Safety switches to control in vitro and in VIVO proliferation of cell therapy products

Abstract

The present disclosure provides modified cells comprising a nucleic acid encoding an agent (e.g., a detection agent, a selection agent, or a detection agent and a selection agent) inserted at an endogenous proliferation gene or off-target cell marker gene, as well as compositions, methods, uses, and kits related thereto.

Description

SAFETY SWITCHES TO CONTROL IN VITRO AND IN VIVO PROLIFERATION OF CELL THERAPY PRODUCTSCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This patent application claims priority benefits of U.S. Provisional Application No. 63 / 620,745 filed on January 12, 2024 and U.S. Provisional Application No. 63 / 624,262 filed on January 23, 2024, the content of each of which is incorporated herein by reference in their entirety.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (186152007740SEQLIST.xml; Size: 154,521 bytes; and Date of Creation: January 8, 2025) is herein incorporated by reference in its entirety.FIELD OF THE INVENTION

[0003] Provided herein are modified cells comprising a nucleic acid encoding an agent (e.g., a detection agent or selection agent) inserted at an endogenous proliferation gene or off- target cell marker gene, as well as compositions, methods, uses, and kits related thereto.BACKGROUND OF THE INVENTION

[0004] The ability to prevent or mitigate an adverse clinical event in a reliable and safe manner is key for successful development of stem cell-derived therapies. One challenge to stem cell-derived therapies is the growth of cancerous or other unwanted cells. Accordingly, there is an unmet need for developing safe and efficient methods for preserving differentiated therapeutic cells.BRIEF SUMMARY OF THE INVENTION

[0005] The present invention in one aspect provides a cell comprising a nucleic acid encoding a selection agent or a detection agent integrated at an endogenous proliferation gene locus and operably linked to the promoter of the endogenous proliferation gene, wherein expression of the nucleic acid encoding the selection agent or the detection agent is regulated by the promoter of the endogenous proliferation gene and / or regulatory elements of the endogenous proliferation gene and methods of uses thereof. The present invention in another aspect provides a cell comprising a nucleic acid encoding a selection agent or a detection agent integrated at an off-target cell type gene locus and operably linked to the promoter of the off-target cell marker gene, wherein expression of the nucleic acid encoding the selectionagent or the detection agent is regulated by the promoter of the off-target cell marker gene and / or regulatory elements of the off-target cell marker gene and methods of uses thereof.BRIEF DESCRIPTION OF THE DRAWINGS

[0006] 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.

[0007] FIG. 1 shows a schematic representation of pluripotent stem cell (PSC) and the subsequent implantation of differentiated cells into a human. Differentiated cells are a mixed pool, with most PSCs progressing to the post-mitotic stage during differentiation, while a small subset of PSCs and other cells with proliferative potential remain. These residual proliferating cells may cause tumors or teratomas that could evade the immune system upon transplantation.

[0008] FIG. 2 depicts a workflow for identification of endogenous proliferation gene candidates in iPSCs by bulk RNA sequencing, cross-referencing candidates with cancer- associated genes from internal and public datasets, and single cell RNA (scRNA) sequencing to interrogate proliferative gene specificity across cell types (e.g., beta islet cells and cardiomyocytes).

[0009] FIG. 3A depicts a heat map of bulk RNA expression of endogenous proliferation genes in sorted cadaveric islets (n=7 adult cadaveric beta cell batches; n=6 adult cadaveric alpha cell batches) and the average transcripts per million (TPM) calculated for each gene (data unsealed). Candidate genes demonstrated the largest fold change in expression among the datasets and had higher absolute TPM values sorted under the three batches of representative iPSC than the adult cadaveric beta and alpha cell batches. The genes from top to bottom are BIRC5, CDK1, TOP2A, PTTG1, CCNB1, TPX2, KIF11, SPC25, RRM2, CENPK, SMC4, TYMS, CDC20, AURKB, H2AZ1, TMSB15A, CENPF, and MKI67. FIG. 3B depicts a heatmap of bulk RNA expression of endogenous proliferation genes in sorted PSC cell lines (n=28) and differentiated batches (n=24) and the average TPM calculated for each gene (data unsealed). Fold change in expression of a gene was calculated as the average TPM of a gene in PSC batches divided by the average TPM of the same gene in differentiated batches. The genes from top to bottom are BIRC5, CDK1, TOP2A, PTTG1, CCNB1, TPX2, KIF11, CENPK, SMC4, TYSM, CDC20, AURKB, CENPF, and MKI67. FIG. 3C depicts a single-cell RNA (scRNA) sequencing map of two different PSC-derived cell types (n=22). The cells from scRNA sequencing were annotated based on their gene expression profiles to identify the proliferative subset of cells, as outlined by a circular shapein the figure. The proliferative subset of cells was then analyzed to identify endogenous proliferation genes specifically expressed in the proliferative subset and not in terminally differentiated cells, agnostic of cell type. The maps on the right depict the expression of two exemplary endogenous proliferation genes, MKI67 and T0P2A.

[0010] FIG. 4A depicts a clustered dotplot for scRNA sequencing of beta islet cell differentiation. FIG. 4B depicts a clustered dotplot for scRNA sequencing of cardiomyocyte differentiation.

[0011] FIG. 5A depicts a homology-directed repair (HDR) donor design for insertion of a nucleic acid encoding an agent into an endogenous proliferation gene locus. The agent can be, for example, a selection agent. FIG. 5B depicts an example of HDR introduction of iCaspase 9, cytosine deaminase, or a promotor-less GFP reporter into the 3’ UTR of the endogenous AURKB locus.

[0012] FIG. 6A depicts a graph of the knock-in (KI) efficiency of inducible Caspase 9- GFP (iC9-GFP) into candidate endogenous proliferation gene loci. KI efficiency was assessed by percentage of GFP-positive cells. KI efficiency of iC9-CD47 was assessed by percentage of CD47-positive cells. The lower dashed, horizontal line represents 2% of cells were GFP-positive or CD47 -positive. The upper dashed, horizontal line represents 5% of cells were GFP-positive or CD47 -positive. FIG. 6B depicts a graph of the clonal copy number by ddPCR. Plasmid backbone genomic copy number is plotted on the y-axis. bGH (polyA signal) genomic copy number is plotted on the x-axis. Integration of a nucleic acid encoding an agent was assessed by the bGH genomic copy number. Clones expressing monoallelic or biallelic integration (1 or 2 bGH genomic copy number) and without plasmid backbone (0 plasmid backbone genomic copy number) were selected as the “correct clones”. Clones with a plasmid backbone genomic copy number greater than 0 were excluded (labeled “excluded clones”).

[0013] FIG. 7 depicts the percentage of positivity for pluripotency markers. Each clone, which had iC9-GFP knocked into the specific locus, is marked as “mono” for monoallelic or “bi” for biallelic. Each set of bars from left to right represents the percentage of Oct4-positive, Sox2-positive, Nanog-positive, and Oct4 / Sox2 / Nanog-positive cells. The parental iPSC line (dko Dl l) and AAVS1 line (iC9-GFP knocked into the AAVS1 safe harbor site in iPSCs) are controls, dko DI 1 is a pluripotent cell line-derived clone with a double knock-out for genes B2M and CIITA.

[0014] FIG. 8A depicts the gene expression level of the specified, edited endogenous proliferation locus (locus with KI), compared to the gene’s expression level in AAVSl-editedclones. The dashed, horizontal line represents the expression level of the specific endogenous proliferation gene in AAVS-1 edited clones. The expression level of all endogenous proliferation genes in AAVS1 -edited clones is set to 1. FIG. 8B depicts the growth rate over 24 hours for the specified, edited endogenous proliferation locus (locus with KI). The parental iPSC line (dko DI 1) is the control, dko DI 1 is a pluripotent cell line-derived clone with a double knock-out for genes B2M and CIITA. Each edited locus is marked as “mono” for monoallelic and “bi” for biallelic.

[0015] FIGs. 9A-9B depicts AP20187 (also named “BB”) dosing kill curves of edited iPSCs. Twenty-four hours after the formation of embryoid bodies (EB), the EB were plated in DMEM, which was day 0 of the spontaneous differentiation timeline. Edited iPSCs at various points of an 18-day spontaneous differentiation timeline (undifferentiated iPSC and differentiated iPSCs at day 5, day 12, and day 18) were subjected to increasing concentrations of AP20187. Cell titer viability was determined 24 hours after dosing. FIG. 9A shows AP20187 dosing kill curves for iPSCs with iC9 KI at the AURKB locus (iC9_AURKB). FIG. 9B shows AP20187 dosing kill curves for iPSCs with iC9 KI at the CDK1 locus (iC9_CDKl). FIG. 9C shows AP20187 dosing kill curves for iPSCs with iC9 KI at the AAVS1 locus (iC9_AAVSl). iC9_AAVSl is the control.

[0016] FIG. 10 depicts quantification of western blotting results for iC9 expression in the iC9_AURKB, iC9_CDKl, and dko Dl l cell lines at the undifferentiated iPSC stage and day 14 (D14) of differentiation. The parental iPSC line (dko DI 1) is the control, dko DI 1 is a pluripotent cell line-derived clone with a double knock-out for genes B2M and CIITA.

[0017] FIGs. 11A-11M depict AP20187 (also named “BB dimerizer”) dosing kill curves of edited iPSCs. Twenty-four hours after the formation of embryoid bodies (EB), the EB were plated in DMEM, which was day 0 of the spontaneous differentiation timeline. Edited iPSCs at various points of an 18-day spontaneous differentiation timeline (undifferentiated iPSC and differentiated iPSCs at day 5, day 12, and day 18) were subjected to increasing concentrations of AP20187. Cell titer viability was determined 24 hours after dosing. FIG. 11A shows AP20187 dosing kill curves for iPSC clones with iC9-GFP KI at the CENPF locus. FIG. 11B shows AP20187 dosing kill curves for iPSC clones with iC9-GFP KI at the TOP2A locus. FIG. 11C shows AP20187 dosing kill curves for iPSC clones with iC9-GFP KI at the SMC4 locus. FIG. 11D shows AP20187 dosing kill curves for iPSC clones with iC9-GFP KI at the AURKB locus. FIG. HE shows AP20187 dosing kill curves for iPSC clones with iC9-GFP KI at the CDK1 locus. FIG. HF shows AP20187 dosing kill curves for iPSC clones with iC9-GFP KI at the BIRC5 locus. FIG. 11G shows AP20187dosing kill curves for iPSC clones with iC9-GFP KI at the TPX2 locus. FIG. 11H shows AP20187 dosing kill curves for iPSC clones with iC9-GFP KI at the MKI67 locus. FIG. Ill shows AP20187 dosing kill curves for iPSC clones with iC9-GFP KI at the CDC20 locus. FIG. 11J shows AP20187 dosing kill curves for iPSC clones with iC9-GFP KI at the PTTG1 locus. FIG. 11K shows AP20187 dosing kill curves for iPSC clones with iC9-GFP KI at the KIF11 locus. FIG. 11L shows AP20187 dosing kill curves for iPSC clones with iC9-GFP KI at the CENPK locus. FIG. 11M shows AP20187 dosing kill curves for iPSC clones with iC9- GFP KI at the TYMS locus. For the graphs with a horizontal line at about 100% cell viability with circles to denote the data points, the horizontal line represents the parental iPSC line (dko Dl l) control, dko DI 1 is a pluripotent cell line-derived clone with a double knock-out for genes B2M and CIITA.

[0018] FIG. 12 is a plot of the IC50 for a specific proliferation gene KI clone against the iC9 expression as measured by mean fluorescence intensity (MFI) of GFP. The dotted horizontal line represents incomplete killing. The dotted vertical line represents the minimum threshold for iC9-GFP expression. The 2 in parenthesis denotes biallelic. All other loci are monoallelic.

[0019] FIG. 13 is a bar graph of the growth rate over 24 hours of specific iC9 KI clones on day 0, day 5, and day 18 of differentiation. For each day, the bars from left to right are as follows: dko Dl l (parental iPSC control), AVSS1 (monoallelic), AAVS1 (biallelic), CENPF (biallelic), TOP2A (monoallelic), TOP2A (biallelic), SMC4 (monoallelic), AURKB (monoallelic), AURKB (biallelic), CDK1 (monoallelic), BIRC5 (biallelic), TPX2 (monoallelic), MKI67 (biallelic), CDC20 (monoallelic), PTTG1 (monoallelic), KIF11 (monoallelic), and TYMS (monoallelic). dko Dl l is a pluripotent cell line-derived clone with a double knock-out for genes B2M and CIITA.

[0020] FIGs. 14A-14O depict AP20187 (also named “BB dimerizer”) dosing kill curves of edited iPSCs with iC9 inserted at proliferation loci. Twenty-four hours after the formation of embryoid bodies (EB), the EB were plated in DMEM, which was day 0 of the spontaneous differentiation timeline. Edited iPSCs at various points (undifferentiated iPSC, differentiated iPSCs at day 5, and differentiated iPSCs at day 18) of an 18-day spontaneous differentiation timeline were subjected to increasing concentrations of AP20187. Cell titer viability was determined 24 hours after dosing. FIG. 14A shows AP20187 dosing kill curves for the parental iPSC control (dko), AAVS1 monoallelic KI clone A (control), and AAVS1 biallelic KI clone B (control), dko DI 1 is a pluripotent cell line-derived clone with a double knock-out for genes B2M and CIITA. FIG. 14B shows AP20187 dosing kill curves forCDK1 monoallelic KI clones A and B. FIG. 14C shows AP20187 dosing kill curves for CENPF biallelic KI clones A and B. FIG. 14D shows AP20187 dosing kill curves for TOP2A monoallelic KI clones A and B. FIG. 14E shows AP20187 dosing kill curves for TOP2A biallelic KI clones C and D. FIG. 14F shows AP20187 dosing kill curves for SMC4 monoallelic KI clones A and B. FIG. 14G shows AP20187 dosing kill curves for AURKB monoallelic KI clones A and B. FIG. 14H shows AP20187 dosing kill curves for AURKB biallelic KI clones C and D. FIG. 141 shows AP20187 dosing kill curves for BIRC5 biallelic KI clone A. FIG. 14J shows AP20187 dosing kill curves for TPX2 monoallelic KI clones A and B. FIG. 14K shows AP20187 dosing kill curves for MKI67 biallelic KI clones A and B. FIG. 14L shows AP20187 dosing kill curves for CDC20 monoallelic KI clones A and B. FIG. 14M shows AP20187 dosing kill curves for PTTG1 monoallelic KI clones A and B. FIG. 14N shows AP20187 dosing kill curves for KIF11 monoallelic KI clone A. FIG. 140 shows AP20187 dosing kill curves for TYMS monoallelic KI clone A.

[0021] FIGs. 15A and 15B is a graph plotting the IC50 for the kill curves (bars) against the percentage of resistant cells on day 18 (triangles) of the specified proliferation gene locus. The vertical dashed line in FIG. 15B delineates gene loci that were further investigated (left of vertical dashed line) and gene loci that did not fit the criteria (right of the vertical dashed line). The 2 in parenthesis denotes biallelic. All other loci are monoallelic.

[0022] FIGs. 16A-16C depict 5-flurocytosine (5-FC) dosing kill curves of edited iPSCs and pancreatic progenitors. Cytosine deaminase (CDA) was knocked into the AAVS1, AURKB, or CDK1 loci in iPSCs that were differentiated into pancreatic progenitors.Undifferentiated iPSCs and day 12 differentiated pancreatic progenitors (“D12”) were subjected to increasing concentrations of 5-FC. Cell titers were assessed 72 hours after dosing. FIG. 16A shows 5-FC dosing kill curves for CDA_AURKB iPSCs and CDA_AURKB pancreatic progenitors. FIG. 16B shows 5-FC dosing kill curves for CDA_CDK1 iPSCs and CDA_CDK1 pancreatic progenitors. FIG. 16C shows 5-FC dosing kill curves for the following controls: dko parental iPSC line (a pluripotent cell line-derived clone with a double knock-out for genes B2M and CIITA), dko pancreatic progenitors (day 12 pancreatic progenitors differentiated from the dko parental iPSC line), CDA_AAVS1 iPSCs (CDA knocked into the AAVS1 safe harbor site in iPSCs), and CDA_AAVS1 pancreatic progenitors (CDA knocked into the AAVS1 safe harbor site in pancreatic progenitors).

[0023] FIGs. 17A-17D depict flow cytometry histograms of Ki67-positive cells in pancreatic progenitors 72 hours after dosing with or without 100 pM 5-FC. CDA wasknocked into the AAVS1 (CDA_AAVS1), AURKB (CDA_AURKB), or CDK1 (CDA_CDK1) loci in pancreatic progenitors, and pancreatic progenitors at day 12 of beta islet differentiation were used. FIG. 17A shows a comparison of Ki67-positive cell count in untreated and treated CDA_AURKB pancreatic progenitor cultures. FIG. 17B shows a comparison of Ki67-positive cell count in untreated and treated CDA_CDK1 pancreatic progenitor cultures. FIG. 17C shows a comparison of Ki67-positive cell count in untreated and treated CDA_AAVS1 pancreatic progenitor cultures (control). FIG. 17D shows a comparison of Ki67-positive cell count in untreated and treated dko pancreatic progenitor cultures (control).

[0024] FIGs. 18A-18C depict AP20187 (also named “BB dimerizer”) dosing kill curves of edited iPSCs and pancreatic progenitors. iCaspase 9 was knocked into the AAVS1, AURKB, or CDK1 loci in iPSCs that were differentiated into pancreatic progenitors, and undifferentiated iPSCs and day 12 differentiated pancreatic progenitors (“D12”) were subjected to increasing concentrations of AP20187. Cell titers were assessed 48 hours after dosing. FIG. 18A shows AP20187 dosing kill curves for iC9_AURKB iPSCs and iC9_AURKB pancreatic progenitors. FIG. 18B shows AP20187 dosing kill curves for iC9_CDKl iPSCs and iC9_CDKl pancreatic progenitors. FIG. 18C shows AP20187 dosing kill curves for the following controls: dko parental iPSC line (dko DI 1 is a pluripotent cell line-derived clone with a double knock-out for genes B2M and OITA), dko pancreatic progenitors (double knock-out for genes B2M and CIITA in pancreatic progenitors), iC9_AAVSl iPSCs (iC9 knocked into the AAVS1 safe harbor site in iPSCs), and iC9_AAVSl pancreatic progenitors (iC9 knocked into the AAVS1 safe harbor site in pancreatic progenitors).

[0025] FIG. 19 depicts a clustered dotplot for scRNA sequencing of beta islet cell differentiation. Top ductal cell markers are identified.

[0026] FIG. 20 depicts a homology-directed repair (HDR) donor design for insertion of a nucleic acid encoding an agent into an off-target cell marker gene locus. The agent can be, for example, a selection agent.

[0027] FIG. 21 depicts a homology-directed repair design for insertion of a nucleic acid encoding an agent into an endogenous proliferation gene or off-target cell marker gene locus. The agent can be, for example, a detection agent.DETAILED DESCRIPTION OF THE INVENTION

[0028] The present disclosure relates to modified cells comprising a nucleic acid encoding an agent inserted at an endogenous proliferation gene or off-target cell marker gene, as well as compositions, methods, uses, and kits related thereto.I. Definitions

[0029] 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.

[0030] The term “locus” refers to a fixed position on a chromosome where a particular gene or genetic marker is located. Reference to a “target locus” refers to a particular locus of a desired gene in which it is desired to target a genetic modification, such as a gene edit or integration of an exogenous polynucleotide.

[0031] As used herein, the term “and / or” includes any and all combinations of one or more of the associated listed items.

[0032] The term “expression” with reference to a gene or “gene expression” refers to the conversion of the information, contained in a gene, into a gene product. A gene product can be the direct transcriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, structural RNA or any other type of RNA) or can be a protein produced by translation of an mRNA. Gene products also include RNAs which are modified, by processes such as capping, polyadenylation, methylation, and editing, and proteins modified by, for example, methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristoylation, and glycosylation. Hence, reference to expression or gene expression includes protein (or polypeptide) expression or expression of a transcribable product of 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.

[0033] 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, exogenous sequence,or exogenous transgene, can be introduced, for example, by introduction of an exogenous encoding nucleic acid into the genetic material of the cells such as by integration into a chromosome or as non-chromosomal genetic material such as a plasmid or expression vector. Therefore, the term as it is used in reference to expression of an encoding nucleic acid refers to introduction of the encoding nucleic acid in an expressible form into the cell. In some cases, an "exogenous" molecule is a molecule, construct, factor and the like that is not normally present in a cell but can be introduced into a cell by one or more genetic, biochemical, or other methods.

[0034] The term “endogenous” refers to a referenced molecule, such as a polynucleotide (e.g., gene), or polypeptide, that is present in a native or unmodified cell. 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 and not exogenously introduced. 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.

[0035] The term “operably linked” refers to two or more genetic elements, such as a polynucleotide coding sequence and a promoter, placed in relative positions that permit the proper biological functioning of the elements, such as the promoter directing transcription of the coding sequence.

[0036] The term “wild-type” or “WT” as used herein, refers to an entity having a structure and / or activity as found in nature in a “normal” (as contrasted with mutant, diseased, altered, engineered, etc.) state or context. A wild-type amino acid sequence is an amino acid sequence that is found in nature, including allelic variations. A wild-type protein or polypeptide has an amino acid sequence that has not been intentionally modified.

[0037] As used herein, the term “modification” with reference to a cell refers to any change or alteration of a nucleic acid in the genome of a cell, which may impact gene expression in the cell. For example, a modification includes a genetic modification that results in alterations, additions, and / or deletion of genes or portions of genes or other nucleic acid sequences. A modified cell, such as a genetically modified cell, can also refer to a cellwith an added, deleted and / or altered gene or portion of a gene. In some embodiments, the modification is a genetic modification that directly changes the gene or regulatory elements thereof encoding a protein product in a cell, such as by gene editing, mutagenesis or by genetic engineering of an exogenous polynucleotide or transgene. Genetic modifications include, for example, both transient knock-in or knock-down mechanisms, and mechanisms that result in permanent knock-in, knock-down, or knock-out of target genes or portions of genes or nucleic acid sequences. Genetic modifications include, for example, both transient knock-in and mechanisms that result in permanent knock-in of nucleic acids sequences. Genetic modifications also include, for example, reduced or increased transcription, reduced or increased mRNA stability, reduced or increased translation, and reduced or increased protein stability.

[0038] As used herein, the terms “effective amount” and “pharmaceutically effective amount” refer to a nontoxic but sufficient amount of an agent or drug to provide the desired biological result. That result can be reduction and / or alleviation of the signs, symptoms, or causes of a disease or disorder, imaging or monitoring of an in vitro or in vivo system (including a living organism), or any other desired alteration of a biological system. An appropriate effective amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation.

[0039] As used herein, the term “pharmaceutically acceptable” refers to a material, such as a carrier or diluent, which does not abrogate the biological activity or properties of the compound, and is relatively nontoxic, i.e., the material may be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

[0040] As used herein, the term “pharmaceutical composition” refers to a mixture of at least one particle with other chemical components, such as carriers, stabilizers, diluents, dispersing agents, suspending agents, thickening agents, and / or excipients. The pharmaceutical composition facilitates administration of the compound to an organism. Multiple techniques of administering a compound exist in the art including, but not limited to, intravenous, oral, aerosol, parenteral, ophthalmic, pulmonary and topical administration.

[0041] A “disease” or “disorder” as used herein refers to a condition where treatment is needed and / or desired.

[0042] As used herein, the terms “treat,” “treating,” or “treatment” refer to ameliorating a disease or disorder, e.g., slowing or arresting or reducing the development of the disease or disorder or reducing at least one of the clinical symptoms thereof. For purposes of thisdisclosure, ameliorating a disease or disorder can include obtaining a beneficial or desired clinical result that includes, but is not limited to, any one or more of: alleviation of one or more symptoms, diminishment of extent of disease, preventing or delaying spread (for example, metastasis, for example metastasis to the lung or to the lymph node) of disease, preventing or delaying recurrence of disease, delay or slowing of disease progression, amelioration of the disease state, inhibiting the disease or progression of the disease, inhibiting or slowing the disease or its progression, arresting its development, and remission (whether partial or total).

[0043] The terms “individual” and “subject” are used interchangeably herein to refer to an animal, for example a mammal. The term patient includes human and veterinary subjects. In some embodiments, methods of treating mammals, including, but not limited to, humans, rodents, simians, felines, canines, equines, bovines, porcines, ovines, caprines, mammalian laboratory animals, mammalian farm animals, mammalian sport animals, and mammalian pets, are provided. The subject can be male or female and can be any suitable age, including infant, juvenile, adolescent, adult, and geriatric subjects. In some examples, an “individual” or “subject” refers to an individual or subject in need of treatment for a disease or disorder. In some embodiments, the subject to receive the treatment can be a patient, designating the fact that the subject has been identified as having a disorder of relevance to the treatment, or being at adequate risk of contracting the disorder. In particular embodiments, the subject is a human, such as a human patient.

[0044] The term “donor subject” or “donor individual” refers to an animal, for example, a human from whom cells can be obtained. The “non-human animals” and “non-human mammals” as used interchangeably herein, includes mammals such as rats, mice, rabbits, sheep, cats, dogs, cows, pigs, and non-human primates. The term “donor subject” also encompasses any vertebrate including but not limited to mammals, reptiles, amphibians and fish. However, advantageously, the donor subject is a mammal such as a human, or other mammals such as a domesticated mammal, e.g., dog, cat, horse, and the like, or production mammal, e.g., cow, sheep, pig, and the like. A “donor subject” can also refer to more than one donor, for example one or more humans or non-human animals or non-human mammals.

[0045] The term “recipient”, “recipient patient”, or “recipient individual” refers to an animal, for example, a human to whom treatment, including prophylactic treatment, with the cells as described herein, is provided. For treatment of those infections, conditions, or disease states, which are specific for a specific animal such as a human patient, the term patient refers to that specific animal. The term “recipient patient” also encompasses any vertebrateincluding but not limited to mammals, reptiles, amphibians, and fish. However, advantageously, the recipient patient is a mammal such as a human, or other mammals such as a domesticated mammal, e.g., dog, cat, horse, and the like, or production mammal, e.g., cow, sheep, pig, and the like.

[0046] As used herein, “percent (%) amino acid sequence identity” and “homology” with respect to a peptide, polypeptide or antibody sequence are defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGNTM (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0047] As used herein, “percent (%) nucleic acid sequence identity” and “homology” with respect to a nucleic acid, nucleotide, DNA, or RNA sequence are defined as the percentage of nucleotides in a candidate sequence that are identical with the nucleotides in the specific nucleic acid, DNA, or RNA sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity.Alignment for purposes of determining percent nucleic acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGNTM (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0048] The term “comparable”, as used herein, refers to two or more agents, entities, situations, sets of conditions, etc. that may not be identical to one another but that are sufficiently similar to permit comparison there between so that conclusions may reasonably be drawn based on differences or similarities observed. Persons of ordinary skill in the art will understand, in context, what degree of identity is required in any given circumstance for two or more such agents, entities, situations, sets of conditions, etc. to be considered comparable. A control cell, for example, can be a comparable cell (e.g., same cell type) that does not comprise the relative modifications.

[0049] The term “vector,” as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a selfreplicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors.”

[0050] The term “antibody” is used to denote, in addition to natural antibodies, genetically engineered or otherwise modified forms of immunoglobulins or portions thereof, including chimeric antibodies, human antibodies, humanized antibodies, or synthetic antibodies. The antibodies may be monoclonal or polyclonal antibodies. In those embodiments wherein an antibody is an immunogenically active portion of an immunoglobulin molecule, the antibody may include, but is not limited to, a single chain variable fragment antibody (scFv), disulfide linked Fv, single domain antibody (sdAb), VHH antibody, antigen-binding fragment (Fab), Fab', F(ab')2 fragment, or diabody. An scFv antibody is derived from an antibody by linking the variable regions of the heavy (VH) and light (VL) chains of the immunoglobulin with a short linker peptide. Similarly, a disulfide linked Fv antibody can be generated by linking the VH and VL using an interdomain disulfide bond. On the other hand, sdAbs consist of only the variable region from either the heavy or light chain and usually are the smallest antigen-binding fragments of antibodies. A VHH antibody is the antigen binding fragment of heavy chain only. A diabody is a dimer of scFv fragment that consists of the VH and VL regions noncovalent connected by a small peptide linker or covalently linked to each other. The antibodies disclosed herein, including those that comprise an immunogenically active portion of an immunoglobulin molecule, retain the ability to bind a specific antigen.

[0051] The term “antigen”, as used herein, refers to a molecule capable of provoking an immune response. Antigens include but are not limited to cells, cell extracts, proteins, polypeptides, peptides, polysaccharides, polysaccharide conjugates, peptide and non-peptide mimics of polysaccharides and other molecules, small molecules, lipids, glycolipids, carbohydrates, viruses and viral extracts and multicellular organisms such as parasites and allergens. The term antigen broadly includes any type of molecule which is recognized by a host immune system as being foreign.

[0052] A “binding domain,” also referred to as a “binding region,” refers to an antibody or portion thereof that possesses the ability to specifically and non-covalently associate, unite, or combine with a target. A binding domain includes any naturally occurring, synthetic,semi-synthetic, or recombinantly produced binding partner for a biological molecule, a molecular complex, or other target of interest. Exemplary binding domains include receptor ectodomains, ligands, scFvs, disulfide linked Fvs, sdAbs, VHH antibodies, Fab fragments, Fab' fragments, F(ab')2 fragments, diabodies, or other synthetic polypeptides selected for their specific ability to bind to a biological molecule, a molecular complex, or other target of interest.

[0053] ‘Immune signaling molecule” as used herein refers to, in some cases, a molecule, protein, peptide and the like that activates immune signaling pathways.

[0054] As used herein, “fusogen” refers to an agent or molecule that creates an interaction between two membranes, including membrane enclosed lumens. In embodiments, the fusogen facilitates fusion of the membranes. In other embodiments, the fusogen creates a connection, e.g., a pore, between two membranes or lumens (e.g., a lumen of a retroviral particle and a cytoplasm of a target cell). In some embodiments, the fusogen comprises a complex of two or more proteins, e.g., wherein neither protein has fusogenic activity alone. In some embodiments, the fusogen comprises a targeting domain.

[0055] 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 a modified primary cell or stem cell (or cell derived from a stem cell) so that the modified primary cell or stem cell (or cell derived from a stem 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.

[0056] The term “hypoimmunogenic” refers to a cell that is less prone to immune rejection by a subject to which such cells are transplanted. For example, relative to a comparable cell that does not contain certain modifications (e.g., a wild-type cell or a cell without one or more modifications intended to address immunogenicity (e.g., a modification to an HEA-I, HEA-II and / or tolerogenic factor gene)), such a hypoimmunogenic cell may be about 2.5%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99% or more less prone to immune rejection by a subject into which such cells are transplanted. Typically, the hypoimmunogenic cells are allogenic to the subject and a hypoimmunogenic cell evades immune rejection in an MHC-mismatched allogeneic recipient. In someembodiments, a hypoimmunogenic cell is protected from T cell-mediated adaptive immune rejection and / or innate immune cell rejection.

[0057] As used herein, “hypoimmunogenicity” of a cell can be determined by evaluating the immunogenicity of the cell such as the cell’s ability to elicit adaptive and innate immune responses.

[0058] The term “kill switch” refers to a system for expressing a protein linked to a gene of interest that, when the gene of interest is expressed or upregulated and when an inducer is provided, the cell expressing the gene of interest will be eliminated. An activated kill switch leads to clearance or death of the cell, e.g., through apoptosis. A kill switch can be designed to be or include an exogenous molecule administered to prevent or mitigate an adverse clinical event. A kill switch may include a protein or molecule that allows for the control of cellular activity in response to an adverse event. A kill switch may be used to eliminate cells that overproliferate or cells that are of an unwanted cell type. A kill switch can be expressed in an inactive state and is fatal to a cell expressing the kill switch upon activation of the switch by a selective, externally provided inducer. In some embodiments, the selection agent is a kill switch. In some embodiments, the kill switch gene is cis-acting in relation to the gene of interest in a construct. Activation of the kill switch causes the cell to kill solely itself or itself and neighboring cells through apoptosis or necrosis.

