Cell for ectopic expression of FOXP3

Abstract

Expression cassettes comprising FOXP3 proteins, chimeric receptors, and / or cell surface receptors are provided. Also provided herein are Treg cells comprising the expression cassettes and methods of use.

Description

Cross-references to related applications

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

[0002] Reference to the electronic sequence list The contents of the electronic sequence list (237752001040seqlist.xml; size: 95,606 bytes; and creation date: August 31, 2024) are incorporated herein by reference in their entirety. Background Technology

[0003] The immune system plays a crucial role in maintaining homeostasis, balancing the elimination of foreign antigens with the tolerance of autoantigens. Specifically, an overactive immune system can lead to various autoimmune disorders (e.g., irritable bowel syndrome, systemic lupus erythematosus, alopecia areata, multiple sclerosis), often resulting from an overactivity of effector T lymphocytes or an underactivity of regulatory T lymphocytes (Tregs). Current treatments for autoimmune diseases include the administration of steroids, which can cause serious side effects and typically provide little relief.

[0004] Regulatory T cells (Tregs) are key players in maintaining organismal homeostasis and preventing damage to other healthy tissues. Tregs are a unique subset of T cells that suppress the cytotoxic or pro-inflammatory activity of effector CD4+ or effector CD8+ T cells. Tregs differentiate from parental T lymphocyte lineages after upregulation of key Treg genes, particularly CD25 (i.e., IL-2Rα) and FOXP3 (see, for example, 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, which is incorporated herein by reference in its entirety. These Tregs are responsible for the production of cytokines (e.g., TGF-β and IL-10) after T cell receptor (TCR) activation (see, for example, Chen, J et al. (2019)). Trends Mol Med ; 25(11):1010-1023, which is incorporated herein by reference in its entirety) and binding to immune checkpoint receptors (e.g., TIGIT or CTLA-4 binding) (see, for example, Knochelmann, HM et al. (2018)). Cell Mol Immunol; 15(5):458-469, which is incorporated hereby by reference) to directly inhibit effector T cell activity.

[0005] FOXP3 is a key determinant of Treg lineage, stability, and function. FOXP3 acts as the master transcription factor driving the genetic programming of Tregs, primarily serving as a transcriptional repressor for key genes involved in T cell activation and effector functions, including the synthesis of proliferative and pro-inflammatory cytokines such as IL-2, IL-4, IL-17A, and IFNγ. FOXP3 is initially expressed during the initial expansion of T cells but is subsequently lost upon polyclonal stimulation. This contrasts with Tregs, where FOXP3 expression increases over time and is maintained. Maintaining the Treg immunophenotype and suppressive function will enhance the efficacy and safety of pharmaceutical products. Summary of the Invention

[0006] In one aspect, this application relates to exogenous expression cassettes comprising modified nucleic acids encoding a FOXP3 protein (e.g., full-length FOXP3 protein and / or delta(Δ)2 FOXP3 lacking the amino acid corresponding to exon 2). This document also provides cells (e.g., Treg or effector T cells) expressing the exogenous expression cassettes and methods of use, said exogenous expression cassettes comprising modified nucleic acids encoding a FOXP3 protein operatively linked to a promoter.

[0007] In one aspect, this document provides an engineered regulatory T cell (Treg) comprising an exogenous expression cassette containing a modified nucleic acid encoding a FOXP3 protein operatively linked to a promoter, wherein the nucleic acid encoding the FOXP3 protein is modified to produce a full-length FOXP3 protein isotype and an alternative splice isotype lacking one or more exons. In some embodiments, the nucleic acid encoding the FOXP3 protein includes an intron sequence flanking the 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 isotype and a FOXP3 protein isotype lacking exon 2.

[0008] In some embodiments of any engineered Treg provided herein, the engineered Treg further comprises 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 operatively 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 jumping 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 located at the 5' or 3' of the nucleic acid encoding the chimeric receptor.

[0009] In some embodiments of any engineered Treg 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 at the 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 ribosomal jumping 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 ribosomal jumping between the nucleic acid encoding the cell surface receptor and the nucleic acid encoding the chimeric receptor.

[0010] In some embodiments of any engineered Treg provided herein, the exogenous expression cassette from 5' to 3' comprises: 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 contains an intron sequence flanking the nucleic acid encoding exon 2; c) an IRES or a nucleic acid encoding a self-cleaving peptide or a peptide that induces ribosome jumping; and d) the nucleic acid encoding the cell surface receptor.

[0011] In some embodiments of any engineered Treg provided herein, the exogenous expression cassette from 5' to 3' comprises: 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 induces ribosome jumping; and d) the modified nucleic acid encoding the FOXP3 protein, wherein the modified nucleic acid encoding the FOXP3 protein comprises an intron sequence flanking the nucleic acid encoding exon 2.

[0012] In some embodiments of any engineered Treg provided herein, the exogenous expression cassette from 5' to 3' comprises: 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 induces ribosome jumping; 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 induces ribosome jumping; and f) the modified nucleic acid encoding the FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein comprises an intron sequence flanking the nucleic acid encoding exon 2.

[0013] In some embodiments of any engineered Treg provided herein, the exogenous expression cassette from 5' to 3' comprises: 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 induces ribosome jumping; d) the modified nucleic acid encoding the FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein contains an intron sequence 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 induces ribosome jumping; and f) the nucleic acid encoding the cell surface receptor.

[0014] In some embodiments of any engineered Treg provided herein, the exogenous expression cassette from 5' to 3' comprises: a) the promoter; b) the modified nucleic acid encoding the FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein comprises an intron sequence 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 induces ribosome jumping; 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 induces ribosome jumping; and f) the nucleic acid encoding the cell surface receptor.

[0015] On the other hand, this paper provides 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 induces ribosome jumping; 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 induces ribosome jumping; and f) a nucleic acid encoding a cell surface receptor.

[0016] This document also provides an engineered Treg comprising an exogenous expression cassette, the 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 induces ribosome jumping; and d) a nucleic acid encoding a cell surface receptor.

[0017] This document also provides an engineered Treg comprising an exogenous 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 induces ribosome jumping; and d) a nucleic acid encoding a FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein is FOXP3 cDNA.

[0018] This document also provides an engineered Treg comprising an exogenous expression cassette, the 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 induces ribosome jumping; 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 induces ribosome jumping; and f) a nucleic acid encoding a cell surface receptor.

[0019] This document also provides 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 induces ribosome jumping; 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 induces ribosome jumping; and f) a nucleic acid encoding a FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein is FOXP3 cDNA.

[0020] This article also provides an engineered Treg comprising an exogenous expression cassette, the 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 contain a nucleic acid sequence encoding exon 2; c) an IRES or a nucleic acid encoding a self-cleaving peptide or a peptide that induces ribosome jumping; and d) a nucleic acid encoding a cell surface receptor.

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

[0022] In some embodiments of any engineered Treg provided herein, the cell surface receptor lacks a functional intracellular signal transduction domain or is unable to mediate intracellular signal transduction. In some embodiments, the cell surface receptor is selected from the group consisting of EGFR, HER2, CD2, H-2Kk, IL-5Rα, and their derivatives or mutants.

[0023] In some embodiments of any engineered Treg provided herein, the chimeric receptor includes 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-β, citrullinated vimentin (CV), TNF-like ligand 1A (TL1A), CD2, CD23 (IgE receptor), IL-6R, IL-4Rα, IL-12R, IL-13Rα1, IL-13Rα2, IL-15R, IL-23R, CD19, CD20, CD22, CD3, CD4, CD5, IFN-γ, IgE, IL-1α, IL-1β, 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-α.

