Cells ectopically expressing FOXP3

Abstract

Provided are expression cassettes that comprise a FOXP3 protein, a chimeric receptor, and / or a cell surface receptor. Also provided herein are Treg cells comprising the expression cassettes and methods of use.

Description

CELLS ECTOPICALLY EXPRESSING FOXP3Cross-Reference To Related Applications

[0001] This application claims the benefit of, and priority to, U.S. Provisional Application 63 / 581,378, filed on September 8, 2023, and U.S. Provisional Application 63 / 594,346, filed on October 30, 2023, the contents of each of which are hereby incorporated herein by reference in their entirety.Reference to an Electronic Sequence Listing

[0002] The contents of the electronic sequence listing (237752001040seqlist.xml; Size: 95,606 bytes; and Date of Creation: August 31, 2024) is herein incorporated by reference in its entirety.Background

[0003] The immune system plays a critical role in maintaining organismal homeostasis, poised between the elimination of foreign antigens and the self-tolerance of autoantigens. In particular, hyperactive immune dysregulation can lead to various autoimmune disorders (e.g., irritable bowel syndrome, systemic lupus erythematosus, alopecia areata, multiple sclerosis), which is often the result of overactive effector T lymphocytes or underactive regulatory T lymphocytes (z.e., Tregs). Current therapies for autoimmune diseases involve the administration of steroids, which can cause serious side-effects in patients and often offer little relief to patients.

[0004] Regulatory T cells (Tregs) are a key player in the maintenance of organismal homeostasis to prevent the destruction of otherwise healthy tissues. Tregs are a unique subset of T cells that inhibit the cytotoxic or pro-inflammatory activity of effector CD4+ or effector CD8+ T cells. Tregs differentiate from the parent T lymphocyte lineage upon the upregulation of key Treg genes, in particular CD25 (z.e., IL-2Ra) and FOXP3 (see, e.g., Chen, ML et al. (2005), Proc Natl Accad Sci USA; 102(2):419-424 and Liu, VC et al. (2007), J Immunol; 178(5):2883- 2892, hereby incorporated by reference in their entirety). Upon T cell receptor (TCR) activation, these Tregs are responsible for directly suppressing effector T cell activity via cytokine production, e.g., TGF-P and IL-10 (see, e.g., Chen, J et al. (2019), Trends Mol Med;25(11): 1010-1023, hereby incorporated by reference in its entirety), and the engagement of immune checkpoint receptors, e.g., TIGIT- or CTLA-4-engagement (see, e.g., Knochelmann,HM et al. (2018), Cell Mol Immunol', 15(5):458-469, hereby incorporated by reference in its entirety).

[0005] FOXP3 is a key determinant for Treg lineage, stability, and function. FOXP3 acts as a master transcription factor driving the genetic programming of Tregs, wherein it primarily acts as a transcriptional repressor of key genes involved in T cell activation and effector functions, including proliferation and synthesis of proinflammatory cytokines (e.g., IL-2, IL-4, IL-17A, and IFNy). FOXP3 is initially expressed during the expansion of T cells for a first time but is followed by the loss of FOXP3 expression after polyclonal stimulation. This is in contrast to Tregs, where FOXP3 expression rises and is maintained over time. Maintenance of Treg immunophenotypes and suppressive functions would enhance the efficacy and safety of the drug products.Summary

[0006] In one aspect, the present application relates to exogenous expression cassettes comprising a modified nucleic acid encoding a FOXP3 protein (e.g., full-length FOXP3 protein and / or delta (A)2 FOXP3 that lacks the amino acids corresponding to exon 2). Also provided herein are cells (e.g., Tregs or effector T cells) that express the exogenous expression cassette comprising a modified nucleic acid encoding a FOXP3 protein operably linked to a promoter and methods of use.

[0007] In one aspect provided herein is an engineered regulatory T cell (Treg) comprising an exogenous expression cassette comprising a modified nucleic acid encoding a FOXP3 protein operably linked to a promoter, wherein the nucleic acid encoding the FOXP3 protein is modified to produce a full-length FOXP3 protein isoform and an alternatively spliced isoform lacking one or more exons. In some embodiments, the nucleic acid encoding the FOXP3 protein comprises intron sequences flanking nucleic acid encoding exon 2. In some embodiments, the nucleic acid encoding the FOXP3 protein is alternatively spliced to produce a full-length FOXP3 protein isoform and a FOXP3 protein isoform lacking exon 2.

[0008] In some embodiments according to any of the engineered Tregs provided herein, the engineered Treg further comprising a nucleic acid encoding a chimeric receptor. In some embodiments, the exogenous expression cassette comprises the nucleic acid encoding the chimeric receptor. In some embodiments, the nucleic acid encoding the FOXP3 protein and the nucleic acid encoding the chimeric receptor are operably linked to the same promoter. In some embodiments, the exogenous expression cassette comprises an internal ribosome entry site(IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping between the nucleic acid encoding the FOXP3 protein and the nucleic acid encoding the chimeric receptor. In some embodiments, the nucleic acid encoding the FOXP3 protein is 5’ or 3’ of the nucleic acid encoding the chimeric receptor.

[0009] In some embodiments according to any of the engineered Tregs provided herein, the exogenous expression cassette further comprises a nucleic acid encoding a cell surface receptor. In some embodiments, the nucleic acid encoding the cell surface receptor is located 5’ or 3’ of the nucleic acid encoding the FOXP3 protein. In some embodiments, the exogenous expression cassette comprises an IRES, or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping between the nucleic acid encoding the cell surface receptor and the nucleic acid encoding the FOXP3 protein. In some embodiments, the exogenous expression cassette comprises an IRES, or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping between the nucleic acid encoding the cell surface receptor and the nucleic acid encoding the chimeric receptor.

[0010] In some embodiments according to any of the engineered Tregs provided herein, the exogenous expression cassette comprises from 5’ to 3’: a) the promoter; b) the modified nucleic acid encoding the FOXP3 protein optionally linked to a detection tag, wherein the nucleic acid encoding the FOXP3 protein comprises intron sequences flanking the nucleic acid encoding exon 2; c) an IRES, or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; and d) the nucleic acid encoding the cell surface receptor.

[0011] In some embodiments according to any of the engineered Tregs provided herein, the exogenous expression cassette comprises from 5’ to 3’: a) the promoter; b) the nucleic acid encoding the cell surface receptor; c) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; and d) the modified nucleic acid encoding the FOXP3 protein, wherein the modified nucleic acid encoding the FOXP3 protein comprises intron sequences flanking the nucleic acid encoding exon 2.

[0012] In some embodiments according to any of the engineered Tregs provided herein, the exogenous expression cassette comprises from 5’ to 3’: a) the promoter; b) the nucleic acid encoding the chimeric receptor; c) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; d) the nucleic acid encoding the cell surface receptor; e) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; and f) the modifiednucleic acid encoding the FOXP3 protein, wherein the nucleic acid encoding a FOXP3 protein comprises intron sequences flanking the nucleic acid encoding exon 2.

[0013] In some embodiments according to any of the engineered Tregs provided herein, the exogenous expression cassette comprises from 5’ to 3’: a) the promoter; b) the nucleic acid encoding the chimeric receptor; c) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; d) the modified nucleic acid encoding the FOXP3 protein, wherein the nucleic acid encoding a FOXP3 protein comprises intron sequences flanking the nucleic acid encoding exon 2; e) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; and f) the nucleic acid encoding the cell surface receptor.

[0014] In some embodiments according to any of the engineered Tregs provided herein, the exogenous expression cassette comprises from 5’ to 3’: a) the promoter; b) the modified nucleic acid encoding the FOXP3 protein, wherein the nucleic acid encoding a FOXP3 protein comprises intron sequences flanking the nucleic acid encoding exon 2; c) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; d) the nucleic acid encoding the chimeric receptor; e) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; and f) the nucleic acid encoding the cell surface receptor.

[0015] In another aspect provided herein is an engineered Treg comprising an exogenous expression cassette comprising from 5’ to 3’: a) a promoter; b) a nucleic acid encoding a chimeric receptor; c) an internal ribosome entry site (IRES), or a nucleic acid encoding a selfcleaving peptide or a peptide that causes ribosome skipping; d) a nucleic acid encoding a FOXP3 protein, wherein the nucleic acid encoding a FOXP3 protein is FOXP3 cDNA; e) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; and f) a nucleic acid encoding a cell surface receptor.

[0016] Also provided herein is an engineered Treg comprising an exogenous expression cassette comprising from 5’ to 3’: a) a promoter; b) a nucleic acid encoding a FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein is FOXP3 cDNA; c) an IRES, or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; and d) a nucleic acid encoding a cell surface receptor.

[0017] Also provided herein is an engineered Treg comprising an exogenous expression cassette comprising from 5’ to 3’: a) a promoter; b) nucleic acid encoding a cell surface receptor; c) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleavingpeptide or a peptide that causes ribosome skipping; and d) a nucleic acid encoding a FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein is FOXP3 cDNA.

[0018] Also provided herein is an engineered Treg comprising an exogenous expression cassette comprising from 5’ to 3’: a) a promoter; b) a nucleic acid encoding a FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein is FOXP3 cDNA; c) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; d) a nucleic acid encoding a chimeric receptor; e) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; and f) a nucleic acid encoding a cell surface receptor.

[0019] Also provided herein is an engineered Treg comprising an exogenous expression cassette comprising from 5’ to 3’: a) a promoter; b) a nucleic acid encoding a chimeric receptor; c) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; d) a nucleic acid encoding a cell surface receptor; e) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; and f) a nucleic acid encoding a FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein is FOXP3 cDNA.

[0020] Also provided herein is an engineered Treg comprising an exogenous expression cassette comprising from 5’ to 3’: a) a promoter; b) a nucleic acid encoding a FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein does not comprise a nucleic acid sequence encoding exon 2; c) an IRES, or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; and d) a nucleic acid encoding a cell surface receptor.

[0021] In some embodiments according to any of the engineered Tregs provided herein, the nucleic acid encoding the FOXP3 protein does not contain intron sequences other than the intron sequences flanking exon 2.

[0022] In some embodiments according to any of the engineered Tregs provided herein, the cell surface receptor lacks a functional intracellular signaling domain or is not capable of mediating intracellular signaling. In some embodiments, the cell surface receptor is selected from the group consisting of EGFR, HER2, CD2, H-2Kk, IL-5Ra, and derivatives or mutants thereof.

[0023] In some embodiments according to any of the engineered Tregs provided herein, the chimeric receptor comprises a chimeric antigen receptor, a T cell Receptor, or a PiG receptor. In some embodiments, the chimeric receptor binds to an antigen selected from the group consisting of MBP, MOG, PLP, PDE4, VEGFR, S 100- , Citrullinated vimentin (CV), TNF-like ligand 1A(TL1A), CD2, CD23 (IgE receptor), IL-6R, IL-4Ra, IL-12R, IL-13Ral, IL-13Ra2, IL-15R, IL- 23R, CD19, CD20, CD22, CD3, CD4, CD5, IFN-y, IgE, IL-la, IL-ip, IL-6, IL-12, IL-23, IL-15, IL-17, IL-17A, IL-22, IL-13, IL-33, TSLP, OX-40, OX-40L, p53, CD38, CD40L, CD28, ICOS, RIPK1, BTK, Bet v 1, GLP2R, JAK1, C5, and TNE-a.

[0024] In some embodiments according to any of the engineered Tregs provided herein, the nucleic acid encoding the self-cleaving peptide is selected from the group consisting of a P2A, T2A, E2A, and E2A sequence.

[0025] In some embodiments according to any of the engineered Tregs provided herein, the promoter is an inducible or a constitutive promoter. In some embodiments, the promoter is a cell-specific promoter. In some embodiments, the promoter is specific for T cells.

[0026] In some embodiments according to any of the engineered Tregs provided herein, the engineered Treg expresses endogenous EOXP3 protein. In some embodiments, the endogenous EOXP3 protein does not comprise a detection tag.

[0027] In some embodiments according to any of the engineered Tregs provided herein, the exogenous expression cassette is in a vector. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a lentiviral vector. In some embodiments, the exogenous expression cassette is in a chromosome of the Treg.

[0028] In some embodiments according to any of the engineered Tregs provided herein, the engineered Treg expresses a higher level of EOXP3 protein compared to a Treg lacking the exogenous expression cassette.

[0029] In some embodiments according to any of the engineered Tregs provided herein, the engineered Treg expresses one or more markers that are expressed by a wildtype Treg or a control Treg lacking exogenous EOXP3 protein; and / or the engineered Treg does not express one or more markers that are not expressed by a wildtype Treg or a control Treg lacking exogenous EOXP3 protein. In some embodiments, the one or more markers is expressed at similar protein and / or gene levels as in a wildtype Treg or a control Treg lacking exogenous LOXP3 protein. In some embodiments, the engineered Treg expresses a similar ratio of full- length LOXP3 protein to an alternatively spliced LOXP3 protein isoform lacking one or more exons as a wildtype Treg or a control Treg lacking exogenous LOXP3 protein.

[0030] In another aspect is provided herein an expression cassette comprising a nucleic acid encoding a LOXP3 protein and a cell surface receptor, wherein the nucleic acid encoding the LOXP3 protein is modified to produce a full-length LOXP3 protein isoform and an alternativelyspliced isoform lacking one or more exons. In some embodiments, the nucleic acid encoding the FOXP3 protein comprises intron sequences flanking the nucleic acid encoding exon 2.

[0031] In some embodiments according to any of the expression cassettes provided herein, the expression cassette further comprises a nucleic acid encoding a chimeric receptor. In some embodiments, the nucleic acid encoding the FOXP3 protein and the nucleic acid encoding the chimeric receptor are operably linked to the same promoter. In some embodiments, the expression cassette comprises an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping between the nucleic acid encoding the FOXP3 protein and the nucleic acid encoding the chimeric receptor.

[0032] In some embodiments according to any of the expression cassettes provided herein, the expression cassette further comprises a nucleic acid encoding a cell surface receptor. In some embodiments, the expression cassette comprises an IRES, or a nucleic acid encoding a selfcleaving peptide or a peptide that causes ribosome skipping between the nucleic acid encoding the cell surface receptor and the nucleic acid encoding the FOXP3 protein. In some embodiments, the expression cassette comprises an IRES, or a nucleic acid encoding a selfcleaving peptide or a peptide that causes ribosome skipping between the nucleic acid encoding the cell surface receptor and the nucleic acid encoding the chimeric receptor.

[0033] In some embodiments according to any of the expression cassettes provided herein, the expression cassette comprises from 5’ to 3’: a) the promoter; b) the modified nucleic acid encoding the FOXP3 protein optionally linked to a detection tag, wherein the nucleic acid encoding the FOXP3 protein comprises intron sequences flanking the nucleic acid encoding exon 2; c) an IRES, or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; and d) the nucleic acid encoding the cell surface receptor.

[0034] In some embodiments according to any of the expression cassettes provided herein, the expression cassette comprises from 5’ to 3’: a) the promoter; b) the nucleic acid encoding the cell surface receptor; c) an internal ribosome entry site (IRES), or a nucleic acid encoding a selfcleaving peptide or a peptide that causes ribosome skipping; and d) the modified nucleic acid encoding the FOXP3 protein, wherein the modified nucleic acid encoding the FOXP3 protein comprises intron sequences flanking the nucleic acid encoding exon 2.

[0035] In some embodiments according to any of the expression cassettes provided herein, the expression cassette comprises from 5’ to 3’: a) the promoter; b) the nucleic acid encoding the chimeric receptor; c) an internal ribosome entry site (IRES), or a nucleic acid encoding a selfcleaving peptide or a peptide that causes ribosome skipping; d) the nucleic acid encoding the cellsurface receptor; e) an internal ribosome entry site (IRES), or a nucleic acid encoding a selfcleaving peptide or a peptide that causes ribosome skipping; and f) the modified nucleic acid encoding the FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein comprises intron sequences flanking the nucleic acid encoding exon 2.

[0036] In some embodiments according to any of the expression cassettes provided herein, the expression cassette comprises from 5’ to 3’: a) the promoter; b) the nucleic acid encoding the chimeric receptor; c) an internal ribosome entry site (IRES), or a nucleic acid encoding a selfcleaving peptide or a peptide that causes ribosome skipping; d) the modified nucleic acid encoding the FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein comprises intron sequences flanking the nucleic acid encoding exon 2; e) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; and f) the nucleic acid encoding the cell surface receptor.

[0037] In some embodiments according to any of the expression cassettes provided herein, the expression cassette comprises from 5’ to 3’: a) the promoter; b) the modified nucleic acid encoding the FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein comprises intron sequences flanking the nucleic acid encoding exon 2; c) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; d) the nucleic acid encoding the chimeric receptor; e) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; and f) the nucleic acid encoding the cell surface receptor.

[0038] In another aspect provided herein is an expression cassette comprising from 5’ to 3’: a) a promoter; b) a nucleic acid encoding a chimeric receptor; c) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; d) a nucleic acid encoding a FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein is FOXP3 cDNA; e) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; and f) a nucleic acid encoding a cell surface receptor.

[0039] Also provided herein is an expression cassette comprising from 5’ to 3’: a) a promoter; b) a nucleic acid encoding a FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein is FOXP3 cDNA; c) an IRES, or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; and d) a nucleic acid encoding a cell surface receptor.

[0040] Also provided herein is an expression cassette comprising from 5’ to 3’: a) a promoter; b) a nucleic acid encoding a cell surface receptor; c) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; and d) a nucleic acid encoding a FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein is FOXP3 cDNA.

[0041] Also provided herein is an expression cassette comprising from 5’ to 3’: a) a promoter; b) a nucleic acid encoding a FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein is FOXP3 cDNA; c) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; d) a nucleic acid encoding a chimeric receptor; e) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; and f) a nucleic acid encoding a cell surface receptor.

[0042] Also provided herein is an expression cassette comprising from 5’ to 3’: a) a promoter; b) a nucleic acid encoding a chimeric receptor; c) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; d) a nucleic acid encoding a cell surface receptor; e) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; and f) a nucleic acid encoding a FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein is FOXP3 cDNA.

[0043] Also provided herein is an expression cassette comprising from 5’ to 3’: a) a promoter; b) a nucleic acid encoding a FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein does not comprise a nucleic acid sequence encoding exon 2; c) an IRES, or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; and d) a nucleic acid encoding a cell surface receptor.

[0044] In some embodiments according to any of the expression cassettes provided herein, the nucleic acid encoding the FOXP3 protein does not contain intron sequences other than the intron sequences flanking exon 2. In some embodiments, the cell surface receptor lacks a functional intracellular signaling domain or is not capable of mediating intracellular signaling, optionally wherein the cell surface receptor is selected from the group consisting of EGFR, HER2, CD2, H-2Kk, IL-5Ra, and derivatives or mutants thereof. In some embodiments, the chimeric receptor comprises a chimeric antigen receptor, a T cell Receptor, or a PiG receptor, optionally wherein the chimeric receptor binds to an antigen selected from the group consisting of MBP, MOG, PLP, PDE4, VEGFR, S 100- , Citrullinated vimentin (CV), TNF-like ligand 1A(TL1A), CD2, CD23 (IgE receptor), IL-6R, IL-4Ra, IL-12R, IL-13Ral, IL-13Ra2, IL-15R, IL- 23R, CD19, CD20, CD22, CD3, CD4, CD5, IFN-y, IgE, IL-la, IL-ip, IL-6, IL-12, IL-23, IL-15, IL-17, IL-17A, IL-22, IL-13, IL-33, TSLP, OX-40, OX-40L, p53, CD38, CD40L, CD28, ICOS, RIPK1, BTK, Bet v 1, GLP2R, JAK1, C5, and TNL-a. In some embodiments, the nucleic acid encoding the self-cleaving peptide is selected from the group consisting of a P2A, T2A, E2A, and E2A sequence. In some embodiments, the promoter is an inducible promoter or a constitutive promoter. In some embodiments, the promoter is a cell- specific promoter, optionally wherein the promoter is specific for T cells.

[0045] In some embodiments according to any of the expression cassettes and / or engineered Tregs provided herein, a) the EOXP3 protein comprises the amino acid sequence of SEQ ID NO:20 or 22; b) the EOXP3 protein comprises the amino acid sequence of SEQ ID NO:21 or 23; c) the chimeric receptor comprises an antigen binding domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:73-76; d) the cell surface receptor comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:17-19; and / or e) the IRES, or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:2-10 and 89-91.

[0046] In some embodiments according to any of the expression cassettes provided herein, the expression cassette is in a vector, optionally, wherein the vector is a viral vector. In some embodiments, the expression cassette is integrated into a chromosome of a Treg.

[0047] Also provided herein are methods of increasing persistence and / or cytokine signaling of a Treg, comprising providing any of the expression cassettes described herein to the Treg, wherein the persistence and / or cytokine signaling of the Treg is increased compared to a Treg that does not comprise the expression cassette. In some embodiments, the expression cassette is provided to the Treg by transducing the Treg with a vector comprising the expression cassette. In some embodiments, the method further comprises measuring the level of one or more cytokines produced by the engineered Treg and comparing the level to the level of the one or more cytokines produced by a wildtype Treg or a control Treg lacking exogenous FOXP3 protein. In some embodiments, the one or more cytokines comprises TNF-a, IFN-y, IL- 10, IL-4, IL-2, IL- 17 A, any functional derivatives thereof, and any combination thereof. In some embodiments, the one or more cytokines comprises at least IL- 10, and wherein the level of IL- 10 protein and / or gene expression is about the same or increased in the engineered Tregcompared to the wildtype Treg or the control Treg lacking exogenous FOXP3 protein under non-inflammatory conditions.

[0048] Also provided herein are methods of treating an immune related disorder in an individual, comprising administering any of the Tregs described herein to an individual in need thereof.

[0049] Also provided herein are methods of generating a Treg, comprising transducing an effector T cell (Teff) with any of the expression cassettes described herein. Also provided herein are methods of overexpressing FOXP3 protein in a Treg, comprising transducing the Treg with any of the expression cassettes described herein. In some embodiments, the methods described herein further comprises measuring the level of one or more markers in the engineered Treg and comparing the level of the one or more markers to a wildtype Treg or a control Treg lacking exogenous FOXP3 protein. In some embodiments, the one or more markers comprises CD27, CD70, CD71, Helios, glucocorticoid-induced TNFR-related protein (GITR), latency-associated peptide (LAP), glycoprotein A repetitions predominant (GARP), CD137, and any combination thereof. In some embodiments, the methods described herein further comprise measuring the level of one or more cytokines produced by the engineered Treg and comparing the level to the level of the one or more cytokines produced by a wildtype Treg or a control Treg lacking exogenous FOXP3 protein. In some embodiments, the one or more cytokines comprises TNF-a, IFN-y, IL- 10, IL-4, IL-2, IL-17A, any functional derivatives thereof, and any combination thereof. In some embodiments, the one or more cytokines comprises at least IL- 10, and wherein the level of IL- 10 protein and / or gene expression is about the same or increased in the engineered Treg compared to the wildtype Treg or the control Treg lacking exogenous FOXP3 protein under non-inflammatory conditions.Brief Description of the Drawings

[0050] FIGs. 1A-1E show structures of the FOXP3 gene and exemplary multicistronic constructs. FIG. 1A depicts the functions and gene structure of human FOXP3, its different splicing isoforms (full length (FL), lacking exon 2 (A2), lacking exon 2 and exon 7 (A2A7), and lacking exon 7 (A7)), and the relative abundances of each isoform. FIG. IB depicts an engineered FOXP3 construct to allow for alternative splicing (FL + A2; test), the full length FOXP3 isoform (FL; control), and a FOXP3 isoform lacking exon 2 (A2; control). FIG. 1C depicts a T cell transduced with a multi-cistronic transgene expression vector carrying FOXP3. The vector facilitates ectopic expression of FOXP3 (ecFOXP3), distinct from endogenous levelsof F0XP3 naturally expressed in T cells. The vector encodes an epitope-tagged, engineered F0XP3 construct and optionally a CAR molecule. FIG. ID depicts an exemplary full-length multicistronic cassette that can be transduced into a cell (e.g., T cell, such as a Treg cell). FIG. IE depicts exemplary multicistronic cassettes comprising an engineered FOXP3 construct to allow for alternative splicing (FL + A2; test), the full length FOXP3 isoform (FL; control), or a chimeric antigen receptor without a FOXP3 construct (z.e., negative control).

[0051] FIGs. 2A-2C depict a method of detecting ectopic FOXP3 expression to distinguish between endogenous FOXP3 and ectopic FOXP3 after vector transduction into T cells. The vector encodes an epitope-tagged FOXP3 construct that can be detected with either an anti-HA (only ectopic FOXP3) or anti-FOXP3 (endogenous and ectopic FOXP3) antibody. FIG. 2A depicts a mock flow plot of untransduced T cells indicating levels of only endogenous FOXP3 expression. FIG. 2B depicts a mock flow plot of transduced T cells indicating levels of both endogenous FOXP3 expression and ectopic FOXP3 expression. FIG. 2C depicts a mock Western blot indicating the specificity of the anti-HA and anti-FOXP3 antibodies for ectopic and endogenous variants of FOXP3. FIGs. 2D-2E depict a method of detecting different isoforms of FOXP3 using different anti-FOXP3 antibody clones. FIG. 2D depicts that all clones except 150D / E4 (PCH101, FJK-16s, eBio7979, 236A / E7, 206D, 259D) can detect all isoforms of FOXP3. 150D / E4 cannot detect the FOXP3 A2 isoform. FIG. 2E depicts a mock flow plot of transduced T cells indicating detection of different FOXP3 isoforms with different anti-FOXP3 antibodies (150D / E4, PCH101). FIG. 2F depicts the results of immunoblotting with different anti-FOXP3 antibodies (236A / E7, 259D, 206D, 150D / E4) indicating that the 150D / E4 clone is capable of detecting only the full length FOXP3 isoform, not A2.

[0052] FIGs. 3A-3S depict the results of immuno staining and flow cytometry following transduction of Treg cells with vectors encoding different constructs of FOXP3 (FIGs. 3A-3D: pSB_0521, FIGs. 3E-3H: pSB_0858; FIGs. 3L3L: pSB_0859; FIGs. 3M-3P: pSB_0965). FIG. 3Q depicts an overlay plot representing data from FIG. 3D, FIG. 3H, FIG. 3L, and FIG. 3P. FIG. 3R depicts mean fluorescence intensity data from FIG. 3B (pSB_0521), FIG. 3F (pSB_0858), FIG. 3J (pSB_0859), and FIG. 3N (pSB_0965). FIG. 3S depicts mean fluorescence intensity data from HA+ cells in FIG. 3A (pSB_0521), FIG. 3E (pSB_0858), FIG. 31 (pSB_0859), and FIG. 3M (pSB_0965), and from EGFR+ cells in FIG. 3C (pSB_0521), FIG. 3G (pSB_0858), FIG. 3K (pSB_0859), and FIG. 30 (pSB_0965).

[0053] FIGs. 4A-4Q depict the results of immuno staining and flow cytometry following transduction of Teff cells with vectors encoding different constructs of FOXP3 (FIGs. 4A-4E:pSB_0858; FIGs 4F-4J: pSB_0859; FIGs. 4K-4O: pSB_0965). FIG. 4P depicts an overlay plot representing data from FIG. 4D, FIG. 41, and FIG. 4N. FIG. 4Q depicts an overlay plot representing data from FIG. 4E, FIG. 4J, and FIG. 40.

[0054] FIGs. 5A-5B depict the results of transduction of T cells with vectors encoding different constructs of FOXP3, and resulting expression levels of FOXP3 measured via immuno staining and flow cytometry. FIG. 5A depicts the results of transduction of Teff cells with vectors encoding different constructs of FOXP3. FIG. 5B depicts the results of transduction of Treg cells with the same set of expression vectors, as in FIG. 5A.

[0055] FIGs. 6A-6B depict the results of transduction of T cells with vectors encoding different constructs of FOXP3, and resulting expression levels of cytokines. FIG. 6A depicts the results of transduction of Teff cells with vectors encoding different constructs of FOXP3. FIG. 6B depicts the results of transduction of Treg cells with the same set of expression vectors, as in FIG. 6A.

[0056] FIG. 7 depicts the results of transduction of Teff and Treg cells with vectors encoding different constructs of FOXP3, placed in different locations within the plasmid compared to the location of CAR. Bar graphs depict cytokine production in transduced cells after staining for FOXP3 using the PCH101 antibody clone.

[0057] FIGs. 8A-8E depict the results of transduction of Treg cells with vectors encoding different constructs of FOXP3, and resulting expression levels of FOXP3 measured via immuno staining and flow cytometry. FIG. 8A depicts the levels of FOXP3 and HA in untransduced control Tregs. FIG. 8B shows the levels of FOXP3 and HA (left panel) and FOXP3 levels in HA -positive (HA+) (right panel) Treg cells transduced with a vector encoding full-length FOXP3 construct. FIG. 8C shows the levels of FOXP3 and HA (left panel) and FOXP3 levels in HA -positive (HA+) (right panel) Treg cells transduced with a vector encoding the dual full-length / delta 2 FOXP3 construct. FIG. 8D shows the levels of FOXP3 and HA (left panel) and FOXP3 levels in HA-positive (HA+) (right panel) Treg cells transduced with a vector encoding the delta 2 FOXP3 construct. FIG. 8E provides graphical representation of the geometric mean fluorescence intensity (gMFI) of FOXP3 (PCH101 or 150D / E)-positive staining as well as representative histogram plots overlaying the full-length FOXP3 construct, dual FOXP3 construct, and A2 FOXP3 construct per each antibody (z.e., PCH101 or 150D / E).

[0058] FIGs. 9A-9B depict the ratio of full-length to delta 2 FOXP3 isoforms in Tregs in non-inflammation and inflammation conditions. FIG. 9A provides an overview of the experimental design and the vectors used. FIG. 9B shows the resulting ratio as depicted bypercentage breakdown of full-length (FL) to delta 2 (A2) F0XP3 isoforms produced in Tregs transduced with a CV CAR control vector, CV CAR with full-length FOXP3 vectors, and CV CAR with dual FL / A2 FOXP3 vectors under non-inflammation and inflammation conditions. FOXP3 150D clone staining was analyzed in the FOXP3 (PCHlOl)-positive population to determine the ratio between full-length and delta 2 FOXP3 isoform. In PCH101+ FOXP3+ cells, 150D-positive cells indicate the presence of the full-length FOXP3 and 150D-negative cells indicate the presence of the delta 2 FOXP3.

[0059] FIGs. 10A-10D depict the expression level of ectopic FOXP3 in Tregs after multiple rounds of activation and inflammation. FIG. 10A provides an overview of the experimental design and the vectors used. FIG. 10B provides the flow cytometric plots showing FOXP3 levels in TCR-stimulated Tregs transduced with CV CAR control vector, CV CAR with full- length FOXP3 vectors, and CV CAR with dual FL / A2 FOXP3 vectors. FIG. 10C provides the flow cytometric plots showing FOXP3 levels in Tregs transduced with CV CAR control vector, CV CAR with full-length FOXP3 vectors, and CV CAR with dual FL / A2 FOXP3 vectors after TCR- stimulation under inflammation conditions. FIG. 10D provides a heat map detailing the percentage of Tag-positive (Tag+) cells positive for each indicated markers measured at day 17 in transduced CAR Tregs in the presence and absence of inflammation.

[0060] FIGs. 11A-11C depict cytokine expression in Tregs transduced with CV CAR control vector, CV CAR with full-length FOXP3 vectors, and CV CAR with dual FL / A2 FOXP3 vectors and stimulated by PMA / ionomycin. FIG. 11A shows the percent of transduced Tregs expressing IL-2, IL-4, IL- 10, IL-17A, TNF-a, or IFN-y after being twice-activated by anti- CD3 / anti-CD28 antibodies for 14 days and then further subjected to PMA / ionomycin stimulation and monensin treatment. FIG. 11B shows the percent of transduced Tregs expressing IL-2, IL-4, IL- 10, IL-17A, TNF-a, or IFN-y after being cultured with inflammatory cytokines and twice-activated by anti-CD3 / anti-CD28 antibodies for 14 days and then further subjected to PMA / ionomycin stimulation and monensin treatment. FIG. 11C shows the percent of untransduced effector T cells that were positive for IL-2, IL-4, IL- 10, IL-17A, TNF-a, or IFN-y cytokine expression after PMA-stimulation on day 14.

[0061] FIGs. 12A-12D depict the Treg phenotype and expression levels of FOXP3 and Helios in transduced Tregs after multiple rounds of activation and inflammation. FIG. 12A shows the level of FOXP3 over time in Tregs transduced with multicistronic cassettes comprising an engineered FOXP3 construct to allow for alternative splicing (“CAR-dual”; test), the full length FOXP3 isoform (“CAR-FL”; control), or CAR without a FOXP3 construct (z.e.,“CAR-tag”; negative control) in the presence of high inflammation. FIG. 12B provides a heat map detailing the percentage of Tag-positive (Tag+) cells positive for each indicated markers measured at day 21 in transduced CAR Tregs in the presence of high inflammation. FIG. 12C shows the level of Helios over time in Tregs transduced with multicistronic cassettes comprising an engineered FOXP3 construct to allow for alternative splicing (“CAR-dual”; test), the full length FOXP3 isoform (“CAR- FL”; control), or CAR without a FOXP3 construct (z.e., “CAR- tag”; negative control) in the presence of high inflammation. FIG. 12D shows the percent of transduced Tregs expressing IL-2, IL-4, IL-17A, TNF-a, IFN-y, or IL- 10 after being twice- activated by anti-CD3 / anti-CD28 antibodies for 14 days and then further subjected to PMA / Ionomycin stimulation and monensin treatment in the presence of high inflammation.

[0062] FIGs. 13A-13D depict the Treg phenotype and expression levels of FOXP3 and Helios in transduced Tregs comprising an exemplary CAR with high tonicity after multiple rounds of activation and inflammation. FIG. 13A shows the level of FOXP3 over time in Tregs transduced with multicistronic cassettes comprising an engineered FOXP3 construct to allow for alternative splicing (“CAR-dual”; test), the full length FOXP3 isoform (“CAR-FL”; control), or CAR without a FOXP3 construct (z.e., “CAR-tag”; negative control) in the presence of high inflammation. FIG. 13B shows the level of Helios over time in Tregs transduced with multicistronic cassettes comprising an engineered FOXP3 construct to allow for alternative splicing (“CAR-dual”; test), the full length FOXP3 isoform (“CAR-FL”; control), or CAR without a FOXP3 construct (z.e., “CAR-tag”; negative control) in the presence of high inflammation. FIG. 13C provides a heat map detailing the percentage of Tag-positive (Tag+) cells positive for each indicated markers measured at day 21 in transduced CAR Tregs in the presence of high inflammation. FIG. 13D shows the percent of transduced Tregs expressing IL- 2, IL-4, IL- 10, IL-17A, TNF-a, or IFN-y, after being twice-activated by anti-CD3 / anti-CD28 antibodies for 21 days and then further subjected to PMA / Ionomycin stimulation and monensin treatment in the presence of high inflammation.