[0059] The term “cell” includes the subject cell and its progeny.

[0060] It will be understood by one of ordinary skill in the art that uracil and thymine can both be represented by ‘t’, instead of ‘u’ for uracil and ‘t’ for thymine; in the context of a ribonucleic acid, it will be understood that ‘t’ is used to represent uracil unless otherwise indicated.

[0061] It is understood that embodiments of the invention described herein include “consisting” and / or “consisting essentially of’ embodiments.

[0062] As used herein, the term “about” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which it is used. As used herein, “about” when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods. Reference to “about” a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X”.

[0063] 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. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For instance, where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit, unless the context clearly dictate otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure. In some embodiments, two opposing and open-ended ranges are provided for a feature, and in such description, it is envisioned that combinations of those two ranges are provided herein. 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.

[0064] As used herein, reference to “not” a value or parameter generally means and describes “other than” a value or parameter. For example, the method is not used to treat cancer of type X means the method is used to treat cancer of types other than X.

[0065] The term “about X-Y” used herein has the same meaning as “about X to about Y.”

[0066] As used herein and in the appended claims, the singular forms “a,” “or,” and “the” include plural referents unless the context clearly dictates otherwise.

[0067] Before the technology is further described, it is to be understood that this technology is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims. It should also be understood that the headers used herein are not limiting and are merely intended to orient the reader, but the subject matter generally applies to the technology disclosed herein.II. Cells

[0068] Certain aspects of the present disclosure relate to modified cells comprising a nucleic acid encoding a selection agent or a detection agent integrated at a gene of interest locus and operably linked to the promoter of the gene of interest, wherein expression of the nucleic acid encoding the selection agent or the detection agent is regulated by the promoter of the gene of interest and / or regulatory elements of the gene of interest. In some aspects, when the promoter of the gene of interest is “turned on” the selection or detection agent is expressed allowing selection or detection of certain cell types.A. Endogenous Proliferation Genes

[0069] When normal tissue and tumor samples are compared by microarray or sequencing analysis, the biggest differences most often occur in the expression levels of genes that control cell proliferation. Cell proliferation is an increase in cell number due to cell division. Although cell proliferation is necessary for normal tissue development and maintenance over a lifespan and is a tightly regulated process, with many different proteins controlling cell cycle checkpoints, genetic mutations found in cancer cells can cause uncontrolled cellular proliferation. Accordingly, various endogenous proliferation genes are upregulated in cancer cells. Proliferation genes can be screened using next-generation sequencing methods 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. In some instances, the sequencing may comprise, for example, bulk RNA- sequencing or single-cell RNA sequencing. Endogenous proliferation genes may be identified by sequencing iPSC cells versus differentiated cells, identifying a subset of genes with largest fold change between datasets, and cross-referencing with open databases of essential cancer genes to identify gene candidates that are essential to proliferation but not expressed in terminally differentiated cell types of interest. The genes that are strong markers of proliferation (overexpressed in iPSC cells) but are not essential are identified as endogenous proliferation genes.

[0070] In some embodiments, there is provided a modified cell comprising a nucleic acid encoding a selection agent or a detection agent integrated at an endogenous proliferation gene locus and operably linked to the promoter of the endogenous proliferation gene, wherein expression of the nucleic acid encoding the selection agent or the detection agent is regulated by the promoter of the endogenous proliferation gene and / or regulatory elements of the endogenous proliferation gene. In some embodiments, there is provided a modified cellcomprising a nucleic acid encoding a selection agent and a detection agent integrated at an endogenous proliferation gene locus and operably linked to the promoter of the endogenous proliferation gene, wherein expression of the nucleic acid encoding the selection agent and the detection agent is regulated by the promoter of the endogenous proliferation gene and / or regulatory elements of the endogenous proliferation gene. In some embodiments, there is provided a modified cell comprising a nucleic acid encoding a first detection agent and a second detection agent, wherein the nucleic acid is integrated at an endogenous proliferation gene locus and operably linked to the promoter of the endogenous proliferation gene, and wherein expression of the nucleic acid encoding the first detection agent and the second detection agent is regulated by the promoter of the endogenous proliferation gene and / or regulatory elements of the endogenous proliferation gene. In some embodiments, there is provided a modified cell comprising a nucleic acid encoding a first selection agent and a second selection agent, wherein the nucleic acid is integrated at an endogenous proliferation gene locus and operably linked to the promoter of the endogenous proliferation gene, and wherein expression of the nucleic acid encoding the first selection agent and the second selection agent is regulated by the promoter of the endogenous proliferation gene and / or regulatory elements of the endogenous proliferation gene. In some embodiments, the first selection agent and the second selection agent are operably linked. In some embodiments, the first selection agent and the second selection agent are operably linked by an internal ribosome entry site. In some embodiments, the first selection agent and the second selection agent are operably linked by a self-cleaving peptide. In some embodiments, there is provided a modified cell comprising a first nucleic acid encoding a selection agent and a second nucleic acid encoding a detection agent, wherein the first nucleic acid is integrated at a first endogenous proliferation gene locus and operably linked to the promoter of the first endogenous proliferation gene, wherein expression of the first nucleic acid encoding the selection agent is regulated by the promoter of the first endogenous proliferation gene and / or regulatory elements of the first endogenous proliferation gene, wherein the second nucleic acid is integrated at a second endogenous proliferation gene locus and operably linked to the promoter of the second endogenous proliferation gene, and wherein expression of the second nucleic acid encoding the detection agent is regulated by the promoter of the second endogenous proliferation gene and / or regulatory elements of the endogenous proliferation gene. In some embodiments, there is provided a modified cell comprising a first nucleic acid encoding a first selection agent and a second nucleic acid encoding a second selection agent, wherein the first nucleic acid is integrated at a first endogenous proliferation gene locus andoperably linked to the promoter of the first endogenous proliferation gene, wherein expression of the first nucleic acid encoding the first selection agent is regulated by the promoter of the first endogenous proliferation gene and / or regulatory elements of the first endogenous proliferation gene, wherein the second nucleic acid is integrated at a second endogenous proliferation gene locus and operably linked to the promoter of the second endogenous proliferation gene, and wherein expression of the second nucleic acid encoding the second selection agent is regulated by the promoter of the second endogenous proliferation gene and / or regulatory elements of the endogenous proliferation gene. In some embodiments, there is provided a modified cell comprising a first nucleic acid encoding a first detection agent and a second nucleic acid encoding a second detection agent, wherein the first nucleic acid is integrated at a first endogenous proliferation gene locus and operably linked to the promoter of the first endogenous proliferation gene, wherein expression of the first nucleic acid encoding the first detection agent is regulated by the promoter of the first endogenous proliferation gene and / or regulatory elements of the first endogenous proliferation gene, wherein the second nucleic acid is integrated at a second endogenous proliferation gene locus and operably linked to the promoter of the endogenous proliferation gene, and wherein expression of the second nucleic acid encoding the second detection agent is regulated by the promoter of the second endogenous proliferation gene and / or regulatory elements of the second endogenous proliferation gene. In some embodiments, the first endogenous proliferation gene and / or the second endogenous proliferation gene are expressed in a cell that is proliferating. In some embodiments, the first endogenous proliferation gene and / or the second endogenous proliferation gene are expressed in a cell that is proliferating. In some embodiments, the first endogenous proliferation gene and / or the second endogenous proliferation gene are expressed in a cell that is included in a population of therapeutic cells. In some embodiments, the first endogenous proliferation gene and / or the second endogenous proliferation gene are expressed in a cell that is included in a population of therapeutic cells but is not a therapeutic cell. In some embodiments, the cell is a pluripotent cell, a progenitor cell, or a differentiated cell that is proliferating. In some embodiments, the first endogenous proliferation gene and the second endogenous proliferation gene are the same, and the first and second nucleic acids are integrated at different alleles. In some embodiments, the first endogenous proliferation gene and the second endogenous proliferation gene are different. In some embodiments, the endogenous proliferation gene is expressed at a higher level from day 0 to about day 5 of differentiation, compared to expression of the endogenous proliferation gene at day 18 of differentiation, or wherein the endogenous proliferation gene is highlyexpressed from day 1 to about day 5 of differentiation. In some embodiments, the nucleic acid encoding the selection agent or the detection agent is integrated in an exon or an intron of the endogenous proliferation gene. In some embodiments, the nucleic acid encoding the selection agent or the detection agent is integrated in a 5’ UTR or a 3’ UTR of the endogenous proliferation gene. In some embodiments, following integration, the endogenous proliferation gene locus comprises i) nucleic acid encoding the endogenous proliferation gene, ii) an internal ribosomal entry site or a self-cleaving RNA site, and iii) the nucleic acid encoding the selection agent or a detection agent. Examples of endogenous proliferation genes include, but are not limited to, AURKB, CDK1, CDC20, RRM2, BIRC5, TOP2A, PTTG1, CCNB1, TPX2, KIF11, SPC25, CENPK, SMC4, TYMS, H2AZ1, TMSB15A, CENPF, and MKI67.

[0071] In some embodiments, the endogenous proliferation gene is AURKB. In some embodiments, the nucleic acid encoding a selection agent or a detection agent is integrated at an exon of AURKB, such as any one of exons 1, 2, 3, 4, 5, 6, 7, 8, or 9. In some embodiments, the nucleic acid encoding a selection agent or a detection agent is integrated at an intron of AURKB, such as any one of introns 1, 2, 3, 4, 5, 6, 7, or 8. In some embodiments, the nucleic acid encoding the selection agent or the detection agent is integrated in the 5’ UTR of AURKB. In some embodiments, the nucleic acid encoding the selection agent or the detection agent is integrated in the 3’ UTR of AURKB.

[0072] In some embodiments, the endogenous proliferation gene is CDK1. In some embodiments, the nucleic acid encoding a selection agent or a detection agent is integrated at an exon of CDK1, such as any one of exons 1, 2, 3, 4, 5, 6, 7, or 8. In some embodiments, the nucleic acid encoding a selection agent or a detection agent is integrated at an intron of CDK1, such as any one of introns 1, 2, 3, 4, 5, 6, or 7. In some embodiments, the nucleic acid encoding the selection agent or the detection agent is integrated in the 5’ UTR of CDK1. In some embodiments, the nucleic acid encoding the selection agent or the detection agent is integrated in the 3’ UTR of CDK1.

[0073] In some embodiments, the endogenous proliferation gene is CDC20. In some embodiments, the nucleic acid encoding a selection agent or a detection agent is integrated at an exon of CDC20, such as any one of exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11. In some embodiments, the nucleic acid encoding a selection agent or a detection agent is integrated at an intron of CDC20, such as any one of introns 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the nucleic acid encoding the selection agent or the detection agent isintegrated in the 5’ UTR of CDC20. In some embodiments, the nucleic acid encoding the selection agent or the detection agent is integrated in the 3’ UTR of CDC20.

[0074] In some embodiments, the endogenous proliferation gene is RRM2. In some embodiments, the nucleic acid encoding a selection agent or a detection agent is integrated at an exon of RRM2, such as any one of exons 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the nucleic acid encoding a selection agent or a detection agent is integrated at an intron of RRM2, such as any one of introns 1, 2, 3, 4, 5, 6, 7, 8, or 9. In some embodiments, the nucleic acid encoding the selection agent or the detection agent is integrated in the 5’ UTR of RRM2. In some embodiments, the nucleic acid encoding the selection agent or the detection agent is integrated in the 3’ UTR of RRM2.

[0075] In some embodiments, the endogenous proliferation gene is BIRC5. In some embodiments, the nucleic acid encoding a selection agent or a detection agent is integrated at an exon of BIRC5, such as any one of exons 1, 2, 3, or 4. In some embodiments, the nucleic acid encoding a selection agent or a detection agent is integrated at an intron of BIRC5, such as any one of introns 1, 2, or 3. In some embodiments, the nucleic acid encoding the selection agent or the detection agent is integrated in the 5’ UTR of BIRC5. In some embodiments, the nucleic acid encoding the selection agent or the detection agent is integrated in the 3’ UTR of BIRC5.

[0076] In some embodiments, the endogenous proliferation gene is TOP2A. In some embodiments, the nucleic acid encoding a selection agent or a detection agent is integrated at an exon of TOP2A, such as any one of exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35. In some embodiments, the nucleic acid encoding a selection agent or a detection agent is integrated at an intron of TOP2A, such as any one of introns 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34. In some embodiments, the nucleic acid encoding the selection agent or the detection agent is integrated in the 5’ UTR of TOP2A. In some embodiments, the nucleic acid encoding the selection agent or the detection agent is integrated in the 3’ UTR of TOP2A.

[0077] In some embodiments, the endogenous proliferation gene is PTTG1. In some embodiments, the nucleic acid encoding a selection agent or a detection agent is integrated at an exon of PTTG1, such as any one of exons 1, 2, 3, 4, 5, or 6. In some embodiments, the nucleic acid encoding a selection agent or a detection agent is integrated at an intron of PTTG1, such as any one of introns 1, 2, 3, 4, or 5. In some embodiments, the nucleic acid encoding the selection agent or the detection agent is integrated in the 5’ UTR of PTTG1. Insome embodiments, the nucleic acid encoding the selection agent or the detection agent is integrated in the 3’ UTR of PTTG1.

[0078] In some embodiments, the endogenous proliferation gene is CCNB1. In some embodiments, the nucleic acid encoding a selection agent or a detection agent is integrated at an exon of CCNB1, such as any one of exons 1, 2, 3, 4, 5, 6, 7, 8, or 9. In some embodiments, the nucleic acid encoding a selection agent or a detection agent is integrated at an intron of CCNB1, such as any one of introns 1, 2, 3, 4, 5, 6, 7, or 8. In some embodiments, the nucleic acid encoding the selection agent or the detection agent is integrated in the 5’ UTR of CCNB1. In some embodiments, the nucleic acid encoding the selection agent or the detection agent is integrated in the 3’ UTR of CCNB1.

[0079] In some embodiments, the endogenous proliferation gene is TPX2. In some embodiments, the nucleic acid encoding a selection agent or a detection agent is integrated at an exon of TPX2, such as any one of exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18. In some embodiments, the nucleic acid encoding a selection agent or a detection agent is integrated at an intron of TPX2, such as any one of introns 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17. In some embodiments, the nucleic acid encoding the selection agent or the detection agent is integrated in the 5’ UTR of TPX2. In some embodiments, the nucleic acid encoding the selection agent or the detection agent is integrated in the 3’ UTR of TPX2.

[0080] In some embodiments, the endogenous proliferation gene is KIF11. In some embodiments, the nucleic acid encoding a selection agent or a detection agent is integrated at an exon of KIF11, such as any one of exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22. In some embodiments, the nucleic acid encoding a selection agent or a detection agent is integrated at an intron of KIF11, such as any one of introns 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21. In some embodiments, the nucleic acid encoding the selection agent or the detection agent is integrated in the 5’ UTR of KIF11. In some embodiments, the nucleic acid encoding the selection agent or the detection agent is integrated in the 3’ UTR of KIF11.

[0081] In some embodiments, the endogenous proliferation gene is SPC25. In some embodiments, the nucleic acid encoding a selection agent or a detection agent is integrated at an exon of SPC25, such as any one of exons 1, 2, 3, 4, 5, 6, or 7. In some embodiments, the nucleic acid encoding a selection agent or a detection agent is integrated at an intron of SPC25, such as any one of introns 1, 2, 3, 4, 5, or 6. In some embodiments, the nucleic acid encoding the selection agent or the detection agent is integrated in the 5’ UTR of SPC25. Insome embodiments, the nucleic acid encoding the selection agent or the detection agent is integrated in the 3’ UTR of SPC25.

[0082] In some embodiments, the endogenous proliferation gene is CENPK. In some embodiments, the nucleic acid encoding a selection agent or a detection agent is integrated at an exon of CENPK, such as any one of exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11. In some embodiments, the nucleic acid encoding a selection agent or a detection agent is integrated at an intron of CENPK, such as any one of introns 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the nucleic acid encoding the selection agent or the detection agent is integrated in the 5’ UTR of CENPK. In some embodiments, the nucleic acid encoding the selection agent or the detection agent is integrated in the 3’ UTR of CENPK.

[0083] In some embodiments, the endogenous proliferation gene is SMC4. In some embodiments, the nucleic acid encoding a selection agent or a detection agent is integrated at an exon of SMC4, such as any one of exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24. In some embodiments, the nucleic acid encoding a selection agent or a detection agent is integrated at an intron of SMC4, such as any one of introns 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23. In some embodiments, the nucleic acid encoding the selection agent or the detection agent is integrated in the 5’ UTR of SMC4. In some embodiments, the nucleic acid encoding the selection agent or the detection agent is integrated in the 3’ UTR of SMC4.

[0084] In some embodiments, the endogenous proliferation gene is TYMS. In some embodiments, the nucleic acid encoding a selection agent or a detection agent is integrated at an exon of TYMS, such as any one of exons 1, 2, 3, 4, 5, 6, or 7. In some embodiments, the nucleic acid encoding a selection agent or a detection agent is integrated at an intron of TYMS, such as any one of introns 1, 2, 3, 4, 5, or 6. In some embodiments, the nucleic acid encoding the selection agent or the detection agent is integrated in the 5’ UTR of TYMS. In some embodiments, the nucleic acid encoding the selection agent or the detection agent is integrated in the 3’ UTR of TYMS.

[0085] In some embodiments, the endogenous proliferation gene is H2AZ1. In some embodiments, the nucleic acid encoding a selection agent or a detection agent is integrated at an exon of H2AZ1, such as any one of exons 1, 2, 3, 4, or 5. In some embodiments, the nucleic acid encoding a selection agent or a detection agent is integrated at an intron of H2AZ1, such as any one of introns 1, 2, 3, or 4. In some embodiments, the nucleic acid encoding the selection agent or the detection agent is integrated in the 5’ UTR of H2AZ1. Insome embodiments, the nucleic acid encoding the selection agent or the detection agent is integrated in the 3’ UTR of H2AZ1.

[0086] In some embodiments, the endogenous proliferation gene is TMSB15A. In some embodiments, the nucleic acid encoding a selection agent or a detection agent is integrated at an exon of TMSB15A, such as any one of exons 1, 2, or 3. In some embodiments, the nucleic acid encoding a selection agent or a detection agent is integrated at an intron of TMSB 15A, such as any one of introns 1 or 2. In some embodiments, the nucleic acid encoding the selection agent or the detection agent is integrated in the 5’ UTR of TMSB 15 A. In some embodiments, the nucleic acid encoding the selection agent or the detection agent is integrated in the 3’ UTR of TMSB 15 A.

[0087] In some embodiments, the endogenous proliferation gene is CENPF. In some embodiments, the nucleic acid encoding a selection agent or a detection agent is integrated at an exon of CENPF, such as any one of exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some embodiments, the nucleic acid encoding a selection agent or a detection agent is integrated at an intron of CENPF, such as any one of introns 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19. In some embodiments, the nucleic acid encoding the selection agent or the detection agent is integrated in the 5’ UTR of CENPF. In some embodiments, the nucleic acid encoding the selection agent or the detection agent is integrated in the 3’ UTR of CENPF.

[0088] In some embodiments, the endogenous proliferation gene is MKI67. In some embodiments, the nucleic acid encoding a selection agent or a detection agent is integrated at an exon of MKI67, such as any one of exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, the nucleic acid encoding a selection agent or a detection agent is integrated at an intron of MKI67, such as any one of introns 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14. In some embodiments, the nucleic acid encoding the selection agent or the detection agent is integrated in the 5’ UTR of MKI67. In some embodiments, the nucleic acid encoding the selection agent or the detection agent is integrated in the 3’ UTR of MKI67.

[0089] In some embodiments, the modified cell described herein has at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% gene expression at the modified locus relative to the unmodified locus. In some embodiments, the modified cell described herein has 100% gene expression at the modified locus relative to that of the unmodified locus. In some embodiments, the modified cell described herein has a growth rate equal to or faster than that of unmodified cells.B. Off -Target Cell Marker Genes

[0090] Cell types in the body exhibit diverse properties in many modalities, including molecular, morphological, physiological, and functional. Cellular properties at the individual cell level are highly heterogeneous. Variations in different modalities do not necessarily exhibit high degrees of unity. However, single-cell transcriptomics has allowed robust cell type classification. Molecular approaches for profiling a cell type using single-cell or singlenucleus RNA- sequencing are used to generate comprehensive atlases of cell types and their markers. Off-target cell marker genes are genes that are highly expressed in one cell type, but are lowly expressed in other types. Off-target cell marker genes define cellular identity. The off-target cell marker gene may be specific to sample type and / or species. Off-target cell marker genes provide insights into the core set of genes whose expression is shared among all cell of a given type and have been used to annotate cell clusters and to examine the cellular composition of bulk tissues. Measurement of specific off-target cell marker genes allows for the identification and verification of the desired differentiated cell type needed for gene or cell therapy.

[0091] In some embodiments, there is provided a modified cell comprising a nucleic acid encoding a selection agent or a detection agent integrated at an off-target cell type gene locus and operably linked to the promoter of the off-target cell marker gene, wherein expression of the nucleic acid encoding the selection agent or the detection agent is regulated by the promoter of the off-target cell marker gene and / or regulatory elements of the off-target cell marker gene. In some embodiments, there is provided a modified cell comprising a nucleic acid encoding a selection agent and a detection agent integrated at an off-target cell type gene locus and operably linked to the promoter of the off-target cell type gene, wherein expression of the nucleic acid encoding the selection agent and the detection agent is regulated by the promoter of the off-target cell type gene and / or regulatory elements of the off-target cell type gene. In some embodiments, there is provided a modified cell comprising a nucleic acid encoding a first detection agent and a second detection agent, wherein the nucleic acid is integrated at an off-target cell type gene locus and operably linked to the promoter of the off-target cell type gene, and wherein expression of the nucleic acid encoding the first detection agent and the second detection agent is regulated by the promoter of the off-target cell type gene and / or regulatory elements of the off-target cell type gene. In some embodiments, there is provided a modified cell comprising a nucleic acid encoding a first selection agent and a second selection agent, wherein the nucleic acid is integrated at an off-target cell type gene locus and operably linked to the promoter of the off-target cell type gene, and wherein expression of the nucleic acid encoding the first selection agent and the second selection agent is regulated by the promoter of the off-target cell type gene and / or regulatory elements of the off-target cell type gene. In some embodiments, the first selection agent and the second selection agent are operably linked. In some embodiments, the first selection agent and the second selection agent are operably linked by an internal ribosome entry site. In some embodiments, the first selection agent and the second selection agent are operably linked by a self-cleaving peptide. In some embodiments, there is provided a modified cell comprising a first nucleic acid encoding a selection agent and a second nucleic acid encoding a detection agent, wherein the first nucleic acid is integrated at a first off-target cell type gene locus and operably linked to the promoter of the first off-target cell type gene, wherein expression of the first nucleic acid encoding the selection agent is regulated by the promoter of the first off-target cell type gene and / or regulatory elements of the first off-target cell type gene, wherein the second nucleic acid is integrated at a second off-target cell type gene locus and operably linked to the promoter of the second off-target cell type gene, and wherein expression of the second nucleic acid encoding the detection agent is regulated by the promoter of the second off-target cell type gene and / or regulatory elements of the off- target cell type gene. In some embodiments, there is provided a modified cell comprising a first nucleic acid encoding a first selection agent and a second nucleic acid encoding a second selection agent, wherein the first nucleic acid is integrated at a first off-target cell type gene locus and operably linked to the promoter of the first off-target cell type gene, wherein expression of the first nucleic acid encoding the first selection agent is regulated by the promoter of the first off-target cell type gene and / or regulatory elements of the first off-target cell type gene, wherein the second nucleic acid is integrated at a second off-target cell type gene locus and operably linked to the promoter of the second off-target cell type gene, and wherein expression of the second nucleic acid encoding the second selection agent is regulated by the promoter of the second off-target cell type gene and / or regulatory elements of the off-target cell type gene. In some embodiments, there is provided a modified cell comprising a first nucleic acid encoding a first detection agent and a second nucleic acid encoding a second detection agent, wherein the first nucleic acid is integrated at a first off- target cell type gene locus and operably linked to the promoter of the first off-target cell type gene, wherein expression of the first nucleic acid encoding the first detection agent is regulated by the promoter of the first off-target cell type gene and / or regulatory elements of the first off-target cell type gene, wherein the second nucleic acid is integrated at a secondoff-target cell type gene locus and operably linked to the promoter of the off-target cell type gene, and wherein expression of the second nucleic acid encoding the second detection agent is regulated by the promoter of the second off-target cell type gene and / or regulatory elements of the second off-target cell type gene. In some embodiments, the first endogenous proliferation gene and / or the second endogenous proliferation gene are expressed in a cell that is included in a population of therapeutic cells. In some embodiments, the first endogenous proliferation gene and / or the second endogenous proliferation gene are expressed in a cell that is included in a population of therapeutic cells but is not a therapeutic cell. In some embodiments, the cell is an off-target cell. In some embodiments, the first off-target cell type gene and the second off-target cell type gene are the same, and the first and second nucleic acids are integrated at different alleles. In some embodiments, first off-target cell type gene and the second off-target cell type gene are different. In some embodiments, the nucleic acid encoding the selection agent or the detection agent is integrated in an exon or an intron of the off-target cell marker gene. In some embodiments, the nucleic acid encoding the selection agent or the detection agent is integrated in a 5’ UTR or a 3’ UTR of the off-target cell marker gene. In some embodiments, following integration, the off-target cell marker gene locus comprises i) nucleic acid encoding the off-target cell marker gene, ii) an internal ribosomal entry site or a self-cleaving RNA site, and iii) the nucleic acid encoding the selection agent or a detection agent. Examples of off-target cell marker genes include, but are not limited to, ANXA1, KRT19, CTSC, DSC2, ARHGAP29, KRT18, KRT8, CD9, PLK2, and KRT17.

[0092] In some embodiments, the off-target cell marker gene is ANXA1. In some embodiments, the nucleic acid encoding a selection agent or a detection agent is integrated at an exon of ANXA1, such as any one of exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13. In some embodiments, the nucleic acid encoding a selection agent or a detection agent is integrated at an intron of ANXA1, such as any one of introns 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. In some embodiments, the nucleic acid encoding the selection agent or the detection agent is integrated in the 5’ UTR of ANXA1. In some embodiments, the nucleic acid encoding the selection agent or the detection agent is integrated in the 3’ UTR of ANXA1.

[0093] In some embodiments, the off-target cell marker gene is KRT19. In some embodiments, the nucleic acid encoding a selection agent or a detection agent is integrated at an exon of KRT19, such as any one of exons 1, 2, 3, 4, 5, or 6. In some embodiments, the nucleic acid encoding a selection agent or a detection agent is integrated at an intron of 1KRT19, such as any one of introns 1, 2, 3, 4, or 5. In some embodiments, the nucleic acid encoding the selection agent or the detection agent is integrated in the 5’ UTR of KRT19. In some embodiments, the nucleic acid encoding the selection agent or the detection agent is integrated in the 3’ UTR of KRT19.

[0094] In some embodiments, the off-target cell marker gene is CTSC. In some embodiments, the nucleic acid encoding a selection agent or a detection agent is integrated at an exon of CTSC, such as any one of exons 1, 2, 3, 4, 5, 6, or 7. In some embodiments, the nucleic acid encoding a selection agent or a detection agent is integrated at an intron of CTSC, such as any one of introns 1, 2, 3, 4, 5, or 6. In some embodiments, the nucleic acid encoding the selection agent or the detection agent is integrated in the 5’ UTR of CTSC. In some embodiments, the nucleic acid encoding the selection agent or the detection agent is integrated in the 3’ UTR of CTSC.

[0095] In some embodiments, the off-target cell marker gene is DSC2. In some embodiments, the nucleic acid encoding a selection agent or a detection agent is integrated at an exon of DSC2, such as any one of exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some embodiments, the nucleic acid encoding a selection agent or a detection agent is integrated at an intron of DSC2, such as any one of introns 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, the nucleic acid encoding the selection agent or the detection agent is integrated in the 5’ UTR of DSC2. In some embodiments, the nucleic acid encoding the selection agent or the detection agent is integrated in the 3’ UTR of DSC2.

[0096] In some embodiments, the off-target cell marker gene is ARHGAP29. In some embodiments, the nucleic acid encoding a selection agent or a detection agent is integrated at an exon of ARHGAP29, such as any one of exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some embodiments, the nucleic acid encoding a selection agent or a detection agent is integrated at an intron of ARHGAP29, such as any one of introns 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, the nucleic acid encoding the selection agent or the detection agent is integrated in the 5’ UTR of ARHGAP29. In some embodiments, the nucleic acid encoding the selection agent or the detection agent is integrated in the 3’ UTR of ARHGAP29.

[0097] In some embodiments, the off-target cell marker gene is KRT18. In some embodiments, the nucleic acid encoding a selection agent or a detection agent is integrated at an exon of KRT18, such as any one of exons 1, 2, 3, 4, 5, 6, or 7. In some embodiments, the nucleic acid encoding a selection agent or a detection agent is integrated at an intron ofKRT18, such as any one of introns 1, 2, 3, 4, 5, or 6. In some embodiments, the nucleic acidencoding the selection agent or the detection agent is integrated in the 5’ UTR of KRT18. In some embodiments, the nucleic acid encoding the selection agent or the detection agent is integrated in the 3’ UTR of KRT18.

[0098] In some embodiments, the off-target cell marker gene is KRT8. In some embodiments, the nucleic acid encoding a selection agent or a detection agent is integrated at an exon of KRT8, such as any one of exons 1, 2, 3, 4, 5, 6, 7, or 8. In some embodiments, the nucleic acid encoding a selection agent or a detection agent is integrated at an intron of KRT8, such as any one of introns 1, 2, 3, 4, 5, 6, or 7. In some embodiments, the nucleic acid encoding the selection agent or the detection agent is integrated in the 5’ UTR of KRT8. In some embodiments, the nucleic acid encoding the selection agent or the detection agent is integrated in the 3’ UTR of KRT8.

[0099] In some embodiments, the off-target cell marker gene is CD9. In some embodiments, the nucleic acid encoding a selection agent or a detection agent is integrated at an exon of CD9, such as any one of exons 1, 2, 3, 4, 5, 6, 7, or 8. In some embodiments, the nucleic acid encoding a selection agent or a detection agent is integrated at an intron of CD9, such as any one of introns 1, 2, 3, 4, 5, 6, or 7. In some embodiments, the nucleic acid encoding the selection agent or the detection agent is integrated in the 5’ UTR of CD9. In some embodiments, the nucleic acid encoding the selection agent or the detection agent is integrated in the 3’ UTR of CD9.

[0100] In some embodiments, the off-target cell marker gene is PLK2. In some embodiments, the nucleic acid encoding a selection agent or a detection agent is integrated at an exon of PLK2, such as any one of exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14. In some embodiments, the nucleic acid encoding a selection agent or a detection agent is integrated at an intron of PLK2, such as any one of introns 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13. In some embodiments, the nucleic acid encoding the selection agent or the detection agent is integrated in the 5’ UTR of PLK2. In some embodiments, the nucleic acid encoding the selection agent or the detection agent is integrated in the 3’ UTR of PLK2.

[0101] In some embodiments, the off-target cell marker gene is KRT17. In some embodiments, the nucleic acid encoding a selection agent or a detection agent is integrated at an exon of KRT17, such as any one of exons 1, 2, 3, 4, 5, 6, 7, or 8. In some embodiments, the nucleic acid encoding a selection agent or a detection agent is integrated at an intron of KRT17, such as any one of introns 1, 2, 3, 4, 5, 6, or 7. In some embodiments, the nucleic acid encoding the selection agent or the detection agent is integrated in the 5’ UTR ofKRT17. In some embodiments, the nucleic acid encoding the selection agent or the detection agent is integrated in the 3’ UTR of KRT17.

