and/or wherein the second identifying region is located 5' to the second promoter within about 1 to about 200 base pairs.

139. The method of claim 128, wherein the first identifying region is located 3' to the first transgene within about 1 to about 200 base pairs and/or wherein the second identifying region is located 3' to the second transgene within about 1 to about 200 base pairs.

140. The method of claim 129, wherein the first identifying region is located 5' to the first promoter within about 1 to about 200 base pairs and/or wherein the second identifying region is located 3' to the second promoter within about 1 to about 200 base pairs.

141. The method of claim 130, wherein the first identifying region is located 3' to the first promoter within about 1 to about 200 base pairs and/or wherein the second identifying region is located 5' to the second promoter within about 1 to about 200 base pairs.

142. The method of any one of claims 99-127, wherein a) the first identifying region of the first vector is inserted i) 5' to the first promoter, 5' to the first transgene, and 5' to a Woodchuck Hepatitis

Virus Posttranscriptional Regulatory Element (WPRE), or ii) 3' to the first promoter, within the first transgene, and 5' to a WPRE; and/or b) the second identifying region of the second vector is inserted i) 5' to the second promoter, 5' to the second transgene, and 5' to a Woodchuck

Hepatitis Virus Posttranscriptional Regulatory Element (WPRE), or ii) 3' to the second promoter, within the second transgene, and 5' to a WPRE.

143. The method of any one of claims 99-142, wherein the first identifying region and the second identifying region are each between about 10 to about 100 nucleotides in length, optionally wherein the first identifying region and the second identifying region are each between about 18 to about 30 nucleotides in length.

144. The method of claim 99-143, wherein the first identifying region and the second identifying region are about the same length.

145. The method of any one of claims 99-144, wherein the first identifying region and/or the second identifying region have more than one barcode sequence.

146. The method of any one of claims 99-145, wherein the first identifying region and/or the second identifying region have two or more probe binding sites.

147. The method of any one of claims 99-146, wherein the first identifying region and/or the second identifying region comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs:24-30.

148. The method of any one of claims 99-147, wherein the first identifying region or the second identifying region comprises a first barcode sequence or a second barcode sequence comprising the nucleotide sequence selected from the group consisting of CATCGGAA, GGACAATT, TGTCAACT, TTACAGTT, ATTCAAGG, and TACAGTTA.

149. The method of any of claims 99-148, wherein the first barcode sequence and/or the second barcode sequence are randomly generated.

150. The method of any one of claims 99-149, wherein the first identifying region or the second identifying region comprises a first probe binding site or a second probe binding site comprising the nucleotide sequence selected from the group consisting of SEQ ID NOs: 18-23 and 31.

151. The method of any one of claims 1-150, wherein the first transgene and/or the second transgene encodes an antibody or an antibody fragment, a chimeric antigen receptor (CAR), a chimeric autoantibody receptor (CAAR), a B-cell autoantibody receptor (BAR), a T cell receptor (TCR), an immunomodulatory factor, a safety switch, one or more transcription factors, a genome-editing complex, one or more fusogens, one or more tolerogenic factors, or any combination thereof.

152. The method of any one of claims 1-151, wherein the first transgene and/or the second transgene encodes a protein selected from the group consisting of: enzymes, structural polypeptides, signaling polypeptides, regulatory polypeptides, transport polypeptides, sensory polypeptides, motor polypeptides, defense polypeptides, storage polypeptides, transcription factors, antibodies, cytokines, hormones, catabolic polypeptides, anabolic polypeptides, proteolytic polypeptides, metabolic polypeptides, kinases, transferases, hydrolases, lyases, isomerases, ligases, enzyme modulator polypeptides, protein binding polypeptides, lipid binding polypeptides, membrane fusion polypeptides, cell differentiation polypeptides, epigenetic polypeptides, cell death polypeptides, nuclear transport polypeptides, nucleic acid binding polypeptides, reprogramming polypeptides, DNA editing polypeptides, DNA repair polypeptides, DNA recombination polypeptides, transposase polypeptides, DNA integration polypeptides, targeted endonucleases, recombinases, transposases, DNA polymerases, RNA polymerases, and reverse transcriptase.

153. The method of claim 151 or claim 152, wherein the first transgene and/or the second transgene encodes a therapeutic antibody.

154. The method of claim 153, wherein the therapeutic antibody is an antibody that binds to an antigen selected from the group consisting of CD47, Sirpa, CD52, amyloid beta, angiopoietin-like protein 3 (ANGPTL3), B cell activating factor (BAFF), A proliferationinducing ligand (APRIL), B cell maturation antigen (BCMA), B. anthracis protective antigen (B. anthracis PA), calcitonin gene-related peptide (CGRP), calcitonin gene-related peptide receptor (CGRP-R), C-C chemokine receptor type 4 (CCR4), CD147, CD19, CD3, CD2, CD20, CD22, CD25, epithelial cell adhesion molecule (EpCAM), Glycoprotein 100 (GP100), CD30, CD33, CD38, CD4, CD52, CD6, CD79b, CD80, CD86, Clostridium difficile Toxin B, coagulation factor IX (Factor IX), coagulation factor X (Factor X), complement Cis (Cis), complement C5 (C5), cytotoxic T-lymphocyte antigen 4 (CTLA4), dabigatran, DNA/Histone Hl, Ebola virus Glycoprotein, epidermal growth factor receptor (EGFR), C-Met, epithelial cell adhesion molecule (EpCAM), Factor IX, Factor VIII, fibroblast growth factor 23 (FGF23), ganglioside GM3 (GM3), GD2 ganglioside (GD2), human epidermal growth factor receptor 2 (HER2), immunoglobulin E (IgE), insulin-like growth factor 1 receptor (IGF-1R), integrin alpha lib beta 3 (Integrin allbp3), integrin alpha4 beta7 (Integrin a4p7), integrin subunit alpha 4 (ITGA4), integrin subunit alpha L (ITGAL), interferon alpha receptor 1 (IFNAR-1), interferon gamma (IFN-y), interleukin 1 alpha (IL- la), interleukin 1 beta (IL-1 P), interleukin 12, (IL- 12), interleukin 23 (IL- 23), interleukin 13 (IL-13), interleukin 17 receptor alpha (IL-17 RA), interleukin 17 A (IL- 17 A), interleukin 17F (IL-17F), interleukin 23 pl9 (IL-23pl9), interleukin 31 receptor subunit alpha (IL-3 IRA), interleukin 4 receptor alpha (IL-4RA), interleukin 5 (IL-5), interleukin 5 receptor subunit alpha (IL-5RA), interleukin 6 (IL-6), interleukin 6 receptor (IL- 6R), interleukin 8 (IL-8), Kallikrein, Lipid A region of endotoxin, lymphocyte activation gene 3 (LAG-3), programmed cell death 1 (PD-1), nectin cell adhesion molecule 4 (Nectin 4), neonatal Fc Receptor (FcRn), platelet-derived growth factor receptor alpha (PDGFRA), programmed cell death 1 ligand 1 (PD-L1), proprotein convertase subtilisin/kexin Type 9 (PCSK9), P-selectin, rabies virus glycoprotein (Rabies virus GP), receptor activator of nuclear factor kappaB ligand (RANKL), respiratory syncytial virus F protein (RSV-F), SARS-Cov-2 spike protein (SARS- Cov-2 S protein), sclerostin, SLAM family member 7 (SLAMF7), thrombopoietin receptor (TPOR), thymic stromal lymphopoietin (TSLP), tissue factor (TF), transferrin receptor protein 1 (TFR1), transforming growth factor-beta (TGF-P), tumor necrosis factor-alpha (TNF-a), tumor- associated calcium signal transducer 2 (TACSTD-2), vascular endothelial growth factor (VEGF), vascular endothelial growth factor A (VEGF-A), angiopoietin 2 (ANG-2), vascular endothelial growth factor receptor 2 (VEGFR-2), or von Willebrand Factor (vWF), and any combination thereof.

155. The method of any one of claims 151-154, wherein the first transgene and/or the second transgene comprises a sequence encoding one or more fusogens.

156. The method of claim 155, wherein the one or more fusogens are selected from the group consisting of NiV-F, NiV-G, Gag-Pol, and Rev.

157. The method of any one of claims 151-156, wherein the first transgene and/or the second transgene comprise a sequence encoding a retroviral vector.

158. The method of claim 157, wherein the retroviral vector is a lentiviral vector.

159. The method of any one of claims 151-158, wherein the first transgene and/or the second transgene comprises a sequence encoding an AAV vector.

160. The method of any one of claims 151-159, wherein the first transgene and/or the second transgene comprises a sequence encoding a virus-like particle.

161. The method of any one of claims 151-160, wherein the first transgene and/or the second transgene encodes a CAR.

162. The method of claim 161, wherein the CAR encoded by the first transgene and/or the second transgene comprises a hinge domain, a transmembrane domain, and one or more signaling domains.

163. The method of claim 161 or claim 162, wherein the hinge domain is selected from the group consisting of: CD8a hinge domain, CD28 hinge domain, IgG4 hinge domain, IgG4 hinge- CH2-CH3 domain, and any functional variant thereof.

164. The method of any one of claims 161-163, wherein the transmembrane domain is selected from the group consisting of: alpha, beta, or zeta chain of a T cell receptor, CD28, CD3s, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CD8a, CD8p, 4-1BB/CD137, CD28, CD34, CD4, FcsRIy, CD16, OX40/CD134, CD3 , CD3s, CD3y, CD35, TCRa, TCRp, TCR^, CD32, CD64, CD64, CD45, CD5, CD9, CD22, CD37, CD80, CD86, CD40, CD40L/CD154, VEGFR2, FAS, FGFR2B, and any functional variant thereof.