[0063] FIGs. 14A-14D depict the expression level of ectopic FOXP3 in Tregs transduced with exemplary multicistronic cassettes that comprise an exemplary CAR construct with high tonicity after multiple rounds of TCR activation in the presence and absence of high inflammation. FIG. 14A shows the level of FOXP3 over time in Tregs transduced with multicistronic cassettes comprising an engineered FOXP3 construct to allow for alternative splicing (“CAR-dual”; test), the full length FOXP3 isoform (“CAR-FL”; control), or CAR without a FOXP3 construct (z.e., “CAR-tag”; negative control) in the absence of highinflammation. FIG. 14B shows the level of FOXP3 over time in Tregs transduced with multicistronic cassettes comprising an engineered FOXP3 construct to allow for alternative splicing (“CAR-dual”; test), the full length FOXP3 isoform (“CAR-FL”; control), or CAR without a FOXP3 construct (z.e., “CAR-tag”; negative control) in the presence of high inflammation. FIG. 14C provides a heat map detailing the percentage of Tag-positive (Tag+) cells positive for each indicated markers measured at day 21 in transduced CAR Tregs in the absence of high inflammation. FIG. 14D provides a heat map detailing the percentage of Tagpositive (Tag+) cells positive for each indicated markers measured at day 21 in transduced CAR Tregs in the presence of high inflammation.

[0064] FIGs. 15A-15B depict cytokine expression in Tregs transduced with a second exemplary CAR control vector, the exemplary CAR with full-length FOXP3 vectors, and the exemplary CAR with dual FL / A2 FOXP3 vectors and stimulated by PMA / ionomycin in the presence and absence of inflammation. FIG. 15A shows the percent of transduced Tregs expressing IL-2, IL-4, IL- 10, IL-17A, TNF-a, or IFN-y after being twice-activated by anti- CD3 / anti-CD28 antibodies for 14 days and then further subjected to PMA / ionomycin stimulation and monensin treatment. FIG. 15B shows the percent of transduced Tregs expressing IL-2, IL-4, IL- 10, IL-17A, TNF-a, or IFN-y after being cultured with inflammatory cytokines and twice-activated by anti-CD3 / anti-CD28 antibodies for 14 days and then further subjected to PMA / ionomycin stimulation and monensin treatment.Detailed Description

[0065] FOXP3 expression is critical to Treg persistence and function. FOXP3 primarily acts as a transcriptional repressor of key genes involved in T cell activation and effector functions, including proliferation and synthesis of proinflammatory cytokines such as IL-2, IL-4, IL-17A, and interferon (IFN)-y. Further, FOXP3 confers potent suppressive functions in Tregs expressing the FOXP3 protein. Sustained expression of FOXP3 in Tregs is required for lineage commitment and stability. There are four naturally-occurring isoforms of FOXP3: full-length (FL), the isoform lacking exon 2 (A2), the isoform lacking exon 2 and exon 7 (A2A7), and the isoform lacking exon 7 (A7), which are expressed at varying levels in Treg cells. The isoforms may have different biological function by virtue of different functional domains in the alternatively spliced exons. Accordingly, providing an expression cassette comprising an engineered FOXP3 protein that can be alternatively spliced may help improve Treg function as compared to an expression cassette comprising a FOXP3 protein that cannot be alternativelyspliced. In some aspects provided here is an engineered Treg comprising an exogenous expression cassette comprising a modified nucleic acid encoding a FOXP3 protein operably linked to a promoter, wherein the nucleic acid encoding the FOXP3 is modified to produce a full-length FOXP3 protein isoform and an alternatively spliced isoform lacking one or more exons, such as exon 2 and / or exon 7. In some aspects, the nucleic acid encoding the FOXP3 protein comprises intronic sequences surrounding exon 2, resulting in a full-length protein and the A2 isoform. In some aspects, the nucleic acid sequence encoding the FOXP3 protein does not comprise other intronic sequences. In another beneficial aspect, the expression cassettes provided herein may be multi-cistronic and comprise multiple protein coding sequences linked to a single promoter. In some embodiments, the expression cassette comprises nucleic acids encoding a FOXP3 protein and a chimeric receptor. In some embodiments, the expression cassette comprises a nucleic acid encoding a cell surface receptor that can used to monitor expression of the expression cassette. In some embodiments, the expression cassette is tri- cistronic comprising a nucleic acid encoding a FOXP3 protein, a nucleic acid encoding a chimeric receptor (e.g., a chimeric antigen receptor (CAR)), and a nucleic acid encoding a cell surface receptor.

[0066] All publications, including patent documents, scientific articles, and databases, referred to in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. If a definition set forth herein is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications, and other publications that are herein incorporated by reference, the definition set forth herein prevails over the definition that is incorporated herein by reference.

[0067] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.I. EXPRESSION CASSETTES

[0068] The present invention provides expression cassettes that express exogenous FOXP3 in the presence or the absence of endogenous FOXP3 expression. In some embodiments, the expression cassettes provided herein are in a vector, such as a viral vector (e.g., a lentiviral vector). In some embodiments, expression cassettes comprise nucleic acids encoding a FOXP3 protein and a cell surface receptor, wherein the nucleic acid encoding the FOXP3 protein is modified to produce a full-length FOXP3 protein isoform and an alternatively spliced isoformlacking one or more exons. In some embodiments, the FOXP3 protein is a naturally occurring protein, e.g., a wild-type FOXP3. In some embodiments, the expression cassette comprises a naturally occurring FOXP3 protein. In some embodiments, the FOXP3 protein comprises an amino acid sequence comprising at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity with the amino acid sequence selected from the group consisting of SEQ ID NOs: 20-23. In some embodiments, the FOXP3 protein comprises one or more amino acid modifications e.g., amino acid substitutions, deletions, and / or additions) compared to a naturally occurring FOXP3 protein. In some embodiments, the FOXP3 protein is an alternatively spliced variant translated from a nucleic acid that lacks the coding sequence for exon 2. In some embodiments, the expression cassette comprises intronic sequences flanking exon 2. In some embodiments, the expression cassette comprises only the intronic sequences flanking exon 2. In some embodiments, the expression cassette does not comprise any intronic sequences except for the intronic sequences flanking exon 2. In some embodiments, the FOXP3 protein is not an alternatively spliced variant translated from a nucleic acid that lacks the coding sequence for exon 7. In some embodiments, the expression cassette encoding the FOXP3 protein encodes only one FOXP3 protein isoform, wherein the FOXP3 protein isoform is full-length FOXP3 protein or a FOXP3 protein isoform lacking exon 2. In some embodiments, the expression cassette encoding the FOXP3 protein encodes two FOXP3 protein isoforms, wherein the FOXP3 protein isoforms are full-length FOXP3 protein and a FOXP3 protein isoform lacking exon 2. In some embodiments, the expression ratio of full-length FOXP3 protein to the FOXP3 isoform lacking exon 2 (A2) within a Treg is about 90:1, about 80:1, about 70:1, about 60:1, about 50:1, about 40:1, about 30:1, about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, about 1:25, about 1:30, about 1:40, about 1:50, about 1:60, about 1:70, about 1:80, or about 1:90. In some embodiments, the expression ratio of full-length FOXP3 protein to the FOXP3 isoform lacking exon 2 (A2) within a Treg comprising the expression cassette is about 90:1, about 80:1, about 70:1, about 60:1, about 50:1, about 40:1, about 30:1, about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, about 1:25, about 1:30, about 1:40, about 1:50, about 1:60, about 1:70, about 1:80, or about 1:90. In some embodiments, theexpression ratio measures F0XP3 gene expression. In some embodiments, the expression ratio measures FOXP3 protein levels. In some embodiments, the expression ratio measures active FOXP3 protein levels.

[0069] In some instances, amino acid modifications may be introduced in the FOXP3 protein, for example to optimize, enhance, promote, increase, etc. , FOXP3 signaling and / or stability, for example to reduce FOXP3 protein degradation. In some embodiments, the FOXP3 protein is linked to a detection tag, for example but not limited to a HIS tag, an HA tag, or the like. Exemplary detection tags are provided in Table 1 below. The attachment of the detection tag to the exogenous FOXP3 allows for separate identification of the levels of expression of the exogenous FOXP3 compared to the endogenous FOXP3 levels. Thus, in some instances, a cell (e.g., a Treg) comprising the exogenous expression cassette can be screened simultaneously for endogenous FOXP3 levels and for exogenous FOXP3 levels.Table 1. Amino acid sequences of exemplary detection tags.

[0070] In some cells, sustained expression of FOXP3 is required for Treg lineage commitment and stability. Therefore, expression cassettes comprising an exogenous FOXP3 protein are able to generate or induce the Treg phenotype.

[0071] In some embodiments, the expression cassettes provided herein have significant advantages including, but not limited to: (1) expression in regulatory T cells (Tregs); (2) increasing Treg phenotype stability; (3) increasing Treg suppressive function; and (4) converting, differentiating, or otherwise transforming Teff cells into Tregs.

[0072] Provided herein are expression cassettes (e.g., exogenous expression cassettes) comprising a nucleic acid encoding an exogenous FOXP3 protein. Also provided herein are vectors comprising the expression cassettes, such as viral vectors (e.g., lentiviral vectors). In some embodiments, the expression cassette comprises a nucleic acid encoding a FOXP3 protein and a nucleic acid encoding a chimeric receptor and / or a nucleic acid encoding a cell surface receptor, wherein each component is separated from another component by a separationelement, for example an IRES, a nucleic acid encoding a self-cleaving peptide, and / or a nucleic acid encoding a peptide that causes ribosome skipping. In some embodiments, the nucleic acid encoding the FOXP3 protein comprises intron sequences flanking the nucleic acid encoding exon 2, wherein the nucleic acid encoding the FOXP3 protein is alternatively spliced to produce a full-length FOXP3 protein isoform and a FOXP3 protein isoform lacking exon 2. In some embodiments, the FOXP3 protein is linked to a detection tag. In some embodiments, the detection tag is linked to the N-terminus of the FOXP3 protein. In some embodiments, the detection tag is linked to the C-terminus of the FOXP3 protein. In some embodiments, the nucleic acid encoding a FOXP3 protein is FOXP3 cDNA. In some embodiments, the exogenous expression cassette further comprises a nucleic acid encoding a chimeric receptor and / or a nucleic acid encoding a cell surface receptor. In some embodiments, the nucleic acid encoding the FOXP3 protein and the nucleic acid encoding the chimeric receptor and / or the nucleic acid encoding the cell surface receptor are operably linked to the same promoter. In some embodiments, the nucleic acid encoding the FOXP3 protein and the nucleic acid encoding the chimeric receptor and / or the nucleic acid encoding the cell surface receptor are each separated by an IRES, a nucleic acid encoding a self-cleaving peptide (e.g., P2A peptide), or a nucleic acid encoding a peptide that causes ribosome skipping. In some embodiments, the expression cassette further comprises a nucleic acid encoding a self-cleaving peptide. In some embodiments, the self-cleaving peptide is a 2A sequence. In some embodiments, the 2A sequence is a P2A sequence. In some embodiments, the 2A sequence is a T2A sequence. In some embodiments, the expression cassette comprises a nucleic acid encoding: a) a promoter; b) a modified nucleic acid encoding the FOXP3 protein linked to a detection tag, wherein the nucleic acid encoding the FOXP3 protein comprises intron sequences flanking the nucleic acid encoding exon 2; c) a nucleic acid encoding a self-cleaving peptide; d) a nucleic acid encoding a chimeric receptor; and / or e) a nucleic acid encoding a cell surface receptor. In some embodiments, the expression cassette comprises a nucleic acid encoding: a) a promoter; b) a modified nucleic acid encoding the FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein comprises intron sequences flanking the nucleic acid encoding exon 2; c) a nucleic acid encoding a self-cleaving peptide; d) a nucleic acid encoding a chimeric receptor; and / or e) a nucleic acid encoding a cell surface receptor.

[0073] In some embodiments, provided herein are expression cassettes comprising a nucleic acid encoding from 5’ to 3’: a) a promoter; b) a modified nucleic acid encoding a FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein comprises intron sequencesflanking a nucleic acid encoding exon 2, wherein the nucleic acid encoding the FOXP3 protein is alternatively spliced to produce a full-length FOXP3 protein isoform and / or a FOXP3 protein isoform lacking exon 2; and c) a nucleic acid encoding a cell surface receptor. In some embodiments, provided herein are expression cassettes comprising a nucleic acid encoding from 5’ to 3’: a) a promoter; b) a modified nucleic acid encoding a FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein comprises intron sequences flanking a nucleic acid encoding exon 2, wherein the nucleic acid encoding the FOXP3 protein is alternatively spliced to produce a full-length FOXP3 protein isoform and / or a FOXP3 protein isoform lacking exon 2; c) an IRES, or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; and d) a nucleic acid encoding a cell surface receptor. In some embodiments, the nucleic acid encoding the FOXP3 protein encodes only one FOXP3 protein isoform, wherein the FOXP3 protein isoform is full-length FOXP3 protein or a FOXP3 protein isoform lacking exon 2. In some embodiments, the nucleic acid encoding the FOXP3 protein encodes two FOXP3 protein isoforms, wherein the FOXP3 protein isoforms are full-length FOXP3 protein and a FOXP3 protein isoform lacking exon 2. In some embodiments, the nucleic acid encoding the FOXP3 protein comprising intron sequences flanking the nucleic acid encoding exon 2 is alternatively spliced to produce a full-length FOXP3 protein isoform and a FOXP3 protein isoform lacking exon 2. In some embodiments, the FOXP3 protein is linked to a detection tag. In some embodiments, the detection tag is linked to the N-terminus of the FOXP3 protein. In some embodiments, the detection tag is linked to the C-terminus of the FOXP3 protein. In some embodiments, the nucleic acid encoding the FOXP3 does not contain intron sequences other than the intron sequences flanking exon 2. In some embodiments, the FOXP3 protein is linked to a detection tag, wherein the detection tag comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 11-16. In some embodiments, the nucleic acid encoding a FOXP3 protein is FOXP3 cDNA. In some embodiments, the expression cassette comprises a nucleic acid encoding a FOXP3 protein having an amino acid sequence comprising at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs:20-23. In some embodiments, the cell surface receptor lacks a functional intracellular signaling domain or is not capable of mediating intracellular signaling. In some embodiments, the cell surface receptor is selected from the group consisting of EGFR, HER2, CD2, H-2Kk, IL-5Ra, and derivatives or mutants thereof. In some embodiments, the nucleic acid encodes a cell surface receptorcomprising an amino acid sequence comprising at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 17-19. In some embodiments, the nucleic acid encoding the FOXP3 protein and the nucleic acid encoding the cell surface receptor are operably linked to the same promoter. In some embodiments, the promoter is an inducible or a constitutive promoter. In some embodiments, the promoter is a cell-specific promoter. In some embodiments, the promoter is specific for T cells (e.g., Tregs). In some embodiments, the nucleic acid encoding the FOXP3 protein and the nucleic acid encoding the cell surface receptor are separated by an IRES, a nucleic acid encoding a self-cleaving peptide (e.g., P2A peptide), or a nucleic acid encoding a peptide that causes ribosome skipping. In some embodiments, the expression cassette further comprises a nucleic acid encoding a self-cleaving peptide. In some embodiments, the self-cleaving peptide is a 2A sequence. In some embodiments, the 2A sequence is a P2A sequence. In some embodiments, the expression cassette comprises a nucleic acid encoding from 5’ to 3’: a) a promoter; b) a modified nucleic acid encoding a FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein comprises intron sequences flanking a nucleic acid encoding exon 2, wherein the nucleic acid encoding the FOXP3 protein is alternatively spliced to produce a full-length FOXP3 protein isoform and / or a FOXP3 protein isoform lacking exon 2, wherein the FOXP3 protein comprises at least about 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid sequence of SEQ ID NO:20 and / or 21; c) a nucleic acid encoding a self-cleaving P2A peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:2-4; and d) a nucleic acid encoding a cell surface receptor. In some embodiments, the expression cassette comprises a nucleic acid encoding from 5’ to 3’: a) a promoter; b) a modified nucleic acid encoding a FOXP3 protein linked to a detection tag, wherein the nucleic acid encoding the FOXP3 protein comprises intron sequences flanking a nucleic acid encoding exon 2, wherein the nucleic acid encoding the FOXP3 protein is alternatively spliced to produce a full-length FOXP3 protein isoform and / or a FOXP3 protein isoform lacking exon 2, wherein the FOXP3 protein comprises at least about 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid sequence of SEQ ID NO:22 and / or 23; c) a nucleic acid encoding a selfcleaving P2A peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:2-4; and d) a nucleic acid encoding a cell surface receptor comprising at leastabout 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 17-19.

[0074] In some embodiments, provided herein are expression cassettes comprising a nucleic acid encoding from 5’ to 3’: a) a promoter; b) a nucleic acid encoding a cell surface receptor; and c) a modified nucleic acid encoding a FOXP3 protein, wherein the modified nucleic acid encoding the FOXP3 protein comprises intron sequences flanking a nucleic acid encoding exon 2, wherein the nucleic acid encoding the FOXP3 protein is alternatively spliced to produce a full-length FOXP3 protein isoform and / or a FOXP3 protein isoform lacking exon 2. In some embodiments, provided herein are expression cassettes comprising a nucleic acid encoding from 5’ to 3’: a) a promoter; b) a nucleic acid encoding a cell surface receptor; c) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; and d) a modified nucleic acid encoding a FOXP3 protein, wherein the modified nucleic acid encoding the FOXP3 protein comprises intron sequences flanking a nucleic acid encoding exon 2, wherein the nucleic acid encoding the FOXP3 protein is alternatively spliced to produce a full-length FOXP3 protein isoform and / or a FOXP3 protein isoform lacking exon 2. In some embodiments, the nucleic acid encoding the FOXP3 protein encodes only one FOXP3 protein isoform, wherein the FOXP3 protein isoform is full-length FOXP3 protein or a FOXP3 protein isoform lacking exon 2. In some embodiments, the nucleic acid encoding the FOXP3 protein encodes two FOXP3 protein isoforms, wherein the FOXP3 protein isoforms are full-length FOXP3 protein and a FOXP3 protein isoform lacking exon 2. In some embodiments, the nucleic acid encoding the FOXP3 protein comprising intron sequences flanking the nucleic acid encoding exon 2 is alternatively spliced to produce a full-length FOXP3 protein isoform and a FOXP3 protein isoform lacking exon 2. In some embodiments, the FOXP3 protein is linked to a detection tag. In some embodiments, the detection tag is linked to the N-terminus of the FOXP3 protein. In some embodiments, the detection tag is linked to the C-terminus of the FOXP3 protein. In some embodiments, the FOXP3 protein is linked to a detection tag, wherein the detection tag comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 11-16. In some embodiments, the nucleic acid encoding the FOXP3 does not contain intron sequences other than the intron sequences flanking exon 2. In some embodiments, the nucleic acid encoding a FOXP3 protein is FOXP3 cDNA. In some embodiments, the expression cassette comprises a nucleic acid encoding a FOXP3 protein having an amino acid sequence comprising at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at leastabout 99% or about 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs:20-23. In some embodiments, the cell surface receptor lacks a functional intracellular signaling domain or is not capable of mediating intracellular signaling. In some embodiments, the cell surface receptor is selected from the group consisting of EGFR, HER2, CD2, H-2Kk, IL-5Ra, and derivatives or mutants thereof. In some embodiments, the nucleic acid encodes a cell surface receptor comprising an amino acid sequence comprising at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs:17-19. In some embodiments, the nucleic acid encoding the FOXP3 protein and the nucleic acid encoding the cell surface receptor are operably linked to the same promoter. In some embodiments, the promoter is an inducible or a constitutive promoter. In some embodiments, the promoter is a cell-specific promoter. In some embodiments, the promoter is specific for T cells (e.g., Tregs). In some embodiments, the nucleic acid encoding the FOXP3 protein and the nucleic acid encoding the cell surface receptor are separated by an IRES, a nucleic acid encoding a selfcleaving peptide (e.g., P2A peptide), or a nucleic acid encoding a peptide that causes ribosome skipping. In some embodiments, the expression cassette further comprises a nucleic acid encoding a self-cleaving peptide. In some embodiments, the self-cleaving peptide is a 2A sequence. In some embodiments, the 2A sequence is a P2A sequence. In some embodiments, the expression cassette comprises a nucleic acid encoding from 5’ to 3’: a) a promoter; b) a nucleic acid encoding a cell surface receptor; c) a nucleic acid encoding a self-cleaving P2A peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:2-4; and d) a modified nucleic acid encoding a FOXP3 protein, wherein the modified nucleic acid encoding the FOXP3 protein comprises intron sequences flanking a nucleic acid encoding exon 2, wherein the nucleic acid encoding the FOXP3 protein is alternatively spliced to produce a full-length FOXP3 protein isoform and / or a FOXP3 protein isoform lacking exon 2, wherein the FOXP3 protein comprises at least about 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid sequence of SEQ ID NO:20 and / or 21. In some embodiments, the expression cassette comprises a nucleic acid encoding from 5’ to 3’: a) a promoter; b) a nucleic acid encoding a cell surface receptor comprising at least about 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs:17-19; c) a nucleic acid encoding a self-cleaving P2A peptide comprising an amino acid sequence selected from thegroup consisting of SEQ ID NOs:2-4; and d) a modified nucleic acid encoding a FOXP3 protein linked to a detection tag, wherein the modified nucleic acid encoding the FOXP3 protein comprises intron sequences flanking a nucleic acid encoding exon 2, wherein the nucleic acid encoding the FOXP3 protein is alternatively spliced to produce a full-length FOXP3 protein isoform and / or a FOXP3 protein isoform lacking exon 2, wherein the FOXP3 protein comprises at least about 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid sequence of SEQ ID NO:22 and / or 23.

[0075] In some embodiments, provided herein expression cassettes comprising a nucleic acid encoding from 5’ to 3’: a) a promoter; b) a nucleic acid encoding a chimeric receptor; c) a nucleic acid encoding a cell surface receptor; and d) a modified nucleic acid encoding a FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein comprises intron sequences flanking a nucleic acid encoding exon 2, wherein the nucleic acid encoding the FOXP3 protein is alternatively spliced to produce a full-length FOXP3 protein isoform and / or a FOXP3 protein isoform lacking exon 2. In some embodiments, provided herein expression cassettes comprising a nucleic acid encoding from 5’ to 3’: a) a promoter; b) a nucleic acid encoding a chimeric receptor; c) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; d) a nucleic acid encoding a cell surface receptor; e) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; and f) a modified nucleic acid encoding a FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein comprises intron sequences flanking a nucleic acid encoding exon 2, wherein the nucleic acid encoding the FOXP3 protein is alternatively spliced to produce a full-length FOXP3 protein isoform and / or a FOXP3 protein isoform lacking exon 2. In some embodiments, the nucleic acid encoding the FOXP3 protein encodes only one FOXP3 protein isoform, wherein the FOXP3 protein isoform is full-length FOXP3 protein or a FOXP3 protein isoform lacking exon 2. In some embodiments, the nucleic acid encoding the FOXP3 protein encodes two FOXP3 protein isoforms, wherein the FOXP3 protein isoforms are full-length FOXP3 protein and a FOXP3 protein isoform lacking exon 2. In some embodiments, the nucleic acid encoding the FOXP3 protein comprising intron sequences flanking the nucleic acid encoding exon 2 is alternatively spliced to produce a full- length FOXP3 protein isoform and a FOXP3 protein isoform lacking exon 2. In some embodiments, the FOXP3 protein is linked to a detection tag. In some embodiments, the detection tag is linked to the N-terminus of the FOXP3 protein. In some embodiments, the detection tag is linked to the C-terminus of the FOXP3 protein. In some embodiments, theF0XP3 protein is linked to a detection tag, wherein the detection tag comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:l l-16. In some embodiments, the nucleic acid encoding the FOXP3 does not contain intron sequences other than the intron sequences flanking exon 2. In some embodiments, the nucleic acid encoding a FOXP3 protein is FOXP3 cDNA. In some embodiments, the expression cassette comprises a nucleic acid encoding a FOXP3 protein having an amino acid sequence comprising at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs:20-23. In some embodiments, the cell surface receptor lacks a functional intracellular signaling domain or is not capable of mediating intracellular signaling. In some embodiments, the cell surface receptor is selected from the group consisting of EGFR, HER2, CD2, H-2Kk, IE-5Ra, and derivatives or mutants thereof. In some embodiments, the nucleic acid encodes a cell surface receptor comprising an amino acid sequence comprising at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs:17-19. In some embodiments, the chimeric receptor comprises a chimeric antigen receptor, a T cell Receptor, or a PiG receptor. In some embodiments, the chimeric receptor binds to an antigen selected from the group consisting of MBP, MOG, PLP, PDE4, VEGFR, S 100- , Citrullinated vimentin (CV), TNF-like ligand 1A (TL1A), CD2, CD23 (IgE receptor), IL-6R, IL-4Ra, IL-12R, IL-13Ral, IL-13Ra2, IL-15R, IL-23R, CD19, CD20, CD22, CD3, CD4, CD5, IFN-y, IgE, IL-la, IL-10, IL-6, IL-12, IL-23, IL-15, IL-17, IL-17A, IL- 22, IL-13, IL-33, TSLP, OX-40, OX-40L, p53, CD38, CD40L, CD28, ICOS, RIPK1, BTK, Bet v 1, GLP2R, JAK1, C5, and TNF-a. In some embodiments, the nucleic acid encodes a chimeric receptor comprising an antigen binding domain comprising at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs:73-76. In some embodiments, the nucleic acid encoding the FOXP3 protein and the nucleic acid encoding the chimeric receptor and / or the nucleic acid encoding the cell surface receptor are operably linked to the same promoter. In some embodiments, the promoter is an inducible or a constitutive promoter. In some embodiments, the promoter is a cell-specific promoter. In some embodiments, the promoter is specific for T cells (e.g., Tregs). In some embodiments, the nucleic acid encoding the FOXP3protein and the nucleic acid encoding the chimeric receptor and / or the nucleic acid encoding the cell surface receptor are each separated by an IRES, a nucleic acid encoding a self-cleaving peptide (e.g., P2A peptide), or a nucleic acid encoding a peptide that causes ribosome skipping. In some embodiments, the expression cassette further comprises a nucleic acid encoding a selfcleaving peptide. In some embodiments, the self-cleaving peptide is a 2A sequence. In some embodiments, the 2A sequence is a P2A sequence. In some embodiments, the 2A sequence is a T2A sequence. In some embodiments, the expression cassette comprises a nucleic acid encoding from 5’ to 3’: a) a promoter; b) a nucleic acid encoding a chimeric receptor comprising at least about 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 80-84; c) a nucleic acid encoding a self-cleaving P2A peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:2-4; d) a nucleic acid encoding a cell surface receptor comprising at least about 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 17- 19; e) a nucleic acid encoding a self-cleaving P2A or T2A peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:2-7; and f) a modified nucleic acid encoding a FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein comprises intron sequences flanking a nucleic acid encoding exon 2, wherein the nucleic acid encoding the FOXP3 protein is alternatively spliced to produce a full-length FOXP3 protein isoform and / or a FOXP3 protein isoform lacking exon 2, wherein the FOXP3 protein comprises at least about 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid sequence of SEQ ID NO:20 and / or 21. In some embodiments, the expression cassette comprises a nucleic acid encoding from 5’ to 3’: a) a promoter; b) a nucleic acid encoding a chimeric receptor comprising at least about 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs:80-84; c) a nucleic acid encoding a self-cleaving P2A peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:2-4; d) a nucleic acid encoding a cell surface receptor comprising at least about 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 17- 19; e) a nucleic acid encoding a self-cleaving P2A or T2A peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:2-7; and f) a modified nucleic acid encoding a FOXP3 protein linked to a detection tag, wherein the nucleic acid 1encoding the FOXP3 protein comprises intron sequences flanking a nucleic acid encoding exon 2, wherein the nucleic acid encoding the FOXP3 protein is alternatively spliced to produce a full-length FOXP3 protein isoform and / or a FOXP3 protein isoform lacking exon 2, wherein the FOXP3 protein comprises at least about 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid sequence of SEQ ID NO:22 and / or 23.

[0076] In some embodiments, provided herein are expression cassettes comprising a nucleic acid encoding from 5’ to 3’: a) a promoter; b) a nucleic acid encoding a chimeric receptor; c) a modified nucleic acid encoding a FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein comprises intron sequences flanking a nucleic acid encoding exon 2, wherein the nucleic acid encoding the FOXP3 protein is alternatively spliced to produce a full-length FOXP3 protein isoform and / or a FOXP3 protein isoform lacking exon 2; and d) a nucleic acid encoding a cell surface receptor. In some embodiments, provided herein are expression cassettes comprising a nucleic acid encoding from 5’ to 3’: a) a promoter; b) a nucleic acid encoding a chimeric receptor; c) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; d) a modified nucleic acid encoding a FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein comprises intron sequences flanking a nucleic acid encoding exon 2, wherein the nucleic acid encoding the FOXP3 protein is alternatively spliced to produce a full-length FOXP3 protein isoform and / or a FOXP3 protein isoform lacking exon 2; e) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; and f) a nucleic acid encoding a cell surface receptor. In some embodiments, the nucleic acid encoding the FOXP3 protein encodes only one FOXP3 protein isoform, wherein the FOXP3 protein isoform is full-length FOXP3 protein or a FOXP3 protein isoform lacking exon 2. In some embodiments, the nucleic acid encoding the FOXP3 protein encodes two FOXP3 protein isoforms, wherein the FOXP3 protein isoforms are full-length FOXP3 protein and a FOXP3 protein isoform lacking exon 2. In some embodiments, the nucleic acid encoding the FOXP3 protein comprising intron sequences flanking the nucleic acid encoding exon 2 is alternatively spliced to produce a full-length FOXP3 protein isoform and a FOXP3 protein isoform lacking exon 2. In some embodiments, the FOXP3 protein is linked to a detection tag. In some embodiments, the detection tag is linked to the N-terminus of the FOXP3 protein. In some embodiments, the detection tag is linked to the C-terminus of the FOXP3 protein. In some embodiments, the FOXP3 protein is linked to a detection tag, wherein the detection tag comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:l l-16. Insome embodiments, the nucleic acid encoding the FOXP3 does not contain intron sequences other than the intron sequences flanking exon 2. In some embodiments, the nucleic acid encoding a FOXP3 protein is FOXP3 cDNA. In some embodiments, the expression cassette comprises a nucleic acid encoding a FOXP3 protein having an amino acid sequence comprising at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs:20-23. In some embodiments, the cell surface receptor lacks a functional intracellular signaling domain or is not capable of mediating intracellular signaling. In some embodiments, wherein the cell surface receptor is selected from the group consisting of EGFR, HER2, CD2, H-2Kk, IL-5Ra, and derivatives or mutants thereof. In some embodiments, the nucleic acid encodes a cell surface receptor comprising an amino acid sequence comprising at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 17-19. In some embodiments, the chimeric receptor comprises a chimeric antigen receptor, a T cell Receptor, or a PiG receptor. In some embodiments, the chimeric receptor binds to an antigen selected from the group consisting of MBP, MOG, PLP, PDE4, VEGFR, S 100- , Citrullinated vimentin (CV), TNF-like ligand 1A (TL1A), CD2, CD23 (IgE receptor), IL-6R, IL-4Ra, IL-12R, IL-13Ral, IL-13Ra2, IL-15R, IL- 23R, CD19, CD20, CD22, CD3, CD4, CD5, IFN-y, IgE, IL-la, IL-10, IL-6, IL-12, IL-23, IL-15, IL-17, IL-17A, IL-22, IL-13, IL-33, TSLP, OX-40, OX-40L, p53, CD38, CD40L, CD28, ICOS, RIPK1, BTK, Bet v 1, GLP2R, JAK1, C5, and TNE-a. In some embodiments, the nucleic acid encodes a chimeric receptor comprising an antigen binding domain comprising at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs:73-76. In some embodiments, the nucleic acid encoding the EOXP3 protein and the nucleic acid encoding the chimeric receptor and / or the nucleic acid encoding the cell surface receptor are operably linked to the same promoter. In some embodiments, the promoter is an inducible or a constitutive promoter. In some embodiments, the promoter is a cell- specific promoter. In some embodiments, the promoter is specific for T cells (e.g., Tregs). In some embodiments, the nucleic acid encoding the EOXP3 protein and the nucleic acid encoding the chimeric receptor and / or the nucleic acid encoding the cell surface receptor are each separated by an IRES, anucleic acid encoding a self-cleaving peptide (e.g. , P2A peptide), or a nucleic acid encoding a peptide that causes ribosome skipping. In some embodiments, the expression cassette further comprises a nucleic acid encoding a self-cleaving peptide. In some embodiments, the selfcleaving peptide is a 2A sequence. In some embodiments, the 2A sequence is a P2A sequence. In some embodiments, the 2A sequence is a T2A sequence. In some embodiments, the expression cassette comprises a nucleic acid encoding from 5’ to 3’: a) a promoter; b) a nucleic acid encoding a chimeric receptor comprising at least about 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 80-84; c) a nucleic acid encoding a selfcleaving P2A peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:2-4; d) a modified nucleic acid encoding a FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein comprises intron sequences flanking a nucleic acid encoding exon 2, wherein the nucleic acid encoding the FOXP3 protein is alternatively spliced to produce a full-length FOXP3 protein isoform and / or a FOXP3 protein isoform lacking exon 2, wherein the FOXP3 protein comprises at least about 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid sequence of SEQ ID NO:20 and / or 21; e) a nucleic acid encoding a self-cleaving P2A or T2A peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:2-7; and f) a nucleic acid encoding a cell surface receptor comprising at least about 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 17-19. In some embodiments, the expression cassette comprises a nucleic acid encoding from 5’ to 3’: a) a promoter; b) a nucleic acid encoding a chimeric receptor comprising at least about 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs:80-84; c) a nucleic acid encoding a self-cleaving P2A peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:2-4; d) a modified nucleic acid encoding a FOXP3 protein linked to a detection tag, wherein the nucleic acid encoding the FOXP3 protein comprises intron sequences flanking a nucleic acid encoding exon 2, wherein the nucleic acid encoding the FOXP3 protein is alternatively spliced to produce a full-length FOXP3 protein isoform and / or a FOXP3 protein isoform lacking exon 2, wherein the FOXP3 protein comprises at least about 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid sequence of SEQ ID NO:22 and / or 23; e) a nucleic acid encoding a self-cleaving P2A or T2A peptide comprising an aminoacid sequence selected from the group consisting of SEQ ID NOs:2-7; and f) a nucleic acid encoding a cell surface receptor comprising at least about 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 17-19.