[0102] As used herein, an “IC50” is used to indicate the effective concentration of an inducer or activator needed to achieve 50% cell viability in vitro. IC50 can be measured by bioassays such as a dosing kill curve. The values are typically expressed as molar concentration. In some embodiments, the gene candidate was chosen based on the IC50 in a dosing kill curve assay. In some embodiments, the IC50 value of the inducer in causing 50% cell death at the iPSC stage is less than about 50 pM, about 49 pM, about 48 pM, about 47 pM, about 46 pM, about 45 pM, about 44 pM, about 43 pM, about 42 pM, about 41 pM, about 40 pM, about 39 pM, about 38 pM, about 37 pM, about 36 pM, about 35 pM, about 34 pM, about 33 pM, about 32 pM, about 31 pM, about 30 pM, about 29 pM, about 28 pM, about 27 pM, about 26 pM, about 25 pM, about 24 pM, about 23 pM, about 22 pM, about 21 pM, about 20 pM, about 19 pM, about 18 pM, about 17 pM, about 16 pM , about 15 pM, about 14 pM, about 13 pM, about 12 pM, about 11 pM, about 10 pM, about 9 pM, about 8 pM, about 7 pM, about 6 pM, about 5 pM, about 4 pM, about 3 pM, about 2 pM, or about 1 pM.

[0103] In some embodiments, the gene candidate was chosen based on the percentage of viable cells in the non-proliferative state (e.g., day 18 of differentiation). In some embodiments, the percentage of resistant cells in the non-proliferative state (e.g., day 18 of differentiation) is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%.C. Exogenous Sequences

[0104] The cells of the present disclosure may be modified to include any exogenous nucleic acid sequence of interest at an endogenous proliferation gene or an off-target cell marker gene, e.g., as described above. In some embodiments, the exogenous sequence(s) inserted at an endogenous proliferation gene or an off-target cell marker gene comprise of selection agents, detection agents, genome editing complexes, and combinations thereof, as described in greater detail below.(1) Transgenes i. Kill Switches

[0105] In some embodiments, the one or more transgenes comprise a kill switch. In some embodiments, the selection agent is a kill switch. A “kill switch” can cause the death of the cell, such as after the modified cell is administered to a subject and if the cells should grow and divide in an undesired manner. A kill switch can be activated by a specific compound. The result is specifically eliminating cells expressing a specific gene (e.g., an endogenous proliferation gene or an off-target cell marker gene). Inclusion of a kill switch allows for controlled killing of the cells in the event of cytotoxicity or other negative consequences to the recipient, thus increasing the safety of cell-based therapies.

[0106] In some embodiments, a kill switch can be incorporated into, such as introduced, into a modified cell provided herein to provide the ability to induce death or apoptosis of modified cell containing the kill switch, for example if the cells grow and divide in an undesired manner or cause excessive toxicity to the host. Thus, the use of kill switches enables one to conditionally eliminate aberrant cells in vivo and can be a critical step for the application of cell therapies in the clinic. Kill switches and their uses thereof are described in, for example, Duzgune§, Origins of Suicide Gene Therapy (2019); Duzgune§ (eds), Suicide Gene Therapy. Methods in Molecular Biology, vol. 1895 (Humana Press, New York, NY) (for HSV-tk, cytosine deaminase, nitroreductase, purine nucleoside phosphorylase, and horseradish peroxidase); Zhou and Brenner, Exp Hematol 44(11): 1013-1019 (2016) (for iCaspase9); Wang et al., Blood 18(5): 1255- 1263 (2001) (for huEGFR); U.S. Patent Application Publication No. 20180002397 (for HER1); and Philip et al., Bloodl24(8):1277- 1287 (2014) (for RQR8).

[0107] In some embodiments, the kill switch can cause cell death in a controlled manner, for example, in the presence of a drug or prodrug or upon activation by a selective exogenous compound. In some embodiments, the kill 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), chemically regulated-SH2-delivered inhibitory tail (CRASH-IT), CCR4, CD16, CD19, CD20, CD30, EGFR, GD2, HER1, HER2, MUC1, PSMA, and RQR8.

[0108] In some embodiments, the kill switch may be a transgene encoding a product with cell killing capabilities when activated by a drug or prodrug, for example, by turning a non-toxic prodrug to a toxic metabolite inside the cell. In these embodiments, cell killing is activated by contacting a modified cell with the drug or prodrug. In some cases, the kill switch is HSV-tk, which converts ganciclovir (GCV) to GCV-triphosphate, therebyinterfering with DNA synthesis and killing dividing cells. In some cases, the kill switch is CyD or a variant thereof, which converts the antifungal drug 5-fluorocytosine (5-FC) to cytotoxic 5-fluorouracil (5-FU) by catalyzing the hydrolytic deamination of cytosine into uracil. 5-FU is further converted to potent anti-metabolites (5- FdUMP, 5-FdUTP, 5-FUTP) by cellular enzymes. These compounds inhibit thymidylate synthase and the production of RNA and DNA, resulting in cell death. In some cases, the kill switch is NTR or a variant thereof, which can act on the prodrug CB 1954 via reduction of the nitro groups to reactive N-hydroxylamine intermediates that are toxic in proliferating and nonproliferating cells. In some cases, the kill switch is PNP or a variant thereof, which can turn prodrug 6- methylpurine deoxyriboside or fludarabine into toxic metabolites to both proliferating and nonproliferating cells. In some cases, the kill switch is horseradish peroxidase or a variant thereof, which can catalyze indole-3-acetic acid (IAA) to a potent cytotoxin and thus achieve cell killing. In other embodiments, the suicide gene is a cytosine deaminase (e.g., the Escherichia coli cytosine deaminase (EC-CD)) gene, and the trigger is 5-fluorocytosine (5- FC) (Barese et al., Mol. Therap. 20(10): 1932-1943 (2012) and Xu et al., Cell Res. 8:73-8 (1998), both incorporated herein by reference in their entirety). In some embodiments, the kill switch and genes associated with the kill switch are expressed from a bicistronic cassette. In some embodiments, the kill switch and one or more tolerogenic factors are expressed from a bicistronic cassette.

[0109] In some embodiments, the kill switch may be an iCasp9. Caspase 9 is a component of the intrinsic mitochondrial apoptotic pathway which, under physiological conditions, is activated by the release of cytochrome C from damaged mitochondria. Activated caspase 9 then activates caspase 3, which triggers terminal effector molecules leading to apoptosis. The iCasp9 may be generated by fusing a truncated caspase 9 (without its physiological dimerization domain or caspase activation domain) to a FK506 binding protein (FKBP), FKBP12-F36V, via a peptide linker. The iCasp9 has low dimer-independent basal activity and can be stably expressed in host cells (e.g., human T cells) without impairing their phenotype, function, or antigen specificity. However, in the presence of chemical inducer of dimerization (CID), such as rimiducid (AP1903), AP20187, and rapamycin, iCasp9 can undergo inducible dimerization and activate the downstream caspase molecules, resulting in apoptosis of cells expressing the iCasp9. See, e.g., PCT Application Publication No. WO2011 / 146862; Stasi et al., N. Engl. J. Med. 365; 18 (2011); Tey et al., Biol. Blood Marrow Transplant 13:913-924 (2007). In particular, the rapamycin-induciblecaspase 9 variant is called rapaCasp9. See Stavrou et al., Mai. Ther. 26(5): 1266- 1276 (2018). Thus, iCasp9 can be used as a kill switch to achieve controlled killing of the host cells.

[0110] In some embodiments, the kill switch may be a membrane-expressed protein, which allows for cell depletion after administration of a specific antibody to that protein. Kill switches of this category may include, for example, one or more transgene encoding CCR4, CD16, CD19, CD20, CD30, EGFR, GD2, HER1, HER2, MUC1, PSMA, or RQR8 for surface expression thereof. These proteins may have surface epitopes that can be targeted by specific antibodies. In some embodiments, the kill switch comprises CCR4, which can be recognized by an anti-CCR4 antibody. Non-limiting examples of suitable anti-CCR4 antibodies include mogamulizumab and biosimilars thereof. In some embodiments, the kill switch comprises CD 16 or CD30, which can be recognized by an anti-CD16 or anti-CD30 antibody. Non-limiting examples of such antiCD 16 or anti-CD30 antibody include AFM13 and biosimilars thereof. In some embodiments, the kill switch comprises CD19, which can be recognized by an anti-CD19 antibody. Non-limiting examples of such anti-CD19 antibody include MOR208 and biosimilars thereof. In some embodiments, the kill switch comprises CD20, which can be recognized by an anti-CD20 antibody. Non-limiting examples of such anti-CD20 antibody include obinutuzumab, ublituximab, ocaratuzumab, rituximab, rituximab-Rllb, and biosimilars thereof. Cells that express the kill switch are thus CD20- positive and can be targeted for killing through administration of an anti-CD20 antibody as described. In some embodiments, the kill switch comprises EGFR, which can be recognized by an anti-EGFR antibody. Non-limiting examples of such anti-EGFR antibody include tomuzotuximab, RO5083945 (GA201), cetuximab, and biosimilars thereof. In some embodiments, the kill switch comprises GD2, which can be recognized by an anti-GD2 antibody. Non-limiting examples of such anti-GD2 antibody include Hul4.18K322A, Hul4.18-IL2, Hu3F8, dinituximab, c.60C3-Rllc, and biosimilars thereof.

[0111] In some embodiments, the expression of a detection agent acts as a signal for the administration of an antibody directed against or specific to a tolerogenic agent, e.g., an anti- CD47 antibody. In some embodiments, the kill switch may be an exogenously administered agent that recognizes one or more tolerogenic factor on the surface of the modified cells. In some embodiments, the exogenously administered agent is an antibody directed against or specific to a tolerogenic agent, e.g., an anti-CD47 antibody. By recognizing and blocking a tolerogenic factor on modified cells, an exogenously administered antibody may block the immune inhibitory functions of the tolerogenic factor thereby re- sensitizing the immune system to the modified cells. For instance, for modified cells that overexpresses CD47, anexogenously administered anti-CD47 antibody may be administered to the subject, resulting in masking of CD47 on the modified cells and triggering of an immune response to the modified cells. In some embodiments, the anti-CD47 antibody is Magrolimab.

[0112] In some the safety switch comprises an anti-CD47 antibody. In some embodiments, the anti-CD47 antibody is Magrolimab. In some embodiments, the safety switch is Magrolimab.

[0113] In some embodiments, the method further comprises introducing an expression vector comprising an inducible suicide switch into the cell.

[0114] In some embodiments, the tolerogenic factor is CD47 and the cell includes an exogenous polynucleotide encoding a CD47 protein. In some embodiments, the cell expresses an exogenous CD47 polypeptide.

[0115] In some embodiments, a method disclosed herein comprises administering to a subject in need thereof a CD47-SIRPa blockade agent, wherein the subject was previously administered modified cells engineered to express an exogenous CD47 polypeptide. In some embodiments, the CD47-SIRPa blockade agent comprises a CD47-binding domain. In some embodiments, the CD47-binding domain comprises signal regulatory protein alpha (SIRPa) or a fragment thereof. In some embodiments, the CD47-SIRPa blockade agent comprises an immunoglobulin G (IgG) Fc domain. In some embodiments, the IgG Fc domain comprises an IgGl Fc domain. In some embodiments, the IgGl Fc domain comprises a fragment of a human antibody. In some embodiments, the CD47-SIRPa blockade agent is selected from the group consisting of TTI-621, TTI-622, and ALX148. In some embodiments, the CD47- SIRPa blockade agent is TTI-621, TTI-622, and ALX148. In some embodiments, the CD47- SIRPa blockade agent is TTI-622. In some embodiments, the CD47-SIRPa blockade agent is ALX148. In some embodiments, the IgG Fc domain comprises an IgG4 Fc domain. In some embodiments, the CD47-SIRPa blockade agent is an antibody. In some embodiments, the antibody is selected from the group consisting of MIAP410, B6H12, and Magrolimab. In some embodiments, the antibody is MIAP410. In some embodiments, the antibody is B6H12. In some embodiments, the antibody is Magrolimab. In some embodiments, the antibody is selected from the group consisting of AO-176, IBI188 (letaplimab), STI-6643, and ZL-1201. In some embodiments, the antibody is AO- 176 (Arch). In some embodiments, the antibody is IBI188 (letaplimab) (Innovent). In some embodiments, the antibody is STI-6643 (Sorrento). In some embodiments, the antibody is ZL-1201 (Zai).

[0116] In some embodiments, useful antibodies or fragments thereof that bind CD47 can be selected from a group that includes magrolimab ((Hu5F9-G4)) (Forty Seven, Inc.;Gilead Sciences, Inc.), urabrelimab, CC-90002 (Celgene; Bristol-Myers Squibb), IBI-188 (Innovent Biologies), IBI-322 (Innovent Biologies), TG-1801 (TG Therapeutics; also known as NI-1701, Novimmune SA), ALX148 (ALX Oncology), TJ011133 (also known as TJC4, 1- Mab Biopharma), FA3M3, ZL-1201 (Zai Lab Co., Ltd), AK117 (Akesbio Australia Pty, Ltd.), AO- 176 (Arch Oncology), SRF231 (Surface Oncology), GenSci-059 (GeneScience), C47B157 (Janssen Research and Development), C47B161 (Janssen Research and Development), C47B167 (Janssen Research and Development), C47B222 (Janssen Research and Development), C47B227 (Janssen Research and Development), Vx-1004 (Corvus Pharmaceuticals), HMBD004 (Hummingbird Bioscience Pte Ltd), SHR-1603 (Hengrui), AMMS4-G4 (Beijing Institute of Biotechnology), RTX-CD47 (University of Groningen), and IMC-002. (Samsung Biologies; ImmuneOncia Therapeutics). In some embodiments, the antibody or fragment thereof does not compete for CD47 binding with an antibody selected from a group that includes magrolimab, urabrelimab, CC-90002, IBI-188, IBI-322, TG-1801 (NI-1701), ALX148, TJ011133, FA3M3, ZL1201, AK117, AO-176, SRF231, GenSci-059, C47B157, C47B161, C47B167, C47B222, C47B227, Vx-1004, HMBD004, SHR-1603, AMMS4-G4, RTX-CD47, and IMC-002. In some embodiments, the antibody or fragment thereof competes for CD47 binding with an antibody selected from magrolimab, urabrelimab, CC-90002, IBI-188, IBI-322, TG-1801 (NI-1701), ALX148, TJ011133, FA3M3, ZL1201, AK117, AO-176, SRF231, GenSci-059, C47B157, C47B161, C47B167, C47B222, C47B227, Vx-1004, HMBD004, SHR-1603, AMMS4-G4, RTX-CD47, and IMC-002. In some embodiments, the antibody or fragment thereof that binds CD47 is selected from a group that includes a single-chain Fv fragment (scFv) against CD47, a Fab against CD47, a VHH nanobody against CD47, a DARPin against CD47, and variants thereof. In some embodiments, the scFv against CD47, a Fab against CD47, and variants thereof are based on the antigen binding domains of any of the antibodies selected from a group that includes magrolimab, urabrelimab, CC-90002, IBI-188, IBI-322, TG-1801 (NI-1701), ALX148, TJ011133, FA3M3, ZL1201, AK117, AO-176, SRF231, GenSci-059, C47B157, C47B161, C47B167, C47B222, C47B227, Vx-1004, HMBD004, SHR-1603, AMMS4-G4, RTX-CD47, and IMC-002.

[0117] In some embodiments, the CD47 antagonist provides CD47 blockade. Methods and agents for CD47 blockade are described in PCT / US2021 / 054326, which is incorporated by reference in its entirety.

[0118] In some embodiments, the modified cells are derived from a source cell already comprising one or more of the desired modifications. In some embodiments, in view of theteachings provided herein one of ordinary skill in the art will readily appreciate how to assess what modifications are required to arrive at the desired final form of a modified cell and that not all reduced or increased levels of target components are achieved via active engineering. In some embodiments, the modifications of the modified cells may be in any order, and not necessarily the order listed in the descriptive language provided herein.

[0119] Once altered, the presence of expression of any of the molecule described herein can be assayed using known techniques, such as Western blots, ELISA assays, FACS assays, flow cytometry, and the like.

[0120] In some embodiments, the kill switch can include any of the strategies as described in WO2021146627A1, which is incorporated by reference in its entirety.

[0121] In some embodiments, a kill switch may be introduced into a modified cell of the disclosure as part of an expression vector comprising, e.g., an inducible kill switch.

[0122] In some embodiments, there is provided a cell comprising a nucleic acid encoding a selection agent or a detection agent integrated at an endogenous proliferation gene locus and operably linked to the promoter of the endogenous proliferation gene, wherein expression of the nucleic acid encoding the selection agent or the detection agent is regulated by the promoter of the endogenous proliferation gene and / or regulatory elements of the endogenous proliferation gene. In some embodiments, the endogenous proliferation gene is selected from the group consisting of AURKB, CDK1, CDC20, RRM2, BIRC5, TOP2A, PTTG1, CCNB1, TPX2, KIF11, SPC25, CENPK, SMC4, TYMS, H2AZ1, TMSB15A, CENPF, and MKI67. In some embodiments, the selection agent is iCaspase 9 or cytosine deaminase. In some embodiments, (i) the endogenous proliferation gene is AURKB and the selection agent is iCaspase9; (ii) the endogenous proliferation gene is CDK1 and the selection agent is iCaspase9; (iii) the endogenous proliferation gene is CDC20 and the selection agent is iCaspase9; (iv) the endogenous proliferation gene is RRM2 and the selection agent is iCaspase9; (v) the endogenous proliferation gene is BIRC5 and the selection agent is iCaspase9; (vi) the endogenous proliferation gene is TOP2A and the selection agent is iCaspase9; (vii) the endogenous proliferation gene is PTTG1 and the selection agent is iCaspase9; (viii) the endogenous proliferation gene is CCNB1 and the selection agent is iCaspase9; (ix) the endogenous proliferation gene is TPX2 and the selection agent is iCaspase9; (x) the endogenous proliferation gene is KIF11 and the selection agent is iCaspase9; (xi) the endogenous proliferation gene is SPC25 and the selection agent is iCaspase9; (xii) the endogenous proliferation gene is CENPK and the selection agent is iCaspase9; (xiii) the endogenous proliferation gene is SMC4 and the selection agent isiCaspase9; (xiv) the endogenous proliferation gene is TYMS and the selection agent is iCaspase9; (xv) the endogenous proliferation gene is H2AZ1 and the selection agent is iCaspase9; (xvi) the endogenous proliferation gene is TMSB15A and the selection agent is iCaspase9; (xvii) the endogenous proliferation gene is CENPF and the selection agent is iCaspase9; (xviii) the endogenous proliferation gene is MKI67 and the selection agent is iCaspase9; (xix) the endogenous proliferation gene is AURKB and the selection agent is cytosine deaminase; (xx) the endogenous proliferation gene is CDK1 and the selection agent is cytosine deaminase; (xxi) the endogenous proliferation gene is CDC20 and the selection agent is cytosine deaminase; (xxii) the endogenous proliferation gene is RRM2 and the selection agent is cytosine deaminase; (xxiii) the endogenous proliferation gene is BIRC5 and the selection agent is cytosine deaminase; (xxiv) the endogenous proliferation gene is TOP2A and the selection agent is cytosine deaminase; (xxv) the endogenous proliferation gene is PTTG1 and the selection agent is cytosine deaminase; (xxvi) the endogenous proliferation gene is CCNB 1 and the selection agent is cytosine deaminase; (xxvii) the endogenous proliferation gene is TPX2 and the selection agent is cytosine deaminase; (xxviii) the endogenous proliferation gene is KIF11 and the selection agent is cytosine deaminase; (xxix) the endogenous proliferation gene is SPC25 and the selection agent is cytosine deaminase; (xxx) the endogenous proliferation gene is CENPK and the selection agent is cytosine deaminase; (xxxi) the endogenous proliferation gene is SMC4 and the selection agent is cytosine deaminase; (xxxii) the endogenous proliferation gene is TYMS and the selection agent is cytosine deaminase; (xxxiii) the endogenous proliferation gene is H2AZ1 and the selection agent is cytosine deaminase; (xxxiv) the endogenous proliferation gene is TMSB15A and the selection agent is cytosine deaminase; (xxxv) the endogenous proliferation gene is CENPF and the selection agent is cytosine deaminase; or (xxxvi) the endogenous proliferation gene is MKI67 and the selection agent is cytosine deaminase.

[0123] In some embodiments, the selection agent or the detection agent of the cell described herein is expressed in proliferating cells. In some embodiments, the selection agent or the detection agent of the cell described herein is not expressed in non-proliferating cells. In some embodiments, the selection agent or the detection agent of the cell described herein is expressed in partially differentiated cells. In some embodiments, the selection agent or the detection agent of the cell described herein is expressed in an intermediate cell type made during differentiation. In some embodiments, the selection agent or the detection agent of the cell described herein is not expressed in differentiated cells. In some embodiments, the selection agent or the detection agent of the cell described herein is not expressed in atherapeutic cell. In some embodiments, the cell is selected from the group consisting of a pancreatic islet cell, an alpha cell, a beta cell, a gamma cell, a delta cell, an epsilon cell, a T cell, a neuron, a glial cell, a cardiomyocyte, a retinal pigmented epithelial cell, a hematopoietic progenitor cell, a natural killer cell, an endothelial cell, and a lung cell.

[0124] In some embodiments, there is provided a cell comprising a nucleic acid encoding a selection agent or a detection agent integrated at an off-target cell type gene locus and operably linked to the promoter of the off-target cell marker gene, wherein expression of the nucleic acid encoding the selection agent or the detection agent is regulated by the promoter of the off-target cell marker gene and / or regulatory elements of the off-target cell marker gene. In some embodiments, the off-target cell marker gene is selected from the group consisting of a pluripotency cell marker gene, a tumorigenic cell marker gene, a ductal marker gene, an enterochromaffin marker gene, a neural marker gene, acinar marker gene, intestinal marker gene, endothelial marker gene, mesenchymal fibroblast marker gene, muscle marker gene, osteoblast marker gene, and stromal marker gene. In some embodiments, the off-target cell marker gene is selected from the group consisting of ANXA1, KRT19, CTSC, DSC2, ARHGAP29, KRT18, KRT8, CD9, PLK2, and KRT17. In some embodiments, the selection agent is iCaspase 9 or cytosine deaminase. In some embodiments, (i) the off-target cell marker gene is ANXA1 and the selection agent is iCaspase9; (ii) the off-target cell marker gene is KRT19 and the selection agent is iCaspase9; (iii) the off-target cell marker gene is CTSC and the selection agent is iCaspase9; (iv) the off- target cell marker gene is DSC2 and the selection agent is iCaspase9; (v) the off-target cell marker gene is ARHGAP29 and the selection agent is iCaspase9; (vi) the off-target cell marker gene is KRT18 and the selection agent is iCaspase9; (vii) the off-target cell marker gene is KRT8 and the selection agent is iCaspase9; (viii) the off-target cell marker gene is CD9 and the selection agent is iCaspase9; (ix) the off-target cell marker gene is PLK2 and the selection agent is iCaspase9; (x) the off-target cell marker gene is KRT17 and the selection agent is iCaspase9; (xi) the off-target cell marker gene is ANXA1 and the selection agent is cytosine deaminase; (xii) the off-target cell marker gene is KRT19 and the selection agent is cytosine deaminase; (xiii) the off-target cell marker gene is CTSC and the selection agent is cytosine deaminase; (xiv) the off-target cell marker gene is DSC2 and the selection agent is cytosine deaminase; (xv) the off-target cell marker gene is ARHGAP29 and the selection agent is cytosine deaminase; (xvi) the off-target cell marker gene is KRT18 and the selection agent is cytosine deaminase; (xvii) the off-target cell marker gene is KRT8 and the selection agent is cytosine deaminase; (xviii) the off-target cell marker gene is CD9 and the selectionagent is cytosine deaminase; (xix) the off-target cell marker gene is PLK2 and the selection agent is cytosine deaminase; or (xx) the off-target cell marker gene is KRT17 and the selection agent is cytosine deaminase.

[0125] In some embodiments, the selection agent or the detection agent of the cell described herein is expressed in a cell with a high level of expression of an off-target cell marker gene. In some embodiments, the selection agent or the detection agent is expressed in a cell with a high level of expression. In some embodiments, the selection agent or the detection agent is expressed in a cell with greater than about 3 transcripts per million (TPM), about 4 TPM, about 5 TPM, about 6 TPM, about 7 TPM, about 8 TPM, about 9 TPM, about 10 TPM, about 15 TPM, about 20 TPM, about 25 TPM, about 30 TPM, about 35 TPM, about 40 TPM, about 45 TPM, or about 50 TPM. In some embodiments, the selection agent or the detection agent is expressed in an off-target cell with greater than about 3 transcripts per million (TPM), about 4 TPM, about 5 TPM, about 6 TPM, about 7 TPM, about 8 TPM, about 9 TPM, about 10 TPM, about 15 TPM, about 20 TPM, about 25 TPM, about 30 TPM, about 35 TPM, about 40 TPM, about 45 TPM, or about 50 TPM. In some embodiments, the selection agent is expressed in a cell such that the selection agent enables selection of the cell. In some embodiments, the detection agent is expressed in a cell such that the detection agent enables detection of the cell.

[0126] In some embodiments, the selection agent or the detection agent of the cell described herein is expressed in a pluripotent cell. In some embodiments, the selection agent or the detection agent of the cell described herein is expressed in a tumorigenic cell. In some embodiments, the selection agent or the detection agent of the cell described herein is expressed in a ductal cell. In some embodiments, the selection agent or the detection agent of the cell described herein is expressed in an enterochromaffin cell. In some embodiments, the selection agent or the detection agent of the cell described herein is expressed in a neural cell. In some embodiments, the selection agent or the detection agent of the cell described herein is in partially differentiated cells. In some embodiments, the selection agent or the detection agent of the cell described herein is not expressed in differentiated cells. In some embodiments, the selection agent or the detection agent of the cell described herein is not expressed in a cell with a low level of expression or no expression of an off-target cell marker gene. In some embodiments, the selection agent or the detection agent of the cell described herein is not expressed in a therapeutic cell. In some embodiments, the off-target cell marker gene of the cell described herein is not a beta cell marker gene, a T cell marker gene, a glial cell marker gene, a cardiac cell marker gene, or a retinal pigment epithelium (RPE) cellmarker gene. ii. Genome-Editing Complexes

[0127] In some embodiments, the cell provided herein comprises a genome editing complex. Genome editing complexes are described in detail in Section C(2) below. In some embodiments, the one or more transgenes encode any of the genome editing complexes described in Section C(2) below.

[0128] In some specific embodiments, the genome editing complex comprises a genome targeting entity and / or a genome modifying entity. In some embodiments, the genome targeting entity is a nucleic acid-guided targeting entity, such as any of a sequence specific nuclease, a nucleic acid programmable DNA binding protein, an RNA guided nuclease, RNA-guided nuclease comprising a Cas nuclease and a guide RNA (CRISPR-Cas combination), a ribonucleoprotein (RNP) complex comprising a gRNA and a Cas nuclease, a homing endonuclease, a zinc finger nuclease (ZF) nucleic acid binding entity, a transcription 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 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), Cas 12g, Casl2h, Casl2i, Cas 12k (C2c5), Casl3, Casl3a (C2c2), Casl3b, Casl3c, Casl3d, C2c4, C2c8, C2c9, Cmrl, Cmr2, Cmr3, Cmr4, Cmr5, Cmr6, Csdl, Csd2, Cas5d, Csel, Cse2, Cse3, Cse4, Cas5e, Csfl, Csml, Csm2, Csm3, Csm4, Csm5, Csnl, Csn2, Cstl, Cst2, Cas5t, Cshl, Csh2, Cas5h, Csal, Csa2, Csa3, Csa4, Csa5, Cas5a, CsxlO, Csxl l, Csyl, Csy2, Csy3, Csy4, Mad7, SpCas9, eSpCas9, SpCas9-HFl, HypaSpCas9, HeFSpCas9, and evoSpCas9 high-fidelity variants of SpCas9, SaCas9, NmeCas9, CjCas9, StCas9, TdCas9, LbCasl2a, AsCasl2a, AacCasl2b, BhCasl2b v4, TnpB, dCas (D10A), dCas (H840A), dCasl3a, dCasl3b, or a functional portion thereof. In some embodiments, the genome modifying entity comprises one or more of the following genome modifying activities: cleaving, deaminating, nicking, polymerizing, interrogating, integrating, cutting, unwinding, breaking, altering, methylating, demethylating, or otherwise destabilizing a target 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 a sequence specific nuclease, a nucleic acid programmable DNA binding protein, an RNA guided nuclease, RNA-guided nuclease comprising a Cas nuclease and a guide RNA (CRISPR-Cas combination), a ribonucleoprotein (RNP) complex comprising the gRNA and the Cas nuclease, a homing endonuclease, a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a meganuclease, a Cas nuclease, a core Cas protein, a TnpB nuclease, an endonuclease-deficient-Cas protein, an enzymatically inactive Cas protein, a CRIS PR-associated transposase (CAST), a Type II or Type V Cas protein, base editing, prime editing, a Programmable Addition via Site-specific Targeting Elements (PASTE), or a functional portion thereof. In certain specific embodiments, the genome modifying entity is selected from Casl, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8a, Cas8b, Cas8c, Cas9, CaslO, Casl2, Casl2a (Cpfl), Casl2b (C2cl), Cas 12c (C2c3), Casl2d (CasY), Casl2e (CasX), Casl2f (C2cl0), Cas 12g, Casl2h, Casl2i, Casl2k (C2c5), Casl3, Casl3a (C2c2), Casl3b, Casl3c, Casl3d, C2c4, C2c8, C2c9, Cmrl, Cmr2, Cmr3, Cmr4, Cmr5, Cmr6, Csdl, Csd2, Cas5d, Csel, Cse2, Cse3, Cse4, Cas5e, Csfl, Csml, Csm2, Csm3, Csm4, Csm5, Csnl, Csn2, Cstl, Cst2, Cas5t, Cshl, Csh2, Cas5h, Csal, Csa2, Csa3, Csa4, Csa5, Cas5a, CsxlO, Csxll, Csyl, Csy2, Csy3, Csy4, Mad7, SpCas9, eSpCas9, SpCas9-HFl, HypaSpCas9, HeFSpCas9, and evoSpCas9 high-fidelity variants of SpCas9, SaCas9, NmeCas9, CjCas9, StCas9, TdCas9, LbCasl2a, AsCasl2a, AacCasl2b, BhCasl2b v4, TnpB, FokI, dCas (D10A), dCas (H840A), dCasl3a, dCasl3b, a base editor, a prime editor, a target-primed reverse transcription (TPRT) editor, APOBEC1, cytidine deaminase, adenosine deaminase, uracil glycosylase inhibitor (UGI), adenine base editors (ABE), cytosine base editors (CBE), reverse transcriptase, serine integrase, recombinase, transposase, polymerase, adenine-to-thymine or “ATBE” (or thymine-to-adenine or “TABE”) transversion base editor, ten-eleven translocation methylcytosine dioxygenases (TETs), TET1, TET3, TET1CD, histone acetyltransferase p300, histone methyltransferase SMYD3, histone methyltransferase PRDM9, H3K79 methyltransferase D0T1L, transcriptional repressor, or a functional portion thereof. In certain embodiments, the genome modifying entity is a Cas protein such as Cas9, or Mad7. In some embodiments, the genome targeting entity and the genome modifying entity are (a) different domains of a single polypeptide; (b) two different polypeptides that are operably linked together; or (c) two different polypeptides that are not linked together. In some embodiments, the genome editing complex comprises aguide 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 cases, the genome editing complex is an RNA-guided nuclease, for example, a Cas nuclease and a guide RNA (CRISPR-Cas combination). In some embodiments, the CRISPR-Cas combination is a ribonucleoprotein (RNP) complex comprising the gRNA and the Cas nuclease. In some embodiments, the Cas nuclease is a Type II or Type V Cas protein. In some embodiments, the Cas nuclease is selected from Cas3, Cas4, Cas5, Cas8a, Cas8b, Cas8c, Cas9, 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 1, Csy 1, Csy2, Csy3, or Mad7.Hi. Barcodes

[0129] In some embodiments, the cell provided herein comprises a vector 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. See, e.g., US63 / 580,663, the contents of which are herein incorporated by reference.