[0024] In some embodiments of any engineered Treg provided herein, the nucleic acid encoding the self-cleaving peptide is selected from the group consisting of P2A, T2A, E2A, and F2A sequences.

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

[0026] In some embodiments of any engineered Treg provided herein, the engineered Treg expresses the endogenous FOXP3 protein. In some embodiments, the endogenous FOXP3 protein does not contain a detection tag.

[0027] In some embodiments of any engineered Treg 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 the chromosome of the Treg.

[0028] In some implementations of any engineered Treg provided herein, the engineered Treg expresses higher levels of FOXP3 protein compared to a Treg lacking the exogenous expression cassette.

[0029] In some embodiments of any engineered Treg provided herein, the engineered Treg expresses one or more biomarkers expressed by a wild-type Treg or a control Treg lacking exogenous FOXP3 protein; and / or the engineered Treg does not express one or more biomarkers not expressed by a wild-type Treg or a control Treg lacking exogenous FOXP3 protein. In some embodiments, the one or more biomarkers are expressed at protein and / or gene levels similar to those in wild-type Tregs or control Tregs lacking exogenous FOXP3 protein. In some embodiments, the engineered Treg expresses a similar ratio of full-length FOXP3 protein to that of an alternatively spliced ​​FOXP3 protein lacking one or more exons, similar to that expressed by a wild-type Treg or a control Treg lacking exogenous FOXP3 protein.

[0030] On the other hand, this document provides an expression cassette comprising 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 isotype and an alternative splicing isotype lacking one or more exons. In some embodiments, the nucleic acid encoding the FOXP3 protein includes an intron sequence flanking the nucleic acid encoding exon 2.

[0031] In some embodiments of any expression cassette 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 operatively 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 jumping between the nucleic acid encoding the FOXP3 protein and the nucleic acid encoding the chimeric receptor.

[0032] In some embodiments of any expression cassette 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 self-cleaving peptide or a peptide that causes ribosomal jumping 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 self-cleaving peptide or a peptide that causes ribosomal jumping between the nucleic acid encoding the cell surface receptor and the nucleic acid encoding the chimeric receptor.

[0033] In some embodiments of any expression cassette provided herein, the expression cassette from 5' to 3' comprises: 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 an intron sequence flanking the nucleic acid encoding exon 2; c) an IRES or a nucleic acid encoding a self-cleaving peptide or a peptide that induces ribosome jumping; and d) the nucleic acid encoding the cell surface receptor.

[0034] In some embodiments of any expression cassette provided herein, the expression cassette from 5' to 3' comprises: 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 jumping; and d) the modified nucleic acid encoding the FOXP3 protein, wherein the modified nucleic acid encoding the FOXP3 protein comprises an intron sequence flanking the nucleic acid encoding exon 2.

[0035] In some embodiments of any expression cassette provided herein, the expression cassette from 5' to 3' comprises: 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 induces ribosome jumping; 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 induces ribosome jumping; and f) the modified nucleic acid encoding the FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein comprises an intron sequence flanking the nucleic acid encoding exon 2.

[0036] In some embodiments of any expression cassette provided herein, the expression cassette from 5' to 3' comprises: 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 induces ribosome jumping; d) the modified nucleic acid encoding the FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein contains an intron sequence 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 induces ribosome jumping; and f) the nucleic acid encoding the cell surface receptor.

[0037] In some embodiments of any expression cassette provided herein, the expression cassette from 5' to 3' comprises: a) the promoter; b) the modified nucleic acid encoding the FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein comprises an intron sequence 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 induces ribosome jumping; 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 induces ribosome jumping; and f) the nucleic acid encoding the cell surface receptor.

[0038] On the other hand, this document provides 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 induces ribosome jumping; 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 induces ribosome jumping; and f) a nucleic acid encoding a cell surface receptor.

[0039] This document also provides 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 induces ribosome jumping; and d) a nucleic acid encoding a cell surface receptor.

[0040] This document also provides 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 induces ribosome jumping; and d) a nucleic acid encoding a FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein is FOXP3 cDNA.

[0041] This document also provides 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 induces ribosome jumping; 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 induces ribosome jumping; and f) a nucleic acid encoding a cell surface receptor.

[0042] This document also provides 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 induces ribosome jumping; 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 induces ribosome jumping; and f) a nucleic acid encoding a FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein is FOXP3 cDNA.

[0043] This document also provides 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 contain a nucleic acid sequence encoding exon 2; c) an IRES or a nucleic acid encoding a self-cleaving peptide or a peptide that induces ribosome jumping; and d) a nucleic acid encoding a cell surface receptor.

[0044] In some embodiments of any expression cassette provided herein, the nucleic acid encoding the FOXP3 protein does not contain intron sequences other than the intron sequence flanking exon 2. In some embodiments, the cell surface receptor lacks a functional intracellular signal transduction domain or is unable to mediate intracellular signal transduction, optionally wherein the cell surface receptor is selected from the group consisting of EGFR, HER2, CD2, H-2Kk, IL-5Rα, and their derivatives or mutants. 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, S100-β, citrullinated vimentin (CV), TNF-like ligand 1A (TL1A), CD2, CD23. (IgE receptor), IL-6R, IL-4Rα, IL-12R, IL-13Rα1, IL-13Rα2, IL-15R, IL-23R, CD19, CD20, CD22, CD3, CD4, CD5, IFN-γ, IgE, IL-1α, IL-1β, 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 v1, GLP2R, JAK1, C5, and TNF-α. In some embodiments, the nucleic acid encoding the self-cleaving peptide is selected from the group consisting of P2A, T2A, E2A, and F2A sequences. 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 to T cells.

[0045] In some embodiments of any expression cassette and / or engineered Treg provided herein, 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 NO: 73-76; d) the cell surface receptor comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 17-19; and / or e) the IRES or the nucleic acid encoding a self-cleaving peptide or a peptide that induces ribosome jumping comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2-10 and 89-91.

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

[0047] This document also provides methods for increasing the persistence and / or cytokine signaling of Tregs, comprising providing the Treg with any of the expression cassettes described herein, wherein the persistence and / or cytokine signaling of the Treg are increased compared to a Treg not containing the expression cassette. In some embodiments, the expression cassette is provided to the Treg by transducing the Treg with a vector containing the expression cassette. In some embodiments, the method further includes measuring the levels of one or more cytokines produced by the engineered Treg and comparing the levels of the one or more cytokines produced by a wild-type Treg or a control Treg lacking exogenous FOXP3 protein. In some embodiments, the one or more cytokines include TNF-α, IFN-γ, IL-10, IL-4, IL-2, IL-17A, any functional derivatives thereof, and any combination thereof. In some embodiments, the one or more cytokines include at least IL-10, and wherein, under non-inflammatory conditions, the levels of IL-10 protein and / or gene expression in the engineered Treg are substantially the same or increased compared to the wild-type Treg or the control Treg lacking exogenous FOXP3 protein.

[0048] This article also provides methods for treating individual immune-related conditions, which include administering any of the Tregs described herein to individuals in need.

