WO2025165779A1

WO2025165779A1 - Simultaneous gene knockin and knockout in engineered cell therapies with mrna expressed guide rnas

Info

Publication number: WO2025165779A1
Application number: PCT/US2025/013441
Authority: WO
Inventors: Ansuman Satpathy; Theo ROTH
Original assignee: Leland Stanford Junior University
Current assignee: Leland Stanford Junior University
Priority date: 2024-02-01
Filing date: 2025-01-28
Publication date: 2025-08-07
Anticipated expiration: 2026-08-01

Abstract

Compositions and methods are provided for genetically modifying a cell to introduce both a gene knockin and a gene knockout. The subject methods use a donor polynucleotide comprising a gene knockin sequence and a knockout guide RNA sequence. Transcription of the donor polynucleotide, after integration of the donor polynucleotide into the genome, produces a mature mRNA sequence comprising the knockout guide RNA. An RNA-guided nuclease is used to excise the knockout guide RNA from the mRNA transcript, which then guides the RNA-guided nuclease to a second target locus where the RNA-guided nuclease creates a double stranded DNA break, resulting in gene knockout at the second target locus. Only cells with the gene knockin are able to express the mRNA containing the knockout guide RNA so that the gene knockin at the first genomic target locus and the subsequent gene knockout at the second genomic target locus are linked.

Description

SIMULTANEOUS GENE KNOCKIN AND KNOCKOUT IN ENGINEERED CELL

THERAPIES WITH MRNA EXPRESSED GUIDE RNAS

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of U.S. Provisional Patent Application No. 63/548,730, filed February 1 , 2024, which application is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

[0002] Genetically modified T cell therapies, especially CAR-T cells, are growing in clinical use and have demonstrated remarkable cures in certain hematological indications. To engineer these therapies, a new synthetic gene, usually a Chimeric Antigen Receptor (CAR) must be knocked into the cells genome. However, recent work has demonstrated numerous gene targets that when knocked out in T cells drastically improve their functionality. Technologies that simultaneously engineer gene knockins along with gene knockouts can take advantage of these functional improvements in T cell behavior to build more effective CAR-T cell therapies.

SUMMARY OF THE INVENTION

[0003] Compositions and methods are provided for genetically modifying a cell to introduce both a gene knockin and a gene knockout. The subject methods use a donor polynucleotide comprising a gene knockin sequence and a knockout guide RNA sequence. Transcription of the donor polynucleotide, after integration of the donor polynucleotide into the genome, produces a mature mRNA sequence comprising the knockout guide RNA. By incorporating the knockout guide RNA into an mRNA transcript, the knockout guide RNA is only expressed in cells that have had the gene knockin construct successfully integrated into the genome. An RNA-guided nuclease is used to excise the knockout guide RNA from the mRNA transcript, which then guides the RNA- guided nuclease to a second target locus where the RNA-guided nuclease creates a double stranded DNA break, resulting in gene knockout at the second target locus. Only cells with the gene knockin are able to express the mRNA containing the knockout guide RNA so that the gene knockin at the first genomic target locus and the subsequent gene knockout at the second genomic target locus are linked. [0004] In one aspect, a method of genetically modifying a cell to introduce a gene knockin and a gene knockout is provided, the method comprising: introducing a donor polynucleotide into the cell, wherein the donor polynucleotide comprises a 5' homology arm that hybridizes to a 5' genomic target sequence and a 3' homology arm that hybridizes to a 3' genomic target sequence flanking a nucleotide sequence encoding I) an exogenous polypeptide and ii) a knockout guide RNA; introducing an RNA-guided nuclease into the cell; introducing a knockin guide RNA into the cell, wherein the knockin guide RNA forms a complex with the RNA-guided nuclease such that the knockin guide RNA directs the RNA-guided nuclease to a first genomic target locus, wherein the RNA- guided nuclease creates a double-stranded break in the genomic DNA at the first genomic target locus, wherein the donor polynucleotide is integrated at the first genomic target locus recognized by its 5' homology arm and 3' homology arm by homology directed repair (HDR); and culturing the cell under conditions suitable for transcription of the integrated donor polynucleotide, wherein a first mRNA transcript encoding the exogenous polypeptide and the knockout guide RNA is produced, wherein the RNA- guided nuclease excises the knockout guide RNA from the first mRNA transcript to produce a second mRNA transcript encoding the exogenous polypeptide without the knockout guide RNA; wherein translation of the second mRNA transcript results in production of the exogenous polypeptide in the cell; wherein the excised knockout guide RNA forms a complex with the RNA-guided nuclease such that the knockout guide RNA directs the RNA-guided nuclease to a second genomic target locus, wherein the RNA- guided nuclease creates a double-stranded break in the genomic DNA at the second genomic target locus, wherein DNA repair of the double-stranded break by non- homologous end joining creates an insertion or deletion (indel) resulting in gene knockout at the second genomic target locus.

[0005] In certain embodiments, the exogenous polypeptide is an enzyme, an extracellular matrix protein, a receptor, a transporter, an ion channel, or other membrane protein, a hormone, a neuropeptide, a growth factor, a cytokine, an antibody, a cytoskeletal protein, or a therapeutic protein; or a fragment thereof, or a biologically active domain of interest.

[0006] In certain embodiments, the cell is a mammalian cell.

[0007] In certain embodiments, the mammalian cell is an immune cell such as, but not limited to, a T cell, a B cell, a natural killer cell, a neutrophil, an eosinophil, a mast cell, a basophil, a monocyte, a macrophage, or a dendritic cell. [0008] In certain embodiments, the mammalian cell is a T cell such as, but not limited to, a helper CD4⁺ T cell, a cytotoxic CD8⁺ T cell, a natural killer T cell, or a gamma delta T cell.

[0009] In certain embodiments, the exogenous protein is a chimeric antigen receptor (CAR) that specifically binds to a target antigen. In some embodiments, the chimeric antigen receptor comprises a transmembrane domain linked to an extracellular antigen binding domain and an intracellular signaling domain, wherein the extracellular antigenbinding domain specifically binds to an antigen on the target cell. In some embodiments, the extracellular antigen binding domain comprises a single chain variable fragment (scFv), an antigen-binding fragment (Fab), a nanobody, a heavy chain variable (VH) domain, a light chain variable (VL) domain, a single domain antibody (sdAb), a shark variable domain of a new antigen receptor (VNAR), a single variable domain on a heavy chain (VHH), a bispecific antibody, a diabody, or a functional fragment thereof that binds specifically to the antigen. In some embodiments, the intracellular signaling domain is a CD3-zeta intracellular signaling domain or a ZAP-70 intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises an immunoreceptor tyrosine-based activation motif (ITAM). In some embodiments, the CAR further comprises a costimulatory domain such as, but not limited to a 4-1 BB, CD28, ICOS, OX- 40, BTLA, CD27, CD30, GITR, or HVEM costimulatory domain. In some embodiments, the transmembrane domain is a CD8, Megfl O, FcRy, Bail , MerTK, TIM4, Stabilin-1 , Stabilin-2, RAGE, CD300f, integrin subunit av, integrin subunit |35, CD36, LRP1 , SCARF1 , C1 Qa, Axl, CD45, or CD86 transmembrane domain.

[0010] In certain embodiments, the exogenous protein is a chimeric antigen receptor (CAR) that specifically binds to a target antigen on a target cell. In some embodiments, the target cell is a cancer cell, a tumor cell, an activated fibroblast, an autoreactive immune cell (e.g., autoreactive T cell or B cell), a pathogen (e.g., virus, a bacterium, a fungus, or a parasite), or a diseased cell. In some embodiments, the antigen on the target cell is a tumor antigen or a tumor-associated antigen. In some embodiments, the antigen on the target cell is a viral antigen, a bacterial antigen, a fungal antigen or a parasite antigen. In some embodiments, the antigen on the target cell is an antigen on the autoreactive T cell or B cell.

[0011] In certain embodiments, the second genomic target locus is a T cell receptor alpha chain locus, T cell receptor beta chain locus, T cell receptor delta chain locus, or a T cell receptor gamma chain locus. [0012] In certain embodiments, the second genomic target locus encodes a cytokine. In some embodiments, the cytokine is GM-CSF or IL-6.

[0013] In certain embodiments, the second genomic target locus encodes an alloantigen. In some embodiments, the alloantigen is a major histocompatibility complex (MHO) class I alloantigen or an MNS blood group alloantigen. In some embodiments, the alloantigen is CD1 , CD2, CD3, CD4, CD7, CD8, Ly-6, Qa-2, RT6, CD19, CD22, CD56, CD58 (LFA- 3), CD59, or CDw90 (Thy 1 ).

[0014] In certain embodiments, the second genomic target locus comprises a CD5, CD52, CD70, BATF, LCK, PD-1 , LAG-3, CTLA-4, 2-B2M, PD-1 , HLA-I, Fas, TGFBR2, PDCD-1 , DGK, EZH2, PAX5, or LDLR gene.

[0015] In certain embodiments, the RNA-guided nuclease is provided by a vector (e.g., plasmid or a viral vector). In some embodiments, the vector is introduced into the cell by transient transfection or stable transfection. In some embodiments, expression of the RNA-guided nuclease is inducible.

[0016] In certain embodiments, the RNA-guided nuclease is provided by a mRNA encoding the RNA-guided nuclease, wherein translation of the mRNA results in production of the RNA-guided nuclease in the cell.

[0017] In certain embodiments, the RNA-guided nuclease is a clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Gas) nuclease. In some embodiments, the Cas nuclease is Cas9 or Cas12a.

[0018] In certain embodiments, the donor polynucleotide further comprises a barcode.

[0019] In certain embodiments, the donor polynucleotide further comprises a gene knockin module comprising a plurality of coding sequences encoding a plurality of polypeptides.

[0020] In certain embodiments, the donor polynucleotide further comprises a sequence encoding a knockout module comprising a plurality of knockout guide RNAs. In some embodiments, at least 2 knockout guide RNAs of the plurality bind to different genomic target sequences in different target genes to generate gene knockouts of more than one target gene. In some embodiments, at least 2 knockout guide RNAs of the plurality bind to different genomic target sequences in the same target gene to increase a rate of gene knockout of the target gene compared to the rate of gene knockout using only one knockout guide RNA.

[0021] In certain embodiments, the first mRNA transcript comprises: a protein coding reading frame encoding the exogenous polypeptide followed by a stop codon; and a 3’- untranslated region comprising: a knockout module flanked by a first spacer sequence and a second spacer sequence, wherein the knockout module comprises a plurality of knockout guide RNAs, wherein each guide RNA is preceded by a synthetic separator sequence followed by a direct repeat sequence; a mRNA stabilizing element, wherein the mRNA stabilizing element is positioned between the stop codon and the first spacer sequence; and a polyadenylation sequence. In some embodiments, the mRNA stabilizing element is a triplex stabilizer.

[0022] In certain embodiments, the transcription of the integrated donor polynucleotide is performed by an RNA polymerase II (Pol II).

[0023] In certain embodiments, the knockout guide RNAs are Cas12a guide RNAs.

[0024] In another aspect, a composition is provided, the composition comprising: a donor polynucleotide comprising a 5' homology arm that hybridizes to a 5' genomic target sequence and a 3' homology arm that hybridizes to a 3' genomic target sequence flanking a nucleotide sequence encoding i) an exogenous polypeptide and ii) a knockout guide RNA; an RNA-guided nuclease; and a knockin guide RNA, wherein the knockin guide RNA can form a complex with the RNA-guided nuclease such that the knockin guide RNA directs the RNA-guided nuclease to a first genomic target locus in a cell, wherein the RNA-guided nuclease creates a double-stranded break in the genomic DNA at the first genomic target locus, wherein the donor polynucleotide is integrated at the first genomic target locus recognized by its 5' homology arm and 3' homology arm by homology directed repair (HDR), wherein a first mRNA transcript encoding the exogenous polypeptide and the knockout guide RNA is produced by transcription of the integrated donor polynucleotide, wherein the RNA-guided nuclease excises the knockout guide RNA from the first mRNA transcript to produce a second mRNA transcript encoding the exogenous polypeptide without the knockout guide RNA, wherein translation of the second mRNA transcript results in production of the exogenous polypeptide in the cell, and wherein the excised knockout guide RNA forms a complex with the RNA-guided nuclease such that the knockout guide RNA directs the RNA-guided nuclease to a second genomic target locus in the cell, wherein the RNA-guided nuclease creates a double-stranded break in the genomic DNA at the second genomic target locus, wherein DNA repair of the double-stranded break by non-homologous end joining creates an insertion or deletion (indel) resulting in gene knockout at the second genomic target locus. [0025] In certain embodiments, the RNA-guided nuclease in the composition is a clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Gas) nuclease. In some embodiments, the Cas nuclease is Cas9 or Cas12a.

[0026] In certain embodiments, the donor polynucleotide further comprises a gene knockin module comprising a plurality of coding sequences encoding a plurality of polypeptides.

[0027] In certain embodiments, the donor polynucleotide in the composition further comprises a sequence encoding a knockout module comprising a plurality of knockout guide RNAs. In some embodiments, at least 2 knockout guide RNAs of the plurality bind to different genomic target sequences in different target genes to generate gene knockouts of more than one target gene. In some embodiments, at least 2 knockout guide RNAs of the plurality bind to different genomic target sequences in the same target gene to increase a rate of gene knockout of the target gene compared to the rate of gene knockout using only one knockout guide RNA.

[0028] In certain embodiments, the donor polynucleotide in the composition can produce a first mRNA transcript comprising: a protein coding reading frame encoding the exogenous polypeptide followed by a stop codon; and a 3’-untranslated region comprising: a knockout module flanked by a first spacer sequence and a second spacer sequence, wherein the knockout module comprises a plurality of knockout guide RNAs, wherein each guide RNA is preceded by a synthetic separator sequence followed by a direct repeat sequence; a mRNA stabilizing element, wherein the mRNA stabilizing element is positioned between the stop codon and the first spacer sequence; and a polyadenylation sequence. In some embodiments, the mRNA stabilizing element is a triplex stabilizer. In some embodiments, the knockout guide RNAs are Cas12a guide RNAs.

[0029] In another aspect, a kit comprising a composition, described herein, and instructions for producing a genetically modified cell is provided.

[0030] In certain embodiments, the kit further comprises a transfection agent.

[0031] In another aspect, a genetically modified cell produced according to a method, described herein, is provided.

[0032] In another aspect, a composition comprising a genetically modified cell produced according to a method, described herein, and a pharmaceutically acceptable excipient is provided. [0033] In another aspect, a method of performing cellular therapy is provided, the method comprising administering a therapeutically effective amount of a composition comprising a genetically modified cell to a subject, wherein the genetically modified cell is produced according to a method described herein.

[0034] In certain embodiments, the genetically modified cell is autologous or allogeneic.

BRIEF DESCRIPTION OF THE DRAWINGS

[0035] FIG. 1. Knockout module with Cas9 + U6-sgRNA architecture. The gene editing method involves introducing a DNA cassette to express the gene knockout gRNA on the same DNA template as the gene knockin. Since gRNAs are small RNA sequences, they traditionally must be expressed by an RNA pol III promoter (the human polymerase that makes small RNAs), normally a U6 promoter. However, because the U6 promoter is active, whether its DNA sequence has been incorporated into the genome (successful knockin) or not (episomal plasmid), this method results in loss of linkage between gene knockin and gene knockout, with cells just as likely to have a gene knockout whether they had a knockin or not.

[0036] FIG. 2. Comparison of knockout modules with Cas9 + U6-sgRNA architecture and Cas12a + mRNA architecture. Our new gene editing system overcomes this challenge by having the knockout gRNA incorporated into a mRNA strand, which can only be expressed after the gene knockin occurs. This crucially results in two improvements- first, only cells with the gene knockin are able to express the knockout gRNA, solving the safety issue of having gene knockouts in the cells that do not have a gene knockin. Second, the gene knockout is temporally separated from the gene knockin, meaning that only one double stranded DNA break is present in the cell at a time, preventing the chromosomal translocations and large-scale genetic damage seen when multiple double stranded breaks are simultaneously induced.

[0037] FIG. 3. Genetic architecture for mRNA encoded gene knockin + knockout in primary human cells. Optimized architecture for simultaneous gene knockins + gene knockouts in primary human cells. After the protein coding frame containing any protein coding sequences knocked in, the genetic material to induce a gene knockout are included in the 3’ untranslated region. After the stop codon, an mRNA stabilizing element (e.g. triplex Stabilizer) is included, followed by a spacer element to separate the stabilizer from the gRNA/s. Each gRNA element includes a Synthetic Separator sequence which enhances gRNA extraction, a direct repeat element to enable recognition and excision of the gRNA sequence by the site-specific RNA guided nuclease used (e.g. Cas12a), and then the actual gRNA target sequence. Multiple gRNA elements can be included immediately following each other as part of a gRNA array encoding either multiple gRNAs targeting the same gene to improve knockout rates, or encoding multiple gRNAs targeting different genes to allow for knockout of more than one target gene using a single Knockin + Knockout construct. After the final gRNA element, another spacer element is included before transcription is concluded by a polyA terminator sequence.

[0038] FIG. 4. Simultaneous gene knockin + gene knockout at additional locus. Additional example targeting the surface receptor CD47 for knockout showing improved specificity of Gene Knockin + Gene Knockouts when using an mRNA encoded architecture (expressed by RNA Pol II) compared to traditional U6 (RNA Pol III) based gRNA expression. When using a U6 driven gRNA expression cassette, significant amounts of gene knockout are observed (CD47 negative cells) even in cells that do not have the target gene knockin (bottom left quadrant). In contrast, using the mRNA encoded architecture yields highly efficient gene knockouts but only in cells that have successfully received the gene knockin (Top left quadrant).

[0039] FIG. 5. Simultaneous gene knockin + gene knockout at additional loci. Gene Knockin + Gene Knockout using mRNA encoded architecture in primary human cells is efficient and specific across gene knockout targets. Knockout of four different target surface receptor proteins is demonstrated using gRNAs against CD47, CD2, CD226, and CD45 respectively.

[0040] FIG. 6. Gene knockin + gene knockout of two target genes simultaneously. Inclusion of multiple gRNAs in the mRNA architecture enables knockout of multiple target proteins to be encoded in a single Knockin + Knockout construct. gRNAs targeting the surface receptors CD226 and B2M were both included in the same Knockin + Knockout construct, and efficient and specific knockout of both target genes was observed in the same cells.

DETAILED DESCRIPTION OF THE INVENTION

[0041 ] Compositions and methods are provided for genetically modifying a cell to introduce both a gene knockin and a gene knockout. The subject methods use a donor polynucleotide comprising a gene knockin sequence and a knockout guide RNA sequence. Transcription of the donor polynucleotide, after integration of the donor polynucleotide into the genome, produces a mature mRNA sequence comprising the knockout guide RNA. An RNA-guided nuclease is used to excise the knockout guide RNA from the mRNA transcript, which then guides the RNA-guided nuclease to a second target locus where the RNA-guided nuclease creates a double stranded DNA break, resulting in gene knockout at the second target locus. Only cells with the gene knockin are able to express the mRNA containing the knockout guide RNA so that the gene knockin at the first genomic target locus and the subsequent gene knockout at the second genomic target locus are linked.

[0042] Before the present devices, systems, software, and methods are described, it is to be understood that this invention is not limited to the particular devices, systems, software, and methods 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 invention will be limited only by the appended claims.

[0043] 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 dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, 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 invention.

[0044] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, some potential and preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. It is understood that the present disclosure supersedes any disclosure of an incorporated publication to the extent there is a contradiction. [0045] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

[0046] It must be noted that as used herein and in the appended claims, the singular forms "a", "an”, and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells and reference to "the nucleic acid" includes reference to one or more nucleic acids and equivalents thereof, such as polynucleotides, known to those skilled in the art, and so forth.

[0047] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

Definitions

[0048] The term "about," particularly in reference to a given quantity, is meant to encompass deviations of plus or minus five percent. As used herein, the term "immune cells" generally includes white blood cells (leukocytes) which are derived from hematopoietic stem cells (HSC) produced in the bone marrow.

[0049] “Biocompatible” or “cytocompatible” as used herein, refers to a property of a material that allows for prolonged contact with a cell or tissue without causing toxicity or significant damage.

[0050] The terms “engineered” or “recombinant” in reference to a T cell, gene, nucleic acid and/or protein as used herein, refer to a T cell, gene, nucleic acid and/or protein that has been altered through human intervention. Accordingly, the term “naturally occurring” as used herein in reference to a T cell, gene, nucleic acid and/or protein as used herein, refer to a T cell, gene, nucleic acid and/or protein existing in nature and without any human intervention. Exemplary human interventions comprise transfection with a heterologous polynucleotide, molecular cloning resulting in a deletion, insertion, modification and/or rearrangement with respect to a naturally occurring sequence such as a naturally occurring sequence in a T cell, gene, nucleic acid and/or protein herein described.

[0051] The term “unit dosage form,” as used herein, refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of the agents calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle. The specifications for the unit dosage forms for use in the present invention depend on the particular compound employed and the effect to be achieved, the pharmacodynamics associated with each compound in the host, and the like.

[0052] The term “biological sample” encompasses a clinical sample, including, but not limited to, a bodily fluid, tissue obtained by surgical resection, tissue obtained by biopsy, cells in culture, cell supernatants, cell lysates, tissue samples, organs, bone marrow, blood, plasma, serum, fine needle aspirate, lymph node aspirate, cystic aspirate, a paracentesis sample, a thoracentesis sample, and the like.

[0053] The terms “obtained” or “obtaining” as used herein can also include the physical extraction or isolation of a biological sample (e.g., comprising immune cells) from a subject. Accordingly, a biological sample comprising immune cells can be isolated from a subject (and thus “obtained”) by the same person or same entity that subsequently isolates immune cells from the sample. When a biological sample is “extracted” or “isolated” from a first party or entity and then transferred (e.g., delivered, mailed, etc.) to a second party, the sample was “obtained” by the first party (and also “isolated” by the first party), and then subsequently “obtained” (but not “isolated”) by the second party. Accordingly, in some embodiments, the step of obtaining does not comprise the step of isolating a biological sample.

[0054] In some embodiments, the step of obtaining comprises the step of isolating a biological sample. Methods and protocols for isolating various biological samples (e.g., a blood sample, a biopsy sample, an aspirate, etc.) will be known to one of ordinary skill in the art and any convenient method may be used to isolate a biological sample.

[0055] “Isolated” refers to an entity of interest that is in an environment different from that in which it may naturally occur. “Isolated” is meant to include entities that are within samples that are substantially enriched for the entity of interest and/or in which the entity of interest is partially or substantially purified.

[0056] "Substantially" or "essentially" means nearly totally or completely, for instance,

95% or greater of some given quantity. [0057] "Substantially purified" generally refers to isolation of a component of a sample (e.g., cell or substance), such that the component comprises the majority percent of the sample in which it resides. Typically in a sample, a substantially purified component comprises at least 70%, preferably at least 80%-85%, more preferably at least 90-99% of the sample.

[0058] The terms "individual," "subject," and "patient" are used interchangeably herein to refer to an individual to be treated by (e.g., administered) the compositions and methods of the present invention. Subjects include, but are not limited to, mammals, including human and non-human mammals such as non-human primates, including chimpanzees and other apes and monkey species; laboratory animals such as mice, rats, rabbits, hamsters, guinea pigs, and chinchillas; domestic animals such as dogs and cats; farm animals such as sheep, goats, pigs, horses and cows. In some cases, the methods of the invention find use in experimental animals, in veterinary application, and in the development of animal models for disease, including, but not limited to, rodents including mice, rats, and hamsters; primates, and transgenic animals. In the context of the disclosure, the term "subject" generally refers to an individual who will be administered or who has been administered one or more compositions described herein (e.g., cellular therapy with cells screened according to the methods described herein).

[0059] The terms "treatment", "treating", "treat" and the like are used herein to generally refer to obtaining a desired pharmacologic and/or physiologic effect. The effect can be prophylactic in terms of completely or partially preventing a disease or symptom(s) thereof and/or may be therapeutic in terms of a partial or complete stabilization or cure for a disease and/or adverse effect attributable to the disease. The term “treatment" encompasses any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the disease and/or symptom(s) from occurring in a subject who may be predisposed to the disease or symptom but has not yet been diagnosed as having it; (b) inhibiting the disease and/or symptom(s), i.e., arresting their development; or (c) relieving the disease symptom(s), i.e., causing regression of the disease and/or symptom(s). Those in need of treatment include those already inflicted as well as those in which prevention is desired, including those with a genetic predisposition or increased susceptibility to developing a disease.