[0130] 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 byprimer binding sites. In some embodiments, the barcode is flanked by primer binding sites comprising a forward primer and a reverse primer.

[0131] 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.

[0132] 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 nondiverged (e.g., wild-type) 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.

[0133] A barcode can encode the same amino acid sequence as a non-diverged nucleotide sequence, e.g., a reference wild-type amino acid sequence. A barcode can have at least about any of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a wild-type nucleotide sequence. In some embodiments, a barcode is a probe binding site. A barcode can be of any suitable length, for example, in some embodiments, the barcode is an oligonucleotide between about 6 to about 30 nucleotides, such as about any of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some embodiments, the barcode can be between about 6 to about 10 nucleotides in length, between about 10 to about 20 nucleotides in length, between about 15 to about 25 nucleotides in length, or between about 20 to about 30 nucleotides in length. The barcode in the present disclosure is distinct from a sequence present in nature, for example present in a subject who may or may not comprise mutations from the reference wild-type sequence (z.e., alternative alleles), such that the barcode can accurately be used to detect the presence of a transgene. The barcode may be a naturally occurring amino acid sequence provided thenucleotide 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.

[0134] 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 set forth in any one of SEQ ID NOs: 12-17, or a nucleotide sequence comprising 1, 2, 3, 4, or 5 nucleotide substitutions, insertions, or deletions from the nucleotide sequence set forth in any one of SEQ ID NOs: 12-17. In some embodiments, the barcode comprises the nucleotide sequence set forth in any one of SEQ ID NOs: 12-17. In some embodiments, the barcode comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 12-17, or comprises a nucleotide sequence comprising 1, 2, 3, 4, or 5 nucleotide substitutions, insertions, or deletions from the nucleotide sequence set forth in any one of SEQ ID NOs: 12-17, 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 the nucleotide sequence set forth in any one of SEQ ID NOs: 12-17 indicates the presence of a transgene encoded by the vector comprising the barcode. a. Primer Binding Sites

[0135] 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.

[0136] 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 30nucleotides. 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.

[0137] 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.

[0138] Primer binding sites may be complementary to universal primers. A universal primer is able to bind to a sequence found in many commonly used plasmid cloning vectors, many of which are derived from pUC vectors. Examples of universal primers include, but are not limited to M13 Reverse (-27), M13 Forward (-41), M13 Forward (-20), M13 Forward (- 21), M13 Reverse (-48), SP6, T3, T7, T7 EEV, T7 Reverse, T7 Term, pBluescript KS, pBluescript SK, 3'pGEX, 5'pGEX, GST-Tag, pTrcHis-Forward, pTrcHis-Reverse, CMV- Forward, CMV-Reverse, EGFP-C, EGFP-N, BGH-Reverse, pQEproseq, pQErevseq, Intein Forward, 5'-pBabe-Seq, 3'-pBabe-Seq, -96 gill Sequencing Primer, GAE1 Forward, pBAD Forward, pBAD Reverse, pTRE 3', pTRE 5', pYESTrp Forward, pYESTrp Reverse, RVprimer3, Rvprimer4, GEprimer 1, GEprimer 2, SeqE-A (ATTE1), SeqE-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.

[0139] 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. Insome 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.

[0140] 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.

[0141] In some embodiments, the forward primer binding sites of the first barcode and the second barcode comprise the same sequence, and the reverse primer binding sites of the first barcode and the second barcode comprise the same sequence. In some embodiments, the forward primer binding site of the first barcode and / or the second barcode comprise different sequences, and the reverse primer binding site of the first barcode and / or the second barcode comprise different sequences. In some embodiments, the first and second forward primer binding sites and the first and second reverse primer binding sites are complementary to universal primers. In some embodiments, the first forward primer binding site and the second forward primer binding site and the first reverse primer binding site and the second reverse primer binding site is each between about 10 to about 30 nucleotides in length.

[0142] 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.

[0143] 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.

[0144] In some embodiments, the forward primer binding site and the reverse primer binding site are unique compared to nucleotide sequences found within the host genome. In some embodiments, the forward primer binding site and the reverse primer binding site are distinguishable from nucleotide sequences found within the host genome. In some embodiments, the primers that bind to the forward primer binding site and the reverse primer binding site do not substantially bind to nucleotide sequences found within the host genome. In some embodiments, the forward primer binding site comprises the nucleotide sequence of SEQ ID NO:33 or a variant thereof comprising about 1, 2, 3, 4, or 5 nucleotide substitutions, insertions, or deletions. In some embodiments, the reverse primer binding site comprises the nucleotide sequence of SEQ ID NO:34 or a variant thereof comprising about 1, 2, 3, 4, or 5 nucleotide substitutions, insertions, or deletions. In some embodiments, the forward primer binding site comprises the nucleotide sequence of SEQ ID NO:33, and / or the reverse primer binding site comprises the nucleotide sequence of SEQ ID NO:34. b. Identifying Regions

[0145] 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.

[0146] 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 thesame 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.

[0147] 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.

[0148] 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 set forth in any one of SEQ ID NOs: 12- 17, 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: 12-17. 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 of any one of SEQ ID NOs: 12-17, or a variant thereof comprising up to about 6 (such as about any of 1, 2, 3, 4, 5, or 6) nucleotide substitutions.

[0149] 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 sequenceset forth in any one of SEQ ID NOs: 18-23, or a variant thereof having at least about 80% (such as at least about any of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to any one of SEQ ID NOs: 18-23. In some embodiments, the first identifying region and / or the second identifying region comprises a first probe binding site and / or a second probe binding site comprising a nucleotide sequence of any one of SEQ ID NOs: 18-23, or a variant thereof comprising up to about 6 (such as about any of 1, 2, 3, 4, 5, or 6) nucleotide substitutions.

[0150] 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.

[0151] 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 someembodiments, the first identifying region and the second identifying region are located on different vectors.

[0152] In some embodiments, the first identifying region is located within the first transgene and / or the second identifying region is located within the second transgene. In some embodiments, the first identifying region is located outside of the first transgene and the second identifying region is located outside of the second transgene. In some embodiments, the first identifying region is located within the first transgene and the second identifying region is located outside of the second transgene. In some embodiments, the first identifying region is located within the first transgene and the second identifying region is located outside of the second transgene. In some embodiments, the first identifying region and the second identifying region are located on the same vector. In some embodiments, the first identifying region and the second identifying region are located in the same region of the vector that comprises the first transgene and the second transgene. In some embodiments, the first identifying region and the second identifying region are located on different vectors.

[0153] 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.

[0154] 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, poly adenylation signalsequences, 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.

[0155] 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.

[0156] In some embodiments, the first promoter is selected from the group consisting of: an EFla promoter, an EFla short promoter, a CAG promoter, a ubiquitin / S27a promoter, an SV40 early promoter, an adenovirus major late promoter, a mouse metallothionein-I promoter, an RSV promoter, an MMTV promoter, a Moloney murine leukemia virus Long Terminal repeat region, a CMV promoter, an actin promoter, an immunoglobulin promoter, a heat shock promoter, polyoma virus promoter, a fowlpox virus promoter, a bovine papilloma virus promoter, an avian sarcoma virus promoter, a retrovirus promoter, a hepatitis-B virus promoter, a PGK promoter, an adenovirus late promoter, a vaccinia virus 7.5K promoter, a SV40 promoter, a tk promoter of HSV, a mouse mammary tumor virus (MMTV) promoter, an LTR promoter of HIV, a promoter of moloney virus, an Epstein Barr virus (EBV)promoter, a Rous sarcoma virus (RSV) promoter, a U6 promoter, and an UBC promoter. In some embodiments, the second promoter is selected from the group consisting of: an EFla promoter, an EFla short promoter, a CAG promoter, a ubiquitin / S27a promoter, an SV40 early promoter, an adenovirus major late promoter, a mouse metallothionein-I promoter, an RSV promoter, an MMTV promoter, a Moloney murine leukemia virus Long Terminal repeat region, a CMV promoter, an actin promoter, an immunoglobulin promoter, a heat shock promoter, polyoma virus promoter, a fowlpox virus promoter, a bovine papilloma virus promoter, an avian sarcoma virus promoter, a retrovirus promoter, a hepatitis-B virus promoter, a PGK promoter, an adenovirus late promoter, a vaccinia virus 7.5K promoter, a SV40 promoter, a tk promoter of HSV, a mouse mammary tumor virus (MMTV) promoter, an LTR promoter of HIV, a promoter of moloney virus, an Epstein Barr virus (EBV) promoter, a Rous sarcoma virus (RSV) promoter, a U6 promoter, and an UBC promoter. In some embodiments, the first transgene is operably linked to a first promoter. In some embodiments, the second transgene is operably linked to a second promoter. In some embodiments, the first promoter and the second promoter comprise the same sequence.

[0157] 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.

[0158] 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 islocated 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.

[0159] 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).

[0160] 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. iv. Engineered Receptors

[0161] 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)

[0162] 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 totarget a specific protein. The receptors are chimeric because they combine both antigenbinding 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.

[0163] 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.

[0164] In some embodiments, the antigen binding domain binds to a cell surface antigen of a cell. In some embodiments, the antigen binding domain targets an antigen characteristic of a cell type, such as a neoplastic cell, a T cell, a B cell, or a senescent cell. In some embodiments, the antigen binding domain binds to a cell surface antigen of a cell. In some embodiments, a cell surface antigen is characteristic of one type of cell. In some embodiments, a cell surface antigen is characteristic of more than one type of cell.

[0165] 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 guanylylcyclase, 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, NAVI.8, NAVI.9, sphingo sin- 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, CDllc, 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-llRa), PSCA, PRSS21, VEGFR2, Lewis Y, 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), urokinase-type 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, CD151 CD340, CD200, tkrA, trkB, or trkC, or an antigenic fragment or antigenic portion thereof.

[0166] 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.

[0167] 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.

[0168] 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 CD10, 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.

[0169] In some embodiments, the antigen binding domain targets an antigen characteristic of a disease, disorder, injury, or condition.

[0170] 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, CD19, 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, IL-13Ra, Mesothelin, MUC1, MUC16, and ROR1 (associated with solid tumors).

[0171] 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 non-limiting 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, receptor-like tyrosine phosphatase, receptor serine / threonine kinase, receptor guanylyl cyclase, or histidine kinase associated receptor.

[0172] In some embodiments, the antigen binding domain targets an antigen characteristic of an infectious disease. In some embodiments, the CAR binds an antigen associated with an infectious disease. In some instances, the antigen is expressed by a cell affected by an infectious disease. In some embodiments, the infectious disease is selected from HIV, hepatitis B virus, hepatitis C virus, Human herpes virus, Human herpes virus 8 (HHV-8, Kaposi sarcoma-associated herpes virus (KSHV)), Human T-lymphotrophic virus- 1 (HTLV-1), Merkel cell polyomavirus (MCV), Simian virus 40 (SV40), Epstein-Barr virus, CMV, or human papillomavirus. In some embodiments, the antigen characteristic of an infectious disease is selected from a cell surface receptor, an ion channel-linked receptor, an enzyme-linked receptor, a G protein-coupled receptor, receptor tyrosine kinase, tyrosine kinase associated receptor, receptor-like tyrosine phosphatase, receptor serine / threonine kinase, receptor guanylyl cyclase, histidine kinase associated receptor, HIV Env, gpl20, or CD4-induced epitope on HIV-1 Env.

[0173] 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, R0R1, 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, IF-l lRa, KDR, Eewis Y, El -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, BREF1, BAEF4, EBNA3C, EMP1, EMP2, EMP2A, EMP2B, BZEF1, BMEF1, gp350, gH / gE, EBNA1 and LMP1, EBNA1 and LMP2A, EBNA1 and LMP1 and LMP2A, LMP and BARF1 and EBNA1, CD19 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 R0R1, B7H3 and EGFR, B7H3 and IL13Ra, B7H3 and R0R1, EGFR and IL13Ra, and / or EGFR and R0R1.

[0174] In some embodiments, a cell comprises a CAR comprising an antigen binding domain specific for two or more target antigens. In some embodiments, a cell comprises a CAR comprising an antigen binding domain specific to two or more epitopes of the same target antigen. In some embodiments, a cell comprises two CARs each comprising a different antigen binding domain from each other. For example, in some embodiments, the two or more CARs each comprise an antigen binding domain specific for a target antigen such that the cell comprises two or more CARs targeting any combinations of CD19xCD20, CD19xBCMA, CD20xBCMA, CD19xCD22, CD19xBAFFR, CD33xCD123, HER2xB7H3, HER2xEGFR, HER2xIL13Ra, HER2xRORl, B7H3xEGFR, B7H3xIL13Ra, B7H3xRORl, EGFRxIL13Ra, EGFRxRORl, and Her2xB7H3xEGFRxIL13Ra2xRORl.

[0175] 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.

[0176] 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, Lymphotoxin-alpha / TNF-beta, OX40 / TNFRSF4, 0X40 Ligand / TNFSF4, RELT / TNFRSF19L, TACI / TNFRSF 13B, TL1A / TNFSF15, TNF- alpha, 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 / 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 (IT AM), CD27, 4-1BB, CD134 / OX40, CD30, CD40, PD-1, ICOS, LIGHT, NKG2C, a ligand that specifically binds with CD83, or functional fragment thereof.

[0177] 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 4-1BB domain, or a CD134 domain, or functional variant thereof. In some embodiments, the CAR comprises (i) a CD3 zeta domain, or an immunoreceptor tyrosinebased 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 CD134 domain, or functional variant thereof; and (iv) a cytokine or costimulatory ligand transgene.

[0178] 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.

[0179] In some embodiments, the CAR comprises a transmembrane domain and a signaling domain. In some embodiments the signaling domain mediates downstream signaling during T cell activation.

[0180] In some embodiments, the CAR is a second-generation CAR. In some embodiments a second-generation CAR comprises an antigen binding domain, a transmembrane domain, and two signaling domains. In some embodiments a signaling domain mediates downstream signaling during T cell activation. In some embodiments a signaling domain is a costimulatory domain. In some embodiments, a costimulatory domain enhances cytokine production, CAR T cell proliferation, and or CAR T cell persistence during T cell activation.

[0181] In some 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.

[0182] 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.

[0183] 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 (NFAT), 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).

[0184] 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, CD 117 antibody, CD 127 antibody, CD 133 antibody, CD 137 (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.

[0185] 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 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 CD138-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 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 MUC 16- specific CAR, a RORl-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 13Ra2- specific CAR, a MAGEA3-specific CAR, a M AGE A4- specific CAR, a MART1- specific CAR, a NY-ESO-1 -specific CAR, a VEGFR2- specific CAR, a a-Folate receptorspecific 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-M A A- 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.

[0186] 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.

[0187] In some embodiments, the CAR comprises a CD3 zeta domain or an immunoreceptor tyrosine -based activation motif (ITAM), or functional variant thereof. Insome embodiments, the CAR comprises (i) a CD3 zeta domain, or an immunoreceptor tyrosine-based activation motif (IT AM), 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 4-1BB domain, or a CD134 domain, or functional variant thereof. In some embodiments, the CAR comprises (i) a CD3 zeta domain, or an immunoreceptor tyrosinebased 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 CD134 domain, or functional variant thereof; and (iv) a cytokine or costimulatory ligand transgene.

[0188] 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 CD137 (4-1BB, TNFRSF9) co- stimulatory domains, linked to a CD3-zeta intracellular domain.

[0189] 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- IBB.

[0190] 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.

[0191] 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. Insome embodiments, the intracellular signaling domain additionally includes an intracellular signaling domain of a costimulatory molecule (e.g., a costimulatory domain).

[0192] In some embodiments, the CAR contains one or more domains that combine an antigen- or ligand-binding domain (e.g., antibody or antibody fragment) that provides specificity for a desired antigen (e.g., tumor antigen) with intracellular signaling domains. In some embodiments, the intracellular signaling domain is a stimulating or an activating intracellular domain portion, such as a T cell stimulating or activating domain, providing a primary activation signal or a primary signal. In some embodiments, the intracellular signaling domain contains or additionally contains a costimulatory signaling domain to facilitate effector functions. In some embodiments, chimeric receptors when genetically engineered into immune cells can modulate T cell activity, and, in some cases, can modulate T cell differentiation or homeostasis, thereby resulting in genetically engineered cells or populations of cells (e.g., T cells, B cells, NK cells, islet cells such as beta cells, or hypoimmunogenic cells thereof) with improved longevity, survival and / or persistence in vivo, such as for use in adoptive cell therapy methods.

[0193] 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.

[0194] 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.

[0195] 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).

[0196] 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, CD 19, CD22, ROR1, CD45, CD47, CD21, CD5, CD33, Igkappa, Iglambda, CD79a, CD79b, or CD30.

[0197] 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 CD 19 and CD22. In some embodiments, the CAR binds to CD20. In some embodiments, the CAR binds to CD 19 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 R0R1. In some embodiments, the CAR binds to HER2 and R0R1. In some embodiments, the CAR binds to B7H3 and EGFR. In some embodiments, the CAR binds to B7H3 and IL13Ra. In some embodiments, the CAR binds to B7H3 and R0R1. In some embodiments, the CAR binds to EGFR and IL13Ra. In some embodiments, the CAR binds to EGFR and R0R1. For example, in some embodiments, the two or more CARs each comprise an antigen binding domain specific for a target antigen such that the cell comprises two or more CARs targeting any combinations of CD19xCD20, CD19xBCMA, CD20xBCMA, CD19xCD22, CD19xBAFFR, CD33xCD123, HER2xB7H3, HER2xEGFR, HER2xIL13Ra, HER2xR0Rl, 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.

[0198] In some embodiments, the antigen targeted by the antigen-binding domain is CD19. 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 VE derived from an antibody or an antibody fragment specific to CD19. 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 someembodiments, the scFv is derived from FMC63. FMC63 generally refers to a mouse monoclonal IgGl antibody raised against Naim-1 and -16 cells expressing CD 19 of human origin (Fing, N. R., et al., (1987). Leucocyte typing III. 302).

[0199] 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.

[0200] 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.

[0201] 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.

[0202] In some embodiments, the antigen receptor comprises an intracellular domain linked directly or indirectly to the extracellular domain. In some embodiments, the chimeric antigen receptor includes a transmembrane domain linking the extracellular domain and the intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises an IT AM. For example, in some aspects, the antigen recognition domain e.g., extracellular domain) generally is linked to one or more intracellular signaling components, such as signaling components that mimic activation through an antigen receptor complex,such as a TCR complex, in the case of a CAR, and / or signal via another cell surface receptor. In some embodiments, the chimeric receptor comprises a transmembrane domain linked or fused between the extracellular domain (e.g., scFv) and intracellular signaling domain. Thus, in some embodiments, the antigen-binding component (e.g., antibody) is linked to one or more transmembrane and intracellular signaling domains.

[0203] 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.

[0204] 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.

[0205] 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.

[0206] Among the intracellular signaling domains are those that mimic or approximate a signal through a natural antigen receptor, a signal through such a receptor in combination with a costimulatory receptor, and / or a signal through a costimulatory receptor alone. In some embodiments, a short oligo- or polypeptide linker, for example, a linker of between 2 and 10 amino acids in length, such as one containing glycines and serines, e.g., glycine-serine doublet, is present and forms a linkage between the transmembrane domain and the cytoplasmic signaling domain of the CAR.

[0207] 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.

[0208] 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.

[0209] 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.

[0210] 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 antigenreceptor component or costimulatory molecule is used in place of an intact immuno stimulatory 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.

[0211] 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.

[0212] 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, 4-1BB, 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 4-1BB. In some aspects, the T cell costimulatory molecule is 4-1BB.

[0213] In some embodiments, the activating domain is included within one CAR, whereas the costimulatory component is provided by another CAR recognizing another antigen. In some embodiments, the CARs include activating or stimulatory CARs, costimulatory CARs, both expressed on the same cell (see WO2014 / 055668). In some aspects, the cells include one or more stimulatory or activating CAR and / or a costimulatory CAR. In some embodiments, the cells further include inhibitory CARs (iCARs, see Fedorov et al., Sci. 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.

[0214] 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 andCD137 (4-1BB, TNFRSF9) co- stimulatory domains, linked to a CD3-zeta intracellular domain.

[0215] 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- IBB.

[0216] 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.

[0217] In some embodiments, a CD 19 specific CAR includes an anti-CD19 singlechain antibody fragment (scFv), a transmembrane domain such as one derived from human CD8a, a 4-1BB (CD137) 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 4-1BB (CD137) co- stimulatory signaling domain, and a CD3(^ signaling domain. In some embodiments, a CD19 / CD22-bispecific CAR includes an anti-CD19 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.

[0218] In some embodiments, the CAR comprises a commercial CAR construct carried by a T cell. Non-limiting examples of commercial CAR-T cell based therapies include brexucabtagene autoleucel (TECARTUS®), axicabtagene ciloleucel (YESCARTA®), idecabtagene vicleucel (ABECMA®), lisocabtagene maraleucel (BREYANZI®), tisagenlecleucel (KYMRIAH®), Descartes-08 and Descartes- 11 from Cartesian Therapeutics, CTL110 from Novartis, P-BMCA-101 from Poseida Therapeutics, AUTO4 from Autolus Limited, UCARTCS from Cellectis, PBCAR19B and PBCAR269A from Precision Biosciences, FT819 from Fate Therapeutics, and CY AD-211 from Clyad Oncology.

[0219] 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, theintracellular signaling domain additionally includes an intracellular signaling domain of a costimulatory molecule (e.g., a costimulatory domain).Exemplary CAR Components

[0220] Exemplary components of CARs that may be used in the present disclosure are described below.

[0221] 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.

[0222] 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.

[0223] 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.

[0224] 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.

[0225] 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 / TNFRSFI 3B, TL1A / TNFSF15, TNFa, 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, CD2, CD7, CD53, CD82 / Kai-1, CD90 / Thyl, CD96, CD160, CD200, CD300a / LMIRl, HLA Class I, HLA-DR, Ikaros, Integrin alpha 4 / CD49d, Integrin alpha 4 beta 1, Integrin alpha 4 beta 7 / LPAM-l, LAG-3, TCL1A, TCL1B, CRTAM, DAP12, Dectin- 1 / CLEC7 A, DPPIV / CD26, EphB6, TIM-l / KIM-l / HAVCR, TIM-4, TSLP, TSLP R, lymphocyte function associated antigen- 1 (LFA-1), NKG2C, CD3^, an immunoreceptor tyrosine-based activation motif (IT AM), CD27, 4-1BB, 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 IT AM, 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 costimulatory and / or signaling domains.

[0226] Markers: In some embodiments, the antigen receptor further includes a marker and / or cells expressing the CAR or other antigen receptor further includes a surrogate marker, such as a cell surface marker, which may be used to confirm transduction or engineering of the cell to express the receptor. In some embodiments, the marker includes all or part (e.g., truncated form) of CD34, a NGFR, or epidermal growth factor receptor, such as truncated version of such a cell surface receptor (e.g., tEGFR). In some embodiments, the nucleic acid encoding the marker is operably linked to a polynucleotide encoding for a linker sequence, such as a cleavable linker sequence, e.g., T2A. For example, a marker, and optionally a linker sequence, can be any as disclosed in published patent application No. WO2014031687. For example, the marker can be a truncated EGFR (tEGFR) that is, optionally, linked to a linker sequence, such as a T2A cleavable linker sequence. In some embodiments, the marker is a molecule, e.g., cell surface protein, not naturally found on T cells or not naturally found on the surface of T cells, or a portion thereof. In some embodiments, the molecule is a non- self molecule, e.g., non-self protein, i.e., one that is not recognized as “self’ by the immune system of the host into which the cells will be adoptively transferred. In some embodiments, the marker serves no therapeutic function and / or produces no effect other than to be used as a marker for genetic engineering, e.g., for selecting cells successfully engineered. In other embodiments, the marker may be a therapeutic molecule or molecule otherwise exerting some desired effect, such as a ligand for a cell to be encountered in vivo, such as a costimulatory or immune checkpoint molecule to enhance and / or dampen responses of the cells upon adoptive transfer and encounter with ligand.Exemplary CD 19 CARs

[0227] 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 hingedomains 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.

[0228] 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 CD19, for example, human CD19. The extracellular binding domain of the CD19 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 CD19 CAR comprises an scFv derived from the FMC63 monoclonal antibody (FMC63), which comprises the heavy chain variable region (VH) and the light chain variable region (VL) of FMC63 connected by a linker. FMC63 and the derived scFv have been described in Nicholson et al., Mol. Immun. 34(16- 17): 1157- 1165 (1997) and PCT Application Publication No. WO2018 / 213337, the entire contents of each of which are incorporated by reference herein. In some embodiments, the amino acid sequences of the entire FMC63-derived scFv (also referred to as FMC63 scFv) and its different portions are provided in Table 6 below. In some embodiments, the CD19-specific scFv may comprise one or more CDRs having the CDR amino acid sequences set forth in Table 6. In some embodiments, the CD19-specific scFv may comprise a light chain with one or more CDRs having the light chain CDR amino acid sequences set forth in Table 6. In some embodiments, the CD19-specific scFv may comprise a heavy chain with one or more CDRs having the heavy chain CDR amino acid sequences set forth in Table 6. In any of these embodiments, the CD19-specific scFv may comprise one or more CDRs comprising one or more amino acid substitutions or comprising a sequence that is at least about 80% identical (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical), to any of the sequences identified in Table 6. In some embodiments, the extracellular binding domain of the CD19 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 bereplaced 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.

[0229] 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.

[0230] In some embodiments, the hinge domain of the CD19 CAR comprises a CD8a hinge domain, for example, a human CD8a hinge domain. In some embodiments, the hinge domain comprises a CD28 hinge domain, for example, a human CD28 hinge domain. In some embodiments, the hinge domain comprises an IgG4 hinge domain, for example, a human IgG4 hinge domain. In some embodiments, the hinge domain comprises a IgG4 hinge-Ch2-Ch3 domain, for example, a human IgG4 hinge-Ch2-Ch3 domain. In some embodiments, the transmembrane domain of the CD 19 CAR comprises a CD8a transmembrane domain, for example, a human CD8a transmembrane domain. In some embodiments, the transmembrane domain comprises a CD28 transmembrane domain, for example, a human CD28 transmembrane domain.

[0231] 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, theCD28 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 CD19 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 (ITAMs). 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.

[0232] 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 CD 19 CAR may additionally comprise a signal peptide (e.g., a CD8a signal peptide) as described above.

[0233] 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 CD 19 CAR may additionally comprise a signal peptide (e.g., a CD8a signal peptide) as described above.

[0234] 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 (i.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.

[0235] 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 setforth in SEQ ID NO: 285 (see Table 7). The encoded CD19 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 (VL- Whitlow linker-Vn), CD8a hinge domain, CD8a transmembrane domain, 4- IBB costimulatory domain, and CD3^ signaling domain.Table 7. Exemplary sequences of CD19 CARs.Exemplary CD20 CARs

[0236] In some embodiments, the CAR is a CD20 CAR (“CD20-CAR”). CD20 is an antigen found on the surface of B cells as early at the pro-B phase and progressively at increasing levels until B cell maturity, as well as on the cells of most B-cell neoplasms.CD20 positive cells are also sometimes found in cases of Hodgkin’s disease, myeloma, and thymoma. In some embodiments, the CD20 CAR may comprise an extracellular binding domain that specifically binds CD20 and one or more of: any of the signal peptides described herein, any of the hinge domains described herein, any of the transmembrane domains described herein, any of the intracellular costimulatory domains described herein, and / or any of the intracellular signaling domains described herein, e.g., in tandem.

[0237] In some embodiments, the signal peptide of the CD20 CAR comprises a CD8a signal peptide. In some embodiments, the signal peptide comprises an IgK signal peptide. In some embodiments, the signal peptide comprises a GMCSFR-a or CSF2RA signal peptide.

[0238] In some embodiments, the extracellular binding domain of the CD20 CAR is specific to CD20, for example, human CD20. The extracellular binding domain of the CD20 CAR can be codon-optimized for expression in a host cell or to have variant sequences to increase functions of the extracellular binding domain. In some embodiments, the extracellular binding domain comprises an immunogenically active portion of an immunoglobulin molecule, for example, an scFv.

[0239] In some embodiments, the extracellular binding domain of the CD20 CAR is derived from an antibody specific to CD20, including, for example, Leul6, IF5, 1.5.3, rituximab, obinutuzumab, ibritumomab, ofatumumab, tositumumab, odronextamab,veltuzumab, ublituximab, and ocrelizumab. In any of these embodiments, the extracellular binding domain of the CD20 CAR can comprise or consist of the Vn, the VL, and / or one or more CDRs of any of the antibodies.

[0240] In some embodiments, the extracellular binding domain of the CD20 CAR comprises an scFv derived from the Leu 16 monoclonal antibody, which comprises the heavy chain variable region (VH) and the light chain variable region (VL) of Leul6 connected by a linker. See Wu et al., Protein Engineering. 14(12): 1025-1033 (2001). In some embodiments, the linker is a 3xG4S linker. In other embodiments, the linker is a Whitlow linker as described herein. In some embodiments, the amino acid sequences of different portions of the entire Leul6-derived scFv (also referred to as Leu 16 scFv) and its different portions are provided in Table 8 below. In some embodiments, the CD20-specific scFv may comprise one or more CDRs having the CDR amino acid sequences set forth in Table 8. In some embodiments, the CD20-specific scFv may comprise a light chain with one or more CDRs having light chain CDR amino acid sequences set forth in Table 8. In some embodiments, the CD20-specific scFv may comprise a heavy chain with one or more CDRs having heavy chain CDR amino acid sequences set forth in Table 8. In any of these embodiments, the CD20- specific scFv may comprise one or more CDRs comprising one or more amino acid substitutions, or comprising a sequence that is at least about 80% identical (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identity), to any of the sequences identified. In some embodiments, the extracellular binding domain of the CD20 CAR comprises or consists of the one or more CDRs as described herein.Table 8. Exemplary sequences of anti-CD20 scFv and components.

[0241] In some embodiments, the hinge domain of the CD20 CAR comprises a CD8a hinge domain, for example, a human CD8a hinge domain. In some embodiments, the hinge domain comprises a CD28 hinge domain, for example, a human CD28 hinge domain. In some embodiments, the hinge domain comprises an IgG4 hinge domain, for example, a human IgG4 hinge domain. In some embodiments, the hinge domain comprises a IgG4 hinge-Ch2-Ch3 domain, for example, a human IgG4 hinge-Ch2-Ch3 domain.

[0242] In some embodiments, the transmembrane domain of the CD20 CAR comprises a CD8a transmembrane domain, for example, a human CD8a transmembrane domain. In some embodiments, the transmembrane domain comprises a CD28 transmembrane domain, for example, a human CD28 transmembrane domain.

[0243] In some embodiments, the intracellular costimulatory domain of the CD20 CAR comprises a 4- IBB costimulatory domain, for example, a human 4- IBB costimulatory domain. In some embodiments, the intracellular costimulatory domain comprises a CD28 costimulatory domain, for example, a human CD28 costimulatory domain.

[0244] In some embodiments, the intracellular signaling domain of the CD20 CAR comprises a CD3 zeta (Q signaling domain, for example, a human CD3(^ signaling domain.

[0245] In some embodiments, the CAR is a CD20 CAR, including, for example, a CD20 CAR comprising a CD20-specific scFv, a CD8a hinge domain, a CD8a transmembrane domain, a 4- IBB costimulatory domain, a CD3(^ signaling domain, and / or variants (z.e., having a sequence that is at least about 80% identical, for example, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity) thereof. In some embodiments, the first barcode and / or the second barcode is located at the junction of the CD20-specific scFv and the CD8a hinge domain. In some embodiments, the first barcode and / or the second barcode is located at the junction of the CD8a hinge domain and the CD28 transmembrane domain. In some embodiments, the first barcode and / or the second barcode is located at the junction of the CD28 transmembrane domain and the 4- IBB costimulatory domain. In some embodiments, the first barcode and / or the second barcode is located at the junction of the 4- IBB costimulatory domain and the CD3(^ signaling domain.

