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

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 CELLSCROSS-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 asubject; 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, whereindetection 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, administeringone 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 someembodiments, 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 cellswith 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, thecomposition 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 mediastinalB 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 gastrointestinaldiseases, 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 receptorneuropathy, 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 inflammatorydemyelinating 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-neutrophiliccytoplasmic 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 ParvovirusB19 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 sequencecomprises 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 firsttransgene 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 probebinding 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 (FactorX), 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 1comprises 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 anycombination 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 andBCMA, 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, anendonuclease-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 someembodiments, 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 someembodiments, 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 identifyingregion 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, Laantigen, 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 modifyingprotein, 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, theengineered 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 reducesprotein 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 B2Mindel / 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 thegroup 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. 4Fdepicts 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), themethods 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, forexample, 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 (orpolypeptide) 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 toit. 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 desiredclinical 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, and209Bi. 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 theprobe 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 moretransgenes. 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 toabout 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 #1CATCGGAAExemplary barcode nucleic acid sequence #2 GGACAATTExemplary barcode nucleic acid sequence #3 TGTCAACTExemplary barcode nucleic acid sequence #4 TTACAGTTExemplary barcode nucleic acid sequence #5ATTCAAGGExemplary 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 notlimited 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 forwardprimer 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 someembodiments, 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 sitecomprising 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)GAAACTCATCGGAAAGACGGAGSEQ ID NO: 19 (Exemplary probe binding sequence #2; barcode bolded and underlined) GAAACTGGACAATTAGACGGAGSEQ ID NO: 20 (Exemplary probe binding sequence #3; barcode bolded and underlined) GAAACTTGTCAACTAGACGGAGSEQ ID NO:21 (Exemplary probe binding sequence #4; barcode bolded and underlined) GAAACTTTACAGTTAGACGGAGSEQ ID NO: 22 (Exemplary probe binding sequence #5; barcode bolded and underlined) GAAACTATTCAAGGAGACGGAGSEQ 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 CGCAGGAATGAAAGACCCCACSEQ ID NO: 25 (Barcode insert / identifying region #2; exemplary barcode underlined and bolded)ATCGAGTGTAGCCTTATCGCAGCTAGACGAAACTGGACAATTAGACGGAGAAGTAA CGCAGGAATGAAAGACCCCACSEQ ID NO: 26 (Barcode insert / identifying region #3; exemplary barcode underlined and bolded) ATCGAGTGTAGCCTTATCGCAGCTAGACGAAACTTGTCAACTAGACGGAGAAGTAA CGCAGGAATGAAAGACCCCACSEQ ID NO: 27 (Barcode insert / identifying region #4; exemplary barcode underlined and bolded) ATCGAGTGTAGCCTTATCGCAGCTAGACGAAACTTTACAGTTAGACGGAGAAGTAA CGCAGGAATGAAAGACCCCACSEQ ID NO:28 (Barcode insert / identifying region #5; exemplary barcode underlined and bolded) ATCGAGTGTAGCCTTATCGCAGCTAGACGAAACTATTCAAGGAGACGGAGAAGTAA CGCAGGAATGAAAGACCCCACSEQ ID NO: 29 (Barcode insert / identifying region #6; exemplary barcode underlined and bolded) ATCGAGTGTAGCCTTATCGCAGCTAGACGAAACTTACAGTTAAGACGGAGAAGTAA CGCAGGAATGAAAGACCCCACSEQ ID NO:30 (Barcode insert / identifying region, Universal Gibson forward primer; randomly generated barcode underlined and bolded)ATCGAGTGTAGCCTTATCGCAGCTAGACGAAACTNNNNNNNNAGACGGAGAAGTAA CGCAGGAATGAAAGACCCCACSEQ 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 firsttransgene 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, aretrovirus 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, enzymemodulator 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 cellsurface 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 infectiousdisease. 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 targetantigen. 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 acostimulatory 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 someembodiments, 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 toB7H3 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-1and -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, suchas 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 someembodiments, 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 cellsfurther 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 AutolusLimited, 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 andCoumarin 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 acell 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 moreamino 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, forexample, 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, CD28transmembrane 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 CARsTable 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 about925 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...

Claims

CLAIMSWhat 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 therapeuticdoses 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, AtypicalTeratoid / 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, contactdermatitis, 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, lamellarichthyosis, 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 injurysyndrome 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, autoimmuneear 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, immunofluorescenceanalysis, 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 / orii) 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 HepatitisVirus 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 WoodchuckHepatitis 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, structuralpolypeptides, 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,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.

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 agRNA 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,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,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.

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 genotype355. 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 subject378. 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 solidtumor 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.

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