[0060] A therapeutic treatment is one in which the subject is inflicted prior to administration and a prophylactic treatment is one in which the subject is not inflicted prior to administration. In some embodiments, the subject has an increased likelihood of becoming inflicted or is suspected of being inflicted prior to treatment. In some embodiments, the subject is suspected of having an increased likelihood of becoming inflicted.

[0061] A "therapeutically effective amount" or ‘‘therapeutic dose” is an amount sufficient to effect desired clinical results (i.e., achieve therapeutic efficacy). A therapeutically effective dose or amount can be administered in one or more administrations.

[0062] "Pharmaceutically acceptable excipient or carrier" refers to an excipient that may optionally be included in the compositions of the invention and that causes no significant adverse toxicological effects to the patient.

[0063] "Pharmaceutically acceptable salt" includes, but is not limited to, amino acid salts, salts prepared with inorganic acids, such as chloride, sulfate, phosphate, diphosphate, bromide, and nitrate salts, or salts prepared from the corresponding inorganic acid form of any of the preceding, e.g., hydrochloride, etc., or salts prepared with an organic acid, such as malate, maleate, fumarate, tartrate, succinate, ethylsuccinate, citrate, acetate, lactate, methanesulfonate, benzoate, ascorbate, para-toluenesulfonate, palmoate, salicylate and stearate, as well as estolate, gluceptate and lactobionate salts. Similarly salts containing pharmaceutically acceptable cations include, but are not limited to, sodium, potassium, calcium, aluminum, lithium, and ammonium (including substituted ammonium).

[0064] The terms "polynucleotide" and "nucleic acid," used interchangeably herein, refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, this term includes, but is not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.

[0065] "Homology" refers to the percent identity between two polynucleotide or two polypeptide molecules. Two nucleic acid, or two polypeptide sequences are “substantially homologous” to each other when the sequences exhibit at least about 50% sequence identity, preferably at least about 75% sequence identity, more preferably at least about 80% 85% sequence identity, more preferably at least about 90% sequence identity, and most preferably at least about 95% 98% sequence identity over a defined length of the molecules. As used herein, substantially homologous also refers to sequences showing complete identity to the specified sequence. [0066] In general, "identity" refers to an exact nucleotide to nucleotide or amino acid to amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. Percent identity can be determined by a direct comparison of the sequence information between two molecules by aligning the sequences, counting the exact number of matches between the two aligned sequences, dividing by the length of the shorter sequence, and multiplying the result by 100. Readily available computer programs can be used to aid in the analysis, such as ALIGN, Dayhoff, M.O. in Atlas of Protein Sequence and Structure M.O. Dayhoff ed., 5 Suppl. 3:353 358, National biomedical Research Foundation, Washington, DC, which adapts the local homology algorithm of Smith and Waterman Advances in AppL Math. 2:482 489, 1981 for peptide analysis. Programs for determining nucleotide sequence identity are available in the Wisconsin Sequence Analysis Package, Version 8 (available from Genetics Computer Group, Madison, Wl) for example, the BESTFIT, FASTA and GAP programs, which also rely on the Smith and Waterman algorithm. These programs are readily utilized with the default parameters recommended by the manufacturer and described in the Wisconsin Sequence Analysis Package referred to above. For example, percent identity of a particular nucleotide sequence to a reference sequence can be determined using the homology algorithm of Smith and Waterman with a default scoring table and a gap penalty of six nucleotide positions.

[0067] Another method of establishing percent identity in the context of the present invention is to use the MPSRCH package of programs copyrighted by the University of Edinburgh, developed by John F. Collins and Shane S. Sturrok, and distributed by IntelliGenetics, Inc. (Mountain View, CA). From this suite of packages, the Smith Waterman algorithm can be employed where default parameters are used for the scoring table (for example, gap open penalty of 12, gap extension penalty of one, and a gap of six). From the data generated the “Match” value reflects "sequence identity." Other suitable programs for calculating the percent identity or similarity between sequences are generally known in the art, for example, another alignment program is BLAST, used with default parameters. For example, BLASTN and BLASTP can be used using the following default parameters: genetic code = standard; filter = none; strand = both; cutoff = 60; expect = 10; Matrix = BLOSUM62; Descriptions = 50 sequences; sort by = HIGH SCORE; Databases = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translations + Swiss protein + Spupdate + PIR. Details of these programs are readily available. [0068] Alternatively, homology can be determined by hybridization of polynucleotides under conditions which form stable duplexes between homologous regions, followed by digestion with single stranded specific nuclease(s), and size determination of the digested fragments. DNA sequences that are substantially homologous can be identified in a Southern hybridization experiment under, for example, stringent conditions, as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Sambrook et al., supra', DNA Cloning, supra; Nucleic Acid Hybridization, supra.

[0069] "Recombinant" as used herein to describe a nucleic acid molecule means a polynucleotide of genomic, cDNA, viral, semisynthetic, or synthetic origin which, by virtue of its origin or manipulation, is not associated with all or a portion of the polynucleotide with which it is associated in nature. The term "recombinant" as used with respect to a protein or polypeptide means a polypeptide produced by expression of a recombinant polynucleotide. In general, the gene of interest is cloned and then expressed in transformed organisms, as described further below. The host organism expresses the foreign gene to produce the protein under expression conditions.

[0070] The term "transformation" refers to the insertion of an exogenous polynucleotide into a host cell, irrespective of the method used for the insertion. For example, direct uptake, transduction or f-mating are included. The exogenous polynucleotide may be maintained as a non-integrated vector, for example, a plasmid, or alternatively, may be integrated into the host genome.

[0071] "Recombinant host cells," "host cells," "cells", "cell lines," "cell cultures," and other such terms denoting microorganisms or higher eukaryotic cell lines cultured as unicellular entities refer to cells which can be, or have been, used as recipients for recombinant vector or other transferred DNA, and include the original progeny of the original cell which has been transfected.

[0072] A "coding sequence" or a sequence which "encodes" a selected polypeptide, is a nucleic acid molecule which is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vivo when placed under the control of appropriate regulatory sequences (or "control elements"). The boundaries of the coding sequence can be determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. A coding sequence can include, but is not limited to, cDNA from viral, prokaryotic or eukaryotic mRNA, genomic DNA sequences from viral or prokaryotic DNA, and even synthetic DNA sequences. A transcription termination sequence may be located 3' to the coding sequence.

[0073] Typical "control elements," include, but are not limited to, transcription promoters, transcription enhancer elements, transcription termination signals, polyadenylation sequences (located 3' to the translation stop codon), sequences for optimization of initiation of translation (located 5’ to the coding sequence), and translation termination sequences.

[0074] "Operably linked" refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function. Thus, a given promoter operably linked to a coding sequence is capable of effecting the expression of the coding sequence when the proper enzymes are present. The promoter need not be contiguous with the coding sequence, so long as it functions to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between the promoter sequence and the coding sequence and the promoter sequence can still be considered "operably linked" to the coding sequence.

[0075] "Expression cassette" or "expression construct" refers to an assembly which is capable of directing the expression of the sequence(s) or gene(s) of interest. An expression cassette generally includes control elements, as described above, such as a promoter which is operably linked to (so as to direct transcription of) the sequence(s) or gene(s) of interest, and often includes a polyadenylation sequence as well. Within certain embodiments of the invention, the expression cassette described herein may be contained within a plasmid construct. In addition to the components of the expression cassette, the plasmid construct may also include, one or more selectable markers, a signal which allows the plasmid construct to exist as single stranded DNA (e.g., a M13 origin of replication), at least one multiple cloning site, and a "mammalian" origin of replication (e.g., a SV40 or adenovirus origin of replication).

[0076] "Purified polynucleotide" refers to a polynucleotide of interest or fragment thereof which is essentially free, e.g., contains less than about 50%, preferably less than about 70%, and more preferably less than about at least 90%, of the protein with which the polynucleotide is naturally associated. Techniques for purifying polynucleotides of interest are well-known in the art and include, for example, disruption of the cell containing the polynucleotide with a chaotropic agent and separation of the polynucleotide(s) and proteins by ion-exchange chromatography, affinity chromatography and sedimentation according to density. [0077] The term "transfection" is used to refer to the uptake of foreign DNA by a cell. A cell has been "transfected" when exogenous DNA has been introduced inside the cell membrane. A number of transfection techniques are generally known in the art. See, e.g., Graham et al. (1973) Virology, 52:456, Sambrook et al. (2001 ) Molecular Cloning, a laboratory manual, 3rd edition, Cold Spring Harbor Laboratories, New York, Davis et al. (1995) Basic Methods in Molecular Biology, 2nd edition, McGraw-Hill, and Chu et al. (1981 ) Gene 13:197. Such techniques can be used to introduce one or more exogenous DNA moieties into suitable host cells. The term refers to both stable and transient uptake of the genetic material, and includes uptake of peptide- or antibody-linked DNAs.

[0078] A "vector" is capable of transferring nucleic acid sequences to target cells (e.g., viral vectors, non-viral vectors, particulate carriers, and liposomes). Typically, "vector construct," "expression vector," and "gene transfer vector," mean any nucleic acid construct capable of directing the expression of a nucleic acid of interest and which can transfer nucleic acid sequences to target cells. Thus, the term includes cloning and expression vehicles, as well as viral vectors.

[0079] The term “hybridization” refers to the specific binding of a nucleic acid to a complementary nucleic acid via Watson-Crick base pairing.

[0080] "Gene transfer" or "gene delivery" refers to methods or systems for reliably inserting DNA or RNA of interest into a host cell. Such methods can result in transient expression of non-integrated transferred DNA, extrachromosomal replication and expression of transferred replicons (e.g., episomes), or integration of transferred genetic material into the genomic DNA of host cells. Gene delivery expression vectors include, but are not limited to, vectors derived from bacterial plasmid vectors, viral vectors, non- viral vectors, adenoviruses, lentiviruses, alphaviruses, pox viruses, and vaccinia viruses.

[0081] A polynucleotide "derived from" a designated sequence refers to a polynucleotide sequence which comprises a contiguous sequence of approximately at least about 6 nucleotides, preferably at least about 8 nucleotides, more preferably at least about 10-12 nucleotides, and even more preferably at least about 15-20 nucleotides corresponding, i.e. , identical or complementary to, a region of the designated nucleotide sequence. The derived polynucleotide will not necessarily be derived physically from the nucleotide sequence of interest, but may be generated in any manner, including, but not limited to, chemical synthesis, replication, reverse transcription or transcription, which is based on the information provided by the sequence of bases in the region(s) from which the polynucleotide is derived. As such, it may represent either a sense or an antisense orientation of the original polynucleotide.

[0082] A “CRISPR system" refers collectively to transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated ("Cas") genes. In some embodiments, one or more elements of a CRISPR system is derived from a type I, type II, or type III CRISPR system. In some embodiments, one or more elements of a CRISPR system is derived from a particular organism comprising an endogenous CRISPR system, such as Streptococcus pyogenes. In general, a CRISPR system is characterized by elements that promote the formation of a CRISPR complex at the site of a target sequence.

[0083] The term "Cas9" as used herein encompasses type II clustered regularly interspaced short palindromic repeats (CRISPR) system Cas9 endonucleases from any species, and also includes biologically active fragments, variants, analogs, and derivatives thereof that retain Cas9 endonuclease activity (i.e., catalyze site-directed cleavage of DNA to generate double-strand breaks).

[0084] A Cas9 endonuclease binds to and cleaves DNA at a site comprising a sequence complementary to its bound guide RNA (gRNA). For purposes of Cas9 targeting, a gRNA may comprise a sequence "complementary" to a target sequence (e.g., in an intron of a TCR gene), capable of sufficient base-pairing to form a duplex (i.e., the gRNA hybridizes with the target sequence). Additionally, the gRNA may comprise a sequence complementary to a PAM sequence, wherein the gRNA also hybridizes with the PAM sequence in a target DNA.

[0085] A Cas9 polynucleotide, nucleic acid, oligonucleotide, protein, polypeptide, or peptide refers to a molecule derived from any source. The molecule need not be physically derived from an organism, but may be synthetically or recombinantly produced. Cas9 sequences from a number of bacterial species are well known in the art and listed in the National Center for Biotechnology Information (NCBI) database. See, for example, NCBI entries for Cas9 from: Streptococcus pyogenes (WP_002989955, WP_038434062, WP_01 1528583); Campylobacter jejuni (WP_022552435, YP_002344900),

Campylobacter coll (WP 060786116); Campylobacter fetus (WP 059434633); Corynebacterium ulcerans (NC_015683, NC_017317); Corynebacterium diphtheria (NC 016782, NC 016786); Enterococcus faecalis (WP 033919308); Spiroplasma syrphidicola (NC_021284); Prevotella intermedia (NC_017861 ); Spiroplasma taiwanense (NC_021846); Streptococcus iniae (NC_021314); Belliella baltica (NC_018010); Psychroflexus torquisl (NG 018721 ); Streptococcus thermophilus (YP_820832), Streptococcus mutans (WP_061046374, WP_024786433); Listeria innocua (NP 472073); Listeria monocytogenes (WP 061665472); Legionella pneumophila (WP 062726656); Staphylococcus aureus (WP 001573634); Francisella tularensis (WP_032729892, WP_014548420), Enterococcus faecalis (WP_033919308); Lactobacillus rhamnosus (WP_048482595, WP_032965177); and Neisseria meningitidis (WP 061704949, YP 002342100); all of which sequences (as entered by the date of filing of this application) are herein incorporated by reference. Any of these sequences or a variant thereof comprising a sequence having at least about 70-100% sequence identity thereto, including any percent identity within this range, such as 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto, can be used for genome editing, as described herein, wherein the variant retains biological activity, such as Cas9 site-directed endonuclease activity. See also Fonfara et al. (2014) Nucleic Acids Res. 42(4):2577-90; Kapitonov et al. (2015) J. Bacteriol. 198(5):797-807, Shmakov et al. (2015) Mol. Cell. 60(3):385-397, and Chylinski et al. (2014) Nucleic Acids Res. 42(10):6091 -6105); for sequence comparisons and a discussion of genetic diversity and phylogenetic analysis of Cas9.

[0086] By "selectively binds" with reference to a guide RNA is meant that the guide RNA binds preferentially to a target sequence of interest or binds with greater affinity to the target sequence than to other genomic sequences. For example, a gRNA will bind to a substantially complementary sequence and not to unrelated sequences. A gRNA that selectively binds to a particular target DNA sequence will selectively direct binding of Cas9 to a substantially complementary sequence at the target site and not to unrelated sequences.

[0087] The term "donor polynucleotide" refers to a polynucleotide that provides a sequence of an intended edit to be integrated into the genome at a target locus by homology directed repair (HDR).

[0088] A "target site" or "target sequence" is the nucleic acid sequence recognized (i.e., sufficiently complementary for hybridization) by a guide RNA (gRNA) or a homology arm of a donor polynucleotide. The target site may be in an exon or an intron or a specific allele.

[0089] By "homology arm" is meant a portion of a donor polynucleotide that is responsible for targeting the donor polynucleotide to the genomic sequence to be edited in a cell. The donor polynucleotide typically comprises a 5' homology arm that hybridizes to a 5' genomic target sequence and a 3' homology arm that hybridizes to a 3' genomic target sequence flanking a nucleotide sequence comprising the intended edit to the genomic DNA. The homology arms are referred to herein as 5' and 3' (i.e., upstream and downstream) homology arms, which relates to the relative position of the homology arms to the nucleotide sequence comprising the intended edit within the donor polynucleotide. The 5' and 3' homology arms hybridize to regions within the target locus in the genomic DNA to be modified, which are referred to herein as the "5' target sequence" and "3' target sequence," respectively. The nucleotide sequence comprising the intended edit is integrated into the genomic DNA by HDR or recombineering at the genomic target locus recognized (i.e., sufficiently complementary for hybridization) by the 5' and 3' homology arms.

[0090] As used herein, the terms "complementary" or "complementarity" refers to polynucleotides that are able to form base pairs with one another. Base pairs are typically formed by hydrogen bonds between nucleotide units in an anti-parallel orientation between polynucleotide strands. Complementary polynucleotide strands can base pair in a Watson-Crick manner (e.g., A to T, A to U, C to G), or in any other manner that allows for the formation of duplexes. As persons skilled in the art are aware, when using RNA as opposed to DNA, uracil (U) rather than thymine (T) is the base that is considered to be complementary to adenosine. However, when a uracil is denoted in the context of the present invention, the ability to substitute a thymine is implied, unless otherwise stated. "Complementarity" may exist between two RNA strands, two DNA strands, or between a RNA strand and a DNA strand. It is generally understood that two or more polynucleotides may be "complementary" and able to form a duplex despite having less than perfect or less than 100% complementarity. Two sequences are "perfectly complementary" or "100% complementary" if at least a contiguous portion of each polynucleotide sequence, comprising a region of complementarity, perfectly base pairs with the other polynucleotide without any mismatches or interruptions within such region. Two or more sequences are considered "perfectly complementary" or "100% complementary" even if either or both polynucleotides contain additional non- complementary sequences as long as the contiguous region of complementarity within each polynucleotide is able to perfectly hybridize with the other. "Less than perfect" complementarity refers to situations where less than all of the contiguous nucleotides within such region of complementarity are able to base pair with each other. Determining the percentage of complementarity between two polynucleotide sequences is a matter of ordinary skill in the art. For purposes of Cas nuclease (e.g., Cas9 or Cas12a) targeting, a gRNA may comprise a sequence "complementary" to a target sequence, capable of sufficient base-pairing to form a duplex (i.e., the gRNA hybridizes with the target sequence). Additionally, the gRNA may comprise a sequence complementary to a PAM sequence, wherein the gRNA also hybridizes with the PAM sequence in a target DNA.

[0091] "Administering" a nucleic acid, such as a viral vector or a CRISPR system (expressing, e.g., a donor polynucleotide, guide RNA, Cas protein (e.g., Cas9, Cas12a (Cpf 1 ), Cas12d, or Cast 3)) to a cell comprises transducing, transfecting, electroporating, translocating, fusing, phagocytosing, shooting or ballistic methods, etc., i.e., any means by which a nucleic acid can be transported across a cell membrane.

[0092] A "barcode" refers to one or more nucleotide sequences that are used to identify a nucleic acid or cell with which the barcode is associated. Barcodes can be 3-1000 or more nucleotides in length, preferably 10-250 nucleotides in length, and more preferably 10-30 nucleotides in length, including any length within these ranges, such as 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, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides in length. Barcodes may be used, for example, to identify a single cell, subpopulation of cells, colony, or sample from which a nucleic acid originated. Barcodes may also be used to identify the position (i.e., positional barcode) of a cell, colony, or sample from which a nucleic acid originated, such as the position of a colony in a cellular array, the position of a well in a multi-well plate, or the position of a tube, flask, or other container in a rack. In particular, a barcode may be used to identify a genetically modified cell from which a nucleic acid originated. In some embodiments, a barcode is used to identify a donor T cell from which a CAR-T cell originated. Alternatively, a unique barcode may be used to identify each guide-RNA and donor polynucleotide used in multiplexed or multi-step genome editing. Furthermore, multiple barcodes can be used in combination to identify different features of a nucleic acid or cell. For example, positional barcoding (e.g., to identify the position of a cell, colony, culture, or sample in an array, multi-well plate, or rack) can be combined with barcodes identifying a T cell donor and/or barcodes identifying guide-RNAs or donor polynucleotides used in genome editing.

[0093] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to any compound comprising naturally occurring or synthetic amino acid polymers or amino acid-like molecules including but not limited to compounds comprising amino and/or imino molecules. No particular size is implied by use of the terms “polypeptide,” “peptide,” and “protein” and these terms are used interchangeably. The “terms include post-expression modifications of the polypeptide, peptide, or protein such as glycosylation, acetylation, phosphorylation, and the like. Further, polypeptides, peptides, or proteins, as described herein may include additional molecules such as labels (e.g., fluorescent, bioluminescent, or radioactive), tags (e.g., histidine tag, epitope tag), or other chemical moieties.

[0094] The terms “antibodies” and “immunoglobulin” include antibodies or immunoglobulins of any isotype, fragments of antibodies which retain specific binding to an antigen, including, but not limited to, Fab, Fv, scFv, and Fd fragments, monoclonal antibodies, hybrid antibodies, chimeric antibodies, humanized antibodies, single-chain antibodies, single-domain antibodies, nanobodies, bispecific antibodies, tri-specific antibodies, and other multi-specific antibodies, and fusion proteins comprising an antigen-binding portion of an antibody and a non-antibody protein.

[0095] “Antibody fragments” comprise a portion of an intact antibody, for example, the antigen binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, each with a single antigen-binding site, and a residual “Fc” fragment, a designation reflecting the ability to crystallize readily. Pepsin treatment yields an F(ab')2 fragment that has two antigen-combining sites and is still capable of cross-linking antigen.

[0096] “Single-chain Fv” or “sFv” antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. In some embodiments, the Fv polypeptide further comprises a polypeptide linker between the Vn and VL domains, which enables the sFv to form the desired structure for antigen binding.

[0097] The terms “specific binding,” “specifically binds,” and the like, refer to non-covalent or covalent preferential binding to a molecule relative to other molecules or moieties in a solution or reaction. In some embodiments, the affinity of one molecule for another molecule to which it specifically binds is characterized by a KD (dissociation constant) of 10^-5 M or less (e.g., 1 O’⁶ M or less, 10’⁷ M or less, 10‘⁸ M or less, 10’⁹ M or less, 10^-1° M or less, 10^-11 M or less, 10^-12 M or less). "Affinity" refers to the strength of binding, increased binding affinity being correlated with a lower KD. In an embodiment, affinity is determined by surface plasmon resonance (SPR), e.g., as used by Biacore systems. The affinity of one molecule for another molecule is determined by measuring the binding kinetics of the interaction, e.g., at 25°C.

[0098] The term “antigen-binding fragment” as used herein refers to any antibody fragment that specifically binds to a target antigen including, but not limited to, a diabody, a Fab, a Fab', a F(ab')2, an Fv fragment, a disulfide stabilized Fv fragment (dsFv), a (dsFv)2, a bispecific dsFv (dsFv-dsFv'), a disulfide stabilized diabody (ds diabody), a single-chain antibody molecule (scFv), an scFv dimer (bivalent diabody), a multispecific antibody formed from a portion of an antibody including one or more complementarity determining regions (CDRs).

[0099] The term "variable" refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions both in the light-chain and the heavy-chain variable domains. The more highly conserved portions of variable domains are called the framework (FR). The variable domains of native heavy and light chains each comprise four FR regions, largely adopting a D-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the D-sheet structure. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991 )). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity. VL and VH sequences can be reformatted as fragments, as single chain binding domains, linked to chimeric antigen receptors, and the like.

[00100] The term “antigen binding domain (ABD)” refers to a domain that specifically binds to a target antigen. The antigen binding domain region of an antibody may comprise a heavy-chain variable domain (VH) and a light-chain variable domain (VL) in non-covalent association as a single polypeptide or as a dimer. The three complementarity-determining regions of the heavy chain variable domain (CDR H1 , H2, H3) and three complementarity-determining regions of the light chain variable domain (CDR L1 , L2, L3) interact to define an antigen-binding site on the surface of an antibody. Collectively, the six CDRs of the light chain and heavy chain variable domains confer antigen-binding specificity to an antibody. An antigen binding domain region of a CAR may comprise all six CDRs of an antibody or a single variable domain or half of an Fv fragment comprising only three CDRs specific for an antigen, which still retains the ability to recognize and bind the target antigen. In some embodiments, the antigen-binding domain binds to one or more target antigens expressed on the surface of a target cell (e.g., cell surface markers).

[00101] The term "T cell" includes all types of immune cells expressing CD3 including T- helper cells (CD4⁺ cells), cytotoxic T-cells (CD8⁺ cells), natural killer T cells, T-regulatory cells (Treg) and gamma-delta T cells. The term "T cell" also includes genetically modified T cells, including T cells engineered to express a chimeric antigen receptor (CAR) and T cells from which the gene encoding the endogenous T cell receptor has been inactivated or deleted (i.e., TCR gene knockout).