[0246] In some embodiments, the CAR is a CD20 CAR, including, for example, a CD20 CAR comprising a CD20-specific scFv, a CD28 hinge domain, a CD8a transmembrane domain, a 4- IBB costimulatory domain, a CD3(^ signaling domain, and / or variants (z.e., having a sequence that is at least about 80% identical, for example, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity) thereof. In some embodiments, the first barcode and / or the second barcode is located at the junction of the CD20-specific scFv and the CD28 hinge domain. In some embodiments, the first barcode and / or the second barcode is located at the junction of the CD28 hinge domain and the CD8a transmembrane domain. In some embodiments, the first barcode and / or the second barcode is located at the junction of the CD8a transmembrane domain and the 4- IBB costimulatory domain. In some embodiments, the first barcode and / or the second barcode is located at the junction of the 4- IBB costimulatory domain and the CD3(^ signaling domain.

[0247] In some embodiments, the CAR is a CD20 CAR, including, for example, a CD20 CAR comprising a CD20-specific scFv, an IgG4 hinge domain, a CD8a transmembrane domain, a 4- IBB costimulatory domain, a CD3(^ signaling domain, and / or variants (z.e., having a sequence that is at least about 80% identical, for example, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity) thereof. In some embodiments, the first barcode and / or the second barcode is located at the junction of the CD20-specific scFv and the IgG4 hinge domain. In some embodiments, the first barcodeand / or the second barcode is located at the junction of the IgG4 hinge domain and the CD8a transmembrane domain. In some embodiments, the first barcode and / or the second barcode is located at the junction of the CD8a transmembrane domain and the 4- IBB costimulatory domain. In some embodiments, the first barcode and / or the second barcode is located at the junction of the 4- IBB costimulatory domain and the CD3(^ signaling domain.

[0248] In some embodiments, the CAR is a CD20 CAR, including, for example, a CD20 CAR comprising a CD20-specific scFv2, a CD8a hinge domain, a CD28 transmembrane domain, a 4- IBB costimulatory domain, a CD3(^ signaling domain, and / or variants (z.e., having a sequence that is at least about 80% identical, for example, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity) thereof. In some embodiments, the first barcode and / or the second barcode is located at the junction of the CD20-specific scFv2 and the CD8a hinge domain. In some embodiments, the first barcode and / or the second barcode is located at the junction of the CD8a hinge domain and the CD28 transmembrane domain. In some embodiments, the first barcode and / or the second barcode is located at the junction of the CD28 transmembrane domain and the 4- IBB costimulatory domain. In some embodiments, the first barcode and / or the second barcode is located at the junction of the 4- IBB costimulatory domain and the CD3(^ signaling domain.

[0249] In some embodiments, the CAR is a CD20 CAR, including, for example, a CD20 CAR comprising a CD20-specific scFv, a CD28 hinge domain, a CD28 transmembrane domain, a 4- IBB costimulatory domain, a CD3(^ signaling domain, and / or variants (z.e., having a sequence that is at least about 80% identical, for example, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity) thereof. In some embodiments, the first barcode and / or the second barcode is located at the junction of the CD20-specific scFv and the CD28 hinge domain. In some embodiments, the first barcode and / or the second barcode is located at the junction of the CD28 hinge domain and the CD28 transmembrane domain. In some embodiments, the first barcode and / or the second barcode is located at the junction of the CD28 transmembrane domain and the 4- IBB costimulatory domain. In some embodiments, the first barcode and / or the second barcode is located at the junction of the 4- IBB costimulatory domain and the CD3(^ signaling domain.

[0250] In some embodiments, the CAR is a CD20 CAR, including, for example, a CD20 CAR comprising a CD20-specific scFv, an IgG4 hinge domain, a CD28 transmembrane domain, a 4- IBB costimulatory domain, a CD3(^ signaling domain, and / orvariants (z.e., having a sequence that is at least about 80% identical, for example, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity) thereof. In some embodiments, the first barcode and / or the second barcode is located at the junction of the CD20-specific scFv and the IgG4 hinge domain. In some embodiments, the first barcode and / or the second barcode is located at the junction of the IgG4 hinge domain and the CD28 transmembrane domain. In some embodiments, the first barcode and / or the second barcode is located at the junction of the CD28 transmembrane domain and the 4- IBB costimulatory domain. In some embodiments, the first barcode and / or the second barcode is located at the junction of the 4- IBB co stimulatory domain and the CD3^ signaling domain.Exemplary CD22 CARs

[0251] In some embodiments, the CAR is a CD22 CAR (“CD22-CAR”). CD22 is a transmembrane protein found mostly on the surface of mature B cells that functions as an inhibitory receptor for B cell receptor (BCR) signaling. CD22 is expressed in 60-70% of B cell lymphomas and leukemias (e.g., B -chronic lymphocytic leukemia, hairy cell leukemia, acute lymphocytic leukemia (ALL), and Burkitt's lymphoma) and is not present on the cell surface in early stages of B cell development or on stem cells. In some embodiments, the CD22 CAR may comprise an extracellular binding domain that specifically binds CD22 and one or more of: any of the signal peptides described herein, any of the hinge domains described herein, any of the transmembrane domains described herein, any of the intracellular costimulatory domains described herein, and / or any of the intracellular signaling domains described herein, e.g., in tandem.

[0252] In some embodiments, the signal peptide of the CD22 CAR comprises a CD8a signal peptide. In some embodiments, the signal peptide comprises an IgK signal peptide. In some embodiments, the signal peptide comprises a GMCSFR-a or CSF2RA signal peptide.

[0253] In some embodiments, the extracellular binding domain of the CD22 CAR is specific to CD22, for example, human CD22. The extracellular binding domain of the CD22 CAR can be codon-optimized for expression in a host cell or to have variant sequences to increase functions of the extracellular binding domain. In some embodiments, the extracellular binding domain comprises an immunogenically active portion of an immunoglobulin molecule, for example, an scFv.

[0254] In some embodiments, the extracellular binding domain of the CD22 CAR is derived from an antibody specific to CD22, including, for example, SM03, inotuzumab,epratuzumab, moxetumomab, or pinatuzumab. In any of these embodiments, the extracellular binding domain of the CD22 CAR can comprise or consist of the VH, the VL, and / or one or more CDRs of any of the antibodies.

[0255] In some embodiments, the extracellular binding domain of the CD22 CAR comprises an scFv derived from the m971 monoclonal antibody (m971), which comprises the heavy chain variable region (VH) and the light chain variable region (VL) of m971 connected by a linker. In some embodiments, the linker is a 3xG4S linker. In other embodiments, the Whitlow linker may be used instead. In some embodiments, the amino acid sequences of the entire m971 -derived scFv (also referred to as m971 scFv) and its different portions are provided in Table 9 below. In some embodiments, the extracellular binding domain of the CD22 CAR comprises an scFv derived from m971-L7, which is an affinity matured variant of m971 with significantly improved CD22 binding affinity compared to the parental antibody m971 (improved from about 2 nM to less than 50 pM). In some embodiments, the scFv derived from m971-L7 comprises the VH and the VL of m971-L7 connected by a 3xG4S linker. In other embodiments, the Whitlow linker may be used instead. In some embodiments, the amino acid sequences of the entire m971-L7-derived scFv (also referred to as m971-L7 scFv) and its different portions are provided in Table 9 below. In some embodiments, the CD22-specific scFv may comprise one or more CDRs having CDR amino acid sequences set forth in Table 9. In some embodiments, the CD22-specific scFv may comprise a heavy chain with one or more CDRs having heavy chain CDR amino acid sequences set forth in Table 9. In some embodiments, the CD22-specific scFv may comprise a light chain with one or more CDRs having light chain CDR amino acid sequences set forth in Table 9. In any of these embodiments, the CD22-specific scFv may comprise one or more CDRs comprising one or more amino acid substitutions, or comprising a sequence that is at least about 80% identical (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical), to any of the sequences identified. In some embodiments, the extracellular binding domain of the CD22 CAR comprises or consists of the one or more CDRs as described herein.Table 9. Exemplary sequences of anti-CD22 scFv and components.

[0256] In some embodiments, the extracellular binding domain of the CD22 CAR comprises immunotoxins HA22 or BL22. Immunotoxins BL22 and HA22 are therapeutic agents that comprise an scFv specific for CD22 fused to a bacterial toxin, and thus can bind to the surface of the cancer cells that express CD22 and kill the cancer cells. BL22 comprises a dsFv of an anti-CD22 antibody, RFB4, fused to a 38-kDa truncated form of Pseudomonas exotoxin A (Bang et al., Clin. Cancer Res., 11:1545-50 (2005)). HA22 (CAT8015, moxetumomab pasudotox) is a mutated, higher affinity version of BL22 (Ho et al., J. Biol. Chem., 280(1): 607-17 (2005)). Suitable sequences of antigen binding domains of HA22 and BL22 specific to CD22 are disclosed in, for example, U.S. Patent Nos. 7,541,034; 7,355,012; and 7,982,011, which are hereby incorporated by reference in their entirety.

[0257] In some embodiments, the hinge domain of the CD22 CAR comprises a CD8a hinge domain, for example, a human CD8a hinge domain. In some embodiments, the hinge domain comprises a CD28 hinge domain, for example, a human CD28 hinge domain. In some embodiments, the hinge domain comprises an IgG4 hinge domain, for example, ahuman 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.

[0258] In some embodiments, the transmembrane domain of the CD22 CAR comprises a CD8a transmembrane domain, for example, a human CD8a transmembrane domain. In some embodiments, the transmembrane domain comprises a CD28 transmembrane domain, for example, a human CD28 transmembrane domain.

[0259] In some embodiments, the intracellular costimulatory domain of the CD22 CAR comprises a 4- IBB costimulatory domain, for example, a human 4- IBB costimulatory domain. In some embodiments, the intracellular costimulatory domain comprises a CD28 costimulatory domain, for example, a human CD28 costimulatory domain.

[0260] In some embodiments, the intracellular signaling domain of the CD22 CAR comprises a CD3-zeta (Q signaling domain, for example, a human CD3(^ signaling domain.

[0261] In some embodiments, the CAR is a CD22 CAR, including, for example, a CD22 CAR comprising a CD22-specific scFv, a CD8a hinge domain, a CD8a transmembrane domain a 4- IBB costimulatory domain, a CD3(^ signaling domain, and / or variants (z.e., having a sequence that is at least about 80% identical, for example, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity) thereof. In some embodiments, the first barcode and / or the second barcode is located at the junction of the CD22-specific scFv and the CD8a hinge domain. In some embodiments, the first barcode and / or the second barcode is located at the junction of the CD8a hinge domain and the CD8a transmembrane domain. In some embodiments, the first barcode and / or the second barcode is located at the junction of the CD8a transmembrane domain and the 4- IBB costimulatory domain. In some embodiments, the first barcode and / or the second barcode is located at the junction of the 4- IBB costimulatory domain and the CD3(^ signaling domain.

[0262] In some embodiments, the CAR is a CD22 CAR, including, for example, a CD22 CAR comprising a CD22-specific scFv, a CD28 hinge domain, a CD8a transmembrane domain, a 4- IBB costimulatory domain, a CD3(^ signaling domain, and / or variants (z.e., having a sequence that is at least about 80% identical, for example, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity) thereof. In some embodiments, the first barcode and / or the second barcode is located at the junction of the CD22-specific scFv and the CD28 hinge domain. In some embodiments, the first barcode and / or the second barcode is located at the junction of the CD28 hinge domain and the CD8atransmembrane domain. In some embodiments, the first barcode and / or the second barcode is located at the junction of the CD8a transmembrane domain and the 4- IBB costimulatory domain. In some embodiments, the first barcode and / or the second barcode is located at the junction of the 4- IBB costimulatory domain and the CD3(^ signaling domain.

[0263] In some embodiments, the CAR is a CD22 CAR, including, for example, a CD22 CAR comprising a CD22-specific scFv, an IgG4 hinge domain, a CD8a transmembrane domain, a 4- IBB costimulatory domain, a CD3(^ signaling domain, and / or variants (z.e., having a sequence that is at least about 80% identical, for example, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity) thereof. In some embodiments, the first barcode and / or the second barcode is located at the junction of the CD22-specific scFv and the IgG4 hinge domain. In some embodiments, the first barcode and / or the second barcode is located at the junction of the IgG4 hinge domain and the CD8a transmembrane domain. In some embodiments, the first barcode and / or the second barcode is located at the junction of the CD8a transmembrane domain and the 4- IBB costimulatory domain. In some embodiments, the first barcode and / or the second barcode is located at the junction of the 4- IBB costimulatory domain and the CD3(^ signaling domain.

[0264] In some embodiments, the CAR is a CD22 CAR, including, for example, a CD22 CAR comprising a CD22-specific scFv, a CD8a hinge domain, a CD28 transmembrane domain, a 4- IBB costimulatory domain, a CD3(^ signaling domain, and / or variants (z.e., having a sequence that is at least about 80% identical, for example, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity) thereof. In some embodiments, the first barcode and / or the second barcode is located at the junction of the CD22-specific scFv and the CD8a hinge domain. In some embodiments, the first barcode and / or the second barcode is located at the junction of the CD8a hinge domain and the CD28 transmembrane domain. In some embodiments, the first barcode and / or the second barcode is located at the junction of the CD28 transmembrane domain and the 4- IBB costimulatory domain. In some embodiments, the first barcode and / or the second barcode is located at the junction of the 4- IBB costimulatory domain and the CD3(^ signaling domain.

[0265] In some embodiments, the CAR is a CD22 CAR, including, for example, a CD22 CAR comprising a CD22-specific scFv, a CD28 hinge domain, a CD28 transmembrane domain, a 4- IBB costimulatory domain, a CD3(^ signaling domain, and / or variants (z.e., having a sequence that is at least about 80% identical, for example, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity) thereof. In some embodiments, the first barcode and / or the second barcode is located at the junction of the CD22-specific scFv and the CD28 hinge domain. In some embodiments, the first barcode and / or the second barcode is located at the junction of the CD28 hinge domain and the CD28 transmembrane domain. In some embodiments, the first barcode and / or the second barcode is located at the junction of the CD28 transmembrane domain and the 4- IBB costimulatory domain. In some embodiments, the first barcode and / or the second barcode is located at the junction of the 4- IBB costimulatory domain and the CD3^ signaling domain.

[0266] In some embodiments, the CAR is a CD22 CAR, including, for example, a CD22 CAR comprising a CD22-specific scFv, an IgG4 hinge domain, a CD28 transmembrane domain, a 4- IBB costimulatory domain, a CD3^ signaling domain, and / or variants (z.e., having a sequence that is at least about 80% identical, for example, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity) thereof. In some embodiments, the first barcode and / or the second barcode is located at the junction of the CD22-specific scFv and the IgG4 hinge domain. In some embodiments, the first barcode and / or the second barcode is located at the junction of the IgG4 hinge domain and the CD28 transmembrane domain. In some embodiments, the first barcode and / or the second barcode is located at the junction of the CD28 transmembrane domain and the 4- IBB costimulatory domain. In some embodiments, the first barcode and / or the second barcode is located at the junction of the 4- IBB costimulatory domain and the CD3^ signaling domain.Exemplary BCMA CARs

[0267] In some embodiments, the CAR is a BCMA CAR (“BCMA-CAR”). BCMA is a tumor necrosis family receptor (TNFR) member expressed on cells of the B cell lineage, with the highest expression on terminally differentiated B cells or mature B lymphocytes. BCMA is involved in mediating the survival of plasma cells for maintaining long-term humoral immunity. The expression of BCMA has been recently linked to a number of cancers, such as multiple myeloma, Hodgkin's and non-Hodgkin's lymphoma, various leukemias, and glioblastoma. In some embodiments, the BCMA CAR may comprise an extracellular binding domain that specifically binds BCMA and one or more of: any of the signal peptides described herein, any of the hinge domains described herein, any of the transmembrane domains described herein, any of the intracellular costimulatory domainsdescribed herein, and / or any of the intracellular signaling domains described herein, e.g., in tandem.

[0268] In some embodiments, the signal peptide of the BCMA CAR comprises a CD8a signal peptide. In some embodiments, the signal peptide comprises an IgK signal peptide. In some embodiments, the signal peptide comprises a GMCSFR-a or CSF2RA signal peptide.

[0269] In some embodiments, the extracellular binding domain of the BCMA CAR is specific to BCMA, for example, human BCMA. The extracellular binding domain of the BCMA CAR can be codon-optimized for expression in a host cell or to have variant sequences to increase functions of the extracellular binding domain.

[0270] In some embodiments, the extracellular binding domain comprises an immunogenically active portion of an immunoglobulin molecule, for example, an scFv. In some embodiments, the extracellular binding domain of the BCMA CAR is derived from an antibody specific to BCMA, including, for example, belantamab, erlanatamab, teclistamab, LCAR-B38M, or ciltacabtagene. In any of these embodiments, the extracellular binding domain of the BCMA CAR can comprise or consist of the Vn, the VL, and / or one or more CDRs of any of the antibodies.

[0271] In some embodiments, the extracellular binding domain of the BCMA CAR comprises an scFv derived from Cl 1D5.3, a murine monoclonal antibody as described in Carpenter et al., Clin. Cancer Res. 19(8):2048-2060 (2013). See also PCT Application Publication No. WO2010 / 104949. The Cl lD5.3-derived scFv may comprise the heavy chain variable region (VH) and the light chain variable region (VL) of Cl 1D5.3 connected by the Whitlow linker, the amino acid sequence of which is provided in Table 10 below. In some embodiments, the extracellular binding domain of the BCMA CAR comprises an scFv derived from another murine monoclonal antibody, C12A3.2, as described in Carpenter et al., Clin. Cancer Res. 19(8):2048-2060 (2013) and PCT Application Publication No.WO2010 / 104949, the amino acid sequence of which is also provided in Table 10 below. In some embodiments, the extracellular binding domain of the BCMA CAR comprises a fully human heavy-chain variable domain (FHVH) as described in Lam et al., Nat. Commun. 11(1):283 (2020), also referred to as FHVH33. See also, PCT Application Publication No. WO20 19 / 006072, and Table 10. In some embodiments, the extracellular binding domain of the BCMA CAR comprises an scFv derived from CT103A (or CAR0085) as described in U.S. Patent No. 11,026,975 B2, the amino acid sequence of which is provided in Table 10 below. In some embodiments, the BCMA-specific extracellular binding domain may comprise one or more CDRs having CDR amino acid sequences set forth in Table 10. Insome embodiments, the BCMA-specific extracellular binding domain may comprise a light chain with one or more CDRs having light chain CDR amino acid sequences set forth in Table 10. In some embodiments, the BCMA-specific extracellular binding domain may comprise a heavy chain with one or more CDRs having heavy chain CDR amino acid sequences set forth in Table 10. In any of these embodiments, the BCMA-specific scFv may comprise one or more CDRs comprising one or more amino acid substitutions, or comprising a sequence that is at least about 80% identical (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical), to any of the sequences identified. In some embodiments, the extracellular binding domain of the BCMA CAR comprises or consists of the one or more CDRs as described herein.

[0272] In some embodiments, the extracellular binding domain of the BCMA CAR comprises a murine monoclonal antibody with high specificity to human BCMA, referred to as BB2121 in Friedman et al., Hum. Gene Ther. 29(5):585-601 (2018)). See also, PCT Application Publication No. WO2012163805. In some embodiments, the extracellular binding domain of the BCMA CAR comprises single variable fragments of two heavy chains (VHH) that can bind to two epitopes of BCMA as described in Zhao et al., J. Hematol.Oncol. 11(1): 141 (2018), also referred to as LCAR-B38M. See also, PCT Application Publication No. WO2018 / 028647.

[0273] Additionally, CARs and binders directed to BCMA have been described in U.S. Application Publication Nos. 2020 / 0246381 Al and 2020 / 0339699 Al, the entire contents of each of which are incorporated by reference herein.Table 10. Exemplary sequences of anti-BCMA binder and components.

[0274] In some embodiments, the hinge domain of the BCMA CAR comprises a CD8a hinge domain, for example, a human CD8a hinge domain. In some embodiments, the hinge domain comprises a CD28 hinge domain, for example, a human CD28 hinge domain. In some embodiments, the hinge domain comprises an IgG4 hinge domain, for example, a human IgG4 hinge domain. In some embodiments, the hinge domain comprises a IgG4 hinge-Ch2-Ch3 domain, for example, a human IgG4 hinge-Ch2-Ch3 domain.

[0275] In some embodiments, the transmembrane domain of the BCMA CAR comprises a CD8a transmembrane domain, for example, a human CD8a transmembrane domain. In some embodiments, the transmembrane domain comprises a CD28 transmembrane domain, for example, a human CD28 transmembrane domain.

[0276] In some embodiments, the intracellular costimulatory domain of the BCMA CAR comprises a 4- IBB costimulatory domain, for example, a human 4- IBB costimulatorydomain. In some embodiments, the intracellular costimulatory domain comprises a CD28 costimulatory domain, for example, a human CD28 costimulatory domain.

[0277] In some embodiments, the intracellular signaling domain of the BCMA CAR comprises a CD3 zeta (Q signaling domain, for example, a human CD3(^ signaling domain.

[0278] In some embodiments, the CAR is a BCMA CAR, including, for example, a BCMA CAR comprising any of the BCMA-specific extracellular binding domains as described, a CD8a hinge domain, a CD8a transmembrane domain, a 4- IBB costimulatory domain, a CD3(^ signaling domain, and / or variants (z.e., having a sequence that is at least about 80% identical, for example, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity) thereof. In any of these embodiments, the BCMA CAR may additionally comprise a signal peptide (e.g., a CD8a signal peptide) as described above. In some embodiments, the first barcode and / or the second barcode is located at the junction of the BCMA-specific extracellular binding domain and the CD8a hinge domain. In some embodiments, the first barcode and / or the second barcode is located at the junction of the CD8a hinge domain and the CD8a transmembrane domain. In some embodiments, the first barcode and / or the second barcode is located at the junction of the CD8a transmembrane domain and the 4- IBB costimulatory domain. In some embodiments, the first barcode and / or the second barcode is located at the junction of the 4- IBB costimulatory domain and the CD3(^ signaling domain.

[0279] In some embodiments, the CAR is a BCMA CAR, including, for example, a BCMA CAR comprising any of the BCMA-specific extracellular binding domains as described, a CD8a hinge domain, a CD8a transmembrane domain, a CD28 costimulatory domain, a CD3(^ signaling domain, and / or variants (z.e., having a sequence that is at least about 80% identical, for example, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity) thereof. In any of these embodiments, the BCMA CAR may additionally comprise a signal peptide as described above. In some embodiments, the first barcode and / or the second barcode is located at the junction of the BCMA-specific extracellular binding domain and the CD8a hinge domain. In some embodiments, the first barcode and / or the second barcode is located at the junction of the CD8a hinge domain and the CD8a transmembrane domain. In some embodiments, the first barcode and / or the second barcode is located at the junction of the CD8a transmembrane domain and the CD28 costimulatorydomain. In some embodiments, the first barcode and / or the second barcode is located at the junction of the CD28 costimulatory domain and the CD3^ signaling domain.

[0280] In some embodiments, the CAR is a BCMA CAR as set forth in SEQ ID NO:346 or is at least about 80% identical (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical) thereto (see Table 10). The encoded BCMA CAR has a corresponding amino acid sequence set forth in SEQ ID NO:347 or is at least about 80% identical (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical) thereto, with the following components: CD8a signal peptide, CT103A scFv (VL- Whitlow linker- VH), CD8a hinge domain, CD8a transmembrane domain, 4- IBB costimulatory domain, and CD3^ signaling domain. In some embodiments, the first barcode and / or the second barcode is located at the junction of the CT103A scFv and the CD8a hinge domain. In some embodiments, the first barcode and / or the second barcode is located at the junction of the CD8a hinge domain and the CD8a transmembrane domain. In some embodiments, the first barcode and / or the second barcode is located at the junction of the CD8a transmembrane domain and the 4- IBB costimulatory domain. In some embodiments, the first barcode and / or the second barcode is located at the junction of the 4- IBB costimulatory domain and the CD3^ signaling domain.

[0281] In some embodiments, the CAR is a commercially available embodiment of BCMA CAR, including, for example, idecabtagene vicleucel (ide-cel, also called bb2121). In some embodiments, the CAR is idecabtagene vicleucel or portions thereof. Idecabtagene vicleucel comprises a BCMA CAR with the following components: the BB2121 binder, CD8a hinge domain, CD8a transmembrane domain, 4- IBB costimulatory domain, and CD3^ signaling domain.

[0282] In some cases, CARs are referred to as first, second, and / or third generation CARs. In some aspects, a first generation CAR is one that solely provides a CD3-chain induced signal upon antigen binding; in some aspects, a second-generation CARs is one that provides such a signal and costimulatory signal, such as one including an intracellular signaling domain from a costimulatory receptor such as CD28 or CD 137; in some aspects, a third generation CAR is one that includes multiple costimulatory domains of different co stimulatory receptors.

[0283] For example, in some embodiments, any of the CARs herein contain an antibody, e.g., an antibody fragment, a transmembrane domain that is or contains atransmembrane portion of CD28 or a functional variant thereof, and an intracellular signaling domain containing a signaling portion of CD28 or functional variant thereof and a signaling portion of CD3-zeta or functional variant thereof. In some embodiments, the CAR contains an antibody, e.g., antibody fragment, a transmembrane domain that is or contains a transmembrane portion of CD28 or a functional variant thereof, and an intracellular signaling domain containing a signaling portion of a 4- IBB or functional variant thereof and a signaling portion of CD3-zeta or functional variant thereof. In some such embodiments, the receptor further includes a spacer containing a portion of an Ig molecule, such as a human Ig molecule, such as an Ig hinge, e.g., an IgG4 hinge, such as a hinge-only spacer.

[0284] In some aspects, the spacer contains only a hinge region of an IgG, such as only a hinge of IgG4 or IgGl. In other embodiments, the spacer is or contains an Ig hinge, e.g., an IgG4-derived hinge, optionally linked to a CH2 and / or CH3 domains. In some embodiments, the spacer is an Ig hinge, e.g., an IgG4 hinge, linked to CH2 and CH3 domains. In some embodiments, the spacer is an Ig hinge, e.g., an IgG4 hinge, linked to a CH3 domain only. In some embodiments, the spacer is or comprises a glycine-serine rich sequence or other flexible linker such as known flexible linkers.

[0285] For example, in some embodiments, any of the CARs herein include an antibody such as an antibody fragment, including scFvs, a spacer, such as a spacer containing a portion of an immunoglobulin molecule, such as a hinge region and / or one or more constant regions of a heavy chain molecule, such as an Ig-hinge containing spacer, a transmembrane domain containing all or a portion of a CD28-derived transmembrane domain, a CD28- derived intracellular signaling domain, and a CD3-zeta signaling domain. In some embodiments, the CAR includes an antibody or fragment, such as scFv, a spacer such as any of the Ig-hinge containing spacers, a CD28-derived transmembrane domain, a 4-lBB-derived intracellular signaling domain, and a CD3-zeta-derived signaling domain.

[0286] The recombinant receptors, such as any of the CARs of the disclosure, expressed by cells administered to a subject generally recognize or specifically bind to a molecule that is expressed in, associated with, and / or specific for the disease or condition or cells thereof being treated. Upon specific binding to the molecule, e.g., antigen, the receptor generally delivers an immuno stimulatory signal, such as an IT AM-transduced signal, into the cell, thereby promoting an immune response targeted to the disease or condition. For example, in some embodiments, the cells express a CAR that specifically binds to an antigen expressed by a cell or tissue of the disease or condition or associated with the disease or condition. In any of these embodiments, the extracellular binding domain of the CAR can becodon-optimized for expression in a host cell or have variant sequences to increase functions of the extracellular binding domain. b. T Cell Receptors

[0287] In some embodiments, the one or more transgenes comprise a T cell receptor (TCR). In some embodiments, the transgene encodes a T cell receptor (TCR) or antigenbinding portion thereof that recognizes a peptide epitope or T cell epitope of a target polypeptide, such as an antigen of a tumor, viral or autoimmune protein.

[0288] In some embodiments, a T cell receptor or TCR is a molecule that contains a variable a and b chain (also known as TCRalpha and TCRbeta, respectively) or a variable g and d chains (also known as TCRalpha and TCRbeta, respectively), or antigen-binding portions thereof, and which is capable of specifically binding to a peptide bound to an MHC molecule. In some embodiments, the TCR is in the alpha-beta (a-P) form. Typically, TCRs that exist in alpha-beta and gamma-delta forms are generally structurally similar, but T cells expressing them may have distinct anatomical locations or functions. A TCR can be found on the surface of a cell or in soluble form. Generally, a TCR is found on the surface of T cells (or T lymphocytes) where it is generally responsible for recognizing antigens bound to major histocompatibility complex (MHC) molecules.

[0289] Unless otherwise stated, the term “TCR” should be understood to encompass full TCRs as well as antigen-binding portions or antigen-binding fragments thereof. In some embodiments, the TCR is an intact or full-length TCR, including TCRs in the a-P form or y-5 form. In some embodiments, the TCR is an antigen-binding portion that is less than a full- length TCR but that binds to a specific peptide bound in an MHC molecule, such as binds to an MHC -peptide complex. In some cases, an antigen-binding portion or fragment of a TCR can contain only a portion of the structural domains of a full-length or intact TCR, but yet is able to bind the peptide epitope, such as MHC -pep tide complex, to which the full TCR binds. In some cases, an antigen-binding portion contains the variable domains of a TCR, such as variable a chain and variable P chain of a TCR, sufficient to form a binding site for binding to a specific MHC-peptide complex. Generally, the variable chains of a TCR contain complementarity determining regions involved in recognition of the peptide, MHC and / or MHC-peptide complex. c. Chimeric Auto-Antibody Receptors (CAAR) and B-cell Autoantibody Receptors (BAR)

[0290] In some embodiments, the one or more transgenes comprise a chimeric autoantibody receptor (CAAR).

[0291] In some embodiments, the CAAR binds, e.g., specifically binds, or recognizes, an autoantibody. In some embodiments, a cell expressing the CAAR, such as a T cell engineered to express a CAAR, can be used to bind to and kill autoantibody-expressing cells, but not normal antibody expressing cells. In some embodiments, CAAR-expressing cells can be used to treat an autoimmune disease associated with expression of self-antigens, such as autoimmune diseases. In some embodiments, CAAR-expressing cells can target B cells that ultimately produce the autoantibodies and display the autoantibodies on their cell surfaces, mark these B cells as disease- specific targets for therapeutic intervention. In some embodiments, CAAR-expressing cells can be used for efficiently targeting and killing the pathogenic B cells in autoimmune diseases by targeting the disease-causing B cells using an antigen- specific chimeric autoantibody receptor. In some embodiments, the recombinant receptor is a CAAR, such as any described in U.S. Patent Application Pub. No. US 2017 / 0051035.

[0292] In some embodiments, the CAAR comprises an antigen selected from a pancreatic P-cell antigen, synovial joint antigen, myelin basic protein, proteolipid protein, myelin oligodendritic glycoprotein, MuSK, keratinocyte adhesion protein desmoglein 3 (Dsg3), Ro-RNP complex, La antigen, myeloperoxidase, proteinase 3, cardiolipin, citrullinated proteins, carbamylated proteins, a3 chain of basement membrane collagen, or any combination thereof.

[0293] In some embodiments, the CAAR comprises an autoantibody binding domain, a transmembrane domain, and one or more intracellular signaling regions or domains (also interchangeably called a cytoplasmic signaling domain or region). In some embodiments, the intracellular signaling region comprises an intracellular signaling domain. In some embodiments, the intracellular signaling domain is or comprises a primary signaling domain, a signaling domain that is capable of stimulating and / or inducing a primary activation signal in a T cell, a signaling domain of a T cell receptor (TCR) component e.g., an intracellular signaling domain or region of a CD3-zeta) chain or a functional variant or signaling portion thereof), and / or a signaling domain comprising an immunoreceptor tyrosine- based activation motif (ITAM).