[0049] This document also provides methods for generating Tregs, including transducing effector T cells (Teffs) using any of the expression cassettes described herein. This document also provides methods for overexpressing FOXP3 protein in Tregs, including transducing the Tregs using any of the expression cassettes described herein. In some embodiments, the methods described herein further include measuring the levels of one or more biomarkers in the engineered Tregs and comparing the levels of said one or more biomarkers to wild-type Tregs or control Tregs lacking exogenous FOXP3 protein. In some embodiments, said one or more biomarkers include CD27, CD70, CD71, Helios, glucocorticoid-induced TNFR-related protein (GITR), latent-related peptide (LAP), glycoprotein A repetitions predominant protein (GARP), CD137, and any combination thereof. In some embodiments, the methods described herein further include measuring the levels of one or more cytokines produced by said engineered Tregs and comparing said levels to the levels of said one or more cytokines produced by wild-type Tregs or control Tregs lacking exogenous FOXP3 protein. In some embodiments, the one or more cytokines include TNF-α, IFN-γ, IL-10, IL-4, IL-2, IL-17A, any functional derivatives thereof, and any combination thereof. In some embodiments, the one or more cytokines include at least IL-10, and wherein, under non-inflammatory conditions, the levels of IL-10 protein and / or gene expression in the engineered Treg are substantially the same or increased compared to the wild-type Treg or the control Treg lacking exogenous FOXP3 protein. Attached Figure Description

[0050] Figures 1A to 1E The structure of the FOXP3 gene and an exemplary polycistronic construct is shown. Figure 1A The function and gene structure of human FOXP3, its different splicing isotypes (full length (FL), exon 2 absence (Δ2), exon 2 and exon 7 absence (Δ2Δ7), and exon 7 absence (Δ7)), and the relative abundance of each isotype were described. Figure 1B The engineered FOXP3 construct that allows for variable splicing (FL+Δ2; test), the full-length FOXP3 isotype (FL; control), and the FOXP3 isotype lacking exon 2 (Δ2; control) were described. Figure 1CT cells transduced with a polycistronic transgenic expression vector carrying FOXP3 were described. This vector promotes ectopic expression of FOXP3 (ecFOXP3), which differs from the endogenous level of naturally expressed FOXP3 in T cells. The vector encodes an epitope-tagged engineered FOXP3 construct and an optional CAR molecule. Figure 1D An exemplary full-length polycistronic cassette that can be transduced into cells (e.g., T cells, such as Treg cells) is depicted. Figure 1E Exemplary polycistronic cassettes are depicted, comprising engineered FOXP3 constructs that allow for alternative splicing (FL+Δ2; test), full-length FOXP3 isotypes (FL; control), or chimeric antigen receptors without FOXP3 constructs (i.e., negative controls).

[0051] Figures 2A to 2C This paper describes a method for detecting ectopic FOXP3 expression after vector transduction into T cells to distinguish between endogenous and ectopic FOXP3. The vector encodes an epitope-tagged FOXP3 construct that can be detected using anti-HA (ectopic FOXP3 only) or anti-FOXP3 (endogenous and ectopic FOXP3) antibodies. Figure 2A Simulated flow cytometry plots of untransduced T cells were depicted, indicating the level of endogenous FOXP3 expression only. Figure 2B Simulated flow cytometry plots of transduced T cells were depicted, indicating the levels of both endogenous and ectopic FOXP3 expression. Figure 2C A simulated protein blot was depicted, indicating the specificity of anti-HA and anti-FOXP3 antibodies for ectopic and endogenous variants of FOXP3. Figures 2D to 2E A method for detecting different isotypes of FOXP3 using different anti-FOXP3 antibody clones is described. Figure 2D All clones except 150D / E4 (PCH101, FJK-16s, eBio7979, 236A / E7, 206D, 259D) were shown to be able to detect all FOXP3 isotypes. 150D / E4 could not detect the FOXP3 Δ2 isotype. Figure 2E Simulated flow cytometry plots of transduced T cells were depicted, indicating different FOXP3 isotypes detected with different anti-FOXP3 antibodies (150D / E4, PCH101). Figure 2F The results of immunoblotting with different anti-FOXP3 antibodies (236A / E7, 259D, 206D, 150D / E4) were described, indicating that the 150D / E4 clone could only detect the full-length FOXP3 isotype, but not Δ2.

[0052] Figures 3A to 3S The results of immunostaining and flow cytometry were described after transducing Treg cells with vectors encoding different FOXP3 constructs. Figures 3A to 3D pSB_0521 Figures 3E to 3H pSB_0858; Figures 3I to 3L pSB_0859; Figures 3M to 3P (pSB_0965). Figure 3Q Depicting the source Figure 3D , Figure 3H , Figure 3L and Figure 3P An overlay of data. Figure 3R Depicting from Figure 3B (pSB_0521) Figure 3F (pSB_0858) Figure 3J (pSB_0859) and Figure 3N Average fluorescence intensity data for (pSB_0965). Figure 3S Depicting from Figure 3A (pSB_0521) Figure 3E (pSB_0858) Figure 3I (pSB_0859) and Figure 3M HA+ cells in (pSB_0965) and from Figure 3C (pSB_0521) Figure 3G (pSB_0858) Figure 3K (pSB_0859) and Figure 3O Mean fluorescence intensity data for EGFR+ cells in (pSB_0965).

[0053] Figures 4A to 4Q The results of immunostaining and flow cytometry following transduction of Teff cells with vectors encoding different FOXP3 constructs are depicted. Figures 4A to 4E pSB_0858; Figures 4F to 4J pSB_0859; Figures 4K to 4O (pSB_0965). Figure 4P Depicting the source Figure 4D , Figure 4I and Figure 4N An overlay of data. Figure 4Q Depicting the source Figure 4E , Figure 4J and Figure 4O An overlay of data.

[0054] Figures 5A to 5B The results of transducing T cells with vectors encoding different FOXP3 constructs are described, along with the resulting FOXP3 expression levels as measured by immunostaining and flow cytometry. Figure 5A The results of transducing Teff cells with vectors encoding different FOXP3 constructs are described. Figure 5B Describes the use of Figure 5AThe results of transducing Treg cells with the same set of expression vectors.

[0055] Figures 6A to 6B The results of transducing T cells with vectors encoding different FOXP3 constructs and the resulting cytokine expression levels were described. Figure 6A The results of transducing Teff cells with vectors encoding different FOXP3 constructs are described. Figure 6B Describes the use of Figure 6A The results of transducing Treg cells with the same set of expression vectors.

[0056] Figure 7 The results of transducing Teff and Treg cells with vectors encoding different FOXP3 constructs, showing the different locations of the FOXP3 constructs within the plasmids compared to the location of the CAR. Bar charts depict cytokine production in cells transduced after FOXP3 staining using a PCH101 antibody clone.

[0057] Figures 8A to 8E The results of transducing Treg cells with vectors encoding different FOXP3 constructs are described, along with the resulting FOXP3 expression levels as measured by immunostaining and flow cytometry. Figure 8A The levels of FOXP3 and HA in the untransduced control Treg were depicted. Figure 8B The left panel shows the levels of FOXP3 and HA in Treg cells transduced with a vector encoding the full-length FOXP3 construct, and the right panel shows the FOXP3 level in HA-positive (HA+) cells. Figure 8C The levels of FOXP3 and HA in Treg cells transduced with a vector encoding a double full-length / Δ2FOXP3 construct are shown in the left panel, and the FOXP3 level in HA-positive (HA+) cells is shown in the right panel. Figure 8D The left panel shows the levels of FOXP3 and HA in Treg cells transduced with a vector encoding the Δ2 FOXP3 construct, and the right panel shows the FOXP3 level in HA-positive (HA+) cells. Figure 8E A graph of geometric mean fluorescence intensity (gMFI) for FOXP3 (PCH101 or 150D / E) positive staining is provided, along with representative histograms of each antibody (i.e., PCH101 or 150D / E) superimposed on the full-length FOXP3 construct, the dual FOXP3 construct, and the Δ2 FOXP3 construct.