165. The method of any one of claims 161-164, wherein the signaling domain is selected from the group consisting of: B7-1/CD80, B7-2/CD86, B7-H1/PD-L1, B7-H2, B7-H3, B7-H4, B7-H6, B7-H7, BTLA/CD272, CD28, CTLA-4, Gi24/VISTA/B7-H5, ICOS/CD278, PD-1, PD-L2/B7- DC, PDCD6, 4-1BB/TNFSF9/CD137, 4-1BB Ligand/TNFSF9, BAFF/BLyS/TNFSF13B, BAFF R/TNFRSF13C, CD27/TNFRSF7, CD27 Ligand/TNFSF7, CD30/TNFRSF8, CD30 Ligand/TNFSF8, CD40/TNFRSF5, CD40/TNFSF5, CD40 Ligand/TNFSF5, DR3/TNFRSF25, GITR/TNFRSF18, GITR Ligand/TNFSF18, HVEM/TNFRSF14, LIGHT/TNFSF14,

Lympho toxin- alpha/TNFp, OX40/TNFRSF4, 0X40 Ligand/TNFSF4, RELT/TNFRSF19L, TACI/TNFRSFI 3B, TL1A/TNFSF15, TNFa, TNF RII/TNFRSF 1 B, 2B4/CD244/SLAMF4, BLAME/SLAMF8, CD2, CD2F-10/SLAMF9, CD48/SLAMF2, CD58/LFA-3, CD84/SLAMF5, CD229/SLAMF3, CRACC/SLAMF7, NTB-A/SLAMF6, SLAM/CD150, CD7, CD53, CD82/Kai-1, CD90/Thyl, CD96, CD160, CD200, CD300a/LMIRl, HLA Class I, HLA-DR, Ikaros, Integrin alpha 4/CD49d, Integrin alpha 4 beta 1, Integrin alpha 4 beta 7/LPAM-l, LAG- 3, TCL1A, TCL1B, CRTAM, DAP12, Dectin- 1/CLEC7 A, DPPIV/CD26, EphB6, TIM-l/KIM- 1/HAVCR, TIM-4, TSLP, TSLP R, lymphocyte function associated antigen-1 (LFA-1), NKG2C, CD3^, an immunoreceptor tyrosine-based activation motif (IT AM), CD27, 4- IBB,

166. The method of any one of claims 161-165, wherein the CAR encoded by the first transgene and/or the second transgene comprises a CD8a hinge domain, a CD8a transmembrane domain, a 4- IBB domain, and a CD3-zeta domain.

167. The method of any one of claims 161-166, wherein the CAR encoded by the first transgene and/or the second transgene is selected from the group consisting of: a CD5-specific CAR, a CD19-specific CAR, a CD20-specific CAR, a CD22-specific CAR, a CD23-specific CAR, a CD30-specific CAR, a CD33-specific CAR, CD38-specific CAR, a CD70-specific CAR, a CD 123 -specific CAR, a CD 138- specific CAR, a Kappa, Lambda, B cell maturation agent (BCMA)-specific CAR, a G-protein coupled receptor family C group 5 member D (GPRC5D)- specific CAR, a CD 123 -specific CAR, a LeY-specific CAR, a NKG2D ligand- specific CAR, a WTl-specific CAR, a GD2-specific CAR, a HER2-specific CAR, a EGFR-specific CAR, a EGFRvIII- specific CAR, a B7H3-specific CAR, a PSMA-specific CAR, a PSCA-specific CAR, a CAIX-specific CAR, a CD 171 -specific CAR, a CEA-specific CAR, a CSPG4- specific CAR, a EPH A2- specific CAR, a FAP-specific CAR, a FRa-specific CAR, a IL- 13Ra- specific CAR, a Mesothelin-specific CAR, a MUC1 -specific CAR, a MUC16-specific CAR, a R0R1 -specific CAR, a C-Met-specific CAR, a CD 133- specific CAR, a Ep-C AM- specific CAR, a GPC3- specific CAR, a HPV16-E6- specific CAR, a IL 13 Ra2- specific CAR, a MAGEA3- specific CAR, a MAGEA4- specific CAR, a MARTI -specific CAR, a NY-ESO-l-specific CAR, a VEGFR2- specific CAR, a a-Folate receptor- specific CAR, a CD24-specific CAR, a CD44v7/8- specific CAR, a EGP-2- specific CAR, a EGP-40-specific CAR, a erb-B2-specific CAR, a erb-B 2,3,4- specific CAR, a FBP- specific CAR, a Fetal acethylcholine e receptor- specific CAR, a GD2- specific CAR, a GD3-specific CAR, a HMW-MAA-specific CAR, a IL- HRa- specific CAR, a KDR-specific CAR, a Lewis Y-specific CAR, a Ll-cell adhesion molecule- specific CAR, a MAGE- Al-specific CAR, a Oncofetal antigen (h5T4)-specific CAR, a TAG-72- specific CAR, and a CD19/CD22-bispecific CAR.

168. The method of any one of claims 152-167, wherein the CAR encoded by the first transgene and/or the second transgene is a CD 19 CAR or a CD22 CAR.

169. The method of any one of claims 152-168, wherein the CAR encoded by the first transgene and/or the second transgene further comprises one or more co-stimulatory domain(s).

170. The method of any one of claims 109-151 and 169, wherein the diverged nucleotide sequence within the first transgene and/or within the second transgene is located at the junction of: i) the signaling domain and the co-stimulatory domain; or ii) the hinge domain and the transmembrane domain.

171. The method of any one of claims 109-170, wherein the diverged nucleotide sequence within the first transgene and/or within the second transgene is located at the junction of the 4- 1BB and CD3-zeta domains.

172. The method of any one of claims 151-171, wherein the one or more transgenes comprise a chimeric autoantibody receptor (CAAR).

173. The method of claim 172, wherein the CAAR comprises an antigen selected from the group consisting of a pancreatic P-cell antigen, synovial joint antigen, myelin basic protein, proteolipid protein, myelin oligodendritic glycoprotein, MuSK, keratinocyte adhesion protein desmoglein 3 (Dsg3), Ro-RNP complex, La antigen, myeloperoxidase, proteinase 3, cardiolipin, citrullinated proteins, carbamylated proteins, and a3 chain of basement membrane collagen.

174. The method of any one of claims 151-173, wherein the one or more transgenes comprise a B-cell autoantibody receptor (BAR).

175. The method of claim 174, wherein the BAR comprises an FVIII antigen.

176. The method of any one of claims 151-175, wherein the one or more transgenes comprise a T cell receptor (TCR).

177. The method of any one of claims 151-171, wherein the one or more transgenes comprise a tolerogenic factor.

178. The method of claim 177, wherein the tolerogenic factor is selected from the group consisting of: CD16, CD24, CD35, CD39, CD46, CD47, CD52, CD55, CD59, CD64, CD200, CCL22, CTLA4-Ig, Cl inhibitor, FASL, IDO1, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, IL-10, IL-35, PD-L1, SERPINB9, CCL21, MFGE8, DUX4, B2M-HLA-E, CD27, IL-39, CD16 Fc Receptor, IL15-RF, H2-M3 (HLA-G), A20/TNFAIP3, CR1, HLA-F, and MANF.

179. The method of any one of claims 151-178, wherein the one or more transgenes comprise a safety switch.

180. The method of claim 179, wherein the safety switch is selected from the group consisting of herpes simplex virus thymidine kinase (HSV-tk), cytosine deaminase (CyD), nitroreductase (NTR), purine nucleoside phosphorylase (PNP), horseradish peroxidase, inducible caspase 9 (iCasp9), rapamycin-activated caspase 9 (rapaCasp9), CCR4, CD16, CD19, CD20, CD30, EGFR, GD2, HER1, HER2, MUC1, PSMA, and RQR8.

181. The method of claim 180, wherein the safety switch is a system wherein, upon activation, cells downregulate expression of one or more tolerogenic factors and/or upregulate expression of one or more immune signaling molecules, thereby marking the cell for elimination by the host immune system.

182. The method of claim 181, wherein the one or more tolerogenic factors are selected from the group consisting of: CD16, CD24, CD35, CD39, CD46, CD47, CD52, CD55, CD59, CD64, CD200, CCL22, CTLA4-Ig, Cl inhibitor, FASL, IDO1, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, IL-10, IL-35, PD-L1, SERPINB9, CCL21, MFGE8, DUX4, B2M-HLA-E, CD27, IL- 39, CD16 Fc Receptor, IL15-RF, H2-M3 (HLA-G), A20/TNFAIP3, CR1, HLA-F, and MANF.

183. The method of claim 181 or claim 182, wherein the one or more immune signaling molecules are selected from the group consisting of B2M, HLA-A, HLA-B, HLA-C, HLA-D, HLA-E, RFXANK, CIITA, CTLA-4, PD-1, RAET1E/ULBP4, RAET1G/ULBP5, RAET1H/ULBP2, RAET1/ULBP1, RAET1L/ULBP6, RAET1N/ULBP3, and ligands of NKG2D.

184. The method of any one of claims 151-183, wherein the one or more transgenes encode a genome editing complex.

185. The method of claim 184, wherein the genome editing complex comprises a genome targeting entity and a genome modifying entity.

186. The method of claim 185, wherein the genome targeting entity is a nucleic acid-guided targeting entity.

187. The method of claim 185 or claim 186, wherein the genome targeting entity is selected from the group consisting of: a sequence specific nuclease, a nucleic acid programmable DNA binding protein, an RNA guided nuclease, RNA-guided nuclease comprising a Cas nuclease and a guide RNA (CRISPR-Cas combination), a ribonucleoprotein (RNP) complex comprising a gRNA and a Cas nuclease, a homing endonuclease, a zinc finger nuclease (ZF) nucleic acid binding entity, a transcription activator-like effector (TALE) nucleic acid binding entity, a meganuclease, a Cas nuclease, a core Cas protein, a homing endonuclease, an endonuclease- deficient-Cas protein, an enzymatically inactive Cas protein, a CRISPR-associated transposase (CAST), a Type II or Type V Cas protein, or a functional portion thereof.