[0077] In some embodiments, provided herein are expression cassettes comprising a nucleic acid encoding from 5’ to 3’: a) a promoter; b) a modified nucleic acid encoding a FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein comprises intron sequences flanking a nucleic acid encoding exon 2, wherein the nucleic acid encoding the FOXP3 protein is alternatively spliced to produce a full-length FOXP3 protein isoform and / or a FOXP3 protein isoform lacking exon 2; c) a nucleic acid encoding a chimeric receptor; and d) a nucleic acid encoding a cell surface receptor. In some embodiments, provided herein are expression cassettes comprising a nucleic acid encoding from 5’ to 3’: a) a promoter; b) a modified nucleic acid encoding a FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein comprises intron sequences flanking a nucleic acid encoding exon 2, wherein the nucleic acid encoding the FOXP3 protein is alternatively spliced to produce a full-length FOXP3 protein isoform and / or a FOXP3 protein isoform lacking exon 2; c) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; d) a nucleic acid encoding a chimeric receptor; e) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; and f) a nucleic acid encoding a cell surface receptor. In some embodiments, the nucleic acid encoding the FOXP3 protein encodes only one FOXP3 protein isoform, wherein the FOXP3 protein isoform is full-length FOXP3 protein or a FOXP3 protein isoform lacking exon 2. In some embodiments, the nucleic acid encoding the FOXP3 protein encodes two FOXP3 protein isoforms, wherein the FOXP3 protein isoforms are full-length FOXP3 protein and a FOXP3 protein isoform lacking exon 2. In some embodiments, the nucleic acid encoding the FOXP3 protein comprising intron sequences flanking the nucleic acid encoding exon 2 is alternatively spliced to produce a full- length FOXP3 protein isoform and a FOXP3 protein isoform lacking exon 2. In some embodiments, the FOXP3 protein is linked to a detection tag. In some embodiments, the detection tag is linked to the N-terminus of the FOXP3 protein. In some embodiments, the detection tag is linked to the C-terminus of the FOXP3 protein. In some embodiments, the FOXP3 protein is linked to a detection tag, wherein the detection tag comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 11- 16. In some embodiments, the nucleic acid encoding the FOXP3 does not contain intron sequences other than the intronsequences flanking exon 2. In some embodiments, the nucleic acid encoding a FOXP3 protein is FOXP3 cDNA. In some embodiments, the expression cassette comprises a nucleic acid encoding a FOXP3 protein having an amino acid sequence comprising at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs:20-23. In some embodiments, the cell surface receptor lacks a functional intracellular signaling domain or is not capable of mediating intracellular signaling. In some embodiments, the cell surface receptor is selected from the group consisting of EGFR, HER2, CD2, H-2Kk, IL-5Ra, and derivatives or mutants thereof. In some embodiments, the nucleic acid encodes a cell surface receptor comprising an amino acid sequence comprising at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs:17-19. In some embodiments, the chimeric receptor comprises a chimeric antigen receptor, a T cell Receptor, or a PiG receptor. In some embodiments, the chimeric receptor binds to an antigen selected from the group consisting of MBP, MOG, PLP, PDE4, VEGFR, S 100- , Citrullinated vimentin (CV), TNF-like ligand 1A (TL1A), CD2, CD23 (IgE receptor), IL-6R, IL-4Ra, IL-12R, IL-13Ral, IL-13Ra2, IL-15R, IL-23R, CD19, CD20, CD22, CD3, CD4, CD5, ILN-y, IgE, IL-la, IL-10, IL-6, IL-12, IL-23, IL-15, IL-17, IL-17A, IL- 22, IL-13, IL-33, TSLP, OX-40, OX-40L, p53, CD38, CD40L, CD28, ICOS, RIPK1, BTK, Bet v 1, GLP2R, JAK1, C5, and TNE-a. In some embodiments, the nucleic acid encodes a chimeric receptor comprising an antigen binding domain comprising at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs:73-76. In some embodiments, the nucleic acid encoding the EOXP3 protein and the nucleic acid encoding the chimeric receptor and / or the nucleic acid encoding the cell surface receptor are operably linked to the same promoter. In some embodiments, the promoter is an inducible or a constitutive promoter. In some embodiments, the promoter is a cell-specific promoter. In some embodiments, the promoter is specific for T cells (e.g., Tregs). In some embodiments, the nucleic acid encoding the EOXP3 protein and the nucleic acid encoding the chimeric receptor and / or the nucleic acid encoding the cell surface receptor are each separated by an IRES, a nucleic acid encoding a self-cleaving peptide (e.g., P2A peptide), or a nucleic acid encoding a peptide that causes ribosome skipping.In some embodiments, the expression cassette further comprises a nucleic acid encoding a selfcleaving peptide. In some embodiments, the self-cleaving peptide is a 2A sequence. In some embodiments, the 2A sequence is a P2A sequence. In some embodiments, the 2A sequence is a T2A sequence. In some embodiments, the expression cassette comprises a nucleic acid encoding from 5’ to 3’: a) a promoter; b) a modified nucleic acid encoding a FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein comprises intron sequences flanking a nucleic acid encoding exon 2, wherein the nucleic acid encoding the FOXP3 protein is alternatively spliced to produce a full-length FOXP3 protein isoform and / or a FOXP3 protein isoform lacking exon 2, wherein the FOXP3 protein comprises at least about 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid sequence of SEQ ID NO:20 and / or 21; c) a nucleic acid encoding a self-cleaving P2A peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:2-4; d) a nucleic acid encoding a chimeric receptor comprising at least about 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs:80-84; e) a nucleic acid encoding a self-cleaving P2A or T2A peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:2-7; and f) a nucleic acid encoding a cell surface receptor comprising at least about 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs:17-19. In some embodiments, the expression cassette comprises a nucleic acid encoding from 5’ to 3’: a) a promoter; b) a modified nucleic acid encoding a FOXP3 protein linked to a detection tag, wherein the nucleic acid encoding the FOXP3 protein comprises intron sequences flanking a nucleic acid encoding exon 2, wherein the nucleic acid encoding the FOXP3 protein is alternatively spliced to produce a full-length FOXP3 protein isoform and / or a FOXP3 protein isoform lacking exon 2, wherein the FOXP3 protein comprises at least about 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid sequence of SEQ ID NO:22 and / or 23; c) a nucleic acid encoding a self-cleaving P2A peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:2-4; d) a nucleic acid encoding a chimeric receptor comprising at least about 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs:80-84; e) a nucleic acid encoding a self-cleaving P2A or T2A peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:2-7; and f) a nucleic acid encoding a cell surface receptor comprising at least about 80% (such as aboutany of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs:17-19.

[0078] In some embodiments, the level of FOXP3 expression in Tregs comprising the exogenous expression cassette(s) described herein is increased at least about 1.1-fold, at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 3.2-fold, at least about 4- fold, at least about 10-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 45-fold, at least about 50-fold, or more compared to Tregs lacking the exogenous expression cassette(s).

[0079] In some embodiments, cell (e.g., Treg) viability, transduction of FOXP3 signaling, activation of FOXP3 signaling, or the ability to suppress effector T cell cytokine production is increased upon transduction with the exogenous expression cassette(s) described herein.

[0080] In some embodiments, the expression cassette has the capability to increase FOXP3 expression in cells (e.g., Tregs) that are transduced with and express the expression cassette. FOXP3 gene expression and / or protein levels can be measured using any suitable method known in the art. For example, FOXP3 protein levels can be measured using antibodies specific for the FOXP3 protein, including specific isoform variants, in combination with flow cytometry analysis as described herein.

[0081] In some embodiments, the expression cassette described herein is expressed in cells. In some embodiments, the expression cassette described herein is expressed in immune cells. In some embodiments, the immune cells are T cells. In some embodiments, the immune cells are Treg cells.

[0082] In some embodiments, the expression cassette described herein is expressed in Tregs. In some embodiments, the Treg cells are CD4+, CD25+, and CD1271ow. In some embodiments, the Tregs that comprise the expression cassette described herein also express endogenous FOXP3. In some embodiments, the Tregs that express the expression cassette described herein also express high cellular levels of FOXP3.

[0083] In some embodiments, the population of Tregs transduced with the expression cassette maintains expression of least one Treg marker selected from the group consisting of CD25+, and FOXP3.A. C MEXIC XECEPTOXS

[0084] In some embodiments, the expression cassettes of the present invention comprise a nucleic acid encoding a chimeric receptor. In some embodiments, the chimeric receptorcomprises a chimeric antigen receptor (CAR), a T cell Receptor (TCR), or a polymeric immunoglobulin receptor (PiG) receptor. In some embodiments, the chimeric receptor comprises a CAR, such as any CAR known in the art. In some embodiments, the CAR targets an autoimmune antigen, a cancer antigen, a bacterial antigen, a fungal antigen, a pathogenic antigen, etc. For example, in some embodiments, the chimeric receptor (such as a CAR) binds to an antigen selected from the group consisting of MBP, MOG, PLP, PDE4, VEGFR, S1OO-P, Citrullinated vimentin (CV), TNF-like ligand 1A (TL1A), CD2, CD23 (IgE receptor), IL-6R, IL-4Ra, IL-12R, IL-13Ral, IL-13Ra2, IL-15R, IL-23R, CD19, CD20, CD22, CD3, CD4, CD5, IFN-y, IgE, IL-la, IL-ip, IL-6, IL-12, IL-23, IL-15, IL-17, IL-17A, IL-22, IL-13, IL-33, TSLP, OX-40, OX-40L, p53, CD38, CD40L, CD28, ICOS, RIPK1, BTK, Bet v 1, GLP2R, JAK1, C5, and TNE-a.

[0085] T cell Receptor (TCR) -engineered effector T cells use a naturally occurring (or minimally modified) TCR that is able to recognize tumor- specific epitopes presented by the major histocompatibility complex (MHC) molecules on the cell (e.g., tumor cell) surface. There are far more antigen- specific sequences within a cell and presented in the MHC than there are antigen- specific proteins on the cell surface. TCRs are able to target intracellular antigens. Accordingly, in some embodiments, the chimeric receptor is a T cell receptor (TCR).

[0086] Polymeric immunoglobulin receptors are members of the Pc receptor family, are widely expressed on epithelial cells, and are responsible for transcytosis of IgA / IgM at mucosal surfaces. (PiGRs or plgRs) transport polymeric immunoglobulin (pig, such as plgA or plgM) at mucosal barriers from the basolateral to luminal surface as part of a mucosal surface defense against microbes. After transport, a portion of PiGR (or “plgR”), called secretory component (SC), is cleaved off to release Ig, forming secreted Ig (“Sig”). Through antigen- specific and nonspecific binding, Sig can modulate microbial communities and pathogenic microbes via several mechanisms: Sig and SC mediate innate protection of the host via immune exclusion, neutralization, and complement activation, and also aid in adaptive immunity by modulating immune cell activation and function, and by maintaining homeostasis. See, e.g., H. Turula and C.E. Wobus (2018), Viruses 10(5):237. Thus, in some embodiments, the chimeric receptor is a PiG receptor.

[0087] ‘Chimeric antigen receptors” or “CARs” are engineered molecules comprising an optional signal peptide, a target binding domain, an optional hinge region, a transmembrane domain, an intracellular signaling domain and an optional co-stimulatory domain. CARs are based on the structure of T cell receptors, which are expressed on T cells and which are involvedin the cell-mediated immune response. The “target binding domain” is also referred to herein as an “antigen binding domain”, and as such the term “target” encompasses an “antigen.”

[0088] So-called “first-generation” CARs had a targeting domain and a CD3^ signal transduction domain. So-called “second generation” CARs further included a co-stimulatory domain, such as a CD28 or 4- IBB domain. So-called “third generation” CARs comprise multiple co-stimulatory domains. So-called “fourth generation” CARs, also referred to as “TRUCKS” are engineered to release a transgenic cytokine upon CAR signaling.

[0089] Chimeric antigen receptors (“CARs”) include the following elements: (1) an optional signal peptide, (2) a target binding domain, (3) an optional hinge region; (4) a transmembrane region; and (5) an intracellular domain comprising a signal transduction domain. Optionally, the CAR can include any of: a CD3^ signal transduction domain, an Fc receptor signal transduction domain, and / or a co-stimulatory (signal transduction) domain. That is, these optional elements can be included in addition to or instead of other optional elements. The target binding domain is heterologous to at least one of the other domains. That is, the target binding domain does not naturally occur on a T cell receptor.

[0090] The “target binding domain” provides binding specificity to the CAR. The “signal peptide” guides the polypeptide through the cell membrane. The target binding domain can bind to a domain of an antibody that binds to the target antigen for a so-called “Universal CAR”. The “hinge region” is a flexible connector region, e.g., a natural or synthetic polypeptide, or any other type of molecule, providing structural flexibility and spacing to flanking polypeptide regions. The “transmembrane domain” is a membrane- spanning protein domain, typically hydrophobic. The “signal transduction domain” or “signaling domain” transmits a signal through a signal transduction pathway into the cell upon binding. Such signaling activates an activity of the cell. “Co-stimulatory domains” are accessory signaling domains that further transmit signals.7. SIGNAL PEPT / DE

[0091] A signal peptide can be any peptide having the function of allowing a polypeptide to traverse a cell membrane. The signal peptide can be derived from CD4, CD8, CD28, TLR, or immunoglobulin family of receptors.

[0092] Exemplary signal peptides are:MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO:1) MALPVTALLLPLALLLHAAR (SEQ ID NO:26).2. TARGET BEVD / NG DOMAINA. STRUCTURE

[0093] The target binding domain can include any polypeptide comprising a target binding function, e.g., an antibody as defined herein. In one embodiment, the target binding domain can comprise a single chain antibody (scFV). The scFv can be connected to the transmembrane domain via a hinge domain whose length, flexibility and origin provides variability in the CAR’s design, and can, along with the transmembrane domain, contribute to the interaction with antigen, building of the immunologic synapse and impact the CAR’s association with additional proteins needed to impart a robust activation signal.B. TARGETS

[0094] Chimeric antigen receptors disclosed herein comprise a target binding domain that binds to target antigens, e.g., autoimmune antigens, including but not limited to any one or more of MBP, MOG, PEP, PDE4, VEGFR, S 100- , Citrullinated vimentin (CV), TNF-like ligand 1A (TL1A), CD2, CD23 (IgE receptor), IL-6R, IL-4Ra, IL-12R, IL-13Ral, IL-13Ra2, IL-15R, IL- 23R, CD19, CD20, CD22, CD3, CD4, CD5, IFN-y, IgE, IL-la, IL-10, IL-6, IL-12, IL-23, IL-15, IL-17, IL-17A, IL-22, IL-13, IL-33, TSLP, OX-40, OX-40L, p53, CD38, CD40L, CD28, ICOS, RIPK1, BTK, Bet v 1, GLP2R, JAK1, C5, and TNF-a. For example, in some embodiments, the CAR binds to a target antigen selected from the group consisting of CD 19, CD20, CD22, and citrullinated vimentin (CV). In particular, the CV target binding domain can bind to one or more of (i) citrullinated vimentin, (ii) citrullinated filaggrin, (iii) citrullinated fibrinogen and (iv) citrullinated peptides thereof. In some embodiments, the target binding domain can bind to a citrullinated peptide fragment of (i)-(iii), wherein the peptide fragment is of at least about 10 amino acids in length, e.g., of at least about 12 amino acids, at least about 14 amino acids or at least about 16 amino acids in length. In some embodiments, the target binding domain further binds to tenascin C. In some embodiments, the target binding domain can bind to two or more of (i) citrullinated vimentin, (ii) citrullinated filaggrin, (iii) citrullinated fibrinogen, and (iv) tenascin C, or citrullinated peptide fragments thereof.

[0095] The target binding domain can comprise sequences from an antibody VH and VL domain. This includes particular CDR sets from VH and VL domains. In some embodiments the target binding domain comprises the CDRs from the VH domains of SEQ ID NO:67 or SEQ ID NO:69 or SEQ ID NO:71. In some embodiments the target binding domain comprises the CDRs from the VL domains of SEQ ID NO:68 or SEQ ID NO:70 or SEQ ID NO:72. In someembodiments the target binding domain comprises the CDRs from VH and VL domains of SEQ ID NO:67 and SEQ ID NO:68, respectively. In some embodiments the target binding domain comprises the CDRs from VH and VL domains of SEQ ID NO:69 and SEQ ID NO:70, respectively. In some embodiments the target binding domain comprises the CDRs from VH and VL domains of SEQ ID NO:71 and SEQ ID NO:72, respectively. In some embodiments the target binding domain comprises the CDRs from VH and VL domains of SEQ ID NO:67 and SEQ ID NO:70, respectively. In some embodiments the target binding domain comprises the CDRs from VH and VL domains of SEQ ID NO:67 and SEQ ID NO:72, respectively. In some embodiments the target binding domain comprises the CDRs from VH and VL domains of SEQ ID NO:69 and SEQ ID NO:68, respectively. In some embodiments the target binding domain comprises the CDRs from VH and VL domains of SEQ ID NO:69 and SEQ ID NO:72, respectively. In some embodiments the target binding domain comprises the CDRs from VH and VL domains of SEQ ID NO:71 and SEQ ID NO:68, respectively. In some embodiments the target binding domain comprises the CDRs from VH and VL domains of SEQ ID NO:71 and SEQ ID NO:70, respectively.

[0096] In some embodiments the target binding domain comprises the complementaritydetermining regions (CDRs) from VH and VL domains of SEQ ID NO:67 and SEQ ID NO:68, respectively. In some embodiments, the VH domain of the target-binding domain comprises a VH-CDR1 comprising the amino acid sequence of SEQ ID NO:34, a VH-CDR2 comprising the amino acid sequence of SEQ ID NO:35, and a VH-CDR3 comprising the amino acid sequence of SEQ ID NO:36, and the VL domain of the target-binding domain comprises a VL-CDR1 comprising the amino acid sequence of SEQ ID NO:92, a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 93, and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO:94.

[0097] In some embodiments the target binding domain comprises the complementaritydetermining regions (CDRs) from VH and VL domains of SEQ ID NO:67 and SEQ ID NO:70, respectively. In some embodiments, the VH domain of the target-binding domain comprises a VH-CDR1 comprising the amino acid sequence of SEQ ID NO:34, a VH-CDR2 comprising the amino acid sequence of SEQ ID NO:35, and a VH-CDR3 comprising the amino acid sequence of SEQ ID NO:36, and the VL domain of the target-binding domain comprises a VL-CDR1 comprising the amino acid sequence of SEQ ID NO:37, a VL-CDR2 comprising the amino acid sequence of SEQ ID NO:38, and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO:39.

[0098] In some embodiments the target binding domain comprises the complementaritydetermining regions (CDRs) from VH and VL domains of SEQ ID NO:69 and SEQ ID NO:68, respectively. In some embodiments, the VH domain of the target-binding domain comprises a VH-CDR1 comprising the amino acid sequence of SEQ ID NO:40, a VH-CDR2 comprising the amino acid sequence of SEQ ID NO:41, and a VH-CDR3 comprising the amino acid sequence of SEQ ID NO:36, and the VL domain of the target-binding domain comprises a VL-CDR1 comprising the amino acid sequence of SEQ ID NO:92, a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 93, and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO:94.

[0099] In some embodiments the target binding domain comprises the complementaritydetermining regions (CDRs) from VH and VL domains of SEQ ID NO:69 and SEQ ID NO:70, respectively. In some embodiments, the VH domain of the target-binding domain comprises a VH-CDR1 comprising the amino acid sequence of SEQ ID NO:40, a VH-CDR2 comprising the amino acid sequence of SEQ ID NO:41, and a VH-CDR3 comprising the amino acid sequence of SEQ ID NO:36, and the VL domain of the target-binding domain comprises a VL-CDR1 comprising the amino acid sequence of SEQ ID NO:37, a VL-CDR2 comprising the amino acid sequence of SEQ ID NO:38, and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO:39.

[0100] In some embodiments the target binding domain comprises the complementaritydetermining regions (CDRs) from VH and VL domains of SEQ ID NO:71 and SEQ ID NO:72, respectively. In some embodiments, the VH domain of the target-binding domain comprises a VH-CDR1 comprising the amino acid sequence of SEQ ID NO:42, a VH-CDR2 comprising the amino acid sequence of SEQ ID NO:43, and a VH-CDR3 comprising the amino acid sequence of SEQ ID NO:44, and the VL domain of the target-binding domain comprises a VL-CDR1 comprising the amino acid sequence of SEQ ID NO:45, a VL-CDR2 comprising the amino acid sequence of SEQ ID NO:46, and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO:47.Table 2. Framework (FR) and complementarity determining regions (CDRs) of the heavy chain variable domain (VH). CV, citrullinated vimentin.Table 3. Framework (FR) and complementarity determining regions (CDRs) of the light chain variable domain (VL). CV, citrullinated vimentin.

[0101] In certain embodiments, the target binding domain comprises VH sequences selected from:CV #1 VHHLHLQESGPGLVKPSETLSLTCTVSGGSINDTTYYWGWIRQPPGKGLEWIGSIYYRGNTHYNSSLRSRVTMSVDTSKNRFSLKVTSVTAADTAVYYCARLDPFDYWGRGTLVTVSS (SEQ ID NO:67);CV #2 / #4 VHQLQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLEWIGSIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARLDP FDYWGRGTLVTVSS (SEQ ID NO:69);CV #3 VHEVKLIESGGGLVEPGRSLRLACTTSGFTFADYGLSWFRQGPGKGLEWVG FTGPKHLGETTECAPSVEDRCTISRDDSKSTVYLQMHRLQHEDTAVYFCV GPWFGDLLMWGQGTLVTVSS (SEQ ID N0:71); or a sequence having at least any of 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.5% sequence identify with the aforementioned sequences, provided target binding domain binds to a citrullinated antigen as described herein.

[0102] The target binding region can comprise VL sequences selected from:CV #l / #4 VLSYVLTQPPSVSLAPGETATITCGGDDIENQNVNWYQQKSGQAPMLLIFFD TRRPSGIPERFSGSRSEDTANLTITRVEAGDDADYFCQVYDRKTDHQVFG PGTTVTVL (SEQ ID NO:68);CV #2 VLSYVLTQPPSVSVAPGKTARITCGGNNIGSKSVHWYQQKPGQAPVLVIYY DSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHQVF GTGTKVTVL (SEQ ID NO:70);CV #3 VLAIQMTQSPSSLSASVGDRVSITCRATQDISTSLGWYHQRPGKAPRLLIYGA SKVQTGVPSRFSGNGSGTEFTLTISSLQPEDIGTYYCLQDDGFPFTVGQGT KLDIKRAA (SEQ ID NO:72); or a sequence having at least any of 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.5% sequence identify with the aforementioned sequences, provided target binding domain binds to a citrullinated antigen as described herein.

[0103] In another embodiment, the antigen binding region comprises an scFV comprising an amino acid sequence selected from:CV #1 scFv - VHVL - GGGSx3 LinkerHLHLQESGPGLVKPSETLSLTCTVSGGSINDTTYYWGWIRQPPGKGLEWI GSIYYRGNTHYNSSLRSRVTMSVDTSKNRFSLKVTSVTAADTAVYYCAR LDPFDYWGRGTLVTVSSGGGGSGGGGSGGGGSSYVLTQPPSVSVAPGK TARITCGGNNIGSKSVHWYQQKPGQAPVLVIYYDSDRPSGIPERFSGSNS GNTATLTISRVEAGDEADYYCQVWDSSSDHQVFGTGTKVTVLR (SEQ ID NO:73) (GGGSx3 linker underlined);CV #2 scFv - VHVL - Whitlow 218 LinkerHLHLQESGPGLVKPSETLSLTCTVSGGSINDTTYYWGWIRQPPGKGLEWI GSIYYRGNTHYNSSLRSRVTMSVDTSKNRFSLKVTSVTAADTAVYYCAR LDPFDYWGRGTLVTVSSGSTSGSGKPGSGEGSTKGSYVLTQPPSVSVAP GKTARITCGGNNIGSKSVHWYQQKPGQAPVLVIYYDSDRPSGIPERFSGS NSGNTATLTISRVEAGDEADYYCQVWDSSSDHQVFGTGTKVTVLR (SEQ ID NO:74) (Whitlow 218 Linker underlined);CV #3 scFv - VHVL - AB pur LinkerHLHLQESGPGLVKPSETLSLTCTVSGGSINDTTYYWGWIRQPPGKGLEWI GSIYYRGNTHYNSSLRSRVTMSVDTSKNRFSLKVTSVTAADTAVYYCAR LDPFDYWGRGTLVTVSSASSGGSTSGSGKPGSGEGSSGSARSYVLTQPP SVSVAPGKTARITCGGNNIGSKSVHWYQQKPGQAPVLVIYYDSDRPSGIP ERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHQVFGTGTKVTV LR (SEQ ID NO:75) (AB pur Linker underlined);CV #4 scFv - VHVL - AB pur LinkerEVKLIESGGGLVEPGRSLRLACTTSGFTFADYGLSWFRQGPGKGLEWVG FTGPKHLGETTECAPSVEDRCTISRDDSKSTVYLQMHRLQHEDTAVYFCV GPWFGDLLMWGQGTLVTVSSASSGGSTSGSGKPGSGEGSSGSARAIQMT QSPSSLSASVGDRVSITCRATQDISTSLGWYHQRPGKAPRLLIYGASKVQT GVPSRFSGNGSGTEFTLTISSLQPEDIGTYYCLQDDGFPFTVGQGTKLDIK RAAA (SEQ ID NO:76) (AB pur Linker underlined); or a sequence having at least any of 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.5% sequence identify with the aforementioned sequences, provided target binding domain binds to a citrullinated antigen as described herein.

[0104] Accordingly, exemplary linkers are:GGGGSGGGGSGGGGS (SEQ ID NO:77) GGGSx3 Linker;GSTSGSGKPGSGEGSTKG (SEQ ID NO:78) Whitlow 218 Linker; or ASSGGSTSGSGKPGSGEGSSGSAR (SEQ ID NO:79) AB pur Linker.

[0105] Optionally, any of the foregoing sequences can include the CDR sets described above.. HINGE EEGION

[0106] In some embodiments, the hinge region of the disclosed CARs can be selected from the CD8, CD4, or CD28 extracellular domain, the Fc region of an IgGl antibody, or the extracellular domain of any of the TLR receptors as is known to one of skill in the art and can be found in the GenBank database. In some embodiments, the hinge region is any suitable hinge region known in the art.

[0107] For example, the hinge region can comprise the sequence:IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO:27) (CD28);TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTC GVLLLSLVITLYC (SEQ ID NO:28) (CD8); or a sequence having at least any of 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.5% sequence identify with the aforementioned hinge region sequences.■Z TEANSMEM EANE DOMAIN

[0108] The transmembrane domain can comprise a transmembrane domain of an immunoglobulin family receptor, such as CD8. The intracellular domain can be selected from any membrane- spanning molecule on a T cell. For example, the transmembrane (TM) domain of the disclosed CAR can comprise the TM domain of CD2, CD3, CD16, CD32, CD64, CD28, CD247, 4-1BBL, CD4, or CD8.

[0109] For example, the transmembrane domain can comprise a sequence selected from:FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO:29);IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO:30); or a sequence having at least any of 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.5% sequence identify with the aforementioned hinge region sequences.5. SIGNAL TEANSDOCTION DOMAINA. CD3Z SIGNAL TRANSDUCTION DOMAIN

[0110] In some embodiments, the signal transduction domain comprises a CD3(^ signaling domain. The CD3(^ signaling domain of the disclosed CAR molecule can comprise a CD3^ amino acid sequence, e.g., a signal transduction domain of CD3 zeta.

[0111] For example, the CD3^ signal transduction domain can have a sequence selected from:RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRK NPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDAL HMQALPPR (SEQ ID NO:31); or a sequence having at least any of 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.5% sequence identify with the aforementioned CD3 zeta signal transduction domain sequence.

[0112] For example, the CD3 zeta signal transduction domain can include amino acids 21- 163, 31-142, 68-89 and / or 138-158 of the sequence shown in SEQ ID NO:31, or functional variants thereof (e.g., with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 10-20 amino acid substitutions, deletions, or additions).B. FC RECEPTOR SIGNAL TRANSDUCTION DOMAIN

[0113] In some embodiments, the signal transduction domain comprises an Fc signaling domain. The Fc signaling domain can be any one of the Fc-alpha, Fc-gamma, Fc-epsilon, Fc- mu, and Fc-delta receptors. For example, the Fc receptor signaling domain can comprise amino acids involved in interaction with Src (e.g., Fgr, Fyn, Hck, Lyn, Yes, and Src) and ZAP-70 family kinases, e.g., one or more IT AM domains (see, e.g., Sanchez-Mejorada et al. (1998) J. Leukocyte Biol. 63:531; Garcia-Garcia et al. (2002) J. Leukocyte Biol. 72:1092). In some embodiments, the Fc receptor signal transduction domain includes at least one ITAM domain, e.g., from any one of the Fc-alpha, Fc-gamma, Fc-epsilon, Fc-mu, and Fc-delta receptors, or substantially identical thereto.

[0114] Sequences also can be found as follows:6. CO-STLMULATOXY DOMAEV

[0115] The CARs of this disclosure can include one or more co- stimulatory domains in addition to a signal transduction domain of CD3^ or an Fc receptor. Co-stimulatory domains can be derived from, for example, CD28, 4- IBB, CD2, CD27, CD30, 0X40, CD40, PD-1, PD- Ll, PD-L2, ICOS, LFA-1, CD7, LIGHT, NKG2C, B7-H3, CD83L, B7-1 (CD80), B7-2 (CD86), B7-H3, B7-H4 and others. The CAR constructs can contain two or more co-stimulatory signaling domains (e.g., CD28 + 4-1BB).

[0116] The co-stimulatory domain or domains can be positioned between the signal transduction domain and the transmembrane region.

[0117] In certain embodiments, a CD28 co-stimulatory domain can comprise a sequence selected from:RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO:32); or a sequence having at least any of 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.5% sequence identify with the aforementioned sequence.

[0118] In certain embodiments, a 41BB co-stimulatory domain can comprise a sequence selected from:KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO:33); or a sequence having at least any of 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.5% sequence identify with the aforementioned sequence.E. CELL SEE FACE XECEPTOXS

[0119] In some embodiments, the expression cassettes of the present invention comprise a nucleic acid encoding a cell surface receptor. The cell surface receptor can be used to identify cells that express the expression cassettes provided herein using standard methods in the field, for example, but not limited to, flow cytometry, western blotting, immunohistochemistry and microscopy (e.g., fluorescence microscopy), radioligand binding assay, ELISA, etc.

[0120] In some embodiments, the cell surface receptor lacks a functional intracellular signaling domain or is not capable of mediating intracellular signaling. In some embodiments, the cell surface receptor binds its native ligand (z.e., wildtype ligand). In some embodiments, the cell surface receptor binds to a non-native ligand, z.e., a ligand not naturally found in theorganism comprising a cell comprising any of the expression cassettes described herein. In some embodiments, the cell surface receptor comprises an extracellular domain and a transmembrane domain. In some embodiments, the extracellular domain is a wildtype extracellular domain or a modified extracellular domain. In some embodiments, the modified extracellular domain comprises one or more (e.g., at least about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acid modifications (e.g., substitution, deletion, or insertion) compared to the amino acid sequence of a wildtype extracellular domain. In some embodiments, the modified extracellular domain binds to a non-native ligand, thereby differentiating between cells expressing a wildtype cell surface receptor only and cells expressing both a wildtype cell surface receptor and an expression cassette comprising a nucleic acid encoding a modified cell surface receptor.

[0121] In some embodiments, the cell surface receptor comprises an intracellular signaling domain. In some embodiments, the cell surface receptor comprises no intracellular signaling domain. In some embodiments, the cell surface receptor comprises a truncated intracellular signaling domain. In some embodiments, the cell surface receptor comprises an intracellular signaling domain that is at least about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acids in length, wherein the intracellular signaling domain comprises at least about 80% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence homology to the N-terminus of the intracellular domain of the wildtype cell surface receptor. In some embodiments, the cell surface receptor comprises a modified intracellular signaling domain comprising one or more (e.g., at least about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acid modifications (e.g., substitution, deletion, or insertion) compared to a wildtype intracellular signaling domain, wherein the modified intracellular signaling domain is not capable of mediating intracellular signaling.

[0122] In some embodiments, the cell surface receptor is not endogenously expressed in the cell transduced with any of the expression cassettes expressing exogenous FOXP3 described herein. In some embodiments, the cell surface receptor is not endogenously expressed in T cells, for example Treg cells. In some embodiments, the cell surface receptor is not endogenously expressed in Treg cells. In some embodiments, the cell surface receptor that is endogenously expressed in Treg cells is modified. In some embodiments, the modified cell surface receptor is distinguishable from the endogenous cell surface receptor. In some embodiments, the cell surface receptor is selected from the group consisting of EGFR, HER2, CD2, H-2Kk, IL-5Ra, and derivatives or mutants thereof. In some embodiments, the cell surface receptor comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:17-19.C. B / C / STRO / C CASSETTES

[0123] In some embodiments, the expression cassettes of the present invention comprise two nucleic acids encoding unique polypeptides, wherein the nucleic acids may be separated by a separation element, for example by an IRES, a nucleic acid encoding a self-cleaving peptide (e.g., P2A peptide), or a nucleic acid encoding a peptide that causes ribosome skipping. Thus, upon translation, the expression cassette comprising the two nucleic acids yield two independent polypeptides, e.g., full-length proteins or derivatives thereof, such as full-length FOXP3 and / or an alternatively spliced isoform that lacks one or more exons, e.g., exon 2, and a cell surface receptor. For example, the expression cassette provided herein may yield, but is not limited to, one or more isoforms of the FOXP3 protein, and a cell surface receptor e.g., the cell membrane portion of EGFR, HER2, CD2, H-2Kk, or IE-5Ra protein or a derivative or mutant thereof).