[0058] Figures 9A to 9B The ratio of full-length Treg to Δ2 FOXP3 isotype was depicted under non-inflammatory and inflammatory conditions. Figure 9A An overview of the experimental design and the vectors used is provided. Figure 9BThe resulting ratios are illustrated by the percentage allocation of full-length (FL) to delta 2 (Δ2) FOXP3 isotypes generated in Tregs transduced with CV CAR control vectors, CV CAR vectors with full-length FOXP3, and CV CAR vectors with dual FL / Δ2 FOXP3 under both non-inflammatory and inflammatory conditions. FOXP3 150D clone staining was analyzed in the FOXP3 (PCH101) positive population to determine the ratio between full-length and Δ2 FOXP3 isotypes. In PCH101+ FOXP3+ cells, 150D positive cells indicated the presence of full-length FOXP3, and 150D negative cells indicated the presence of Δ2 FOXP3.

[0059] Figures 10A to 10D The expression levels of ectopic FOXP3 in Tregs following multiple rounds of activation and inflammation were depicted. Figure 10A An overview of the experimental design and the vectors used is provided. Figure 10B Flow cytometry plots are provided, showing the FOXP3 levels in Tregs stimulated with TCR transduced with a CV CAR control vector, a CV CAR vector with full-length FOXP3, and a CV CAR vector with dual FL / Δ2 FOXP3. Figure 10C Flow cytometry plots are provided showing the FOXP3 levels in Tregs transduced with CV CAR control vector, CV CAR vector with full-length FOXP3, and CV CAR vector with dual FL / Δ2 FOXP3 after TCR stimulation under inflammatory conditions. Figure 10D A heatmap was provided detailing the percentage of Tag-positive (Tag+) cells in the transduced CAR Treg measured on day 17, with and without inflammation.

[0060] Figures 11A to 11C Cytokine expression in Tregs transduced and stimulated by PMA / ionomycin was described using a CV CAR control vector, a CV CAR vector with full-length FOXP3, and a CV CAR vector with dual FL / Δ2 FOXP3. Figure 11A The percentage of transduced Tregs expressing IL-2, IL-4, IL-10, IL-17A, TNF-α, or IFN-γ is shown after two activations with anti-CD3 / anti-CD28 antibodies for 14 days, followed by further PMA / ionomycin stimulation and monensin treatment. Figure 11BThe percentage of transducing Tregs expressing IL-2, IL-4, IL-10, IL-17A, TNF-α, or IFN-γ after being cultured with inflammatory cytokines and activated twice with anti-CD3 / anti-CD28 antibodies for 14 days, followed by further PMA / ionomycin stimulation and monensin treatment. Figure 11C The percentage of untransduced effector T cells that were positive for IL-2, IL-4, IL-10, IL-17A, TNF-α, or IFN-γ cytokines on day 14 after PMA stimulation is shown.

[0061] Figures 12A to 12D The Treg phenotype and expression levels of FOXP3 and Helios in transduced Tregs after multiple rounds of activation and inflammation were depicted. Figure 12A This study shows the levels of FOXP3 over time in Tregs transduced with a multicistron box containing an engineered FOXP3 construct to allow alternative splicing (“CAR-Double”; test), a full-length FOXP3 isotype (“CAR-FL”; control), or a CAR without a FOXP3 construct (i.e., “CAR-Label”; negative control) in the presence of high inflammation. Figure 12B A heatmap was provided detailing the percentage of Tag-positive (Tag+) cells in the transduced CAR Treg measured on day 21 in the presence of high inflammation. Figure 12C The levels of Helios in Treg transduced with the following methods over time are shown in the presence of high inflammation: multicistron boxes containing engineered FOXP3 constructs to allow alternative splicing (“CAR-Double”; test), full-length FOXP3 isoforms (“CAR-FL”; control), or CARs without FOXP3 constructs (i.e., “CAR-Label”; negative control). Figure 12D The percentage of transduced Tregs expressing IL-2, IL-4, IL-17A, TNF-α, IFN-γ, or IL-10 is shown after two 14-day activations with anti-CD3 / anti-CD28 antibodies, followed by further PMA / ionomycin stimulation and monensin treatment in the presence of high inflammation.

[0062] Figures 13A to 13D The Treg phenotype and expression levels of FOXP3 and Helios in transduced Tregs containing an exemplary CAR with high tonicity were depicted after multiple rounds of activation and inflammation. Figure 13AThe following are examples of FOXP3 levels over time in Treg transduced with the following in the presence of high inflammation: multicistron boxes containing engineered FOXP3 constructs to allow alternative splicing (“CAR-Double”; test), full-length FOXP3 isotypes (“CAR-FL”; control), or CARs without FOXP3 constructs (i.e., “CAR-Label”; negative control). Figure 13B The levels of Helios in Treg transduced with the following methods over time are shown in the presence of high inflammation: multicistron boxes containing engineered FOXP3 constructs to allow alternative splicing (“CAR-Double”; test), full-length FOXP3 isoforms (“CAR-FL”; control), or CARs without FOXP3 constructs (i.e., “CAR-Label”; negative control). Figure 13C A heatmap was provided detailing the percentage of Tag-positive (Tag+) cells in the transduced CAR Treg measured on day 21 in the presence of high inflammation. Figure 13D The percentage of transduced Tregs expressing IL-2, IL-4, IL-10, IL-17A, TNF-α, or IFN-γ is shown after two 21-day activations with anti-CD3 / anti-CD28 antibodies, followed by further PMA / ionomycin stimulation and monensin treatment in the presence of high inflammation.

[0063] Figures 14A to 14D The expression levels of ectopic FOXP3 in Tregs transduced with an exemplary polycistronic cassette containing an exemplary CAR construct with a high base were depicted after multiple rounds of TCR activation, in the presence and absence of high inflammation. Figure 14A The following are examples of FOXP3 levels over time in Treg transduced with the following in the absence of high inflammation: multicistron cassettes containing engineered FOXP3 constructs to allow alternative splicing (“CAR-Double”; test), full-length FOXP3 isoforms (“CAR-FL”; control), or CARs without FOXP3 constructs (i.e., “CAR-Label”; negative control). Figure 14B The following are examples of FOXP3 levels over time in Treg transduced with the following in the presence of high inflammation: multicistron boxes containing engineered FOXP3 constructs to allow alternative splicing (“CAR-Double”; test), full-length FOXP3 isotypes (“CAR-FL”; control), or CARs without FOXP3 constructs (i.e., “CAR-Label”; negative control). Figure 14C A heatmap was provided detailing the percentage of Tag-positive (Tag+) cells in the transduced CAR Treg measured on day 21, in the absence of high inflammation, that were positive for each indicator marker. Figure 14D A heatmap was provided detailing the percentage of Tag-positive (Tag+) cells in the transduced CAR Treg measured on day 21 in the presence of high inflammation.