188. The method of any one of claims 185-187, wherein the genome targeting entity is selected from the group consisting of Casl, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8a, Cas8b, Cas8c, Cas9, CaslO, Casl2, Casl2a (Cpfl), Casl2b (C2cl), Casl2c (C2c3), Casl2d (CasY), Casl2e (CasX), Casl2f (C2cl0), Casl2g, Casl2h, Casl2i, Casl2k (C2c5), Casl3, Casl3a (C2c2), Casl3b, Casl3c, Casl3d, C2c4, C2c8, C2c9, Cmrl, Cmr2, Cmr3, Cmr4, Cmr5, Cmr6, Csdl, Csd2, Cas5d, Csel, Cse2, Cse3, Cse4, Cas5e, Csfl, Csml, Csm2, Csm3, Csm4, Csm5, Csnl, Csn2, Cstl, Cst2, Cas5t, Cshl, Csh2, Cas5h, Csal, Csa2, Csa3, Csa4, Csa5, Cas5a, CsxlO, Csxl l, Csyl, Csy2, Csy3, Csy4, Mad7, SpCas9, eSpCas9, SpCas9-HFl, HypaSpCas9, HeFSpCas9, and evoSpCas9 high-fidelity variants of SpCas9, SaCas9, NmeCas9, CjCas9, StCas9, TdCas9, LbCasl2a, AsCasl2a, AacCasl2b, BhCasl2b v4, TnpB, dCas (D10A), dCas (H840A), dCasl3a, dCasl3b, or a functional portion thereof.

189. The method of any one of claims 185-188, wherein the genome modifying entity cleaves, deaminates, nicks, polymerizes, interrogates, integrates, cuts, unwinds, breaks, alters, methylates, demethylates, or otherwise destabilizes the target locus.

190. The method of any one of claims 185-189, wherein the genome modifying entity comprises a recombinase, integrase, transposase, endonuclease, exonuclease, nickase, helicase, DNA polymerase, RNA polymerase, reverse transcriptase, deaminase, flippase, methylase, demethylase, acetylase, a nucleic acid modifying protein, an RNA modifying protein, a DNA modifying protein, an Argonaute protein, an epigenetic modifying protein, a histone modifying protein, or a functional portion thereof.

191. The method of any one of claims 185-190, wherein the genome modifying entity is selected from the group consisting of: a sequence specific nuclease, a nucleic acid programmable DNA binding protein, an RNA guided nuclease, RNA-guided nuclease comprising a Cas nuclease and a guide RNA (CRISPR-Cas combination), a ribonucleoprotein (RNP) complex comprising the gRNA and the Cas nuclease, a homing endonuclease, a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a meganuclease, a Cas nuclease, a core Cas protein, a TnpB nuclease, an endonuclease-deficient-Cas protein, an enzymatically inactive Cas protein, a CRIS PR-associated transposase (CAST), a Type II or Type V Cas protein, base editing, prime editing, a Programmable Addition via Site-specific Targeting Elements (PASTE), or a functional portion thereof.

192. The method of any one of claims 185-191, wherein the genome modifying entity is selected from the group consisting of Casl, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8a, Cas8b, Cas8c, Cas9, CaslO, Casl2, Casl2a (Cpfl), Casl2b (C2cl), Casl2c (C2c3), Casl2d (CasY), Casl2e (CasX), Casl2f (C2cl0), Casl2g, Casl2h, Casl2i, Casl2k (C2c5), Casl3, Casl3a (C2c2), Casl3b, Casl3c, Casl3d, C2c4, C2c8, C2c9, Cmrl, Cmr2, Cmr3, Cmr4, Cmr5, Cmr6, Csdl, Csd2, Cas5d, Csel, Cse2, Cse3, Cse4, Cas5e, Csfl, Csml, Csm2, Csm3, Csm4, Csm5, Csnl, Csn2, Cstl, Cst2, Cas5t, Cshl, Csh2, Cas5h, Csal, Csa2, Csa3, Csa4, Csa5, Cas5a, CsxlO, Csxl l, Csyl, Csy2, Csy3, Csy4, Mad7, SpCas9, eSpCas9, SpCas9-HFl, HypaSpCas9, HeFSpCas9, and evoSpCas9 high-fidelity variants of SpCas9, SaCas9, NmeCas9, CjCas9, StCas9, TdCas9, LbCasl2a, AsCasl2a, AacCasl2b, BhCasl2b v4, TnpB, FokI, dCas (D10A), dCas (H840A), dCasl3a, dCasl3b, a base editor, a prime editor, a target-primed reverse transcription (TPRT) editor, APOBEC1, cytidine deaminase, adenosine deaminase, uracil glycosylase inhibitor (UGI), adenine base editors (ABE), cytosine base editors (CBE), reverse transcriptase, serine integrase, recombinase, transposase, polymerase, adenine-to-thymine or “ATBE” (or thymine-to-adenine or “TABE”) transversion base editor, ten-eleven translocation methylcytosine dioxygenases (TETs), TET1, TET3, TET1CD, histone acetyltransferase p300, histone methyltransferase SMYD3, histone methyltransferase PRDM9, H3K79 methyltransferase DOT IL, transcriptional repressor, or a functional portion thereof.

193. The method of any one of claims 185-192, wherein the genome targeting entity and the genome modifying entity are different domains of a single polypeptide.

194. The method of any one of claims 185-193, wherein the genome targeting entity and genome modifying entity are two different polypeptides that are operably linked together.

195. The method of any one of claims 185-192, wherein the genome targeting entity and genome modifying entity are two different polypeptides that are not linked together.

196. The method of any one of claims 185-195, wherein the genome editing complex comprises a guide nucleic acid having a targeting domain that is complementary to at least one target locus, optionally wherein the guide nucleic acid is a guide RNA (gRNA).

197. The method of any one of claims 185-196, wherein the genome editing complex is an RNA-guided nuclease.

198. The method of claim 197, wherein the RNA-guided nuclease comprises a Cas nuclease and a guide RNA (CRISPR-Cas combination).

199. The method of claim 198, wherein the CRISPR-Cas combination is a ribonucleoprotein (RNP) complex comprising the gRNA and the Cas nuclease.

200. The method of claim 198 or claim 199, wherein the Cas nuclease is a Type II or Type V Cas protein.

201. The method of any one of claims 198-200, wherein the Cas nuclease is selected from the group consisting of Cas3, Cas4, Cas5, Cas8a, Cas8b, Cas8c, Cas9, CaslO, Casl2, Casl2a (Cpfl), Casl2b (C2cl), Casl2c (C2c3), Casl2d (CasY), Casl2e (CasX), Casl2f (C2cl0), Casl2g, Casl2h, Casl2i, Casl2k (C2c5), Casl3, Casl3a (C2c2), Casl3b, Casl3c, Casl3d, C2c4, C2c8, C2c9, Cmr5, Csel, Cse2, Csfl, Csm2, Csn2, CsxlO, Csxl l, Csyl, Csy2, Csy3, and Mad7.

202. The method of any one of claims 151-201, wherein the one or more transgenes encode one or more transcription factors.

203. The method of claim 202, wherein the one or more transcription factors are selected from the group consisting of OCT4, SOX2, NANOG, KLF4, LIN28, C-MYC, ECAT1, UTF1, ESRRB, SV40LT, HESRG, CDH1, TDGF1, DPPA4, DNMT3B, ZIC3, p53DD, and L1TD1.

204. The method of any one of claims 151-203, wherein the one or more transgenes encode a recombinant protein.

205. The method of any one of claims 151-204, wherein the one or more transgenes comprise a multi-cistronic construct comprising two or more sequences encoding two or more transgenes.

206. The method of any one of claims 151-205, wherein the one or more transgenes are operably linked to one or more regulatory elements.

207. The method of any one of claims 1-206, wherein the first transgene is operably linked to a first promoter.

208. The method of any one of claims 1-207, wherein the second transgene is operably linked to a second promoter.

209. The method of any one of claims 1-208, wherein the first promoter and the second promoter comprise the same sequence.

210. The method of any one of claims 1-209, wherein the first promoter is selected from the group consisting of: an EFla promoter, an EFla short promoter, a CAG promoter, a ubiquitin/S27a promoter, an SV40 early promoter, an adenovirus major late promoter, a mouse metallothionein-I promoter, an RSV promoter, an MMTV promoter, a Moloney murine leukemia virus Long Terminal repeat region, a CMV promoter, an actin promoter, an immunoglobulin promoter, a heat shock promoter, polyoma virus promoter, a fowlpox virus promoter, a bovine papilloma virus promoter, an avian sarcoma virus promoter, a retrovirus promoter, a hepatitis-B virus promoter, a PGK promoter, an adenovirus late promoter, a vaccinia virus 7.5K promoter, a SV40 promoter, a tk promoter of HSV, a mouse mammary tumor virus (MMTV) promoter, an LTR promoter of HIV, a promoter of moloney virus, an Epstein Barr virus (EBV) promoter, a Rous sarcoma virus (RSV) promoter, a U6 promoter, and an UBC promoter.

211. The method of any one of claims 1-209, wherein the second promoter is selected from the group consisting of: an EFla promoter, an EFla short promoter, a CAG promoter, a ubiquitin/S27a promoter, an SV40 early promoter, an adenovirus major late promoter, a mouse metallothionein-I promoter, an RSV promoter, an MMTV promoter, a Moloney murine leukemia virus Long Terminal repeat region, a CMV promoter, an actin promoter, an immunoglobulin promoter, a heat shock promoter, polyoma virus promoter, a fowlpox virus promoter, a bovine papilloma virus promoter, an avian sarcoma virus promoter, a retrovirus promoter, a hepatitis-B virus promoter, a PGK promoter, an adenovirus late promoter, a vaccinia virus 7.5K promoter, a SV40 promoter, a tk promoter of HSV, a mouse mammary tumor virus (MMTV) promoter, an LTR promoter of HIV, a promoter of moloney virus, an Epstein Barr virus (EBV) promoter, a Rous sarcoma virus (RSV) promoter, a U6 promoter, and an UBC promoter.