[0124] In some embodiments, provided herein are expression cassettes comprising a nucleic acid encoding from 5’ to 3’: a) a promoter; b) a modified nucleic acid encoding a FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein comprises intron sequences flanking a nucleic acid encoding exon 2, wherein the nucleic acid encoding the FOXP3 protein is alternatively spliced to produce a full-length FOXP3 protein isoform and a FOXP3 protein isoform lacking exon 2; and c) a nucleic acid encoding a cell surface receptor. In some embodiments, provided herein are expression cassettes comprising a nucleic acid encoding from 5’ to 3’: a) a promoter; b) a modified nucleic acid encoding a FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein comprises intron sequences flanking a nucleic acid encoding exon 2, wherein the nucleic acid encoding the FOXP3 protein is alternatively spliced to produce a full-length FOXP3 protein isoform and a FOXP3 protein isoform lacking exon 2; c) an IRES, or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; and d) a nucleic acid encoding a cell surface receptor. In some embodiments, the nucleic acid encoding the FOXP3 protein encodes only one FOXP3 protein isoform, wherein the FOXP3 protein isoform is full-length FOXP3 protein or a FOXP3 protein isoform lacking exon 2. In some embodiments, the nucleic acid encoding the FOXP3 protein encodes two FOXP3 protein isoforms, wherein the FOXP3 protein isoforms are full-length FOXP3 protein and a FOXP3 protein isoform lacking exon 2. In some embodiments, the nucleic acid encoding the FOXP3 protein comprising intron sequences flanking the nucleic acid encoding exon 2 is alternatively spliced to produce a full-length FOXP3 protein isoform and a FOXP3 protein isoform lacking exon 2. In some embodiments, the nucleic acid encoding the FOXP3 protein is alternatively spliced to produce a full-length FOXP3 protein isoform and a FOXP3 proteinisoform lacking exon 2, wherein the FOXP3 protein comprises the amino acid sequences of SEQ ID NOs:20 and 21. In some embodiments, the nucleic acid encoding the FOXP3 protein is alternatively spliced to produce a full-length FOXP3 protein isoform and a FOXP3 protein isoform lacking exon 2, wherein the FOXP3 protein comprises the amino acid sequences of SEQ ID NOs:22 and 23. In some embodiments, the FOXP3 protein is linked to a detection tag. In some embodiments, the detection tag is linked to the N-terminus of the FOXP3 protein. In some embodiments, the detection tag is linked to the C-terminus of the FOXP3 protein. In some embodiments, the nucleic acid encoding the FOXP3 does not contain intron sequences other than the intron sequences flanking exon 2. In some embodiments, the nucleic acid encoding a FOXP3 protein is FOXP3 cDNA. In some embodiments, the cell surface receptor lacks a functional intracellular signaling domain or is not capable of mediating intracellular signaling. In some embodiments, the cell surface receptor is selected from the group consisting of EGFR, HER2, CD2, H-2Kk, IE-5Ra, and derivatives or mutants thereof. In some embodiments, the nucleic acid encoding the FOXP3 protein and the nucleic acid encoding the cell surface receptor are operably linked to the same promoter. In some embodiments, the promoter is an inducible or a constitutive promoter. In some embodiments, the promoter is a cell-specific promoter. In some embodiments, the promoter is specific for T cells (e.g., Tregs). In some embodiments, the nucleic acid encoding the FOXP3 protein and the nucleic acid encoding the cell surface receptor are separated by an IRES, a nucleic acid encoding a self-cleaving peptide (e.g., P2A peptide), or a nucleic acid encoding a peptide that causes ribosome skipping. In some embodiments, the expression cassette further comprises a nucleic acid encoding a self-cleaving peptide. In some embodiments, the self-cleaving peptide is a 2A sequence. In some embodiments, the 2A sequence is a P2A sequence. In some embodiments, the expression cassette comprises a nucleic acid encoding from 5’ to 3’: a) a promoter; b) a modified nucleic acid encoding a FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein comprises intron sequences flanking a nucleic acid encoding exon 2, wherein the nucleic acid encoding the FOXP3 protein is alternatively spliced to produce a full-length FOXP3 protein isoform and a FOXP3 protein isoform lacking exon 2, wherein the FOXP3 protein comprises the amino acid sequences of SEQ ID NOs:20 and 21; c) a nucleic acid encoding a self-cleaving 2A peptide (e.g., P2A peptide), wherein the 2A peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:2-10 and 89-91; and d) a nucleic acid encoding a cell surface receptor comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:17-19. In some embodiments, the expression cassette comprises a nucleic acid encoding from 5’ to 3’: a) apromoter; b) a modified nucleic acid encoding a FOXP3 protein linked to a detection tag, wherein the nucleic acid encoding the FOXP3 protein comprises intron sequences flanking a nucleic acid encoding exon 2, wherein the nucleic acid encoding the FOXP3 protein is alternatively spliced to produce a full-length FOXP3 protein isoform and a FOXP3 protein isoform lacking exon 2, wherein the FOXP3 protein comprises the amino acid sequences of SEQ ID NOs:22 and 23; c) a nucleic acid encoding a self-cleaving 2A peptide (e.g., P2A peptide), wherein the 2A peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:2-10 and 89-91; and d) a nucleic acid encoding a cell surface receptor comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:17-19.

[0125] In some embodiments, provided herein are expression cassettes comprising a nucleic acid encoding from 5’ to 3’: a) a promoter; b) a modified nucleic acid encoding a full-length FOXP3 protein; and c) a nucleic acid encoding a cell surface receptor. In some embodiments, provided herein are expression cassettes comprising a nucleic acid encoding from 5’ to 3’: a) a promoter; b) a modified nucleic acid encoding a full-length FOXP3 protein; c) an IRES, or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; and d) a nucleic acid encoding a cell surface receptor. In some embodiments, the nucleic acid encoding the FOXP3 protein encodes only one FOXP3 protein isoform, wherein the FOXP3 protein isoform is full-length FOXP3 protein. In some embodiments, the nucleic acid encoding the full- length FOXP3 protein comprises the amino acid sequence of SEQ ID NO:20 or 22. In some embodiments, the FOXP3 protein is linked to a detection tag. In some embodiments, the detection tag is linked to the N-terminus of the FOXP3 protein. In some embodiments, the detection tag is linked to the C-terminus of the FOXP3 protein. In some embodiments, the nucleic acid encoding a FOXP3 protein is FOXP3 cDNA. In some embodiments, the cell surface receptor lacks a functional intracellular signaling domain or is not capable of mediating intracellular signaling. In some embodiments, the cell surface receptor is selected from the group consisting of EGFR, HER2, CD2, H-2Kk, IL-5Ra, and derivatives or mutants thereof. In some embodiments, the nucleic acid encoding the FOXP3 protein and the nucleic acid encoding the cell surface receptor are operably linked to the same promoter. In some embodiments, the promoter is an inducible or a constitutive promoter. In some embodiments, the promoter is a cell-specific promoter. In some embodiments, the promoter is specific for T cells (e.g., Tregs). In some embodiments, the nucleic acid encoding the FOXP3 protein and the nucleic acid encoding the cell surface receptor are separated by an IRES, a nucleic acid encoding a selfcleaving peptide (e.g., P2A peptide), or a nucleic acid encoding a peptide that causes ribosomeskipping. In some embodiments, the expression cassette further comprises a nucleic acid encoding a self-cleaving peptide. In some embodiments, the self-cleaving peptide is a 2A sequence. In some embodiments, the 2A sequence is a P2A sequence. In some embodiments, the expression cassette comprises a nucleic acid encoding from 5’ to 3’: a) a promoter; b) a modified nucleic acid encoding a FOXP3 protein comprising the amino sequence of SEQ ID NO:20 or 22; c) a nucleic acid encoding a self-cleaving 2A peptide; and d) a nucleic acid encoding a cell surface receptor.

[0126] In some embodiments, provided herein are expression cassettes comprising a nucleic acid encoding from 5’ to 3’: a) a promoter; b) a modified nucleic acid encoding a FOXP3 protein isoform lacking exon 2; and c) a nucleic acid encoding a cell surface receptor. In some embodiments, provided herein are expression cassettes comprising a nucleic acid encoding from 5’ to 3’: a) a promoter; b) a modified nucleic acid encoding a FOXP3 protein isoform lacking exon 2; c) an IRES, or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; and d) a nucleic acid encoding a cell surface receptor. In some embodiments, the nucleic acid encoding the FOXP3 protein encodes only one FOXP3 protein isoform, wherein the FOXP3 protein isoform is a FOXP3 protein isoform lacking exon 2. In some embodiments, the modified nucleic acid encoding the FOXP3 protein isoform lacking exon 2 comprises the amino acid sequence of SEQ ID NO:21 or 23. In some embodiments, the FOXP3 protein is linked to a detection tag. In some embodiments, the detection tag is linked to the N-terminus of the FOXP3 protein. In some embodiments, the detection tag is linked to the C-terminus of the FOXP3 protein. In some embodiments, the nucleic acid encoding a FOXP3 protein is FOXP3 cDNA. In some embodiments, the cell surface receptor lacks a functional intracellular signaling domain or is not capable of mediating intracellular signaling. In some embodiments, the cell surface receptor is selected from the group consisting of EGFR, HER2, CD2, H-2Kk, IL-5Ra, and derivatives or mutants thereof. In some embodiments, the nucleic acid encoding the FOXP3 protein and the nucleic acid encoding the cell surface receptor are operably linked to the same promoter. In some embodiments, the promoter is an inducible or a constitutive promoter. In some embodiments, the promoter is a cell-specific promoter. In some embodiments, the promoter is specific for T cells (e.g., Tregs). In some embodiments, the nucleic acid encoding the FOXP3 protein and the nucleic acid encoding the cell surface receptor are separated by an IRES, a nucleic acid encoding a self-cleaving peptide (e.g., P2A peptide), or a nucleic acid encoding a peptide that causes ribosome skipping. In some embodiments, the expression cassette further comprises a nucleic acid encoding a self-cleaving peptide. In some embodiments, theself-cleaving peptide is a 2A sequence. In some embodiments, the 2A sequence is a P2A sequence. In some embodiments, the expression cassette comprises a nucleic acid encoding from 5’ to 3’: a) a promoter; b) a modified nucleic acid encoding a FOXP3 Protein isoform lacking exon 2 comprising the amino acid sequence of SEQ ID NO:21 or 23; c) a nucleic acid encoding a self-cleaving 2A peptide; and d) a nucleic acid encoding a cell surface receptor.

[0127] In some embodiments, provided herein are expression cassettes comprising a nucleic acid encoding from 5’ to 3’: a) a promoter; b) a nucleic acid encoding a cell surface receptor; and c) a modified nucleic acid encoding a FOXP3 protein, wherein the modified nucleic acid encoding the FOXP3 protein comprises intron sequences flanking a nucleic acid encoding exon 2, wherein the nucleic acid encoding the FOXP3 protein is alternatively spliced to produce a full-length FOXP3 protein isoform and a FOXP3 protein isoform lacking exon 2. In some embodiments, provided herein are expression cassettes comprising a nucleic acid encoding from 5’ to 3’: a) a promoter; b) a nucleic acid encoding a cell surface receptor; c) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; and d) a modified nucleic acid encoding a FOXP3 protein, wherein the modified nucleic acid encoding the FOXP3 protein comprises intron sequences flanking a nucleic acid encoding exon 2, wherein the nucleic acid encoding the FOXP3 protein is alternatively spliced to produce a full-length FOXP3 protein isoform and a FOXP3 protein isoform lacking exon 2. In some embodiments, the nucleic acid encoding the FOXP3 protein encodes only one FOXP3 protein isoform, wherein the FOXP3 protein isoform is full-length FOXP3 protein or a FOXP3 protein isoform lacking exon 2. In some embodiments, the nucleic acid encoding the FOXP3 protein encodes two FOXP3 protein isoforms, wherein the FOXP3 protein isoforms are full-length FOXP3 protein and a FOXP3 protein isoform lacking exon 2. In some embodiments, the nucleic acid encoding the FOXP3 protein comprising intron sequences flanking the nucleic acid encoding exon 2 is alternatively spliced to produce a full-length FOXP3 protein isoform and a FOXP3 protein isoform lacking exon 2. In some embodiments, the nucleic acid encoding the FOXP3 protein is alternatively spliced to produce a full-length FOXP3 protein isoform and a FOXP3 protein isoform lacking exon 2, wherein the FOXP3 protein comprises the amino acid sequences of SEQ ID NOs:20 and 21. In some embodiments, the nucleic acid encoding the FOXP3 protein is alternatively spliced to produce a full-length FOXP3 protein isoform and a FOXP3 protein isoform lacking exon 2, wherein the FOXP3 protein comprises the amino acid sequences of SEQ ID NOs:22 and 23. In some embodiments, the FOXP3 protein is linked to a detection tag. In some embodiments, the detection tag is linkedto the N-terminus of the F0XP3 protein. In some embodiments, the detection tag is linked to the C-terminus of the F0XP3 protein. In some embodiments, the nucleic acid encoding the FOXP3 does not contain intron sequences other than the intron sequences flanking exon 2. In some embodiments, the nucleic acid encoding a FOXP3 protein is FOXP3 cDNA. In some embodiments, the cell surface receptor lacks a functional intracellular signaling domain or is not capable of mediating intracellular signaling. In some embodiments, the cell surface receptor is selected from the group consisting of EGFR, HER2, CD2, H-2Kk, IL-5Ra, and derivatives or mutants thereof. In some embodiments, the nucleic acid encoding the FOXP3 protein and the nucleic acid encoding the cell surface receptor are operably linked to the same promoter. In some embodiments, the promoter is an inducible or a constitutive promoter. In some embodiments, the promoter is a cell-specific promoter. In some embodiments, the promoter is specific for T cells (e.g., Tregs). In some embodiments, the nucleic acid encoding the FOXP3 protein and the nucleic acid encoding the cell surface receptor are separated by an IRES, a nucleic acid encoding a self-cleaving peptide (e.g. , P2A peptide), or a nucleic acid encoding a peptide that causes ribosome skipping. In some embodiments, the expression cassette further comprises a nucleic acid encoding a self-cleaving peptide. In some embodiments, the selfcleaving peptide is a 2A sequence. In some embodiments, the 2A sequence is a P2A sequence. In some embodiments, the expression cassette comprises a nucleic acid encoding from 5’ to 3’: a) a promoter; b) a nucleic acid encoding a cell surface receptor comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:17-19; c) a nucleic acid encoding a self-cleaving 2A peptide (e.g., P2A peptide), wherein the 2A peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:2-10 and 89-91; and d) a modified nucleic acid encoding a FOXP3 protein, wherein the modified nucleic acid encoding the FOXP3 protein comprises intron sequences flanking a nucleic acid encoding exon 2, wherein the nucleic acid encoding the FOXP3 protein is alternatively spliced to produce a full-length FOXP3 protein isoform and a FOXP3 protein isoform lacking exon 2, wherein the FOXP3 protein comprises the amino acid sequences of SEQ ID NOs:20 and 21. In some embodiments, the expression cassette comprises a nucleic acid encoding from 5’ to 3’: a) a promoter; b) a nucleic acid encoding a cell surface receptor comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:17-19; c) a nucleic acid encoding a self-cleaving 2A peptide (e.g., P2A peptide), wherein the 2A peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:2-10 and 89-91; and d) a modified nucleic acid encoding a FOXP3 protein linked to a detection tag, wherein the modified nucleic acid encoding the FOXP3 protein comprisesintron sequences flanking a nucleic acid encoding exon 2, wherein the nucleic acid encoding the FOXP3 protein is alternatively spliced to produce a full-length FOXP3 protein isoform and a FOXP3 protein isoform lacking exon 2, wherein the FOXP3 protein comprises the amino acid sequences of SEQ ID NOs:22 and 23.

[0128] In some embodiments, provided herein are expression cassettes comprising a nucleic acid encoding from 5’ to 3’: a) a promoter; b) a nucleic acid encoding a cell surface receptor; and c) a modified nucleic acid encoding a full-length FOXP3 protein. In some embodiments, provided herein are expression cassettes comprising a nucleic acid encoding from 5’ to 3’: a) a promoter; b) a nucleic acid encoding a cell surface receptor; c) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; and d) a modified nucleic acid encoding a full-length FOXP3 protein. In some embodiments, the nucleic acid encoding the FOXP3 protein encodes only one FOXP3 protein isoform, wherein the FOXP3 protein isoform is full-length FOXP3. In some embodiments, the nucleic acid encoding the full-length FOXP3 protein comprises the amino acid sequence of SEQ ID NO:20 or 22. In some embodiments, the FOXP3 protein is linked to a detection tag. In some embodiments, the detection tag is linked to the N-terminus of the FOXP3 protein. In some embodiments, the detection tag is linked to the C-terminus of the FOXP3 protein. In some embodiments, the nucleic acid encoding a FOXP3 protein is FOXP3 cDNA. In some embodiments, the cell surface receptor lacks a functional intracellular signaling domain or is not capable of mediating intracellular signaling. In some embodiments, the cell surface receptor is selected from the group consisting of EGFR, HER2, CD2, H-2Kk, IL-5Ra, and derivatives or mutants thereof. In some embodiments, the nucleic acid encoding the FOXP3 protein and the nucleic acid encoding the cell surface receptor are operably linked to the same promoter. In some embodiments, the promoter is an inducible or a constitutive promoter. In some embodiments, the promoter is a cell-specific promoter. In some embodiments, the promoter is specific for T cells (e.g., Tregs). In some embodiments, the nucleic acid encoding the FOXP3 protein and the nucleic acid encoding the cell surface receptor are separated by an IRES, a nucleic acid encoding a self-cleaving peptide (e.g. , P2A peptide), or a nucleic acid encoding a peptide that causes ribosome skipping. In some embodiments, the expression cassette further comprises a nucleic acid encoding a self-cleaving peptide. In some embodiments, the selfcleaving peptide is a 2A sequence. In some embodiments, the 2A sequence is a P2A sequence. In some embodiments, the expression cassette comprises a nucleic acid encoding from 5’ to 3’: a) a promoter; b) a nucleic acid encoding a cell surface receptor; c) a nucleic acid encoding aself-cleaving 2A peptide; and d) a modified nucleic acid encoding a full-length FOXP3 protein comprising the amino acid sequence of SEQ ID NO:20 or 22.

[0129] In some embodiments, provided herein are expression cassettes comprising a nucleic acid encoding from 5’ to 3’: a) a promoter; b) a nucleic acid encoding a cell surface receptor; and c) a modified nucleic acid encoding a FOXP3 protein isoform lacking exon 2. In some embodiments, provided herein are expression cassettes comprising a nucleic acid encoding from 5’ to 3’: a) a promoter; b) a nucleic acid encoding a cell surface receptor; c) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; and d) a modified nucleic acid encoding a FOXP3 protein isoform lacking exon 2. In some embodiments, the nucleic acid encoding the FOXP3 protein encodes only one FOXP3 protein isoform, wherein the FOXP3 protein isoform is a FOXP3 protein isoform lacking exon 2. In some embodiments, the modified nucleic acid encoding the FOXP3 protein isoform lacking exon 2 comprises the amino acid sequence of SEQ ID NO:21 or 23. In some embodiments, the FOXP3 protein is linked to a detection tag. In some embodiments, the detection tag is linked to the N-terminus of the FOXP3 protein. In some embodiments, the detection tag is linked to the C-terminus of the FOXP3 protein. In some embodiments, the nucleic acid encoding a FOXP3 protein is FOXP3 cDNA. In some embodiments, the cell surface receptor lacks a functional intracellular signaling domain or is not capable of mediating intracellular signaling. In some embodiments, the cell surface receptor is selected from the group consisting of EGFR, HER2, CD2, H-2Kk, IL-5Ra, and derivatives or mutants thereof. In some embodiments, the nucleic acid encoding the FOXP3 protein and the nucleic acid encoding the cell surface receptor are operably linked to the same promoter. In some embodiments, the promoter is an inducible or a constitutive promoter. In some embodiments, the promoter is a cell-specific promoter. In some embodiments, the promoter is specific for T cells (e.g., Tregs). In some embodiments, the nucleic acid encoding the FOXP3 protein and the nucleic acid encoding the cell surface receptor are separated by an IRES, a nucleic acid encoding a selfcleaving peptide (e.g., P2A peptide), or a nucleic acid encoding a peptide that causes ribosome skipping. In some embodiments, the expression cassette further comprises a nucleic acid encoding a self-cleaving peptide. In some embodiments, the self-cleaving peptide is a 2A sequence. In some embodiments, the 2A sequence is a P2A sequence. In some embodiments, the expression cassette comprises a nucleic acid encoding from 5’ to 3’: a) a promoter; b) a nucleic acid encoding a cell surface receptor; c) a nucleic acid encoding a self-cleaving 2A peptide; andd) a modified nucleic acid encoding a FOXP3 protein isoform lacking exon 2 comprising the amino acid sequence of SEQ ID NO:21 or 23.D. TE / C / STEOJV / C / MULT / -C / STEOJV / C CASSETTES

[0130] In some embodiments, the expression cassettes of the present invention comprise three or more nucleic acids encoding unique polypeptides, wherein each nucleic acid may be separated by a separation element, for example an IRES, a nucleic acid encoding a self-cleaving peptide (e.g., P2A peptide), or a nucleic acid encoding a peptide that causes ribosome skipping. Thus, upon translation, the expression cassette comprising the three or more nucleic acids yield three or more independent polypeptides, e.g., full-length proteins or derivatives thereof, such as full-length FOXP3 and / or an alternatively spliced isoform that lacks one or more exons, e.g., exon 2. For example, the expression cassette provided herein may yield, but is not limited to, one or more isoforms of the FOXP3 protein, a cell surface receptor (e.g., the cell membrane portion of the EGFR, HER2, CD2, H-2Kk, or IE-5Ra protein or a derivative or mutant thereof), and a chimeric receptor, such as a chimeric antigen receptor (CAR), for example a CAR that targets (e.g.) MBP, MOG, PEP, PDE4, VEGFR, S 100- , Citrullinated vimentin (CV), TNF-like ligand 1A (TL1A), CD2, CD23 (IgE receptor), IL-6R, IL-4Ra, IL-12R, IL-13Ral, IL-13Ra2, IL-15R, IL-23R, CD19, CD20, CD22, CD3, CD4, CD5, IFN-y, IgE, IL-la, IL-10, IL-6, IL-12, IL-23, IL- 15, IL- 17, IL-17A, IL-22, IL- 13, IL-33, TSLP, OX-40, OX-40L, p53, CD38, CD40L, CD28, ICOS, RIPK1, BTK, Bet v 1, GLP2R, JAK1, C5, or TNF-a (such as citrullinated vimentin or CD 19). Nucleic acids encoding additional proteins can be further incorporated into the expression cassette.

[0131] In some embodiments, provided herein expression cassettes comprising a nucleic acid encoding from 5’ to 3’: a) a promoter; b) a nucleic acid encoding a chimeric receptor; c) a nucleic acid encoding a cell surface receptor; and d) a modified nucleic acid encoding a FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein comprises intron sequences flanking a nucleic acid encoding exon 2, wherein the nucleic acid encoding the FOXP3 protein is alternatively spliced to produce a full-length FOXP3 protein isoform and a FOXP3 protein isoform lacking exon 2. In some embodiments, provided herein expression cassettes comprising a nucleic acid encoding from 5’ to 3’: a) a promoter; b) a nucleic acid encoding a chimeric receptor; c) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; d) a nucleic acid encoding a cell surface receptor; e) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleavingpeptide or a peptide that causes ribosome skipping; and f) a modified nucleic acid encoding a FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein comprises intron sequences flanking a nucleic acid encoding exon 2, wherein the nucleic acid encoding the FOXP3 protein is alternatively spliced to produce a full-length FOXP3 protein isoform and a FOXP3 protein isoform lacking exon 2. In some embodiments, the nucleic acid encoding the FOXP3 protein encodes only one FOXP3 isoform, wherein the FOXP3 protein isoform is full- length FOXP3 protein or a FOXP3 protein isoform lacking exon 2. In some embodiments, the nucleic acid encoding the FOXP3 protein encodes two FOXP3 protein isoforms, wherein the FOXP3 protein isoforms are full-length FOXP3 protein and a FOXP3 protein isoform lacking exon 2. In some embodiments, the nucleic acid encoding the FOXP3 protein comprising intron sequences flanking the nucleic acid encoding exon 2 is alternatively spliced to produce a full- length FOXP3 protein isoform and a FOXP3 protein isoform lacking exon 2. In some embodiments, the nucleic acid encoding the FOXP3 protein is alternatively spliced to produce a full-length FOXP3 protein isoform and a FOXP3 protein isoform lacking exon 2, wherein the FOXP3 protein comprises the amino acid sequences of SEQ ID NOs:20 and 21. In some embodiments, the nucleic acid encoding the FOXP3 protein is alternatively spliced to produce a full-length FOXP3 protein isoform and a FOXP3 protein isoform lacking exon 2, wherein the FOXP3 protein comprises the amino acid sequences of SEQ ID NOs:22 and 23. In some embodiments, the FOXP3 protein is linked to a detection tag. In some embodiments, the detection tag is linked to the N-terminus of the FOXP3 protein. In some embodiments, the detection tag is linked to the C-terminus of the FOXP3 protein. In some embodiments, the nucleic acid encoding the FOXP3 does not contain intron sequences other than the intron sequences flanking exon 2. In some embodiments, the nucleic acid encoding a FOXP3 protein is FOXP3 cDNA. In some embodiments, the cell surface receptor lacks a functional intracellular signaling domain or is not capable of mediating intracellular signaling. In some embodiments, the cell surface receptor is selected from the group consisting of EGFR, HER2, CD2, H-2Kk, IL- 5Ra, and derivatives or mutants thereof. In some embodiments, the chimeric receptor comprises a chimeric antigen receptor, a T cell Receptor, or a PiG receptor. In some embodiments, the chimeric receptor binds to an antigen selected from the group consisting of MBP, MOG, PLP, PDE4, VEGFR, S 100- , Citrullinated vimentin (CV), TNF-like ligand 1A (TL1A), CD2, CD23 (IgE receptor), IL-6R, IL-4Ra, IL-12R, IL-13Ral, IL-13Ra2, IL-15R, IL-23R, CD19, CD20, CD22, CD3, CD4, CD5, ILN-y, IgE, IL-la, IL-10, IL-6, IL-12, IL-23, IL-15, IL-17, IL-17A, IL- 22, IL-13, IL-33, TSLP, OX-40, OX-40L, p53, CD38, CD40L, CD28, ICOS, RIPK1, BTK, Betv 1, GLP2R, JAK1, C5, and TNF-a. In some embodiments, the nucleic acid encoding the FOXP3 protein and the nucleic acid encoding the chimeric receptor and / or the nucleic acid encoding the cell surface receptor are operably linked to the same promoter. In some embodiments, the promoter is an inducible or a constitutive promoter. In some embodiments, the promoter is a cell-specific promoter. In some embodiments, the promoter is specific for T cells (e.g., Tregs). In some embodiments, the nucleic acid encoding the FOXP3 protein and the nucleic acid encoding the chimeric receptor and / or the nucleic acid encoding the cell surface receptor are each separated by an IRES, a nucleic acid encoding a self-cleaving peptide (e.g., P2A peptide), or a nucleic acid encoding a peptide that causes ribosome skipping. In some embodiments, the expression cassette further comprises a nucleic acid encoding a self-cleaving peptide. In some embodiments, the self-cleaving peptide is a 2A sequence. In some embodiments, the 2A sequence is a P2A sequence. In some embodiments, the 2A sequence is a T2A sequence. In some embodiments, the expression cassette comprises a nucleic acid encoding from 5’ to 3’: a) a promoter; b) a nucleic acid encoding a chimeric receptor (e.g., a chimeric receptor comprising an antigen binding domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:73-76); c) a nucleic acid encoding a self-cleaving 2A peptide (e.g., P2A peptide), wherein the 2A peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:2-10 and 89-91; d) a nucleic acid encoding a cell surface receptor comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:17-19; e) a nucleic acid encoding a self-cleaving 2A peptide (e.g., P2A or T2A peptide), wherein the 2A peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:2-10 and 89-91; and f) a modified nucleic acid encoding a FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein comprises intron sequences flanking a nucleic acid encoding exon 2, wherein the nucleic acid encoding the FOXP3 protein is alternatively spliced to produce a full-length FOXP3 protein isoform and a FOXP3 protein isoform lacking exon 2, wherein the FOXP3 protein comprises the amino acid sequences of SEQ ID NOs:20 and 21. In some embodiments, the expression cassette comprises a nucleic acid encoding from 5’ to 3’: a) a promoter; b) a nucleic acid encoding a chimeric receptor (e.g., a chimeric receptor comprising an antigen binding domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:73-76); c) a nucleic acid encoding a selfcleaving 2A peptide (e.g., P2A peptide), wherein the 2A peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:2-10 and 89-91; d) a nucleic acid encoding a cell surface receptor comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs:17-19; e) a nucleic acid encoding a self-cleaving 2A peptide (e.g., P2A or T2A peptide), wherein the 2A peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:2-10 and 89-91; and f) a modified nucleic acid encoding a FOXP3 protein linked to a detection tag, wherein the nucleic acid encoding the FOXP3 protein comprises intron sequences flanking a nucleic acid encoding exon 2, wherein the nucleic acid encoding the FOXP3 protein is alternatively spliced to produce a full-length FOXP3 protein isoform and a FOXP3 protein isoform lacking exon 2, wherein the FOXP3 protein comprises the amino acid sequences of SEQ ID NOs:22 and 23.

[0132] In some embodiments, provided herein expression cassettes comprising a nucleic acid encoding from 5’ to 3’: a) a promoter; b) a nucleic acid encoding a chimeric receptor; c) a nucleic acid encoding a cell surface receptor; and d) a modified nucleic acid encoding a full- length FOXP3 protein. In some embodiments, provided herein expression cassettes comprising a nucleic acid encoding from 5’ to 3’: a) a promoter; b) a nucleic acid encoding a chimeric receptor; c) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; d) a nucleic acid encoding a cell surface receptor; e) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; and f) a modified nucleic acid encoding a full-length FOXP3 protein. In some embodiments, the nucleic acid encoding the FOXP3 protein encodes only one FOXP3 isoform, wherein the FOXP3 protein isoform is full-length FOXP3 protein. In some embodiments, the nucleic acid encoding the full-length FOXP3 protein comprises the amino acid sequence of SEQ ID NO:20 or 22. In some embodiments, the FOXP3 protein is linked to a detection tag. In some embodiments, the detection tag is linked to the N- terminus of the FOXP3 protein. In some embodiments, the detection tag is linked to the C- terminus of the FOXP3 protein. In some embodiments, the nucleic acid encoding the FOXP3 protein is FOXP3 cDNA. In some embodiments, the cell surface receptor lacks a functional intracellular signaling domain or is not capable of mediating intracellular signaling. In some embodiments, the cell surface receptor is selected from the group consisting of EGFR, HER2, CD2, H-2Kk, IL-5Ra, and derivatives or mutants thereof. In some embodiments, the chimeric receptor comprises a chimeric antigen receptor, a T cell Receptor, or a PiG receptor. In some embodiments, the chimeric receptor binds to an antigen selected from the group consisting of MBP, MOG, PLP, PDE4, VEGFR, S 100- , Citrullinated vimentin (CV), TNF-like ligand 1A (TL1A), CD2, CD23 (IgE receptor), IL-6R, IL-4Ra, IL-12R, IL-13Ral, IL-13Ra2, IL-15R, IL- 23R, CD19, CD20, CD22, CD3, CD4, CD5, IFN-y, IgE, IL-la, IL-10, IL-6, IL-12, IL-23, IL-15,IL-17, IL-17A, IL-22, IL-13, IL-33, TSLP, OX-40, OX-40L, p53, CD38, CD40L, CD28, ICOS, RIPK1, BTK, Bet v 1, GLP2R, JAK1, C5, and TNF-a. In some embodiments, the nucleic acid encoding the FOXP3 protein and the nucleic acid encoding the chimeric receptor and / or the nucleic acid encoding the cell surface receptor are operably linked to the same promoter. In some embodiments, the promoter is an inducible or a constitutive promoter. In some embodiments, the promoter is a cell-specific promoter. In some embodiments, the promoter is specific for T cells (e.g., Tregs). In some embodiments, the nucleic acid encoding the FOXP3 protein and the nucleic acid encoding the chimeric receptor and / or the nucleic acid encoding the cell surface receptor are each separated by an IRES, a nucleic acid encoding a self-cleaving peptide (e.g., P2A peptide), or a nucleic acid encoding a peptide that causes ribosome skipping. In some embodiments, the expression cassette further comprises a nucleic acid encoding a selfcleaving peptide. In some embodiments, the self-cleaving peptide is a 2A sequence. In some embodiments, the 2A sequence is a P2A sequence. In some embodiments, the 2A sequence is a T2A sequence. In some embodiments, the expression cassette comprises a nucleic acid encoding from 5’ to 3’: a) a promoter; b) a nucleic acid encoding a chimeric receptor; c) a nucleic acid encoding a self-cleaving 2A peptide; d) a nucleic acid encoding a cell surface receptor; e) a nucleic acid encoding a self-cleaving 2A peptide; and f) a modified nucleic acid encoding a full- length FOXP3 protein comprising the amino acid sequence of SEQ ID NO:20 or 22.

[0133] In some embodiments, provided herein expression cassettes comprising a nucleic acid encoding from 5’ to 3’: a) a promoter; b) a nucleic acid encoding a chimeric receptor; c) a nucleic acid encoding a cell surface receptor; and d) a modified nucleic acid encoding a FOXP3 protein isoform lacking exon 2. In some embodiments, provided herein expression cassettes comprising a nucleic acid encoding from 5’ to 3’: a) a promoter; b) a nucleic acid encoding a chimeric receptor; c) an internal ribosome entry site (IRES), or a nucleic acid encoding a selfcleaving peptide or a peptide that causes ribosome skipping; d) a nucleic acid encoding a cell surface receptor; e) an internal ribosome entry site (IRES), or a nucleic acid encoding a selfcleaving peptide or a peptide that causes ribosome skipping; and f) a modified nucleic acid encoding a FOXP3 protein isoform lacking exon 2. In some embodiments, the nucleic acid encoding the FOXP3 protein encodes only one FOXP3 isoform, wherein the FOXP3 protein isoform is a FOXP3 protein isoform lacking exon 2. In some embodiments, the modified nucleic acid encoding the FOXP3 protein isoform lacking exon 2 comprises the amino acid sequence of SEQ ID NO:21 or 23. In some embodiments, the FOXP3 protein is linked to a detection tag. In some embodiments, the detection tag is linked to the N-terminus of the FOXP3 protein. In someembodiments, the detection tag is linked to the C-terminus of the F0XP3 protein. In some embodiments, the nucleic acid encoding the FOXP3 protein is FOXP3 cDNA. In some embodiments, the cell surface receptor lacks a functional intracellular signaling domain or is not capable of mediating intracellular signaling. In some embodiments, the cell surface receptor is selected from the group consisting of EGFR, HER2, CD2, H-2Kk, IL-5Ra, and derivatives or mutants thereof. In some embodiments, the chimeric receptor comprises a chimeric antigen receptor, a T cell Receptor, or a PiG receptor. In some embodiments, the chimeric receptor binds to an antigen selected from the group consisting of MBP, MOG, PLP, PDE4, VEGFR, S1OO-P, Citrullinated vimentin (CV), TNF-like ligand 1A (TL1A), CD2, CD23 (IgE receptor), IL-6R, IL-4Ra, IL-12R, IL-13Ral, IL-13Ra2, IL-15R, IL-23R, CD19, CD20, CD22, CD3, CD4, CD5, ILN-y, IgE, IL-la, IL-10, IL-6, IL-12, IL-23, IL-15, IL-17, IL-17A, IL-22, IL-13, IL-33, TSLP, OX-40, OX-40L, p53, CD38, CD40L, CD28, ICOS, RIPK1, BTK, Bet v 1, GLP2R, JAK1, C5, and TNE-a. In some embodiments, the nucleic acid encoding the EOXP3 protein and the nucleic acid encoding the chimeric receptor and / or the nucleic acid encoding the cell surface receptor are operably linked to the same promoter. In some embodiments, the promoter is an inducible or a constitutive promoter. In some embodiments, the promoter is a cell-specific promoter. In some embodiments, the promoter is specific for T cells (e.g., Tregs). In some embodiments, the nucleic acid encoding the EOXP3 protein and the nucleic acid encoding the chimeric receptor and / or the nucleic acid encoding the cell surface receptor are each separated by an IRES, a nucleic acid encoding a self-cleaving peptide (e.g., P2A peptide), or a nucleic acid encoding a peptide that causes ribosome skipping. In some embodiments, the expression cassette further comprises a nucleic acid encoding a self-cleaving peptide. In some embodiments, the selfcleaving peptide is a 2A sequence. In some embodiments, the 2A sequence is a P2A sequence. In some embodiments, the 2A sequence is a T2A sequence. In some embodiments, the expression cassette comprises a nucleic acid encoding from 5’ to 3’: a) a promoter; b) a nucleic acid encoding a chimeric receptor; c) a nucleic acid encoding a self-cleaving 2A peptide; d) a nucleic acid encoding a cell surface receptor; e) a nucleic acid encoding a self-cleaving 2A peptide; and f) a modified nucleic acid encoding a EOXP3 protein isoform lacking exon 2 comprising the amino acid sequence of SEQ ID NO:21 or 23.