[00102] The terms “T cell receptor” and “TCR” are used interchangeably and generally refer to a receptor found on the surface of T cells or T lymphocytes that is responsible for recognizing antigenic peptides bound to major histocompatibility complex (MHC) molecules. The TCR is a membrane-anchored heterodimeric protein comprising two different protein chains. In the majority of human T cells, the TCR consists of an alpha (a) chain and a beta (b) chain (encoded by TRA and TRB genes, respectively). In about 5% of human T cells, the TCR consists of gamma and delta (g/d) chains (encoded by TRG and TRD genes, respectively). T cells expressing a TCR comprising alpha and beta chains are referred to as ab T cells, and T cells expressing a TCR comprising gamma and delta chains are referred to as gd T cells The ratio of ab T cells to gd T cells differs between species and may be altered by disease (such as leukemia). The variable domains of the TCR a-chain and p-chain each have three hypervariable or complementarity-determining regions (CDRs). CDR 1 and CDR3 bind to the antigenic peptide. CDR2 recognizes the MHC. The constants domains of the TCR a-chain and p- chain each have a cysteine that forms a disulfide bond that links the two chains. The TCR receptor a and p chains associate with six additional adaptor proteins, including a delta chain, a gamma chain, two epsilon chains, and two zeta chains to form an octameric complex. The adaptor proteins comprise signaling motifs involved in TCR signaling. [00103] Chimeric antigen receptor (CAR). A CAR may have any suitable architecture, as known in the art, comprising an antigen binding domain, usually provided in an scFv format, linked to T cell receptor effector functions. The term refers to artificial multimodule molecules capable of triggering or inhibiting the activation of an immune cell. A CAR will generally comprise an antigen binding domain, linker, transmembrane domain and cytoplasmic signaling domain. In some instances, a CAR will include one or more co-stimulatory domains and/or one or more co-inhibitory domains.

[00104] The antigen-binding domain of the CAR may include any naturally occurring, synthetic, semi-synthetic, or recombinantly produced binding partner for a target antigen of interest. In some embodiments, the binding region is an antigen-binding region, such as an antibody or functional binding domain or antigen-binding fragment thereof. The antigen-binding region of the CAR can include any domain that binds to the antigen and may include, but is not limited to, a monoclonal antibody, a polyclonal antibody, a synthetic antibody, a human antibody, a humanized antibody, a non-human antibody, a single-chain antibody, and any antigen-binding fragment thereof. Thus, in some embodiments, the antigen binding domain portion includes a mammalian antibody or an antigen-binding fragment thereof. An antigen-binding domain may comprise an antigenbinding fragment (Fab), a single-chain variable fragment (scFv), a nanobody, a VH domain, a VL domain, a single domain antibody (sdAb), a shark variable domain of a new antigen receptor (VNAR), a single variable domain on a heavy chain (VHH), a bispecific antibody, or a diabody; or a functional antigen-binding fragment thereof. In some embodiments, the antigen-binding domain is derived from the same cell type or the same species in which the CAR will ultimately be used. For example, for use in humans, the antigen-binding domain of the CAR may include a human antibody, a humanized antibody, or an antigen-binding fragment thereof.

[00105] In some embodiments, the antigen binding domain is derived from a single chain antibody that selectively binds to a target antigen. In some embodiments, the antigen binding domain is provided by a single chain variable fragment (scFv). A scFv is a recombinant molecule in which the variable regions of the light and heavy immunoglobulin chains are connected in a single fusion polypeptide. Generally, the VH and VL sequences are joined by a linker sequence. See, for example, Ahmad (2012) Clinical and Developmental Immunology Article ID 980250, herein specifically incorporated by reference. In principle, there are no particular limitations to the length and/or amino acid composition of the linker peptide joining the VH and VL sequences. In some embodiments, any arbitrary single-chain peptide including about 1 to 100 amino acid residues (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, etc. amino acid residues) can be used as a peptide linker. In some embodiments, the linker peptide sequence includes about 5 to 50, about 10 to 60, about 20 to 70, about 30 to 80, about 40 to 90, about 50 to 100, about 60 to 80, about 70 to 100, about 30 to 60, about 20 to 80, about 30 to 90 amino acid residues. In some embodiments, the linker peptide sequence includes about 1 to 10, about 5 to 15, about 10 to 20, about 15 to 25, about 20 to 40, about 30 to 50, about 40 to 60, about 50 to 70 amino acid residues. In some embodiments, the linker peptide sequence includes about 40 to 70, about 50 to 80, about 60 to 80, about 70 to 90, or about 80 to 100 amino acid residues. In some embodiments, the linker peptide sequence includes about 1 to 10, about 5 to 15, about 10 to 20, about 15 to 25 amino acid residues.

[00106] The transmembrane domain may be derived either from a natural or a synthetic source. Where the source is natural, the domain may be derived from any membranebound or transmembrane protein. In some embodiments, the transmembrane domain comprises at least the stalk and/or transmembrane region(s) of CD8, Megf10, FcRy, Bail , MerTK, TIM4, Stabilin-1 , Stabilin-2, RAGE, CD300f, integrin subunit av, Integrin subunit |35, CD36, LRP1 , SCARF1 , C1 Qa, Axl, CD45, and/or CD86. In some embodiments, the CAR transmembrane domain is derived from a type I membrane protein, such as, but not limited to, CD3£, CD4, CD8, or CD28. In other embodiments, the transmembrane domain is synthetic, in which case it will include predominantly hydrophobic residues such as leucine, isoleucine, valine, phenylalanine, tryptophan, and alanine. In some embodiments, a triplet of phenylalanine, tryptophan and valine will be inserted at each end of a synthetic transmembrane domain.

[00107] In some embodiments, the CAR further comprises one or more linkers/spacers. For example, an extracellular spacer region may link the antigen binding domain to the transmembrane domain and/or an intracellular spacer region may link an intracellular signaling domain to the transmembrane domain. A spacer (linker) region linking the antigen binding domain to the transmembrane domain should be flexible enough to allow the antigen binding domain to orient in different directions to facilitate antigen recognition.

[00108] Various types of linkers may be used in the CARs described herein. In some embodiments, the linker includes a peptide linker/spacer sequence. In some embodiments, the spacer comprises the hinge region from an immunoglobulin, e.g., the hinge from any one of lgG1 , lgG2a, lgG2b, lgG3, lgG4, particularly the human protein sequences. Alternatives include the CH2CH3 region of immunoglobulin and portions of CD3. For many scFv based constructs, an IgG hinge is effective.

[00109] In principle, there are no particular limitations to the length and/or amino acid composition of a linker peptide sequence. In some embodiments, a linker peptide sequence comprises about 1 to 100 amino acid residues, including any number of residues within this range such as 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues. In some embodiments, the linker peptide sequence includes about 5 to 50, about 10 to 60, about 20 to 70, about 30 to 80, about 40 to 90, about 50 to 100, about 60 to 80, about 70 to 100, about 30 to 60, about 20 to 80, about 30 to 90 amino acid residues. In some embodiments, the linker peptide sequence includes about 1 to 10, about 5 to 15, about 10 to 20, about 15 to 25, about 20 to 40, about 30 to 50, about 40 to 60, about 50 to 70 amino acid residues. In some embodiments, the linker peptide sequence includes about 40 to 70, about 50 to 80, about 60 to 80, about 70 to 90, or about 80 to 100 amino acid residues. In some embodiments, the linker peptide sequence includes about 1 to 10, about 5 to 15, about 10 to 20, about 15 to 25 amino acid residues. In some embodiments, the linker peptide sequence may include up to 300 amino acids, preferably 10 to 100 amino acids and most preferably 25 to 50 amino acids. In some embodiments, a short oligo- or polypeptide linker, preferably between 2 and 10 amino acids in length may form the linkage between the transmembrane domain and the intracellular engulfment signaling domain or extracellular antigen binding domain of the CAR. In some embodiments the linker comprises the amino acid sequence (G4S)n where n is 1 , 2, 3, 4, 5, etc., and in some embodiments, n is 3.

[00110] A cytoplasmic signaling domain, such as those derived from the T cell receptor □- chain, is employed as part of the CAR in order to produce stimulatory signals for T lymphocyte proliferation and effector function following engagement of the chimeric receptor with the target antigen. Endodomains from co-stimulatory molecules may be included in the cytoplasmic signaling portion of the CAR.

[oom] The term “co-stimulatory domain”, refers to a stimulatory domain, typically an endodomain, of a CAR that provides a secondary non-specific activation mechanism through which a primary specific stimulation is propagated. Examples of co-stimulation include antigen nonspecific T cell co-stimulation following antigen specific signaling through the T cell receptor and antigen nonspecific B cell co-stimulation following signaling through the B cell receptor. Co-stimulation, e.g., T cell co-stimulation, and the factors involved have been described in Chen & Flies. Nat Rev Immunol (2013) 13(4):227-42, the disclosure of which are incorporated herein by reference in their entirety. Non-limiting examples of suitable co-stimulatory polypeptides include, but are not limited to, 4-1 BB (CD137), CD28, ICOS, OX-40, BTLA, CD27, CD30, GITR, and HVEM.

[00112] The term “co-inhibitory domain” refers to an inhibitory domain, typically an endodomain, derived from a receptor that provides secondary inhibition of primary antigen-specific activation mechanisms which prevents co-stimulation. Co-inhibition, e.g., T cell co-inhibition, and the factors involved have been described in Chen & Flies. Nat Rev Immunol (2013) 13(4):227-42 and Thaventhiran et al. J Clin Cell Immunol (2012) S12. In some embodiments, co-inhibitory domains homodimerize. A co-inhibitory domain can be an intracellular portion of a transmembrane protein. Non-limiting examples of suitable co-inhibitory polypeptides include, but are not limited to, CTLA-4 and PD-1 .

[00113] A first-generation CAR transmits the signal from antigen binding through only a single signaling domain, for example a signaling domain derived from the high-affinity receptor for IgE FCERI D D or the CD3 chain. The domain contains one or three immunoreceptor tyrosine-based activating motif(s) [ITAM(s)] for antigen-dependent T- cell activation. The ITAM-based activating signal endows T-cells with the ability to lyse the target tumor cells and secret cytokines in response to antigen binding.

[00114] Second-generation CARs include a co-stimulatory signal in addition to the CD3D signal. Coincidental delivery of the delivered co-stimulatory signal enhances cytokine secretion and antitumor activity induced by CAR-transduced T-cells. The co-stimulatory domain will usually be membrane proximal relative to the CD3D domain. Third- generation CARs include a tripartite signaling domain, comprising for example a CD28, CD3 , 0X40 or 4-1 BB signaling region. In fourth generation, or “armored car” CAR-T cells, CAR-T cells are further genetically modified to express or block molecules and/or receptors to enhance immune activity.

[00115] CAR variants include split CARs wherein the extracellular portion, the ABD and the cytoplasmic signaling domain of a CAR are present on two separate molecules. CAR variants also include ON-switch CARs which are conditionally activatable CARs, e.g., comprising a split CAR wherein conditional hetero-dimerization of the two portions of the split CAR is pharmacologically controlled. CAR molecules and derivatives thereof (i.e., CAR variants) are described, e.g., in PCT Application Nos. US2014/016527, US1996/017060, US2013/063083; Fedorov et al. Sci Trans! Med (2013) ;5(215) :215ra172; Glienke et al. Front Pharmacol (2015) 6:21 ; Kakarla & Gottschalk 52 Cancer J (2014) 20(2):151 -5; Riddell et al. Cancer J (2014) 20(2):141 -4; Pegram et al. Cancer J (2014) 20(2):127-33; Cheadle et al. Immunol Rev (2Q14) 257(1 ):91 -106; Barrett et al. Annu Rev Med (2014) 65:333-47; Sadelain et al. Cancer Discov (2013) 3(4):388- 98; Cartellieri et al., J Biomed Biotechnol (2010) 956304; the disclosures of which are incorporated herein by reference in their entirety.

[00116] CAR variants also include bispecific or tandem CARs, which include a secondary CAR binding domain that can either amplify or inhibit the activity of a primary CAR. CAR variants also include inhibitory chimeric antigen receptors (iCARs) which may, e.g., be used as a component of a bispecific CAR system, where binding of a secondary CAR binding domain results in inhibition of primary CAR activation. Tandem CARs (TanCAR) mediate bispecific activation of T cells through the engagement of two chimeric receptors designed to deliver stimulatory or costimulatory signals in response to an independent engagement of two different tumor associated antigens. iCARs use the dual antigen targeting to shout down the activation of an active CAR through the engagement of a second suppressive receptor equipped with inhibitory signaling domains

[00117] The dual recognition of different epitopes by two CARs diversely designed to either deliver killing through -chain or costimulatory signals, e.g., through CD28 allows a more selective activation of the reprogrammed T cells by restricting Tandem CAR's activity to cancer cell expressing simultaneously two antigens rather than one. The potency of delivered signals in engineered T cells will remain below threshold of activation and thus ineffective in absence of the engagement of costimulatory receptor. The combinatorial antigen recognition enhances selective tumor eradication and protects normal tissues expressing only one antigen from unwanted reactions.

[00118] Inhibitory CARs (iCARs) are designed to regulate CAR-T cells activity through inhibitory receptor signaling module activation. This approach combines the activity of two CARs, one of which generates dominant negative signals limiting the responses of CAR-T cells activated by the activating receptor. iCARs can switch off the response of the counteracting activator CAR when bound to a specific antigen expressed only by normal tissues. In this way, iCARs-T cells can distinguish cancer cells from healthy ones, and reversibly block functionalities of transduced T cells in an antigen-selective fashion. CTLA-4 or PD-1 intracellular domains in iCARs trigger inhibitory signals on T lymphocytes, leading to less cytokine production, less efficient target cell lysis, and altered lymphocyte motility. [00119] An ABD can be provided as a “chimeric bispecific binding member”, i.e., a chimeric polypeptide having dual specificity to two different binding partners (e.g., two different antigens). Non-limiting examples of chimeric bispecific binding members include bispecific antibodies, bispecific conjugated monoclonal antibodies (mab)2, bispecific antibody fragments (e.g., F(ab)2, bispecific scFv, bispecific diabodies, single chain bispecific diabodies, etc.), bispecific T cell engagers (BiTE), bispecific conjugated single domain antibodies, micabodies and mutants thereof, and the like. Non-limiting examples of chimeric bispecific binding members also include those chimeric bispecific agents described in Kontermann. MAbs. (2012) 4(2): 182-197; Stamova et al. Antibodies 2012, 1 (2), 172-198; Farhadfar et al. Leuk Res. (2016) 49:13-21 ; Benjamin et al. Ther Adv Hematol. (2016) 7(3):142-56; Kiefer et al. Immunol Rev. (2016) 270(1 ):178-92; Fan et al. J Hematol Oncol. (2015) 8:130; May et al. Am J Health Syst Pharm. (2016) 73(1 ):e6-e13; the disclosures of which are incorporated herein by reference in their entirety.

[00120] In some instances, a chimeric bispecific binding member may be a bispecific T cell engager (BiTE). A BiTE is generally made by fusing a specific binding member (e.g., a scFv) that binds an antigen to a specific binding member (e.g., a scFv) with a second binding domain specific for a T cell molecule such as CD3.

[00121] In some instances, a chimeric bispecific binding member may be a CAR-T cell adapter. As used herein, by “CAR-T cell adapter” is meant an expressed bispecific polypeptide that binds the antigen recognition domain of a CAR and redirects the CAR to a second antigen. Generally, a CAR-T cell adapter will have two binding regions, one specific for an epitope on the CAR to which it is directed and a second epitope directed to a binding partner which, when bound, transduces the binding signal activating the CAR. Useful CAR-T cell adapters include but are not limited to e.g., those described in Kim et al. J Am Chem Soc. (2015) 137(8):2832-5; Ma et al. Proc Natl Acad Sci U S A. (2016) 1 13(4):E450-8 and Cao et al. Angew Chem Int Ed Engl. (2016) 55(26) :7520-4; the disclosures of which are incorporated herein by reference in their entirety.

[00122] Effector CAR-T cells include autologous or allogeneic immune cells having cytolytic activity against a target cell. In some embodiments, a T cell is engineered to express a CAR. The term “T cells” refers to mammalian immune effector cells that may be characterized by expression of CD3 and/or a T cell antigen receptor.

[00123] In some embodiments, the CAR-T cells are engineered from a complex mixture of immune cells, e.g., tumor infiltrating lymphocytes (TILs) isolated from an individual in need of treatment. See, for example, Yang and Rosenberg (2016) Adv Immunol. 130:279-94, “Adoptive T Cell Therapy for Cancer; Feldman et al (2015) Semin Oncol. 42(4):626-39 “Adoptive Cell Therapy-Tumor-Infiltrating Lymphocytes, T-Cell Receptors, and Chimeric Antigen Receptors”; Clinical Trial NCT01 174121 , “Immunotherapy Using Tumor Infiltrating Lymphocytes for Patients With Metastatic Cancer”; Tran et al. (2014) Science 344(6184)641 -645, “Cancer immunotherapy based on mutation-specific CD4+ T cells in a patient with epithelial cancer”.

[00124] In other embodiments, the engineered T cell is allogeneic with respect to the individual that is treated, e.g. see clinical trials NCT03121625; NCT03016377; NCT02476734; NCT02746952; NCT02808442. See for review Graham et al. (2018) Cells. 7(10) E155. In some embodiments an allogeneic engineered T cell is fully HLA matched. However not all patients have a fully matched donor, and a cellular product suitable for all patients independent of HLA type provides an alternative.

[00125] Allogeneic T cells may be administered in combination with intensification of lymphodepletion to allow CAR-T cells to expand and clear malignant cells prior to host immune recovery, e.g., by administration of Alemtuzumab (monoclonal anti-CD52), purine analogs, etc. The allogeneic T cells may be modified for resistance to Alemtuzumab. Gene editing can be used to prevent expression of HLA class I molecules on CAR-T cells, e.g. by deletion of □2-microglobulin.

[00126] In addition to modifying T cells, induced pluripotent stem (iPS) cell-derived CAR- T cells can be used. For example, donor T cells can be transduced with reprogramming factors to restore pluripotency, and then re-differentiated into T effector cells.

[00127] T cells for engineering, as described above, collected from a subject or a donor, may be separated from a mixture of cells by techniques that enrich for desired cells, or may be engineered and cultured without separation. An appropriate solution may be used for dispersion or suspension. Such solution will generally be a balanced salt solution, e.g. normal saline, PBS, Hank’s balanced salt solution, etc., conveniently supplemented with fetal calf serum or other naturally occurring factors, in conjunction with an acceptable buffer at low concentration, generally from 5-25 mM. Convenient buffers include HEPES, phosphate buffers, lactate buffers, etc.

[00128] Techniques for affinity separation may include magnetic separation, using antibody-coated magnetic beads, affinity chromatography, cytotoxic agents joined to a monoclonal antibody or used in conjunction with a monoclonal antibody, e.g., complement and cytotoxins, and "panning" with antibody attached to a solid matrix, e.g., a plate, or other convenient technique. Techniques providing accurate separation include fluorescence activated cell sorters, which can have varying degrees of sophistication, such as multiple color channels, low angle and obtuse light scattering detecting channels, impedance channels, etc. The cells may be selected against dead cells by employing dyes associated with dead cells (e.g., propidium iodide). Any technique may be employed which is not unduly detrimental to the viability of the selected cells. The affinity reagents may be specific receptors or ligands for the cell surface molecules indicated above. In addition to antibody reagents, peptide-MHC antigen and T cell receptor pairs may be used; peptide ligands and receptor; effector and receptor molecules, and the like. [00129] The separated cells may be collected in any appropriate medium that maintains the viability of the cells, usually having a cushion of serum at the bottom of the collection tube. Various media are commercially available and may be used according to the nature of the cells, including dMEM, HBSS, dPBS, RPMI, Iscove's medium, etc., frequently supplemented with fetal calf serum (FCS).

[00130] The collected and optionally enriched cell population may be used immediately for genetic modification, or may be frozen at liquid nitrogen temperatures and stored, being thawed and capable of being reused. The cells will usually be stored in 10% DMSO, 50% FCS, 40% RPMI 1640 medium.

[00131] Engineered CAR-T cells may be infused into a subject in any physiologically acceptable medium by any convenient route of administration, normally intravascularly, though CAR-T cells may also be introduced by other routes, where the cells may find an appropriate site for growth. Usually, at least 1 x10⁶ cells/kg will be administered, at least 1 x10⁷ cells/kg, at least 1 x10⁸ cells/kg, at least 1 x10⁹ cells/kg, at least 1 x10¹° cells/kg, or more, usually being limited by the number of T cells that are obtained during collection.

[00132] By “genetically engineered” or “genetically modified”, it is intended to mean that the genome of a cell has been altered. In some cases, the genome of the cell has been manipulated to express an expression product that is not normally naturally expressed by the cell. Examples of cells that have been genetically engineered include chimeric antigen receptor (CAR)-T cells that are T-cells that have been genetically engineered to express a CAR. A coding sequence encoding a CAR may be introduced on an expression vector into a cell to be engineered. For example, a CAR coding sequence may be introduced into the genome at the site of an endogenous T cell receptor gene. In some cases, cells are further engineered to delete an endogenous T cell receptor (i.e., TCR knockout). In some cases, a CRISPR/Cas9 system is used to genetically modify a T cell. A CRISPR/Cas9 system can be introduced into cells by transfection with mRNA or a plasmid that encodes Gas9 and a gRNA or by viral delivery of CRISPR components, e.g., using lentiviral, retroviral vectors, or non-integrating viruses, such as adenovirus and adeno-associated virus (AAV).

[00133] By “binding-triggered transcriptional switch” or “BTSS”, it is intended to mean a synthetic modular polypeptide or system of interacting polypeptides having an extracellular domain that includes a second member of a specific binding pair that binds a first member of the specific binding pair (e.g., an antigen), a binding-transducer and an intracellular domain. Upon binding of the first member of the specific binding pair to the BTTS the binding signal is transduced to the intracellular domain such that the intracellular domain becomes activated and performs a function, e.g., transcription activation, within the cell that it does not perform in the absence of the binding signal.

[00134] Examples of BTSS include the synNotch system, the MESA system, the TANGO system, the A2 Notch system, etc. The synNotch receptor may be for example as described in U.S. Patent No. 9,670,281 and described in more detail below. The MESA system may be as described in WO 2018/081039 A1 and comprises a self-containing sensing and signal transduction system, such that binding of a ligand (first member of the specific binding pair) to the receptor (second member of the specific binding pair) induces signaling to regulate expression of a target gene. In the MESA system, binding of the ligand to the receptor induces dimerization that results in proteolytic trans-cleavage of the system to release a transcriptional activator previously sequestered at the plasma membrane. The TANGO system may be as described in Barnea et al., 2008 Proc. Natl. Acad. Sci. U.S.A., 105(1 ): 64-9. Briefly, the TANGO system sequesters a transcription factor to the cell membrane by physically linking it to a membrane-bound receptor (e.g., GPCRs, receptor kinases, Notch, steroid hormone receptors, etc.). Activation of the receptor fusion results in the recruitment of a signaling protein fused to a protease that then cleaves and releases the transcription factor to activate genes in the cell. The A2 Notch system may be as described in WO 2019099689 A1 . Briefly, the A2 Notch system incorporates a force sensor cleavage domain which, upon cleavage induced upon binding of a ligand to the receptor, releases the intracellular domain into the cell.

[00135] In certain embodiments, the second binding member may be present on the surface of a genetically engineered cell, such as, a cell expressing a BTTS and a CAR under the control of the BTTS. In certain embodiments, the second binding member may be present on the surface of a genetically engineered cell, such as, a cell expressing the

BTTS and a CAR under control of the [00136] In certain cases, the first binding member may bind to a synNotch receptor as described in U.S. Patent No. 9,670,281 . For example, the synNotch receptor may include an extracellular domain that includes the second binding member, where the second binding member is a single-chain Fv (scFv) or a nanobody and the first binding member present on the particles is an antigen to which the single-chain Fv (scFv) or a nanobody binds. In certain cases, the second binding member may be an anti-CD19, anti- mesothelin, anti-GFP antibody, scFv, or a nanobody and the first binding member may be CD19, mesothelin, GFP, respectively.