212. The method of any one of claims 1-211, wherein the first vector and/or the second vector is a plasmid, a phagemid, a cosmid, a transposon, or a viral vector.

213. The method of claim 212, wherein the viral vector is selected from the group consisting of adenovirus, Chimpanzee adenovirus, lentivirus, alphavirus, retrovirus, poxvirus, flavivirus, adeno-associated virus, rhabdoviridae, herpes simplex virus, chimeric virus, arenavirus, fowlpox virus, vesicular stomatitis virus, vaccinia virus, modified vaccinia virus Ankara, human cytomegalovirus, Sendai virus, measles virus, and Newcastle disease virus.

214. A vector comprising an identifying region and a transgene sequence, wherein the identifying region comprises a barcode that identifies the vector, wherein the identifying region is located upstream of the transgene sequence within about 300 bp, downstream of the transgene sequence within about 300 bp, or within the transgene sequence.

215. The vector of claim 188, wherein the first barcode sequence and/or the second barcode sequence comprises primer binding sites, wherein the forward primer binding site is 5' to the barcode sequence, and wherein the reverse primer binding site is 3' to the barcode sequence.

216. The vector of claim 214 or 215, wherein the forward primer binding sites of the first barcode sequence and of the second barcode sequence comprise the same sequence.

217. The vector of claim 214 or 215, wherein the forward primer binding sites of the first barcode sequence and of the second barcode sequence comprise different sequences.

218. The vector of any one of claims 214-217, wherein the reverse primer binding sites of the first barcode sequence and of the second barcode sequence comprise the same sequence.

219. The vector of any one of claims 214-217, wherein the reverse primer binding sites of the first barcode sequence and of the second barcode sequence comprise different sequences.

220. The vector of any one of claims 214-219, wherein the first and second forward primer binding sites and the first and second reverse primer binding sites are complementary to universal primers.

221. The vector of any one of claims 214-220, wherein the first forward primer binding site and the second forward primer binding site is each between about 10 to about 30 nucleotides in length.

222. The vector of any one of claims 214-220, wherein the first reverse primer binding site and the second reverse primer binding site is each between about 10 to about 30 nucleotides in length.

223. The vector of claim 214 or 215, wherein the first barcode sequence and/or the second barcode sequence is located outside of the first and/or second transgene sequence.

224. The vector of claim 214 or 215, wherein the first barcode sequence and/or the second barcode sequence is located within the first and/or the second transgene sequence.

225. The vector of claim 214 or 215, wherein a portion of the first barcode sequence and/or the second barcode sequence is located within the first and/or the second transgene sequence, and a portion of the first barcode sequence and/or the second barcode sequence is located outside of the first and/or second transgene sequence.

226. The vector of any one of claims 214-225, wherein the first barcode sequence and the second barcode sequence are each between about 6 to about 30 nucleotides in length.

227. The vector of any one of claims 214-226, wherein the first barcode sequence and the second barcode sequence are about the same nucleotide length.

228. The vector of any one of claims 214-227, wherein the vector further comprises a promoter operably linked to the first transgene and/or the second transgene.

229. The vector of any one of claims 214-228, wherein the first identifying region is located in a non-coding region or a coding region of the vector comprising the first transgene.

230. The vector of claim 229, wherein the second identifying region is located in a second non-coding region or a second coding region of the vector comprising the second transgene.

231. The vector of any one of claims 214-230, wherein the first identifying region and the second identifying region are located in the same region of the vector comprising the first transgene and the second transgene.

232. The vector of any one of claims 214-231, wherein the first identifying region is upstream of the first promoter and/or the second identifying region is upstream of the second promoter.

233. The vector of claim 232, wherein the first identifying region is located 5' to the first promoter within about 1 to about 200 base pairs and/or the second identifying region is located 5' to the second promoter within about 1 to about 200 base pairs.

234. The vector of any one of claims 214-231, wherein the first identifying region is downstream of the first promoter and/or the second identifying region is downstream of the second promoter.

235. The vector of any one of claims 214-231, wherein the first identifying region is located 3' to the first transgene within about 1 to about 200 base pairs and/or the second identifying region is located 3' to the second transgene within about 1 to about 200 base pairs.

236. The vector of any one of claims 214-235, wherein the first identifying region is upstream of the first promoter and the second identifying region is downstream of the second promoter.

237. The vector of any one of claims 214-235, wherein the first identifying region is downstream of the first promoter and the second identifying region is upstream of the second promoter.

238. The vector of any one of claims 214-237, wherein the first identifying region and/or the second identifying region is upstream of one or more additional regulatory elements.

239. The vector of any one of claims 214-237, wherein the first identifying region and/or second identifying region is downstream of one or more additional regulatory elements.

240. The vector of claim 238 or 239, wherein the one or more additional regulatory elements is selected from the group consisting of: promoter sequences, enhancer sequences, intron sequences, terminator sequences, translation initiation signal sequences, polyadenylation signal sequences, replication element sequences, RNA processing and export element sequences, transposon sequences, transposase sequences, insulator sequences, 5' UTR sequences, 3' UTR sequences, mRNA 3' end processing sequences, boundary element sequences, locus control region (LCR) sequences, matrix attachment region (MAR) sequences, ubiquitous chromatin opening elements, linker sequences, secretion signal sequences, anchoring peptide sequences, localization signal sequences, fusion tag sequences, affinity tag sequences, chaperonin sequences, protease sequences, posttranscriptional regulatory element sequences, and any combination thereof.

241. The vector of any one of claims 214-231, wherein the first identifying region is located within the first transgene sequence and the second identifying region is located within the second transgene sequence.

242. The vector of any one of claims 214-231, wherein the first identifying region is located outside of the first transgene sequence and the second identifying region is located outside of the second transgene sequence.

243. The vector of any one of claims 214-231, wherein the first identifying region is located within the first transgene sequence and the second identifying region is located outside of the second transgene sequence.

244. The vector of any one of claims 214-231, wherein the first identifying region is located outside of the first transgene sequence and the second identifying region is located within the second transgene sequence.

245. The vector of claim 214-222, wherein the first barcode sequence and/or the second barcode sequence of the first identifying region and/or the second identifying region comprises a diverged nucleotide sequence.

246. The vector of claim 245, wherein the diverged nucleotide sequence within the first transgene and/or the second transgene encodes the same amino acid sequence as a non-diverged nucleotide sequence.

247. The vector of claim 245 or 246, wherein the diverged nucleotide sequence is located at a junction between one or more transgene domains.

248. The vector of any one of claims 214-247, wherein the forward primer binding site and the reverse primer binding site are complementary to universal primers.

249. The vector of any one of claims 214-248, wherein the forward primer binding sites and/or the reverse primer binding sites are each between about 10 to about 30 nucleotides in length.

250. The vector of any one of claims 214-249, wherein the first identifying region and/or the second identifying region is each between about 10 to about 100 nucleotides in length.

251. The vector of any one of claims 214-250, wherein the first identifying region and/or the second identifying region is each between about 18 and about 30 nucleotides in length.

252. The vector of any one of claims 214-251, wherein the first identifying region and/or the second identifying region has more than one barcode sequence.

253. The vector of any one of claims 214-252, wherein the first identifying region and/or the second identifying region has more than one probe binding site.

254. The vector of any one of claims 214-253, wherein the first identifying region and/or the second identifying region comprises a nucleotide sequence set forth in SEQ ID NOs:24-30.

255. The vector of any one of claims 214-254, wherein the first identifying region and/or the second identifying region comprises a first barcode sequence and/or a second barcode sequence each comprising a nucleotide sequence selected from the group consisting of CATCGGAA, GGACAATT, TGTCAACT, TTACAGTT, ATTCAAGG, and TACAGTTA.

256. The vector of any one of claims 214-255, wherein the first identifying region and/or the second identifying region comprises a first probe binding site and/or a second probe binding site comprising the nucleotide sequence set forth in SEQ ID NO: 18-23 and 31.

257. The vector of any one of claims 214-256, wherein the vector comprises a plasmid, a phagemid, a viral vector, a cosmid, or a transposon.

258. The method of claim 257, wherein the viral vector is selected from the group consisting of adenovirus, Chimpanzee adenovirus, lentivirus, alphavirus, retrovirus, poxvirus, flavivirus, adeno-associated virus, rhabdoviridae, herpes simplex virus, chimeric virus, arenavirus, fowlpox virus, vesicular stomatitis virus, vaccinia virus, modified vaccinia virus Ankara, human cytomegalovirus, Sendai virus, measles virus, and Newcastle disease virus.

259. The vector of any one of claims 214-258, wherein the first transgene and/or the second transgene encodes an antibody or an antibody fragment, a chimeric antigen receptor (CAR), a chimeric autoantibody receptor (CAAR), a B-cell autoantibody receptor (BAR), a T cell receptor (TCR), an immunomodulatory factor, a safety switch, one or more transcription factors, a genome-editing complex, one or more fusogens, or one or more tolerogenic factors.

260. The vector of any one of claims 214-259, wherein the first transgene and/or the second transgene encodes a protein selected from the group consisting of: enzymes, structural polypeptides, signaling polypeptides, regulatory polypeptides, transport polypeptides, sensory polypeptides, motor polypeptides, defense polypeptides, storage polypeptides, transcription factors, antibodies, cytokines, hormones, catabolic polypeptides, anabolic polypeptides, proteolytic polypeptides, metabolic polypeptides, kinases, transferases, hydrolases, lyases, isomerases, ligases, enzyme modulator polypeptides, protein binding polypeptides, lipid binding polypeptides, membrane fusion polypeptides, cell differentiation polypeptides, epigenetic polypeptides, cell death polypeptides, nuclear transport polypeptides, nucleic acid binding polypeptides, reprogramming polypeptides, DNA editing polypeptides, DNA repair polypeptides, DNA recombination polypeptides, transposase polypeptides, DNA integration polypeptides, targeted endonucleases, recombinases, transposases, DNA polymerases, RNA polymerases, and reverse transcriptase.