[0134] In some embodiments, provided herein are expression cassettes comprising a nucleic acid encoding from 5’ to 3’: a) a promoter; b) a nucleic acid encoding a chimeric receptor; c) a modified nucleic acid encoding a EOXP3 protein, wherein the nucleic acid encoding the EOXP3 protein comprises intron sequences flanking a nucleic acid encoding exon 2, wherein the nucleicacid encoding the FOXP3 protein is alternatively spliced to produce a full-length F0XP3 protein isoform and a FOXP3 protein isoform lacking exon 2; and d) a nucleic acid encoding a cell surface receptor. In some embodiments, provided herein are expression cassettes comprising a nucleic acid encoding from 5’ to 3’: a) a promoter; b) a nucleic acid encoding a chimeric receptor; c) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; d) a modified nucleic acid encoding a FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein comprises intron sequences flanking a nucleic acid encoding exon 2, wherein the nucleic acid encoding the FOXP3 protein is alternatively spliced to produce a full-length FOXP3 protein isoform and a FOXP3 protein isoform lacking exon 2; e) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; and f) a nucleic acid encoding a cell surface receptor. In some embodiments, the nucleic acid encoding the FOXP3 protein encodes only one FOXP3 isoform, wherein the FOXP3 protein isoform is full-length FOXP3 protein or a FOXP3 protein isoform lacking exon 2. In some embodiments, the nucleic acid encoding the FOXP3 protein encodes two FOXP3 protein isoforms, wherein the FOXP3 protein isoforms are full-length FOXP3 protein and a FOXP3 protein isoform lacking exon 2. In some embodiments, the nucleic acid encoding the FOXP3 protein comprising intron sequences flanking the nucleic acid encoding exon 2 is alternatively spliced to produce a full- length FOXP3 protein isoform and a FOXP3 protein isoform lacking exon 2. In some embodiments, the nucleic acid encoding the FOXP3 protein is alternatively spliced to produce a full-length FOXP3 protein isoform and a FOXP3 protein isoform lacking exon 2, wherein the FOXP3 protein comprises the amino acid sequences of SEQ ID NOs:20 and 21. In some embodiments, the nucleic acid encoding the FOXP3 protein is alternatively spliced to produce a full-length FOXP3 protein isoform and a FOXP3 protein isoform lacking exon 2, wherein the FOXP3 protein comprises the amino acid sequences of SEQ ID NOs:22 and 23. In some embodiments, the FOXP3 protein is linked to a detection tag. In some embodiments, the detection tag is linked to the N-terminus of the FOXP3 protein. In some embodiments, the detection tag is linked to the C-terminus of the FOXP3 protein. In some embodiments, the nucleic acid encoding the FOXP3 does not contain intron sequences other than the intron sequences flanking exon 2. In some embodiments, the nucleic acid encoding a FOXP3 protein is FOXP3 cDNA. In some embodiments, the cell surface receptor lacks a functional intracellular signaling domain or is not capable of mediating intracellular signaling. In some embodiments, the cell surface receptor is selected from the group consisting of EGFR, HER2, CD2, H-2Kk, IL-5Ra, and derivatives or mutants thereof. In some embodiments, the chimeric receptor comprises a chimeric antigen receptor, a T cell Receptor, or a PiG receptor. In some embodiments, the chimeric receptor binds to an antigen selected from the group consisting of MBP, MOG, PLP, PDE4, VEGFR, S 100- , Citrullinated vimentin (CV), TNF-like ligand 1A (TL1A), CD2, CD23 (IgE receptor), IL-6R, IL-4Ra, IL-12R, IL-13Ral, IL-13Ra2, IL-15R, IL-23R, CD19, CD20, CD22, CD3, CD4, CD5, IFN-y, IgE, IL-la, IL-10, IL-6, IL-12, IL-23, IL-15, IL-17, IL-17A, IL- 22, IL-13, IL-33, TSLP, OX-40, OX-40L, p53, CD38, CD40L, CD28, ICOS, RIPK1, BTK, Bet v 1, GLP2R, JAK1, C5, and TNF-a. In some embodiments, the nucleic acid encoding the FOXP3 protein and the nucleic acid encoding the chimeric receptor and / or the nucleic acid encoding the cell surface receptor are operably linked to the same promoter. In some embodiments, the promoter is an inducible or a constitutive promoter. In some embodiments, the promoter is a cell-specific promoter. In some embodiments, the promoter is specific for T cells (e.g., Tregs). In some embodiments, the nucleic acid encoding the FOXP3 protein and the nucleic acid encoding the chimeric receptor and / or the nucleic acid encoding the cell surface receptor are each separated by an IRES, a nucleic acid encoding a self-cleaving peptide (e.g., P2A peptide), or a nucleic acid encoding a peptide that causes ribosome skipping. In some embodiments, the expression cassette further comprises a nucleic acid encoding a self-cleaving peptide. In some embodiments, the self-cleaving peptide is a 2A sequence. In some embodiments, the 2A sequence is a P2A sequence. In some embodiments, the 2A sequence is a T2A sequence. In some embodiments, the expression cassette comprises a nucleic acid encoding from 5’ to 3’: a) a promoter; b) a nucleic acid encoding a chimeric receptor (e.g., a chimeric receptor comprising an antigen binding domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:73-76); c) a nucleic acid encoding a self-cleaving 2A peptide (e.g., P2A peptide), wherein the 2A sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:2-10 and 89-91; d) a modified nucleic acid encoding a FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein comprises intron sequences flanking a nucleic acid encoding exon 2, wherein the nucleic acid encoding the FOXP3 protein is alternatively spliced to produce a full-length FOXP3 protein isoform and a FOXP3 protein isoform lacking exon 2, wherein the FOXP3 protein comprises the amino acid sequences of SEQ ID NOs:20 and 21; e) a nucleic acid encoding a self-cleaving 2A peptide (e.g., P2A or T2A peptide), wherein the 2A sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:2-10 and 89-91; and f) a nucleic acid encoding a cell surface receptor comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs:17-19. In some embodiments, the expression cassette comprises a nucleic acid encoding from 5’ to 3’: a) a promoter; b) a nucleic acid encoding a chimeric receptor (e.g., a chimeric receptor comprising an antigen binding domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:73-76); c) a nucleic acid encoding a self-cleaving 2A peptide (e.g., P2A peptide), wherein the 2A sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:2-10 and 89-91; d) a modified nucleic acid encoding a FOXP3 protein linked to a detection tag, wherein the nucleic acid encoding the FOXP3 protein comprises intron sequences flanking a nucleic acid encoding exon 2, wherein the nucleic acid encoding the FOXP3 protein is alternatively spliced to produce a full-length FOXP3 protein isoform and a FOXP3 protein isoform lacking exon 2, wherein the FOXP3 protein comprises the amino acid sequences of SEQ ID NOs:22 and 23; e) a nucleic acid encoding a self-cleaving 2A peptide (e.g., P2A or T2A peptide), wherein the 2A sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:2-10 and 89-91; and f) a nucleic acid encoding a cell surface receptor comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 17-19.

[0135] In some embodiments, provided herein are expression cassettes comprising a nucleic acid encoding from 5’ to 3’: a) a promoter; b) a nucleic acid encoding a chimeric receptor; c) a modified nucleic acid encoding a full-length FOXP3 protein; and d) a nucleic acid encoding a cell surface receptor. In some embodiments, provided herein are expression cassettes comprising a nucleic acid encoding from 5’ to 3’: a) a promoter; b) a nucleic acid encoding a chimeric receptor; c) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; d) a modified nucleic acid encoding a full- length FOXP3 protein; e) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; and f) a nucleic acid encoding a cell surface receptor. In some embodiments, the nucleic acid encoding the FOXP3 protein encodes only one FOXP3 isoform, wherein the FOXP3 protein isoform is full-length FOXP3 protein. In some embodiments, the nucleic acid encoding the full-length FOXP3 protein comprises the amino acid sequence of SEQ ID NO:20 or 22. In some embodiments, the FOXP3 protein is linked to a detection tag. In some embodiments, the detection tag is linked to the N- terminus of the FOXP3 protein. In some embodiments, the detection tag is linked to the C- terminus of the FOXP3 protein. In some embodiments, the nucleic acid encoding a FOXP3 protein is FOXP3 cDNA. In some embodiments, the cell surface receptor lacks a functional intracellular signaling domain or is not capable of mediating intracellular signaling. In someembodiments, the cell surface receptor is selected from the group consisting of EGFR, HER2, CD2, H-2Kk, IL-5Ra, and derivatives or mutants thereof. In some embodiments, the chimeric receptor comprises a chimeric antigen receptor, a T cell Receptor, or a PiG receptor. In some embodiments, the chimeric receptor binds to an antigen selected from the group consisting of MBP, MOG, PLP, PDE4, VEGFR, S 100- , Citrullinated vimentin (CV), TNF-like ligand 1A (TL1A), CD2, CD23 (IgE receptor), IL-6R, IL-4Ra, IL-12R, IL-13Ral, IL-13Ra2, IL-15R, IL- 23R, CD19, CD20, CD22, CD3, CD4, CD5, IFN-y, IgE, IL-la, IL-10, IL-6, IL-12, IL-23, IL-15, IL-17, IL-17A, IL-22, IL-13, IL-33, TSLP, OX-40, OX-40L, p53, CD38, CD40L, CD28, ICOS, RIPK1, BTK, Bet v 1, GLP2R, JAK1, C5, and TNF-a. In some embodiments, the nucleic acid encoding the FOXP3 protein and the nucleic acid encoding the chimeric receptor and / or the nucleic acid encoding the cell surface receptor are operably linked to the same promoter. In some embodiments, the promoter is an inducible or a constitutive promoter. In some embodiments, the promoter is a cell-specific promoter. In some embodiments, the promoter is specific for T cells (e.g., Tregs). In some embodiments, the nucleic acid encoding the FOXP3 protein and the nucleic acid encoding the chimeric receptor and / or the nucleic acid encoding the cell surface receptor are each separated by an IRES, a nucleic acid encoding a self-cleaving peptide (e.g., P2A peptide), or a nucleic acid encoding a peptide that causes ribosome skipping. In some embodiments, the expression cassette further comprises a nucleic acid encoding a selfcleaving peptide. In some embodiments, the self-cleaving peptide is a 2A sequence. In some embodiments, the 2A sequence is a P2A sequence. In some embodiments, the 2A sequence is a T2A sequence. In some embodiments, the expression cassette comprises a nucleic acid encoding from 5’ to 3’: a) a promoter; b) a nucleic acid encoding a chimeric receptor; c) a nucleic acid encoding a self-cleaving 2A peptide; d) a modified nucleic acid encoding a full-length FOXP3 protein comprising the amino acid sequence of SEQ ID NO:20 or 22; e) a nucleic acid encoding a self-cleaving 2A peptide; and f) a nucleic acid encoding a cell surface receptor.

[0136] In some embodiments, provided herein are expression cassettes comprising a nucleic acid encoding from 5’ to 3’: a) a promoter; b) a nucleic acid encoding a chimeric receptor; c) a modified nucleic acid encoding a FOXP3 protein isoform lacking exon 2; and d) a nucleic acid encoding a cell surface receptor. In some embodiments, provided herein are expression cassettes comprising a nucleic acid encoding from 5’ to 3’: a) a promoter; b) a nucleic acid encoding a chimeric receptor; c) an internal ribosome entry site (IRES), or a nucleic acid encoding a selfcleaving peptide or a peptide that causes ribosome skipping; d) a modified nucleic acid encoding a FOXP3 protein isoform lacking exon 2; e) an internal ribosome entry site (IRES), or a nucleicacid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; and f) a nucleic acid encoding a cell surface receptor. In some embodiments, the nucleic acid encoding the FOXP3 protein encodes only one FOXP3 isoform, wherein the FOXP3 protein isoform is a FOXP3 protein isoform lacking exon 2. In some embodiments, the modified nucleic acid encoding the FOXP3 protein isoform lacking exon 2 comprises the amino acid sequence of SEQ ID NO:21 or 23. In some embodiments, the FOXP3 protein is linked to a detection tag. In some embodiments, the detection tag is linked to the N-terminus of the FOXP3 protein. In some embodiments, the detection tag is linked to the C-terminus of the FOXP3 protein. In some embodiments, the nucleic acid encoding a FOXP3 protein is FOXP3 cDNA. In some embodiments, the cell surface receptor lacks a functional intracellular signaling domain or is not capable of mediating intracellular signaling. In some embodiments, the cell surface receptor is selected from the group consisting of EGFR, HER2, CD2, H-2Kk, IL-5Ra, and derivatives or mutants thereof. In some embodiments, the chimeric receptor comprises a chimeric antigen receptor, a T cell Receptor, or a PiG receptor. In some embodiments, the chimeric receptor binds to an antigen selected from the group consisting of MBP, MOG, PLP, PDE4, VEGFR, S1OO-P, Citrullinated vimentin (CV), TNF-like ligand 1A (TL1A), CD2, CD23 (IgE receptor), IL-6R, IL-4Ra, IL-12R, IL-13Ral, IL-13Ra2, IL-15R, IL-23R, CD19, CD20, CD22, CD3, CD4, CD5, ILN-y, IgE, IL-la, IL-10, IL-6, IL-12, IL-23, IL-15, IL-17, IL-17A, IL-22, IL-13, IL-33, TSLP, OX-40, OX-40L, p53, CD38, CD40L, CD28, ICOS, RIPK1, BTK, Bet v 1, GLP2R, JAK1, C5, and TNE-a. In some embodiments, the nucleic acid encoding the EOXP3 protein and the nucleic acid encoding the chimeric receptor and / or the nucleic acid encoding the cell surface receptor are operably linked to the same promoter. In some embodiments, the promoter is an inducible or a constitutive promoter. In some embodiments, the promoter is a cell-specific promoter. In some embodiments, the promoter is specific for T cells (e.g., Tregs). In some embodiments, the nucleic acid encoding the EOXP3 protein and the nucleic acid encoding the chimeric receptor and / or the nucleic acid encoding the cell surface receptor are each separated by an IRES, a nucleic acid encoding a self-cleaving peptide (e.g., P2A peptide), or a nucleic acid encoding a peptide that causes ribosome skipping. In some embodiments, the expression cassette further comprises a nucleic acid encoding a self-cleaving peptide. In some embodiments, the selfcleaving peptide is a 2A sequence. In some embodiments, the 2A sequence is a P2A sequence. In some embodiments, the 2A sequence is a T2A sequence. In some embodiments, the expression cassette comprises a nucleic acid encoding from 5’ to 3’: a) a promoter; b) a nucleic acid encoding a chimeric receptor; c) a nucleic acid encoding a self-cleaving 2A peptide; d) amodified nucleic acid encoding a FOXP3 protein isoform lacking exon 2 comprising the amino acid sequence of SEQ ID NO:21 or 23; e) a nucleic acid encoding a self-cleaving 2A peptide; and f) a nucleic acid encoding a cell surface receptor.

[0137] In some embodiments, provided herein are expression cassettes comprising a nucleic acid encoding from 5’ to 3’: a) a promoter; b) a modified nucleic acid encoding a FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein comprises intron sequences flanking a nucleic acid encoding exon 2, wherein the nucleic acid encoding the FOXP3 protein is alternatively spliced to produce a full-length FOXP3 protein isoform and a FOXP3 protein isoform lacking exon 2; c) a nucleic acid encoding a chimeric receptor; and d) a nucleic acid encoding a cell surface receptor. In some embodiments, provided herein are expression cassettes comprising a nucleic acid encoding from 5’ to 3’: a) a promoter; b) a modified nucleic acid encoding a FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein comprises intron sequences flanking a nucleic acid encoding exon 2, wherein the nucleic acid encoding the FOXP3 protein is alternatively spliced to produce a full-length FOXP3 protein isoform and a FOXP3 protein isoform lacking exon 2; c) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; d) a nucleic acid encoding a chimeric receptor; e) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; and f) a nucleic acid encoding a cell surface receptor. In some embodiments, the nucleic acid encoding the FOXP3 protein encodes only one FOXP3 isoform, wherein the FOXP3 protein isoform is full- length FOXP3 protein or a FOXP3 protein isoform lacking exon 2. In some embodiments, the nucleic acid encoding the FOXP3 protein encodes two FOXP3 protein isoforms, wherein the FOXP3 protein isoforms are full-length FOXP3 protein and a FOXP3 protein isoform lacking exon 2. In some embodiments, the nucleic acid encoding the FOXP3 protein comprising intron sequences flanking the nucleic acid encoding exon 2 is alternatively spliced to produce a full- length FOXP3 protein isoform and a FOXP3 protein isoform lacking exon 2. In some embodiments, the nucleic acid encoding the FOXP3 protein is alternatively spliced to produce a full-length FOXP3 protein isoform and a FOXP3 protein isoform lacking exon 2, wherein the FOXP3 protein comprises the amino acid sequences of SEQ ID NOs:20 and 21. In some embodiments, the nucleic acid encoding the FOXP3 protein is alternatively spliced to produce a full-length FOXP3 protein isoform and a FOXP3 protein isoform lacking exon 2, wherein the FOXP3 protein comprises the amino acid sequences of SEQ ID NOs:22 and 23. In some embodiments, the FOXP3 protein is linked to a detection tag. In some embodiments, thedetection tag is linked to the N-terminus of the F0XP3 protein. In some embodiments, the detection tag is linked to the C-terminus of the F0XP3 protein. In some embodiments, the nucleic acid encoding the FOXP3 does not contain intron sequences other than the intron sequences flanking exon 2. In some embodiments, the nucleic acid encoding a FOXP3 protein is FOXP3 cDNA. In some embodiments, the cell surface receptor lacks a functional intracellular signaling domain or is not capable of mediating intracellular signaling. In some embodiments, the cell surface receptor is selected from the group consisting of EGFR, HER2, CD2, H-2Kk, IL- 5Ra, and derivatives or mutants thereof. In some embodiments, the chimeric receptor comprises a chimeric antigen receptor, a T cell Receptor, or a PiG receptor. In some embodiments, the chimeric receptor binds to an antigen selected from the group consisting of MBP, MOG, PLP, PDE4, VEGFR, S 100- , Citrullinated vimentin (CV), TNF-like ligand 1A (TL1A), CD2, CD23 (IgE receptor), IL-6R, IL-4Ra, IL-12R, IL-13Ral, IL-13Ra2, IL-15R, IL-23R, CD19, CD20, CD22, CD3, CD4, CD5, ILN-y, IgE, IL-la, IL-10, IL-6, IL-12, IL-23, IL-15, IL-17, IL-17A, IL- 22, IL-13, IL-33, TSLP, OX-40, OX-40L, p53, CD38, CD40L, CD28, ICOS, RIPK1, BTK, Bet v 1, GLP2R, JAK1, C5, and TNE-a. In some embodiments, the nucleic acid encoding the EOXP3 protein and the nucleic acid encoding the chimeric receptor and / or the nucleic acid encoding the cell surface receptor are operably linked to the same promoter. In some embodiments, the promoter is an inducible or a constitutive promoter. In some embodiments, the promoter is a cell-specific promoter. In some embodiments, the promoter is specific for T cells (e.g., Tregs). In some embodiments, the nucleic acid encoding the EOXP3 protein and the nucleic acid encoding the chimeric receptor and / or the nucleic acid encoding the cell surface receptor are each separated by an IRES, a nucleic acid encoding a self-cleaving peptide (e.g., P2A peptide), or a nucleic acid encoding a peptide that causes ribosome skipping. In some embodiments, the expression cassette further comprises a nucleic acid encoding a self-cleaving peptide. In some embodiments, the self-cleaving peptide is a 2A sequence. In some embodiments, the 2A sequence is a P2A sequence. In some embodiments, the 2A sequence is a T2A sequence. In some embodiments, the expression cassette comprises a nucleic acid encoding from 5’ to 3’: a) a promoter; b) a modified nucleic acid encoding a EOXP3 protein, wherein the nucleic acid encoding the LOXP3 protein comprises intron sequences flanking a nucleic acid encoding exon 2, wherein the nucleic acid encoding the LOXP3 protein is alternatively spliced to produce a full-length LOXP3 protein isoform and a LOXP3 protein isoform lacking exon 2, wherein the LOXP3 protein comprises the amino acid sequences of SEQ ID NOs:20 and 21; c) a nucleic acid encoding a self-cleaving 2A peptide (e.g., P2A peptide), wherein the 2A peptidecomprises an amino acid sequence selected from the group consisting of SEQ ID NOs:2-10 and 89-91; d) a nucleic acid encoding a chimeric receptor (e.g., a chimeric receptor comprising an antigen binding domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:73-76); e) a nucleic acid encoding a self-cleaving 2A peptide (e.g., P2A or T2A peptide), wherein the 2A peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:2-10 and 89-91; and f) a nucleic acid encoding a cell surface receptor comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 17-19. In some embodiments, the expression cassette comprises a nucleic acid encoding from 5’ to 3’: a) a promoter; b) a modified nucleic acid encoding a FOXP3 protein linked to a detection tag, wherein the nucleic acid encoding the FOXP3 protein comprises intron sequences flanking a nucleic acid encoding exon 2, wherein the nucleic acid encoding the FOXP3 protein is alternatively spliced to produce a full-length FOXP3 protein isoform and a FOXP3 protein isoform lacking exon 2, wherein the FOXP3 protein comprises the amino acid sequences of SEQ ID NOs:22 and 23; c) a nucleic acid encoding a self-cleaving 2A peptide (e.g., P2A peptide), wherein the 2A peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:2-10 and 89-91; d) a nucleic acid encoding a chimeric receptor (e.g., a chimeric receptor comprising an antigen binding domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:73-76); e) a nucleic acid encoding a self-cleaving 2A peptide (e.g., P2A or T2A peptide), wherein the 2A peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:2-10 and 89-91; and f) a nucleic acid encoding a cell surface receptor comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 17- 19.

[0138] In some embodiments, provided herein are expression cassettes comprising a nucleic acid encoding from 5’ to 3’: a) a promoter; b) a modified nucleic acid encoding a full-length FOXP3 protein; c) a nucleic acid encoding a chimeric receptor; and d) a nucleic acid encoding a cell surface receptor. In some embodiments, provided herein are expression cassettes comprising a nucleic acid encoding from 5’ to 3’: a) a promoter; b) a modified nucleic acid encoding a full- length FOXP3 protein; c) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; d) a nucleic acid encoding a chimeric receptor; e) an internal ribosome entry site (IRES), or a nucleic acid encoding a selfcleaving peptide or a peptide that causes ribosome skipping; and f) a nucleic acid encoding a cell surface receptor. In some embodiments, the nucleic acid encoding the FOXP3 protein encodes only one FOXP3 isoform, wherein the FOXP3 protein isoform is full-length FOXP3 protein. Insome embodiments, the nucleic acid encoding the full-length FOXP3 protein comprises the amino acid sequence of SEQ ID NO:20 or 22. In some embodiments, the FOXP3 protein is linked to a detection tag. In some embodiments, the detection tag is linked to the N-terminus of the FOXP3 protein. In some embodiments, the detection tag is linked to the C-terminus of the FOXP3 protein. In some embodiments, the nucleic acid encoding a FOXP3 protein is FOXP3 cDNA. In some embodiments, the cell surface receptor lacks a functional intracellular signaling domain or is not capable of mediating intracellular signaling. In some embodiments, the cell surface receptor is selected from the group consisting of EGFR, HER2, CD2, H-2Kk, IE-5Ra, and derivatives or mutants thereof. In some embodiments, the chimeric receptor comprises a chimeric antigen receptor, a T cell Receptor, or a PiG receptor. In some embodiments, the chimeric receptor binds to an antigen selected from the group consisting of MBP, MOG, PEP, PDE4, VEGFR, S 100- , Citrullinated vimentin (CV), TNF-like ligand 1A (TL1A), CD2, CD23 (IgE receptor), IL-6R, IL-4Ra, IL-12R, IL-13Ral, IL-13Ra2, IL-15R, IL-23R, CD19, CD20, CD22, CD3, CD4, CD5, IFN-y, IgE, IL-la, IL-10, IL-6, IL-12, IL-23, IL-15, IL-17, IL-17A, IL- 22, IL-13, IL-33, TSLP, OX-40, OX-40L, p53, CD38, CD40L, CD28, ICOS, RIPK1, BTK, Bet v 1, GLP2R, JAK1, C5, and TNF-a. In some embodiments, the nucleic acid encoding the FOXP3 protein and the nucleic acid encoding the chimeric receptor and / or the nucleic acid encoding the cell surface receptor are operably linked to the same promoter. In some embodiments, the promoter is an inducible or a constitutive promoter. In some embodiments, the promoter is a cell-specific promoter. In some embodiments, the promoter is specific for T cells (e.g., Tregs). In some embodiments, the nucleic acid encoding the FOXP3 protein and the nucleic acid encoding the chimeric receptor and / or the nucleic acid encoding the cell surface receptor are each separated by an IRES, a nucleic acid encoding a self-cleaving peptide (e.g., P2A peptide), or a nucleic acid encoding a peptide that causes ribosome skipping. In some embodiments, the expression cassette further comprises a nucleic acid encoding a self-cleaving peptide. In some embodiments, the self-cleaving peptide is a 2A sequence. In some embodiments, the 2A sequence is a P2A sequence. In some embodiments, the 2A sequence is a T2A sequence. In some embodiments, the expression cassette comprises a nucleic acid encoding from 5’ to 3’: a) a promoter; b) a modified nucleic acid encoding a full-length FOXP3 protein comprising the amino acid sequence of SEQ ID NO:20 or 22; c) a nucleic acid encoding a selfcleaving 2 A peptide; d) a nucleic acid encoding a chimeric receptor; e) a nucleic acid encoding a self-cleaving 2A peptide; and f) a nucleic acid encoding a cell surface receptor.

[0139] In some embodiments, provided herein are expression cassettes comprising a nucleic acid encoding from 5’ to 3’: a) a promoter; b) a modified nucleic acid encoding a FOXP3 protein isoform lacking exon 2; c) a nucleic acid encoding a chimeric receptor; and d) a nucleic acid encoding a cell surface receptor. In some embodiments, provided herein are expression cassettes comprising a nucleic acid encoding from 5’ to 3’: a) a promoter; b) a modified nucleic acid encoding a FOXP3 protein isoform lacking exon 2; c) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; d) a nucleic acid encoding a chimeric receptor; e) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; and f) a nucleic acid encoding a cell surface receptor. In some embodiments, the nucleic acid encoding the FOXP3 protein encodes only one FOXP3 isoform, wherein the FOXP3 protein isoform is a FOXP3 protein isoform lacking exon 2. In some embodiments, the modified nucleic acid encoding the FOXP3 protein isoform lacking exon 2 comprises the amino acid sequence of SEQ ID NO:21 or 23. In some embodiments, the FOXP3 protein is linked to a detection tag. In some embodiments, the detection tag is linked to the N-terminus of the FOXP3 protein. In some embodiments, the detection tag is linked to the C-terminus of the FOXP3 protein. In some embodiments, the nucleic acid encoding a FOXP3 protein is FOXP3 cDNA. In some embodiments, the cell surface receptor lacks a functional intracellular signaling domain or is not capable of mediating intracellular signaling. In some embodiments, the cell surface receptor is selected from the group consisting of EGFR, HER2, CD2, H-2Kk, IL-5Ra, and derivatives or mutants thereof. In some embodiments, the chimeric receptor comprises a chimeric antigen receptor, a T cell Receptor, or a PiG receptor. In some embodiments, the chimeric receptor binds to an antigen selected from the group consisting of MBP, MOG, PLP, PDE4, VEGFR, S 100- , Citrullinated vimentin (CV), TNF-like ligand 1A (TL1A), CD2, CD23 (IgE receptor), IL-6R, IL-4Ra, IL-12R, IL-13Ral, IL-13Ra2, IL-15R, IL-23R, CD19, CD20, CD22, CD3, CD4, CD5, ILN-y, IgE, IL-la, IL-10, IL-6, IL-12, IL-23, IL-15, IL-17, IL-17A, IL- 22, IL-13, IL-33, TSLP, OX-40, OX-40L, p53, CD38, CD40L, CD28, ICOS, RIPK1, BTK, Bet v 1, GLP2R, JAK1, C5, and TNE-a. In some embodiments, the nucleic acid encoding the EOXP3 protein and the nucleic acid encoding the chimeric receptor and / or the nucleic acid encoding the cell surface receptor are operably linked to the same promoter. In some embodiments, the promoter is an inducible or a constitutive promoter. In some embodiments, the promoter is a cell-specific promoter. In some embodiments, the promoter is specific for T cells (e.g., Tregs). In some embodiments, the nucleic acid encoding the EOXP3 protein and thenucleic acid encoding the chimeric receptor and / or the nucleic acid encoding the cell surface receptor are each separated by an IRES, a nucleic acid encoding a self-cleaving peptide (e.g., P2A peptide), or a nucleic acid encoding a peptide that causes ribosome skipping. In some embodiments, the expression cassette further comprises a nucleic acid encoding a self-cleaving peptide. In some embodiments, the self-cleaving peptide is a 2A sequence. In some embodiments, the 2A sequence is a P2A sequence. In some embodiments, the 2A sequence is a T2A sequence. In some embodiments, the expression cassette comprises a nucleic acid encoding from 5’ to 3’: a) a promoter; b) a modified nucleic acid encoding a FOXP3 protein isoform lacking exon 2 comprising the amino acid sequence of SEQ ID NO:21 or 23; c) a nucleic acid encoding a self-cleaving 2A peptide; d) a nucleic acid encoding a chimeric receptor; e) a nucleic acid encoding a self-cleaving 2A peptide; and f) a nucleic acid encoding a cell surface receptor.Z. SEPA AT / O ELEMENTS

[0140] In some embodiments, the expression cassettes provided herein include polycistronic expression systems. Separation elements are part of such polycistronic systems and allow each component of the expression cassette to be produced as independent proteins. Expression of the gene products in a polycistronic system is co-regulated by the same promoter and terminator, so differential expression and competition for transcription factors is minimized. Further, the controlling of additional gene products by a single promoter and terminator also decreases the required size of transgene expression systems, improving cloning and transfection efficiency. Polycistronic expression can be achieved using multiple mechanisms, with the most common being viral-derived sequences such as internal ribosome entry site (IRES) sequences or 2A sequences.

[0141] In some embodiments, each component of the expression cassette is translated into individual polypeptides. In some embodiments, each component of the expression cassette is translated into individual polypeptides due to ribosomal skipping. In some embodiments, each component of the expression cassette is translated into one polypeptide and then further cleaved into individual polypeptides, z.e., undergoes proteolytic cleavage. In some embodiments, the presence of the separation element does not impact gene expression levels within a cell (e.g., a T cell, such as a regulatory T (Treg) cell) that is transduced with and expresses any of the expression cassettes described herein.

[0142] Self-cleaving peptides were originally thought to act by proteolytic cleavage, however 2A self-cleaving peptides in particular act by direct interference with the translationalprocess by causing the ribosome to skip the formation of a peptide bond. Accordingly, nucleic acids encoding a self-cleaving peptide can be used to separate two or more components of any of the expression cassettes described herein while permitting translation initiation of two genes under the control of the same promoter and termination sequences.

[0143] An internal ribosome entry site (IRES) sequence is an internal ribosome entry site, which allows for translation initiation of two genes under the control of the same promoter such that the two genes are expressed as two independent proteins. IRES sequences permit ribosomes to initiate translation at a second open reading frame inside an mRNA strand, and can thus be used to express multiple completely separate proteins. In some embodiments, each component of the expression cassette is separated from another component by an IRES.

[0144] In some embodiments, the expression cassettes described herein further comprise a cleavable linker. In some embodiments, the cleavable linker is a 2A peptide. In some embodiments, the expression cassette comprising the FOXP3 protein comprises a nucleic acid encoding a P2A linker between a nucleic acid encoding the FOXP3 protein and a cell surface receptor (such as EGFR, HER2, CD2, H-2Kk, IL-5Ra, or derivatives or mutants thereof). In some embodiments, the expression cassette comprising the FOXP3 protein comprises a nucleic acid encoding a P2A linker between a nucleic acid encoding the FOXP3 protein and a chimeric receptor. In some embodiments, the expression cassette comprising the FOXP3 protein comprises a nucleic acid encoding a P2A linker between a nucleic acid encoding chimeric receptor and a cell surface receptor (such as EGFR, HER2, CD2, H-2Kk, IL-5Ra, or derivatives or mutants thereof). In some embodiments, 2A-like sequences, or "peptide bond- skipping" 2A sequences, are derived from, for example, many different viruses, including, for example, from Thosea asigna. These sequences are sometimes also known as "peptide skipping sequences." When this type of sequence is placed within a cistron between two polypeptides that are intended to be separated, the ribosome appears to skip a peptide bond; in the case of the Thosea asigna sequence, the bond between the Gly and Pro amino acids at the carboxy terminal "P-G-P" is omitted. This may leave two to three polypeptides, for example, an inducible chimeric pro- apoptotic polypeptide and a chimeric antigen receptor, or, for example, a marker polypeptide and an inducible chimeric pro-apoptotic polypeptide. When this sequence is used, the polypeptide that is encoded 5' of the 2A sequence may end up with additional amino acids at the carboxy terminus, including the Gly residue and any upstream residues in the 2A sequence. The peptide that is encoded 3' of the 2A sequence may end up with additional amino acids at the amino terminus, including the Pro residue and any downstream residues following the 2Asequence. In some embodiments, the cleavable linker is a 2A peptide derived from porcine teschovirus- 1 (P2A). In some embodiments, the 2A cotranslational sequence is a 2A-like sequence. In some embodiments, the 2A cotranslational sequence is T2A (Thosea asigna virus 2A), F2A (foot and mouth disease virus 2A), P2A (porcine teschovirus- 1 2A), BmCPV 2A (cytoplasmic polyhedrosis virus 2A), BmIFV 2A (flacherie virus of B. mori 2A), or E2A (equine rhinitis A virus 2A). In some embodiments, the 2A cotranslational sequence is T2A-GSG, F2A- GSG, P2A-GSG, or E2A-GSG. In some embodiments, the 2A cotranslational sequence is selected from the group consisting of T2A, P2A, and F2A. By "cleavable linker" is meant that the linker is cleaved by any means, including, for example, nonenzymatic means, such as peptide skipping, or enzymatic means (see, e.g., Donnelly, ML (2001), J. Gen. Virol. 82:1013- 25, hereby incorporated by reference in its entirety). In a specific embodiment, a P2A comprises (or consists of) a sequence disclosed herein. In a specific embodiment, a P2A comprises (or consists of) a sequence disclosed herein (e.g., a sequence disclosed in the Examples below).

[0145] In some embodiments, the separation element is a 2A linker. In some embodiments, the 2A linker is selected from the group consisting of a P2A sequence, a T2A sequence, an E2A sequence, an F2A sequence, a self-cleaving derivative or mutant thereof, and any combination thereof. In some embodiments, the 2A sequence is selected from the group consisting of SEQ ID NOs:2-10 and 89-91. In certain embodiments, a 2A linker is a P2A linker that includes the amino acid sequence of SEQ ID NO:2 (ATNFSLLKQAGDVEENPGP), SEQ ID NOG (SGATNFSLLKQAGDVEENPGP), or SEQ ID NO:4 (GSGATNFSLLKQAGDVEENPGP). In certain embodiments, the 2 A linker further includes a GSG amino acid sequence at the amino terminus of the peptide; in other embodiments, the 2A linker includes a GSGPR amino acid sequence (SEQ ID NO:24) at the amino terminus of the peptide. Thus, by a "2A" sequence, the term may refer to a 2A sequence in an example described herein or may also refer to a 2A sequence as listed herein further comprising an SG, GSG, or GSGPR sequence (SEQ ID NO:24) at the amino terminus of the linker. See Table 4 below for exemplary 2A peptide sequences.