[0294] In some embodiments, the one or more transgenes comprise a B-cell autoantibody receptor (BAR). In some embodiments, the BAR comprises an FVIII antigen.

[0295] In some embodiments, the autoantibody binding domain comprises an autoantigen or a fragment thereof. The choice of autoantigen can depend upon the type of autoantibody being targeted. For example, the autoantigen may be chosen because itrecognizes an autoantibody on a target cell, such as a B cell, associated with a particular disease state, e.g., an autoimmune disease, such as an autoantibody-mediated autoimmune disease. In some embodiments, the autoimmune disease includes pemphigus vulgaris (PV). Exemplary autoantigens include desmoglein 1 (Dsgl) and Dsg3.

[0296] In some embodiments, the first transgene and / or the second transgene encodes an antibody or an antibody fragment, a chimeric antigen receptor (CAR), a chimeric autoantibody receptor (CAAR), a B-cell autoantibody receptor (BAR), a T cell receptor (TCR), or one or more tolerogenic factors. In some embodiments, the first transgene and / or the second transgene encodes a CAR.

[0297] In some embodiments, the CAR encoded by the first transgene and / or the second transgene comprises a hinge domain, a transmembrane domain, and one or more signaling domains. In some embodiments, the hinge domain is a hinge domain of a naturally occurring protein. Hinge domains of any protein known in the art to comprise a hinge domain are compatible for use in the CARs as described herein. In some embodiments, the hinge domain is at least a portion of a hinge domain of a naturally occurring protein and confers flexibility to the CAR as described herein. In some embodiments, the hinge domain is a variant of a hinge domain of a naturally occurring protein (z.e., having a sequence that is at least about 80% identical, for example, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity).

[0298] In some embodiments, the hinge domain is selected from the group consisting of: CD8a hinge domain, CD28 hinge domain, IgG4 hinge domain, IgG4 hinge-CH2-CH3 domain, and any functional variant thereof. In some embodiments, the hinge domain is derived from CD8a. In some embodiments, the hinge domain is a portion of the hinge domain of CD8a, or any functional variant thereof. In some embodiments, the hinge domain is derived from CD28. In some embodiments, the hinge domain is a portion of the hinge domain of CD28, or any functional variant thereof. In some embodiments, the hinge domain is derived from IgG4. In some embodiments, the hinge domain is a portion of the hinge domain of IgG4, or any functional variant thereof. In some embodiments, the hinge domain is derived from IgG4 hinge-CH2-CH3. In some embodiments, the hinge domain is a portion of the hinge domain of IgG4 hinge-CH2-CH3, or any functional variant thereof.

[0299] In some embodiments, the transmembrane domain is selected from the group consisting of: alpha, beta, or zeta chain of a T cell receptor, CD28, CD3s, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154,CD8a, CD8P, 4-1BB / CD137, CD28, CD34, CD4, FcsRIy, CD16, OX40 / CD134, CD3^, CD3s, CD3y, CD35, TCRa, TCRp, TCR^, CD32, CD64, CD64, CD45, CD5, CD9, CD22, CD37, CD80, CD86, CD40, CD40L / CD154, VEGFR2, FAS, FGFR2B, and any functional variant thereof. In some embodiments, the signaling domain is selected from the group consisting of: B7-1 / CD80, B7-2 / CD86, B7-H1 / PD-L1, B7-H2, B7-H3, B7-H4, B7-H6, B7- H7, BTLA / CD272, CD28, CTLA-4, Gi24 / VISTA / B7-H5, ICOS / CD278, PD-1, PD-L2 / B7- DC, PDCD6, 4-1BB / TNFSF9 / CD137, 4-1BB Ligand / TNFSF9, BAFF / BLyS / TNFSF13B, BAFF R / TNFRSF13C, CD27 / TNFRSF7, CD27 Ligand / TNFSF7, CD30 / TNFRSF8, CD30 Ligand / TNFSF8, CD40 / TNFRSF5, CD40 / TNFSF5, CD40 Ligand / TNFSF5, DR3 / TNFRSF25, GITR / TNFRSF18, GITR Ligand / TNFSF18, HVEM / TNFRSF14, LIGHT / TNFSF14, Lymphotoxin-alpha / TNFp, OX40 / TNFRSF4, 0X40 Ligand / TNFSF4, RELT / TNFRSF19L, TACI / TNFRSFI 3B, TL1A / TNFSF15, TNFa, TNF RII / 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 / 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.

[0300] In some embodiments, the CAR encoded by the first transgene and / or the second transgene comprises a CD8a hinge domain, a CD8a transmembrane domain, a 4- IBB domain, and a CD3zeta domain. In some embodiments, the CAR encoded by the first transgene and / or the second transgene is selected from the group consisting of: a CD5- specific CAR, a 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 CD138-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 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 IE-13Ra-specific CAR, a Mesothelin-specific CAR, a MUC1 -specific CAR, a MUC 16- specific CAR, a RORl-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 IE 13Ra2- specific CAR, a MAGEA3-specific CAR, a M AGE A4- specific CAR, a MART1- specific CAR, a NY-ESO-1 -specific CAR, a VEGFR2- specific CAR, a a-Folate receptorspecific 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-M A A- 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. In some embodiments, the CAR encoded by the first transgene and / or the second transgene further comprises one or more costimulatory domain(s).

[0301] In some embodiments, the diverged nucleotide sequence within the transgene is located at the junction of: (i) the signaling domain and the co-stimulatory domain; or (ii) the hinge domain and the transmembrane domain. In some embodiments, the diverged nucleotide sequence within the transgene is located at the junction of the 4- IBB and CD3-zeta domains. v. Tolerogenic Factors

[0302] In some embodiments, the one or more transgenes comprise a tolerogenic factor. Tolerogenic factors include any factors that promote or contribute to promoting or inducing tolerance to an engineered cell or population of cells (e.g., a hypoimmunogenic islet cell such as a hypoimmunogenic beta cell) of the disclosure by the immune system e.g., innate or adaptive immune system). In some embodiments, the expression of a detection agent acts as a signal for the administration of an antibody directed against or specific to a tolerogenic agent or kill switch, e.g., an anti-CD47 antibody. In some embodiments, the kill switch may be an exogenously administered agent that recognizes one or more tolerogenic factor on the surface of the modified cells. In some embodiments, the exogenously administered agent is an antibody directed against or specific to a tolerogenic agent, e.g., an anti-CD47 antibody. By recognizing and blocking a tolerogenic factor on modified cells, an exogenously administered antibody may block the immune inhibitory functions of the tolerogenic factor thereby re-sensitizing the immune system to the modified cells. For instance, for modified cells that overexpresses CD47, an exogenously administered anti-CD47 antibody may be administered to the subject, resulting in masking of CD47 on the modified cells and triggering of an immune response to the modified cells. In some embodiments, the anti-CD47 antibody is Magrolimab.

[0303] In some embodiments, the tolerogenic factor comprises one or more of CD16, CD24, CD35, CD39, CD46, CD47, CD52, CD55, CD59, CD64, CD200, CCL22, CTLA4-Ig, Cl inhibitor, FASL, IDO1, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, IL- 10, IL-35, PD- Ll, SERPINB9, CCL21, MFGE8, DUX4, B2M-HLA-E, CD27, IL-39, CD16 Fc Receptor, IL15-RF, H2-M3 (HLA-G), A20 / TNFAIP3, CR1, HLA-F, DUX4, MANF, or any combinations, functional fragments, or variants thereof. In some embodiments, the tolerogenic factor is CD47, PD-L1, HLA-E or HLA-G, CCL21, FasL, Serpinb9, CD200, Mfge8, or any combinations, functional fragments, or variants thereof. In some embodiments, expression of the tolerogenic e.g., immune) factor affects immune recognition and tolerance of an engineered cell or population of cells (e.g., T cells, B cells, NK cells, islet cells such as beta cells, or hypoimmunogenic cells thereof) of the disclosure in a recipient. In some embodiments, the engineered cell or population of cells contains an exogenous sequence that encodes the one or more tolerogenic factors. In some embodiments, the tolerogenic factor is overexpressed in the cell. In some embodiments, the expression of the tolerogenic factor is overexpressed or increased in the engineered cell or population of cells, e.g., compared to a similar cell of the same cell type that has not been engineered with the modification, such as a reference or non-engineered cell, e.g., a cell not engineered with a transgene encoding the tolerogenic factor.

[0304] In some embodiments, the tolerogenic factor is CD47, or a functional fragment or variant thereof. CD47 is a leukocyte surface antigen and has a role in cell adhesion and modulation of integrins. It is normally expressed on the surface of a cell and signals to circulating macrophages not to eat the cell. In some embodiments, the engineered cell or population of cells (e.g., T cells, B cells, NK cells, islet cells such as beta cells, or hypoimmunogenic cells thereof) contains a transgene sequence that encodes CD47, such as human CD47, or a functional fragment or variant thereof. In some embodiments, CD47, or a functional fragment or variant thereof, is overexpressed in the cell. In some embodiments, the expression of CD47, or a functional fragment or variant thereof, is overexpressed or increased in the engineered cell or population of cells compared to a similar cell of the same cell type that has not been engineered, such as a reference or non-engineered cell, e.g., a cellnot engineered with a transgene encoding CD47. In some embodiments, the engineered cell or population of cells contains an overexpressed transgene that encodes CD47, or a functional fragment or variant thereof, such as human CD47. Useful genomic, polynucleotide and polypeptide information about human CD47 are provided in, for example, the NP_001768.1, NP_942088.1, NM_001777.3 and NM_198793.2.

[0305] In some embodiments, the cell outlined herein comprises a transgene sequence encoding a CD47 polypeptide that has at least 95% sequence identity (e.g., 95%, 96%, 97%, 98%, 99%, or more) to an amino acid sequence as set forth in NCBI Ref. Sequence Nos. NP_001768.1 and NP_942088.1, or to fragments thereof. In some embodiments, the cell outlined herein comprises a transgene sequence encoding a CD47 polypeptide having an amino acid sequence as set forth in NCBI Ref. Sequence Nos. NP_001768.1 and NP_942088.1, or a functional fragment or variant thereof. In some embodiments, the cell comprises a transgene sequence encoding a CD47 polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) to the sequence set forth in NCBI Ref. Nos. NM_001777.3 and NM_198793.2, or to fragments thereof. In some embodiments, the cell comprises a transgene sequence encoding a CD47 polynucleotide as set forth in NCBI Ref. Sequence Nos. NM_001777.3 and NM_198793.2, or fragments thereof.

[0306] In some embodiments, the cell outlined herein comprises a transgene sequence encoding a CD47 polypeptide has at least 95% sequence identity (e.g., 95%, 96%, 97%, 98%, 99%, or more) to an amino acid sequence as set forth in NCBI Ref. Sequence Nos. NP_001768.1 and NP_942088.1, or to fragments thereof. In some embodiments, the cell outlined herein comprises a transgene sequence encoding a CD47 polypeptide having an amino acid sequence as set forth in NCBI Ref. Sequence Nos. NP_001768.1 and NP_942088.1, or a functional fragment or variant thereof. In some embodiments, the cell comprises a transgene sequence encoding a CD47 polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) to the sequence set forth in NCBI Ref. Nos. NM_001777.3 and NM_198793.2, or to a functional fragment thereof. In some embodiments, the cell comprises a transgene sequence encoding a CD47 polynucleotide as set forth in NCBI Ref. Sequence Nos. NM_001777.3 and NM_198793.2, or fragments thereof.

[0307] In some embodiments, the cell comprises a CD47 polypeptide having at least 95% sequence identity (e.g., 95%, 96%, 97%, 98%, 99%, or more) to an amino acid sequence as set forth in NCBI Ref. Sequence Nos. NP_001768.1 and NP_942088.1, or a functionalI l lfragment thereof. In some embodiments, the cell outlined herein comprises a CD47 polypeptide having an amino acid sequence as set forth in NCBI Ref. Sequence Nos. NP_001768.1 and NP_942088.1, or a functional fragment or variant thereof.

[0308] In some embodiments, the cell comprises a CD47 polypeptide having at least 95% sequence identity (e.g., 95%, 96%, 97%, 98%, 99%, or more) to an amino acid sequence selected from the group consisting of SEQ ID NOs:l-5, or a functional fragment thereof. In some embodiments, the cell outlined herein comprises a CD47 polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs:l-5, or a functional fragment or variant thereof, as laid out in Table 11 below.Table 11. CD47 polypeptide sequences.

[0309] In some embodiments, the cell comprises a CD47 polypeptide encoded by a nucleic acid sequence having at least 95% sequence identity (e.g., 95%, 96%, 97%, 98%, 99%, or more) to a nucleic acid sequence selected from the group consisting of SEQ ID NOs:6-l 1, or a functional fragment thereof. In some embodiments, the cell outlined herein comprises a CD47 polypeptide encoded by a nucleic acid sequence having a nucleic acid sequence selected from the group consisting of SEQ ID NOs:6-l 1, or a functional fragment or variant thereof, as laid out in Table 12 below.Table 12. CD47 nucleic acid sequences.

[0310] In some embodiments, all or a functional portion of CD47 can be linked to other components such as a signal peptide, a leader sequence, a secretory signal, a label (e.g., a reporter gene), or any combination thereof. In some embodiments, the nucleic acid sequence encoding a signal peptide of CD47 is replaced with a nucleic acid sequence encoding a signal peptide from a heterologous protein. The heterologous protein can be, for example, CD8a, CD28, tissue plasminogen activator (tPA), growth hormone, granulocyte-macrophage colony stimulating factor (GM-CSF), GM-CSF receptor (GM-CSFRa), or an immunoglobulin (e.g., IgE or IgK). In some embodiments, the signal peptide is a signal peptide from an immunoglobulin (such as IgG heavy chain or IgG-kappa light chain), a cytokine (such as interleukin-2 (IL-2), or CD33), a serum albumin protein (e.g., HSA or albumin), a human azurocidin preprotein signal sequence, a luciferase, a trypsinogen (e.g., chymotrypsinogen or trypsinogen) or other signal peptide able to efficiently express a protein by or on a cell.

[0311] In some embodiments, the engineered cell or population of cells (e.g., T cells, B cells, NK cells, islet cells such as beta cells, or hypoimmunogenic cells thereof) contains a transgene that encodes CD200, such as human CD200, or a functional fragment or variant thereof. In some embodiments, CD200 is overexpressed in the cell. In some embodiments, the expression of CD200 is increased in the engineered cell or population of cells compared to a similar reference or non-engineered cell (including with any other modifications) except that the reference or non-engineered cell does not include the transgene encoding CD200. Useful genomic, polynucleotide and polypeptide information about human CD200 are provided in, for example, the GeneCard Identifier GC03P112332, HGNC No. 7203, NCBI Gene ID 4345, Uniprot No. P41217, and NCBI RefSeq Nos. NP_001004196.2, NM_001004196.3, NP_001305757.1, NM_001318828.1, NP_005935.4, NM_005944.6, XP_005247539.1, and XM_005247482.2. In certain embodiments, the transgene encoding CD200, or a functional fragment or variant thereof, is operably linked to a promoter.

[0312] In some embodiments, the engineered cell or population of cells (e.g., T cells, B cells, NK cells, islet cells such as beta cells, or hypoimmunogenic cells thereof) contains a transgene that encodes HLA-E, such as human HLA-E, or a functional fragment or variant thereof. In some embodiments, HLA-E is overexpressed in the cell. In some embodiments, the expression of HLA-E is increased in the engineered cell or population of cells compared to a similar reference or non-engineered cell (including with any other modifications) exceptthat the reference or non-engineered cell does not include the transgene encoding HLA-E. Useful genomic, polynucleotide, and polypeptide information about human HLA-E are provided in, for example, the GeneCard Identifier GC06P047281, HGNC No. 4962, NCBI Gene ID 3133, Uniprot No. P13747, and NCBI RefSeq Nos. NP_005507.3 and NM_005516.5. In certain embodiments, the transgene encoding HLA-E, or a functional fragment or variant thereof, is operably linked to a promoter.

[0313] In some embodiments, the engineered cell or population of cells (e.g., T cells, B cells, NK cells, islet cells such as beta cells, or hypoimmunogenic cells thereof) contains a transgene that encodes HLA-G, such as human HLA-G, or a functional fragment or variant thereof. In some embodiments, HLA-G is overexpressed in the cell. In some embodiments, the expression of HLA-G is increased in the engineered cell or population of cells compared to a similar reference or non-engineered cell (including with any other modifications) except that the reference or non-engineered cell does not include the transgene encoding HLA-G. Useful genomic, polynucleotide and polypeptide information about human HLA-G are provided in, for example, the GeneCard Identifier GC06P047256, HGNC No. 4964, NCBI Gene ID 3135, Uniprot No. P17693, and NCBI RefSeq Nos. NP_002118.1 and NM_002127.5. In certain embodiments, the transgene encoding HLA-G, or a functional fragment or variant thereof, is operably linked to a promoter.

[0314] In some embodiments, the engineered cell or population of cells (e.g., T cells, B cells, NK cells, islet cells such as beta cells, or hypoimmunogenic cells thereof) contains a transgene that encodes PD-L1, such as human PD-L1, or a functional fragment or variant thereof. In some embodiments, PD-L1 is overexpressed in the cell. In some embodiments, the expression of PD-L1 is increased in the engineered cell or population of cells compared to a similar reference or non-engineered cell (including with any other modifications) except that the reference or non-engineered cell does not include the transgene encoding PD-L1. Useful genomic, polynucleotide and polypeptide information about human PD-L1 or CD274 are provided in, for example, the GeneCard Identifier GC09P005450, HGNC No. 17635, NCBI Gene ID 29126, Uniprot No. Q9NZQ7, and NCBI RefSeq Nos. NP_001254635.1, NM_001267706.1, NP_054862.1, and NM_014143.3. In certain embodiments, the polynucleotide encoding PD-L1, or a functional fragment or variant thereof, is operably linked to a promoter.

[0315] In some embodiments, the engineered cell or population of cells (e.g., T cells, B cells, NK cells, islet cells such as beta cells, or hypoimmunogenic cells thereof) contains a transgene that encodes FasL, such as human FasL, or a functional fragment or variant thereofIn some embodiments, FasL is overexpressed in the cell. In some embodiments, the expression of FasL is increased in the engineered cell or population of cells compared to a similar reference or non-engineered cell (including with any other modifications) except that the reference or non-engineered cell does not include the transgene encoding FasL. Useful genomic, polynucleotide and polypeptide information about human Fas ligand (which is known as FasL, FASLG, CD178, TNFSF6, and the like) are provided in, for example, the GeneCard Identifier GC01P172628, HGNC No. 11936, NCBI Gene ID 356, Uniprot No. P48023, and NCBI RefSeq Nos. NP_000630.1, NM_000639.2, NP_001289675.1, and NM_001302746.1. In certain embodiments, the transgene encoding Fas-L is operably linked to a promoter.

[0316] In some embodiments, the engineered cell or population of cells (e.g., T cells, B cells, NK cells, islet cells such as beta cells, or hypoimmunogenic cells thereof) contains a transgene that encodes CCL21, such as human CCL21, or a functional fragment or variant thereof. In some embodiments, CCL21 is overexpressed in the cell. In some embodiments, the expression of CCL21 is increased in the engineered cell or population of cells compared to a similar reference or non-engineered cell (including with any other modifications) except that the reference or non-engineered cell does not include the transgene encoding CCL21. Useful genomic, polynucleotide and polypeptide information about human CCL21 are provided in, for example, the GeneCard Identifier GC09M034709, HGNC No. 10620, NCBI Gene ID 6366, Uniprot No. 000585, and NCBI RefSeq Nos. NP_002980.1 and NM_002989.3. In certain embodiments, the transgene encoding CCL21, or a functional fragment or variant thereof, is operably linked to a promoter.

[0317] In some embodiments, the engineered cell or population of cells (e.g., T cells, B cells, NK cells, islet cells such as beta cells, or hypoimmunogenic cells thereof) contains a transgene that encodes CCL22, such as human CCL22, or a functional fragment or variant thereof. In some embodiments, CCL22 is overexpressed in the cell. In some embodiments, the expression of CCL22 is increased in the engineered cell or population of cells compared to a similar reference or non-engineered cell (including with any other modifications) except that the reference or non-engineered cell does not include the transgene encoding CCL22. Useful genomic, polynucleotide and polypeptide information about human CCL22 are provided in, for example, the GeneCard Identifier GC16P057359, HGNC No. 10621, NCBI Gene ID 6367, Uniprot No. 000626, and NCBI RefSeq Nos. NP_002981.2, NM_002990.4, XP_016879020.1, and XM_017023531.1. In certain embodiments, the transgene encoding CCL22, or a functional fragment or variant thereof, is operably linked to a promoter.

[0318] In some embodiments, the engineered cell or population of cells (e.g., T cells, B cells, NK cells, islet cells such as beta cells, or hypoimmunogenic cells thereof) contains a transgene that encodes Mfge8, such as human Mfge8, or a functional fragment or variant thereof. In some embodiments, Mfge8 is overexpressed in the cell. In some embodiments, the expression of Mfge8 is increased in the engineered cell or population of cells compared to a similar reference or non-engineered cell or population of cells (including with any other modifications) except that the reference or non-engineered cell or population of cells does not include the transgene encoding Mfge8. Useful genomic, polynucleotide and polypeptide information about human Mfge8 are provided in, for example, the GeneCard Identifier GC15M088898, HGNC No. 7036, NCBI Gene ID 4240, Uniprot No. Q08431, and NCBI RefSeq Nos. NP.001108086.1, NM_001114614.2, NP_001297248.1, NM_001310319.1, NP_001297249.1, NM_001310320.1, NP_001297250.1, NM_001310321.1, NP_005919.2, and NM_005928.3. In certain embodiments, the transgene encoding Mfge8, or a functional fragment or variant thereof , is operably linked to a promoter.

[0319] In some embodiments, the engineered cell or population of cells (e.g., T cells, B cells, NK cells, islet cells such as beta cells, or hypoimmunogenic cells thereof) contains a transgene that encodes SerpinB9, such as human SerpinB9, or a functional fragment or variant thereof. In some embodiments, SerpinB9 is overexpressed in the cell. In some embodiments, the expression of SerpinB9 is increased in the engineered cell or population of cells compared to a similar reference or non-engineered cell or population of cells (including with any other modifications) except that the reference or non-engineered cell or population of cells does not include the transgene encoding SerpinB9. Useful genomic, polynucleotide and polypeptide information about human SerpinB9 are provided in, for example, the GeneCard Identifier GC06M002887, HGNC No. 8955, NCBI Gene ID 5272, Uniprot No. P50453, and NCBI RefSeq Nos. NP_004146.1, NM_004155.5, XP_005249241.1, and XM_005249184.4. In certain embodiments, the transgene encoding SerpinB9, or a functional fragment or variant thereof, is operably linked to a promoter.(2) Insertion of Exogenous Sequences at Endogenous Proliferation Genes or Off- Target Cell Marker Genes

[0320] In some embodiments, provided herein is a modified cell comprising one or more exogenous sequences inserted at endogenous proliferation genes or off-target cell marker genes, such as any of endogenous proliferation genes or off-target cell marker genes described above or known in the art. Exogenous sequences, such as any of the exogenoussequences described below, may be inserted at endogenous proliferation genes or off-target cell marker genes by various methods and agents, for example, using zinc finger nucleases, restriction enzymes, transcription activator-like effector nucleases (TALENs), Programmable Addition via Site-specific Targeting Elements (PASTE), nucleic acid-guided nuclease editing, and the like. In some embodiments, exogenous sequences, such as any of the exogenous sequences described below, may be inserted at endogenous proliferation genes or off-target cell marker genes using a genome editing complex, as described further below. a. Genome editing complexes

[0321] In some embodiments, one or more exogenous sequences, such as any of the exogenous sequences described below, may be inserted at endogenous proliferation genes or off-target cell marker genes using a genome editing complex.

[0322] Any of a variety of agents associated with gene editing technologies can be included in a genome editing complex. The genome editing complex can be used for knock- in or integration of DNA sequences into a region of the genome. In some embodiments, the genome editing complex mediates single-strand breaks (SSB). In some embodiments, the genome editing complex mediates double-strand breaks (DSB), including in connection with non-homologous end-joining (NHEJ) or homology-directed repair (HDR). In some embodiments, the genome editing complex does not mediate SSB. In some embodiments, the genome editing complex does not mediate DSB. In some embodiments, the genome editing complex can be used for DNA base editing or prime-editing. In some embodiments, the genome editing complex can be used for Programmable Addition via Site- specific Targeting Elements (PASTE). In some embodiments, the genome editing complex can cleave, deaminate, nick, polymerize, interrogate, integrate, cut, unwind, break, alter, methylate, demethylate, or otherwise destabilize a target locus.

[0323] In some embodiments, the one or more exogenous sequences are inserted at endogenous proliferation genes or off-target cell marker genes by one or more gene edits. In some embodiments, said one or more gene edits are made by a genome editing complex. In some embodiments, a cell of the disclosure comprises a genome editing complex, e.g., which can insert one or more exogenous sequences at endogenous proliferation genes or off-target cell marker genes.

[0324] In some embodiments, the genome editing complex is or encodes one or more polypeptides having an activity selected from nuclease activity e.g., programmable nucleaseactivity); nickase activity (e.g., programmable nickase activity); homing activity (e.g., programmable DNA binding activity); nucleic acid polymerase activity (e.g., DNA polymerase or RNA polymerase activity); integrase activity; recombinase activity; or base editing activity (e.g., cytidine deaminase or adenosine deaminase activity).

[0325] In some embodiments, the genome editing complex is one for use in target- primed reverse transcription (TPRT) or “prime editing”. In some embodiments, prime editing mediates targeted insertions, deletions, all 12 possible base-to-base conversions, and combinations thereof in human cells without requiring DSBs or donor DNA templates. Prime editing is a genome editing method that directly writes new genetic information into a specified DNA site using a nucleic acid programmable DNA binding protein (“napDNAbp”) working in association with a polymerase (i.e., in the form of a fusion protein or otherwise provided in trans with the napDNAbp), wherein the prime editing system is programmed with a prime editing (PE) guide RNA (“PEgRNA”) that both specifies the target site and templates the synthesis of the desired edit in the form of a replacement DNA strand by way of an extension (either DNA or RNA) engineered onto a guide RNA (e.g., at the 5' or 3' end, or at an internal portion of a guide RNA). The replacement strand containing the desired edit (e.g., a single nucleobase substitution) shares the same sequence as the endogenous strand of the target site to be edited (with the exception that it includes the desired edit). Through DNA repair and / or replication machinery, the endogenous strand of the target site is replaced by the newly synthesized replacement strand containing the desired edit. In some cases, prime editing may be thought of as a “search-and-replace” genome editing technology since the prime editors search and locate the desired target site to be edited, and encode a replacement strand containing a desired edit which is installed in place of the corresponding target site endogenous DNA strand at the same time. For example, prime editing can be adapted for conducting precision CRISPR / Cas-based genome editing in order to bypass double stranded breaks. In some embodiments, the genome editing complex is or encodes for a primer editor that is a reverse transcriptase, or any DNA polymerase known in the art. Thus, in one aspect, the prime editor may comprise Cas9 (or an equivalent napDNAbp) which is programmed to target a DNA sequence by associating it with a specialized guide RNA (i.e., PEgRNA) containing a spacer sequence that anneals to a complementary protospacer in the target DNA. Such methods include any disclosed in Anzalone et al., (doi.org / 10.1038 / s41586-019-1711- 4), or in PCT publication Nos. WO2020191248, WO2021226558, or W02022067130, which are hereby incorporated in their entirety. In some embodiments, the genome editing complex is or encodes for a Cas protein-reverse transcriptase fusion or related systems to target aspecific DNA sequence with a guide RNA, generate a single strand nick at the target site, and use the nicked DNA as a primer for reverse transcription of an engineered reverse transcriptase template that is integrated with the guide RNA. In some embodiments, the prime editor protein is paired with two prime editing guide RNAs (pegRNAs) that template the synthesis of complementary DNA flaps on opposing strands of genomic DNA, resulting in the replacement of endogenous DNA sequence between the PE-induced nick sites with pegRNA-encoded sequences.

[0326] In some embodiments, the genome editing complex is or encodes a base editor (e.g., a nucleobase editor). Base editors (BEs) are typically fusions of a Cas (“CRISPR- associated”) domain and a nucleobase modification domain (e.g., a natural or evolved deaminase, such as a cytidine deaminase that include APOBEC1 (“apolipoprotein B mRNA editing enzyme, catalytic polypeptide 1”), CDA (“cytidine deaminase”), and AID (“activation-induced cytidine deaminase”) domains. In some cases, base editors may also include proteins or domains that alter cellular DNA repair processes to increase the efficiency and / or stability of the resulting single-nucleotide change. Currently available base editors include cytidine base editors (e.g., BE4) that convert target C*G to T«A and adenine base editors (e.g., ABE7.10) that convert target A«T to G*C. Cas9-targeted deamination was first demonstrated in connection with a Base Editor (BE) system designed to induce base changes without introducing double-strand DNA breaks. Further Rat deaminase APOBEC1 (rAPOBECl) fused to deactivated Cas9 (dCas9) was used to successfully convert cytidines to thymidines upstream of the PAM of the sgRNA. This first BE system was optimized by changing the dCas9 to a “nickase” Cas9 D10A, which nicks the strand opposite the deaminated cytidine. Without being bound by theory, this is expected to initiate long-patch base excision repair (BER), where the deaminated strand is preferentially used to template the repair to produce a U:A base pair, which is then converted to T:A during DNA replication. In some embodiments, the genome editing complex is a nucleobase editor containing a first DNA binding protein domain that is catalytically inactive, a domain having base editing activity, and a second DNA binding protein domain having nickase activity, where the DNA binding protein domains are expressed on a single fusion protein or are expressed separately (e.g., on separate expression vectors). In some embodiments, the base editor is a fusion protein comprising a domain having base editing activity (e.g., cytidine deaminase or adenosine deaminase), and two nucleic acid programmable DNA binding protein domains (napDNAbp), a first comprising nickase activity and a second napDNAbp that is catalytically inactive, wherein at least the two napDNAbp are joined by a linker. In some embodiments,the base editor is a fusion protein that comprises a DNA domain of a CRISPR-Cas e.g., Cas9) having nickase activity (nCas; nCas9), a catalytically inactive domain of a CRISPR- Cas protein (e.g., Cas9) having nucleic acid programmable DNA binding activity (dCas; e.g., dCas9), and a deaminase domain, wherein the dCas is joined to the nCas by a linker, and the dCas is immediately adjacent to the deaminase domain. In some embodiments, the base editor is an adenine-to-thymine or “ATBE” (or thymine-to-adenine or “TABE”) transversion base editor. Exemplary base editor and base editor systems include any as described in patent publication Nos. US20220127622, US20210079366, US20200248169, US20210093667, US20210071163, W02020181202, WO2021158921, WO2019126709, W02020181178, W02020181195, W02020214842, W02020181193, which are hereby incorporated in their entirety.

[0327] In some embodiments, the genome editing complex is for use in Programmable Addition via Site-specific Targeting Elements (PASTE). In some aspects, PASTE is a platform in which genomic insertion is directed via a CRISPR-Cas9 nickase fused to both a reverse transcriptase and serine integrase. As described in loannidi et al.(doi.org / 10.1101 / 2021.11.01.466786), PASTE does not generate double stranded breaks but allows for integration of sequences as large as ~36 kb. In some embodiments, the serine integrase can be any known in the art. In some embodiments, the serine integrase has sufficient orthogonality such that PASTE can be used for multiplexed gene integration, simultaneously integrating at least two different genes in at least two genomic loci. In some embodiments, PASTE has editing efficiencies comparable to or better than those of homology directed repair or non-homologous end joining based integration, with activity in nondividing cells and fewer detectable off-target events.

[0328] In some embodiments, the genome editing complex comprises a genome targeting entity and / or a genome modifying entity.

[0329] In some embodiments, the genome targeting entity is a nucleic acid- guided targeting entity, such as any of a sequence specific nuclease, a nucleic acid programmable DNA binding protein, an RNA guided nuclease, RNA-guided nuclease comprising a Cas nuclease and a guide RNA (CRISPR-Cas combination), a ribonucleo...