[0064] Figures 15A to 15B Cytokine expression in Tregs transduced and stimulated by PMA / ionomycin was depicted in the presence and absence of inflammation using a second exemplary CAR control vector, an exemplary CAR vector with full-length FOXP3, and an exemplary CAR vector with dual FL / Δ2 FOXP3. Figure 15A The percentage of transduced Tregs expressing IL-2, IL-4, IL-10, IL-17A, TNF-α, or IFN-γ is shown after two activations with anti-CD3 / anti-CD28 antibodies for 14 days, followed by further PMA / ionomycin stimulation and monensin treatment. Figure 15B The percentage of transducing Tregs expressing IL-2, IL-4, IL-10, IL-17A, TNF-α, or IFN-γ after being cultured with inflammatory cytokines and activated twice with anti-CD3 / anti-CD28 antibodies for 14 days, followed by further PMA / ionomycin stimulation and monensin treatment. Detailed Implementation

[0065] FOXP3 expression is crucial for Treg persistence and function. FOXP3 primarily acts as a transcriptional repressor for key genes involved in T cell activation and effector functions, including the synthesis of proliferative and pro-inflammatory cytokines such as IL-2, IL-4, IL-17A, and interferon (IFN)-γ. Furthermore, FOXP3 confers potent repressive function in Tregs expressing the FOXP3 protein. Sustained FOXP3 expression in Tregs is essential for lineage morphology and stability. Four naturally occurring FOXP3 isoforms exist: full-length (FL), isoform lacking exon 2 (Δ2), isoform lacking both exons 2 and 7 (Δ2Δ7), and isoform lacking exon 7 (Δ7), which are expressed at varying levels in Treg cells. Due to the different functional domains in alternatively spliced ​​exons, these isoforms can have different biological functions. Therefore, providing an expression cassette containing an engineered FOXP3 protein capable of alternative splicing can help improve Treg function compared to expression cassettes containing a non-allexically spliced ​​FOXP3 protein. In some aspects, this document provides an engineered Treg containing a foreign expression cassette comprising a modified nucleic acid encoding a FOXP3 protein operatively linked to a promoter, wherein the nucleic acid encoding 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 contains an intron sequence surrounding exon 2, thereby producing a full-length protein and a Δ2 isoform. In some aspects, the nucleic acid sequence encoding the FOXP3 protein does not contain other intron sequences. In another advantageous aspect, the expression cassette provided herein can be polycistronic and contains multiple protein-coding sequences linked to a single promoter. In some embodiments, the expression cassette contains nucleic acids encoding both the FOXP3 protein and a chimeric receptor. In some embodiments, the expression cassette contains nucleic acid encoding a cell surface receptor that can be used to monitor the expression of the expression cassette. In some embodiments, the expression cassette is tricistronic and contains nucleic acid encoding a FOXP3 protein, nucleic acid encoding a chimeric receptor (e.g., a chimeric antigen receptor (CAR)), and nucleic acid encoding a cell surface receptor.

[0066] All publications mentioned in this application, including patent documents, scientific articles, and databases, are incorporated in their entirety by reference for all purposes, to the extent that each individual publication is individually incorporated by reference. Where a definition presented herein contradicts or is inconsistent with a definition presented in a patent, application, published application, or other publication incorporated herein by reference, the definition presented herein shall prevail over the definition presented herein by reference.

[0067] The section headings used in this article are for organizational purposes only and should not be construed as limiting the topics described.

[0068] I. Expression Box This invention provides an expression cassette for expressing exogenous FOXP3 in the presence or absence of endogenous FOXP3 expression. In some embodiments, the expression cassette provided herein is in a vector, such as a viral vector (e.g., a lentiviral vector). In some embodiments, the expression cassette comprises 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 isotype and an alternative splicing isotype lacking one or more exons. In some embodiments, the FOXP3 protein is a naturally occurring protein, such as 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 having 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 NO: 20-23. In some embodiments, the FOXP3 protein comprises one or more amino acid modifications (e.g., amino acid substitution, deletion, and / or addition) compared to the naturally occurring FOXP3 protein. In some embodiments, the FOXP3 protein is an alternative splicing variant translated from a nucleic acid lacking the coding sequence of exon 2. In some embodiments, the expression cassette contains intron sequences flanking exon 2. In some embodiments, the expression cassette contains only intron sequences flanking exon 2. In some embodiments, the expression cassette contains no intron sequences other than those flanking exon 2. In some embodiments, the FOXP3 protein is not an alternative splicing variant translated from a nucleic acid lacking the coding sequence of exon 7. In some embodiments, the expression cassette encoding the FOXP3 protein encodes only one FOXP3 protein isotype, wherein the FOXP3 protein isotype is either the full-length FOXP3 protein or a FOXP3 protein isotype lacking exon 2. In some implementations, the expression cassette encoding the FOXP3 protein encodes two FOXP3 protein isotypes, wherein the FOXP3 protein isotypes are the full-length FOXP3 protein and the FOXP3 protein isotype lacking exon 2.In some implementations, the expression ratio of full-length FOXP3 protein in Tregs to the exon 2-deficient FOXP3 isoform (Δ2) is approximately 90:1, 80:1, 70:1, 60:1, 50:1, 40:1, 30:1, 25:1, 20:1, 15:1, 10:1, 9:1, 8:1, 7:1, and 6:1. 1. Approximately 5:1, Approximately 4:1, Approximately 3:1, Approximately 2:1, Approximately 1:1, Approximately 1:2, Approximately 1:3, Approximately 1:4, Approximately 1:5, Approximately 1:6, Approximately 1:7, Approximately 1:8, Approximately 1:9, Approximately 1:10, Approximately 1:15, Approximately 1:20, Approximately 1:25, Approximately 1:30, Approximately 1:40, Approximately 1:50, Approximately 1:60, Approximately 1:70, Approximately 1:80, or Approximately 1:90. In some implementations, the expression ratio of full-length FOXP3 protein to the exon 2-deficient FOXP3 isoform (Δ2) within the Treg containing the expression cassette is approximately 90:1, approximately 80:1, approximately 70:1, approximately 60:1, approximately 50:1, approximately 40:1, approximately 30:1, approximately 25:1, approximately 20:1, approximately 15:1, approximately 10:1, approximately 9:1, approximately 8:1, and approximately 7:1. The ratios are approximately 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:25, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, or 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.

[0069] In some cases, amino acid modifications can be introduced into the FOXP3 protein, for example, to optimize, enhance, promote, or increase FOXP3 signaling and / or stability, such as to reduce FOXP3 protein degradation. In some embodiments, the FOXP3 protein is linked to a detection tag (e.g., but not limited to HIS tags, HA tags, etc.). Exemplary detection tags are provided in Table 1 below. Attachment of a detection tag to exogenous FOXP3 allows for the separate identification of exogenous FOXP3 expression levels compared to endogenous FOXP3 levels. Therefore, in some cases, both endogenous and exogenous FOXP3 levels in cells containing exogenous expression cassettes (e.g., Tregs) can be screened simultaneously.

[0070] Table 1. Amino acid sequences of exemplary detection tags.

[0071] In some cells, sustained expression of FOXP3 is essential for Treg lineage typology and stability. Therefore, expression cassettes containing exogenous FOXP3 protein can generate or induce the Treg phenotype.

[0072] In some implementations, the expression cassettes provided herein have significant advantages, including but not limited to: (1) expression in regulatory T cells (Tregs); (2) increased Treg phenotypic stability; (3) increased Treg repressive function; and (4) conversion, differentiation, or otherwise transformation of Teff cells into Tregs.

[0073] This document provides expression cassettes (e.g., exogenous expression cassettes) comprising nucleic acids encoding exogenous FOXP3 protein. This document also provides vectors comprising expression cassettes, such as viral vectors (e.g., lentiviral vectors). In some embodiments, the expression cassette comprises nucleic acids encoding FOXP3 protein and nucleic acids encoding chimeric receptors and / or cell surface receptors, wherein each component is separated from another component by a separating element (e.g., an IRES, nucleic acid encoding a self-cleaving peptide, and / or nucleic acid encoding a peptide that induces ribosome jumping). In some embodiments, the nucleic acid encoding FOXP3 protein comprises intron sequences flanking the nucleic acid encoding exon 2, wherein the nucleic acid encoding FOXP3 protein is alternatively spliced ​​to produce a full-length FOXP3 protein isotype and an exon 2-deficient FOXP3 protein isotype. 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 exogenous expression cassette also contains nucleic acids encoding a chimeric receptor and / or a cell surface receptor. In some embodiments, the nucleic acids encoding the FOXP3 protein and the nucleic acids encoding the chimeric receptor and / or the cell surface receptor are operatively linked to the same promoter. In some embodiments, the nucleic acids encoding the FOXP3 protein and the nucleic acids encoding the chimeric receptor and / or the cell surface receptor are each separated by an IRES, a nucleic acid encoding a self-cleaving peptide (e.g., a P2A peptide), or a nucleic acid encoding a peptide that induces ribosome jumping. In some embodiments, the expression cassette also contains 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 nucleic acids encoding: a) a promoter; b) a modified nucleic acid encoding a FOXP3 protein linked to the detection tag, wherein the nucleic acid encoding the FOXP3 protein includes an intron sequence flanking a 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.