261. The vector of claim 259 or claim 260, wherein the first transgene and/or the second transgene encodes a therapeutic antibody.

262. The vector of claim 261, wherein the therapeutic antibody is an antibody that binds to an antigen selected from the group consisting of CD47, Sirpa, CD52, amyloid beta, angiopoietin- like protein 3 (ANGPTL3), B cell activating factor (BAFF), A proliferation-inducing ligand (APRIL), B cell maturation antigen (BCMA), B. anthracis protective antigen (B. anthracis PA), calcitonin gene-related peptide (CGRP), calcitonin gene-related peptide receptor (CGRP-R), C- C chemokine receptor type 4 (CCR4), CD147, CD19, CD3, CD2, CD20, CD22, CD25, epithelial cell adhesion molecule (EpCAM), Glycoprotein 100 (GP100), CD30, CD33, CD38, CD4, CD52, CD6, CD79b, CD80, CD86, Clostridium difficile Toxin B, coagulation factor IX (Factor IX), coagulation factor X (Factor X), complement Cis (Cis), complement C5 (C5), cytotoxic T- lymphocyte antigen 4 (CTLA4), dabigatran, DNA/Histone Hl, Ebola virus Glycoprotein, epidermal growth factor receptor (EGFR), C-Met, epithelial cell adhesion molecule (EpCAM), Factor IX, Factor VIII, fibroblast growth factor 23 (FGF23), ganglioside GM3 (GM3), GD2 ganglioside (GD2), human epidermal growth factor receptor 2 (HER2), immunoglobulin E (IgE), insulin-like growth factor 1 receptor (IGF-1R), integrin alpha lib beta 3 (Integrin allbp3), integrin alpha4 beta7 (Integrin a4p7), integrin subunit alpha 4 (ITGA4), integrin subunit alpha L (ITGAL), interferon alpha receptor 1 (IFNAR-1), interferon gamma (IFN-y), interleukin 1 alpha (IL-la), interleukin 1 beta (IL-1 P), interleukin 12, (IL-12), interleukin 23 (IL- 23), interleukin 13 (IL-13), interleukin 17 receptor alpha (IL-17 RA), interleukin 17A (IL-17A), interleukin 17F (IL-17F), interleukin 23 pl9 (IL-23pl9), interleukin 31 receptor subunit alpha (IL-31RA), interleukin 4 receptor alpha (IL-4RA), interleukin 5 (IL-5), interleukin 5 receptor subunit alpha (IL-5RA), interleukin 6 (IL-6), interleukin 6 receptor (IL-6R), interleukin 8 (IL-8), Kallikrein, Lipid A region of endotoxin, lymphocyte activation gene 3 (LAG-3), programmed cell death 1 (PD-1), nectin cell adhesion molecule 4 (Nectin 4), neonatal Fc Receptor (FcRn), platelet- derived growth factor receptor alpha (PDGFRA), programmed cell death 1 ligand 1 (PD-L1), proprotein convertase subtilisin/kexin Type 9 (PCSK9), P-selectin, rabies virus glycoprotein (Rabies virus GP), receptor activator of nuclear factor kappaB ligand (RANKL), respiratory syncytial virus F protein (RSV-F), SARS-Cov-2 spike protein (SARS-Cov-2 S protein), sclerostin, SLAM family member 7 (SLAMF7), thrombopoietin receptor (TPOR), thymic stromal lymphopoietin (TSLP), tissue factor (TF), transferrin receptor protein 1 (TFR1), transforming growth factor-beta (TGF-P), tumor necrosis factor-alpha (TNF-a), tumor-associated calcium signal transducer 2 (TACSTD-2), vascular endothelial growth factor (VEGF), vascular endothelial growth factor A (VEGF-A), angiopoietin 2 (ANG-2), vascular endothelial growth factor receptor 2 (VEGFR-2), or von Willebrand Factor (vWF), and any combination thereof.

263. The vector of any one of claims 259-262, wherein the first transgene and/or the second transgene comprises a sequence encoding one or more fusogens.

264. The vector of claim 263, wherein the one or more fusogens are selected from the group consisting of NiV-F, NiV-G, Gag-Pol, and Rev.

265. The vector of any one of claims 259-264, wherein the first transgene and/or the second transgene comprise a sequence encoding a retroviral vector.

266. The vector of 265, wherein the retroviral vector is a lentiviral vector.

267. The vector of any one of claims 259-266, wherein the first transgene and/or the second transgene comprises a sequence encoding an AAV vector.

268. The vector of any one of claims 259-267, wherein the first transgene and/or the second transgene comprises a sequence encoding a virus-like particle.

269. The vector of any one of claims 259-268, wherein the first transgene and/or the second transgene encodes a CAR.

270. The vector of claim 269, wherein the CAR encoded by the first transgene and/or the second transgene comprises a hinge domain, a transmembrane domain, and one or more signaling domains.

271. The vector of claim 269 or claim 270, wherein the hinge domain is selected from the group consisting of: CD8a hinge domain, CD28 hinge domain, IgG4 hinge domain, IgG4 hinge- CH2-CH3 domain, and any functional variant thereof.

272. The vector of any one of claims 269-271, wherein the transmembrane domain is selected from the group consisting of: alpha, beta, or zeta chain of a T cell receptor, CD28, CD3s, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CD8a, CD8p, 4-1BB/CD137, CD28, CD34, CD4, FcsRIy, CD16, OX40/CD134, CD3^, CD3s, CD3y, CD35, TCRa, TCRp, TCR^, CD32, CD64, CD64, CD45, CD5, CD9, CD22, CD37, CD80, CD86, CD40, CD40L/CD154, VEGFR2, FAS, FGFR2B, and any functional variant thereof.

273. The vector of any one of claims 269-272, wherein the signaling domain is selected from the group consisting of: B7-1/CD80, B7-2/CD86, B7-H1/PD-L1, B7-H2, B7-H3, B7-H4, B7-H6, B7-H7, BTLA/CD272, CD28, CTLA-4, Gi24/VISTA/B7-H5, ICOS/CD278, PD-1, PD-L2/B7- DC, PDCD6, 4-1BB/TNFSF9/CD137, 4-1BB Ligand/TNFSF9, BAFF/BLyS/TNFSF13B, BAFF R/TNFRSF13C, CD27/TNFRSF7, CD27 Ligand/TNFSF7, CD30/TNFRSF8, CD30 Ligand/TNFSF8, CD40/TNFRSF5, CD40/TNFSF5, CD40 Ligand/TNFSF5, DR3/TNFRSF25, GITR/TNFRSF18, GITR Ligand/TNFSF18, HVEM/TNFRSF14, LIGHT/TNFSF14,

274. The vector of any one of claims 269-273, wherein the CAR encoded by the first transgene and/or the second transgene comprises a CD8a hinge domain, a CD8a transmembrane domain, a 4- IBB domain, and a CD3-zeta domain.

275. The vector of any one of claims 269-274, wherein the CAR encoded by the first transgene and/or the second transgene is selected from the group consisting of: a CD5-specific CAR, a CD19-specific CAR, a CD20-specific CAR, a CD22-specific CAR, a CD23-specific CAR, a CD30-specific CAR, a CD33-specific CAR, CD38-specific CAR, a CD70-specific CAR, a CD123-specific CAR, a CD 138- specific CAR, a Kappa, Lambda, B cell maturation agent (BCMA)-specific CAR, a G-protein coupled receptor family C group 5 member D (GPRC5D)- specific CAR, a CD 123 -specific CAR, a LeY-specific CAR, a NKG2D ligand- specific CAR, a WTl-specific CAR, a GD2-specific CAR, a HER2-specific CAR, a EGFR-specific CAR, a EGFRv III- specific CAR, a B7H3-specific CAR, a PSMA-specific CAR, a PSCA-specific CAR, a CAIX-specific CAR, a CD 171 -specific CAR, a CEA-specific CAR, a CSPG4- specific CAR, a EPH A2- specific CAR, a FAP-specific CAR, a FRa-specific CAR, a IL-13Ra-specific CAR, a Mesothelin-specific CAR, a MUC1 -specific CAR, a MUC16-specific CAR, a ROR1 -specific CAR, a C-Met-specific CAR, a CD 133- specific CAR, a Ep-C AM- specific CAR, a GPC3- specific CAR, a HPV16-E6- specific CAR, a IL 13 Ra2- specific CAR, a MAGEA3- specific CAR, a MAGEA4- specific CAR, a MARTI -specific CAR, a NY-ESO-l-specific CAR, a VEGFR2- specific CAR, a a-Folate receptor- specific CAR, a CD24-specific CAR, a CD44v7/8- specific CAR, a EGP-2- specific CAR, a EGP-40-specific CAR, a erb-B2-specific CAR, a erb-B 2,3,4- specific CAR, a FBP- specific CAR, a Fetal acethylcholine e receptor- specific CAR, a GD2- specific CAR, a GD3-specific CAR, a HMW-MAA-specific CAR, a IL- HRa- specific CAR, a KDR-specific CAR, a Lewis Y-specific CAR, a Ll-cell adhesion molecule- specific CAR, a MAGE- Al-specific CAR, a Oncofetal antigen (h5T4)-specific CAR, a TAG-72- specific CAR, and a CD19/CD22-bispecific CAR.

276. The vector of any one of claims 259-275, wherein the CAR encoded by the first transgene and/or the second transgene is a CD 19 CAR or a CD22 CAR.