Claims

CLAIMSWhat is claimed is:

1. A cell comprising a nucleic acid encoding a selection agent or a detection agent integrated at an endogenous proliferation gene locus and operably linked to the promoter of the endogenous proliferation gene, wherein expression of the nucleic acid encoding the selection agent or the detection agent is regulated by the promoter of the endogenous proliferation gene and / or regulatory elements of the endogenous proliferation gene.

2. A cell comprising a nucleic acid encoding a selection agent and a detection agent integrated at an endogenous proliferation gene locus and operably linked to the promoter of the endogenous proliferation gene, wherein expression of the nucleic acid encoding the selection agent and the detection agent is regulated by the promoter of the endogenous proliferation gene and / or regulatory elements of the endogenous proliferation gene.

3. A cell comprising a first nucleic acid encoding a selection agent and a second nucleic acid encoding a detection agent, wherein the first nucleic acid is integrated at a first endogenous proliferation gene locus and operably linked to the promoter of the first endogenous proliferation gene, wherein expression of the first nucleic acid encoding the selection agent is regulated by the promoter of the first endogenous proliferation gene and / or regulatory elements of the first endogenous proliferation gene, wherein the second nucleic acid is integrated at a second endogenous proliferation gene locus and operably linked to the promoter of the second endogenous proliferation gene, and wherein expression of the second nucleic acid encoding the detection agent is regulated by the promoter of the second endogenous proliferation gene and / or regulatory elements of the endogenous proliferation gene.

4. The cell of any one of claims 1-3, wherein the first endogenous proliferation gene and / or the second endogenous proliferation gene are expressed in a cell that is proliferating.

5. The cell of any one of claims 1-4, wherein the first endogenous proliferation gene and / or the second endogenous proliferation gene are expressed in a cell that is included in a population of therapeutic cells.

6. The cell of any one of claims 1-5, wherein the first endogenous proliferation gene and / or the second endogenous proliferation gene are expressed in a cell that is included in a population of therapeutic cells but is not a therapeutic cell.

7. The cell of any one of claims 1-6, wherein the cell is a pluripotent cell, a progenitor cell, or a differentiated cell that is proliferating.

8. The cell of any one of claims 1-7, wherein the first endogenous proliferation gene and the second endogenous proliferation gene are the same, and the first and second nucleic acids are integrated at different alleles.

9. The cell of any one of claims 1-8, wherein the first endogenous proliferation gene and the second endogenous proliferation gene are different.

10. The cell of any one of claims 1-9, wherein the endogenous proliferation gene is expressed at a higher level from day 0 to about day 5 of differentiation, compared to expression of the endogenous proliferation gene at day 18 of differentiation, or wherein the endogenous proliferation gene is highly expressed from day 1 to about day 5 of differentiation.

11. The cell of any one of claims 1-10, wherein the endogenous proliferation gene is selected from the group consisting of AURKB, CDK1, CDC20, RRM2, BIRC5, TOP2A, PTTG1, CCNB1, TPX2, KIF11, SPC25, CENPK, SMC4, TYMS, H2AZ1, TMSB15A, CENPF, and MKI67.

12. The cell of any one of claims 1-11, wherein the nucleic acid encoding the selection agent or the detection agent is integrated in a 5’ UTR or a 3’ UTR of the endogenous proliferation gene.

13. The cell of any one of claims 1-12, wherein the nucleic acid encoding the selection agent or the detection agent is integrated in an exon or an intron of the endogenous proliferation gene.

14. The cell of any one of claims 1-13, wherein following integration, the endogenous proliferation gene locus comprises i) nucleic acid encoding the endogenous proliferation gene, ii) an internal ribosomal entry site or a self-cleaving RNA site, and iii) the nucleic acid encoding the selection agent or a detection agent.

15. The cell of any one of claims 1-14, wherein the selection agent or the detection agent is expressed in proliferating cells.

16. The cell of any one of claims 1-15, wherein the selection agent or the detection agent is not expressed in non-proliferating cells.

17. The cell of any one of claims 1-16, wherein the selection agent or the detection agent is expressed in partially differentiated cells.

18. The cell of any one of claims 1-17, wherein the selection agent or the detection agent is expressed in an intermediate cell type made during differentiation.

19. The cell of any one of claims 1-18, wherein the selection agent or the detection agent is not expressed in differentiated cells.

20. The cell of any one of claims 1-19, wherein the selection agent or the detection agent is not expressed in a therapeutic cell.

21. The cell of any one of claims 1-20, wherein the cell is selected from the group consisting of a pluripotent stem cell, an induced pluripotent stem cell, a primary cell, a progenitor cell, an embryonic stem cell, a hematopoietic stem cell, a mesenchymal stem cell, an epithelial stem cell, a germline stem cell, a mammary stem cell, an olfactory adult stem cell, a hair follicle stem cell, a multipotent stem cell, an amniotic stem cell, a cord blood stem cell, a neural stem cell, a somatic stem cell, a totipotent stem cell, a fibroblast, a monocytic precursor, an exocrine cell, a pancreatic progenitor, an endocrine progenitor, a hepatoblast, amyoblast, a preadipocyte, a chondrocyte, a bone cell, a synovial cell, a tendon cell, a ligament cell, a meniscus cell, an adipose cell, a dendritic cell, a natural killer cell, a muscle cell, an erythroid-megakaryocytic cell, an eosinophil, an islet beta cell, a neuron, a cardiomyocyte, a blood cell, an exocrine progenitor, a ductal cell, an acinar cell, an alpha cell, a beta cell, a delta cell, a cholangiocyte, a brown adipocyte, a cardiac muscle cell, a PP cell, an epidermal keratinocyte, an epithelial cell, a germ cell, a skeletal joint synovium cell, a periosteum cell, a bone cell, a perichondrium cell, a pericardium cell, a meningeal cell, a keratinocyte precursor cell, a keratinocyte stem cell, a pericyte, a glial cell, an ependymal cell, a serosal cell, a heart cell, a brain cell, a spinal cord cell, a lung cell, a pancreas cell, a bladder cell, a bone marrow cell, a spleen cell, an intestine cell, a stomach cell, an islet cell, a pancreatic islet cell, an immune cell, a B cell, a T cell, a natural killer (NK) cell, a natural killer T (NKT) cell, a macrophage, an endothelial cell, a muscle cell, a smooth muscle cell, a skeletal muscle cell, a dopaminergic neuron, a retinal pigmented epithelium cell, an optic cell, a hepatocyte, a thyroid cell, a skin cell, a glial progenitor cell, a neural cell, a stem cell, an endothelial stem cell, an adipose stem cell, an adipose progenitor cell, a lung stem cell, a lung progenitor cell, a neural progenitor cell, a T cell, a CAR-T cell, a CD 14+ cell, a dendritic cell, a PBMC cell, an exocrine secretory epithelial cell, a thyroid epithelial cell, a keratinizing epithelial cell, a gall bladder epithelial cell, a surface epithelial cell, a kidney cell, a primary T cell, a CD3+ T cell, a CD4+ T cell, a CD8+ T cell, a naive T cell, a regulatory T (Treg) cell, a non-regulatory T cell, a Thl cell, a Th2 cell, a Th9 cell, a Thl7 cell, a T-follicular helper (Tfh) cell, a cytotoxic T lymphocyte (CTL), an effector T (Teff) cell, a central memory T (Tcm) cell, an effector memory T (Tern) cell, a TEMRA cells, a tissue-resident memory (Trm) cell, a virtual memory T cell, an innate memory T cell, a memory stem cell (Tsc), a y5 T cell, a helper T cell, a central memory T cell, an effector memory T cell, an effector memory RA T cell, a tissue infiltrating lymphocyte, a capillary endothelial cell, a vascular endothelial cell, an aortic endothelial cell, an arterial endothelial cell, a venous endothelial cell, a renal endothelial cell, a brain endothelial cell, a liver endothelial cell, a gamma cell, an epsilon cell, hematopoietic progenitor cell, a nodal cardiomyocyte, a conducting cardiomyocyte, a working cardiomyocyte, a cardiomyocyte precursor cell, a cardiomyocyte progenitor cell, a cardiac stem cell, an atrial cardiac stem cell, a ventricular cardiac stem cell, an epicardial cell, a vascular endothelial cell, an endocardial endothelial cell, a cardiac valve interstitial cell, a cardiac pacemaker cell, a neuroectodermal cell, a neuronal cell, a neuroendocrine cell, a cholinergic cell, a serotonergic (5-HT) cell, a glutamatergic cell, a GABAergic cell, an adrenergic cell, a noradrenergic cell, a sympathetic neuronal cell, a parasympathetic neuronalcell, a sympathetic peripheral neuronal cell, an astrocyte, an oligodendrocyte, an ependymal cell, a radial glia cell, a Schwann cell, a cerebral endothelial cells (ECs), a neuronal stem cell, a neuronal progenitor cell, a white blood cell, a red blood cell, a platelet cell, a tumor cell, an enterochromaffin cell, a mesenchymal fibroblast, an osteoblast, a stromal cell, and any combination thereof.

22. A cell comprising a nucleic acid encoding a selection agent or a detection agent integrated at an off-target cell type gene locus and operably linked to the promoter of the off- target cell marker gene, wherein expression of the nucleic acid encoding the selection agent or the detection agent is regulated by the promoter of the off-target cell marker gene and / or regulatory elements of the off-target cell marker gene.

23. A cell comprising a nucleic acid encoding a selection agent and a detection agent integrated at an off-target cell type gene locus and operably linked to the promoter of the off- target cell type gene, wherein expression of the nucleic acid encoding the selection agent and the detection agent is regulated by the promoter of the off-target cell type gene and / or regulatory elements of the off-target cell type gene.

24. A cell comprising a first nucleic acid encoding a selection agent and a second nucleic acid encoding a detection agent, wherein the first nucleic acid is integrated at a first off-target cell type gene locus and operably linked to the promoter of the first off-target cell type gene, wherein expression of the first nucleic acid encoding the selection agent is regulated by the promoter of the first off-target cell type gene and / or regulatory elements of the first off-target cell type gene, wherein the second nucleic acid is integrated at a second off-target cell type gene locus and operably linked to the promoter of the second off-target cell type gene, and wherein expression of the second nucleic acid encoding the detection agent is regulated by the promoter of the second off-target cell type gene and / or regulatory elements of the off-target cell type gene.

25. The cell of claim 24, wherein the first off-target cell type gene and / or the second off- target cell type gene are expressed in a cell that is included in a population of therapeutic cells.

26. The cell of claim 24 or 25, wherein the first off-target cell type gene and / or the second off-target cell type gene are expressed in a cell that is included in a population of therapeutic cells but is not a therapeutic cell.

27. The cell of any one of claims 24-26, wherein the cell is an off-target cell.

28. The cell of any one of claims 24-27, wherein the first off-target cell type gene and the second off-target cell type gene are the same, and the first and second nucleic acids are integrated at different alleles.

29. The cell of any one of claims 24-28, wherein the first off-target cell type gene and the second off-target cell type gene are different.

30. The cell of any one of claims 22-29, wherein the off-target cell marker gene is selected from the group consisting of a pluripotent stem cell marker gene, an induced pluripotent stem cell marker gene, a primary cell marker gene, a progenitor cell marker gene, an embryonic stem cell marker gene, a hematopoietic stem cell marker gene, a mesenchymal stem cell marker gene, an epithelial stem cell marker gene, a germline stem cell marker gene, a mammary stem cell marker gene, an olfactory adult stem cell marker gene, a hair follicle stem cell marker gene, a multipotent stem cell marker gene, an amniotic stem cell marker gene, a cord blood stem cell marker gene, a neural stem cell marker gene, a somatic stem cell marker gene, a totipotent stem cell marker gene, a fibroblast marker gene, a monocytic precursor marker gene, an exocrine cell marker gene, a pancreatic progenitor marker gene, an endocrine progenitor marker gene, a hepatoblast marker gene, a myoblast marker gene, a preadipocyte marker gene, a chondrocyte marker gene, a synovial cell marker gene, a tendon cell marker gene, a ligament cell marker gene, a meniscus cell marker gene, an adipose cell marker gene, a dendritic cell marker gene, a natural killer cell marker gene, a muscle cell marker gene, an erythroid-megakaryocytic cell marker gene, an eosinophil marker gene, an islet beta cell marker gene, a neuron marker gene, a cardiomyocyte marker gene, a blood cell marker gene, an exocrine progenitor marker gene, a ductal cell marker gene, an acinar cell marker gene, an alpha cell marker gene, a beta cell marker gene, a delta cell marker gene, a cholangiocyte marker gene, a brown adipocyte marker gene, a cardiac muscle cell marker gene, a PP cell marker gene, an epidermal keratinocyte marker gene, an epithelial cell markergene, a germ cell marker gene, a skeletal joint synovium cell marker gene, a periosteum cell marker gene, a bone cell marker gene, a perichondrium cell marker gene, a pericardium cell marker gene, a meningeal cell marker gene, a keratinocyte precursor cell marker gene, a keratinocyte stem cell marker gene, a pericyte marker gene, a glial cell marker gene, an ependymal cell marker gene, a serosal cell marker gene, a heart cell marker gene, a brain cell marker gene, a spinal cord cell marker gene, a lung cell marker gene, a pancreas cell marker gene, a bladder cell marker gene, a bone marrow cell marker gene, a spleen cell marker gene, an intestine cell marker gene, a stomach cell marker gene, an islet cell marker gene, a pancreatic islet cell marker gene, an immune cell marker gene, a B cell marker gene, a T cell marker gene, a natural killer (NK) cell marker gene, a natural killer T (NKT) cell marker gene, a macrophage marker gene, an endothelial cell marker gene, a muscle cell marker gene, a smooth muscle cell marker gene, a skeletal muscle cell marker gene, a dopaminergic neuron marker gene, a retinal pigmented epithelium cell marker gene, an optic cell marker gene, a hepatocyte marker gene, a thyroid cell marker gene, a skin cell marker gene, a glial progenitor cell marker gene, a neural cell marker gene, a stem cell marker gene, an endothelial stem cell marker gene, an adipose stem cell marker gene, an adipose progenitor cell marker gene, a lung stem cell marker gene, a lung progenitor cell marker gene, a neural progenitor cell marker gene, a T cell marker gene, a CAR-T cell marker gene, a CD 14+ cell marker gene, a dendritic cell marker gene, a PBMC cell marker gene, an exocrine secretory epithelial cell marker gene, a thyroid epithelial cell marker gene, a keratinizing epithelial cell marker gene, a gall bladder epithelial cell marker gene, a surface epithelial cell marker gene, a kidney cell marker gene, a primary T cell marker gene, a CD3+ T cell marker gene, a CD4+ T cell marker gene, a CD8+ T cell marker gene, a naive T cell marker gene, a regulatory T (Treg) cell marker gene, a non-regulatory T cell marker gene, a Thl cell marker gene, a Th2 cell marker gene, a Th9 cell marker gene, a Th 17 cell marker gene, a T-follicular helper (Tfh) cell marker gene, a cytotoxic T lymphocyte (CTL) marker gene, an effector T (Teff) cell marker gene, a central memory T (Tcm) cell marker gene, an effector memory T (Tern) cell marker gene, a TEMRA cell marker gene, a tissue-resident memory (Trm) cell marker gene, a virtual memory T cell marker gene, an innate memory T cell marker gene, a memory stem cell (Tsc) marker gene, a y5 T cell marker gene, a helper T cell marker gene, a central memory T cell marker gene, an effector memory T cell marker gene, an effector memory RA T cell marker gene, a tissue infiltrating lymphocyte marker gene, a capillary endothelial cell marker gene, a vascular endothelial cell marker gene, an aortic endothelial cell marker gene, an arterial endothelial cell marker gene, a venous endothelial cell marker gene, a renalendothelial cell marker gene, a brain endothelial cell marker gene, a liver endothelial cell marker gene, a gamma cell marker gene, an epsilon cell marker gene, hematopoietic progenitor cell marker gene, a nodal cardiomyocyte marker gene, a conducting cardiomyocyte marker gene, a working cardiomyocyte marker gene, a cardiomyocyte precursor cell marker gene, a cardiomyocyte progenitor cell marker gene, a cardiac stem cell marker gene, an atrial cardiac stem cell marker gene, a ventricular cardiac stem cell marker gene, an epicardial cell marker gene, a vascular endothelial cell marker gene, an endocardial endothelial cell marker gene, a cardiac valve interstitial cell marker gene, a cardiac pacemaker cell marker gene, a neuroectodermal cell marker gene, a neuronal cell marker gene, a neuroendocrine cell marker gene, a cholinergic cell marker gene, a serotonergic (5- HT) cell marker gene, a glutamatergic cell marker gene, a GABAergic cell marker gene, an adrenergic cell marker gene, a noradrenergic cell marker gene, a sympathetic neuronal cell marker gene, a parasympathetic neuronal cell marker gene, a sympathetic peripheral neuronal cell marker gene, an astrocyte marker gene, an oligodendrocyte marker gene, an ependymal cell marker gene, a radial glia cell marker gene, a Schwann cell marker gene, a cerebral endothelial cells (ECs) marker gene, a neuronal stem cell marker gene, a neuronal progenitor cell marker gene, a white blood cell marker gene, a red blood cell marker gene, a platelet cell marker gene, a tumor cell marker gene, an enterochromaffin cell marker gene, a mesenchymal fibroblast marker gene, an osteoblast marker gene, a stromal cell marker gene, and any combination thereof.

31. The cell of any one of claims 22-30, wherein the off-target cell marker gene is identified using flow cytometry and / or single-cell RNA-sequencing.

32. The cell of any one of claims 22-31, wherein the off-target cell marker gene is selected from the group consisting of ANXA1, KRT19, CTSC, DSC2, ARHGAP29, KRT18, KRT8, CD9, PLK2, and KRT17.

33. The cell of any one of claims 22-32, wherein the nucleic acid encoding the selection agent or the detection agent is integrated in a 5’ UTR or a 3’ UTR of the off-target cell marker gene.

34. The cell of any one of claims 22-33, wherein the nucleic acid encoding the selection agent or the detection agent is integrated in an exon or an intron of the off-target cell marker gene.

35. The cell of any one of claims 22-34, wherein the selection agent or the detection agent is expressed in a cell with a high level of expression of an off-target cell marker gene.

36. The cell of any one of claims 22-35, wherein the selection agent or the detection agent is expressed in a cell with greater than about 3 transcripts per million (TPM), about 4 TPM, about 5 TPM, about 6 TPM, about 7 TPM, about 8 TPM, about 9 TPM, about 10 TPM, about 15 TPM, about 20 TPM, about 25 TPM, about 30 TPM, about 35 TPM, about 40 TPM, about 45 TPM, or about 50 TPM.

37. The cell of any one of claims 22-36, wherein the selection agent or the detection agent is expressed in an off-target cell with greater than about 3 transcripts per million (TPM), about 4 TPM, about 5 TPM, about 6 TPM, about 7 TPM, about 8 TPM, about 9 TPM, about 10 TPM, about 15 TPM, about 20 TPM, about 25 TPM, about 30 TPM, about 35 TPM, about 40 TPM, about 45 TPM, or about 50 TPM.

38. The cell of any one of claims 22, 23, and 25-37, wherein the selection agent is expressed in a cell such that the selection agent enables selection of the cell.

39. The cell of claim 38, wherein the detection agent is expressed in a cell such that the detection agent enables detection of the cell.

40. The cell of any one of claims 22-38, wherein the selection agent or the detection agent is expressed in a pluripotent cell.

41. The cell of any one of claims 22-40, wherein the selection agent or the detection agent is expressed in a tumorigenic cell.

42. The cell of any one of claims 22-41, wherein the selection agent or the detection agent is expressed in a ductal cell.

43. The cell of any one of claims 22-42, wherein the selection agent or the detection agent is expressed in an enterochromaffin cell.

44. The cell of any one of claims 22-43, wherein the selection agent or the detection agent is expressed in partially differentiated cells.

45. The cell of any one of claims 22-44, wherein the selection agent or the detection agent is not expressed in differentiated cells.

46. The cell of any one of claims 22-45, wherein the selection agent or the detection agent is not expressed in a cell with a low level of expression or no expression of an off-target cell marker gene.

47. The cell of any one of claims 22-46, wherein the selection agent or the detection agent is not expressed in a therapeutic cell.

48. The cell of any one of claims 22-47, wherein the off-target cell marker gene is not a beta cell marker gene, a T cell marker gene, a neuronal cell marker gene, a glial cell marker gene, a cardiac cell marker gene, a retinal pigment epithelium (RPE) cell marker gene, a hematopoietic progenitor cell marker gene, a natural killer cell marker gene, an endothelial cell marker gene, or a lung cell marker gene.

49. The cell of any one of claims 1-48, wherein the cell further comprises a genome editing complex.

50. A composition comprising the cell of any one of the preceding claims.

51. The composition of claim 50, wherein the cell is an allogeneic cell.

52. The composition of claim 50, wherein the cell is an autologous cell.

53. A method of eliminating proliferating cells comprising providing a population of cells with an inducer, wherein the population of cells express a nucleic acid encoding a selection agent integrated at an endogenous proliferation gene locus and operably linked to thepromoter of the endogenous proliferation gene, and wherein providing the inducer causes elimination of proliferating cells.

54. The method of claim 53, wherein the providing is administering the inducer to an individual harboring the population of cells.

55. The method of claim 53 or 54, wherein the method further comprises administering the population of cells to a subject either before or after the inducer is provided to the subject.

56. The method of claim 55, wherein the population of cells is incubated with the inducer prior to administration to the subject.

57. The method of claim 56, wherein the population of cells are administered to a subject before the inducer is provided to the subject.

58. The method of claim 53, wherein the providing comprises incubating the population of cells in vitro.

59. The method of any one of claims 53-58, wherein the endogenous proliferation gene locus is selected from the group consisting of AURKB, CDC20, RRM2, CDK1, BIRC5, T0P2A, PTTG1, CCNB1, TPX2, KIF11, SPC25, CENPK, SMC4, TYMS, H2AZ1, TMSB15A, CENPF, and MKI67.

60. The method of any one of claims 53-59, wherein the method selectively eliminates proliferating cells, cancer cells, pluripotent cells, multipotent stem cells, progenitor cells, dedifferentiated cells, undifferentiated cells, and / or partially differentiated cells.

61. The method of any one of claims 53-60, wherein the population of cells comprises proliferating cells and non-proliferating cells.

62. The method of any one of claims 53-61, wherein the differentiated cells and / or nonproliferating cells are not eliminated.

63. A method of eliminating a specific cell type comprising providing a population of cells with an inducer, wherein the population of cells express a nucleic acid encoding a kill switch from an off-target cell marker gene locus and operably linked to the promoter of the off-target cell marker gene, and wherein providing the inducer causes elimination of an off- target cell.

64. The method of claim 63, wherein the providing is administering the inducer to an individual harboring the population of cells.

65. The method of claim 63 or 64, wherein the method further comprises administering the population of cells to a subject either before or after the inducer is provided to the subject.

66. The method of claim 65, wherein the population of cells is incubated with the inducer prior to administration to the subject.

67. The method of claim 65, wherein the population of cells are administered to a subject before the inducer is provided to the subject.

68. The method of claim 63, wherein the providing comprises incubating the population of cells in vitro.

69. The method of any one of claims 63-68, wherein the off-target cell marker gene is selected from the group consisting of ANXA1, KRT19, CTSC, DSC2, ARHGAP29, KRT18, KRT8, CD9, PLK2, and KRT17.

70. The method of any one of claims 63-69, wherein the method selectively eliminates cancer cells, pluripotent cells, multipotent stem cells, progenitor cells, de-differentiated cells, undifferentiated cells, partially differentiated cells, and / or off-target cells.

71. The method of any one of claims 63-70, wherein the inducer does not cause elimination of a cell type other than the off-target cell.

72. The method of any one of claims 63-71, wherein on-target cells are not eliminated.

73. The method of any one of claims 63-72, wherein the population of cells comprises on- target cells and off-target cells.

74. The method of any one of claims 63-73, wherein the population of cells comprises therapeutic cells and off-target cells.

75. The method of any one of claims 63-74, wherein therapeutic cells are not eliminated.

76. The method of any one of claims 63-75, wherein the population of cells are stem cell derived cells or primary cells.

77. The method of any one of claims 63-76, wherein the stem cells are pluripotent stem cells, embryonic stem cells, induced pluripotent stem cells, multipotent stem cells, or adult stem cells.

78. The method of any one of claims 63-77, wherein the cells are autologous cells.

79. The method of any one of claims 63-78, wherein the cells are allogeneic cells.

80. The method of any one of claims 53-79, wherein the inducer is administered if cell proliferation is detected or if information indicating cell proliferation is obtained.

81. The method of any one of claims 53-80, wherein the inducer is administered if expression of proliferation markers is detected or if information indicating expression of proliferation markers is obtained.

82. The method of any one of claims 53-81, wherein the inducer is administered if an excess number of cells is detected or if information indicating an excess number of cells is obtained.

83. The method of any one of claims 53-82, further comprising detecting proliferation using flow cytometry for a proliferation marker.

84. The method of any one of claims 53-83, further comprising detecting proliferation using single-cell RNA-sequencing for expression of proliferation markers.

85. A method of detecting a proliferating cell expressing a detection agent in a population of cells, wherein the population of cells comprises a nucleic acid encoding the detection agent integrated at an endogenous proliferation gene locus and operably linked to the promoter of the endogenous proliferation gene, and wherein expression of the nucleic acid encoding the detection agent is regulated by the promoter of the endogenous proliferation gene and / or regulatory elements of the endogenous proliferation gene.

86. A method of detecting a proliferating cell expressing a detection agent in a population of cells in an individual, wherein the population of cells comprises a nucleic acid encoding the detection agent integrated at an endogenous proliferation gene locus and operably linked to the promoter of the endogenous proliferation gene, and wherein expression of the nucleic acid encoding the detection agent is regulated by the promoter of the endogenous proliferation gene and / or regulatory elements of the endogenous proliferation gene.

87. A method of detecting an off-target cell expressing a detection agent in a population of cells, wherein the population of cells comprises a nucleic acid encoding the detection agent integrated at an off-target cell marker gene locus and operably linked to the promoter of the off-target cell marker gene, and wherein expression of the nucleic acid encoding the detection agent is regulated by the promoter of the off-target cell marker gene and / or regulatory elements of the off-target cell marker gene.

88. A method of detecting an off-target cell expressing a detection agent in a population of cells in an individual, wherein the population of cells comprises a nucleic acid encoding the detection agent integrated at an off-target cell marker gene locus and operably linked to the promoter of the off-target cell marker gene, and wherein expression of the nucleic acid encoding the detection agent is regulated by the promoter of the off-target cell marker gene and / or regulatory elements of the off-target cell marker gene.

89. A method of selecting a cell population that is suitable for cell therapy, comprising detecting a proliferating cell expressing a detection agent in a population of cells, wherein the population of cells comprises a nucleic acid encoding the detection agent integrated at anendogenous proliferation gene locus and operably linked to the promoter of the endogenous proliferation gene.

90. The method of claim 89, wherein the selected cell population comprises less than about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, or about 1% of cells expressing the detection agent.

91. The method of claim 89 or 90, wherein the selected cell population comprises less than about 30-50%, about 20-40%, about 10-30%, about 5-20%, about 1-20%, about 1-15%, about 1-10%, about 1-9%, about 1-8%, about 1-7%, about 1-6%, about 1-5%, about 1-4%, about 1-3%, or about 1-2% of the total cell population.

92. The method of any one of claims 89-91, wherein the selected cell population comprises less than about 50%, about 49%, about 48%, about 47%, about 46%, about 45%, about 44%, about 43%, about 42%, about 41%, about 40%, about 39%, about 38%, about 37%, about 36%, about 35%, about 34%, about 33%, about 32%, about 31%, about 30%, about 29%, about 28%, about 27%, about 26%, about 25%, about 24%, about 23%, about 22%, about 21%, about 20%, about 19%, about 18%, about 17%, about 16%, about 15%, about 14%, about 13%, about 12%, about 11%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, or about 1% of the total cell population.

93. A method of selecting a cell population that is suitable for cell therapy, comprising detecting an off-target cell expressing a detection agent in a population of cells, wherein the population of cells comprises a nucleic acid encoding the detection agent integrated at an off- target cell marker gene locus and operably linked to the promoter of the off-target cell marker gene.

94. The method of claim 93, wherein the selected cell population comprises less than about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, or about 1% of cells expressing the detection agent.

95. The method of claim 93 or 94, wherein the selected cell population comprises less than about 30-50%, 20-40%, 10-30%, 5-20%, 1-20%, 1-15%, 1-10%, about 1-9%, about 1- 8%, about 1-7%, about 1-6%, about 1-5%, about 1-4%, about 1-3%, or about 1-2% of the total cell population.

96. The method of any one of claims 93-95, wherein the selected cell population comprises less than about 50%, about 49%, about 48%, about 47%, about 46%, about 45%, about 44%, about 43%, about 42%, about 41%, about 40%, about 39%, about 38%, about 37%, about 36%, about 35%, about 34%, about 33%, about 32%, about 31%, about 30%, about 29%, about 28%, about 27%, about 26%, about 25%, about 24%, about 23%, about 22%, about 21%, about 20%, about 19%, about 18%, about 17%, about 16%, about 15%, about 14%, about 13%, about 12%, about 11%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, or about 1% of the total cell population.

97. A method of making a cell comprising a selection agent or a detection agent integrated at an endogenous proliferation gene locus and operably linked to the promoter of the endogenous proliferation gene, the method comprising introducing a nucleic acid encoding a selection agent or a detection agent into the cell, wherein the nucleic acid encoding the selection agent or the detection agent is integrated in the genome of the cell at the endogenous proliferation gene locus.

98. A method of making a cell comprising a selection agent or a detection agent integrated at an off-target cell marker gene locus and operably linked to the promoter of the off-target marker gene, the method comprising introducing a nucleic acid encoding a selection agent or a detection agent into the cell, wherein the nucleic acid encoding the selection agent or the detection agent is integrated in the genome of the cell at the off-target cell marker gene locus.

99. The method of claim 97 or 98, wherein the integration of a nucleic acid comprises a gene knock-in.

100. The method of claim 97 or 98, wherein the integration of a nucleic acid comprises a gene knock-out.

101. The method of any one of claims 97-100, wherein the integration comprises a targeted integration method.

102. The method of any one of claims 97-101, wherein the method further comprises a genome editing complex.

103. The method of claim 101 or 102, wherein the targeted integration method is a Cas- directed homology-directed repair (HDR).

104. A method of treating a patient in need thereof comprising,(c) administering a population of cells to the patient, wherein the population of cells comprise a nucleic acid encoding a selection agent integrated at an endogenous proliferation gene locus and operably linked to the promoter of the endogenous proliferation gene, and(d) activating the selection agent to eliminate one or more cells in the population of cells.

105. A method of treating a patient in need thereof comprising,(a) administering a population of cells to the patient, wherein the population of cells comprise a nucleic acid encoding a selection agent integrated at an off-target cell marker gene locus and operably linked to the promoter of the off-target cell marker gene, and(b) activating the selection agent to eliminate one or more cells in the population of cells.

106. The method of claim 104 or 105, wherein the step of activating the selection agent takes place before the population of cells are administered to the patient and / or after the population of cells are administered to the patient.

107. The method of claim 106, wherein the step of activating the selection agent takes place before the population of cells are administered to the patient.

108. The method of claim 106, wherein the step of activating the selection agent takes place after the population of cells are administered to the patient.

109. The method of claim 106, wherein the step of activating the selection agent takes place before the population of cells are administered to the patient and after the population of cells are administered to the patient.

110. A method of treating a patient in need thereof comprising activating a selection agent to eliminate one or more cells in a population of cells, wherein the patient was previously administered the population of cells, wherein the population of cells comprise a nucleic acid encoding a selection agent integrated at an endogenous proliferation gene locus and operably linked to the promoter of the endogenous proliferation gene.

111. A method of treating a patient in need thereof comprising activating a selection agent to eliminate one or more cells in a population of cells, wherein the patient was previously administered the population of cells, wherein the population of cells comprise a nucleic acid encoding a selection agent integrated at an off-target cell marker gene locus and operably linked to the promoter of the off-target cell marker gene.