[0074] In some embodiments, this document provides an expression cassette containing nucleic acids 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 contains an intron sequence 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 / or a FOXP3 protein isoform lacking exon 2; and c) a nucleic acid encoding a cell surface receptor. In some embodiments, this document provides an expression cassette comprising nucleic acids 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 contains an intron sequence 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 an exon 2-deficient FOXP3 protein isoform; c) an IRES or a nucleic acid encoding a self-cleaving peptide or a peptide that induces ribosome jumping; 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 either a full-length FOXP3 protein or an exon 2-deficient FOXP3 protein isoform. In some embodiments, the nucleic acid encoding the FOXP3 protein encodes two FOXP3 protein isotypes, wherein the FOXP3 protein isotypes are a full-length FOXP3 protein and a FOXP3 protein isotype lacking exon 2. In some embodiments, the nucleic acid encoding the FOXP3 protein, including an intron sequence flanking the nucleic acid encoding exon 2, is alternatively spliced ​​to produce a full-length FOXP3 protein isotype and a FOXP3 protein isotype 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 FOXP3 does not contain any intron sequences other than the intron sequence flanking exon 2. In some embodiments, the FOXP3 protein is linked to a detection tag, wherein the detection tag contains an amino acid sequence selected from the group consisting of SEQ ID NO:11-16. In some embodiments, the nucleic acid encoding the FOXP3 protein is FOXP3 cDNA. In some embodiments, the expression cassette contains a nucleic acid encoding the FOXP3 protein having an amino acid sequence having 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 NO:20-23.In some embodiments, the cell surface receptor lacks a functional intracellular signal transduction domain or is unable to mediate intracellular signal transduction. In some embodiments, the cell surface receptor is selected from EGFR, HER2, CD2, and H-2K. kThe group consists of IL-5Rα and its derivatives or mutants. In some embodiments, the nucleic acid encodes a cell surface receptor comprising an amino acid sequence having 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 NO:17-19. In some embodiments, the nucleic acid encoding the FOXP3 protein and the nucleic acid encoding the cell surface receptor are operatively linked to the same promoter. In some embodiments, the promoter is an inducible promoter or a constitutive promoter. In some embodiments, the promoter is a cell-specific promoter. In some embodiments, the promoter is specific to 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 induces ribosome jumping. In some embodiments, the expression cassette also 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 an intron sequence 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 isotype and / or an exon 2-deficient FOXP3 protein isotype, wherein the FOXP3 protein has at least about 80% (such as about 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher) sequence identity with the amino acid sequences 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 NO: 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 includes an intron sequence 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 isotype and / or a FOXP3 protein isotype lacking exon 2, wherein the FOXP3 protein has at least about 80% (such as about 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher) sequence identity with the amino acid sequences 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 NO: 2-4; and d) a nucleic acid encoding a cell surface receptor having at least about 80% of the amino acid sequence selected from the group consisting of SEQ ID NO: 17-19. Sequence identity (such as approximately 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher).

[0075] In some embodiments, this document provides an expression cassette 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 contains an intron sequence 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 / or a FOXP3 protein isoform lacking exon 2. In some embodiments, this document provides an expression cassette comprising nucleic acids 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 induces ribosome jumping; and d) a modified nucleic acid encoding a FOXP3 protein, wherein the modified nucleic acid encoding the FOXP3 protein contains an intron sequence 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 isotype and / or an exon 2-deficient FOXP3 protein isotype. In some embodiments, the nucleic acid encoding the FOXP3 protein encodes only one FOXP3 protein isotype, wherein the FOXP3 protein isotype is either a full-length FOXP3 protein or an exon 2-deficient FOXP3 protein isotype. In some embodiments, the nucleic acid encoding the FOXP3 protein encodes two FOXP3 protein isotypes, wherein the FOXP3 protein isotypes are a full-length FOXP3 protein and a FOXP3 protein isotype lacking exon 2. In some embodiments, the nucleic acid encoding the FOXP3 protein, including an intron sequence flanking the nucleic acid encoding exon 2, is alternatively spliced ​​to produce a full-length FOXP3 protein isotype and a FOXP3 protein isotype 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 NO:11-16. In some embodiments, the nucleic acid encoding FOXP3 does not contain an intron sequence other than the intron sequence flanking exon 2. In some embodiments, the nucleic acid encoding the FOXP3 protein is FOXP3 cDNA. In some embodiments, the expression cassette contains a nucleic acid encoding the FOXP3 protein having an amino acid sequence having 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 NO:20-23.In some embodiments, the cell surface receptor lacks a functional intracellular signal transduction domain or is unable to mediate intracellular signal transduction. In some embodiments, the cell surface receptor is selected from EGFR, HER2, CD2, and H-2K. kThe group consists of IL-5Rα and its derivatives or mutants. In some embodiments, the nucleic acid encodes a cell surface receptor comprising an amino acid sequence having 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 NO:17-19. In some embodiments, the nucleic acid encoding the FOXP3 protein and the nucleic acid encoding the cell surface receptor are operatively linked to the same promoter. In some embodiments, the promoter is an inducible promoter or a constitutive promoter. In some embodiments, the promoter is a cell-specific promoter. In some embodiments, the promoter is specific to 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 induces ribosome jumping. In some embodiments, the expression cassette also 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 NO:2-4; and d) a modified nucleic acid encoding a FOXP3 protein, wherein the modified nucleic acid encoding the FOXP3 protein comprises an intron sequence 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 isotype and / or an exon 2-deficient FOXP3 protein isotype, wherein the FOXP3 protein has at least about 80% (such as about 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher) sequence identity with the amino acid sequences of SEQ ID NO:20 and / or 21.In some embodiments, the expression cassette comprises nucleic acids encoding from 5' to 3': a) a promoter; b) a nucleic acid encoding a cell surface receptor having at least about 80% (such as about 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher) sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 17-19; c) a nucleic acid encoding a self-cleaved P2A peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2-4; and b) a modified nucleic acid encoding a FOXP3 protein linked to a detection tag, wherein the modified nucleic acid encoding the FOXP3 protein comprises an intron sequence 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 isotype and / or a FOXP3 protein isotype lacking exon 2, wherein the FOXP3 protein has at least about 80% amino acid identity with the group consisting of SEQ ID NO: 22 and / or 23. Sequence identity (such as approximately 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher).