[00137] In certain embodiments, the BTTS is a chimeric Notch polypeptide comprising, from N-terminus to C-terminus and in covalent linkage: a) an extracellular domain comprising the second member of the specific-binding pair that is not naturally present in a Notch receptor polypeptide and that specifically binds to the first member of the specific-binding pair; b) a Notch regulatory region comprising a Lin 12-Notch repeat, an S2 proteolytic cleavage site, and a transmembrane domain comprising an S3 proteolytic cleavage site; c) an intracellular domain comprising a transcriptional activator or a transcriptional repressor that is heterologous to the Notch regulatory region and replaces a naturally-occurring intracellular Notch domain, wherein binding of the first member of the specific-binding pair to the second member of the specific-binding pair induces cleavage at the S2 and S3 proteolytic cleavage sites, thereby releasing the intracellular domain; and a transcriptional control element, responsive to the transcriptional activator, operably linked to a nucleotide sequence encoding a chimeric antigen receptor (CAR). In certain cases, the cell may be a T-cell, such as, those described in U.S. Patent No. 9,670,281 , which is herein incorporated by reference.

Genetically Modifying a Cell with Both a Gene Knockin and Gene Knockout

[00138] Compositions and methods are provided for genetically modifying a cell to introduce both a gene knockin and a gene knockout. The subject methods use a donor polynucleotide comprising a gene knockin sequence and a knockout guide RNA sequence. Transcription of the donor polynucleotide, after integration of the donor polynucleotide into the genome, produces a mature messenger RNA sequence comprising the knockout guide RNA. By incorporating the knockout guide RNA into a messenger RNA transcript, the knockout guide RNA is only expressed in cells that have had the gene knockin construct successfully integrated into the genome. An RNA-guided nuclease is used to excise the knockout guide RNA from the messenger RNA transcript, which then guides the RNA-guided nuclease to a second target locus where the RNA- guided nuclease creates a double stranded DNA break, resulting in gene knockout at the second target locus. Only cells with the gene knockin are able to express the messenger RNA containing the knockout guide RNA so that the gene knockin at the first genomic target locus and the subsequent gene knockout at the second genomic target locus are linked.

[00139] In certain embodiments, the method comprises introducing a donor polynucleotide into a cell, wherein the donor polynucleotide comprises a 5' homology arm that hybridizes to a 5' genomic target sequence and a 3' homology arm that hybridizes to a 3' genomic target sequence flanking a nucleotide sequence encoding i) an exogenous polypeptide and ii) a knockout guide RNA. An RNA-guided nuclease and a knockin guide RNA are also introduced into the cell, wherein the knockin guide RNA forms a complex with the RNA-guided nuclease such that the knockin guide RNA directs the RNA-guided nuclease to a first genomic target locus, wherein the RNA-guided nuclease creates a doublestranded break in the genomic DNA at the first genomic target locus, wherein the donor polynucleotide is integrated at the first genomic target locus recognized by its 5' homology arm and 3' homology arm by homology directed repair (HDR). The cell is cultured under conditions suitable for transcription of the integrated donor polynucleotide, wherein a first mRNA transcript encoding the exogenous polypeptide and the knockout guide RNA is produced, wherein the RNA-guided nuclease excises the knockout guide RNA from the first mRNA transcript to produce a second mRNA transcript encoding the exogenous polypeptide without the knockout guide RNA. Translation of the second mRNA transcript results in production of the exogenous polypeptide in the cell. The excised knockout guide RNA forms a complex with the RNA-guided nuclease such that the knockout guide RNA directs the RNA-guided nuclease to a second genomic target locus, wherein the RNA-guided nuclease creates a double-stranded break in the genomic DNA at the second genomic target locus. DNA repair of the double-stranded break by non- homologous end joining creates an indel resulting in gene knockout at the second genomic target locus

[00140] In the donor polynucleotide, the nucleotide sequence encoding the exogenous polypeptide and the knockout guide RNA is flanked by a pair of homology arms responsible for targeting the donor polynucleotide to the first genomic target locus where the donor polynucleotide is integrated into the genome of the cell. The donor polynucleotide typically comprises a 5' homology arm that hybridizes to a 5' genomic target sequence and a 3' homology arm that hybridizes to a 3' genomic target sequence. The homology arms are referred to herein as 5' and 3' (i.e., upstream and downstream) homology arms, which relates to the relative position of the homology arms to the nucleotide sequence encoding the exogenous polypeptide and the knockout guide RNA within the donor polynucleotide. The 5' and 3' homology arms hybridize to regions within the first genomic target locus in the genomic DNA, which are referred to herein as the "5' target sequence" and "3' target sequence," respectively.

[00141] The homology arm must be sufficiently complementary for hybridization to the target sequence to mediate homologous recombination between the donor polynucleotide and genomic DNA at the first genomic target locus where gene knockin is desired. For example, a homology arm may comprise a nucleotide sequence having at least about 80-100% sequence identity to the corresponding genomic target sequence, including any percent identity within this range, such as at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto, wherein the nucleotide sequence encoding the exogenous polypeptide and the knockout guide RNA is integrated into the genomic DNA by HDR at the genomic target locus recognized (i.e., sufficiently complementary for hybridization) by the 5' and 3' homology arms.

[00142] In certain embodiments, the corresponding homologous nucleotide sequences in the genomic target sequence (i.e., the "5' target sequence" and "3' target sequence") flank a specific site for cleavage and/or a specific site for introducing the nucleotide sequence encoding the exogenous polypeptide and the knockout guide RNA. The distance between the specific cleavage site and the homologous nucleotide sequences (e.g., each homology arm) can be several hundred nucleotides. In some embodiments, the distance between a homology arm and the cleavage site is 200 nucleotides or less (e.g., 0, 10, 20, 30, 50, 75, 100, 125, 150, 175, and 200 nucleotides). In most cases, a smaller distance may give rise to a higher gene targeting rate. In a preferred embodiment, the donor polynucleotide is substantially identical to the target genomic sequence, across its entire length except for the sequence changes to be introduced to a portion of the genome that encompasses both the specific cleavage site and the portions of the genomic target sequence to be altered.

[00143] A homology arm can be of any length, e.g., 10 nucleotides or more, 50 nucleotides or more, 100 nucleotides or more, 250 nucleotides or more, 300 nucleotides or more, 350 nucleotides or more, 400 nucleotides or more, 450 nucleotides or more, 500 nucleotides or more, 1000 nucleotides (1 kb) or more, 5000 nucleotides (5 kb) or more, 10000 nucleotides (10 kb) or more, etc. In some instances, the 5' and 3' homology arms are substantially equal in length to one another, e.g. one may be 30% shorter or less than the other homology arm, 20% shorter or less than the other homology arm, 10% shorter or less than the other homology arm, 5% shorter or less than the other homology arm, 2% shorter or less than the other homology arm, or only a few nucleotides less than the other homology arm. In other instances, the 5' and 3' homology arms are substantially different in length from one another, e.g., one may be 40% shorter or more, 50% shorter or more, sometimes 60% shorter or more, 70% shorter or more, 80% shorter or more, 90% shorter or more, or 95% shorter or more than the other homology arm.

[00144] An RNA-guided nuclease can be targeted to a particular genomic sequence (i.e., genomic target sequence to be modified) by altering its guide RNA sequence. A targetspecific guide RNA comprises a nucleotide sequence that is complementary to a genomic target sequence, and thereby mediates binding of the nuclease-guide RNA complex by hybridization at the target site. For example, the knockin guide RNA can be designed with a sequence complementary to a sequence of the first genomic target locus to target the nuclease-knockin guide RNA complex to a first target site where the gene knockin is desired. The knockout guide RNA can be designed with a sequence complementary to a sequence of the second genomic target locus to target the nuclease-knockout guide RNA complex to a second target site where the gene knockout is desired.

[00145] In certain embodiments, the RNA-guided nuclease used for genome modification is a clustered regularly interspersed short palindromic repeats (CRISPR) system Cas nuclease. Any RNA-guided Cas nuclease capable of catalyzing site-directed cleavage of DNA to allow integration of donor polynucleotides by the HDR mechanism can be used in genome editing, including CRISPR system type I, type II, or type III Cas nucleases. Examples of Cas proteins include Cas1 , Cas1 B, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a1 , Cas8a2, Cas8b, Cas8c, Cas9 (Csn1 or Csx12), Casi o, Cas10d, CasF, CasG, CasH, Csy1 , Csy2, Csy3, Cse1 (CasA), Cse2 (CasB), Cse3 (CasE), Cse4 (CasC), Csc1 , Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1 , Cmr3, Cmr4, Cmr5, Cmr6, Csb1 , Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1 , Csx15, Csf1 , Csf2, Csf3, Csf4, and Cu1966, and homologs or modified versions thereof.

[00146] In certain embodiments, a type II CRISPR system Cas9 endonuclease is used. Cas9 nucleases from any species, or biologically active fragments, variants, analogs, or derivatives thereof that retain Cas9 endonuclease activity (i.e., catalyze site-directed cleavage of DNA to generate double-strand breaks) may be used to perform genome modification as described herein. The Cas9 need not be physically derived from an organism, but may be synthetically or recombinantly produced. Cas9 sequences from a number of bacterial species are well known in the art and listed in the National Center for Biotechnology Information (NCBI) database. See, for example, NCBI entries for Cas9 from: Streptococcus pyogenes (WP 002989955, WP 038434062, WP 01 1528583); Campylobacter jejuni (WP 022552435, YP 002344900), Campylobacter coll (WP 0607861 16); Campylobacter fetus (WP 059434633); Corynebacterium ulcerans (NC 015683, NC 017317); Corynebacterium diphtheria (NC_016782, NC_016786); Enterococcus faecalis (WP_033919308); Spiroplasma syrphidicola (NC_021284); Prevotella intermedia (NC_017861 ); Spiroplasma taiwanense (NC_021846); Streptococcus iniae (NC_021314); Belliella baltica (NC_018010); Psychroflexus torquisl (NC_018721 ); Streptococcus thermophilus (YP_820832), Streptococcus mutans (WP 061046374, WP_024786433); Listeria innocua (NP_472073); Listeria monocytogenes (WP 061665472); Legionella pneumophila (WP 062726656); Staphylococcus aureus (WP 001573634); Francisella tularensis (WP 032729892, WP 014548420), Enterococcus faecalis (WP 033919308); Lactobacillus rhamnosus (WP 048482595, WP_032965177); and Neisseria meningitidis (WP_061704949, YP 002342100); all of which sequences (as entered by the date of filing of this application) are herein incorporated by reference. Any of these sequences or a variant thereof comprising a sequence having at least about 70-100% sequence identity thereto, including any percent identity within this range, such as 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto, can be used for genome editing, as described herein. See also Fonfara et al. (2014) Nucleic Acids Res. 42(4):2577-90; Kapitonov et al. (2015) J. Bacteriol. 198(5):797-807, Shmakov et al. (2015) Mol. Cell. 60(3):385-397, and Chylinski et al. (2014) Nucleic Acids Res. 42(10):6091 -6105); for sequence comparisons and a discussion of genetic diversity and phylogenetic analysis of Cas9.

[00147] The CRISPR-Cas system naturally occurs in bacteria and archaea where it plays a role in RNA-mediated adaptive immunity against foreign DNA. The bacterial type II CRISPR system uses the endonuclease, Cas9, which forms a complex with a guide RNA (gRNA) that specifically hybridizes to a complementary genomic target sequence, where the Cas9 endonuclease catalyzes cleavage to produce a double-stranded break. Targeting of Cas9 typically further relies on the presence of a 5' protospacer-adjacent motif (PAM) in the DNA at or near the gRNA-binding site.

[00148] The genomic target site will typically comprise a nucleotide sequence that is complementary to the guide RNA and may further comprise a protospacer adjacent motif (PAM). In certain embodiments, the target site comprises 20-30 base pairs in addition to a 3 base pair PAM. Typically, the first nucleotide of a PAM can be any nucleotide, while the two other nucleotides will depend on the specific Cas9 protein that is chosen. Exemplary PAM sequences are known to those of skill in the art and include, without limitation, NNG, NGN, NAG, and NGG, wherein N represents any nucleotide. In certain embodiments, the intron sequence of the TOR gene targeted by a guide RNA comprises a mutation that creates a PAM within the intron, wherein the PAM promotes binding of the Cas9-gRNA complex to the intron.

[00149] In certain embodiments, the guide RNA is 5-50 nucleotides, 10-30 nucleotides, 15-25 nucleotides, 18-22 nucleotides, or 19-21 nucleotides in length, or any length between the stated ranges, including, for example, 10, 1 1 , 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 nucleotides in length. The guide RNA may be a single guide RNA comprising crRNA and tracrRNA sequences in a single RNA molecule, or the guide RNA may comprise two RNA molecules with crRNA and tracrRNA sequences residing in separate RNA molecules.

[00150] In another embodiment, the CRISPR nuclease from Prevotella and Francisella 1 (Cpf1 ), also referred to as CRISPR associated protein 12a (Cas12a), may be used. Cas12a is another class II CRISPR/Cas system RNA-guided nuclease with similarities to Cas9 and may be used analogously. Unlike Cas9, Cas12a does not require a tracrRNA and only depends on a crRNA in its guide RNA, which provides the advantage that shorter guide RNAs can be used with Cas12a for targeting than Cas9. Cas12a is capable of cleaving either DNA or RNA. The PAM sites recognized by Cas12a have the sequences 5’-YTN-3' (where "Y" is a pyrimidine and "N" is any nucleobase) or 5'-TTN-3', in contrast to the G-rich PAM site recognized by Cas9. Cas12a cleavage of DNA produces double-stranded breaks with a sticky-ends having a 4 or 5 nucleotide overhang. For a discussion of Cas12a, see, e.g., Ledford et al. (2015) Nature. 526 (7571 ):17-17, Zetsche et al. (2015) Cell. 163 (3):759-771 , Murovec et al. (2017) Plant Biotechnol. J. 15(8):917-926, Zhang et al. (2017) Front. Plant Sci. 8:177, Fernandes et al. (2016) Postepy Biochem. 62(3):315-326; herein incorporated by reference. [00151] C2c1 is another class II CRISPR/Cas system RNA-guided nuclease that may be used. C2c1 , similarly to Cas9, depends on both a crRNA and tracrRNA for guidance to target sites. For a description of C2c1 , see, e.g., Shmakov et al. (2015) Mol Cell. 60(3):385-397, Zhang et al. (2017) Front Plant Sci. 8:177; herein incorporated by reference.

[00152] In yet another embodiment, an engineered RNA-guided Fokl nuclease may be used. RNA-guided Fokl nucleases comprise fusions of inactive Cas9 (dCas9) and the Fokl endonuclease (Fokl-dCas9), wherein the dCas9 portion confers guide RNA- dependent targeting on Fokl. For a description of engineered RNA-guided Fokl nucleases, see, e.g., Havlicek et al. (2017) Mol. Ther. 25(2):342-355, Pan et al. (2016) Sci Rep. 6:35794, Tsai et al. (2014) Nat Biotechnol. 32(6):569-576; herein incorporated by reference.

[00153] In certain embodiments, the donor polynucleotide comprises a gene knockin module comprising a plurality of coding sequences encoding a plurality of polypeptides. In certain embodiments, the donor polynucleotide comprises a sequence encoding a knockout module comprising a plurality of knockout guide RNAs. In some embodiments, at least 2 knockout guide RNAs of the plurality bind to different genomic target sequences in different target genes to generate gene knockouts of more than one target gene. In some embodiments, at least 2 knockout guide RNAs of the plurality bind to different genomic target sequences in the same target gene to increase a rate of gene knockout of the target gene compared to the rate of gene knockout using only one knockout guide RNA.

[00154] In certain embodiments, the first mRNA transcript comprises: a protein coding reading frame encoding the exogenous polypeptide followed by a stop codon; and a 3'- untranslated region comprising: a knockout module flanked by a first spacer sequence and a second spacer sequence, wherein the knockout module comprises a plurality of knockout guide RNAs, wherein each guide RNA is preceded by a synthetic separator sequence followed by a direct repeat sequence; a mRNA stabilizing element, wherein the mRNA stabilizing element is positioned between the stop codon and the first spacer sequence; and a polyadenylation sequence. In some embodiments, the mRNA stabilizing element is a triplex stabilizer. In some embodiments, the transcription of the integrated donor polynucleotide is performed by an RNA polymerase II (Pol II). In some embodiments, the knockout guide RNAs are Cas12a guide RNAs. [00155] The RNA-guided nuclease can be provided in the form of a protein, such as the nuclease complexed with a guide RNA, or provided by a nucleic acid encoding the RNA- guided nuclease, such as an RNA (e.g., messenger RNA) or DNA (expression vector such as a plasmid or viral vector). Codon usage may be optimized to improve production of an RNA-guided nuclease in a particular cell or organism. For example, a nucleic acid encoding an RNA-guided nuclease can be modified to substitute codons having a higher frequency of usage in a human cell or a non-human mammalian cell, such as a nonhuman primate cell, a rodent cell, a mouse cell, a rat cell, or any other host cell of interest, as compared to the naturally occurring polynucleotide sequence. When a nucleic acid encoding the guide RNA and/or RNA-guided nuclease is introduced into cells, the guide RNA and/or RNA-guided nuclease can be transiently, conditionally, or constitutively expressed in the cell. Recombinant nucleic acids encoding the guide RNA, RNA-guided nuclease, and/or donor polynucleotide can be introduced into a cell using any suitable transfection technique such as, but not limited to electroporation, nucleofection, or lipofection. Alternatively, a ribonucleoprotein complex of the guide RNA and the RNA- guided nuclease may be introduced into a cell by microinjection into the cytoplasm or nucleus.

[00156] A CRISPR-Cas system can be introduced into cells with a viral vector that encodes a Cas nuclease (e.g., Cas9 or Cas12a) and a guide RNA (gRNA). Viral delivery of CRISPR components has been demonstrated using lentiviral, retroviral, adenovirus, and adeno-associated virus (AAV) vectors. For a description of methods of introducing a CRISPR system into cells with various viral vectors, see, e.g., Shalem et al. (2014) Science 343:84-87, Williams et al. (2016) Sci Rep. 6:2561 1 , Ran et al. (2015) Nature 520:186-191 , Swiech et al. (2015) Nat Biotechnol. 33:102-106; herein incorporated by reference.

[00157] Alternatively, a gRNA and a messenger RNA encoding the Cas nuclease can be introduced into cells, wherein the Cas nuclease is produced by translation of the mRNA in the cytoplasm. The gRNA and Cas nuclease then form a complex in the cytoplasm and enter the nucleus. RNA transfection of T cells can be performed using electroporation, cationic-lipid-mediated transfection, or using liposomes or lipid nanoparticles (LNPs) encapsulating the gRNA and mRNA. See, e.g., Billingsley et al. (2022) Nano Lett 22(1 ):533-542, Tchou et al. (2017) Cancer Immunol Res. 5(12):1 152- 1161 , Ye et al. (2022) ACS Biomater Sci Eng. 8(2):722-733, Guevara et al. (2020) Front. Chem. 8:589959; herein incorporated by reference. [00158] Donor polynucleotides and guide RNAs are readily synthesized by standard techniques, e.g., solid phase synthesis via phosphoramidite chemistry, as disclosed in U.S. Patent Nos. 4,458,066 and 4,415,732, incorporated herein by reference; Beaucage et al., Tetrahedron (1992) 48:2223-2311 ; and Applied Biosystems User Bulletin No. 13 (1 April 1987). Other chemical synthesis methods include, for example, the phosphotriester method described by Narang et al., Meth. Enzymol. (1979) 68:90 and the phosphodiester method disclosed by Brown et al., Meth. Enzymol. (1979) 68:109. In view of the short lengths of gRNAs (typically about 20 nucleotides in length) and donor polynucleotides (typically about 100-150 nucleotides), gRNA-donor polynucleotide cassettes can be produced by standard oligonucleotide synthesis techniques and subsequently ligated into vectors.

[00159] The method steps using an RNA-guided nuclease and a donor polynucleotide including a knockout guide RNA, as described herein, can be repeated to provide any desired number of DNA modifications. Accordingly, genetically modified cells may have multiple gene knockins and knockouts.

[00160] In some embodiments, the cell is genetically modified to produce a therapeutic cell that may be used in cellular therapy. The cell may be any suitable type of cell for transplanting to an individual in need. For example, the cell may be a stem cell, progenitor cell, or mature cell. The cell may be autologous, allogeneic, xenogeneic.

[00161] In some cases, the therapeutic cells include cells whose activity is conditional, e.g., cells that modulate their function based on the physiological state of the host and/or the environment of the host tissue. The therapeutic cell may be a type of cell that specifically possesses the functional activity by virtue of its cell type (e.g., by differentiating or having differentiated into a cell type that exhibits the functional activity) or may be genetically modified to exhibit the functional activity that was not exhibited by the cell before being genetically modified.

[00162] In some embodiments, the cell secretes a biological agent, e.g., a signaling molecule, a hormone, a growth factor, a cytokine, a chemokine, a neuropeptide, an enzyme, an antibody, etc. In some cases, the therapeutic cells include cells (e.g., immune cells, such as cytotoxic T lymphocytes) that interact with targets at or in the vicinity in the host tissue in which cells are transplanted.

[00163] Exemplary therapeutic molecules that can be secreted by a therapeutic cell include, without limitation, insulin, human growth hormone, thyroxine, glucagon-like peptide-1 (GLP-1 ), GLP-1 (7-37), GLP-1 (7-36), and like GLP-1 receptor agonist polypeptides, GLP-2, interleukins 1 to 33 (e.g., IL-1 , IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11 , IL-12, IL-13, IL-17, IL-18, IL-21 , IL-22, IL-27, IL-33), interferon (a, 0, y), GM-CSF, G-CSF, M-CSF, SCF, FAS ligands, TRAIL, leptin, adiponectin, blood coagulation factor Vlll/blood coagulation factor IX, von Willebrand factor, glucocerebrosidase, lipoprotein lipase (LPL), lecithin-cholesterol acyltransferase (LCAT), erythropoietin, apoA-l, albumin, atrial natriuretic peptide (ANP), luteinizing hormone releasing hormone (LHRH), angiostatin/endostatin, endogenous opioid peptides (enkephalins, endorphins, etc.), calcitonin/bone morphogenetic protein (BMP), pancreatic secretory trypsin inhibitors, catalase, superoxide dismutase, anti-TNF-a antibody, soluble IL-6 receptor, IL-1 receptor antagonist, a2 antitrypsin, etc.

[00164] In some cases, the cell is a stem cell or stem cell-derived cell. Stem cells of interest include, without limitation, hematopoietic stem cells, embryonic stem cells, adult stem cells, mesenchymal stem cells, neural stem cells, epidermal stem cells, endothelial stem cells, gastrointestinal stem cells, liver stem cells, cord blood stem cells, amniotic fluid stem cells, skeletal muscle stem cells, smooth muscle stem cells (e.g., cardiac smooth muscle stem cells), pancreatic stem cells, olfactory stem cells, induced pluripotent stem cells; and the like; as well as differentiated cells that can be cultured in vitro and used in a therapeutic regimen, where such cells include, but are not limited to, keratinocytes, adipocytes, cardiomyocytes, neurons, osteoblasts, pancreatic islet cells, retinal cells, and the like. The cell that is used will depend in part on the nature of the disorder or condition to be treated.

[00165] Suitable human embryonic stem (ES) cells include, but are not limited to, any of a variety of available human ES lines, e.g., BG01 (hESBGN-01 ), BG02 (hESBGN-02), BG03 (hESBGN-03) (BresaGen, Inc.; Athens, Ga.); SA01 (Sahlgrenska 1 ), SA02 (Sahlgrenska 2) (Cellartis AB; Goeteborg, Sweden); ES01 (HES-1 ), ES01 (HES-2), ES03 (HES-3), ES04 (HES-4), ES05 (HES-5), ES06 (HES-6) (ES Cell International; Singapore); UC01 (HSF-1 ), UC06 (HSF-6) (University of California, San Francisco; San Francisco, Calif.); WA01 (H1 ), WA07 (H7), WA09 (H9), WA09/Oct4D10 (H9-hOct4-pGZ), WA13 (H13), WA14 (H14) (Wisconsin Alumni Research Foundation; WARF; Madison, Wis.). Cell line designations are given as the National Institutes of Health (NIII) code, followed in parentheses by the provider code.

[00166] Hematopoietic stem cells (HSCs) are mesoderm-derived cells that can be isolated from bone marrow, blood, cord blood, fetal liver and yolk sac. HSCs are characterized as CD34⁺ and CD3-. HSCs can repopulate the erythroid, neutrophil-macrophage, megakaryocyte and lymphoid hematopoietic cell lineages in vivo. In vitro, HSCs can be induced to undergo at least some self- renewing cell divisions and can be induced to differentiate to the same lineages as is seen in vivo. As such, HSCs can be induced to differentiate into one or more of erythroid cells, megakaryocytes, neutrophils, macrophages, and lymphoid cells.