277. The vector of any one of claims 259-276, wherein the CAR encoded by the first transgene and/or the second transgene further comprises one or more co-stimulatory domain(s).

278. The vector of claim 277, wherein the diverged nucleotide sequence within the first transgene and/or within the second transgene is located at the junction of: i) the signaling domain and the co-stimulatory domain; or ii) the hinge domain and the transmembrane domain.

279. The vector of claim 277 or claim 278, wherein the diverged nucleotide sequence within the first transgene and/or within the second transgene is located at the junction of the 4- IBB and CD3-zeta domains.

280. The vector of any one of claims 259-279, wherein the one or more transgenes comprise a chimeric autoantibody receptor (CAAR).

281. The vector of claim 280, wherein the CAAR comprises an antigen selected from the group consisting of a pancreatic P-cell antigen, synovial joint antigen, myelin basic protein, proteolipid protein, myelin oligodendritic glycoprotein, MuSK, keratinocyte adhesion protein desmoglein 3 (Dsg3), Ro-RNP complex, La antigen, myeloperoxidase, proteinase 3, cardiolipin, citrullinated proteins, carbamylated proteins, and a3 chain of basement membrane collagen.

282. The vector of any one of claims 259-281, wherein the one or more transgenes comprise a B-cell autoantibody receptor (BAR).

283. The vector of claim 282, wherein the BAR comprises an FVIII antigen.

284. The vector of any one of claims 259-283, wherein the one or more transgenes comprise a T cell receptor (TCR).

285. The vector of any one of claims 259-284, wherein the one or more transgenes comprise a tolerogenic factor.

286. The vector of claim 285, wherein the tolerogenic factor is selected from the group consisting of: CD16, CD24, CD35, CD39, CD46, CD47, CD52, CD55, CD59, CD64, CD200, CCL22, CTLA4-Ig, Cl inhibitor, FASL, IDO1, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, IL-10, IL-35, PD-L1, SERPINB9, CCL21, MFGE8, DUX4, B2M-HLA-E, CD27, IL-39, CD16 Fc Receptor, IL15-RF, H2-M3 (HLA-G), A20/TNFAIP3, CR1, HLA-F, and MANF.

287. The vector of any one of claims 259-286, wherein the one or more transgenes comprise a safety switch.

288. The vector of claim 287, wherein the safety switch is selected from the group consisting of herpes simplex virus thymidine kinase (HSV-tk), cytosine deaminase (CyD), nitroreductase (NTR), purine nucleoside phosphorylase (PNP), horseradish peroxidase, inducible caspase 9 (iCasp9), rapamycin-activated caspase 9 (rapaCasp9), CCR4, CD16, CD19, CD20, CD30, EGFR, GD2, HER1, HER2, MUC1, PSMA, and RQR8.

289. The vector of claim 287, wherein the safety switch is a system wherein, upon activation, cells downregulate expression of one or more tolerogenic factors and/or upregulate expression of one or more immune signaling molecules, thereby marking the cell for elimination by the host immune system.

290. The vector of claim 289, wherein the one or more tolerogenic factors are selected from the group consisting of: CD16, CD24, CD35, CD39, CD46, CD47, CD52, CD55, CD59, CD64, CD200, CCL22, CTLA4-Ig, Cl inhibitor, FASL, IDO1, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, IL-10, IL-35, PD-L1, SERPINB9, CCL21, MFGE8, DUX4, B2M-HLA-E, CD27, IL- 39, CD16 Fc Receptor, IL15-RF, H2-M3 (HLA-G), A20/TNFAIP3, CR1, HLA-F, and MANF.

291. The vector of claim 289 or claim 290, wherein the one or more immune signaling molecules are selected from the group consisting of B2M, HLA-A, HLA-B, HLA-C, HLA-D, HLA-E, RFXANK, CIITA, CTLA-4, PD-1, RAET1E/ULBP4, RAET1G/ULBP5, RAET1H/ULBP2, RAET1/ULBP1, RAET1L/ULBP6, RAET1N/ULBP3, and ligands of NKG2D.

292. The vector of any one of claims 259-291, wherein the one or more transgenes encode a genome editing complex.

293. The vector of claim 292, wherein the genome editing complex comprises a genome targeting entity and a genome modifying entity.

294. The vector of claim 293, wherein the genome targeting entity is a nucleic acid-guided targeting entity.

295. The vector of claim 293 or claim 294, wherein the genome targeting entity is selected from the group consisting of: a sequence specific nuclease, a nucleic acid programmable DNA binding protein, an RNA guided nuclease, RNA-guided nuclease comprising a Cas nuclease and a guide RNA (CRISPR-Cas combination), a ribonucleoprotein (RNP) complex comprising a gRNA and a Cas nuclease, a homing endonuclease, a zinc finger nuclease (ZF) nucleic acid binding entity, a transcription activator-like effector (TALE) nucleic acid binding entity, a meganuclease, a Cas nuclease, a core Cas protein, a homing endonuclease, an endonuclease- deficient-Cas protein, an enzymatically inactive Cas protein, a CRISPR-associated transposase (CAST), a Type II or Type V Cas protein, or a functional portion thereof.

296. The vector of any one of claims 293-295, wherein the genome targeting entity is selected from the group consisting of Casl, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8a, Cas8b, Cas8c, Cas9, CaslO, Casl2, Casl2a (Cpfl), Casl2b (C2cl), Casl2c (C2c3), Casl2d (CasY), Casl2e (CasX), Casl2f (C2cl0), Casl2g, Casl2h, Casl2i, Casl2k (C2c5), Casl3, Casl3a (C2c2), Casl3b, Casl3c, Casl3d, C2c4, C2c8, C2c9, Cmrl, Cmr2, Cmr3, Cmr4, Cmr5, Cmr6, Csdl, Csd2, Cas5d, Csel, Cse2, Cse3, Cse4, Cas5e, Csfl, Csml, Csm2, Csm3, Csm4, Csm5, Csnl, Csn2, Cstl, Cst2, Cas5t, Cshl, Csh2, Cas5h, Csal, Csa2, Csa3, Csa4, Csa5, Cas5a, CsxlO, Csxl l, Csyl, Csy2, Csy3, Csy4, Mad7, SpCas9, eSpCas9, SpCas9-HFl, HypaSpCas9, HeFSpCas9, and evoSpCas9 high-fidelity variants of SpCas9, SaCas9, NmeCas9, CjCas9, StCas9, TdCas9, LbCasl2a, AsCasl2a, AacCasl2b, BhCasl2b v4, TnpB, dCas (D10A), dCas (H840A), dCasl3a, dCasl3b, or a functional portion thereof.

297. The vector of any one of claims 293-296, wherein the genome modifying entity cleaves, deaminates, nicks, polymerizes, interrogates, integrates, cuts, unwinds, breaks, alters, methylates, demethylates, or otherwise destabilizes the target locus.

298. The vector of any one of claims 293-297, wherein the genome modifying entity comprises a recombinase, integrase, transposase, endonuclease, exonuclease, nickase, helicase, DNA polymerase, RNA polymerase, reverse transcriptase, deaminase, flippase, methylase, demethylase, acetylase, a nucleic acid modifying protein, an RNA modifying protein, a DNA modifying protein, an Argonaute protein, an epigenetic modifying protein, a histone modifying protein, or a functional portion thereof.

299. The vector of any one of claims 293-298, wherein the genome modifying entity is selected from the group consisting of: a sequence specific nuclease, a nucleic acid programmable DNA binding protein, an RNA guided nuclease, RNA-guided nuclease comprising a Cas nuclease and a guide RNA (CRISPR-Cas combination), a ribonucleoprotein (RNP) complex comprising the gRNA and the Cas nuclease, a homing endonuclease, a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a meganuclease, a Cas nuclease, a core Cas protein, a TnpB nuclease, an endonuclease-deficient-Cas protein, an enzymatically inactive Cas protein, a CRIS PR-associated transposase (CAST), a Type II or Type V Cas protein, base editing, prime editing, a Programmable Addition via Site-specific Targeting Elements (PASTE), or a functional portion thereof.

300. The vector of any one of claims 293-299, wherein the genome modifying entity is selected from the group consisting of Casl, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8a, Cas8b, Cas8c, Cas9, CaslO, Casl2, Casl2a (Cpfl), Casl2b (C2cl), Casl2c (C2c3), Casl2d (CasY), Casl2e (CasX), Casl2f (C2cl0), Casl2g, Casl2h, Casl2i, Casl2k (C2c5), Casl3, Casl3a (C2c2), Casl3b, Casl3c, Casl3d, C2c4, C2c8, C2c9, Cmrl, Cmr2, Cmr3, Cmr4, Cmr5, Cmr6, Csdl, Csd2, Cas5d, Csel, Cse2, Cse3, Cse4, Cas5e, Csfl, Csml, Csm2, Csm3, Csm4, Csm5, Csnl, Csn2, Cstl, Cst2, Cas5t, Cshl, Csh2, Cas5h, Csal, Csa2, Csa3, Csa4, Csa5, Cas5a, CsxlO, Csxl l, Csyl, Csy2, Csy3, Csy4, Mad7, SpCas9, eSpCas9, SpCas9-HFl, HypaSpCas9, HeFSpCas9, and evoSpCas9 high-fidelity variants of SpCas9, SaCas9, NmeCas9, CjCas9, StCas9, TdCas9, LbCasl2a, AsCasl2a, AacCasl2b, BhCasl2b v4, TnpB, FokI, dCas (D10A), dCas (H840A), dCasl3a, dCasl3b, a base editor, a prime editor, a target-primed reverse transcription (TPRT) editor, APOBEC1, cytidine deaminase, adenosine deaminase, uracil glycosylase inhibitor (UGI), adenine base editors (ABE), cytosine base editors (CBE), reverse transcriptase, serine integrase, recombinase, transposase, polymerase, adenine-to-thymine or “ATBE” (or thymine-to-adenine or “TABE”) transversion base editor, ten-eleven translocation methylcytosine dioxygenases (TETs), TET1, TET3, TET1CD, histone acetyltransferase p300, histone methyltransferase SMYD3, histone methyltransferase PRDM9, H3K79 methyltransferase DOT IL, transcriptional repressor, or a functional portion thereof.