[0146] Table 4. Exemplary 2A peptide sequences.IL CELLS

[0147] The disclosure provides for an adoptive cellular immunotherapy composition comprising a genetically modified cell preparation as described herein, e.g., genetically modified immune cells, e.g., lymphocytes. These cells are, for example, multipotent cells such as hematopoietic stem cells, progenitor or precursor cells of hematopoietic lineages, or immune cells (e.g., human autologous or allogeneic T, natural killer (NK), dendritic, or B cells). These cells may also be pluripotent stem cells (PSCs) such as human embryonic stem cells and induced PSCs, which can be used to generate therapeutic cell populations. In some embodiments, pluripotent and multipotent cells are differentiated into a desired cell type in vitro before being implanted into the patient.

[0148] In some alternatives, the genetically modified cell preparation is a T lymphocyte cell preparation. In some embodiments the T lymphocyte cell preparation comprises CD4+ T cells that have a chimeric receptor comprising an extracellular antibody variable domain specific for a ligand associated with the disease or disorder, a spacer region, a transmembrane domain, and an intracellular signaling domain of a T cell receptor and an exogenous FOXP3 protein as described herein. In some alternatives, the chimeric receptor modified T cell population of the disclosure persists in vivo for at least about 3 days or longer. In some alternatives, each of these populations can be combined with one another or other cell types to provide a composition.

[0149] In some alternatives, the genetically modified cell preparation is a Treg cell preparation. Tregs comprise both natural and induced subtypes. Natural Tregs (nTregs) are cells which originate as a separate cell lineage during development. Peripheral or induced Tregs (iTregs) differentiate from conventional T cells. In some embodiments, CD4+ T cells that are not nTregs and / or not iTregs are engineered into Tregs using the methods and compositions of the disclosure. In some embodiments, the CD4+ T cells are used to make Tregs expressing exogenous FOXP3 protein using the methods and compositions of the disclosure.

[0150] In some embodiments, the cell expressing the exogenous FOXP3 protein of the disclosure is not naturally occurring. In some embodiments, the cell is an immune cell. In some embodiments, the cell is a lymphocyte. In some embodiments, the cell is a T cell. In someembodiment, the cell is a regulatory T cell (Treg). In some embodiments, the Treg is compared to a control Treg that lacks the exogenous FOXP3 protein. For example, in other embodiments, the Treg is compared to a control Treg that is transduced with an empty vector lacking a FOXP3 protein and / or a control Treg that is not transduced.

[0151] Thus, provided herein are engineered regulatory T cells (Tregs) comprising exogenous expression cassettes comprising a modified nucleic acid encoding a FOXP3 protein operably linked to a promoter, wherein the nucleic acid encoding the FOXP3 is modified to produce a full-length FOXP3 protein isoform and an alternatively spliced isoform lacking one or more exons. In some embodiments, the nucleic acid encoding the FOXP3 protein comprises intron sequences flanking the nucleic acid encoding exon 2. In some embodiments, the nucleic acid encoding the FOXP3 protein comprising intron sequences flanking the nucleic acid encoding exon 2 is alternatively spliced to produce a full-length FOXP3 protein isoform and a FOXP3 protein isoform lacking exon 2. In some embodiments, the FOXP3 protein is linked to a detection tag. In some embodiments, the detection tag is linked to the N-terminus of the FOXP3 protein. In some embodiments, the detection tag is linked to the C-terminus of the FOXP3 protein. In some embodiments, the nucleic acid encoding a FOXP3 protein is FOXP3 cDNA. In some embodiments, the exogenous expression cassette further comprises a nucleic acid encoding a chimeric receptor and / or a nucleic acid encoding a cell surface receptor. In some embodiments, the nucleic acid encoding the FOXP3 protein and the nucleic acid encoding the chimeric receptor and / or the nucleic acid encoding the cell surface receptor are operably linked to the same promoter. In some embodiments, the nucleic acid encoding the FOXP3 protein and the nucleic acid encoding the chimeric receptor and / or the nucleic acid encoding the cell surface receptor are each separated by an IRES, a nucleic acid encoding a self-cleaving peptide (e.g., P2A peptide), or a nucleic acid encoding a peptide that causes ribosome skipping. In some embodiments, the engineered Treg comprises an exogenous expression cassette further comprising a nucleic acid encoding a self-cleaving peptide. In some embodiments, the selfcleaving peptide is a 2A sequence. In some embodiments, the 2A sequence is a P2A sequence. In some embodiments, the engineered Treg comprises an exogenous expression cassette comprising a nucleic acid encoding: a) a promoter; b) a modified nucleic acid encoding the FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein comprises intron sequences flanking the nucleic acid encoding exon 2, wherein the FOXP3 protein comprises the amino acid sequence of SEQ ID NOs:20 and 21; c) a nucleic acid encoding a self-cleaving P2A peptide; d) a nucleic acid encoding a chimeric receptor; and / or e) a nucleic acid encoding a cellsurface receptor. In some embodiments, the engineered Treg comprises an exogenous expression cassette comprising a nucleic acid encoding: a) a promoter; b) a modified nucleic acid encoding the FOXP3 protein linked to a detection tag, wherein the nucleic acid encoding the FOXP3 protein comprises intron sequences flanking the nucleic acid encoding exon 2, wherein the FOXP3 protein comprises the amino acid sequence of SEQ ID NOs:22 and 23; c) a nucleic acid encoding a self-cleaving P2A peptide; d) a nucleic acid encoding a chimeric receptor; and / or e) a nucleic acid encoding a cell surface receptor.

[0152] In some embodiments, provided herein are engineered Tregs comprising an exogenous expression cassette comprising a nucleic acid encoding from 5’ to 3’: a) a promoter; b) a modified nucleic acid encoding a FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein comprises intron sequences flanking a nucleic acid encoding exon 2; c) an IRES, or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; and d) a nucleic acid encoding a cell surface receptor. In some embodiments, the nucleic acid encoding the FOXP3 protein comprising intron sequences flanking the nucleic acid encoding exon 2 is alternatively spliced to produce a full-length FOXP3 protein isoform and a FOXP3 protein isoform lacking exon 2. In some embodiments, the FOXP3 protein is linked to a detection tag. In some embodiments, the detection tag is linked to the N-terminus of the FOXP3 protein. In some embodiments, the detection tag is linked to the C-terminus of the FOXP3 protein. In some embodiments, the nucleic acid encoding the FOXP3 does not contain intron sequences other than the intron sequences flanking exon 2. In some embodiments, the nucleic acid encoding a FOXP3 protein is FOXP3 cDNA. In some embodiments, the cell surface receptor lacks a functional intracellular signaling domain or is not capable of mediating intracellular signaling. In some embodiments, the cell surface receptor is selected from the group consisting of EGFR, HER2, CD2, H-2Kk, IL-5Ra, and derivatives or mutants thereof. In some embodiments, the nucleic acid encoding the FOXP3 protein and the nucleic acid encoding the nucleic acid encoding the cell surface receptor are operably linked to the same promoter. In some embodiments, the promoter is an inducible or a constitutive promoter. In some embodiments, the promoter is a cell-specific promoter. In some embodiments, the promoter is specific for T cells (e.g., Tregs). In some embodiments, the nucleic acid encoding the FOXP3 protein and the nucleic acid encoding the nucleic acid encoding the cell surface receptor are each separated by an IRES, a nucleic acid encoding a self-cleaving peptide (e.g., P2A peptide), or a nucleic acid encoding a peptide that causes ribosome skipping. In some embodiments, the engineered Treg comprises an exogenous expression cassette further comprising a nucleic acidencoding a self-cleaving peptide. In some embodiments, the self-cleaving peptide is a 2A sequence. In some embodiments, the 2A sequence is a P2A sequence. In some embodiments, the engineered Treg comprising an exogenous expression cassette comprising a nucleic acid encoding from 5’ to 3’: a) a promoter; b) a modified nucleic acid encoding a FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein comprises intron sequences flanking a nucleic acid encoding exon 2, wherein the FOXP3 protein comprises the amino acid sequence of SEQ ID NOs:20 and 21; c) a nucleic acid encoding a self-cleaving P2A peptide; and d) a nucleic acid encoding a cell surface. In some embodiments, the engineered Treg comprising an exogenous expression cassette comprising a nucleic acid encoding from 5’ to 3’: a) a promoter; b) a modified nucleic acid encoding a FOXP3 protein linked to a detection tag, wherein the nucleic acid encoding the FOXP3 protein comprises intron sequences flanking a nucleic acid encoding exon 2, wherein the FOXP3 protein comprises the amino acid sequence of SEQ ID NOs:22 and 23; c) a nucleic acid encoding a self-cleaving P2A peptide; and d) a nucleic acid encoding a cell surface.

[0153] In some embodiments, provided herein are engineered Tregs comprising an exogenous expression cassette comprising a nucleic acid encoding from 5’ to 3’: a) a promoter; b) a nucleic acid encoding a cell surface receptor; c) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; and d) a modified nucleic acid encoding a FOXP3 protein, wherein the modified nucleic acid encoding the FOXP3 protein comprises intron sequences flanking a nucleic acid encoding exon 2. In some embodiments, the nucleic acid encoding the FOXP3 protein comprising intron sequences flanking the nucleic acid encoding exon 2 is alternatively spliced to produce a full-length FOXP3 protein isoform and a FOXP3 protein isoform lacking exon 2. In some embodiments, the FOXP3 protein is linked to a detection tag. In some embodiments, the detection tag is linked to the N-terminus of the FOXP3 protein. In some embodiments, the detection tag is linked to the C-terminus of the FOXP3 protein. In some embodiments, the nucleic acid encoding the FOXP3 does not contain intron sequences other than the intron sequences flanking exon 2. In some embodiments, the nucleic acid encoding a FOXP3 protein is FOXP3 cDNA. In some embodiments, the cell surface receptor lacks a functional intracellular signaling domain or is not capable of mediating intracellular signaling. In some embodiments, the cell surface receptor is selected from the group consisting of EGFR, HER2, CD2, H-2Kk, IL-5Ra, and derivatives or mutants thereof. In some embodiments, the nucleic acid encoding the FOXP3 protein and the nucleic acid encoding the nucleic acid encoding the cell surface receptor are operably linked tothe same promoter. In some embodiments, the promoter is an inducible or a constitutive promoter. In some embodiments, the promoter is a cell- specific promoter. In some embodiments, the promoter is specific for T cells (e.g., Tregs). In some embodiments, the nucleic acid encoding the FOXP3 protein and the nucleic acid encoding the nucleic acid encoding the cell surface receptor are each separated by an IRES, a nucleic acid encoding a selfcleaving peptide (e.g., P2A peptide), or a nucleic acid encoding a peptide that causes ribosome skipping. In some embodiments, the engineered Treg comprises an exogenous expression cassette further comprising a nucleic acid encoding a self-cleaving peptide. In some embodiments, the self-cleaving peptide is a 2A sequence. In some embodiments, the 2A sequence is a P2A sequence. In some embodiments, the engineered Treg comprising an exogenous expression cassette comprising a nucleic acid encoding from 5’ to 3’: a) a promoter; b) a nucleic acid encoding a cell surface receptor; c) a nucleic acid encoding a self-cleaving P2A peptide; and d) a modified nucleic acid encoding a FOXP3 protein, wherein the modified nucleic acid encoding the FOXP3 protein comprises intron sequences flanking a nucleic acid encoding exon 2, wherein the FOXP3 protein comprises the amino acid sequence of SEQ ID NOs:20 and 21. In some embodiments, the engineered Treg comprising an exogenous expression cassette comprising a nucleic acid encoding from 5’ to 3’: a) a promoter; b) a nucleic acid encoding a cell surface receptor; c) a nucleic acid encoding a self-cleaving P2A peptide; and d) a modified nucleic acid encoding a FOXP3 protein linked to a detection tag, wherein the modified nucleic acid encoding the FOXP3 protein comprises intron sequences flanking a nucleic acid encoding exon 2, wherein the FOXP3 protein comprises the amino acid sequence of SEQ ID NOs:22 and 23.

[0154] In some embodiments, provided herein are engineered Tregs comprising an exogenous expression cassette comprising a nucleic acid encoding from 5’ to 3’: a) a promoter; b) a nucleic acid encoding a chimeric receptor; c) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; d) a nucleic acid encoding a cell surface receptor; e) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; and f) a modified nucleic acid encoding a FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein comprises intron sequences flanking a nucleic acid encoding exon 2. In some embodiments, the nucleic acid encoding the FOXP3 protein comprising intron sequences flanking the nucleic acid encoding exon 2 is alternatively spliced to produce a full-length FOXP3 protein isoform and a FOXP3 protein isoform lacking exon 2. In some embodiments,the F0XP3 protein is linked to a detection tag. In some embodiments, the detection tag is linked to the N-terminus of the F0XP3 protein. In some embodiments, the detection tag is linked to the C-terminus of the F0XP3 protein. In some embodiments, the nucleic acid encoding the FOXP3 does not contain intron sequences other than the intron sequences flanking exon 2. In some embodiments, the nucleic acid encoding a FOXP3 protein is FOXP3 cDNA. In some embodiments, the cell surface receptor lacks a functional intracellular signaling domain or is not capable of mediating intracellular signaling. In some embodiments, the cell surface receptor is selected from the group consisting of EGFR, HER2, CD2, H-2Kk, IL-5Ra, and derivatives or mutants thereof. In some embodiments, the chimeric receptor comprises a chimeric antigen receptor, a T cell Receptor, or a PiG receptor. In some embodiments, the chimeric receptor binds to an antigen selected from the group consisting of MBP, MOG, PLP, PDE4, VEGFR, S1OO-P, Citrullinated vimentin (CV), TNF-like ligand 1A (TL1A), CD2, CD23 (IgE receptor), IL-6R, IL-4Ra, IL-12R, IL-13Ral, IL-13Ra2, IL-15R, IL-23R, CD19, CD20, CD22, CD3, CD4, CD5, IFN-y, IgE, IL-la, IL-10, IL-6, IL-12, IL-23, IL-15, IL-17, IL-17A, IL-22, IL-13, IL-33, TSLP, OX-40, OX-40L, p53, CD38, CD40L, CD28, ICOS, RIPK1, BTK, Bet v 1, GLP2R, JAK1, C5, and TNE-a. In some embodiments, the nucleic acid encoding the EOXP3 protein and the nucleic acid encoding the chimeric receptor and / or the nucleic acid encoding the cell surface receptor are operably linked to the same promoter. In some embodiments, the promoter is an inducible or a constitutive promoter. In some embodiments, the promoter is a cell-specific promoter. In some embodiments, the promoter is specific for T cells (e.g., Tregs). In some embodiments, the nucleic acid encoding the EOXP3 protein and the nucleic acid encoding the chimeric receptor and / or the nucleic acid encoding the cell surface receptor are each separated by an IRES, a nucleic acid encoding a self-cleaving peptide (e.g., P2A peptide), or a nucleic acid encoding a peptide that causes ribosome skipping. In some embodiments, the engineered Treg comprises an exogenous expression cassette further comprising a nucleic acid encoding a self-cleaving peptide. In some embodiments, the self-cleaving peptide is a 2A sequence. In some embodiments, the 2A sequence is a P2A sequence. In some embodiments, the 2A sequence is a T2A sequence. In some embodiments, the engineered Treg comprising an exogenous expression cassette comprising a nucleic acid encoding from 5’ to 3’: a) a promoter; b) a nucleic acid encoding a chimeric receptor; c) a nucleic acid encoding a self-cleaving P2A peptide; d) a nucleic acid encoding a cell surface receptor; e) a nucleic acid encoding a self-cleaving 2A (e.g., T2A or P2A) peptide; and f) a modified nucleic acid encoding a LOXP3 protein, wherein the nucleic acid encoding the LOXP3 protein comprises intron sequences flanking a nucleic acidencoding exon 2, wherein the FOXP3 protein comprises the amino acid sequence of SEQ ID NOs:20 and 21. In some embodiments, the engineered Treg comprising an exogenous expression cassette comprising a nucleic acid encoding from 5’ to 3’: a) a promoter; b) a nucleic acid encoding a chimeric receptor; c) a nucleic acid encoding a self-cleaving P2A peptide; d) a nucleic acid encoding a cell surface receptor; e) a nucleic acid encoding a self-cleaving 2A (e.g., T2A or P2A) peptide; and f) a modified nucleic acid encoding a FOXP3 protein linked to a detection tag, wherein the nucleic acid encoding the FOXP3 protein comprises intron sequences flanking a nucleic acid encoding exon 2, wherein the FOXP3 protein comprises the amino acid sequence of SEQ ID NOs:22 and 23.

[0155] In some embodiments, provided herein are engineered Tregs comprising an exogenous expression cassette comprising a nucleic acid encoding from 5’ to 3’: a) a promoter; b) a nucleic acid encoding a chimeric receptor; c) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; d) a modified nucleic acid encoding a FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein comprises intron sequences flanking a nucleic acid encoding exon 2; e) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; and f) a nucleic acid encoding a cell surface receptor. In some embodiments, the nucleic acid encoding the FOXP3 protein comprising intron sequences flanking the nucleic acid encoding exon 2 is alternatively spliced to produce a full-length FOXP3 protein isoform and a FOXP3 protein isoform lacking exon 2. In some embodiments, the FOXP3 protein is linked to a detection tag. In some embodiments, the detection tag is linked to the N-terminus of the FOXP3 protein. In some embodiments, the detection tag is linked to the C-terminus of the FOXP3 protein. In some embodiments, the nucleic acid encoding the FOXP3 does not contain intron sequences other than the intron sequences flanking exon 2. In some embodiments, the nucleic acid encoding a FOXP3 protein is FOXP3 cDNA. In some embodiments, the cell surface receptor lacks a functional intracellular signaling domain or is not capable of mediating intracellular signaling. In some embodiments, the cell surface receptor is selected from the group consisting of EGFR, HER2, CD2, H-2Kk, IL-5Ra, and derivatives or mutants thereof. In some embodiments, the chimeric receptor comprises a chimeric antigen receptor, a T cell Receptor, or a PiG receptor. In some embodiments, the chimeric receptor binds to an antigen selected from the group consisting of MBP, MOG, PLP, PDE4, VEGFR, S100-P, Citrullinated vimentin (CV), TNF-like ligand 1A (TL1A), CD2, CD23 (IgE receptor), IL-6R, IL-4Ra, IL-12R, IL-13Ral, IL-13Ra2, IL-15R, IL-23R, CD19, CD20, CD22, CD3, CD4, CD5,ILN-y, IgE, IL-la, IL-10, IL-6, IL-12, IL-23, IL-15, IL-17, IL-17A, IL-22, IL-13, IL-33, TSLP, OX-40, OX-40L, p53, CD38, CD40L, CD28, ICOS, RIPK1, BTK, Bet v 1, GLP2R, JAK1, C5, and TNL-a. In some embodiments, the nucleic acid encoding the LOXP3 protein and the nucleic acid encoding the chimeric receptor and / or the nucleic acid encoding the cell surface receptor are operably linked to the same promoter. In some embodiments, the promoter is an inducible or a constitutive promoter. In some embodiments, the promoter is a cell-specific promoter. In some embodiments, the promoter is specific for T cells (e.g., Tregs). In some embodiments, the nucleic acid encoding the LOXP3 protein and the nucleic acid encoding the chimeric receptor and / or the nucleic acid encoding the cell surface receptor are each separated by an IRES, a nucleic acid encoding a self-cleaving peptide (e.g., P2A peptide), or a nucleic acid encoding a peptide that causes ribosome skipping. In some embodiments, the engineered Treg comprises an exogenous expression cassette further comprising a nucleic acid encoding a self-cleaving peptide. In some embodiments, the self-cleaving peptide is a 2A sequence. In some embodiments, the 2A sequence is a P2A sequence. In some embodiments, the 2A sequence is a T2A sequence. In some embodiments, the engineered Treg comprising an exogenous expression cassette comprising a nucleic acid encoding from 5’ to 3’: a) a promoter; b) a nucleic acid encoding a chimeric receptor; c) a nucleic acid encoding a self-cleaving P2A peptide; d) a modified nucleic acid encoding a LOXP3 protein, wherein the nucleic acid encoding the LOXP3 protein comprises intron sequences flanking a nucleic acid encoding exon 2, wherein the LOXP3 protein comprises the amino acid sequence of SEQ ID NOs:20 and 21; e) a nucleic acid encoding a self-cleaving 2A (e.g., T2A or P2A) peptide; and f) a nucleic acid encoding a cell surface receptor. In some embodiments, the engineered Treg comprising an exogenous expression cassette comprising a nucleic acid encoding from 5’ to 3’: a) a promoter; b) a nucleic acid encoding a chimeric receptor; c) a nucleic acid encoding a self-cleaving P2A peptide; d) a modified nucleic acid encoding a EOXP3 protein linked to a detection tag, wherein the nucleic acid encoding the EOXP3 protein comprises intron sequences flanking a nucleic acid encoding exon 2, wherein the EOXP3 protein comprises the amino acid sequence of SEQ ID NOs:22 and 23; e) a nucleic acid encoding a self-cleaving 2A (e.g., T2A or P2A) peptide; and f) a nucleic acid encoding a cell surface receptor.

[0156] In some embodiments, provided herein are engineered Tregs comprising an exogenous expression cassette comprising a nucleic acid encoding from 5’ to 3’: a) a promoter; b) a modified nucleic acid encoding a EOXP3 protein, wherein the nucleic acid encoding the EOXP3 protein comprises intron sequences flanking a nucleic acid encoding exon 2; c) aninternal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; d) a nucleic acid encoding a chimeric receptor; e) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; and f) a nucleic acid encoding a cell surface receptor. In some embodiments, the nucleic acid encoding the FOXP3 protein comprising intron sequences flanking the nucleic acid encoding exon 2 is alternatively spliced to produce a full-length FOXP3 protein isoform and a FOXP3 protein isoform lacking exon 2. In some embodiments, the FOXP3 protein is linked to a detection tag. In some embodiments, the detection tag is linked to the N-terminus of the FOXP3 protein. In some embodiments, the detection tag is linked to the C-terminus of the FOXP3 protein. In some embodiments, the nucleic acid encoding the FOXP3 does not contain intron sequences other than the intron sequences flanking exon 2. In some embodiments, the nucleic acid encoding a FOXP3 protein is FOXP3 cDNA. In some embodiments, the cell surface receptor lacks a functional intracellular signaling domain or is not capable of mediating intracellular signaling. In some embodiments, the cell surface receptor is selected from the group consisting of EGFR, HER2, CD2, H-2Kk, IL-5Ra, and derivatives or mutants thereof. In some embodiments, the chimeric receptor comprises a chimeric antigen receptor, a T cell Receptor, or a PiG receptor. In some embodiments, the chimeric receptor binds to an antigen selected from the group consisting of MBP, MOG, PLP, PDE4, VEGFR, S1OO-P, Citrullinated vimentin (CV), TNF-like ligand 1A (TL1A), CD2, CD23 (IgE receptor), IL-6R, IL-4Ra, IL-12R, IL-13Ral, IL-13Ra2, IL-15R, IL-23R, CD19, CD20, CD22, CD3, CD4, CD5, IFN-y, IgE, IL-la, IL-1 , IL-6, IL-12, IL-23, IL-15, IL-17, IL-17A, IL-22, IL-13, IL-33, TSLP, OX-40, OX-40L, p53, CD38, CD40L, CD28, ICOS, RIPK1, BTK, Bet v 1, GLP2R, JAK1, C5, and TNF-a. In some embodiments, the nucleic acid encoding the FOXP3 protein and the nucleic acid encoding the chimeric receptor and / or the nucleic acid encoding the cell surface receptor are operably linked to the same promoter. In some embodiments, the promoter is an inducible or a constitutive promoter. In some embodiments, the promoter is a cell-specific promoter. In some embodiments, the promoter is specific for T cells (e.g., Tregs). In some embodiments, the nucleic acid encoding the FOXP3 protein and the nucleic acid encoding the chimeric receptor and / or the nucleic acid encoding the cell surface receptor are each separated by an IRES, a nucleic acid encoding a self-cleaving peptide (e.g., P2A peptide), or a nucleic acid encoding a peptide that causes ribosome skipping. In some embodiments, the engineered Treg comprises an exogenous expression cassette further comprising a nucleic acid encoding a self-cleaving peptide. In some embodiments, the self-cleaving peptide is a 2A sequence. In someembodiments, the 2A sequence is a P2A sequence. In some embodiments, the 2A sequence is a T2A sequence. In some embodiments, the engineered Treg comprising an exogenous expression cassette comprising a nucleic acid encoding from 5’ to 3’: a) a promoter; b) a modified nucleic acid encoding a FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein comprises intron sequences flanking a nucleic acid encoding exon 2, wherein the FOXP3 protein comprises the amino acid sequence of SEQ ID NOs:20 and 21; c) a nucleic acid encoding a selfcleaving P2A peptide; d) a nucleic acid encoding a chimeric receptor; e) a nucleic acid encoding a self-cleaving 2A (e.g., P2A or T2A) peptide; and f) a nucleic acid encoding a cell surface receptor. In some embodiments, the engineered Treg comprising an exogenous expression cassette comprising a nucleic acid encoding from 5’ to 3’: a) a promoter; b) a modified nucleic acid encoding a FOXP3 protein linked to a detection tag, wherein the nucleic acid encoding the FOXP3 protein comprises intron sequences flanking a nucleic acid encoding exon 2, wherein the FOXP3 protein comprises the amino acid sequence of SEQ ID NOs:22 and 23; c) a nucleic acid encoding a self-cleaving P2A peptide; d) a nucleic acid encoding a chimeric receptor; e) a nucleic acid encoding a self-cleaving 2A (e.g., P2A or T2A) peptide; and f) a nucleic acid encoding a cell surface receptor.

[0157] In some embodiments, the Treg expressing the exogenous expression cassette of the disclosure is not naturally occurring (not an nTreg and / or not an iTreg). In some embodiments, the cell expresses one or more markers characteristic of a Treg. Treg markers include high levels of CD25 (CD25+), low levels of CD127 (CD1271ow), or both high CD25 and low CD127. The levels of CD25 and CD 127 are compared, for example to a CD4+ T cell that is not a Treg. In some embodiments, the Treg cell expressing the exogenous expression cassette has a high CD25, high CD4, and low CD 127 phenotype.

[0158] In some embodiments, the Treg expresses one or more proteins associated with a Treg phenotype. In some embodiments, the Treg expresses FOXP3. In some embodiments, the Treg expresses endogenous FOXP3. In some embodiments, the Treg further expresses exogenous FOXP3. Expression levels of FOXP3 may be assessed by conventional methods such as Western blotting, flow cytometry, or ELISA. Expression levels may also be assessed by analyzing mRNA expression using techniques such as RT-qPCR. In some embodiments, expression of FOXP3 increases compared to untransduced cells. In some embodiments, expression of FOXP3 increases compared to cells without exogenous expression cassettes.

[0159] In some embodiments, FOXP3 expression increases at least about 1.1-fold, at least about 1.25-fold, at least about 1.5-fold, at least about 1.75-fold, at least about 2-fold, at leastabout 3-fold, at least about 4-fold, at least about 5-fold, or more compared to cells lacking the exogenous expression cassette(s). In some embodiments, FOXP3 expression increases at least about 1.1-fold, at least about 1.25-fold at least about 1.5-fold, at least about 1.75-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, or more compared to cells lacking the exogenous expression cassette(s). In some embodiments, FOXP3 expression is maintained at approximately the same level as the day of highest FOXP3 expression, following transduction with the exogenous expression cassette(s).

[0160] In some embodiments, the expression ratio of full-length FOXP3 protein to the FOXP3 isoform lacking exon 2 (A2) within a Treg is about 90:1, about 80:1, about 70:1, about 60:1, about 50:1, about 40:1, about 30:1, about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, about 1:25, about 1:30, about 1:40, about 1:50, about 1:60, about 1:70, about 1:80, or about 1:90. In some embodiments, the expression ratio of full-length FOXP3 protein to the FOXP3 isoform lacking exon 2 (A2) within a Treg comprising the expression cassette is about 90:1, about 80:1, about 70:1, about 60:1, about 50:1, about 40:1, about 30:1, about 25:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, about 1:25, about 1:30, about 1:40, about 1:50, about 1:60, about 1:70, about 1:80, or about 1:90. In some embodiments, the expression ratio measures FOXP3 gene expression. In some embodiments, the expression ratio measures FOXP3 protein levels. In some embodiments, the expression ratio measures active FOXP3 protein levels.

[0161] In some embodiments, the Treg is CD4 positive (CD4+). In some embodiments, the Treg is CD4+ / CD25+. In some embodiments, the Treg is CD4+ / CD127 low. In some embodiments, the Treg is CD4+ / CD25+ / CD127 low.

[0162] Tregs can be characterized by expression of CD25+. In some embodiments, the Treg is CD25+. In some embodiments, the Treg is CD4+ / CD25+. In some embodiments, the Treg is CD25+ / CD127 low. In some embodiments, the Treg is CD4+ / CD25+ / CD127 low.

[0163] In some embodiments, the Treg expresses a low level of CD 127 (CD1271ow). In some embodiments, the Treg is CD4+ / CD127 low. In some embodiments, the Treg is CD25 / CD127 low. In some embodiments, the Treg is CD4+ / CD25+ / CD127 low. In someembodiments, Tregs transduced with the exogenous expression cassette(s) provided herein maintain CD 127 low status upon transduction.

[0164] Treg populations may be further characterized by additional markers. Such markers may be indicative of a Treg phenotype, may be absent in Tregs, or may be present or absent in subpopulations of Tregs (e.g., activated Tregs, tissue-resident Tregs, senescent Tregs, functionally exhausted Tregs). For example, CD27 is a marker of highly suppressive Tregs, whereas CD70 marks a profound loss of regulatory function in Tregs. Exemplary markers can include, but are not limited to, CD27, CD70, CD71 (Transferrin Receptor, TfR), FOXP3, Helios, glucocorticoid-induced TNFR-related protein (GITR), latency-associated peptide (LAP), glycoprotein A repetitions predominant (GARP), and CD137 (4-1BB). In some embodiments, the genetically modified cell preparation is a Treg cell preparation (z.e., engineered Treg), wherein the engineered Treg comprises Treg cells comprising expression cassettes that express exogenous FOXP3. In some embodiments, the engineered Treg comprises the same set of markers as a wild-type Treg. In some embodiments, the engineered Treg comprises the same set of markers as a control Treg lacking exogenous FOXP3. In some embodiments, the engineered Treg lacks the same set of markers as a wild-type Treg. In some embodiments, the engineered Treg lacks the same set of markers as a control Treg lacking an expression cassette that expresses exogenous FOXP3. In some embodiments, the additional markers are selected from the group consisting of CD27, CD70, CD71, CD154, FOXP3, Helios, CD69, CD57, TIGIT, GITR, LAP, GARP, CD137 (4-1BB), and any combination thereof.

[0165] Treg populations may be further characterized by the presence or absence of specific cytokines and / or chemokines. For example, Treg cells generally do not express or minimally express the cytokine IL-2. In some embodiments, the Treg cell comprising an expression cassette that expresses exogenous FOXP3 further expresses TNF-a. In some embodiments, the Treg cell comprising an expression cassette that expresses exogenous FOXP3 further expresses IFN-y. In some embodiments, the Treg cell comprising an expression cassette that expresses exogenous FOXP3 further expresses IL- 10. In some embodiments, the Treg cell comprising an expression cassette that expresses exogenous FOXP3 further expresses TGF-p. In some embodiments, the Treg cell comprising an expression cassette that expresses exogenous FOXP3 further expresses IL-35. In some embodiments, the Treg cell comprising an expression cassette that expresses exogenous FOXP3 does not express or minimally expresses IL-2. In some embodiments, the Treg cell comprising an expression cassette that expresses exogenous FOXP3 does not express or minimally expresses IL-17 (z.e., IL-17A). In some embodiments, the Tregcell comprising an expression cassette that expresses exogenous FOXP3 does not express or minimally expresses IL-4. In some embodiments, the Treg cell comprising an expression cassette that expresses exogenous FOXP3 further expresses the same cytokines as a wild-type Treg. In some embodiments, the Treg cell comprising an expression cassette that expresses exogenous FOXP3 further expresses the same cytokines as a control Treg lacking an expression cassette that expresses exogenous FOXP3. In some embodiments, the Treg cell comprising an expression cassette that expresses exogenous FOXP3 lacks expression of the same cytokines as a wild-type Treg. In some embodiments, the Treg cell comprising an expression cassette that expresses exogenous FOXP3 lacks expression of the same cytokines as a control Treg lacking an expression cassette that expresses exogenous FOXP3. In some embodiments, the Treg cell comprising an expression cassette that expresses exogenous FOXP3 comprises the same cytokine phenotype as a wild-type Treg. In some embodiments, the Treg cell comprising an expression cassette that expresses exogenous FOXP3 comprises the same cytokine phenotype as a control Treg lacking an expression cassette that expresses exogenous FOXP3. In some embodiments, the cytokines are selected from the group consisting of TNF-a, IFN-y, TGF-P, IL- 10, IL-35, IL- 17 (z.e., IL-17A), IL-2, IL-4, and any combination thereof. In some embodiments, the Treg cell comprising the expression cassette that expresses exogenous FOXP3 expresses an increased level (e.g., at least about any of 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, or more increased level) of IL- 10 production compared to a control Treg lacking an expression cassette that expresses exogenous FOXP3.

[0166] In some embodiments, the Treg has an immunosuppressive phenotype. In one embodiment, the Treg generates an immunosuppressive effect in an individual having an immune-related disorder. In some embodiments, the individual is human. In some embodiments, the Treg is autologous to the individual.

[0167] In some embodiment, the Treg suppresses, blocks, or inhibits Graft-vs-host disease (GvHD) in the individual. In one embodiment, the Treg suppresses, blocks, or inhibits an immune-related disorder in the individual. In some embodiments, the Treg is administered prior to onset of an immune-related disorder. In some embodiments, the Treg is administered after the onset of an immune-related disorder. In some embodiments, the individual is human. In some embodiments, the Treg is autologous to the individual. In some embodiments, the Treg is allogeneic to the individual.