[0076] In some embodiments, this document provides an expression cassette containing nucleic acids 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 contains an intron sequence 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 / or a FOXP3 protein isoform lacking exon 2. In some embodiments, this document provides an 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 induces ribosome jumping; 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 induces ribosome jumping; and f) a modified nucleic acid encoding a FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein contains an intron sequence 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 / or a FOXP3 protein isoform lacking exon 2. In some embodiments, the nucleic acid encoding the FOXP3 protein encodes only one FOXP3 protein isotype, wherein the FOXP3 protein isotype is either the full-length FOXP3 protein or a FOXP3 protein isotype lacking exon 2. In some embodiments, the nucleic acid encoding the FOXP3 protein encodes two FOXP3 protein isotypes, wherein the FOXP3 protein isotype is either the full-length FOXP3 protein or a FOXP3 protein isotype lacking exon 2. In some embodiments, the nucleic acid encoding the FOXP3 protein, including an intron sequence flanking the nucleic acid encoding exon 2, is alternatively spliced ​​to produce both the full-length FOXP3 protein isotype and the FOXP3 protein isotype 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 contains an amino acid sequence selected from the group consisting of SEQ ID NO:11-16. In some embodiments, the nucleic acid encoding FOXP3 does not contain intron sequences other than those flanking exon 2. In some embodiments, the nucleic acid encoding the FOXP3 protein is FOXP3 cDNA.In some embodiments, the expression cassette comprises nucleic acid encoding a FOXP3 protein having an amino acid sequence having 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 NO:20-23. In some embodiments, the cell surface receptor lacks a functional intracellular signal transduction domain or is unable to mediate intracellular signal transduction. In some embodiments, the cell surface receptor is selected from EGFR, HER2, CD2, and H-2K. kThe group consists of IL-5Rα and its derivatives or mutants. In some embodiments, the nucleic acid encodes a cell surface receptor comprising an amino acid sequence having 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 NO:17-19. In some embodiments, the chimeric receptor includes 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-β, citrullinated vimentin (CV), TNF-like ligand 1A (TL1A), CD2, CD23 (IgE receptor), IL-6R, IL-4Rα, IL-12R, IL-13Rα1, IL-13Rα2, IL-15R, IL-23R, CD19, CD20, CD22, CD3, CD4, CD5, IFN-γ, IgE, IL-1α, IL-1β, 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 v1, GLP2R, JAK1, C5, and TNF-α. In some embodiments, the nucleic acid encodes a chimeric receptor comprising an antigen-binding domain having 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 NO: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 operatively linked to the same promoter. In some embodiments, the promoter is an inducible promoter or a constitutive promoter. In some embodiments, the promoter is a cell-specific promoter. In some embodiments, the promoter is specific to 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., a P2A peptide), or a nucleic acid encoding a peptide that induces ribosome jumping. In some embodiments, the expression cassette also contains 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 having at least about 80% (such as about 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher) sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 80-84; c) a nucleic acid encoding a self-cleaved P2A peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2-4; d) a nucleic acid encoding a cell surface receptor having at least about 80% (such as about 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher) sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 17-19; e) a nucleic acid encoding a self-cleaved P2A or T2A peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 80-84. The amino acid sequence of group NO:2-7; and f) a modified nucleic acid encoding the FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein contains an intron sequence 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 / or a FOXP3 protein isoform lacking exon 2, wherein the FOXP3 protein has at least about 80% (such as about 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher) sequence identity with the amino acid sequences 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 having at least about 80% (such as about 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher) sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 80-84; c) a nucleic acid encoding a self-cleaved P2A peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2-4; d) a nucleic acid encoding a cell surface receptor having at least about 80% (such as about 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher) sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 17-19; e) a nucleic acid encoding a self-cleaved P2A or T2A peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 80-84. The amino acid sequence of group NO:2-7; and f) the modified nucleic acid encoding the FOXP3 protein linked to the detection tag, wherein the nucleic acid encoding the FOXP3 protein contains an intron sequence 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 / or a FOXP3 protein isoform lacking exon 2, wherein the FOXP3 protein has at least about 80% (such as about 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher) sequence identity with the amino acid sequences of SEQ ID NO:22 and / or 23.

[0077] In some embodiments, this document provides an expression cassette containing nucleic acids 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 contains an intron sequence 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 / or a FOXP3 protein isoform lacking exon 2; and d) a nucleic acid encoding a cell surface receptor. In some embodiments, this document provides an expression cassette containing nucleic acids 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 induces ribosome jumping; d) a modified nucleic acid encoding a FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein contains an intron sequence 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 / 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 induces ribosome jumping; 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 isotype, wherein the FOXP3 protein isotype is either the full-length FOXP3 protein or a FOXP3 protein isotype lacking exon 2. In some embodiments, the nucleic acid encoding the FOXP3 protein encodes two FOXP3 protein isotypes, wherein the FOXP3 protein isotype is either the full-length FOXP3 protein or a FOXP3 protein isotype lacking exon 2. In some embodiments, the nucleic acid encoding the FOXP3 protein, including an intron sequence flanking the nucleic acid encoding exon 2, is alternatively spliced ​​to produce both the full-length FOXP3 protein isotype and the FOXP3 protein isotype 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 contains an amino acid sequence selected from the group consisting of SEQ ID NO:11-16. In some embodiments, the nucleic acid encoding FOXP3 does not contain intron sequences other than those flanking exon 2. In some embodiments, the nucleic acid encoding the FOXP3 protein is FOXP3 cDNA.In some embodiments, the expression cassette comprises nucleic acid encoding a FOXP3 protein having an amino acid sequence having 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 NO:20-23. In some embodiments, the cell surface receptor lacks a functional intracellular signal transduction domain or is unable to mediate intracellular signal transduction. In some embodiments, the cell surface receptor is selected from EGFR, HER2, CD2, and H-2K. kThe group consists of IL-5Rα and its derivatives or mutants. In some embodiments, the nucleic acid encodes a cell surface receptor comprising an amino acid sequence having 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 NO:17-19. In some embodiments, the chimeric receptor includes 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-β, citrullinated vimentin (CV), TNF-like ligand 1A (TL1A), CD2, CD23 (IgE receptor), IL-6R, IL-4Rα, IL-12R, IL-13Rα1, IL-13Rα2, IL-15R, IL-23R, CD19, CD20, CD22, CD3, CD4, CD5, IFN-γ, IgE, IL-1α, IL-1β, 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 v1, GLP2R, JAK1, C5, and TNF-α. In some embodiments, the nucleic acid encodes a chimeric receptor comprising an antigen-binding domain having 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 NO: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 operatively linked to the same promoter. In some embodiments, the promoter is an inducible promoter or a constitutive promoter. In some embodiments, the promoter is a cell-specific promoter. In some embodiments, the promoter is specific to 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., a P2A peptide), or a nucleic acid encoding a peptide that induces ribosome jumping. In some embodiments, the expression cassette also contains 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 having at least about 80% (such as about 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher) sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 80-84; c) a nucleic acid encoding a self-cleaved P2A peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2-4; d) a modified nucleic acid encoding a FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein comprises an intron sequence 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 / or a FOXP3 protein isoform lacking exon 2, wherein the FOXP3 protein has at least about 80% amino acid identity with the amino acid sequences of SEQ ID NO: 20 and / or 21. (e) sequence identity of at least about 80% (such as about 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher); e) nucleic acid encoding a self-cleaved P2A or T2A peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2-7; and f) nucleic acid encoding a cell surface receptor having at least about 80% (such as about 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher) sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: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 having at least about 80% (such as about 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher) sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 80-84; c) a nucleic acid encoding a self-cleaved P2A peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 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 an intron sequence 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 isotype and / or an exon 2-deficient FOXP3 protein isotype, wherein the FOXP3 protein has at least about 80% amino acid identity with the amino acid sequences of SEQ ID NO: 22 and / or 23. (e) sequence identity of at least about 80% (such as about 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher); e) nucleic acid encoding a self-cleaved P2A or T2A peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2-7; and f) nucleic acid encoding a cell surface receptor having at least about 80% (such as about 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher) sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO:17-19.