301. The vector of any one of claims 293-300, wherein the genome targeting entity and the genome modifying entity are different domains of a single polypeptide.

302. The vector of any one of claims 293-301, wherein the genome targeting entity and genome modifying entity are two different polypeptides that are operably linked together.

303. The vector of any one of claims 293-302, wherein the genome targeting entity and genome modifying entity are two different polypeptides that are not linked together.

304. The vector of any one of claims 292-303, wherein the genome editing complex comprises a guide nucleic acid having a targeting domain that is complementary to at least one target locus, optionally wherein the guide nucleic acid is a guide RNA (gRNA).

305. The vector of any one of claims 292-304, wherein the genome editing complex is an RNA-guided nuclease.

306. The vector of claim 305, wherein the RNA-guided nuclease comprises a Cas nuclease and a guide RNA (CRISPR-Cas combination).

307. The vector of claim 306, wherein the CRISPR-Cas combination is a ribonucleoprotein (RNP) complex comprising the gRNA and the Cas nuclease.

308. The vector of claim 306 or claim 307, wherein the Cas nuclease is a Type II or Type V Cas protein.

309. The vector of any one of claims 306-308, wherein the Cas nuclease is selected from the group consisting of Cas3, Cas4, Cas5, Cas8a, Cas8b, Cas8c, Cas9, CaslO, Casl2, Casl2a (Cpfl), Casl2b (C2cl), Casl2c (C2c3), Casl2d (CasY), Casl2e (CasX), Casl2f (C2cl0), Casl2g, Casl2h, Casl2i, Casl2k (C2c5), Casl3, Casl3a (C2c2), Casl3b, Casl3c, Casl3d, C2c4, C2c8, C2c9, Cmr5, Csel, Cse2, Csfl, Csm2, Csn2, CsxlO, Csxll, Csyl, Csy2, Csy3, and Mad7.

310. The vector of any one of claims 259-309, wherein the one or more transgenes encode one or more transcription factors.

311. The vector of claim 310, wherein the one or more transcription factors are selected from the group consisting of OCT4, SOX2, NANOG, KLF4, LIN28, C-MYC, ECAT1, UTF1, ESRRB, SV40LT, HESRG, CDH1, TDGF1, DPPA4, DNMT3B, ZIC3, p53DD, and L1TD1.

312. The vector of any one of claims 259-311, wherein the one or more transgenes encode a recombinant protein.

313. The vector of any one of claims 259-312, wherein the one or more transgenes comprise a multi-cistronic construct comprising two or more sequences encoding two or more transgenes.

314. The vector of any one of claims 259-313, wherein the one or more transgenes are operably linked to one or more regulatory elements.

315. A cell comprising the vector of any one of claims 214-314.

316. The cell of claim 315, wherein the cell is a mammalian cell.

317. The cell of claim 316, wherein the mammalian cell is a human cell.

318. The cell of any one of claims 315-317, wherein the cell is an immune cell.

319. The cell of claim 317, wherein the cell is selected from the group consisting of: islet cells, beta islet cells, pancreatic islet cells, immune cells, B cells, T cells, natural killer (NK) cells, natural killer T (NKT) cells, macrophage cells, endothelial cells, muscle cells, cardiac muscle cells, smooth muscle cells, skeletal muscle cells, dopaminergic neurons, retinal pigmented epithelium cells, optic cells, hepatocytes, thyroid cells, skin cells, glial progenitor cells, neural cells, cardiac cells, stem cells, hematopoietic stem cells, induced pluripotent stem cells (iPSCs), mesenchymal stem cells (MSCs), embryonic stem cells (ESCs), pluripotent stem cell (PSCs), blood cells, endothelial stem cells, epithelial stem cells, adipose stem or progenitor cells, germline stem cells, lung stem or progenitor cells, mammary stem cells, olfactory adult stem cells, hair follicle stem cells, multipotent stem cells, amniotic stem cells, cord blood stem cells, neural stem or progenitor cells, HKB-11, CAP, BOSC 23 cells, WI-38 cells, MRC5 cells, A549 cells, HT1080 cells, HEK293 cells, HEK293T cells, HepG2 cells, Saos-2 cells, Huh7 cells, HeLa cells, MSTO-211 cells, PerC6, and MSTO-211 A cells.

320. The cell of any one of claims 315-319, wherein the cell is a primary human cell.

321. The cell of claim 320, wherein the primary human cell is a pluripotent stem cell.

322. The cell of claim 321, wherein the pluripotent stem cell is derived or differentiated into islet cells.

323. The cell of claim 321 or claim 322, wherein the pluripotent stem cell or the derived stem cells are engineered to be hypoimmunogenic islet cells, wherein the cells are engineered to comprise the vector of any one of claims 214-314.

324. The cell of claim 323, wherein the engineered hypoimmunogenic islet cells comprise engineered beta islet cells.

325. The cell of claim 324, wherein the engineered hypoimmunogenic islet cells further comprise additional engineered islet cells, wherein the additional engineered islet cells comprise alpha cells and/or delta cells.

326. The cell of claim 325, wherein the additional engineered islet cells comprise cells that express the same vector as the engineered beta islet cells.

327. The cell of any one of claims 323-326, wherein the engineered hypoimmunogenic islets is an islet cluster.

328. The cell of claim 321, wherein the pluripotent stem cell is derived or differentiated into an immune cell.

329. The cell of claim 321 or claim 328, wherein the pluripotent stem cell or the derived stem cells are engineered to be hypoimmunogenic immune cells, wherein the cells are engineered to comprise the vector of any one of claims 214-314.

330. The cell of claim 329, wherein the engineered hypoimmunogenic immune cells comprise engineered hypoimmunogenic T cells.

331. The cell of claim 330, wherein the engineered hypoimmunogenic T cells further comprise additional engineered hypoimmunogenic immune cells, wherein the additional engineered hypoimmunogenic immune cells comprise B cells and/or NK cells.

332. The cell of claim 331, wherein the additional engineered hypoimmunogenic immune cells comprise cells that express the same vector as the engineered hypoimmunogenic T cells.

333. The cell of any one of claims 329-332, wherein the engineered hypoimmunogenic T cell is a conventional T cell, a regulatory T cell, a CD4+ T cell, a CD8+ T cell, an a/p T cell, a y/8 T cell, a tissue resident T cell, a tumor infiltrating T cell, a naive T cell, a memory T cell, an effector T cell, a cytolytic T cell, a helper T cell, a primed T cell, an activated T cell, a proliferating T cell, or a combination of one or more of the above T cells.

334. The cell of any of claims 323-327, wherein the engineered hypoimmunogenic islets comprise the vector of any one of claims 214-314, wherein the vector generates germline modifications that:

(a) inactivate or disrupt one or more alleles of: (i) one or more major histocompatibility complex (MHC) class I molecules or one or more molecules that regulate expression of the one or more MHC class I molecules, and/or (ii) one or more MHC class II molecules or one or more molecules that regulate expression of the one or more MHC class II molecules; and/or

(b) increase expression of one or more tolerogenic factors, wherein the increased expression is relative to a control or wild- type islet that does not comprise the modifications.

335. The cell of claim 334, wherein the one or more molecules that regulate expression of the one or more MHC class I molecules is B2M.

336. The cell of claim 334 or claim 335, wherein the modifications comprise a modification that regulates the expression of the one or more MHC class I molecules, and the modification inactivates or disrupts one or more alleles of B2M.

337. The cell of any of claims 334-336, wherein the modification that inactivates or disrupts one or more alleles of B2M reduces mRNA expression of the B2M gene.

338. The cell of any of claims 334-337, wherein the modification that inactivates or disrupts one or more alleles of B2M reduces protein expression of B2M.

339. The cell of any of claims 334-338, wherein the modification that inactivates or disrupts one or more alleles of B2M comprises: inactivation or disruption of one allele of the B2M gene; inactivation or disruption of both alleles of the B2M gene; or inactivation or disruption of all B2M coding alleles in the cell.

340. The cell of any of claims 334-339, wherein the inactivation or disruption comprises an indel in the B2M gene.

341. The cell of any of claims 334-340, wherein the inactivation or disruption comprises a frameshift mutation or a deletion of a contiguous stretch of genomic DNA of the B2M gene.

342. The cell of claim 334 or claim 335, wherein the modification is a modification that regulates expression of the one or more MHC class II molecules, and the modification inactivates or disrupts one or more alleles of CIITA.

343. The cell of claim 342, wherein the modification that inactivates or disrupts one or more alleles of CIITA reduces protein expression of CIITA.

344. The cell of claim 342 or claim 343, wherein the modification that inactivates or disrupts one or more alleles of CIITA comprises: inactivation or disruption of one allele of the CIITA gene; inactivation or disruption of both alleles of the CIITA gene; or inactivation or disruption of all CIITA coding alleles in the cell.

345. The method of any of claims 342-344, wherein the inactivation or disruption comprises an indel in the CIITA gene.

346. The method of any of claims 342-345, wherein the inactivation or disruption is a frameshift mutation or a deletion of a contiguous stretch of genomic DNA of the CIITA gene.

347. The method of any of claims 334-346, wherein expression of HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, and HLA-DR are reduced in the engineered hypoimmunogenic islets.

348. The cell of any one of claims 334-347, wherein the vector comprises one or more transgenes comprising one or more tolerogenic factors selected from the group consisting of CD16, CD24, CD35, CD39, CD46, CD47, CD52, CD55, CD59, CD64, CD200, CCL22, CTLA4-Ig, Cl inhibitor, FASL, IDO1, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, IL- 10, IL-35, PD-L1, SERPINB9, CCL21, MFGE8, DUX4, B2M-HLA-E, CD27, IL-39, CD16 Fc Receptor, IL15-RF, H2-M3 (HLA-G), A20/TNFAIP3, CR1, HLA-F, and MANF.