[0168] Methods of measuring markers used to characterize Tregs will be readily apparent to the person of ordinary skill in the art. For example, Tregs or populations of T cells comprisingTregs can be cultured using the methods described herein. In some embodiments, the method of measuring markers comprises measuring Treg markers selected from the group consisting of CD4+, CD25+, CD1271ow, and FOXP3. In some embodiments, the method further comprises measuring one or more additional markers and comparing the presence / absence and / or level of the one or more additional markers to the presence / absence and / or level in a wildtype Treg and / or a control Treg that does not comprise any of the expression cassettes expressing exogenous FOXP3 described herein. In some embodiments, the additional markers are selected from the group consisting of CD27, CD70, CD71, CD154, FOXP3, Helios, CD69, CD57, TIGIT, GITR, LAP, GARP, CD137 (4-1BB), and any combination thereof. In some embodiments, the additional markers are Treg markers. In some embodiments, the additional markers are Treg subset (e.g., activated Treg) markers. In some embodiments, the additional markers are markers of non-Treg cell populations (e.g., markers of conventional T cells). Following culturing, Tregs can be collected and stained using antibodies, e.g., fluorophore- conjugated antibodies, that recognize and bind to target Treg markers (such as FOXP3, CD25, and CD 127) and analyzed for the expression level of such Treg markers using fluorescence activated flow cytometry (FACS) or fluorescence microscopy. Similarly, Tregs can be cultured using the methods described herein with (e.g.) Golgi-Stop or Golgi-Plug, and then Tregs can be collected, fixed (e.g., with paraformaldehyde), permeabilized, and stained for intracellular production of cytokines, such as TNF-a, IFN-y, and IL- 10. Treg production of cytokines can also be tested by ELISA or ELISpot to assess the levels of cytokines secreted into culture media by the Tregs. Gene expression of Treg markers and / or cytokine expression may be measured by methods such as RT-qPCR, in situ hybridization, etc.III. METHODS OF GENERATING TREGS OVEREXPRESSING FOXP3

[0169] Also provided herein is a method of generating a Treg overexpressing FOXP3. For example, in some embodiments, there is provided a method comprising transducing the Treg with any of the expression cassettes described herein. In other embodiments, there is provided a method of generating a Treg overexpressing FOXP3, comprising transducing an effector T cell (Teff) with any of the expression cassettes described herein.

[0170] In some embodiments, there is provided a method of generating a Treg overexpressing FOXP3 comprising transducing the Treg with an expression cassette comprising a modified nucleic acid encoding a FOXP3 protein operably linked to a promoter, wherein the nucleic acid encoding the FOXP3 protein is modified to produce a full-length FOXP3 proteinisoform and an alternatively spliced isoform lacking one or more exons. In some embodiments, the nucleic acid encoding the FOXP3 protein comprises intron sequences flanking the nucleic acid encoding exon 2. In some embodiments, the nucleic acid encoding the FOXP3 protein comprising intron sequences flanking the nucleic acid encoding exon 2 is alternatively spliced to produce a full-length FOXP3 protein isoform and a FOXP3 protein isoform lacking exon 2. In some embodiments, the FOXP3 protein is linked to a detection tag. In some embodiments, the detection tag is linked to the N-terminus of the FOXP3 protein. In some embodiments, the detection tag is linked to the C-terminus of the FOXP3 protein. In some embodiments, the nucleic acid encoding a FOXP3 protein is FOXP3 cDNA. In some embodiments, the exogenous expression cassette further comprises a nucleic acid encoding a chimeric receptor and / or a nucleic acid encoding a cell surface receptor. In some embodiments, the nucleic acid encoding the FOXP3 protein and the nucleic acid encoding the chimeric receptor and / or the nucleic acid encoding the cell surface receptor are operably linked to the same promoter. In some embodiments, the nucleic acid encoding the FOXP3 protein and the nucleic acid encoding the chimeric receptor and / or the nucleic acid encoding the cell surface receptor are each separated by an IRES, a nucleic acid encoding a self-cleaving peptide (e.g., P2A peptide), or a nucleic acid encoding a peptide that causes ribosome skipping. Thus, in some embodiments, there is provided a method of overexpressing FOXP3 in a Treg, comprising transducing the Treg with an exogenous expression cassette comprising a nucleic acid encoding: (I) a promoter; (II) a nucleic acid encoding a FOXP3 protein; (III) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; (IV) a nucleic acid encoding a cell surface receptor; or (I) a promoter; (II) a nucleic acid encoding a FOXP3 protein; (III) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; (IV) a nucleic acid encoding a chimeric receptor; (IV) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; and (VI) a nucleic acid encoding a cell surface receptor, wherein the order of FOXP3 protein, chimeric receptor, and / or cell surface receptor from 5’ to 3’ is interchangeable and wherein each component is separated from the other by a self-cleaving peptide (e.g., P2A) or the like. In some embodiments, the method further comprises measuring Treg markers selected from the group consisting of CD4+, CD25+, CD1271ow, and FOXP3. In some embodiments, the method further comprises measuring one or more additional markers and comparing the presence / absence and level of the one or more additional markers to the presence / absence and level in a wildtype Treg and / or a control Tregthat does not comprise any of the expression cassettes expressing exogenous FOXP3 described herein. For example, specific markers may be indicative of a Treg population whereas other markers may be absent in Tregs (e.g., instead indicative of an effector T cell population), and the combination of the presence of specific Treg markers and absence of specific non-Treg markers can be used to assess the cell phenotype of the Treg overexpressing F0XP3 described herein. In some embodiments, the Treg maintains expression of at least one Treg marker selected from the group consisting of CD4+, CD25+, CD1271ow, and FOXP3. In some embodiments, the Treg maintains expression of FOXP3 and one or more Treg markers selected from the group consisting of CD4+, CD25+, and CD1271ow. In some embodiments, the Treg expresses similar additional markers at similar protein and / or gene expression levels as wildtype Tregs or control Tregs that do not comprise any of the expression cassettes expressing exogenous FOXP3 described herein. In some embodiments, the additional markers are selected from the group consisting of CD27, CD70, CD71, CD154, FOXP3, Helios, CD69, CD57, TIGIT, GITR, LAP, GARP, CD137 (4-1BB), and any combination thereof. In some embodiments, the additional markers are Treg markers. In some embodiments, the additional markers are Treg subset (e.g., activated Treg) markers. In some embodiments, the additional markers are markers of non-Treg cell populations (e.g., markers of conventional T cells). In some embodiments, the Treg maintains a Treg functional phenotype, wherein the Treg functional phenotype is characterized by one or more Treg activities selected from the group consisting of suppression of effector T cell activity, production of suppressive cytokines (e.g., IL- 10), suppression of effector T cell cytokines (e.g., IL-2, IFNy, TNFa), and reduction in the rate of division of effector T cells. In some embodiments, the Treg expresses similar one or more cytokine(s) expressed by wildtype Tregs or control Tregs that do not comprise any of the expression cassettes expressing exogenous FOXP3 described herein. In some embodiments, the Treg does not express similar one or more cytokine(s) that are not expressed by wildtype Tregs nor control Tregs that do not comprise any of the expression cassettes expressing exogenous FOXP3 described herein. In some embodiments, the one or more cytokine(s) are selected from the group consisting of TNF- a, IFN-y, TGF-P, IL-10, IL-35, IL-17 (i.e., IL-17A), IL-2, IL-4, and any combination thereof. In some embodiments, the Treg expresses an increased level (e.g., at least about any of 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, or more increased level) of IL- 10 production compared to a wildtype Treg or a control Treg lacking an expression cassette that expresses exogenous FOXP3.

[0171] In some embodiments, there is provided a method of generating a Treg, comprising transducing an effector T cell (Teff) with an expression cassette comprising a modified nucleic acid encoding a FOXP3 protein operably linked to a promoter, wherein the nucleic acid encoding the FOXP3 protein is modified to produce a full-length FOXP3 protein isoform and an alternatively spliced isoform lacking one or more exons, wherein the expression of the FOXP3 protein causes the Teff to differentiate or transform into a Treg. In some embodiments, the nucleic acid encoding the FOXP3 protein comprises intron sequences flanking the nucleic acid encoding exon 2. In some embodiments, the nucleic acid encoding the FOXP3 protein comprising intron sequences flanking the nucleic acid encoding exon 2 is alternatively spliced to produce a full-length FOXP3 protein isoform and a FOXP3 protein isoform lacking exon 2. In some embodiments, the FOXP3 protein is linked to a detection tag. In some embodiments, the detection tag is linked to the N-terminus of the FOXP3 protein. In some embodiments, the detection tag is linked to the C-terminus of the FOXP3 protein. In some embodiments, the exogenous expression cassette further comprises a nucleic acid encoding a chimeric receptor and / or a nucleic acid encoding a cell surface receptor. In some embodiments, the nucleic acid encoding the FOXP3 protein and the nucleic acid encoding the chimeric receptor and / or the nucleic acid encoding the cell surface receptor are operably linked to the same promoter. In some embodiments, the nucleic acid encoding the FOXP3 protein and the nucleic acid encoding the chimeric receptor and / or the nucleic acid encoding the cell surface receptor are each separated by an IRES, a nucleic acid encoding a self-cleaving peptide (e.g., P2A peptide), or a nucleic acid encoding a peptide that causes ribosome skipping. Thus, in some embodiments, there is provided a method of generating a Treg, comprising transducing an effector T cell (Teff) with an exogenous expression cassette encoding: (I) a promoter; (II) a nucleic acid encoding a FOXP3 protein; (III) an internal ribosome entry site (IRES), or a nucleic acid encoding a selfcleaving peptide or a peptide that causes ribosome skipping; (IV) a nucleic acid encoding a cell surface receptor; or (I) a promoter; (II) a nucleic acid encoding a FOXP3 protein; (III) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; (IV) a nucleic acid encoding a chimeric receptor; (IV) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; and (VI) a nucleic acid encoding a cell surface receptor; wherein the order of FOXP3 protein, chimeric receptor, and / or cell surface receptor from 5’ to 3’ is interchangeable and wherein each component is separated from the other by a self-cleaving peptide (e.g., P2A) or the like; and wherein the expression of the FOXP3 protein causes the Teffto differentiate or transform into a Treg. In some embodiments, the nucleic acid encoding a FOXP3 protein is FOXP3 cDNA. In some embodiments, the method further comprises measuring Treg markers selected from the group consisting of CD4+, CD25+, CD1271ow, and FOXP3. In some embodiments, the method further comprises measuring one or more additional markers and comparing the presence / absence and level of the one or more additional markers to the presence / absence and level in a wildtype Treg and / or a control Treg that does not comprise any of the expression cassettes expressing exogenous FOXP3 described herein. In some embodiments, the Teff differentiates or transforms into a Treg after being transduced with the exogenous expression cassette, wherein the differentiated or transformed Treg maintains expression of at least one Treg marker selected from the group consisting of CD4+, CD25+, CD1271ow, and FOXP3. In some embodiments, the differentiated or transformed Treg maintains expression of FOXP3 and one or more Treg markers selected from the group consisting of CD4+, CD25+, and CD1271ow. In some embodiments, the differentiated or transformed Treg expresses similar additional markers at similar protein and / or gene expression levels as wildtype Tregs or control Tregs that do not comprise any of the expression cassettes expressing exogenous FOXP3 described herein. In some embodiments, the additional markers are selected from the group consisting of CD27, CD70, CD71, CD154, FOXP3, Helios, CD69, CD57, TIGIT, GITR, LAP, GARP, CD137 (4-1BB), and any combination thereof. In some embodiments, the additional markers are Treg markers. In some embodiments, the additional markers are Treg subset (e.g., activated Treg) markers. In some embodiments, the additional markers are markers of non-Treg cell populations (e.g., markers of effector T cells). In some embodiments, the presence of one or more additional markers is indicative of a differentiated or transformed Treg as compared to the presence of the one or more additional markers in a wildtype Treg. In some embodiments, the absence of one or more additional markers is indicative of a differentiated or transformed Treg as compared to the absence of the one or more additional markers in a wildtype Treg. In some embodiments, the differentiated or transformed Treg maintains a Treg functional phenotype, wherein the Treg functional phenotype is characterized by one or more Treg activities selected from the group consisting of suppression of effector T cell activity, production of suppressive cytokines (e.g., IL- 10), suppression of effector T cell cytokines (e.g., IL-2, IFNy, TNFa), and reduction in the rate of division of effector T cells. In some embodiments, the differentiated or transformed Treg expresses similar one or more cytokine(s) expressed by wildtype Tregs or control Tregs that do not comprise any of the expression cassettes expressing exogenous FOXP3 described herein. In someembodiments, the differentiated or transformed Treg does not express similar one or more cytokine(s) that are not expressed by wildtype Tregs nor control Tregs that do not comprise any of the expression cassettes expressing exogenous FOXP3 described herein. In some embodiments, the one or more cytokine(s) are selected from the group consisting of TNF-a, IFN-y, TGF-P, IL- 10, IL-35, IL- 17 (z.e., IL-17A), IL-2, IL-4, and any combination thereof. In some embodiments, the differentiated or transformed Treg expresses an increased level (e.g., at least about any of 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, or more increased level) of IL- 10 production compared to a wildtype Treg or a control Treg lacking an expression cassette that expresses exogenous FOXP3.IV. METHODS OF PREPARATION

[0172] The expression cassettes described herein may be prepared and transduced into host cells, e.g., Treg cells. Sequences encoding the expression cassettes can be synthesized. After obtaining said sequence, it is cloned into a suitable expression vector, which is then transduced into a suitable host cell. The transduced host cells are recovered and cultured to obtain viable host cells that stably express the expression cassettes of the present invention.

[0173] In some embodiments, the expression cassettes may be DNA. In some embodiments, the expression cassettes are inserted into a vector, such as an expression vector, a viral vector, or a cloning vector. For expression of the expression cassettes, the vector may be introduced into a host cell to allow expression of the expression cassettes within the host cell. The expression vectors may contain a variety of elements for controlling expression, including without limitation, promoter sequences, transcription initiation sequences, enhancer sequences, selectable markers, and signal sequences. These elements may be selected as appropriate by a person of ordinary skill in the art. For example, the promoter sequences may be selected to promote the transcription of the expression cassette in the vector. Suitable promoter sequences include, without limitation, MND, T7 promoter, T3 promoter, SP6 promoter, beta-actin promoter, EFla promoter, CMV promoter, and SV40 promoter. Enhancer sequences may be selected to enhance the transcription of the expression cassettes. Selectable markers may be included to allow selection of the host cells inserted with the vector from those host cells lacking the vector, for example, a selectable marker may be a transmembrane gene (such as EGFR, HER2, CD2, H-2Kk, IL-5Ra, or any derivatives or mutants thereof) that can be identified by flow cytometry and FACS analysis.

[0174] In some embodiments, the expression cassette (or the vector comprising said expression cassette, such as an expression vector, a viral vector, or a cloning vector) expresses a FOXP3 protein, for example a full-length FOXP3 protein and / or an alternatively spliced isoform lacking one or more exons (e.g., exon 2). In some embodiments, the expression cassette (or the vector comprising said expression cassette, such as an expression vector, a viral vector, or a cloning vector) further expresses a chimeric receptor and / or another additional polypeptide. The chimeric receptor may be, for example, a CAR, an engineered TCR, a TCR mimic or a chimeric antibody-T cell receptor, or a chimeric signaling receptor. The chimeric receptor may target an antigen of interest (e.g., a target in an inflammation site). The antigens may include, without limitation, AFP (alpha- fetoprotein), avP6 or another integrin, BCMA, B7-H3, B7-H6, CA9 (carbonic anhydrase 9), CCL-1 (C-C motif chemokine ligand 1), CD5, CD19, CD20, CD21, CD22, CD23, CD24, CD30, CD33, CD38, CD40, CD44, CD44v6, CD44v7 / 8, CD45, CD47, CD56, CD66e, CD70, CD74, CD79a, CD79b, CD98, CD123, CD138, CD171, CD352, CEA (carcinoembryonic antigen), Claudin, c-MET, DLL3 (delta-like protein 3), DLL4, ENPP3 (ectonucleotide pyrophosphatase / phosphodiesterase family member 3), EpCAM, EPG-2 (epithelial glycoprotein 2), EPG-40, ephrinB2, EPHa2 (ephrine receptor A2), ERBB dimers, estrogen receptor, ETBR (endothelin B receptor), FAP-a (fibroblast activation protein a), fetal AchR (fetal acetylcholine receptor), FBP (a folate binding protein), FCRL5, FR-a (folate receptor alpha), GCC (guanyl cyclase C), GD2, GD3, GPC2 (glypican-2), GPC3, gplOO (glycoprotein 100), GPNMB (glycoprotein NMB), GPRC5D (G Protein Coupled Receptor 5D), HER2, HER3, HER4, hepatitis B surface antigen, HLA-A1 (human leukocyte antigen Al), HLA-A2 (human leukocyte antigen A2), HMW-MAA (human high molecular weight- melanoma-associated antigen), IGF1R (insulin-like growth factor 1 receptor), Ig kappa, Ig lambda, IL-22Ra (IL- 22 receptor alpha), IL-13Ra2 (IL- 13 receptor alpha 2), KDR (kinase insert domain receptor), LI cell adhesion molecule (LI -CAM), Liv-1, LRRC8A (leucine rich repeat containing 8 Family member A), Lewis Y, melanoma-associated antigen (MAGE)-Al, MAGE- A3, MAGE-A6, MART-1 (melan A), murine cytomegalovirus (MCMV), MCSP (melanoma- associated chondroitin sulfate proteoglycan), mesothelin, mucin 1 (MUC1), MUC16, MHC / peptide complexes e.g., HLA-A complexed with peptides derived from AFP, KRAS, NY-ESO, MAGE-A, and WT1), NCAM (neural cell adhesion molecule), Nectin-4, NKG2D (natural killer group 2 member D) ligands, NY-ESO, oncofetal antigen, PD-1, PD-L1, PRAME (preferentially expressed antigen of melanoma), progesterone receptor, PSA (prostate specific antigen), PSCA (prostate stem cell antigen), PSMA (prostate specific membrane antigen),R0R1, ROR2, SIRPa (signal-regulatory protein alpha), SLIT, SLITRK6 (NTRK-like protein 6), STEAP1 (six transmembrane epithelial antigen of the prostate 1), survivin, TAG72 (tumor- associated glycoprotein 72), TPBG (trophoblast glycoprotein), Trop-2, VEGFR1 (vascular endothelial growth factor receptor 1), VEGFR2, and antigens from HIV, HBV, HCV, HPV, and other pathogens. For example, in some embodiments, the target antigen is selected from the group consisting of Carboxypeptidase H, Chromogranin A, Glutamate decarboxylase, Imogen- 38, Insulin, Proinsulin, Insulinoma antigen-2 and 2p, Islet-specific glucose-6-phosphatase catalytic subunit related protein (IGRP), a-enolase, Aquaporin-4, P-arrestin, Myelin basic protein (MBP), Myelin oligodendrocytic glycoprotein (MOG), Proteolipid protein (PLP), phosphodiesterase 4 (PDE4), vascular endothelial growth factor receptor (VEGFR), S1OO-P, Citrullinated vimentin (CV), Collagen II, Human cartilage glycoprotein 39, La antigen, Phospholipid-P-2 glycoprotein I complex, Sm antigens of U-l small ribonucleoprotein complex, tumor necrosis factor (TNF)-like ligand 1A (TL1A), Complement Cis, Cam-3001, CD125, CD154, CD2, CD23 (IgE receptor), IL-6 receptor (IL-6R), IL-4Ra, IL-12R, IL-13Ral, IL- 13Ra2, IL-15R, IL-23R, rhuMAb P7, NKG2D, NKp30, A0C3 (VAP-1), CCL11 (eotaxin-1), CD19, CD20, CD22, CD3, CD4, CD5, IFN-y, IgE, IgE Fc region, IL-1 (z.e., IL- la and / or IL- 1P), IL-12, IL-23, IL-15, IL-17, IL-17A, IL-22, IL-6, IL-13, IL-33, TSLP, integrin a4p7, LFA-1 (CDl la), OX-40, OX-40L, p53, CD38, CD40L, CD28, ICOS, RIPK1, BTK, Bet v 1, GLP2R, JAK1, C5, and TNF-a.

[0175] In some embodiments, the host cell (e.g., Treg cell) contains the vector described above. The vector can be introduced to the cell using any suitable methods known in the art, including, but not limited to, DEAE-dextran mediated delivery, calcium phosphate precipitate method, cationic lipids mediated delivery, liposome mediated transfection, electroporation, microprojectile bombardment, receptor-mediated gene delivery, delivery mediated by polylysine, histone, chitosan, and peptides. Standard methods for transduction of cells for expression of a vector of interest are well known in the art. In some embodiments, the host cells comprise a vector comprising the expression cassette encoding the FOXP3 protein.

[0176] In some embodiments, the present application provides methods of expressing any of the expression cassettes described herein, comprising culturing the isolated host cell containing the vector. The isolated host cells are cultured under conditions that allow expression of the expression cassettes inserted in the vectors. Suitable conditions for expression of the expression cassettes may include, without limitation, suitable medium, suitable density of host cells in the culture medium, presence of necessary nutrients, presence of supplemental factors, suitabletemperatures and humidity, and absence of microorganism contaminants. A person with ordinary skill in the art can select the suitable conditions as appropriate for the purpose of the expression.Vectors

[0177] In certain aspects, the present disclosure provides expression cassettes that encode a FOXP3 protein. Such expression cassettes can be inserted into an appropriate vector (e.g., viral vector or non- viral plasmid vector) for introduction in a host cell (e.g., a Treg) of interest.

[0178] As used herein, the term “recombinant” or “non-natural” refers to an organism, microorganism, cell, nucleic acid molecule, or vector that includes at least one genetic alteration or has been modified by introduction of an exogenous expression cassette, wherein such alterations or modifications are introduced by genetic engineering. Genetic alterations include, for example, modifications introducing expressible expression cassettes encoding one or more proteins, fusion proteins, or enzymes, or other expression cassette additions, deletions, substitutions, or other functional disruption of a cell's genetic material. Additional modifications include, for example, non-coding regulatory regions in which the modifications alter expression of a gene or operon. In some embodiments, a cell, such as a regulatory T cell (Treg) or an effector T cell, obtained from a subject may be converted into a non-natural or recombinant regulatory T cell (Treg) (e.g., a non-natural or recombinant regulatory T cell (Treg)) by introducing an expression cassette as described herein.

[0179] A vector that encodes a core virus is referred to herein as a “viral vector.” There are a large number of available viral vectors suitable for use with the compositions of the instant disclosure, including those identified for human gene therapy applications (see Pfeifer and Verma (2001), Ann. Rev. Genomics Hum. Genet. 2:177, hereby incorporated by reference in its entirety). Suitable viral vectors include vectors based on RNA viruses, such as retrovirus- derived vectors, e.g., Maloney murine leukemia virus (MLV)-derived vectors, and include more complex retrovirus-derived vectors, e.g., lentivirus-derived vectors. HIV-l-derived vectors belong to this category. Other examples include lentivirus vectors derived from HIV-2, FIV, equine infectious anemia virus, SIV, and Maedi-Visna virus (ovine lentivirus). Methods of using retroviral and lentiviral viral vectors and packaging cells for transducing mammalian host regulatory T cells (Treg) with viral particles containing chimeric antigen receptor transgenes are known in the art and have been previously described, for example, in U.S. Pat. No. 8,119,772; Walchli et al. (2011), PLoS One 6:327930; Zhao et al. (2005), J. Immunol. 174:4415; Engels etal. (2003), Hum. Gene Ther. 14:1155; Frecha et al. (2010), Mol. Ther. 18:1748; and Verhoeyen et al. (2009), Methods Mol. Biol. 506:97. Retroviral and lentiviral vector constructs and expression systems are also commercially available.

[0180] In some embodiments, a viral vector is used to introduce any expression cassette described herein. A viral vector may be a retroviral vector or a lentiviral vector. A viral vector may also include nucleic acid sequences encoding a cell surface receptor as a marker for successful cell transduction. Transduction markers for viral vectors are known in the art and include selection markers, which may confer drug resistance, or detectable markers, such as fluorescent markers or cell surface proteins that can be detected by methods such as flow cytometry. For example, in particular embodiments, a viral vector may further comprise a gene marker for transduction comprising green fluorescent protein, or a truncated human EGFR (huEGFRt; see Wang et al. (2011), Blood 118:1255). When a viral vector genome comprises a plurality of nucleic acid sequences to be expressed in a host cell (e.g., T cell such as Treg) as separate transcripts, the viral vector may also comprise additional sequences between the two (or more) transcripts allowing bicistronic or multicistronic expression. Examples of such sequences used in viral vectors include internal ribosome entry sites (IRES), furin cleavage sites, viral 2A peptides, or any combination thereof.

[0181] Other vectors also can be used for expression cassette delivery including DNA viral vectors, including for example adenovirus-based vectors and adeno-associated virus (AAV)- based vectors; and vectors derived from herpes simplex viruses (HSVs), including amplicon vectors, replication-defective HSV and attenuated HSV (Krisky et al. (1998), Gene Ther. 5: 1517).

[0182] Other vectors recently developed for gene therapy uses can also be used with the compositions and methods of this disclosure. Such vectors include those derived from baculoviruses and a-viruses. (Jolly, DJ (1999), Emerging Viral Vectors, pp 209-40 in Friedmann T. ed. The Development of Human Gene Therapy. New York: Cold Spring Harbor Lab), or plasmid vectors (such as sleeping beauty or other transposon vectors).

[0183] In certain embodiments, hematopoietic progenitor cells or embryonic stem cells are modified to comprise an expression cassette of this disclosure. Hematopoietic progenitor cells may comprise induced pluripotent stem cells, which may be derived or originate from fetal liver tissue, bone marrow, cord blood, or peripheral blood. The hematopoietic progenitor cells may be from human, mouse, rat, or other mammals. In certain embodiments, the host cell transfected to express an expression cassette of this disclosure is a functional Treg or an effector T cell.V. METHODS OF EXPANDING AND CULTURING CELLS

[0184] In some embodiments, the cell transfected with the expression cassette of this disclosure is a eukaryotic cell. In some embodiments, the cell transfected with the expression cassette of this disclosure is a human cell. In certain embodiments, the cell transfected with the expression cassette of this disclosure is an immune cell. In certain embodiments, the cell transfected with the expression cassette of this disclosure is a T cell. In certain embodiments, the cell transfected with the expression cassette of this disclosure is a functional regulatory T cell (Treg).

[0185] In some embodiments, the Treg is isolated from peripheral blood mononuclear (PBMC) cells. In some embodiments, the Treg is isolated from PBMCs using density gradient centrifugation. In some embodiments, the Tregs are enriched by positive selection. In some embodiments, the Tregs are enriched by positive selection for CD25+. In some embodiments, the Tregs are enriched by positive selection for CD4+CD25+CD1271ow cells. In some embodiments, the enrichment occurs by FACS. In some embodiments, the Tregs are stimulated by anti-CD3 and / or anti-CD28 antibodies on day 0 after positive selection. In some embodiments, the Tregs are re-stimulated by anti-CD3 and / or anti-CD28 antibodies on day 9 of in vitro culture after positive selection.

[0186] One or more growth factor cytokines may be added to the in vitro culture, wherein the one or more growth factor cytokines promote proliferation of T cells such as Tregs, including Tregs that comprise any of the expression cassettes of this disclosure and / or effector T cells expressing an expression cassette of this disclosure. The cytokines may be human or nonhuman cytokines. Exemplary growth factor cytokines that may be used to promote regulatory T cell (Treg) proliferation include IL-2, IL-4, IL-7, IL-9, IL-15, TGEp, and the like. In some embodiments, the cytokine(s) is added to the media prior to selection. In some embodiments, the cytokine(s) is added to the media after cell isolation. In some embodiments, the cytokine(s) is added to the cell culture media approximately every 12 to 60 hours, such as about 24 to about 48 hours. In some embodiments, the cytokine(s) is added to the media for the duration of the culture. In some embodiments, the Treg is cultured in a medium comprising two, three, four, five or more cytokines.

[0187] In some embodiments, cells expressing any of the expression cassettes of this disclosure are cultured for a sufficient time to induce proliferation or differentiation. The cells are maintained in culture for about 3 days to about 5 days, about 4 days to about 10 days, about 5 days to about 20 days, about 10 days to about 23 days, about 15 days to about 30 days, orabout 23 days to about 30 days. It will be appreciated that the cells may be maintained for an appropriate amount of time required to achieve a desired result, i.e., a desired cellular composition or level of proliferation. For example, to generate a cellular composition comprising primarily Tregs, cells may be maintained in culture for about 30 days.

[0188] In some embodiments, the method further comprises detecting one or more Treg markers provided herein. In some embodiments, the method further comprises detecting FOXP3 levels and / or FOXP3.

[0189] In some embodiments, the suppressive activity of the Treg comprising any of the expression cassettes of this disclosure is increased compared to a Treg lacking the expression cassette(s). In some embodiments, the production of suppressive cytokines by a Treg comprising any of the expression cassettes of this disclosure is increased compared to a Treg lacking the expression cassette(s). In some embodiments, the production of cytotoxic and pro- inflammatory cytokines by a cytotoxic T cell and / or an effector T cell is decreased by the Tregs comprising any of the expression cassettes of this disclosure. In some embodiments, the activity of a cytotoxic T cell and / or an effector T cell is decreased by the Tregs comprising any of the expression cassettes of this disclosure.VI. PHARMACEUTICAL COMPOSITIONS, ARTICLES OF MANUFACTURE, AND KITS

[0190] Further provided by the present application are pharmaceutical compositions comprising a cell, e.g., a T cell such as a Treg cell, comprising a recombinant cytokine receptor described herein.

[0191] The pharmaceutical compositions may be suitable for a variety of modes of administration described herein, including for example systemic or localized administration. In some embodiments, the pharmaceutical composition is formulated for intravenous administration.

[0192] The pharmaceutical compositions to be used for in vivo administration are generally formulated as sterile, substantially isotonic, and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration. Sterility is readily accomplished by filtration through sterile filtration membranes. In some embodiments, the composition is free of pathogens. For injection, the pharmaceutical composition can be in the form of liquid solutions, for example in physiologically compatible buffers such as Hank's solution or Ringer's solution.

[0193] In some embodiments, the pharmaceutical composition is suitable for administration to a human. In some embodiments, the pharmaceutical composition is suitable for administration to a rodent (e.g., mice, rats) or non-human primates (e.g., Cynomolgus monkey). In some embodiments, the pharmaceutical composition is cryopreserved.

[0194] The present application also provides kits comprising compositions (such as pharmaceutical compositions) described herein and may further comprise instruction(s) on methods of using the composition, such as uses described herein. The kits described herein may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for performing any methods described herein.VII. METHODS OF TREATMENT

[0195] In some embodiments, there is provided herein a method of treating an immune- related disorder comprising administering an engineered Treg comprising an exogenous expression cassette comprising a modified nucleic acid encoding a FOXP3 protein operably linked to a promoter, wherein the nucleic acid encoding the FOXP3 protein is modified to produce a full-length FOXP3 protein isoform and an alternatively spliced isoform lacking one or more exons. In some embodiments, the nucleic acid encoding the FOXP3 protein comprises intron sequences flanking the nucleic acid encoding exon 2. In some embodiments, the nucleic acid encoding the FOXP3 protein comprising intron sequences flanking the nucleic acid encoding exon 2 is alternatively spliced to produce a full-length FOXP3 protein isoform and a FOXP3 protein isoform lacking exon 2. In some embodiments, the exogenous expression cassette further comprises a nucleic acid encoding a chimeric receptor and / or a nucleic acid encoding a cell surface receptor. In some embodiments, the nucleic acid encoding the FOXP3 protein and the nucleic acid encoding the chimeric receptor and / or the nucleic acid encoding the cell surface receptor are operably linked to the same promoter. In some embodiments, the nucleic acid encoding the FOXP3 protein and the nucleic acid encoding the chimeric receptor and / or the nucleic acid encoding the cell surface receptor are each separated by an IRES, a nucleic acid encoding a self-cleaving peptide (e.g., P2A peptide), or a nucleic acid encoding a peptide that causes ribosome skipping. Thus, in some embodiments, there is provided a method of treating an immune-related disorder comprising administering a Treg comprising an exogenous expression cassette encoding: (I) a promoter; (II) a nucleic acid encoding a FOXP3 protein; (III) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleavingpeptide or a peptide that causes ribosome skipping; (IV) a nucleic acid encoding a cell surface receptor;; or (I) a promoter; (II) a nucleic acid encoding a FOXP3 protein; (III) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; (IV) a nucleic acid encoding a chimeric receptor; (IV) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; and (VI) a nucleic acid encoding a cell surface receptor; wherein the order of FOXP3 protein, chimeric receptor, and / or cell surface receptor from 5’ to 3’ is interchangeable and wherein each is separated from the other by a self-cleaving peptide (e.g., P2A). In some embodiments, the nucleic acid encoding a FOXP3 protein is FOXP3 cDNA. In some embodiments, the cell is an immune cell. In some embodiments, the cell is a lymphocyte. In some embodiments, the cell is a T cell. In some embodiment, the cell is a Treg.

[0196] In some embodiments, there is provided herein a method of treating an immune- related disorder comprising administering an engineered Treg comprising an exogenous expression cassette comprising a modified nucleic acid encoding the FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein comprises intron sequences flanking the nucleic acid encoding exon 2, and a nucleic acid encoding a cell surface receptor. In some embodiments, the nucleic acid encoding the FOXP3 protein comprising intron sequences flanking the nucleic acid encoding exon 2 is alternatively spliced to produce a full-length FOXP3 protein isoform and a FOXP3 protein isoform lacking exon 2. In some embodiments, the nucleic acid encoding the FOXP3 protein and the nucleic acid encoding the cell surface receptor are operably linked to the same promoter. In some embodiments, the nucleic acid encoding the FOXP3 protein and the nucleic acid encoding the cell surface receptor are each separated by a nucleic acid encoding an IRES, a self-cleaving peptide (e.g., P2A peptide), or a peptide that causes ribosome skipping. Thus, in some embodiments, there is provided a method of treating an immune-related disorder comprising administering a Treg comprising an exogenous expression cassette encoding from 5’ to 3’: (I) a promoter; (II) a modified nucleic acid encoding the FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein comprises intron sequences flanking the nucleic acid encoding exon 2; (III) an IRES, or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; and (IV) a nucleic acid encoding a cell surface receptor. In some embodiments, the nucleic acid encoding a FOXP3 protein is FOXP3 cDNA. In some embodiments, the cell is an immune cell. In some embodiments, the cell is a lymphocyte. In some embodiments, the cell is a T cell. In some embodiment, the cell is a Treg.

[0197] In some embodiments, there is provided herein a method of treating an immune- related disorder comprising administering an engineered Treg comprising an exogenous expression cassette comprising a nucleic acid encoding a cell surface receptor, and a modified nucleic acid encoding the FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein comprises intron sequences flanking the nucleic acid encoding exon 2. In some embodiments, the nucleic acid encoding the FOXP3 protein comprising intron sequences flanking the nucleic acid encoding exon 2 is alternatively spliced to produce a full-length FOXP3 protein isoform and a FOXP3 protein isoform lacking exon 2. In some embodiments, the nucleic acid encoding the FOXP3 protein and the nucleic acid encoding the cell surface receptor are operably linked to the same promoter. In some embodiments, the nucleic acid encoding the FOXP3 protein and the nucleic acid encoding the cell surface receptor are each separated by a nucleic acid encoding an IRES, a self-cleaving peptide (e.g., P2A peptide), or a peptide that causes ribosome skipping. Thus, in some embodiments, there is provided a method of treating an immune-related disorder comprising administering a Treg comprising an exogenous expression cassette encoding from 5’ to 3’: (I) a promoter; (II) a nucleic acid encoding a cell surface receptor; (III) an IRES, or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; and (IV) a modified nucleic acid encoding the FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein comprises intron sequences flanking the nucleic acid encoding exon 2. In some embodiments, the nucleic acid encoding a FOXP3 protein is FOXP3 cDNA. In some embodiments, the cell is an immune cell. In some embodiments, the cell is a lymphocyte. In some embodiments, the cell is a T cell. In some embodiment, the cell is a Treg.