[00167] Neural stem cells (NSCs) are capable of differentiating into neurons, and glia (including oligodendrocytes, and astrocytes). A neural stem cell is a multipotent stem cell which is capable of multiple divisions, and under specific conditions can produce daughter cells which are neural stem cells, or neural progenitor cells that can be neuroblasts or glioblasts, e.g., cells committed to become one or more types of neurons and glial cells respectively. Methods of obtaining NSCs are known in the art.

[00168] Mesenchymal stem cells (MSCs) can be obtained from connective tissue including, without limitation, bone marrow, placenta, umbilical cord blood, adipose tissue, muscle, corneal stroma, and dental pulp of deciduous baby teeth. MSCs can differentiate to form muscle, bone, cartilage, fat, marrow stroma, and tendon. Methods of isolating MSCs are known in the art; and any known method can be used to obtain MSCs.

[00169] An induced pluripotent stem (iPS) cell is a pluripotent stem cell induced from a somatic cell, e.g., a differentiated somatic cell. iPS cells are capable of self-renewal and differentiation into cell fate-committed stem cells, including neural stem cells, as well as various types of mature cells. iPS cells can be generated from somatic cells, including skin fibroblasts, using, e.g., known methods. iPS cells can be generated from somatic cells (e.g., skin fibroblasts) by genetically modifying the somatic cells with one or more expression constructs encoding Oct-3/4 and Sox2. In some embodiments, somatic cells are genetically modified with one or more expression constructs comprising nucleotide sequences encoding Oct-3/4, Sox2, c-myc, and K1 f4. In some embodiments, somatic cells are genetically modified with one or more expression constructs comprising nucleotide sequences encoding Oct-4, Sox2, Nanog, and LIN28. Methods of generating iPS are known in the art, and any such method can be used to generate iPS.

[00170] In some cases, the therapeutic cells are lymphocytes, such as CD4+ and/or CD8+ T lymphocytes, or B lymphocytes. In some embodiments, the therapeutic cells are cytotoxic T lymphocytes.

[00171] In some embodiments, the therapeutic cells include insulin-secreting cells. The insulin-secreting cells may be any suitable type of insulin-secreting cell. In some cases, the insulin-secreting cells are a type of cell that secretes insulin (e.g., pancreatic islet cells, or p-like cells). In some cases, the insulin-secreting cells are primary islet cells (e.g., mature p islet cells isolated from a pancreas). In some cases, the insulin-secreting cells are p cells, or p-like cells that are derived in vitro from immature cells, precursor cells, progenitor cells, or stem cells. The insulin-secreting cells may be derived from (i.e. , obtained by differentiating) stem and/or progenitor cells such as hepatocytes (e.g., transdifferentiated hepatocytes), acinar cells, pancreatic duct cells, stem cells, embryonic stem cells (ES), partially differentiated stem cells, non-pluripotent stem cells, pluripotent stem cells, induced pluripotent stem cells (iPS cells), etc. Suitable insulin-secreting cells and methods of generating the same are described in, e.g., US20030082810; US20120141436; and Raikwar et al. (PLoS One. 2015 Jan 28;10(1 ):e01 16582), each of which are incorporated herein by reference.

[00172] The insulin-secreting cells may produce (e.g., secrete) insulin at a rate independent of the ambient/extracellular glucose concentration (e.g., the concentration of glucose in the host tissue in which the tissue graft is implanted), or may produce (e.g., secrete) insulin at a rate that depends on the ambient/extracellular glucose concentration. In some cases, the insulin-secreting cells constitutively secrete insulin. In some embodiments, the insulin-secreting cells increase insulin secretion when the ambient/extracellular glucose concentration increases, and decreases insulin secretion when the ambient/extracellular glucose concentration decreases.

[00173] In some embodiments, the gene knockin results in expression of an exogenous polypeptide in the cell. The exogenous polypeptide may be any type of protein/peptide of interest, including, without limitation, an enzyme, an extracellular matrix protein, a receptor, a transporter, an ion channel, or other membrane protein, a hormone, a neuropeptide, a growth factor, a cytokine, an antibody, or a cytoskeletal protein; or a fragment thereof, or a biologically active domain of interest.

[00174] In some embodiments, the exogenous protein is a chimeric antigen receptor (CAR) that binds specifically to a target antigen. For example, an immune cell such as a T cell or macrophage can be genetically modified to express a CAR that targets the immune cell to a pathogenic cell or particle in need of eradication. For example, lymphocytes, e.g., cytotoxic T cells, may be engineered to express a CAR that specifically binds to an antigen on a pathogenic cell that is associated with a disease, e.g., cancer or tumor, fibrosis, or antibiotic-resistant infection that is to be treated with the genetically modified cell. [00175] In some embodiments, the gene knockout at the second genomic target locus is used to reduce graft versus host disease and/or improve cell function and survival. In certain embodiments, the second genomic target locus encodes an alloantigen that is knocked out to reduce graft versus host disease. Exemplary alloantigens include, but are not limited to, major histocompatibility complex (MHC) class I alloantigens, MNS blood group alloantigens, CD1 , CD2, CD3, CD4, CD7, CD8, Ly-6, Qa-2, RT6, CD19, CD22, CD56, CD58 (LFA-3), CD59, and CDw90 (Thy 1 ), any one of which or any combination of which may be knocked out in the genetically modified cell using the methods described herein.

[00176] In some embodiments, the method further comprises barcoding the genetically modified cells. A "barcode" refers to one or more nucleotide sequences that are used to identify a nucleic acid or cell with which the barcode is associated. Barcodes can be 3- 1000 or more nucleotides in length, preferably 10-250 nucleotides in length, and more preferably 10-30 nucleotides in length, including any length within these ranges, such as 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, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides in length. Barcodes may be used, for example, to identify a single cell, subpopulation of cells, colony, or sample from which a nucleic acid originated. Barcodes may also be used to identify the position (i.e., positional barcode) of a cell, colony, or sample from which a nucleic acid originated, such as the position of a colony in a cellular array, the position of a well in a multi-well plate, or the position of a tube, flask, or other container in a rack. In particular, a barcode may be used to identify a genetically modified cell from which a nucleic acid originated. In some embodiments, a barcode is used to identify a cell from which a genetically modified cell originated. Alternatively, a unique barcode may be used to identify each guide-RNA and donor polynucleotide used in genome editing. Furthermore, multiple barcodes can be used in combination to identify different features of a nucleic acid or cell. For example, positional barcoding (e.g., to identify the position of a cell, colony, culture, or sample in an array, multi-well plate, or rack) can be combined with barcodes identifying a cell donor and/or barcodes identifying guide-RNAs, and donor polynucleotides used in genome editing.

[00177] In some embodiments, the cells are further engineered to express a fluorescent protein, for example, to allow cells derived from different donors to be distinguished in multiplexed screening. In some embodiments, cells from the same donor are engineered to express the same fluorescent protein, and cells from different donors are engineered to express different fluorescent proteins. Exemplary fluorescent proteins include, without limitation, green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), superfolder GFP, emerald, Azami Green, mWasabi, TagGFP, TurboGFP, red fluorescent protein, blue fluorescent protein (BFP), EBFP, EBFP2, mTagBFP, Azurite, cyan fluorescent protein (CFP), mECFP, Cerulean, mCerulean, mTurquoise, CyPet, AmCyanl , Midori-lshi Cyan, yellow fluorescent protein (YFP), EYFP, Topaz, Venus, YPet, mCitrine, mBanana, orange fluorescent protein (OFP), Kusabira Orange, Kusabira Orange2, mOrange, m0range2, dTomato, TagRFP, DsRed, DsRed2, mTangerine, red fluorescent protein (RFP), mRuby, mRuby2, mApple, mStrawberry, mCherry, mRaspberry, AsRed2, mRFP1 , JRed, dKeima-Tandem, Dronpa, mPlum, E2-Crimson, and aequorin.

[00178] In some instances, a population of cells may be enriched for those comprising a genetic modification by separating the genetically modified cells from the remaining population. Separation of genetically modified cells typically relies upon the expression of a selectable marker co-integrated with the intended edit at the target locus. After integration of a donor polynucleotide by HDR, positive selection is performed to isolate cells from a population, e.g. to create an enriched population of cells comprising the genetic modification.

[00179] Cell separation may be accomplished by any convenient separation technique appropriate for the selection marker used, including, but not limited to flow cytometry, fluorescence activated cell sorting (FACS), magnetic-activated cell sorting (MACS), elutriation, immunopurification, and affinity chromatography. For example, if a fluorescent marker is used, cells may be separated by fluorescence activated cell sorting (FACS), whereas if a cell surface marker is used, cells may be separated from the heterogeneous population by affinity separation techniques, e.g., MACS, affinity chromatography, "panning" with an affinity reagent attached to a solid matrix, immunopurification with an antibody specific for the cell surface marker, or other convenient technique.

[00180] In certain embodiments, positive and/or negative selection of genetically modified cells is performed using a binding agent that specifically binds to a selection marker on a cell (e.g., such as produced from integration of a donor polynucleotide at a target genomic locus). Examples of binding agents include, without limitation, antibodies, antibody fragments, antibody mimetics, aptamers, and ligands. In some embodiments, the binding agent binds to the selection marker with high affinity. [00181] The binding agent may be immobilized on a solid support to facilitate removal of cells having a selection marker from a liquid sample. The binding agent may be associated with the solid support either directly or indirectly. Binding agents may be immobilized on the surface of a solid support, such as, but not limited to, a non-magnetic bead, magnetic bead, rod, particle, plate, slide, wafer, strand, disc, membrane, film, or the inner surface of a tube, channel, column, flow cell device, or microfluidic device. A solid support may comprise various materials, including, but not limited to glass, quartz, silicon, metal, ceramic, plastic, nylon, polyacrylamide, agarose, resin, porous polymer monoliths, hydrogels, and composites thereof. Additionally, a substrate may be added to the surface of a solid support to facilitate attachment of a binding agent.

[00182] In positive selection, cells carrying a selection marker are collected, whereas in negative selection, cells carrying a selection marker are removed from a cell population. For example, in positive selection, a binding agent specific for a surface marker can be immobilized on a solid support (e.g., column or magnetic bead) and used to collect cells of interest on the solid support. Cells that are not of interest do not bind to the solid support (e.g., flow through the column or do not attach to the magnetic beads). In negative selection, the binding agent is used to deplete a cell population of cells that are not of interest. The cells of interest are those that do not bind to the binding agent (e.g., flow through the column or remain after the magnetic beads are removed).

[00183] In certain embodiments, the binding agent comprises an antibody that specifically binds to the selection marker on a cell. Any type of antibody may be used, including polyclonal and monoclonal antibodies, hybrid antibodies, altered antibodies, chimeric antibodies and, humanized antibodies, as well as: hybrid (chimeric) antibody molecules (see, for example, Winter et al. (1991 ) Nature 349:293-299; and U.S. Pat. No. 4,816,567); F(ab')2 and F(ab) fragments; F_v molecules (noncovalent heterodimers, see, for example, Inbar et al. (1972) Proc Natl Acad Sci USA 69:2659-2662; and Ehrlich et al. (1980) Biochem 19:4091 -4096); single-chain Fv molecules (sFv) (see, e.g., Huston et al. (1988) Proc Natl Acad Sci USA 85:5879-5883); nanobodies or single-domain antibodies (sdAb) (see, e.g., Wang et al. (2016) Int J Nanomedicine 11 :3287-3303, Vincke et al. (2012) Methods Mol Biol 91 1 :15-26; dimeric and trimeric antibody fragment constructs; minibodies (see, e.g., Pack et al. (1992) Biochem 31 :1579-1584; Cumber et al. (1992) J Immunology 149B:120-126); humanized antibody molecules (see, e.g., Riechmann et al. (1988) Nature 332:323-327; Verhoeyan et al. (1988) Science 239:1534-1536; and U.K. Patent Publication No. GB 2,276,169, published 21 Sep. 1994); and, any functional fragments obtained from such molecules, wherein such fragments retain specific-binding properties of the parent antibody molecule (i.e., specifically binds to a selection marker on a cell).

[00184] In other embodiments, the binding agent comprises an aptamer that specifically binds to the selection marker on a cell. Any type of aptamer may be used, including a DNA, RNA, xeno-nucleic acid (XNA), or peptide aptamer that specifically binds to the target antibody isotype. Such aptamers can be identified, for example, by screening a combinatorial library. Nucleic acid aptamers (e.g., DNA or RNA aptamers) that bind selectively to a target antibody isotype can be produced by carrying out repeated rounds of in vitro selection or systematic evolution of ligands by exponential enrichment (SELEX). Peptide aptamers that bind to a selection marker on a cell may be isolated from a combinatorial library and improved by directed mutation or repeated rounds of mutagenesis and selection. For a description of methods of producing aptamers, see, e.g., Aptamers: Tools for Nanotherapy and Molecular Imaging (R.N. Veedu ed., Pan Stanford, 2016), Nucleic Acid and Peptide Aptamers: Methods and Protocols (Methods in Molecular Biology, G. Mayer ed., Humana Press, 2009), Nucleic Acid Aptamers: Selection, Characterization, and Application (Methods in Molecular Biology, G. Mayer ed., Humana Press, 2016), Aptamers Selected by Cell-SELEX for Theranostics (W. Tan, X. Fang eds., Springer, 2015), Cox et al. (2001 ) Bioorg. Med. Chem. 9(10):2525-2531 ; Cox et al. (2002) Nucleic Acids Res. 30(20): e108, Kenan et al. (1999) Methods Mol Biol. 118:217-231 ; Platelia et al. (2016) Biochim. Biophys. Acta Nov 16 pii: S0304- 4165(16)30447-0, and Lyu et al. (2016) Theranostics 6(9):1440-1452; herein incorporated by reference in their entireties.

[00185] In yet other embodiments, the binding agent comprises an antibody mimetic. Any type of antibody mimetic may be used, including, but not limited to, affibody molecules (Nygren (2008) FEBS J. 275 (1 1 ):2668-2676), affilins (Ebersbach et al. (2007) J. Mol. Biol. 372 (1 ):172-185), affimers (Johnson et al. (2012) Anal. Chem. 84 (15):6553-6560), affitins (Krehenbrink et al. (2008) J. Mol. Biol. 383 (5):1058-1068), alphabodies (Desmet et al. (2014) Nature Communications 5:5237), anticalins (Skerra (2008) FEBS J. 275 (1 1 ):2677-2683), avimers (Silverman et al. (2005) Nat. Biotechnol. 23 (12):1556-1561 ), darpins (Stumpp et al. (2008) Drug Discov. Today 13 (15-16):695-701 ), fynomers (Grabulovski et al. (2007) J. Biol. Chem. 282 (5):3196-3204), and monobodies (Koide et al. (2007) Methods Mol. Biol. 352:95-109). [00186] Dead cells may be selected against by employing dyes that preferentially stain dead cells (e.g. propidium iodide). Any technique may be employed which is not unduly detrimental to the viability of the genetically modified cells.

[00187] Compositions that are highly enriched for cells having a desired genetic modification can be produced in this manner. By "highly enriched" is meant that the genetically modified cells are 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more, or 98% or more of the cell composition. In other words, the composition may be a substantially pure composition of genetically modified cells.

[00188] Genetically modified cells produced by the methods described herein may be used immediately. Alternatively, the cells may be frozen at liquid nitrogen temperatures and stored for long periods of time before being thawed and used. In such cases, cells may be frozen in 10% DMSO, 50% serum, 40% buffered medium, or some other such solution as is commonly used in the art to preserve cells at such freezing temperatures, and thawed in a manner as commonly known in the art for thawing frozen cultured cells.

Methods of Transplanting Genetically Modified Cells into an Individual

[00189] Also provided herein are methods of transplanting cells that are genetically modified, as described herein, into an individual, for example, to treat a disease. In some embodiments, the cells are encapsulated in a biocompatible carrier, matrix, or scaffold. Suitable matrices include a polymeric mesh or sponge or a polymeric hydrogel.

[00190] A hydrogel is defined as a substance formed when an organic polymer (natural or synthetic) is cross-linked via covalent, ionic, or hydrogen bonds to create a three- dimensional open-lattice structure, which entraps water molecules to form a gel. In general, these polymers are at least partially soluble in aqueous solutions, such as water, buffered salt solutions, or aqueous alcohol solutions that have charged side groups, or a monovalent ionic salt thereof. Any suitable hydrogel polymers can be used to form a hydrogel. Exemplary hydrogel polymers include, without limitation, natural polymers such as polysaccharides, including hyaluronic acid, chitosan, heparin, alginate, cellulose, dextran, and agarose, and proteins, including fibrin, fibrinogen, collagen, elastin, gelatin, silk, laminin, fibronectin, albumin, thrombin, and keratin; modified natural polymers, including hydroxymethylcellulose, hydroxyethylcellulose, gelatin methacrylate, polyanionic N-carboxymethyl chitosan, and polycationic N-trimethyl chitosan; and synthetic polymers, including polyvinyl alcohol, N-vinylpyrrolidone, polyethylene glycol, polyethylene glycol) diacrylate, polyacrylamide, poly(N-isopropylacrylamide), sodium polyacrylate, acrylate polymers and copolymers such as hydroxyethyl methacrylate, ethyl methacrylate, propylene glycol methacrylate, ethylene glycol di-methyl acrylate, methyl methacrylate, glycidyl methacrylate, and glycol methacrylate, poly(N- isopropylacrylamide-co-acrylic acid), polyesters, polyurethanes, nylon, synthetic polyamino acids, prolamines; and combinations thereof, and other such molecules, including recombinant versions of such polymers. In some embodiments, the hydrogel comprises polyacrylamide.

[00191] The method may include implanting (e.g., surgically implanting) a matrix or scaffold containing the therapeutic cells at an implantation site of a host individual. The host individual may be suffering from a condition, e.g., a disease, that may be treated by providing the therapeutic cells to the individual. The therapeutic cells may be any suitable therapeutic cells, as described above, and the type of therapeutic cells may depend on the disease to be treated.

[00192] The implantation site may be any suitable location (e.g., surgically accessible location) in the individual. In some cases, the implantation site is in a kidney, liver, omentum, peritoneum, abdomen, or submuscular or subcutaneous tissue. In some cases, the implantation site is at or in the vicinity of a tissue that is affected by the disease (e.g., a tissue with a solid tumor, fibrotic tissue, infected tissue).

[00193] A medical practitioner may locate the site for transplantation of the therapeutic cells, for example, by medical imaging (e.g., ultrasound, radiography, or MRI). In some embodiments, a contrast agent is included in the composition comprising the therapeutic cells to allow confirmation of the location of the cells by medical imaging after transplantation. In some embodiments, the contrast agent is a microbubble (e.g., for use in ultrasound) or a radiopaque contrast agent (e.g., for use in radiography). The contrast agent may be contained in the same composition as the therapeutic cells or in a different composition and used prior to or after transplantation.

CAR-T Cells

[00194] A T cell can be genetically modified, as described herein, to express a chimeric antigen receptor (CAR) that specifically binds to a target antigen (i.e., a CAR-T cell). The CAR localizes the T cell to sites where target cells are present that express the target antigen. Binding of a CAR-T cell to a target antigen on the surface of a cell activates the T cell resulting in secretion of cytokines, which regulate other immune cells, and killing of target cells. For example, CAR-T cells may be engineered to target an antigen that is expressed on the surface of tumors but not on healthy cells to selectively kill tumor cells. In another example, CAR-T cells may also be engineered to target an antigen that is expressed on the surface of activated fibroblasts or fibrotic tissue, which may be used to selectively eliminate fibrotic tissue. In another example, CAR-T cells may also be engineered to target an antigen that is expressed on the surface of a pathogen (e.g., bacterium, virus, fungus, or parasite) to eradicate a pathogen. In a further example, CAR- T cells may be engineered to target an antigen that is expressed on the surface of an autoreactive immune cell (e.g., autoreactive T cell or B cell) to eliminate autoreactive immune cells. Thus, CAR-T cells may be used for the treatment of various diseases, including cancer, fibrosis, infections such as bacterial infections (e.g., multidrug resistant bacteria), viral infections, fungal infections, and parasitic infections, and autoimmune diseases.

[00195] The T cell, from which the CAR-T cell is derived, may be autologous or allogeneic. In some embodiments, the CAR-T cell is an effector T cell (e.g., a helper CD4⁺ T cell, a cytotoxic CD8⁺ T cell, a natural killer T cell, or a gamma delta T cell) or a regulatory T cell (Treg) that has been genetically modified to express a CAR.

[00196] A CAR may have any suitable architecture, known in the art, wherein the CAR comprises an antigen binding domain linked to T cell receptor effector functions. The term “CAR” refers to an artificial multi-module molecule capable of triggering or inhibiting the activation of an immune cell. A CAR will generally comprise an antigen binding domain, linker, transmembrane domain and cytoplasmic signaling domain. In some instances, a CAR includes one or more co-stimulatory domains and/or one or more co- inhibitory domains.

[00197] The antigen-binding domain of a CAR may include any naturally occurring, synthetic, semi-synthetic, or recombinantly produced binding partner for a target antigen of interest. In some embodiments, the binding region is an antigen-binding region, such as an antibody or functional binding domain or antigen-binding fragment thereof. The antigen-binding region of the CAR can include any domain that binds to the antigen and may include, but is not limited to, a monoclonal antibody, a polyclonal antibody, a synthetic antibody, a human antibody, a humanized antibody, a non-human antibody, a single-chain antibody, and any antigen-binding fragment thereof. Thus, in some embodiments, the antigen binding domain portion includes a mammalian antibody or an antigen-binding fragment thereof. An antigen-binding domain may comprise an antigenbinding fragment (Fab), a single-chain variable fragment (scFv), a nanobody, a VH domain, a VL domain, a single domain antibody (sdAb), a shark variable domain of a new antigen receptor (VNAR), a single variable domain on a heavy chain (VHH), a bispecific antibody, or a diabody; or a functional antigen-binding fragment thereof. In some embodiments, the antigen-binding domain is derived from the same cell type or the same species in which the CAR will ultimately be used. For example, for use in humans, the antigen-binding domain of the CAR may include a human antibody, a humanized antibody, or an antigen-binding fragment thereof.

[00198] In some embodiments, the antigen binding domain is derived from a single chain antibody that selectively binds to a target antigen. In some embodiments, the antigen binding domain is provided by a single chain variable fragment (scFv). A scFv is a recombinant molecule in which the variable regions of the light and heavy immunoglobulin chains are connected in a single fusion polypeptide. Generally, the VH and VL sequences are joined by a linker sequence. See, for example, Ahmad (2012) Clinical and Developmental Immunology Article ID 980250, herein specifically incorporated by reference. In principle, there are no particular limitations to the length and/or amino acid composition of the linker peptide joining the VH and VL sequences. In some embodiments, any arbitrary single-chain peptide including about 1 to 100 amino acid residues (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, etc. amino acid residues) can be used as a peptide linker. In some embodiments, the linker peptide sequence includes about 5 to 50, about 10 to 60, about 20 to 70, about 30 to 80, about 40 to 90, about 50 to 100, about 60 to 80, about 70 to 100, about 30 to 60, about 20 to 80, about 30 to 90 amino acid residues. In some embodiments, the linker peptide sequence includes about 1 to 10, about 5 to 15, about 10 to 20, about 15 to 25, about 20 to 40, about 30 to 50, about 40 to 60, about 50 to 70 amino acid residues. In some embodiments, the linker peptide sequence includes about 40 to 70, about 50 to 80, about 60 to 80, about 70 to 90, or about 80 to 100 amino acid residues. In some embodiments, the linker peptide sequence includes about 1 to 10, about 5 to 15, about 10 to 20, about 15 to 25 amino acid residues.

[00199] The transmembrane domain may be derived either from a natural or a synthetic source. Where the source is natural, the domain may be derived from any membranebound or transmembrane protein. In some embodiments, the transmembrane domain comprises at least the stalk and/or transmembrane region(s) of CD8, Megf10, FcRy, Bail , MerTK, TIM4, Stabilin-1 , Stabilin-2, RAGE, CD300f, integrin subunit av, Integrin subunit [35, CD36, LRP1 , SCARF1 , Axl, CD45, and/or CD86. In some embodiments, the CAR transmembrane domain is derived from a type I membrane protein, such as, but not limited to, CD3 , 0D4, CD8, or CD28. In other embodiments, the transmembrane domain is synthetic, in which case it will include predominantly hydrophobic residues such as leucine, isoleucine, valine, phenylalanine, tryptophan, and alanine. In some embodiments, a triplet of phenylalanine, tryptophan and valine will be inserted at each end of a synthetic transmembrane domain.