349. The cell of claim 348, wherein at least one of the one or more tolerogenic factors is CD47.

350. The cell of claim 348 or 349, wherein at least one of the one or more tolerogenic factors is PD-L1.

351. The cell of any of claims 348-350, wherein at least one of the one or more tolerogenic factors is HLA-E.

352. The cell of any of claims 348-351, wherein at least one of the one or more tolerogenic factors is HLA-G.

353. The cell of any of claims 334-352, wherein the engineered hypoimmunogenic islet cells further comprises a modification to increase expression of an exogenous safety switch.

354. The cell of claim 353, wherein the engineered hypoimmunogenic islet cells have the genotype

355. The cell of claim 353 or claim 354, wherein the safety switch is a system wherein upon activation, cells downregulate expression of the one or more tolerogenic factors and/or upregulate expression of one or more immune signaling molecules, thereby marking the cell for elimination by the host immune system.

356. The cell of claim 355, wherein the one or more immune signaling molecules is selected from the group consisting of B2M, HLA-A, HLA-B, HLA-C, HLA-D, HLA-E, RFXANK, CIITA, CTLA-4, PD-1, RAET1E/ULBP4, RAET1G/ULBP5, RAET1H/ULBP2, RAET1/ULBP1, RAET1L/ULBP6, RAET1N/ULBP3, and other ligands of NKG2D.

357. The cell of any one of claims 353-356, wherein the safety switch is a suicide gene.

358. The cell of claim 357, wherein the suicide gene is selected from the group consisting of cytosine deaminase (CyD), herpesvirus thymidine kinase (HSV-Tk), an inducible caspase 9 (iCaspase9), and rapamycin-activated caspase 9 (rapaCasp9).

359. The cell of any of claims 334-358, wherein the one or more modifications in (a) reduce cell surface protein expression of the one or more MHC class I molecules, optionally wherein the one or more modifications in (a) reduce cell surface trafficking of the one or more MHC class I molecules.

360. The cell of any of claims 1-359, wherein the one or more modifications in (a) reduce a function of the one or more MHC class I molecules, optionally wherein the function is antigen presentation.

361. The cell of any of claims 1-360, wherein the one or more modifications comprise a modification that regulates cell surface protein expression of the one or more MHC class I molecules and the modification inactivates or disrupts one or more alleles of B2M.

362. A population of cells comprising the vector of any one of claims 214-314 or the cell of any one of claims 315-361.

363. The population of cells of claim 362, wherein the population of cells is selected from the group consisting of immune cells, B cells, T cells, natural killer (NK) cells, natural killer T (NKT) cells, macrophage cells, monocytes, and dendritic cells.

364. The population of cells of claim 362 or claim 363, further comprising a first subpopulation of cells and a second subpopulation of cells.

365. The population of cells of claim 364, wherein the first subpopulation of cells and/or the second subpopulation of cells is a population of engineered T cells comprising a chimeric antigen receptor (CAR).

366. The population of cells of claim 364 or claim 365, wherein: i. the first subpopulation of cells comprises a first vector comprising a first barcode and a first transgene encoding a CD 19 CAR, and/or ii. wherein the second subpopulation of cells comprises a second vector comprising a second barcode and a second transgene encoding a CD22 CAR.

367. The population of cells of any one of claims claim 362-366, further comprising a third subpopulation of cells.

368. The population of cells of claim 367, wherein the third subpopulation of cells comprises: i) a vector comprising a first barcode and a first transgene encoding a CD 19 CAR and/or a second barcode and a second transgene encoding a CD22 CAR; ii) a first vector comprising a first barcode and a first transgene encoding a CD 19 CAR and/or a second vector comprising a second barcode and a second transgene encoding a CD22 CAR; or iii) a vector comprising a barcode and a transgene encoding a CD19/CD22-bispecific CAR.

369. The population of cells of any one of claims 362-368, further comprising a fourth population of cells.

370. The population of cells of claim 369, wherein the fourth subpopulation of cells does not comprise a CAR.

371. The population of cells of any one of claims 362-370, wherein the first subpopulation of cells and the second subpopulation of cells comprise the same cell of claim 319 or the same population of cells of claim 363.

372. The population of the cells of any one of claims 362-371, comprising three or more subpopulations of cells, wherein each subpopulation of cells comprises a vector comprising a unique barcode sequence associated with a sequence encoding a transgene, wherein each unique barcode sequence is used to identify a population of cells comprising the transgene.

373. The population of the cells of any one of claims 362-372, comprising a first vector comprising a first transgene encoding CD 19 chimeric antigen receptor (CAR), and a second vector comprising a second transgene encoding CD22 CAR.

374. A composition comprising the cells of any one of claims 315-361 or the population of cells of any one of claims 362-373.

375. The cell of any one of claims 315-361, the population of cells of any one of claims 362- 373, or the composition of claim 374 for use in the method of any one of claims 1-314.

376. The cell of any one of claims 315-375, wherein the cell or population of cells are engineered in vivo by administering a vector comprising the first transgene and the first barcode and/or the second transgene and the second barcode to a subject.

377. The cell of claim 376, wherein the vector is packaged in a fusosome for trafficking to the target cell or target population of cells in vivo within the subject

378. The method of any one of claims 1-213, wherein the sample is a tissue biopsy sample, a liquid biopsy sample, a control sample, a blood sample, a plasma sample, a cerebrospinal fluid sample, a sputum sample, a stool sample, a urine sample, a saliva sample, a cell sample, a tissue sample, a nucleic acid sample, mRNA, DNA, cell-free DNA, or cell-free RNA.

379. The method of any one of claims 1-213 and 376, wherein the sample comprises a cell or population of cells.

380. The method of claim 379, wherein the cell or population of cells comprises the cell of any one of claims 315-362 or the population of cells of any one of claims 363-373.

381. The method of any one of claims 1-213, the vector of any one of claims 214-314, the cell of any one of claims 315-362 and 375-377, or the population of cells of any one of claims 363- 373, further comprising a third, fourth, fifth, or more transgene with a third, fourth, fifth or more barcode, optionally encoded by the same vector, different vectors, or a combination thereof.

382. The method of any one of claims 151-167 or 381, wherein the CARs encoded by the first transgene and the second transgene specifically bind to antigens selected from the group consisting of CD19, CD20, CD22, CD33, CD123, BCMA, BAFFR, HER2, B7H3, EGFR, IL13Ra, and ROR1.

383. The method of any one of claims 151-167 or 381, wherein the CARs encoded by the first transgene and the second transgene specifically bind to antigens selected from the group consisting of CD 19 and CD20, CD 19 and BCMA, CD20 and BCMA, CD 19 and CD22, CD 19 and BAFFR, CD33 and CD123, HER2 and B7H3, HER2 and EGFR, HER2 and IL13Ra, HER2 and ROR1, B7H3 and EGFR, B7H3 and IL13Ra, B7H3 and ROR1, EGFR and IL13Ra, EGFR and ROR1, and IL13Ra2 and ROR1.

384. The method of claim 382 or claim 383, comprising first, second, third, fourth, and fifth transgenes, wherein each transgene is a CAR, wherein each CARs specifically binds a solid tumor antigen selected from the group consisting of HER2, B7H3, EGFR, IL13Ra2, R0R1, and any combination thereof.

385. The method of claim 382 or claim 383, wherein the CARs specifically bind to autoimmune disease antigens selected from the group consisting of CD 19 and CD20, CD 19 and BCMA, CD20 and BCMA, and CD 19 and CD22.

386. The method of claim 382 or claim 383, wherein the CARs specifically bind to leukemia or lymphoma antigens selected from the group consisting of CD 19 and CD20, CD 19 and BAFFR, and CD 19 and CD22.

387. The method of claim 382 or claim 383, wherein the CARs specifically bind to the acute myeloid leukemia antigens CD33 and CD123.

388. The vector of any one of claims 259-275 or 381, wherein the CARs encoded by the first transgene and the second transgene specifically bind to antigens selected from the group consisting of CD19, CD20, CD22, CD33, CD123, BCMA, BAFFR, HER2, B7H3, EGFR, IE13Ra, and R0R1.

389. The vector of any one of claims 259-275 or 381, wherein the CARs encoded by the first transgene and the second transgene specifically bind to antigens selected from the group consisting of CD 19 and CD20, CD 19 and BCMA, CD20 and BCMA, CD 19 and CD22, CD 19 and BAFFR, CD33 and CD123, HER2 and B7H3, HER2 and EGFR, HER2 and IE13Ra, HER2 and R0R1, B7H3 and EGFR, B7H3 and IE13Ra, B7H3 and R0R1, EGFR and IE13Ra, EGFR and R0R1, and IE13Ra2 and R0R1.

390. The vector of claim 388 or claim 389, wherein the vector comprises first, second, third, fourth, and fifth transgenes, wherein each transgene is a CAR, wherein each CARs specifically binds a solid tumor antigen selected from the group consisting of HER2, B7H3, EGFR, IE13Ra2, ROR1, and any combination thereof.

391. The vector of claim 388 or claim 389, wherein the CARs specifically bind to autoimmune disease antigens selected from the group consisting of CD 19 and CD20, CD 19 and BCMA, CD20 and BCMA, and CD 19 and CD22.

392. The vector of claim 388 or claim 389, wherein the CARs specifically bind to leukemia or lymphoma antigens selected from the group consisting of CD 19 and CD20, CD 19 and BAFFR, and CD 19 and CD22.

393. The vector of claim 388 or claim 389, wherein the CARs specifically bind to the acute myeloid leukemia antigens CD33 and CD123.