112. A method of treating a disease in an individual, comprising administering a cell therapy to treat the disease, wherein the cell therapy comprises a nucleic acid encoding a selection agent or a detection agent integrated at an endogenous proliferation gene locus and operably linked to the promoter of the endogenous proliferation gene.

113. A method of treating a disease in an individual, comprising administering a cell therapy to treat the disease, wherein the cell therapy comprises a nucleic acid encoding a selection agent or a detection agent integrated at an off-target cell marker gene locus and operably linked to the promoter of the off-target cell marker gene.

114. The method of claim 112 or 113, wherein the disease is a cellular deficiency.

115. The method of claim 114, wherein the disease is associated with diabetes or is diabetes.

116. The method of claim 115, wherein the diabetes is Type I diabetes.

117. The method of claim 112 or 113, wherein the disease is a cancer, a neurological disease, or an autoimmune disease.

118. The method of any one of claims 85, 86, 89-92, 97, 99, 104, 106, 110, 112 or 114- 117, wherein the endogenous proliferation gene is selected from the group consisting of AURKB, CDK1, CDC20, RRM2, BIRC5, TOP2A, PTTG1, CCNB1, TPX2, KIF11, SPC25, CENPK, SMC4, TYMS, H2AZ1, TMSB15A, CENPF, and MKI67.

119. The method of any one of claims any one of claims 85, 86, 89-92, 97, 99, 104, 106, 110, 112 or 114-118, wherein the nucleic acid encoding the selection agent or the detection agent is integrated in the 5’ UTR or the 3’ UTR of the endogenous proliferation gene.

120. The method of any one of claims 85, 86, 89-92, 97, 99, 104, 106, 110, 112 or 114-119, wherein the nucleic acid encoding the selection agent or the detection agent is integrated in the exon or intron of the endogenous proliferation gene.

121. The method of any one of claims 85, 86, 89-92, 97, 99, 104, 106, 110, 112 or 114-120, wherein expression of the nucleic acid encoding the selection agent or a detection agent is regulated by the endogenous promoter and / or regulatory elements of the endogenous proliferation gene.

122. The method of any one of claims 85, 86, 89-92, 97, 99, 104, 106, 110, 112 or 114-121, wherein following integration, the endogenous proliferation gene locus comprises i) nucleic acid encoding the proliferation gene, ii) an internal ribosomal entry site or a selfcleaving RNA site, and iii) the nucleic acid encoding the selection agent or a detection agent.

123. The method of any one of claims 85, 86, 89-92, 97, 99, 104, 106, 110, 112 or 114-122, wherein the selection agent or the detection agent is expressed in proliferating cells.

124. The method of any one of claims 85, 86, 89-92, 97, 99, 104, 106, 110, 112 or 114-123, wherein the selection agent or the detection agent is not expressed in non-proliferating cells.

125. The method of any one of claims 85, 86, 89-92, 97, 99, 104, 106, 110, 112 or 114-124, wherein the selection agent or the detection agent is expressed in partially differentiated cells.

126. The method of any one of claims 85, 86, 89-92, 97, 99, 104, 106, 110, 112 or 114-125, wherein the selection agent or the detection agent is expressed in an intermediate cell type made during differentiation.

127. The method of any one of claims 85, 86, 89-92, 97, 99, 104, 106, 110, 112 or 114-126, wherein the selection agent or the detection agent is not expressed in differentiated cells.

128. The method of any one of claims 85, 86, 89-92, 97, 99, 104, 106, 110, 112 or 114-127, wherein the selection agent or the detection agent is not expressed in a therapeutic cell.

129. The method of any one of claims 85, 86, 89-92, 97, 99, 104, 106, 110, 112 or 114-128, wherein the cell is selected from the group consisting of a pluripotent stem cell, an induced pluripotent stem cell, a primary cell, a progenitor cell, an embryonic stem cell, a hematopoietic stem cell, a mesenchymal stem cell, an epithelial stem cell, a germline stem cell, a mammary stem cell, an olfactory adult stem cell, a hair follicle stem cell, a multipotent stem cell, an amniotic stem cell, a cord blood stem cell, a neural stem cell, a somatic stem cell, a totipotent stem cell, a fibroblast, a monocytic precursor, an exocrine cell, a pancreatic progenitor, an endocrine progenitor, a hepatoblast, a myoblast, a preadipocyte, a chondrocyte, a bone cell, a synovial cell, a tendon cell, a ligament cell, a meniscus cell, an adipose cell, a dendritic cell, a natural killer cell, a muscle cell, an erythroid-megakaryocytic cell, an eosinophil, an islet beta cell, a neuron, a cardiomyocyte, a blood cell, an exocrine progenitor, a ductal cell, an acinar cell, an alpha cell, a beta cell, a delta cell, a cholangiocyte, a brown adipocyte, a cardiac muscle cell, a PP cell, an epidermal keratinocyte, an epithelial cell, a germ cell, a skeletal joint synovium cell, a periosteum cell, a bone cell, a perichondrium cell, a pericardium cell, a meningeal cell, a keratinocyte precursor cell, a keratinocyte stem cell, a pericyte, a glial cell, an ependymal cell, a serosal cell, a heart cell, a brain cell, a spinal cord cell, a lung cell, a pancreas cell, a bladder cell, a bone marrow cell, a spleen cell, an intestine cell, a stomach cell, an islet cell, a pancreatic islet cell, an immune cell, a B cell, a T cell, a natural killer (NK) cell, a natural killer T (NKT) cell, a macrophage, an endothelial cell, a muscle cell, a smooth muscle cell, a skeletal muscle cell, a dopaminergic neuron, a retinalpigmented epithelium cell, an optic cell, a hepatocyte, a thyroid cell, a skin cell, a glial progenitor cell, a neural cell, a stem cell, an endothelial stem cell, an adipose stem cell, an adipose progenitor cell, a lung stem cell, a lung progenitor cell, a neural progenitor cell, a T cell, a CAR-T cell, a CD 14+ cell, a dendritic cell, a PBMC cell, an exocrine secretory epithelial cell, a thyroid epithelial cell, a keratinizing epithelial cell, a gall bladder epithelial cell, a surface epithelial cell, a kidney cell, a primary T cell, a CD3+ T cell, a CD4+ T cell, a CD8+ T cell, a naive T cell, a regulatory T (Treg) cell, a non-regulatory T cell, a Thl cell, a Th2 cell, a Th9 cell, a Thl7 cell, a T-follicular helper (Tfh) cell, a cytotoxic T lymphocyte (CTL), an effector T (Teff) cell, a central memory T (Tcm) cell, an effector memory T (Tern) cell, a TEMRA cells, a tissue-resident memory (Trm) cell, a virtual memory T cell, an innate memory T cell, a memory stem cell (Tsc), a y5 T cell, a helper T cell, a central memory T cell, an effector memory T cell, an effector memory RA T cell, a tissue infiltrating lymphocyte, a capillary endothelial cell, a vascular endothelial cell, an aortic endothelial cell, an arterial endothelial cell, a venous endothelial cell, a renal endothelial cell, a brain endothelial cell, a liver endothelial cell, a gamma cell, an epsilon cell, hematopoietic progenitor cell, a nodal cardiomyocyte, a conducting cardiomyocyte, a working cardiomyocyte, a cardiomyocyte precursor cell, a cardiomyocyte progenitor cell, a cardiac stem cell, an atrial cardiac stem cell, a ventricular cardiac stem cell, an epicardial cell, a vascular endothelial cell, an endocardial endothelial cell, a cardiac valve interstitial cell, a cardiac pacemaker cell, a neuroectodermal cell, a neuronal cell, a neuroendocrine cell, a cholinergic cell, a serotonergic (5-HT) cell, a glutamatergic cell, a GABAergic cell, an adrenergic cell, a noradrenergic cell, a sympathetic neuronal cell, a parasympathetic neuronal cell, a sympathetic peripheral neuronal cell, an astrocyte, an oligodendrocyte, an ependymal cell, a radial glia cell, a Schwann cell, a cerebral endothelial cells (ECs), a neuronal stem cell, a neuronal progenitor cell, a white blood cell, a red blood cell, a platelet cell, a tumor cell, an enterochromaffin cell, a mesenchymal fibroblast, an osteoblast, a stromal cell, and any combination thereof.

130. The method of claim 129, wherein the cell is selected from the group consisting of a pancreatic islet cell, an alpha cell, a beta cell, a gamma cell, a delta cell, an epsilon cell, a T cell, a neuron, a glial cell, a cardiomyocyte, a retinal pigmented epithelial cell, a hematopoietic progenitor cell, a natural killer cell, an endothelial cell, and a lung cell.

131. The method of claim 130, wherein the off-target cell marker gene is selected from the group consisting of a pluripotent stem cell marker gene, an induced pluripotent stem cell marker gene, a primary cell marker gene, a progenitor cell marker gene, an embryonic stem cell marker gene, a hematopoietic stem cell marker gene, a mesenchymal stem cell marker gene, an epithelial stem cell marker gene, a germline stem cell marker gene, a mammary stem cell marker gene, an olfactory adult stem cell marker gene, a hair follicle stem cell marker gene, a multipotent stem cell marker gene, an amniotic stem cell marker gene, a cord blood stem cell marker gene, a neural stem cell marker gene, a somatic stem cell marker gene, a totipotent stem cell marker gene, a fibroblast marker gene, a monocytic precursor marker gene, an exocrine cell marker gene, a pancreatic progenitor marker gene, an endocrine progenitor marker gene, a hepatoblast marker gene, a myoblast marker gene, a preadipocyte marker gene, a chondrocyte marker gene, a synovial cell marker gene, a tendon cell marker gene, a ligament cell marker gene, a meniscus cell marker gene, an adipose cell marker gene, a dendritic cell marker gene, a natural killer cell marker gene, a muscle cell marker gene, an erythroid-megakaryocytic cell marker gene, an eosinophil marker gene, an islet beta cell marker gene, a neuron marker gene, a cardiomyocyte marker gene, a blood cell marker gene, an exocrine progenitor marker gene, a ductal cell marker gene, an acinar cell marker gene, an alpha cell marker gene, a beta cell marker gene, a delta cell marker gene, a cholangiocyte marker gene, a brown adipocyte marker gene, a cardiac muscle cell marker gene, a PP cell marker gene, an epidermal keratinocyte marker gene, an epithelial cell marker gene, a germ cell marker gene, a skeletal joint synovium cell marker gene, a periosteum cell marker gene, a bone cell marker gene, a perichondrium cell marker gene, a pericardium cell marker gene, a meningeal cell marker gene, a keratinocyte precursor cell marker gene, a keratinocyte stem cell marker gene, a pericyte marker gene, a glial cell marker gene, an ependymal cell marker gene, a serosal cell marker gene, a heart cell marker gene, a brain cell marker gene, a spinal cord cell marker gene, a lung cell marker gene, a pancreas cell marker gene, a bladder cell marker gene, a bone marrow cell marker gene, a spleen cell marker gene, an intestine cell marker gene, a stomach cell marker gene, an islet cell marker gene, a pancreatic islet cell marker gene, an immune cell marker gene, a B cell marker gene, a T cell marker gene, a natural killer (NK) cell marker gene, a natural killer T (NKT) cell marker gene, a macrophage marker gene, an endothelial cell marker gene, a muscle cell marker gene, a smooth muscle cell marker gene, a skeletal muscle cell marker gene, a dopaminergic neuron marker gene, a retinal pigmented epithelium cell marker gene, an optic cell marker gene, a hepatocyte marker gene, a thyroid cell marker gene, a skin cell marker gene, a glial progenitor cellmarker gene, a neural cell marker gene, a stem cell marker gene, an endothelial stem cell marker gene, an adipose stem cell marker gene, an adipose progenitor cell marker gene, a lung stem cell marker gene, a lung progenitor cell marker gene, a neural progenitor cell marker gene, a T cell marker gene, a CAR-T cell marker gene, a CD 14+ cell marker gene, a dendritic cell marker gene, a PBMC cell marker gene, an exocrine secretory epithelial cell marker gene, a thyroid epithelial cell marker gene, a keratinizing epithelial cell marker gene, a gall bladder epithelial cell marker gene, a surface epithelial cell marker gene, a kidney cell marker gene, a primary T cell marker gene, a CD3+ T cell marker gene, a CD4+ T cell marker gene, a CD8+ T cell marker gene, a naive T cell marker gene, a regulatory T (Treg) cell marker gene, a non-regulatory T cell marker gene, a Thl cell marker gene, a Th2 cell marker gene, a Th9 cell marker gene, a Th 17 cell marker gene, a T-follicular helper (Tfh) cell marker gene, a cytotoxic T lymphocyte (CTL) marker gene, an effector T (Teff) cell marker gene, a central memory T (Tcm) cell marker gene, an effector memory T (Tern) cell marker gene, a TEMRA cell marker gene, a tissue-resident memory (Trm) cell marker gene, a virtual memory T cell marker gene, an innate memory T cell marker gene, a memory stem cell (Tsc) marker gene, a y5 T cell marker gene, a helper T cell marker gene, a central memory T cell marker gene, an effector memory T cell marker gene, an effector memory RA T cell marker gene, a tissue infiltrating lymphocyte marker gene, a capillary endothelial cell marker gene, a vascular endothelial cell marker gene, an aortic endothelial cell marker gene, an arterial endothelial cell marker gene, a venous endothelial cell marker gene, a renal endothelial cell marker gene, a brain endothelial cell marker gene, a liver endothelial cell marker gene, a gamma cell marker gene, an epsilon cell marker gene, hematopoietic progenitor cell marker gene, a nodal cardiomyocyte marker gene, a conducting cardiomyocyte marker gene, a working cardiomyocyte marker gene, a cardiomyocyte precursor cell marker gene, a cardiomyocyte progenitor cell marker gene, a cardiac stem cell marker gene, an atrial cardiac stem cell marker gene, a ventricular cardiac stem cell marker gene, an epicardial cell marker gene, a vascular endothelial cell marker gene, an endocardial endothelial cell marker gene, a cardiac valve interstitial cell marker gene, a cardiac pacemaker cell marker gene, a neuroectodermal cell marker gene, a neuronal cell marker gene, a neuroendocrine cell marker gene, a cholinergic cell marker gene, a serotonergic (5-HT) cell marker gene, a glutamatergic cell marker gene, a GABAergic cell marker gene, an adrenergic cell marker gene, a noradrenergic cell marker gene, a sympathetic neuronal cell marker gene, a parasympathetic neuronal cell marker gene, a sympathetic peripheral neuronal cell marker gene, an astrocyte marker gene, an oligodendrocyte marker gene, an ependymal cell marker gene, a radial gliacell marker gene, a Schwann cell marker gene, a cerebral endothelial cells (ECs) marker gene, a neuronal stem cell marker gene, a neuronal progenitor cell marker gene, a white blood cell marker gene, a red blood cell marker gene, a platelet cell marker gene, a tumor cell marker gene, an enterochromaffin cell marker gene, a mesenchymal fibroblast marker gene, an osteoblast marker gene, a stromal cell marker gene, and any combination thereof.

132. The method of claim 131, wherein the off-target cell marker gene is selected from the group consisting of a pluripotency cell marker gene, a tumorigenic cell marker gene, a ductal marker gene, an enterochromaffin marker gene, a neural marker gene, acinar marker gene, intestinal marker gene, endothelial marker gene, mesenchymal fibroblast marker gene, muscle marker gene, osteoblast marker gene, and stromal marker gene.

133. The method of claim 132, wherein the off-target cell marker gene is selected from the group consisting of ANXA1, KRT19, CTSC, DSC2, ARHGAP29, KRT18, KRT8, CD9, PLK2, and KRT17.

134. The method of claim 132, wherein the nucleic acid encoding the selection agent or the detection agent is integrated in the 5’ UTR or 3’ UTR of the off-target cell marker gene.

135. The method of claim 132, wherein the nucleic acid encoding the selection agent or the detection agent is integrated in the exon or intron of the off-target cell marker gene.

136. The method of claim 135, wherein the off-target cell marker gene is not a beta cell marker gene, a T cell marker gene, a neuronal cell marker gene, a glial cell marker gene, a cardiac cell marker gene, a retinal pigment epithelium (RPE) cell marker gene, a hematopoietic progenitor cell marker gene, a natural killer cell marker gene, an endothelial cell marker gene, or a lung cell marker gene.

137. A kit comprising a cell comprising the cell of any one of claims 1-21 and 49 and instructions for use.

138. A kit comprising a cell comprising the cell of any one of claims 22-48 and 49 and instructions for use.

139. The kit of claim 137 or 138, wherein the kit further comprises an inducer.

140. The cell of any one of claims 1-49, the method of any one of claims 53-136, and the kit of any one of claims 137-139, wherein the inducer is selected from the group consisting of rimiducid (AP1903), AP20187, rapamycin, 5-fluorocytosine, ganciclovir, CB 1954, 6- methylpurine deoxyriboside, fludarabine, indole-3 -acetic acid (IAA), tetracycline, doxycycline, tamoxifen, cumate, FKCsA, abscisic acid (ABA), riboswitch, ecdysone, tryptophan, arabinose, isopropyl P-d-1 -thiogalactopyranoside (IPTG), asunaprevir, grazoprevir, dTAG-13, lenalidomide, anti-MHC-I antibodies, anti-MHC-II antibodies, anti- CCR4 antibodies, anti-CD16 antibodies, anti-CD19 antibodies, anti-CD20 antibodies, anti- CD30 antibodies, anti-EGFR antibodies, anti-GD2 antibodies, anti-HERl antibodies, anti- HER2 antibodies, anti-MUCl antibodies, anti-PSMA antibodies, and anti-RQR8 antibodies.

141. The cell of any one of claims 1-49 and 140, the method of any one of claims 53-136 and 140, and the kit of any one of claims 137-140, wherein the selection agent is a kill switch.

142. The cell of any one of claims 1-49, 140, and 141, the method of any one of claims 53- 136, 140, and 141, and the kit of any one of claims 137-141, wherein the kill switch is selected from the group consisting of an inducible caspase 9 (iCasp9), cytosine deaminase (CDA), herpes simplex virus thymidine kinase (HSV-Tk), rapamycin-activated caspase 9 (rapaCasp9), chemically regulated-SH2-delivered inhibitory tail (CRASH-IT), nitroreductase (NTR), purine nucleoside phosphorylase (PNP), horseradish peroxidase, inducible MHC-I, inducible MHC-II, CCR4, CD16, CD19, CD20, CD30, EGFR, GD2, HER1, HER2, MUC1, PSMA, and RQR8.

143. The cell of any one of claims 1-49, and 140-142, the method of any one of claims 53- 136 and 140-142, and the kit of any one of claims 137-142, wherein the kill switch is iCasp9 or CDA.

144. The cell of any one of claims 1-49 and 140-143, the method of any one of claims 53- 136 and 140, and the kit of any one of claims 137-142, wherein the selection agent is degron.

145. The cell of any one of claims 1-49 and 140-144, the method of any one of claims 53- 136 and 140-144, and the kit of any one of claims 137-142, wherein the inducible MHC-I is selected from the group consisting of HLA-A, HLA-B, and HLA-C.

146. The cell of any one of claims 1-49 and 140-145, the method of any one of claims 53- 136 and 140-145, and the kit of any one of claims 137-145, wherein the inducible MHC-II is selected from the group consisting of HLA-DP, HLA-DQ, and HLA-DR.

147. The cell of any one of claims 1-49 and 140-146, the method of any one of claims 53- 136 and 140, and the kit of any one of claims 137-146, wherein the detection agent is a cell surface protein.

148. The cell of any one of claims 1-49 and 140-147, the method of any one of claims 53- 136, 140, and 147, and the kit of any one of claims 137-140, wherein the cell surface protein is selected from the group consisting of EGFR fused to a His-tag, RQRB fused to a His-tag, and CD47 fused to a His-tag.

149. The cell of any one of claims 1-49 and 140, the method of any one of claims 53-136 and 140, and the kit of any one of claims 137-140, wherein the detection agent is a fluorescent protein.

150. The cell of any one of claims 1-49, 140 and 149, the method of any one of claims 53- 136, 140, and 149, and the kit of any one of claims 137-140 and 149, wherein the fluorescent protein is selected from the group consisting of: green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (EYFP), blue fluorescent protein (BFP), enhanced blue fluorescent protein (EBFP), cyan fluorescent protein (CFP), enhanced cyan fluorescent protein (ECFP), superfolder GFP, superfolder YFP, orange fluorescent protein, red fluorescent protein, small ultrared fluorescent protein, FMN-binding fluorescent protein, dsRed, qFP611, Dronpa, TagRFP, KFP, EosFP, IrisFP, Dendra, Kaede, KikGrl, emerald fluorescent protein, Azami Green, mWasabi, TagGFP, TurboGFP, AcGFP, ZsGreen, and T-Sapphire.

151. The cell of any one of claims 1-49 and 140, the method of any one of claims 53-136 and 140, and the kit of any one of claims 137-140, wherein the detection agent is a blood- detectable biomarker.

152. The cell of any one of claims 1-49, 140, and 147-151, the method of any one of claims 53-136, 140, and 147-151, and the kit of any one of claims 137-140 and 147-151, wherein the detection agent is associated with a barcode.

153. The cell of any one of claims 1-49, 140, and 147-151, the method of any one of claims 1-49, 140, and 147-151, and the kit of any one of claims 1-49, 140, and 147-151, wherein the barcode is integrated in a 5’ UTR or a 3’ UTR of the endogenous proliferation gene.

154. The cell of any one of claims 1-49, 140, and 147-151, the method of any one of claims 1-49, 140, and 147-151, and the kit of any one of claims 1-49, 140, and 147-151, wherein the barcode is integrated in a 5’ UTR or a 3’ UTR of the off-target cell marker gene.

155. The cell of any one of claims 1-49 and 140, the method of any one of claims 53-136 and 140, and the kit of any one of claims 137-140, wherein the detection agent is a secreted protein.

156. The cell of any one of claims 1-49, 140, and 155, the method of any one of claims 1- 49, 140, and 155, and the kit of any one of claims 137-140 and 155, wherein the secreted protein is detectable in vitro.

157. The cell of any one of claims 1-49, 140, 155, and 156, the method of any one of claims 53-136, 140, 155, and 156, and the kit of any one of claims 137-140, 155, and 156, wherein in vitro detection comprises detecting the secreted protein in cell culture medium collected from a cell culture comprising the cell of any one of claims 1-49.

158. The cell of any one of claims 1-49, 140, and 155, the method of any one of claims 1- 49, 140, and 155, and the kit of any one of claims 137-140 and 155, wherein the secreted protein is detectable ex vivo.

159. The cell of any one of claims 1-49, 140, 155, and 158, the method of any one of claims 53-136, 140, 156, and 158, and the kit of any one of claims 137-140, 156, and 158, wherein ex vivo detection comprises detecting the secreted protein in a blood sample taken from an individual administered the cells of any one of claims 1-49.

160. The cell of any one of claims 1-49 and 140-159, the method of any one of claims 53-159, and the kit of any one of claims 137-159, wherein the cell is a mammalian cell.

161. The cell of any one of claims 1-49 and 140-160, the method of any one of claims 53-160, and the kit of any one of claims 137-160, wherein the mammalian cell is a human cell.

162. The cell of any one of claims 1-49 and 140-161, the method of any one of claims 53-161, and the kit of any one of claims 137-161, wherein the cell is a stem cell-derived cell.

163. The cell of any one of claims 1-49 and 140-162, the method of any one of claims 53-162, and the kit of any one of claims 137-162, wherein the stem cell-derived cell is derived from a cell selected from the group consisting of embryonic stem cell, induced pluripotent stem cell, multipotent stem cell, adult stem cell, hematopoietic stem cell, mesenchymal stem cell, endothelial stem cell, epithelial stem cell, adipose stem or progenitor cells, germline stem cells, lung stem or progenitor cells, mammary stem cells, olfactory adult stem cells, hair follicle stem cells, multipotent stem cells, amniotic stem cells, cord blood stem cells, neural stem, and progenitor cells.

164. The cell of any one of claims 1-49 and 140-163, the method of any one of claims 53-163, and the kit of any one of claims 137-163, wherein the stem-cell derived cell is selected from the group consisting of a stem cell-derived beta cell, an alpha cell, and a delta cell.

165. The cell of any one of claims 1-49 and 140-164, the method of any one of claims 53-164, and the kit of any one of claims 137-164, wherein the stem-cell derived cell is a stem cell-derived beta cell (SC-beta cell).

166. The cell of any one of claims 1-49 and 140-165, the method of any one of claims 53-165, and the kit of any one of claims 137-165, wherein the stem-cell derived cell is a stem cell-derived beta islet cell.

167. The cell of any one of claims 1-49 and 140-166, the method of any one of claims 53-166, and the kit of any one of claims 137-166, wherein the cell comprises modifications that (i) increase expression of one or more tolerogenic factors, and (ii) reduce expression of one or more major histocompatibility complex (MHC) class I molecules and / or one or more MHC class II molecules, wherein the increased expression of (i) and the reduced expression of (ii) is relative to a cell of the same cell type that does not comprise the modifications.

168. The cell of any one of claims 1-49 and 140-167, the method of any one of claims 53-167, and the kit of any one of claims 137-167, wherein the one or more of the modifications in (ii) reduce expression of: a. one or more MHC class I molecules; b. one or more MHC class II molecules; or c. one or more MHC class I molecules and one or more MHC class II molecules.

169. The cell of any one of claims 1-49 and 140-168, the method of any one of claims 53-168, and the kit of any one of claims 137-168, wherein the one or more modifications reduce expression of one or more molecules selected from the group consisting of B2M, TAP I, NLRC5, CIITA, HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, HLA-DR, RFX5, RFXANK, RFXAP, NFY-A, NFY-B and / or NFY-C and any combination thereof.

170. The cell of any one of claims 1-49 and 140-169, the method of any one of claims 53-169, and the kit of any one of claims 137-169, wherein the engineered cell does not express one or more molecules selected from the group consisting of B2M, TAP I, NLRC5, CIITA, HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, HLA- DR, and combinations thereof.

171. The cell of any one of claims 1-49 and 140-170, the method of any one of claims 53-170, and the kit of any one of claims 137-170, wherein the one or more tolerogenic factors is selected from the group consisting of CD47, A20 / TNFAIP3, Cl -Inhibitor, CCL21, CCL22, CD16, CD16 Fc receptor, CD24, CD27, CD35, CD39, CD46, CD52, CD55, CD59, CD200, CR1, CTLA4-Ig, DUX4, FasL, H2-M3, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, PD-LI, ID01, IL-10, IL15-RF, IL-35, MANF, Mfge8, PD-1, PD-L1 or Serpinb9 , and any combination thereof.

172. The cell of any one of claims 1-49 and 140-171, the method of any one of claims 53-171, and the kit of any one of claims 137-171, wherein the modification(s) that increase expression comprise increased surface expression, and / or the modifications that reduce expression comprise reduced surface expression, optionally wherein there is no detectable surface expression.

173. The cell of any one of claims 1-49 and 140-172, the method of any one of claims 53-172, and the kit of any one of claims 137-172, wherein the modification that increases expression of the one or more tolerogenic factors comprises an exogenous polynucleotide encoding the one or more tolerogenic factors.

174. The cell of any one of claims 1-49 and 140-173, the method of any one of claims 53-173, and the kit of any one of claims 137-173, wherein the one or more tolerogenic factors comprises CD47.

175. The cell of claim 173 or 174, the method of claim 173 or 174, and the kit of claim 173 or 174, wherein the one or more tolerogenic factors is CD47 and the exogenous polynucleotide encoding CD47 encodes a sequence of amino acids having at least 85% identity to the amino acid sequence of SEQ ID NO: 2, and reduces innate immune killing of the engineered primary cell.

176. The cell of claim 175, the method of claim 175, and the kit of claim 175, wherein the exogenous polynucleotide encoding CD47 encodes a sequence set forth in SEQ ID NO: 2.

177. The cell of any one of claims 1-49 and 140-176, the method of any one of claims 53- 176, and the kit of any one of claims of any of claims 137-176, wherein the exogenous polynucleotide encoding the one or more tolerogenic factors is operably linked to a promoter.

178. The cell of claim 177, the method of claim 177, and the kit of claim 177, wherein the promoter is a constitutive promoter.

179. The cell of claim 177 or 178, the method of claim 177 or 178, and the kit of claim 177 or 178, wherein the promoter is selected from the group consisting of the CAG promoter, the cytomegalovirus (CMV) promoter, the EFla promoter, the 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, promoter of moloney virus, Epstein barr virus (EBV) promoter, the Rous sarcoma virus (RSV) promoter and the UBC promoter.

180. The cell of any one of claims 1-49 and 140-179, the method of any one of claims 53- 179, and the kit of any one of claims 137-179, wherein the modification that reduces expression of one or more MHC class I molecules is a modification that reduces expression of B-2 microglobulin (B2M).

181. The cell of claim 180, the method of any one of claims 180, and the kit of any one of claims 180, wherein the modification that reduces expression of one or more MHC class I molecules is a modification that reduces expression of an HLA-A protein, an HLA-B protein, or HLA-C protein, optionally wherein a gene encoding said HLA-A protein, an HLA-B protein, or HLA-C protein is knocked out.

182. The cell of any one of claims 1-49 and 140-181, the method of any one of claims 53- 181, and the kit of any one of claims 137-181, wherein the modification that reduces expression of one or more MHC class II molecules is a modification that reduces expression of CIITA.

183. The cell of any claim 182, the method of claim 182, and the kit of claim 182, wherein the donor subject is healthy or is not suspected of having a disease or condition at the time the donor sample is obtained from the donor subject.

184. The cell of any one of claims 1-49 and 140-183, the method of any one of claims 53- 183, and the kit of any one of claims 137-183, wherein the engineered primary cell is an islet cell, optionally a beta islet cell.

185. The cell of any one of claims 1-49 and 140-184, the method of any one of claims 53-184, and the kit of any one of claims 137-184, wherein the engineered primary cell is ABO blood group type O.

186. The cell of any one of claims 1-49 and 140-185, the method of any one of claims 53-185, and the kit of any one of claims 137-185, wherein the engineered primary cell is Rhesus factor negative (Rh-).

187. The cell of any one of claims 1-49 and 140-186, the method of any one of claims 53-186, and the kit of any one of claims 137-186, wherein the engineered cell is capable of controlled killing of the engineered cell.

188. The cell of any one of claims 1-49 and 140-187, the method of any of claims 53-187, or the kit of any one of claims 137-187, wherein the engineered cell comprises a suicide gene or a suicide switch.

189. The cell of claim 188, the method of claim 188, or the kit of claim 188, wherein the suicide gene or the suicide switch induces controlled cell death in the presence of a drug or prodrug, or upon activation by a selective exogenous compound.

190. The cell of any one of claims 1-49 and 140-189, the method of any of claims 53-189 or the kit of any one of claims 137-189, comprising administering an agent that allows for depletion of an engineered cell of the population of engineered cells.

191. The cell of any one of claims 1-49 and 140-190, the method of any of claims 53-190, or the kit of any one of claims 137-190, comprising administering an agent that recognizes the one or more tolerogenic factors on the surface of the engineered cell.

192. The cell of claim 191, the method of claim 191, or the kit of claim 191, wherein the engineered cell is engineered to express the one or more tolerogenic factors.

193. The cell of claim 191 or 192, the method of claim 191 or 192, or the kit of claim 191 or 192, wherein the one or more tolerogenic factors is CD47.

194. The cell of any one of claims 1-49 and 140-193, the method of any one of claim 53- 193, or the kit of any one of claims 163-193, wherein expression of a detection agent acts as a signal for administration of an exogenous kill switch directed against or specific to a tolerogenic agent.

195. The cell of claim 194, the method of claim 194, or the kit of claim 194, wherein the exogenous kill switch is an anti-CD47 antibody.

196. The cell of any one of claims 1-49 and 140-195, the method of any one of claims 53- 195, and the kit of any one of claims 137-195, wherein the cells are assayed for hypoimmunogenic phenotypes.

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