[00200] In some embodiments, the CAR further comprises one or more linkers/spacers. For example, an extracellular spacer region may link the antigen binding domain to the transmembrane domain and/or an intracellular spacer region may link an intracellular signaling domain to the transmembrane domain. A spacer (linker) region linking the antigen binding domain to the transmembrane domain should be flexible enough to allow the antigen binding domain to orient in different directions to facilitate antigen recognition. [00201] Various types of linkers may be used in the CARs described herein. In some embodiments, the linker includes a peptide linker/spacer sequence. In some embodiments, the spacer comprises the hinge region from an immunoglobulin, e.g., the hinge from any one of lgG1 , lgG2a, lgG2b, lgG3, lgG4, particularly the human protein sequences. Alternatives include the CH2CH3 region of immunoglobulin and portions of CD3. For many scFv based constructs, an IgG hinge is effective.

[00202] In principle, there are no particular limitations to the length and/or amino acid composition of a linker peptide sequence. In some embodiments, a linker peptide sequence comprises about 1 to 100 amino acid residues, including any number of residues within this range such as 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues. In some embodiments, the linker peptide sequence includes about 5 to 50, about 10 to 60, about 20 to 70, about 30 to 80, about 40 to 90, about 50 to 100, about 60 to 80, about 70 to 100, about 30 to 60, about 20 to 80, about 30 to 90 amino acid residues. In some embodiments, the linker peptide sequence includes about 1 to 10, about 5 to 15, about 10 to 20, about 15 to 25, about 20 to 40, about 30 to 50, about 40 to 60, about 50 to 70 amino acid residues. In some embodiments, the linker peptide sequence includes about 40 to 70, about 50 to 80, about 60 to 80, about 70 to 90, or about 80 to 100 amino acid residues. In some embodiments, the linker peptide sequence includes about 1 to 10, about 5 to 15, about 10 to 20, about 15 to 25 amino acid residues. In some embodiments, the linker peptide sequence may include up to 300 amino acids, preferably 10 to 100 amino acids and most preferably 25 to 50 amino acids. In some embodiments, a short oligo- or polypeptide linker, preferably between 2 and 10 amino acids in length may form the linkage between the transmembrane domain and the intracellular engulfment signaling domain or extracellular antigen binding domain of the CAR. In some embodiments the linker comprises the amino acid sequence (G4S)_n where n is 1 , 2, 3, 4, 5, etc., and in some embodiments, n is 3.

[00203] A cytoplasmic signaling domain, such as those derived from the T cell receptor □- chain, is employed as part of the CAR in order to produce stimulatory signals for T lymphocyte proliferation and effector function following engagement of the chimeric receptor with the target antigen. Endodomains from co-stimulatory molecules may be included in the cytoplasmic signaling portion of the CAR.

[00204] The term “co-stimulatory domain”, refers to a stimulatory domain, typically an endodomain, of a CAR that provides a secondary non-specific activation mechanism through which a primary specific stimulation is propagated. Examples of co-stimulation include antigen nonspecific T cell co-stimulation following antigen specific signaling through the T cell receptor and antigen nonspecific B cell co-stimulation following signaling through the B cell receptor. Co-stimulation, e.g., T cell co-stimulation, and the factors involved have been described in Chen & Flies, Nat Rev Immunol (2013) 13(4):227-42, the disclosure of which is incorporated herein by reference in its entirety. Non-limiting examples of suitable co-stimulatory polypeptides include, but are not limited to, 4-1 BB (CD137), CD28, ICOS, OX-40, BTLA, CD27, CD30, GITR, and HVEM.

[00205] The term “co-inhibitory domain” refers to an inhibitory domain, typically an endodomain, derived from a receptor that provides secondary inhibition of primary antigen-specific activation mechanisms which prevents co-stimulation. Co-inhibition, e.g., T cell co-inhibition, and the factors involved have been described in Chen & Flies. Nat Rev Immunol (2013) 13(4):227-42 and Thaventhiran et al. J Clin Cell Immunol (2012) S12. In some embodiments, co-inhibitory domains homodimerize. A co-inhibitory domain can be an intracellular portion of a transmembrane protein. Non-limiting examples of suitable co-inhibitory polypeptides include, but are not limited to, CTLA-4 and PD-1 .

[00206] A first-generation CAR transmits the signal from antigen binding through only a single signaling domain, for example a signaling domain derived from the high-affinity receptor for IgE FccRlD D or the CD3£ chain. The domain contains one or three immunoreceptor tyrosine-based activating motif(s) [ITAM(s)] for antigen-dependent T- cell activation. The ITAM-based activating signal endows T-cells with the ability to lyse the target tumor cells and secret cytokines in response to antigen binding. [00207] Second-generation CARs include a co-stimulatory signal in addition to the CD3D signal. Coincidental delivery of the delivered co-stimulatory signal enhances cytokine secretion and antitumor activity induced by CAR-transduced T-cells. The co-stimulatory domain will usually be membrane proximal relative to the CD3D domain. Third- generation CARs include a tripartite signaling domain, comprising for example a CD28, CD3 , 0X40 or 4-1 BB signaling region. In fourth generation, or “armored car” CAR-T cells, CAR-T cells are further genetically modified to express or block molecules and/or receptors to enhance immune activity.

[00208] CAR variants include split CARs wherein the extracellular portion, the ABD and the cytoplasmic signaling domain of a CAR are present on two separate molecules. CAR variants also include ON-switch CARs which are conditionally activatable CARs, e.g., comprising a split CAR wherein conditional hetero-dimerization of the two portions of the split CAR is pharmacologically controlled. CAR molecules and derivatives thereof (i.e., CAR variants) are described, e.g., in PCT Application Nos. US2014/016527, US1996/017060, US2013/063083; Fedorov et al. Sci Trans! Med (2013) 5(215):215ra172; Glienke et al. Front Pharmacol (2015) 6:21 ; Kakarla & Gottschalk 52 Cancer J (2014) 20(2):151 -5; Riddell et al. Cancer J (2014) 20(2):141 -4; Pegram et al. Cancer J (2014) 20(2):127-33; Cheadle et al. Immunol Rev (2014) 257(1 ) :91 -106; Barrett et al. Annu Rev Med (2014) 65:333-47; Sadelain et al. Cancer Discov (2013) 3(4):388- 98; Cartellieri et al., J Biomed Biotechnol (2010) 956304; herein incorporated by reference in their entireties.

[00209] CAR variants also include bispecific or tandem CARs, which include a secondary CAR binding domain that can either amplify or inhibit the activity of a primary CAR. CAR variants also include inhibitory chimeric antigen receptors (iCARs) which may, e.g., be used as a component of a bispecific CAR system, where binding of a secondary CAR binding domain results in inhibition of primary CAR activation. Tandem CARs (TanCAR) mediate bispecific activation of T cells through the engagement of two chimeric receptors designed to deliver stimulatory or costimulatory signals in response to an independent engagement of two different tumor associated antigens. iCARs use the dual antigen targeting to shout down the activation of an active CAR through the engagement of a second suppressive receptor equipped with inhibitory signaling domains.

[00210] The dual recognition of different epitopes by two CARs diversely designed to either deliver killing through -chain or costimulatory signals, e.g., through CD28 allows a more selective activation of the reprogrammed T cells by restricting Tandem CAR’s activity to cancer cell expressing simultaneously two antigens rather than one. The potency of delivered signals in engineered T cells will remain below threshold of activation and thus ineffective in absence of the engagement of costimulatory receptor. The combinatorial antigen recognition enhances selective tumor eradication and protects normal tissues expressing only one antigen from unwanted reactions.

[00211] Inhibitory CARs (iCARs) are designed to regulate CAR-T cell activity through inhibitory receptor signaling module activation. This approach combines the activity of two CARs, one of which generates dominant negative signals limiting the responses of CAR-T cells activated by the activating receptor. iCARs can switch off the response of the counteracting activator CAR when bound to a specific antigen expressed only by normal tissues. In this way, iCARs-T cells can distinguish cancer cells from healthy ones, and reversibly block functionalities of transduced T cells in an antigen-selective fashion. CTLA-4 or PD-1 intracellular domains in iCARs trigger inhibitory signals on T lymphocytes, leading to less cytokine production, less efficient target cell lysis, and altered lymphocyte motility.

[00212] An ABD can be provided as a “chimeric bispecific binding member”, i.e., a chimeric polypeptide having dual specificity to two different binding partners (e.g., two different antigens). Non-limiting examples of chimeric bispecific binding members include bispecific antibodies, bispecific conjugated monoclonal antibodies (mab)2, bispecific antibody fragments (e.g., F(ab)2, bispecific scFv, bispecific diabodies, single chain bispecific diabodies, etc.), bispecific T cell engagers (BiTE) , bispecific conjugated single domain antibodies, micabodies and mutants thereof, and the like. Non-limiting examples of chimeric bispecific binding members also include those chimeric bispecific agents described in Kontermann. MAbs. (2012) 4(2): 182-197; Stamova et al. Antibodies 2012, 1 (2), 172-198; Farhadfar et al. Leak Res. (2016) 49:13-21 ; Benjamin et al. Ther Adv Hematol. (2016) 7(3):142-56; Kiefer et al. Immunol Rev. (2016) 270(1 ):178-92; Fan et al. J Hematol Oncol. (2015) 8:130; May et al. Am J Health Syst Pharm. (2016) 73(1 ):e6-e13; the disclosures of which are incorporated herein by reference in their entirety.

[00213] In some instances, a chimeric bispecific binding member may be a bispecific T cell engager (BiTE). A BiTE is generally made by fusing a specific binding member (e.g., a scFv) that binds an antigen to a specific binding member (e.g., a scFv) with a second binding domain specific for a T cell molecule such as CD3.

[00214] In some instances, a chimeric bispecific binding member may be a CAR-T cell adapter. As used herein, by “CAR-T cell adapter” is meant an expressed bispecific polypeptide that binds the antigen recognition domain of a CAR and redirects the CAR to a second antigen. Generally, a CAR-T cell adapter will have two binding regions, one specific for an epitope on the CAR to which it is directed and a second epitope directed to a binding partner which, when bound, transduces the binding signal activating the CAR. Useful CAR-T cell adapters include but are not limited to e.g., those described in Kim et al. J Am Chem Soc. (2015) 137(8):2832-5; Ma et al. Proc Natl Acad Sci U S A. (2016) 1 13(4):E450-8 and Cao et al. Angew Chem Int Ed Engl. (2016) 55(26) :7520-4; the disclosures of which are incorporated herein by reference in their entirety.

[00215] Effector CAR-T cells include autologous or allogeneic immune cells having cytolytic activity against a target cell. In some embodiments, a patient's own T cells or T cells from a donor are engineered to express a CAR. In some embodiments, the CAR-T cells are engineered from a complex mixture of immune cells, e.g., tumor infiltrating lymphocytes (TILs) isolated from an individual in need of treatment. See, e.g., Yang and Rosenberg (2016) Adv Immunol. 130:279-94, “Adoptive T Cell Therapy for Cancer; Feldman et al (2015) Semin Oncol. 42(4):626-39 “Adoptive Cell Therapy-Tumor- Infiltrating Lymphocytes, T-Cell Receptors, and Chimeric Antigen Receptors”; Clinical Trial NCT01 174121 , “Immunotherapy Using Tumor Infiltrating Lymphocytes for Patients With Metastatic Cancer”; Tran et al. (2014) Science 344(6184)641 -645, “Cancer immunotherapy based on mutation-specific CD4+ T cells in a patient with epithelial cancer”. In other embodiments, stem cells, differentiated into T cells, are engineered to express a CAR. In some embodiments, induced pluripotent stem cell (iPSC)-derived T cells are engineered to express a CAR. See, e.g., Zhou et al. (2022) Cancers (Basel) 14(9):2266, Nezhad et al. (2021 ) Pharm Res 38(6):931 -945; herein incorporated by reference in their entireties.

[00216] A biological sample comprising T cells, from which CAR-T cells are generated, may be collected from a subject or a donor. The biological sample may include, without limitation, blood, lymphoid tissue (e.g., bone marrow, spleen, tonsils, lymph nodes), mucosal tissue (e.g., lungs, small intestine, and large intestine), skin, or a tissue where T cells have infiltrated. The T cells may be separated from a mixture of cells prior to engineering the T cells to generate CAR-T cells. Alternatively, T cells may be engineered and cultured without separation from other cells.

[00217] T cells may be separated from other cells using any suitable cell separation technique such as, but not limited to, centrifugation-based cell separation, positive or negative selection against surface markers on cells (e.g., with antibody-coated beads), affinity chromatography, panning and immunopanning techniques, fluorescence activated cell sorting (FACS), or magnetic-activated cell sorting (MACS). Affinity reagents may be employed comprising specific receptors or ligands specific for cell surface molecules. The T cells may be separated from dead cells by employing viability dyes (e.g., propidium iodide). Any technique may be employed which is not unduly detrimental to the viability of the T cells.

[00218] The cells may be collected in any appropriate medium that maintains the viability of the cells. Various media are commercially available and may be used according to the nature of the cells, including dMEM, HBSS, dPBS, RPMI, Iscove’s medium, etc., which may be supplemented with fetal calf serum (FCS). The collected cells may be used immediately or frozen (e.g., at liquid nitrogen temperatures) prior to use.

[00219] In some embodiments, CAR-T cells are expanded in culture prior to screening, as described further below, or use in therapy. The CAR-T cells require activation for expansion in vitro or ex vivo, which can be accomplished by co-incubating T cells with natural antigen-presenting cells (e.g., dendritic cells) or artificial antigen-presenting cells or particles that present antigen and/or activating signals to the CAR-T cells. See, e.g., Rhodes et al. (2018) Mol Immunol. 98:13-18, Couture et al. (2019) Front Immunol. 10:1081 , Turtle (2010) Cancer J. 16(4):374-81 , Wang et al. (2017) Theranostics 7(14):3504-3516, Est-Witte et al. (2021 ) Semin Immunol. 56:101541 , Perica et al. (2014) Nanomedicine. 10 (1 ): 1 19-129, Latouche et al. (2000) Nature Biotechnology. 18 (4): 405-409; herein incorporated by reference.

[00220] In some embodiments, the gene knockout at the second genomic target locus is used to reduce graft versus host disease, T-cell exhaustion, and cytokine-related toxicities, and improve T-cell effector function and persistence.

[00221] In certain embodiments, the second genomic target locus encodes a cytokine that is knocked out to reduce cytokine-related toxicity resulting from CAR-T cell therapy. In some embodiments, the cytokine is GM-CSF or IL-6.

[00222] In certain embodiments, the second genomic target locus encodes an alloantigen that is knocked out to reduce graft versus host disease. In some embodiments, the alloantigen is a major histocompatibility complex (MHC) class I alloantigen or an MNS blood group alloantigen. In some embodiments, the alloantigen is CD1 , CD2, CD3, CD4, CD7, CD8, Ly-6, Qa-2, RT6, CD19, CD22, CD56, CD58 (LFA-3), CD59, or CDw90 (Thy 1 ) that is knocked out. [00223] In certain embodiments, the second genomic target locus comprises a CD5, CD52, CD70, BATF, LCK, PD-1 , LAG-3, CTLA-4, 2-B2M, PD-1 , HLA-I, Fas, TGFBR2, PDCD-1 , DGK, EZH2, PAX5, or LDLR gene that is knocked out.

[00224] In certain embodiments, the second genomic target locus is a T cell receptor alpha locus, T cell receptor beta locus, T cell receptor delta locus, or a T cell receptor gamma locus, wherein the endogenous T cell receptor (TCR) of the T cell is knocked out. TCR knockout reduces graft versus host disease and allows T cells from different donors to be pooled for multiplexed screening, as discussed in more detail in co-owned Provisional Patent Application entitled "Massively Parallel Mixed Lymphocyte Reactions," filed even date herewith, the disclosure of which is hereby incorporated by reference herein in its entirety. In addition, TCR knockout in the CAR-T cells provides the advantage that genetically modified cells can be enriched by negative selection methods. A population of T cells is produced in which the successfully edited T cells do not have an endogenous TCR, whereas the unsuccessfully edited T cells have the endogenous TCR. This allows the T cell population to be enriched for the successfully edited T cells expressing the CAR using negative selection with a binding agent (e.g., antibody, antibody mimetic, aptamer, or ligand) that specifically binds to the endogenous TCR to remove the unsuccessfully edited TCR positive T cells from the T cell population, leaving only the successfully edited TCR negative T cells behind for further research or clinical use without having to bind any reagents to the successfully edited T cells expressing the CAR.

Multiplexed Screening

[00225] CAR-T cells from multiple donors, wherein the CAR-T cells have knockouts of their endogenous TCRs, as described herein, can be pooled and tested simultaneously in multiplexed assays. See, e.g., co-owned Provisional Patent Application, entitled "Massively Parallel Mixed Lymphocyte Reactions,” filed even date herewith, the disclosure of which is hereby incorporated by reference herein in its entirety. Activation of CAR-T cells can be determined by measuring cell proliferation, expression of activation markers (e.g., detection of CD69, HLA-DR, IL2RA, and/or CD25), and production of effector cytokines (e.g., IFN-g, TNF-a, TNF-b, IL-1 , IL-2, IL-3, IL-4, IL-5, IL-9, IL-10, IL- 12, IL-13, and IL-25). Multiplexed screening of CAR-T cell cytotoxic activity can be performed in vitro to validate activity against target cells before further testing individual CAR-T cell in vivo in animal models and human clinical trials. [00226] Cytotoxicity of GD8+ CAR-T cells involves exocytosis of granules containing the pore-forming toxin, perforin, proapoptotic serine proteases, and granzymes that lyse target cells. Cytotoxicity of CD4+ CAR-T cells involves secretion of cytokines and apoptotic factors such as TNF-a, INF-g, and TRAIL that induce apoptosis of target cells or activate macrophages to engulf tumor cells. Perforin, proapoptotic serine proteases, granzymes, cytokines, and apoptotic factors can be measured, for example, using a multiplexed enzyme-linked immunosorbent assay (ELISA). Cytolysis can be assayed in vitro based on the release of compounds containing radioactive isotopes such as ⁵¹ Cr from radiolabeled target cells. Alternatively a membrane-permeable live-cell labeling dye such as calcein acetoxymethyl ester of calcein (Calcein/AM) can be used to distinguish live cells from dead cells. In the Calcein/AM assay, intracellular esterases cleave the acetoxymethyl (AM) ester group to produce a membrane-impermeable calcein fluorescent dye that is retained in live cells. Apoptotic and dead cells without intact cell membranes do not retain the calcein fluorescent dye. A lactate dehydrogenase (LDH) assay can also be used to evaluate cytotoxicity. LDH is a cytoplasmic enzyme, which is released into the extracellular space when the plasma membrane is damaged. Cytotoxicity is monitored by detecting LDH release from cells. See, e.g., Lieberman (2003) Nat Rev Immunol 3(5):361 -370, Neri et al. (2001 ) Clin Diagn Lab Immunol 8(6) : 1131 -1135, Smith et al. (2011 ) PLoS One 6(1 1 ):e26908, Chan et al. (2013) Methods Mol Biol 979:65-70; herein incorporated by reference in their entireties.

[00227] Flow cytometry can also be used to assess cell proliferation, activation, and cytotoxicity. The percentage of target cells that are live, apoptotic, or dead can be determined by staining target cells with viability dyes such that the live and dead cell populations can be distinguished based on differences in fluorescence. For example, Annexin V-FITC can be used to label target cells that are at an early stage of apoptosis. Propidium iodide can be used to label target cells that are at a late stage of apoptosis or dead. Lipophilic dyes, such as PKH67 and PKH26 can be used to label the cell membranes of target cells for measuring proliferation of CAR-T cells by flow cytometry. In addition, T cell activation can also be detected by immunofluorescent labeling of activation markers such as CD69, HLA-DR, IL2RA, and CD25. See, e.g., Zaritskaya et al. (2010) Expert Rev Vaccines 9(6):601 -616, Fischer et al. (2002) J Immunol Methods 259(1 -2):159-169, Aubry et al. (1999) Cytometry 37(3):197-204, and Tario et al. (201 1 ) Methods Mol Biol 699:1 19-164; herein incorporated by reference in their entireties. [00228] Cell proliferation can also be detected and quantified, for example, using a cell counter or staining of CAR-T cells with a fluorescent tracking dye, such as carboxyfluorescein succinimidyl ester (CFSE).

[00229] The CAR-T cells may be further tested for efficacy in treating a disease in vivo, e.g., in an animal. For example, CAR-T cells can be tested for cytotoxicity against cancerous cells in an animal with solid tumors. In some embodiments, human xenograft tumors are implanted in animals, followed by administration of CAR-T cells, and evaluation of antitumor responses. An exemplary animal model of cancer is a NOD Scid Gamma (NSG) mouse transplanted with human tumors. NSG mice are completely deficient in adaptive immunity and severely deficient in innate immunity, which avoids transplant rejection of CAR-T cells and patient-derived xenografts.

[00230] Antitumor responses can be evaluated by various methods known in the art. The volume of a subcutaneous tumor can be measured by using a digital caliper. Internal tumors can be measured by x-ray imaging, computed tomography (CT), ultrasound (US), magnetic resonance imaging (MRI), positron emission tomography (PET), or singlephoton emission computed tomography (SPECT). In some cases, the CAR-T cells are further modified to express a bioluminescent protein such as luciferase to allow monitoring of tumors by bioluminescence imaging or a fluorescent protein such as green fluorescent protein to allow monitoring of tumors by fluorescence imaging.

[00231] In addition, tumors can be removed from the animals and measured after the treatment with CAR-T cells is completed. Immunohistochemistry of tumor specimens can be used to detect T cell infiltration into tumors and quantitate target antigen expression. Cytokine profiling of tumors treated with CAR-T cells can also be performed.

[00232] In another example, CAR-T cells can be tested for cytotoxicity against activated fibroblasts or fibrotic tissue in an animal with fibrosis. The extent of fibrosis can be monitored in an animal in vivo, for example, by x-ray imaging, computed tomography (CT), ultrasound (US), magnetic resonance imaging (MRI), positron emission tomography (PET), or single-photon emission computed tomography (SPECT). In addition, fibrotic tissue can be removed from the animals and measured after the treatment with CAR-T cells is completed. Immunohistochemistry of fibrotic tissue specimens can be used to detect T cell infiltration into fibrotic tissue and quantitate target antigen expression. Cytokine profiling of fibrotic tissue treated with CAR-T cells can also be performed. [00233] An animal model can be used not only to determine efficacy but also the toxicity or side effects of treatment with a CAR-T cell. Furthermore, this disclosure pertains to uses of CAR-T cells, identified by the above-described screening assays for treatment of a disease such as, but not limited to, cancer, fibrosis, an infection, or an autoimmune disease. A CAR-T cell, identified by the above-described screening assays for treatment of a disease, may be expanded in culture in the presence of a natural antigen-presenting cell (e.g., dendritic cell) or an artificial antigen-presenting cell or particle under selective conditions prior to formulation into a pharmaceutical composition and administration.

Pharmaceutical Compositions

[00234] Pharmaceutical compositions comprising CAR-T cells, generated as described herein, can be prepared by formulating the CAR-T cells into dosage forms by known pharmaceutical methods. For example, a pharmaceutical composition comprising CAR- T cells can be formulated for parenteral administration, as capsules, liquids, film-coated preparations, suspensions, emulsions, and injections (such as venous injections, drip injections, and the like).

[00235] In formulation into these dosage forms, the CAR-T cells can be combined as appropriate, with pharmaceutically acceptable carriers or media, in particular, sterile water and physiological saline, vegetable oils, resolvents, bases, emulsifiers, suspending agents, surfactants, stabilizers, vehicles, antiseptics, binders, diluents, tonicity agents, soothing agents, bulking agents, disintegrants, buffering agents, coating agents, lubricants, coloring agents, solution adjuvants, or other additives.