[0198] In some embodiments, there is provided herein a method of treating an immune- related disorder comprising administering an engineered Treg comprising an exogenous expression cassette comprising a nucleic acid encoding a chimeric receptor, a nucleic acid encoding a cell surface receptor, and a modified nucleic acid encoding a FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein comprises intron sequences flanking the nucleic acid encoding exon 2. In some embodiments, the nucleic acid encoding the FOXP3 protein comprising intron sequences flanking the nucleic acid encoding exon 2 is alternatively spliced to produce a full-length FOXP3 protein isoform and a FOXP3 protein isoform lacking exon 2. In some embodiments, the nucleic acid encoding the FOXP3 protein, the nucleic acid encoding the chimeric receptor, and the nucleic acid encoding the cell surface receptor are operably linked to the same promoter. In some embodiments, the nucleic acid encoding the FOXP3 protein, the nucleic acid encoding the chimeric receptor, and the nucleic acid encodingthe cell surface receptor are each separated by an IRES, a nucleic acid encoding a self-cleaving peptide (e.g., P2A peptide), or a nucleic acid encoding a peptide that causes ribosome skipping. Thus, in some embodiments, there is provided a method of treating an immune-related disorder comprising administering a Treg comprising an exogenous expression cassette encoding from 5’ to 3’: (I) a promoter; (II) a nucleic acid encoding a chimeric receptor; (III) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; (IV) a nucleic acid encoding a cell surface receptor; (V) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; and (VI) a modified nucleic acid encoding a FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein comprises intron sequences flanking the nucleic acid encoding exon 2. In some embodiments, the nucleic acid encoding a FOXP3 protein is FOXP3 cDNA. In some embodiments, the cell is an immune cell. In some embodiments, the cell is a lymphocyte. In some embodiments, the cell is a T cell. In some embodiment, the cell is a Treg.

[0199] In some embodiments, there is provided herein a method of treating an immune- related disorder comprising administering an engineered Treg comprising an exogenous expression cassette comprising a nucleic acid encoding a chimeric receptor, a modified nucleic acid encoding a FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein comprises intron sequences flanking the nucleic acid encoding exon 2, and a nucleic acid encoding a cell surface receptor. In some embodiments, the nucleic acid encoding the FOXP3 protein comprising intron sequences flanking the nucleic acid encoding exon 2 is alternatively spliced to produce a full-length FOXP3 protein isoform and a FOXP3 protein isoform lacking exon 2. In some embodiments, the nucleic acid encoding the FOXP3 protein, the nucleic acid encoding the chimeric receptor, and the nucleic acid encoding the cell surface receptor are operably linked to the same promoter. In some embodiments, the nucleic acid encoding the FOXP3 protein, the nucleic acid encoding the chimeric receptor, and the nucleic acid encoding the cell surface receptor are each separated by an IRES, a nucleic acid encoding a self-cleaving peptide (e.g., P2A peptide), or a nucleic acid encoding a peptide that causes ribosome skipping. Thus, in some embodiments, there is provided a method of treating an immune-related disorder comprising administering a Treg comprising an exogenous expression cassette encoding from 5’ to 3’: (I) a promoter; (II) a nucleic acid encoding a chimeric receptor; (III) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; (IV) a modified nucleic acid encoding a FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein comprises intron sequences flanking the nucleic acid encodingexon 2; (V) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; and (VI) a nucleic acid encoding a cell surface receptor. In some embodiments, the nucleic acid encoding a FOXP3 protein is FOXP3 cDNA. In some embodiments, the cell is an immune cell. In some embodiments, the cell is a lymphocyte. In some embodiments, the cell is a T cell. In some embodiment, the cell is a Treg.

[0200] In some embodiments, there is provided herein a method of treating an immune- related disorder comprising administering an engineered Treg comprising an exogenous expression cassette comprising a modified nucleic acid encoding a FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein comprises intron sequences flanking the nucleic acid encoding exon 2, a nucleic acid encoding a chimeric receptor, and a nucleic acid encoding a cell surface receptor. In some embodiments, the nucleic acid encoding the FOXP3 protein comprising intron sequences flanking the nucleic acid encoding exon 2 is alternatively spliced to produce a full-length FOXP3 protein isoform and a FOXP3 protein isoform lacking exon 2. In some embodiments, the nucleic acid encoding the FOXP3 protein, the nucleic acid encoding the chimeric receptor, and the nucleic acid encoding the cell surface receptor are operably linked to the same promoter. In some embodiments, the nucleic acid encoding the FOXP3 protein, the nucleic acid encoding the chimeric receptor, and the nucleic acid encoding the cell surface receptor are each separated by an IRES, a nucleic acid encoding a self-cleaving peptide (e.g., P2A peptide), or a nucleic acid encoding a peptide that causes ribosome skipping. Thus, in some embodiments, there is provided a method of treating an immune-related disorder comprising administering a Treg comprising an exogenous expression cassette encoding from 5’ to 3’: (I) a promoter; (II) a modified nucleic acid encoding a FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein comprises intron sequences flanking the nucleic acid encoding exon 2; (III) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; (IV) a nucleic acid encoding a chimeric receptor; (V) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; and (VI) a nucleic acid encoding a cell surface receptor. In some embodiments, the nucleic acid encoding a FOXP3 protein is FOXP3 cDNA. In some embodiments, the cell is an immune cell. In some embodiments, the cell is a lymphocyte. In some embodiments, the cell is a T cell. In some embodiment, the cell is a Treg.

[0201] In some embodiments, there is provided herein a method of treating an immune- related disorder comprising administering an engineered Treg comprising an exogenous expression cassette comprising a nucleic acid encoding a cell surface receptor, and a modifiednucleic acid encoding the FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein comprises intron sequences flanking the nucleic acid encoding exon 2. In some embodiments, the nucleic acid encoding the FOXP3 protein comprising intron sequences flanking the nucleic acid encoding exon 2 is alternatively spliced to produce a full-length FOXP3 protein isoform and a FOXP3 protein isoform lacking exon 2. In some embodiments, the nucleic acid encoding the FOXP3 protein and the nucleic acid encoding the cell surface receptor are operably linked to the same promoter. In some embodiments, the nucleic acid encoding the FOXP3 protein and the nucleic acid encoding the cell surface receptor are each separated by a nucleic acid encoding an IRES, a self-cleaving peptide (e.g., P2A peptide), or a peptide that causes ribosome skipping. Thus, in some embodiments, there is provided a method of treating an immune-related disorder comprising administering a Treg comprising an exogenous expression cassette encoding from 5’ to 3’: (I) a promoter; (II) a nucleic acid encoding a cell surface receptor; (III) an IRES, or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; and (IV) a modified nucleic acid encoding the FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein comprises intron sequences flanking the nucleic acid encoding exon 2. In some embodiments, the nucleic acid encoding a FOXP3 protein is FOXP3 cDNA. In some embodiments, the cell is an immune cell. In some embodiments, the cell is a lymphocyte. In some embodiments, the cell is a T cell. In some embodiment, the cell is a Treg.

[0202] In some embodiments, there is provided herein a method of treating an immune- related disorder comprising administering an engineered Treg comprising an exogenous expression cassette comprising a nucleic acid encoding a FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein does not comprise a nucleic acid sequence encoding exon 2, and a nucleic acid encoding a cell surface receptor. In some embodiments, the nucleic acid encoding the FOXP3 protein and the nucleic acid encoding the cell surface receptor are operably linked to the same promoter. In some embodiments, the nucleic acid encoding the FOXP3 protein and the nucleic acid encoding the cell surface receptor are each separated by an IRES, a nucleic acid encoding a self-cleaving peptide (e.g. , P2A peptide), or a nucleic acid encoding a peptide that causes ribosome skipping. Thus, in some embodiments, there is provided a method of treating an immune-related disorder comprising administering a Treg comprising an exogenous expression cassette encoding from 5’ to 3’: (I) a promoter; (II) a nucleic acid encoding a FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein does not comprise a nucleic acid sequence encoding exon 2; (III) an IRES, or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; and (IV) a nucleic acidencoding a cell surface receptor. In some embodiments, the nucleic acid encoding a FOXP3 protein is FOXP3 cDNA. In some embodiments, the cell is an immune cell. In some embodiments, the cell is a lymphocyte. In some embodiments, the cell is a T cell. In some embodiment, the cell is a Treg.

[0203] In some embodiments, the method of treating an immune-related disorder comprises administering a Treg comprising. In some embodiments, the method comprises transducing the Treg with a vector encoding any one of the exogenous expression cassettes described herein. In some embodiments, the cells are autologous to the individual to be treated. In some embodiments, the nucleic acid encoding a FOXP3 protein is FOXP3 cDNA. In some embodiments, the cell is an immune cell. In some embodiments, the cell is a lymphocyte. In some embodiments, the cell is a T cell. In some embodiment, the cell is a Treg.

[0204] In some embodiments, the expression cassette of the present disclosure is expressed in a T cell, e.g., a Treg. In some embodiments, one or more expression cassettes are expressed in a T cell, e.g., a Treg. In some embodiments, the T cell is a regulatory T cell (Treg). In some embodiments, the Treg is CD4+, CD25+, and CD1271ow. In some embodiments, the Treg expresses endogenous FOXP3. In some embodiments, the expression cassette is expressed in a Treg cell.

[0205] Immune conditions, diseases, disorders and reactions or responses to be treated according to the methods and compositions of the invention means a disease in which the immune system contributes to pathogenesis or can be part of the treatment. These reactions include, but are not limited to, cancers, inflammation, autoimmune conditions, disorders or diseases and persistent and progressive immune reactions to infectious non- self-antigens from bacterial, viral e.g., HCV), fungal, or parasitic organisms which invade and persist within mammals and humans. Such conditions and disorders include allergies and / or asthma. The allergies and asthma may be due to sensitization with foreign or non-self-antigens as pollen, animal dander and food proteins. The source of the provoking foreign antigen can be plant, fungal, mold, or other environmental contaminants.

[0206] Autoimmunity is defined as persistent and progressive immune reactions to noninfectious self-antigens, as distinct from infectious non-self-antigens from bacterial, viral, fungal, or parasitic organisms which invade and persist within mammals and humans. Autoimmune conditions include graft-versus-host disease, autoimmune polyendocrinopathy syndromes, Type I diabetes mellitus (TIDM), autoimmune gastritis, autoimmune uveoretinitis, autoimmune vasculitis, colitis, thyroiditis, Addison's disease, agammaglobulinemia, alopeciaareata, amyloidosis, ankylosing spondylitis, anti-GBM / anti-TBM nephritis, antiphospholipid syndrome, autoimmune hepatitis, autoimmune inner ear disease, axonal and neuronal neuropathy, Behqet's disease, bullous pemphigoid, Castleman disease, celiac disease, Chagas disease, chronic inflammatory demyelinating polyneuropathy(CIDP), chronic recurrent multifocal osteomyelitis, Churg-Strauss, cicatrical pemphigold / benign mucosal pemphigoid, Cogan's syndrome, cold agglutinin disease, congenital heart block, coxsackie myocarditis, CREST syndrome, Crohn's disease, dermatitis herpetiformis, dermatomyositis, Devic's disease, discoid lupus, Dressier's syndrome, endometriosis, eosinophilic esophagitis, eosinophilic fasciitis, erythema nodosum, essential mixed cryoglobulinemia, Evans syndrome, fibromyalgia, fibrosing alveolitits, giant cell arteritis (temporal arteritis), giant cell myocarditis, glomerulonephritis, Goodpasture's syndrome, granulomatosis with polyangitis, Grave's disease, Guillian-Barre syndrome, Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schonnlien purpura, herpes gestationis or pemphigoid gestationis, hypogammaglobulinemia, IgA nephropathy, IgG4-related sclerosing disease, inclusion body myositis, interstitial cystitis, juvenile arthritis, juvenile diabetes (Typeldiabetes), juvenile myositis, Kawasaki disease, Lambert-Eaton syndrome, leukocyteclastic vasculitis, lichen planus, lichen sclerosis, ligneous conjunctivitis, linear IgA disease, lupus, Lyme disease, chronic Meniere's disease, microscopic polyangiitis, mixed connective tissue disease, Mooren's ulcer, Mucha-Habermann disease, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neuromyelitis opica, neutropenia, ocular cicatrical pemphigoid, optic neuritis, palindromic rheumatism, PANDAS (pediatric autoimmune neuropsychiatric disorders associated with streptococcus), paraneoplastic cerebellar degeneration, paroxysmal nocturnal hemoglobinuria, Parry Romber syndrome, pars planitis (peripheral uveitis), Parsonnage-Tumer syndrome, pemphigus, peripheral neuropathy, perivenous encephalomyelitis, pernicious anemia, POEMS syndrome, polyarteritis nodosa, polymyalgia rheumatic, polymyositis, postmycoardial infarction syndrome, postpericadiotomy syndrome, primary biliary cirrhosis, primary sclerosing cholangitis, progesterone dermatitis, psoriasis, psoriatic arthritis, pure red cell aplasia, pyoderma gangrenosum, Raynaud's phenomenon, reactive arthritis, reflex sympathetic dystrophy, Reiter's syndrome, relapsing polychondritis, restless leg syndrome, retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt syndrome, scleritis, scleroderma, Sjogren's syndrome, sperm and testicular autoimmunity, stiff person syndrome, subacute bacterial endocarditis, Susac's syndrome, sympathetic ophtalmia, Takayasu's arteritis, temporal arteritis / giant cell arteritis, thrombocytopenic purpura, Tolosa-Hunt syndrome, transverse myelitis, ulcerative colitis,undifferentiated connective tissue disease, uveitis, vasculitis, vitiligo, and Wegner's granulomatosis (granulomatosis with polyangitis). “Autoantigen” or “self-antigen” as used herein refers to an antigen or epitope which is native to the mammal, and which is immunogenic in said mammalian disease. One aspect of the present application provides a method of treating an immune-related disorder.

[0207] The cells may be “allogeneic cells” (allogenicity), which are those isolated from one individual (the donor) and infused into another; whereas “autologous cells” (autology) refer to those cells that are isolated and infused back into the same individual. In some embodiments, the cells are autologous to the individual. In some embodiments, the individual is a human. In some embodiments, the cells are T cells. In some embodiments, the cells are Tregs. In some embodiments, the Treg cells are isolated from human peripheral blood mononuclear cells (PBMCs). In some embodiments, the Treg cells are modified ex vivo with a vector encoding one or more expression cassettes. In some embodiments, the Treg cells are modified with a vector comprising the expression cassette. In some embodiments, the Treg cells are modified to express exogenous FOXP3. In some embodiments, the Treg is CD4+, CD25+, and CD1271ow. In some embodiments, the Treg expresses endogenous FOXP3. In some embodiments, the Treg cells are administered to the same individual to treat an immune-related disorder.

[0208] In some embodiments, the method of treating an immune-related disorder comprises administering a Treg to an individual in need thereof. In some embodiments, the Treg is CD4+, CD25+, and CD1271ow. In some embodiments, the Treg expresses endogenous FOXP3.

[0209] In some embodiments, the method of treating an immune-related disorder comprises administering Tregs that are autologous to the individual. In some embodiments, the individual is a human. In some embodiments, the method of treating an immune-related disorder comprises administering Tregs that are isolated from human peripheral blood mononuclear cells (PBMCs). In some embodiments, the method of treating an immune-related disorder comprises administering Treg cells that are modified with a vector comprising the expression cassettes. In some embodiments, the method of treating an immune-related disorder comprises administering Tregs that are modified ex vivo with a vector comprising one or more expression cassettes. In some embodiments, the method of treating an immune-related disorder comprises administering Tregs that are modified to express exogenous FOXP3. In some embodiments, the method of treating an immune-related disorder comprises administering Tregs that are modified to overexpress FOXP3. In some embodiments, the method of treating an immune-related disorder comprises administering the Tregs that are CD4+, CD25+, and CD1271ow. In someembodiments, the method of treating an immune-related disorder comprises administering the Tregs that express endogenous FOXP3.

[0210] In some embodiments, the method of treating an immune-related disorder comprises administering the Tregs comprising the expression cassettes of the present disclosure. In some embodiments, the suppressive activity of the Tregs on effector T cells is increased. In some embodiments, the suppressive activity of the Tregs decreases the production of CD4+ and / or CD8+ T cell-specific cytokines (e.g., IL-2, IFNy, TNFa). In some embodiments, the Tregs display increased production of suppressive cytokines (e.g., IL- 10).

[0211] In some embodiments, the method of treating an immune-related disorder comprises transducing a population of cells with an expression cassette. In some embodiments, the method of treating an immune-related disorder comprises transducing a population of T cells with an expression cassette.. In some embodiments, the population of Treg cells transduced with the expression cassette persist in vivo for at least about any of 1, 2, 3, 5, 6, 8, 10, or 11 months, or at least a year or more.

[0212] In some embodiments, the method of treating an immune-related disorder comprises admini...

Claims

CLAIMS1. An engineered regulatory T cell (Treg) comprising an exogenous expression cassette comprising a modified nucleic acid encoding a FOXP3 protein operably linked to a promoter, wherein the nucleic acid encoding the FOXP3 protein is modified to produce a full-length FOXP3 protein isoform and an alternatively spliced isoform lacking one or more exons.

2. The engineered Treg of claim 1, wherein the nucleic acid encoding the FOXP3 protein comprises intron sequences flanking nucleic acid encoding exon 2.

3. The engineered Treg of claim 2, wherein the nucleic acid encoding the FOXP3 protein is alternatively spliced to produce a full-length FOXP3 protein isoform and a FOXP3 protein isoform lacking exon 2.

4. The engineered Treg of any one of claims 1-3, further comprising a nucleic acid encoding a chimeric receptor.

5. The engineered Treg of claim 4, wherein the exogenous expression cassette comprises the nucleic acid encoding the chimeric receptor.

6. The engineered Treg of claim 5, wherein the nucleic acid encoding the FOXP3 protein and the nucleic acid encoding the chimeric receptor are operably linked to the same promoter.

7. The engineered Treg of claim 6, wherein the exogenous expression cassette comprises an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping between the nucleic acid encoding the FOXP3 protein and the nucleic acid encoding the chimeric receptor.

8. The engineered Treg of any one of claims 4-7, wherein the nucleic acid encoding the FOXP3 protein is 5’ or 3’ of the nucleic acid encoding the chimeric receptor.

9. The engineered Treg of any one of claims 1-8, wherein the exogenous expression cassette further comprises a nucleic acid encoding a cell surface receptor.

10. The engineered Treg of claim 9, wherein the nucleic acid encoding the cell surface receptor is located 5’ or 3’ of the nucleic acid encoding the FOXP3 protein.

11. The engineered Treg of claim 9 or claim 10, wherein the exogenous expression cassette comprises an IRES, or a nucleic acid encoding a self-cleaving peptide or a peptide thatcauses ribosome skipping between the nucleic acid encoding the cell surface receptor and the nucleic acid encoding the FOXP3 protein.

12. The engineered Treg of any one of claims 9-11, wherein the exogenous expression cassette comprises an IRES, or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping between the nucleic acid encoding the cell surface receptor and the nucleic acid encoding the chimeric receptor.

13. The engineered Treg of any one of claims 1-3 and 9-12, wherein the exogenous expression cassette comprises from 5’ to 3’: a) the promoter; b) the modified nucleic acid encoding the FOXP3 protein, optionally linked to a detection tag, wherein the nucleic acid encoding the FOXP3 protein comprises intron sequences flanking the nucleic acid encoding exon 2; c) an IRES, or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; and d) the nucleic acid encoding the cell surface receptor.

14. The engineered Treg cell of any one of claims 1-3 and 9-12, comprising from 5’ to 3’: a) the promoter; b) the nucleic acid encoding the cell surface receptor; c) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; and d) the modified nucleic acid encoding the FOXP3 protein, wherein the modified nucleic acid encoding the FOXP3 protein comprises intron sequences flanking the nucleic acid encoding exon 2.

15. The engineered Treg of any one of claims 1-12, wherein the exogenous expression cassette comprises from 5’ to 3’: a) the promoter; b) the nucleic acid encoding the chimeric receptor; c) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; d) the nucleic acid encoding the cell surface receptor;e) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; and f) the modified nucleic acid encoding the FOXP3 protein, wherein the nucleic acid encoding a FOXP3 protein comprises intron sequences flanking the nucleic acid encoding exon 2.

16. The engineered Treg of any one of claims 1-12, wherein the exogenous expression cassette comprises from 5’ to 3’: a) the promoter; b) the nucleic acid encoding the chimeric receptor; c) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; d) the modified nucleic acid encoding the FOXP3 protein, wherein the nucleic acid encoding a FOXP3 protein comprises intron sequences flanking the nucleic acid encoding exon 2; e) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; and f) the nucleic acid encoding the cell surface receptor.

17. The engineered Treg of any one of claims 1-12, wherein the exogenous expression cassette comprises from 5’ to 3’: a) the promoter; b) the modified nucleic acid encoding the FOXP3 protein, wherein the nucleic acid encoding a FOXP3 protein comprises intron sequences flanking the nucleic acid encoding exon 2; c) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; d) the nucleic acid encoding the chimeric receptor; e) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; and f) the nucleic acid encoding the cell surface receptor.

18. An engineered Treg comprising an exogenous expression cassette comprising from 5’ to 3’: a) a promoter;b) a nucleic acid encoding a chimeric receptor; c) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; d) a nucleic acid encoding a FOXP3 protein, wherein the nucleic acid encoding a FOXP3 protein is FOXP3 cDNA; e) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; and f) a nucleic acid encoding a cell surface receptor.

19. An engineered Treg comprising an exogenous expression cassette comprising from 5’ to 3’: a) a promoter; b) a nucleic acid encoding a FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein is FOXP3 cDNA; c) an IRES, or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; and d) a nucleic acid encoding a cell surface receptor.

20. An engineered Treg comprising an exogenous expression cassette comprising from 5’ to 3’: a) a promoter; b) nucleic acid encoding a cell surface receptor; c) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; and d) a nucleic acid encoding a FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein is FOXP3 cDNA.

21. An engineered Treg comprising an exogenous expression cassette comprising from 5’ to 3’: a) a promoter; b) a nucleic acid encoding a FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein is FOXP3 cDNA; c) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; d) a nucleic acid encoding a chimeric receptor;e) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; and f) a nucleic acid encoding a cell surface receptor.

22. An engineered Treg comprising an exogenous expression cassette comprising from 5’ to 3’: a) a promoter; b) a nucleic acid encoding a chimeric receptor; c) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; d) a nucleic acid encoding a cell surface receptor; e) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; and f) a nucleic acid encoding a FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein is FOXP3 cDNA.

23. An engineered Treg comprising an exogenous expression cassette comprising from 5’ to 3’: a) a promoter; b) a nucleic acid encoding a FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein does not comprise a nucleic acid sequence encoding exon 2; c) an IRES, or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; and d) a nucleic acid encoding a cell surface receptor.

24. The engineered Treg of any one of claims 1-17, wherein the nucleic acid encoding the FOXP3 protein does not contain intron sequences other than the intron sequences flanking exon 2.

25. The engineered Treg of any one of claims 9-24, wherein the cell surface receptor lacks a functional intracellular signaling domain or is not capable of mediating intracellular signaling.

26. The engineered Treg of any one of claims 9-25, wherein the cell surface receptor is selected from the group consisting of EGFR, HER2, CD2, H-2Kk, IL-5Ra, and derivatives or mutants thereof.

27. The engineered Treg of any one of claims 4-26, wherein the chimeric receptor comprises a chimeric antigen receptor, a T cell Receptor, or a PiG receptor.

28. The engineered Treg of any one of claims 1-27, wherein the chimeric receptor binds to an antigen selected from the group consisting of MBP, MOG, PLP, PDE4, VEGFR, S1OO-P, Citrullinated vimentin (CV), TNF-like ligand 1A (TL1A), CD2, CD23 (IgE receptor), IL-6R, IL-4Ra, IL-12R, IL-13Ral, IL-13Ra2, IL-15R, IL-23R, CD19, CD20, CD22, CD3, CD4, CD5, IFN-y, IgE, IL-la, IL-ip, IL-6, IL-12, IL-23, IL-15, IL-17, IL-17A, IL-22, IL-13, IL-33, TSLP, OX-40, OX-40L, p53, CD38, CD40L, CD28, ICOS, RIPK1, BTK, Bet v 1, GLP2R, JAK1, C5, and TNF-a.

29. The engineered Treg of any one of claims 11-28, wherein the nucleic acid encoding the self-cleaving peptide is selected from the group consisting of a P2A, T2A, E2A, and F2A sequence.

30. The engineered Treg of any one of claims 1-29, wherein the promoter is an inducible or a constitutive promoter.

31. The engineered Treg of any one of claims 1-30, wherein the promoter is a cellspecific promoter.

32. The engineered Treg of claim 31, wherein the promoter is specific for T cells.

33. The engineered Treg of any one of claims 1-32, wherein the engineered Treg expresses endogenous FOXP3 protein.

34. The engineered Treg of any one of claims 1-33, wherein the endogenous FOXP3 protein does not comprise a detection tag.

35. The engineered Treg of any one of claims 1-34, wherein the exogenous expression cassette is in a vector.

36. The engineered Treg of claim 35, wherein the vector is a viral vector.

37. The engineered Treg of claim 36, wherein the vector is a lentiviral vector.

38. The engineered Treg of any one of claims 1-37, wherein the exogenous expression cassette is in a chromosome of the Treg.

39. The engineered Treg of any one of claims 1-38, wherein the engineered Treg expresses a higher level of F0XP3 protein compared to a Treg lacking the exogenous expression cassette.

40. An expression cassette comprising a nucleic acid encoding a FOXP3 protein and a cell surface receptor, wherein the nucleic acid encoding the FOXP3 protein is modified to produce a full-length FOXP3 protein isoform and an alternatively spliced isoform lacking one or more exons.

41. The expression cassette of claim 40, wherein the nucleic acid encoding the FOXP3 protein comprises intron sequences flanking the nucleic acid encoding exon 2.

42. The expression cassette of claim 40 or claim 41, further comprising a nucleic acid encoding a chimeric receptor.

43. The expression cassette of claim 42, wherein the nucleic acid encoding the FOXP3 protein and the nucleic acid encoding the chimeric receptor are operably linked to the same promoter.

44. The expression cassette of claim 43, comprising an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping between the nucleic acid encoding the FOXP3 protein and the nucleic acid encoding the chimeric receptor.

45. The expression cassette of any one of claims 40-44, further comprising a nucleic acid encoding a cell surface receptor.

46. The expression cassette of claim 45, wherein the expression cassette comprises an IRES, or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping between the nucleic acid encoding the cell surface receptor and the nucleic acid encoding the FOXP3 protein.

47. The expression cassette of claim 45 or 46, wherein the expression cassette comprises an IRES, or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping between the nucleic acid encoding the cell surface receptor and the nucleic acid encoding the chimeric receptor.

48. The expression cassette of any one of claims 40, 41, 45, and 46, comprising from 5’ to 3’:a) the promoter; b) the modified nucleic acid encoding the FOXP3 protein, optionally linked to a detection tag, wherein the nucleic acid encoding the FOXP3 protein comprises intron sequences flanking the nucleic acid encoding exon 2; c) an IRES, or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; and d) the nucleic acid encoding the cell surface receptor.

49. The expression cassette of any one of claims 40, 41, 45, and 46, comprising from 5’ to 3’: a) the promoter; b) the nucleic acid encoding the cell surface receptor; c) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; and d) the modified nucleic acid encoding the FOXP3 protein, wherein the modified nucleic acid encoding the FOXP3 protein comprises intron sequences flanking the nucleic acid encoding exon 2.

50. The expression cassette of any one of claims 40-47, comprising from 5’ to 3’: a) the promoter; b) the nucleic acid encoding the chimeric receptor; c) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; d) the nucleic acid encoding the cell surface receptor; e) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; and f) the modified nucleic acid encoding the FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein comprises intron sequences flanking the nucleic acid encoding exon 2.

51. The expression cassette of any one of claims 40-47, comprising from 5’ to 3’: a) the promoter; b) the nucleic acid encoding the chimeric receptor; c) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping;d) the modified nucleic acid encoding the FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein comprises intron sequences flanking the nucleic acid encoding exon 2; e) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; and f) the nucleic acid encoding the cell surface receptor.

52. The expression cassette of any one of claims 40-47, comprising from 5’ to 3’: a) the promoter; b) the modified nucleic acid encoding the FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein comprises intron sequences flanking the nucleic acid encoding exon 2; c) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; d) the nucleic acid encoding the chimeric receptor; e) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; and f) the nucleic acid encoding the cell surface receptor.

53. An expression cassette comprising from 5’ to 3’ : a) a promoter; b) a nucleic acid encoding a chimeric receptor; c) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; d) a nucleic acid encoding a FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein is FOXP3 cDNA; e) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; and f) a nucleic acid encoding a cell surface receptor.

54. An expression cassette comprising from 5’ to 3’ : a) a promoter; b) a nucleic acid encoding a FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein is FOXP3 cDNA;c) an IRES, or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; and d) a nucleic acid encoding a cell surface receptor.

55. An expression cassette comprising from 5’ to 3’ : a) a promoter; b) a nucleic acid encoding a cell surface receptor; c) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; and d) a nucleic acid encoding a FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein is FOXP3 cDNA.

56. An expression cassette comprising from 5’ to 3’ : a) a promoter; b) a nucleic acid encoding a FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein is FOXP3 cDNA; c) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; d) a nucleic acid encoding a chimeric receptor; e) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; and f) a nucleic acid encoding a cell surface receptor.

57. An expression cassette comprising from 5’ to 3’ : a) a promoter; b) a nucleic acid encoding a chimeric receptor; c) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; d) a nucleic acid encoding a cell surface receptor; e) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; and f) a nucleic acid encoding a FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein is FOXP3 cDNA.

58. An expression cassette comprising from 5’ to 3’: a) a promoter;b) a nucleic acid encoding a FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein does not comprise a nucleic acid sequence encoding exon 2; c) an IRES, or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping; and d) a nucleic acid encoding a cell surface receptor.

59. The expression cassette of any one of claims 40-52, wherein the nucleic acid encoding the FOXP3 protein does not contain intron sequences other than the intron sequences flanking exon 2.

60. The expression cassette of any one of claims 45-59, wherein the cell surface receptor lacks a functional intracellular signaling domain or is not capable of mediating intracellular signaling, optionally wherein the cell surface receptor is selected from the group consisting of EGFR, HER2, CD2, H-2Kk, IL-5Ra, and derivatives or mutants thereof.

61. The expression cassette of any one of claims 42-47, 50-53, 56, 57, 59, and 60, wherein the chimeric receptor comprises a chimeric antigen receptor, a T cell Receptor, or a PiG receptor, optionally wherein the chimeric receptor binds to an antigen selected from the group consisting of MBP, MOG, PLP, PDE4, VEGFR, S100-P, Citrullinated vimentin (CV), TNF-like ligand 1A (TL1A), CD2, CD23 (IgE receptor), IL-6R, IL-4Ra, IL-12R, IL-13Ral, IL-13Ra2, IL-15R, IL-23R, CD19, CD20, CD22, CD3, CD4, CD5, IFN-y, IgE, IL-la, IL-ip, IL-6, IL-12, IL-23, IL- 15, IL- 17, IL-17A, IL-22, IL- 13, IL-33, TSLP, OX-40, OX-40L, p53, CD38, CD40L, CD28, ICOS, RIPK1, BTK, Bet v 1, GLP2R, JAK1, C5, and TNE-a.

62. The expression cassette of any one of claims 44-61, wherein the nucleic acid encoding the self-cleaving peptide is selected from the group consisting of a P2A, T2A, E2A, and E2A sequence.

63. The expression cassette of any one of claims 40-62, wherein the promoter is an inducible promoter or a constitutive promoter.

64. The expression cassette of any one of claims 40-63, wherein the promoter is a cellspecific promoter, optionally wherein the promoter is specific for T cells.

65. The expression cassette of any one of claims 40-64, wherein the expression cassette is in a vector, optionally, wherein the vector is a viral vector.

66. The expression cassette of any one of claims 40-64, wherein the expression cassette is integrated into a chromosome of a Treg.

67. A method of increasing persistence and / or cytokine signaling of a Treg, comprising providing the expression cassette of any one of claims 40-66 to the Treg, wherein the persistence and / or cytokine signaling of the Treg is increased compared to a Treg that does not comprise the expression cassette.

68. The method of claim 67, wherein the expression cassette is provided to the Treg by transducing the Treg with a vector comprising the expression cassette.

69. A method of treating an immune related disorder in an individual, comprising administering the Treg of any one of claims 1-39 to an individual in need thereof.

70. A method of generating a Treg, comprising transducing an effector T cell (Teff) with the expression cassette of any one of claims 40-66.

71. A method of overexpressing FOXP3 protein in a Treg, comprising transducing the Treg with the expression cassette of any one of claims 40-66.

72. The engineered Treg of any one of claims 1-39 or the expression cassette of any one of claims 40-66, wherein: a) the FOXP3 protein comprises the amino acid sequence of SEQ ID NO:20 or 22; b) the FOXP3 protein comprises the amino acid sequence of SEQ ID NO:21 or 23; c) the chimeric receptor comprises an antigen binding domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:73-76; d) the cell surface receptor comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 17-19; and / or e) the IRES, or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:2-10 and 89-91.

73. The method of claim 70 or claim 71, further comprising measuring the level of one or more markers in the engineered Treg and comparing the level of the one or more markers to a wildtype Treg or a control Treg lacking exogenous FOXP3 protein.

74. The engineered Treg of any one of claims 1-39, wherein: a) the engineered Treg expresses one or more markers that are expressed by a wildtype Treg or a control Treg lacking exogenous FOXP3 protein; and / or b) the engineered Treg does not express one or more markers that are not expressed by a wildtype Treg or a control Treg lacking exogenous FOXP3 protein.

75. The engineered Treg of claim 74, wherein the one or more markers is expressed at similar protein and / or gene levels as in a wildtype Treg or a control Treg lacking exogenous FOXP3 protein.

76. The method of claim 73 or the engineered Treg of claim 74 or claim 75, wherein the one or more markers comprises CD27, CD70, CD71, Helios, glucocorticoid-induced TNFR- related protein (GITR), latency-associated peptide (LAP), glycoprotein A repetitions predominant (GARP), CD137, and any combination thereof.

77. The method of any one of claims 67, 70, or 71, further comprising measuring the level of one or more cytokines produced by the engineered Treg and comparing the level to the level of the one or more cytokines produced by a wildtype Treg or a control Treg lacking exogenous FOXP3 protein.

78. The method of claim 77, wherein the one or more cytokines comprises TNF-a, IFN-y, IL- 10, IL-4, IL-2, IL-17A, any functional derivatives thereof, and any combination thereof.

79. The method of claim 78, wherein the one or more cytokines comprises at least IL- 10, and wherein the level of IL- 10 protein and / or gene expression is about the same or increased in the engineered Treg compared to the wildtype Treg or the control Treg lacking exogenous FOXP3 protein under non-inflammatory conditions.

80. The engineered Treg of any one of claims 1-39, 72, and 74-76, wherein the engineered Treg expresses a similar ratio of full-length FOXP3 protein to an alternatively spliced FOXP3 isoform lacking one or more exons as a wildtype Treg or a control Treg lacking exogenous FOXP3 protein.

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