[00236] The CAR-T cells may also be used in combination with other therapeutic agents for treating a disease. For example, for treatment of cancer, CAR-T cells may be used in combination with anti-cancer agents such as, but not limited to: chemotherapeutic agents such as cyclophosphamide, doxorubicin, vincristine, methotrexate, cytarabine, ifosfamide, etoposide, adriamycin, bleomycin, vinblastine, dacarbazine, chlormethine, oncovin, and procarbazine; immunotherapeutic agents such as antibodies (e.g., rituximab), cytokines (e.g., interferons, including type I (IFNa and IFNb), type II (IFNg) and type III (IFNI) and interleukins, including interleukin-2 (IL-2)), adjuvant immunochemotherapy agents (e.g., polysaccharide-K), adoptive T-cell therapy agents, and immune checkpoint blockade therapy agents; steroids such as prednisolone, biologic therapeutic agents such as tyrosine-kinase inhibitors, such as Imatinib mesylate (Gleevec, also known as STI-571 ), Gefitinib (Iressa, also known as ZD1839), Erlotinib (marketed as Tarceva), Sorafenib (Nexavar), Sunitinib (Sutent), Dasatinib (Sprycel), Lapatinib (Tykerb), Nilotinib (Tasigna), and Bortezomib (Velcade); Janus kinase inhibitors, such as tofacitinib; ALK inhibitors, such as crizotinib; Bcl-2 inhibitors, such as obatoclax and gossypol; PARP inhibitors, such as Iniparib and Olaparib; PI3K inhibitors, such as perifosine; VEGF receptor 2 inhibitors, such as Apatinib; AN-152 (AEZS-108) doxorubicin linked to [D-Lys(6)]-LHRH; Braf inhibitors, such as vemurafenib, dabrafenib, and LGX818; MEK inhibitors, such as trametinib; CDK inhibitors, such as PD-0332991 and LEE01 1 ; Hsp90 inhibitors, such as salinomycin; small molecule drug conjugates, such as Vintafolide; serine/threonine kinase inhibitors, such as Temsirolimus (Torisel), Everolimus (Afinitor), Vemurafenib (Zelboraf), Trametinib (Mekinist), and Dabrafenib (Tafinlar); pro-apoptotic agents such as oblimersen sodium, sodium butyrate, depsipetide, fenretinide, flavipirodol, gossypol, ABT-737, ABT-263 (Navitoclax), GX15- 070 and HA14-1 ; angiogenesis inhibitors such as bevacizumab, ramucirumab, ranibizumab, sorafenib, sunitinib, itraconazole, and carboxyamidotriazole; photoactive agents such as porfimer sodium, chlorins, bacteriochlorins, phthalocyanines, and aminolevulinic acid prodrugs; radiosensitizing agents such as cisplatin, fluoropyrimidines, gemcitabine, misonidazole, metronidazole, and taxanes; radioisotopes such as iodine- 131 , holmium-166, lutetium-177, radium-223, samarium-153, strontium-89, and yttrium- 90; or other therapeutic agents.

[00237] In some embodiments, the pharmaceutical composition comprising the CAR-T cells is a sustained-release formulation, or a formulation that is administered using a sustained-release device. Such devices are well known in the art, and include, for example, transdermal patches, and miniature implantable pumps that can provide for delivery of the CAR-T cells over time in a continuous, steady-state fashion at a variety of doses to achieve a sustained-release effect with a non-sustained-release pharmaceutical composition.

[00238] Usually, but not always, the subject who receives the CAR-T cells (i.e., the recipient) is also the subject from whom the original T cells (i.e., before genetic modification to express a CAR specific for a target cell) are harvested or obtained, which provides the advantage that the cells are autologous. However, T cells can be obtained from another subject (i.e., donor), a culture of cells from a donor, or from established cell culture lines and genetically modified, as described herein. T cells may be obtained from the same or a different species than the subject to be treated, but preferably are of the same species, and more preferably of the same immunological profile as the subject. Such cells can be obtained, for example, from a biological sample comprising T cells from a close relative or matched donor, genetically modified to express a CAR, and administered to a subject in need of treatment. The patients or subjects who donate or receive the T cells are typically mammalian, and usually human. However, this need not always be the case, as veterinary applications are also contemplated. In certain embodiments, the CAR-T cells administered to a subject are autologous or allogeneic.

Cellular Therapy with CAR-T cells

[00239] CAR-T cells are administered to a subject in a therapeutically effective amount. The phrase “therapeutically effective amount” refers to the administration of the CAR-T cells to a subject, either alone or as a part of a pharmaceutical composition and either in a single dose or as part of a series of doses, in an amount that is capable of having any detectable, positive effect on any symptom, aspect, or characteristics of a disease, disorder or condition when administered to a patient. The therapeutically effective amount can be ascertained by measuring relevant physiological effects. For example, in the case of cancer, a therapeutically effective amount of the CAR-T cells provides an anti-tumor effect, as defined herein. Therefore, for example, a positive therapeutic response would refer to one or more of the following improvements in the disease: (1) reduction in tumor size; (2) reduction in the number of cancer cells; (3) inhibition (i.e., slowing to some extent, preferably halting) of tumor growth; (4) inhibition (i.e., slowing to some extent, preferably halting) of cancer cell infiltration into peripheral organs; (5) inhibition (i.e., slowing to some extent, preferably halting) of tumor metastasis; and (6) some extent of relief from one or more symptoms associated with the cancer. Such therapeutic responses may be further characterized as to degree of improvement. Thus, for example, an improvement may be characterized as a complete response. By “complete response” is documentation of the disappearance of all symptoms and signs of all measurable or evaluable disease confirmed by physical examination, laboratory, nuclear and radiographic studies (i.e., CT (computer tomography) and/or MRI (magnetic resonance imaging)), and other non-invasive procedures repeated for all initial abnormalities or sites positive at the time of entry into the study. Alternatively, an improvement in the disease may be categorized as being a partial response. By “partial response” is intended a reduction of greater than 50% in the sum of the products of the perpendicular diameters of all measurable lesions when compared with pretreatment measurements. [00240] In certain embodiments, antigen-presenting cells (e.g., dendritic cells) or artificial antigen-presenting cells or particles are used to stimulate proliferation and expansion of CAR-T cells in vitro or ex vivo prior to administration. In certain embodiments, the ex vivo method comprises contacting a population of T cells comprising a CAR-T cell with the antigen-presenting cells or artificial antigen-presenting cells or particles, wherein the population of T cells have been obtained from the subject to be treated, then genetically modified to express a CAR with an endogenous TCR knockout, as described herein. After one or more rounds of antigen-stimulation with the antigen-presenting cells or artificial antigen-presenting cells or particles and expansion of the CAR-T cells in culture, the autologous CAR-T cells are subsequently administered to the subject.

[00241] In certain embodiments, stimulation of proliferation and expansion of CAR-T cells with antigen-presenting cells (e.g., dendritic cells) or artificial antigen-presenting cells or particles are carried out in vitro. In certain embodiments, the in vitro method comprises contacting a population of T cells comprising a CAR-T cell with the antigen-presenting cells (e.g., dendritic cells) or artificial antigen-presenting cells or particles, wherein the T cells have been obtained from a donor, a culture of cells from a donor, or from established cell culture lines, then genetically modified to express a CAR with an endogenous TCR knockout, as described herein. The T cells may be obtained from the same or a different species than the subject to be treated, but preferably are of the same species, and more preferably of the same immunological profile as the subject. Such cells can be obtained, for example, from a blood sample comprising T cells from a close relative or matched donor. After one or more rounds of antigen-stimulation with the antigen-presenting cells (e.g., dendritic cells) or artificial antigen-presenting cells or particles and expansion of the CAR-T cells in culture, the CAR-T cells may be subsequently administered to a subject.

[00242] In certain embodiments, proliferation and expansion of CAR-T cells occurs in vivo either by stimulation with an endogenous antigen-presenting cell or by coadministration of antigen-presenting cells or artificial antigen-presenting cells or particles with the CAR- T cells to the subject.

[00243] In the in vitro, ex vivo, or in vivo methods described herein, the subject may have cancer, wherein the CAR-T cells comprise a CAR that specifically binds to an antigen expressed on a cancerous cell. In some embodiments, the antigen is a tumor-specific antigen or a tumor-associated antigen expressed on a cancerous cell, wherein the antigen is used to activate a CAR-T cell designed for therapeutic use against a cancerous cell. Exemplary tumor-specific antigens and tumor-associated antigens include, without limitation, oncogene protein products, mutated or dysregulated tumor suppressor proteins, oncovirus proteins, oncofetal antigens, mutated or dysregulated differentiation antigens, overexpressed or aberrantly expressed cellular proteins (e.g., mutated or aberrantly expressed growth factors, mitogens, receptor tyrosine kinases, cytoplasmic tyrosine kinases, serine/threonine kinases and their regulatory subunits, G proteins, and transcription factors), and altered cell surface glycolipids and glycoproteins on cancerous cells. For example, tumor-specific antigens and tumor-associated antigens may include without limitation, dysregulated or mutated RAS, WNT, MYC, ERK, TRK, CTAG1 B, MAGEA1 , Bcr-Abl, p53, c-Sis, epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGFR), vascular endothelial growth factor receptor (VEGFR), HER2/neu, Src-family, Syk-ZAP-70 family proteins, and BTK family of tyrosine kinases, Abl, Raf kinase, cyclin-dependent kinases, alphafetoprotein (AFP), carcinoembryonic antigen (CEA), CA-125, MUC-1 , epithelial tumor antigen (ETA), tyrosinase, melanoma- associated antigen (MAGE), and other abnormal or dysregulated proteins expressed on cancerous cells. In certain embodiments, the subject has leukemia, lymphoma, myeloma, prostate cancer, breast cancer, lung cancer, kidney cancer, lung cancer, ovarian cancer, intestine cancer, or glioblastoma. In other embodiments, the subject has fibrosis, wherein the CAR-T cells comprise a CAR that specifically binds to a fibrosis antigen expressed on activated fibroblasts or fibrotic tissue such as fibroblast activation protein (FAP). In certain embodiments, the subject is undergoing or has previously undergone CAR-T cell immunotherapy.

[00244] The present disclosure contemplates the administration of the CAR-T cells, and compositions thereof, in any appropriate manner. Suitable routes of administration include parenteral (e.g., intramuscular, intravenous, subcutaneous (e.g., injection or implant), intraperitoneal, intracisternal, intraarticular, intraperitoneal, intracerebral (intraparenchymal) and intracerebroventricular), oral, nasal, vaginal, sublingual, intraocular, rectal, topical (e.g., transdermal), sublingual, inhalation, local, e.g., injection directly into a target organ or tissue such as a tumor or fibrotic tissue.

[00245] In some embodiments, the CAR-T cells may comprise a binding-triggered transcriptional switch. In some embodiments, the method may further include activating a T cell such as a T cell expressing a chimeric Notch polypeptide, as described herein. In certain embodiments, the method of the present disclosure may be used for inducing T-cell proliferation without significantly increasing cytokine production by the T cell. For example, the method may include administering a T cell expressing a chimeric Notch polypeptide and CAPP having a protein displayed on the surface, where the protein binds to the Notch polypeptide resulting in expression of a cancer associated CAR on the cell surface. The CAPP further includes an antigen that binds the cancer associated CAR, where binding of the antigen on the particle to the cancer associated CAR results in activation of the T cell in absence of significant expression of cytokines. In certain embodiments, the level of cytokines produced by the T cells in the absence of cancer cells expressing the CAR antigen is substantially lower than the level of the cytokines produced by the T cells in the presence of cancer cells expressing the CAR antigen. Thus, use of particles functionalized with both a protein that binds to the chimeric Notch polypeptide and an antigen that binds to the CAR expressed in response to the binding of the protein to the chimeric Notch polypeptide provides for proliferation of the T-cells while having a substantially lower production of cytokines by the activated T cell.

[00246] In certain aspects, contacting a CAR-T cell expressing a BTTS, e.g., a chimeric Notch receptor polypeptide, as described herein with the CAPP of the present disclosure may modulate an activity of the CAR-T cell. In some cases, release of the intracellular domain modulates proliferation of the cell or of cells surrounding the cell. In some cases, release of the intracellular domain modulates apoptosis in the cell or in cells surrounding the cell. In some cases, release of the intracellular domain induces cell death by a mechanism other than apoptosis. In some cases, release of the intracellular domain modulates gene expression in the cell through transcriptional regulation, chromatin regulation, translation, trafficking or post-translational processing. In some cases, release of the intracellular domain modulates differentiation of the cell. In some cases, release of the intracellular domain modulates migration of the cell or of cells surrounding the cell. In some cases, release of the intracellular domain modulates the expression and secretion of a molecule from the cell. In some cases, release of the intracellular domain modulates adhesion of the cell to a second cell or to an extracellular matrix. In some cases, release of the intracellular domain induces de novo expression a gene product in the cell. In some cases, where release of the intracellular domain induces de novo expression a gene product in the cell, the gene product is a transcriptional activator, a transcriptional repressor, a chimeric antigen receptor, a second chimeric Notch receptor polypeptide, a translation regulator, a cytokine, a hormone, a chemokine, or an antibody.

Kits Kits are provided to perform the subject methods for genetically modifying cells to introduce both a gene knockin and a gene knockout. In some embodiments, the kit comprises a CRISPR system for genetically modifying cells as described herein. For example, the kit may comprise a donor polynucleotide comprising a nucleotide sequence encoding an exogenous polypeptide and a knockout guide RNA, an RNA-guided nuclease, and a knockin guide RNA, as described herein. In some embodiments, the kit comprises a CRISPR system for genetically modifying T cells to produce CAR-T cells, as described herein. A kit may also include a binding agent for performing negative or positive selection to separate successfully edited cells from unsuccessfully edited cells in a sample. In some cases, the binding agent comprises a magnetic bead comprising an antibody specific for a cellular marker that can be used for enrichment of a selected population of cells using magnetic separation. A kit may further comprise media suitable for culturing cells. Additionally, the kit may include cells, transfection agents, buffers, tissue culture plates, flasks, test tubes, vials, and the like, and optionally one or more other factors, such as cytokines (e.g., IL-2, IL-3, IL-6, IL-7, IL-15, TNFD, IFN-D, and GM- CSF), growth factors, antibiotics, or other media supplements, and the like.

[00247] In certain embodiments, the donor polynucleotide in the kit further comprises a sequence encoding a knockout module comprising a plurality of knockout guide RNAs. In some embodiments, at least 2 knockout guide RNAs of the plurality bind to different genomic target sequences in different target genes to generate gene knockouts of more than one target gene. In some embodiments, at least 2 knockout guide RNAs of the plurality bind to different genomic target sequences in the same target gene to increase a rate of gene knockout of the target gene compared to the rate of gene knockout using only one knockout guide RNA.

[00248] In certain embodiments, the donor polynucleotide in the kit can produce a first mRNA transcript comprising: a protein coding reading frame encoding the exogenous polypeptide followed by a stop codon; and a 3’-untranslated region comprising: a knockout module flanked by a first spacer sequence and a second spacer sequence, wherein the knockout module comprises a plurality of knockout guide RNAs, wherein each guide RNA is preceded by a synthetic separator sequence followed by a direct repeat sequence; a mRNA stabilizing element, wherein the mRNA stabilizing element is positioned between the stop codon and the first spacer sequence; and a polyadenylation sequence. In some embodiments, the mRNA stabilizing element is a triplex stabilizer. In some embodiments, the knockout guide RNAs are Cas12a guide RNAs. [00249] Such kits generally will comprise, in suitable means, distinct containers for each individual reagent or solution. Suitable containers for the compositions include, for example, bottles, vials, syringes, and test tubes. Containers can be formed from a variety of materials, including glass or plastic. A container may have a sterile access port (for example, the container may be a vial having a stopper pierceable by a hypodermic injection needle).

[00250] The kit may also provide a delivery device for administration of CAR-T cells to a patient. For example, kits may comprise a container having a sterile access port (e.g., the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The kit can further comprise a container comprising a pharmaceutically-acceptable buffer, such as phosphate-buffered saline, Ringer's solution, or dextrose solution. It can also contain other materials useful to the end-user, including other pharmaceutically acceptable formulating solutions such as buffers, diluents, filters, needles, and syringes or other delivery device.

[00251] In addition to the above components, the subject kits may further include (in certain embodiments) instructions for practicing the subject methods. These instructions may be present in the subject kits in a variety of forms, one or more of which may be present in the kit. One form in which these instructions may be present is as printed information on a suitable medium or substrate, e.g., a piece or pieces of paper on which the information is printed, in the packaging of the kit, in a package insert, and the like. Yet another form of these instructions is a computer readable medium, e.g., diskette, compact disk (CD), DVD, flash drive, and the like, on which the information has been recorded. Yet another form of these instructions that may be present is a website address which may be used via the internet to access the information at a removed site.

Examples of Non-Limiting Aspects of the Disclosure

[00252] Aspects, including embodiments, of the present subject matter described above may be beneficial alone or in combination, with one or more other aspects or embodiments. Without limiting the foregoing description, certain non-limiting aspects of the disclosure numbered 1 -71 are provided below. As will be apparent to those of skill in the art upon reading this disclosure, each of the individually numbered aspects may be used or combined with any of the preceding or following individually numbered aspects. This is intended to provide support for all such combinations of aspects and is not limited to combinations of aspects explicitly provided below. 1 . A method of genetically modifying a cell to introduce a gene knockin and a gene knockout, the method comprising: introducing a donor polynucleotide into the cell, wherein the donor polynucleotide comprises a 5' homology arm that hybridizes to a 5' genomic target sequence and a 3' homology arm that hybridizes to a 3' genomic target sequence flanking a nucleotide sequence encoding i) an exogenous polypeptide and ii) a knockout guide RNA; introducing an RNA-guided nuclease into the cell; introducing a knockin guide RNA into the cell, wherein the knockin guide RNA forms a complex with the RNA-guided nuclease such that the knockin guide RNA directs the RNA-guided nuclease to a first genomic target locus, wherein the RNA-guided nuclease creates a double-stranded break in the genomic DNA at the first genomic target locus, wherein the donor polynucleotide is integrated at the first genomic target locus recognized by its 5' homology arm and 3' homology arm by homology directed repair (HDR); and culturing the cell under conditions suitable for transcription of the integrated donor polynucleotide, wherein a first mRNA transcript encoding the exogenous polypeptide and the knockout guide RNA is produced, wherein the RNA-guided nuclease excises the knockout guide RNA from the first mRNA transcript to produce a second mRNA transcript encoding the exogenous polypeptide without the knockout guide RNA; wherein translation of the second mRNA transcript results in production of the exogenous polypeptide in the cell; wherein the excised knockout guide RNA forms a complex with the RNA-guided nuclease such that the knockout guide RNA directs the RNA-guided nuclease to a second genomic target locus, wherein the RNA-guided nuclease creates a doublestranded break in the genomic DNA at the second genomic target locus, wherein DNA repair of the double-stranded break by non-homologous end joining creates an insertion or deletion (indel) resulting in gene knockout at the second genomic target locus.

2. The method of aspect 1 , wherein the exogenous polypeptide is an enzyme, an extracellular matrix protein, a receptor, a transporter, an ion channel, or other membrane protein, a hormone, a neuropeptide, a growth factor, a cytokine, an antibody, a cytoskeletal protein, or a therapeutic protein; or a fragment thereof, or a biologically active domain of interest. 3. The method of aspect 1 or 2, wherein the cell is a stem cell, progenitor cell, or an adult cell.

4. The method of aspect 3, wherein the stem cell is an induced-pluripotent stem cell or an adult stem cell.

5. The method of any one of aspects 1 -4, wherein the cell secretes a cytokine, a chemokine, a neuropeptide, a growth factor, or a hormone.

6. The method of any one of aspects 1 -5, wherein the cell is a mammalian cell.

7. The method of aspect 6, wherein the mammalian cell is an immune cell.

8. The method of aspect 7, wherein the immune cell is a T cell, a B cell, a natural killer cell, a neutrophil, an eosinophil, a mast cell, a basophil, a monocyte, a macrophage, or a dendritic cell.

9. The method of aspect 8, wherein the T cell is a helper CD4⁺ T cell, a cytotoxic CD8⁺ T cell, a natural killer T cell, or a gamma delta T cell.

10. The method of aspect 8 or 9, wherein the exogenous protein is a chimeric antigen receptor (CAR) that specifically binds to a target antigen.

11 . The method of aspect 10, wherein the chimeric antigen receptor comprises a transmembrane domain linked to an extracellular antigen binding domain and an intracellular signaling domain, wherein the extracellular antigen-binding domain specifically binds to the target antigen.

12. The method of aspect 11 , wherein the extracellular antigen binding domain comprises a single chain variable fragment (scFv), an antigen-binding fragment (Fab), a nanobody, a heavy chain variable (VH) domain, a light chain variable (VL) domain, a single domain antibody (sdAb), a shark variable domain of a new antigen receptor (VNAR), a single variable domain on a heavy chain (VHH), a bispecific antibody, a diabody, or a functional fragment thereof that binds specifically to the antigen.

13. The method of aspect 11 or 12, wherein the intracellular signaling domain is a CD3-zeta intracellular signaling domain or a ZAP-70 intracellular signaling domain.

14. The method of aspect 11 or 12, wherein the intracellular signaling domain comprises an immunoreceptor tyrosine-based activation motif (ITAM).

15. The method of any one of aspects 11 -14, wherein the CAR further comprises a costimulatory domain.

16. The method of aspect 15, wherein the costimulatory domain is a 4-1 BB, CD28, ICOS, OX-40, BTLA, CD27, CD30, GITR, or HVEM costimulatory domain.

17. The method of any one of aspects 11 -16, wherein the transmembrane domain is a CD8, Megfl O, FcRy, Bail , MerTK, TIM4, Stabilin-1 , Stabilin-2, RAGE, CD300f, integrin subunit av, integrin subunit (35, CD36, LRP1 , SCARF1 , C1 Qa, Axl, CD45, or CD86 transmembrane domain.

18. The method of any one of aspects 11 -17, wherein the target antigen is on a cancer cell, a tumor cell, an activated fibroblast, an autoreactive immune cell, a pathogen, or a diseased cell.

19. The method of aspect 18, wherein the target antigen is a tumor antigen or a tumor-associated antigen.

20. The method of aspect 18, wherein the pathogen is a virus, a bacterium, a fungus, or a parasite.

21 . The method of aspect 20, wherein the target antigen is a viral antigen, a bacterial antigen, a fungal antigen or a parasite antigen. 22. The method of aspect 18, wherein the autoreactive immune cell is an autoreactive T cell or B cell.

23. The method of aspect 22, wherein the target antigen is an antigen on the autoreactive T cell or B cell.

24. The method of any one of aspects 1 -23, wherein the second genomic target locus is a T cell receptor alpha chain locus, T cell receptor beta chain locus, T cell receptor delta chain locus, or a T cell receptor gamma chain locus.

25. The method of any one of aspects 1 -23, wherein the second genomic target locus encodes a cytokine.

26. The method of aspect 25, wherein the cytokine is GM-CSF or IL-6

27. The method of any one of aspects 1 -23, wherein the second genomic target locus encodes an alloantigen.

28. The method of aspect 27, wherein the alloantigen is a major histocompatibility complex (MHC) class I alloantigen or an MNS blood group alloantigen.

29. The method of aspect 27, wherein the alloantigen is CD1 , CD2, CD3, CD4, CD7, CD8, Ly-6, Qa-2, RT6, CD19, CD22, CD56, CD58 (LFA-3), OD59, or CDw90 (Thy 1 ).

30. The method of any one of aspects 1 -23, wherein the second genomic target locus comprises a CD5, CD52, CD70, BATF, LCK, PD-1 , LAG-3, CTLA-4, 2-B2M, PD- 1 , HLA-I, Fas, TGFBR2, PDCD-1 , DGK, EZH2, PAX5, or LDLR gene.

31 . The method of any one of aspects 1 -30, wherein the donor polynucleotide and/or the RNA-guided nuclease is provided by a vector.

32. The method of aspect 31 , wherein the vector is a viral vector or a plasmid. 33. The method of aspect 32, wherein the viral vector is a lentivirus vector, retrovirus vector, or adeno-associated virus vector

34. The method of any one of aspects 31 -33, wherein the vector is introduced into the cell by transient transfection or stable transfection.

35. The method of aspect 34, wherein the vector is introduced into the cell by electroporation, nucleofection, or lipofection.

36. The method of any one of aspects 31-35, wherein the donor polynucleotide and the RNA-guided nuclease are provided by separate vectors.

37. The method of any one of aspects 31-35, wherein the donor polynucleotide and the RNA-guided nuclease are provided by the same vector.

38. The method of any one of aspects 31 -37, wherein expression of the RNA- guided nuclease is inducible.

39. The method of any one of aspects 1 -30, wherein the RNA-guided nuclease is provided by a mRNA encoding the RNA-guided nuclease, wherein translation of the mRNA results in production of the RNA-guided nuclease in the cell.

40. The method of any one of aspects 1 -39, wherein the RNA-guided nuclease is a clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) nuclease.

41 . The method of aspect 40, wherein the Cas nuclease is Cas9 or Cas12a.

42. The method of any one of aspects 1 -41 , wherein the donor polynucleotide further comprises a barcode.

43. The method of any one of aspects 1 -41 , wherein the donor polynucleotide further comprises a gene knockin module comprising a plurality of coding sequences encoding a plurality of polypeptides. 44. The method of any one of aspects 1 -43, wherein the donor polynucleotide further comprises a sequence encoding a knockout module comprising a plurality of knockout guide RNAs.

45. The method of aspect 44, wherein at least 2 knockout guide RNAs of the plurality bind to different genomic target sequences in different target genes to generate gene knockouts of more than one target gene.

46. The method of aspect 44 or 45, wherein at least 2 knockout guide RNAs of the plurality bind to different genomic target sequences in the same target gene to increase a rate of gene knockout of the target gene compared to the rate of gene knockout using only one knockout guide RNA.

47. The method of any one of aspects 44-46, wherein the first mRNA transcript comprises: a protein coding reading frame encoding the exogenous polypeptide followed by a stop codon; and a 3’-untranslated region comprising: a knockout module flanked by a first spacer sequence and a second spacer sequence, wherein the knockout module comprises a plurality of knockout guide RNAs, wherein each guide RNA is preceded by a synthetic separator sequence followed by a direct repeat sequence; a mRNA stabilizing element, wherein the mRNA stabilizing element is positioned between the stop codon and the first spacer sequence; and a polyadenylation sequence.

48. The method of aspect 47, wherein the mRNA stabilizing element is a triplex stabilizer.

49. The method of any one of aspects 1 -48, wherein the transcription of the integrated donor polynucleotide is performed by an RNA polymerase II (Pol II). 50. The method of any one of aspects 1 -49, wherein the knockout guide RNAs are Cas12a guide RNAs.

51 . A composition comprising: a donor polynucleotide comprising a 5' homology arm that hybridizes to a 5' genomic target sequence and a 3' homology arm that hybridizes to a 3' genomic target sequence flanking a nucleotide sequence encoding i) an exogenous polypeptide and ii) a knockout guide RNA; an RNA-guided nuclease; and a knockin guide RNA, wherein the knockin guide RNA can form a complex with the RNA-guided nuclease such that the knockin guide RNA directs the RNA-guided nuclease to a first genomic target locus in a cell, wherein the RNA-guided nuclease creates a double-stranded break in the genomic DNA at the first genomic target locus, wherein the donor polynucleotide is integrated at the first genomic target locus recognized by its 5' homology arm and 3' homology arm by homology directed repair (HDR), wherein a first mRNA transcript encoding the exogenous polypeptide and the knockout guide RNA is produced by transcription of the integrated donor polynucleotide, wherein the RNA-guided nuclease excises the knockout guide RNA from the first mRNA transcript to produce a second mRNA transcript encoding the exogenous polypeptide without the knockout guide RNA, wherein translation of the second mRNA transcript results in production of the exogenous polypeptide in the cell, and wherein the excised knockout guide RNA forms a complex with the RNA-guided nuclease such that the knockout guide RNA directs the RNA-guided nuclease to a second genomic target locus in the cell, wherein the RNA-guided nuclease creates a double-stranded break in the genomic DNA at the second genomic target locus, wherein DNA repair of the doublestranded break by non-homologous end joining creates an insertion or deletion (indel) resulting in gene knockout at the second genomic target locus.

52. The composition of aspect 51 , wherein the RNA-guided nuclease is a clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Gas) nuclease.

53. The composition of aspect 52, wherein the Cas nuclease is Cas9 or Cas12a. 54. The composition of any one of aspects 51 -52, wherein the donor polynucleotide further comprises a gene knockin module comprising a plurality of coding sequences encoding a plurality of polypeptides.

55. The composition of any one of aspects 51 -54, wherein the donor polynucleotide further comprises a sequence encoding a knockout module comprising a plurality of knockout guide RNAs.

56. The composition of aspect 55, wherein at least 2 knockout guide RNAs of the plurality bind to different genomic target sequences in different target genes to generate gene knockouts of more than one target gene.

57. The composition of aspect 55 or 56, wherein at least 2 knockout guide RNAs of the plurality bind to different genomic target sequences in the same target gene to increase a rate of gene knockout of the target gene compared to the rate of gene knockout using only one knockout guide RNA.

58. The composition of any one of aspects 55-57, wherein the first mRNA transcript comprises: a protein coding reading frame encoding the exogenous polypeptide followed by a stop codon; and a 3’-untranslated region comprising: a knockout module flanked by a first spacer sequence and a second spacer sequence, wherein the knockout module comprises a plurality of knockout guide RNAs, wherein each guide RNA is preceded by a synthetic separator sequence followed by a direct repeat sequence; a mRNA stabilizing element, wherein the mRNA stabilizing element is positioned between the stop codon and the first spacer sequence; and a polyadenylation sequence.

59. The composition of aspect 58, wherein the mRNA stabilizing element is a triplex stabilizer. 60. The composition of any one of aspects 51 -59, wherein the transcription of the integrated donor polynucleotide is performed by an RNA polymerase II (Pol II).

61 . The composition of any one of aspects 55-60, wherein the knockout guide RNAs are Cas12a guide RNAs.

62. A kit comprising the composition of any one of aspects 51 -61 and instructions for producing a genetically modified T cell expressing a chimeric antigen receptor.

63. The kit of aspect 62, further comprising a transfection agent.

64. A genetically modified cell produced according to the method of any one of aspects 1 -50.

65. A composition comprising the genetically modified cell of aspect 64 and a pharmaceutically acceptable excipient or carrier.

66. A method of performing cellular therapy, the method comprising administering a therapeutically effective amount of the composition of aspect 65 to a subject.

67. The method of aspect 66, wherein the genetically modified cell is autologous or allogeneic.

68. The method of aspect 66 or 67, wherein the genetically modified cell is an immune cell.

69. The method of aspect 68, wherein the immune cell is a T cell, a B cell, a natural killer cell, a neutrophil, an eosinophil, a mast cell, a basophil, a monocyte, a macrophage, or a dendritic cell.

70. The method of aspect 69, wherein the T cell is a helper CD4⁺ T cell, a cytotoxic CD8⁺ T cell, a natural killer T cell, or a gamma delta T cell. 71 . The method of any one of aspects 68-70, wherein the exogenous protein is a chimeric antigen receptor (CAR) that specifically binds to a target antigen.

[00253] It will be apparent to one of ordinary skill in the art that various changes and modifications can be made without departing from the spirit or scope of the invention.

EXPERIMENTAL

[00254] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.

[00255] All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

[00256] The present invention has been described in terms of particular embodiments found or proposed by the present inventor to comprise preferred modes for the practice of the invention. It will be appreciated by those of skill in the art that, in light of the present disclosure, numerous modifications and changes can be made in the particular embodiments exemplified without departing from the intended scope of the invention. For example, due to codon redundancy, changes can be made in the underlying DNA sequence without affecting the protein sequence. Moreover, due to biological functional equivalency considerations, changes can be made in protein structure without affecting the biological action in kind or amount. All such modifications are intended to be included within the scope of the appended claims. Example 1

Simultaneous Gene Knockin + Knockout in Engineered Cell Therapies with mRNA expressed gRNAs

[00257] Genetically modified T cell therapies, especially CAR T cells, are growing in clinical use and have demonstrated remarkable cures in certain hematological indications. To engineer these therapies, a new synthetic gene, usually a Chimeric Antigen Receptor (CAR) must be knocked into the cells genome. However, recent work has demonstrated numerous gene targets that when knocked out in T cells drastically improve their functionality. Technologies that simultaneously engineer gene knockins along with gene knockouts can take advantage of these functional improvements in T cell behavior to build more effective CAR T cell therapies.

[00258] Performing a gene knockin requires a targetable nuclease with a guide RNA (e.g., Cas9 + gRNA, together termed a ribonucleoprotein (RNP) as well as a DNA template containing the new gene (e.g. CAR) that will be inserted at the CRISPR/Cas9 RNPs cut site. To knockout an additional gene at the same time, either a second (or third, fourth, etc.) Cas9 RNP with a different gRNA targeting the additional gene, can be introduced into the cells at the same time. Alternatively, the DNA template can contain an RNA Pol III (e.g. U6 promoter) cassette to drive expression of a second gRNA itself (FIG. 1 ). Both of these current systems have fundamental challenges limiting their use, especially in pre-clinical development applications, as described below.

[00259] We have developed a new technology for combined gene Knockin + Knockout (KI+KO) based on the inclusion of the knockout gRNA sequence within the mature mRNA sequence of the gene knockin construct (CAR). A schematic of this approach is shown in FIG. 2. By incorporating the gRNA into an mRNA transcript, it is only expressed in cells that have had a successful knockin, unlike the two competing technologies where cells without a knockin are able to get the knockout, a potential safety concern. Only after gene knockin is the mRNA sequence successfully expressed (containing both the CAR/additional protein coding genes as well as the gRNA sequence). We next use a Cast 2a nuclease introduced into the cell at the same time as the DNA knockin sequence, as Cas12a is able to excise its own gRNA from a longer mRNA strand. The Cast 2a now loaded with its gRNA is then able to identify its target site in the genome, induce a double stranded DNA break, and cause frameshift mutations resulting in loss of the target protein. Crucially, only cells with the gene knockin are able to express the mRNA containing the gRNA, so there is a tight linkage between cells that have the gene knockin and cells that have the subsequent gene knockout. We have optimized the genetic architecture of the mRNA sequence including the gRNA to maximize efficiency and selectivity of knockout.

[00260] Gene knockin plus gene knockout using mRNA expressed gRNAs in human T cells has two main applications. First, in preclinical discovery efforts, where large scale pooled screening of gene knockins + gene knockouts are not possible using the two existing KI + KO technologies (additional RNPs or U6 expressed gRNAs). Second, in final cell therapy clinical products, the mRNA expressed gRNA based KI+KO technology enables a safer cell product by maintaining a close linkage between cells that acquire the gene knockin and cells that acquire the gene knockout, ensuring that cells with only a gene knockout (potential autoimmunity concern) are not injected back into a patient.

[00261] As described above, there are two existing technologies to generate gene knockins in human cell therapies along with a gene knockout, both with crucial limitations that our mRNA expressed gRNA KI+KO technology overcomes. The first technology involves introducing additional knockout Cas9/gRNA complexes (RNPs) into cells at the same time as the knockin RNP and DNA template. This results in the knockin and knockout being entirely independent, meaning that cells with the knockin are not guaranteed to have a knockout, and cells without a knockin are just as likely to have a knockout as cells with the knockin. While this may be acceptable in some applications, it can pose safety concerns (primarily autoimmune side effects) depending on the knockout target. Additionally, the knockin and knockout RNPs are cutting their target DNA sequences at the same time, resulting in large amounts of chromosomal rearrangements, translocations, and other genomic damage, increasing the risk for cellular transformation/cancer. The second technology involves introducing a DNA cassette to express the gene knockout gRNA on the same DNA template as the gene knockin. Since gRNAs are small RNA sequences, they traditionally must be expressed by an RNA pol III promoter (the human polymerase that makes small RNAs), normally a U6 promoter. However, because the U6 promoter is active, whether its DNA sequence has been incorporated into the genome (successful knockin) or not (episomal plasmid), this method similarly results in loss of linkage between gene knockin and gene knockout, with cells just as likely to have a gene knockout whether they had a knockin or not. [00262] As described above, our new system overcomes this challenge by having the knockout gRNA incorporated into an mRNA strand, which can only be expressed after the gene knockin occurs. This crucially results in two improvements- first, only cells with the gene knockin are able to express the knockout gRNA, solving the safety issue of having gene knockouts in the cells that do not have a gene knockin. Second, the gene knockout is temporally separated from the gene knockin, meaning that only one double stranded DNA break is present in the cell at a time, preventing the chromosomal translocations and large-scale genetic damage seen when multiple double stranded breaks are simultaneously induced.

Claims

What is claimed is:

1 . A method of genetically modifying a cell to introduce a gene knockin and a gene knockout, the method comprising: introducing a donor polynucleotide into the cell, wherein the donor polynucleotide comprises a 5' homology arm that hybridizes to a 5' genomic target sequence and a 3' homology arm that hybridizes to a 3' genomic target sequence flanking a nucleotide sequence encoding i) an exogenous polypeptide and ii) a knockout guide RNA; introducing an RNA-guided nuclease into the cell; introducing a knockin guide RNA into the cell, wherein the knockin guide RNA forms a complex with the RNA-guided nuclease such that the knockin guide RNA directs the RNA-guided nuclease to a first genomic target locus, wherein the RNA-guided nuclease creates a double-stranded break in the genomic DNA at the first genomic target locus, wherein the donor polynucleotide is integrated at the first genomic target locus recognized by its 5' homology arm and 3' homology arm by homology directed repair (HDR); and culturing the cell under conditions suitable for transcription of the integrated donor polynucleotide, wherein a first mRNA transcript encoding the exogenous polypeptide and the knockout guide RNA is produced, wherein the RNA-guided nuclease excises the knockout guide RNA from the first mRNA transcript to produce a second mRNA transcript encoding the exogenous polypeptide without the knockout guide RNA; wherein translation of the second mRNA transcript results in production of the exogenous polypeptide in the cell; wherein the excised knockout guide RNA forms a complex with the RNA-guided nuclease such that the knockout guide RNA directs the RNA-guided nuclease to a second genomic target locus, wherein the RNA-guided nuclease creates a doublestranded break in the genomic DNA at the second genomic target locus, wherein DNA repair of the double-stranded break by non-homologous end joining creates an insertion or deletion (indel) resulting in gene knockout at the second genomic target locus.

2. The method of claim 1 , wherein the exogenous polypeptide is an enzyme, an extracellular matrix protein, a receptor, a transporter, an ion channel, or other membrane protein, a hormone, a neuropeptide, a growth factor, a cytokine, an antibody, a cytoskeletal protein, or a therapeutic protein; or a fragment thereof, or a biologically active domain of interest.

3. The method of claim 1 or 2, wherein the cell is a stem cell, progenitor cell, or an adult cell.

4. The method of claim 3, wherein the stem cell is an induced-pluripotent stem cell or an adult stem cell.

5. The method of any one of claims 1 -4, wherein the cell secretes a cytokine, a chemokine, a neuropeptide, a growth factor, or a hormone.

6. The method of any one of claims 1 -5, wherein the cell is a mammalian cell.

7. The method of claim 6, wherein the mammalian cell is an immune cell.

8. The method of claim 7, wherein the immune cell is a T cell, a B cell, a natural killer cell, a neutrophil, an eosinophil, a mast cell, a basophil, a monocyte, a macrophage, or a dendritic cell.

9. The method of claim 8, wherein the T cell is a helper CD4⁺ T cell, a cytotoxic CD8⁺ T cell, a natural killer T cell, or a gamma delta T cell.

10. The method of claim 8 or 9, wherein the exogenous protein is a chimeric antigen receptor (CAR) that specifically binds to a target antigen.

11 . The method of claim 10, wherein the chimeric antigen receptor comprises a transmembrane domain linked to an extracellular antigen binding domain and an intracellular signaling domain, wherein the extracellular antigen-binding domain specifically binds to the target antigen.

12. The method of claim 11 , wherein the extracellular antigen binding domain comprises a single chain variable fragment (scFv), an antigen-binding fragment (Fab), a nanobody, a heavy chain variable (VH) domain, a light chain variable (VL) domain, a single domain antibody (sdAb), a shark variable domain of a new antigen receptor (VNAR), a single variable domain on a heavy chain (VHH), a bispecific antibody, a diabody, or a functional fragment thereof that binds specifically to the antigen.

13. The method of claim 11 or 12, wherein the intracellular signaling domain is a CD3-zeta intracellular signaling domain or a ZAP-70 intracellular signaling domain.

14. The method of claim 1 1 or 12, wherein the intracellular signaling domain comprises an immunoreceptor tyrosine-based activation motif (ITAM).

15. The method of any one of claims 1 1 -14, wherein the CAR further comprises a costimulatory domain.

16. The method of claim 15, wherein the costimulatory domain is a 4-1 BB, CD28, IGOS, OX-40, BTLA, CD27, CD30, GITR, or HVEM costimulatory domain.

17. The method of any one of claims 11 -16, wherein the transmembrane domain is a CD8, Megfl O, FcRy, Bail , MerTK, TIM4, Stabilin-1 , Stabilin-2, RAGE, CD300f, integrin subunit av, integrin subunit 5, CD36, LRP1 , SCARF1 , C1 Qa, Axl, CD45, or CD86 transmembrane domain.

18. The method of any one of claims 11 -17, wherein the target antigen is on a cancer cell, a tumor cell, an activated fibroblast, an autoreactive immune cell, a pathogen, or a diseased cell.

19. The method of claim 18, wherein the target antigen is a tumor antigen or a tumor-associated antigen.

20. The method of claim 18, wherein the pathogen is a virus, a bacterium, a fungus, or a parasite.

21 . The method of claim 20, wherein the target antigen is a viral antigen, a bacterial antigen, a fungal antigen or a parasite antigen.

22. The method of claim 18, wherein the autoreactive immune cell is an autoreactive T cell or B cell.

23. The method of claim 22, wherein the target antigen is an antigen on the autoreactive T cell or B cell.

24. The method of any one of claims 1 -23, wherein the second genomic target locus is a T cell receptor alpha chain locus, T cell receptor beta chain locus, T cell receptor delta chain locus, or a T cell receptor gamma chain locus.

25. The method of any one of claims 1 -23, wherein the second genomic target locus encodes a cytokine.

26. The method of claim 25, wherein the cytokine is GM-CSF or IL-6

27. The method of any one of claims 1 -23, wherein the second genomic target locus encodes an alloantigen.

28. The method of claim 27, wherein the alloantigen is a major histocompatibility complex (MHC) class I alloantigen or an MNS blood group alloantigen.

29. The method of claim 27, wherein the alloantigen is CD1 , CD2, CD3, CD4, CD7, CD8, Ly-6, Qa-2, RT6, CD19, CD22, CD56, CD58 (LFA-3), OD59, or CDw90 (Thy 1 ).

30. The method of any one of claims 1 -23, wherein the second genomic target locus comprises a CD5, CD52, CD70, BATF, LCK, PD-1 , LAG-3, CTLA-4, 2-B2M, PD- 1 , HLA-I, Fas, TGFBR2, PDCD-1 , DGK, EZH2, PAX5, or LDLR gene.

31. The method of any one of claims 1 -30, wherein the donor polynucleotide and/or the RNA-guided nuclease is provided by a vector.

32. The method of claim 31 , wherein the vector is a viral vector or a plasmid.

33. The method of claim 32, wherein the viral vector is a lentivirus vector, retrovirus vector, or adeno-associated virus vector

34. The method of any one of claims 31 -33, wherein the vector is introduced into the cell by transient transfection or stable transfection.

35. The method of claim 34, wherein the vector is introduced into the cell by electroporation, nucleofection, or lipofection.

36. The method of any one of claims 31 -35, wherein the donor polynucleotide and the RNA-guided nuclease are provided by separate vectors.

37. The method of any one of claims 31 -35, wherein the donor polynucleotide and the RNA-guided nuclease are provided by the same vector.

38. The method of any one of claims 31 -37, wherein expression of the RNA- guided nuclease is inducible.

39. The method of any one of claims 1-30, wherein the RNA-guided nuclease is provided by a mRNA encoding the RNA-guided nuclease, wherein translation of the mRNA results in production of the RNA-guided nuclease in the cell.

40. The method of any one of claims 1-39, wherein the RNA-guided nuclease is a clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) nuclease.

41 . The method of claim 40, wherein the Cas nuclease is Cas9 or Cas12a.

42. The method of any one of claims 1 -41 , wherein the donor polynucleotide further comprises a barcode.

43. The method of any one of claims 1 -41 , wherein the donor polynucleotide further comprises a gene knockin module comprising a plurality of coding sequences encoding a plurality of polypeptides.

44. The method of any one of claims 1 -43, wherein the donor polynucleotide further comprises a sequence encoding a knockout module comprising a plurality of knockout guide RNAs.

45. The method of claim 44, wherein at least 2 knockout guide RNAs of the plurality bind to different genomic target sequences in different target genes to generate gene knockouts of more than one target gene.

46. The method of claim 44 or 45, wherein at least 2 knockout guide RNAs of the plurality bind to different genomic target sequences in the same target gene to increase a rate of gene knockout of the target gene compared to the rate of gene knockout using only one knockout guide RNA.

47. The method of any one of claims 44-46, wherein the first mRNA transcript comprises: a protein coding reading frame encoding the exogenous polypeptide followed by a stop codon; and a 3’-untranslated region comprising: a knockout module flanked by a first spacer sequence and a second spacer sequence, wherein the knockout module comprises a plurality of knockout guide RNAs, wherein each guide RNA is preceded by a synthetic separator sequence followed by a direct repeat sequence; a mRNA stabilizing element, wherein the mRNA stabilizing element is positioned between the stop codon and the first spacer sequence; and a polyadenylation sequence.

48. The method of claim 47, wherein the mRNA stabilizing element is a triplex stabilizer.

49. The method of any one of claims 1 -48, wherein the transcription of the integrated donor polynucleotide is performed by an RNA polymerase II (Pol II).

50. The method of any one of claims 1 -49, wherein the knockout guide RNAs are Cas12a guide RNAs.

52. The composition of claim 51 , wherein the RNA-guided nuclease is a clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Gas) nuclease.

53. The composition of claim 52, wherein the Gas nuclease is Cas9 or Cas12a.

54. The composition of any one of claims 51 -52, wherein the donor polynucleotide further comprises a gene knockin module comprising a plurality of coding sequences encoding a plurality of polypeptides.

55. The composition of any one of claims 51 -54, wherein the donor polynucleotide further comprises a sequence encoding a knockout module comprising a plurality of knockout guide RNAs.

56. The composition of claim 55, wherein at least 2 knockout guide RNAs of the plurality bind to different genomic target sequences in different target genes to generate gene knockouts of more than one target gene.

57. The composition of claim 55 or 56, wherein at least 2 knockout guide RNAs of the plurality bind to different genomic target sequences in the same target gene to increase a rate of gene knockout of the target gene compared to the rate of gene knockout using only one knockout guide RNA.

58. The composition of any one of claims 55-57, wherein the first mRNA transcript comprises: a protein coding reading frame encoding the exogenous polypeptide followed by a stop codon; and a 3’-untranslated region comprising: a knockout module flanked by a first spacer sequence and a second spacer sequence, wherein the knockout module comprises a plurality of knockout guide RNAs, wherein each guide RNA is preceded by a synthetic separator sequence followed by a direct repeat sequence; a mRNA stabilizing element, wherein the mRNA stabilizing element is positioned between the stop codon and the first spacer sequence; and a polyadenylation sequence.

59. The composition of claim 58, wherein the mRNA stabilizing element is a triplex stabilizer.

60. The composition of any one of claims 51 -59, wherein the transcription of the integrated donor polynucleotide is performed by an RNA polymerase II (Pol II).

61 . The composition of any one of claims 55-60, wherein the knockout guide RNAs are Cas12a guide RNAs.

62. A kit comprising the composition of any one of claims 51 -61 and instructions for producing a genetically modified T cell expressing a chimeric antigen receptor.

63. The kit of claim 62, further comprising a transfection agent.

64. A genetically modified cell produced according to the method of any one of claims 1 -50.

65. A composition comprising the genetically modified cell of claim 64 and a pharmaceutically acceptable excipient or carrier.

66. A method of performing cellular therapy, the method comprising administering a therapeutically effective amount of the composition of claim 65 to a subject.

67. The method of claim 66, wherein the genetically modified cell is autologous or allogeneic.

68. The method of claim 66 or 67, wherein the genetically modified cell is an immune cell.

69. The method of claim 68, wherein the immune cell is a T cell, a B cell, a natural killer cell, a neutrophil, an eosinophil, a mast cell, a basophil, a monocyte, a macrophage, or a dendritic cell.

70. The method of claim 69, wherein the T cell is a helper CD4⁺ T cell, a cytotoxic CD8⁺ T cell, a natural killer T cell, or a gamma delta T cell.

71 . The method of any one of claims 68-70, wherein the exogenous protein is a chimeric antigen receptor (CAR) that specifically binds to a target antigen.