[0078] In some embodiments, this document provides an expression cassette containing nucleic acids 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 contains an intron sequence 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 / 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, this document provides an expression cassette containing nucleic acids 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 contains an intron sequence 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 induces ribosome jumping; 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 induces ribosome jumping; 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 isotype, wherein the FOXP3 protein isotype is either the full-length FOXP3 protein or a FOXP3 protein isotype lacking exon 2. In some embodiments, the nucleic acid encoding the FOXP3 protein encodes two FOXP3 protein isotypes, wherein the FOXP3 protein isotype is either the full-length FOXP3 protein or a FOXP3 protein isotype lacking exon 2. In some embodiments, the nucleic acid encoding the FOXP3 protein, including an intron sequence flanking the nucleic acid encoding exon 2, is alternatively spliced ​​to produce both the full-length FOXP3 protein isotype and the FOXP3 protein isotype 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 contains an amino acid sequence selected from the group consisting of SEQ ID NO:11-16. In some embodiments, the nucleic acid encoding FOXP3 does not contain intron sequences other than those flanking exon 2. In some embodiments, the nucleic acid encoding the FOXP3 protein is FOXP3 cDNA.In some embodiments, the expression cassette comprises nucleic acid encoding a FOXP3 protein having an amino acid sequence having 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 NO:20-23. In some embodiments, the cell surface receptor lacks a functional intracellular signal transduction domain or is unable to mediate intracellular signal transduction. In some embodiments, the cell surface receptor is selected from EGFR, HER2, CD2, and H-2K. kThe group consists of IL-5Rα and its derivatives or mutants. In some embodiments, the nucleic acid encodes a cell surface receptor comprising an amino acid sequence having 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 NO:17-19. In some embodiments, the chimeric receptor includes 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-β, citrullinated vimentin (CV), TNF-like ligand 1A (TL1A), CD2, CD23 (IgE receptor), IL-6R, IL-4Rα, IL-12R, IL-13Rα1, IL-13Rα2, IL-15R, IL-23R, CD19, CD20, CD22, CD3, CD4, CD5, IFN-γ, IgE, IL-1α, IL-1β, 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 v1, GLP2R, JAK1, C5, and TNF-α. In some embodiments, the nucleic acid encodes a chimeric receptor comprising an antigen-binding domain having 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 NO: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 operatively linked to the same promoter. In some embodiments, the promoter is an inducible promoter or a constitutive promoter. In some embodiments, the promoter is a cell-specific promoter. In some embodiments, the promoter is specific to 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., a P2A peptide), or a nucleic acid encoding a peptide that induces ribosome jumping. In some embodiments, the expression cassette also contains 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, wherein the nucleic acid encoding the FOXP3 protein contains an intron sequence 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 isotype and / or a FOXP3 protein isotype lacking exon 2, wherein the FOXP3 protein has at least about 80% (such as about 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher) sequence identity with the amino acid sequences of SEQ ID NO: 20 and / or 21; c) a nucleic acid encoding a self-cleaving P2A peptide, the self-cleaving P2A peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2-4; d) a nucleic acid encoding a chimeric receptor, the chimeric receptor having at least about 80% of the amino acid sequence selected from the group consisting of SEQ ID NO: 80-84. (e) sequence identity of at least about 80% (such as about 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher); e) nucleic acid encoding a self-cleaved P2A or T2A peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2-7; and f) nucleic acid encoding a cell surface receptor having at least about 80% (such as about 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher) sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: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 includes an intron sequence 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 isotype and / or a FOXP3 protein isotype lacking exon 2, wherein the FOXP3 protein has at least about 80% (such as about 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher) sequence identity with the amino acid sequences of SEQ ID NO: 22 and / or 23; c) a nucleic acid encoding a self-cleaving P2A peptide, the self-cleaving P2A peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2-4; d) a nucleic acid encoding a chimeric receptor, the chimeric receptor having at least about 80% of the amino acid sequence selected from the group consisting of SEQ ID NO: 80-84. (e) sequence identity of at least about 80% (such as about 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher); e) nucleic acid encoding a self-cleaved P2A or T2A peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2-7; and f) nucleic acid encoding a cell surface receptor having at least about 80% (such as about 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher) sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO:17-19.

[0079] In some implementations, the FOXP3 expression level in a Treg containing the exogenous expression cassette is increased by at least about 1.1 times, at least about 1.5 times, at least about 2 times, at least about 3 times, at least about 3.2 times, at least about 4 times, at least about 10 times, at least about 20 times, at least about 25 times, at least about 30 times, at least about 35 times, at least about 45 times, at least about 50 times or more compared to a Treg lacking the exogenous expression cassette described herein.

[0080] In some implementations, transduction with the exogenous expression cassette described herein increases cell (e.g., Treg) viability, transduction of FOXP3 signaling, and the ability of FOXP3 signaling to activate or inhibit effector T cell cytokine production.

[0081] In some embodiments, the expression cassette has the ability to increase FOXP3 expression in cells (e.g., Tregs) transduced with and expressing 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 an antibody specific to FOXP3 protein (including specific isotype variants) combined with flow cytometry analysis as described herein.

[0082] In some embodiments, the expression cassettes described herein are expressed in cells. In some embodiments, the expression cassettes described herein are expressed in immune cells. In some embodiments, the immune cells are T cells. In some embodiments, the immune cells are Treg cells.

[0083] In some embodiments, the expression cassette described herein is expressed in Treg cells. In some embodiments, the Treg cells are low in CD4+, CD25+, and CD127. In some embodiments, Treg cells containing the expression cassette described herein also express endogenous FOXP3. In some embodiments, Treg cells expressing the expression cassette described herein also express high cellular levels of FOXP3.

[0084] In some implementations, the expression of at least one Treg biomarker selected from the group consisting of CD25+ and FOXP3 is maintained by a population of Tregs transduced by expression cassettes.

[0085] A. Chimeric receptor In some embodiments, the expression cassette of the present invention comprises a nucleic acid encoding a chimeric receptor. In some embodiments, the chimeric receptor includes a chimeric antigen receptor (CAR), a T-cell receptor (TCR), or a polymeric immunoglobulin receptor (PiG) receptor. In some embodiments, the chimeric receptor includes a CAR, such as any CAR known in the art. In some embodiments, the CAR targets autoantigens, cancer antigens, bacterial antigens, fungal antigens, pathogenic antigens, 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, S100-β, citrullinated vimentin (CV), TNF-like ligand 1A (TL1A), CD2, CD23. (IgE receptor), IL-6R, IL-4Rα, IL-12R, IL-13Rα1, IL-13Rα2, IL-15R, IL-23R, CD19, CD20, CD22, CD3, CD4, CD5, IFN-γ, IgE, IL-1α, IL-1β, 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-α.

[0086] Engineered T-cell receptors (TCRs) utilize naturally occurring (or minimally modified) TCRs capable of recognizing tumor-specific epitopes presented by major histocompatibility complex (MHC) molecules on the surface of cells (e.g., tumor cells). There are far more antigen-specific sequences presented intracellularly and within the MHC than antigen-specific proteins on the cell surface. TCRs are able to target intracellular antigens. Therefore, in some embodiments, the chimeric receptor is the T-cell receptor (TCR).

[0087] Polymeric immunoglobulin receptors (PiGRs or pIgRs) are members of the Fc receptor family, widely expressed on epithelial cells, and responsible for transcytosis of IgA / IgM at the mucosal surface. PiGRs transport polymeric immunoglobulins (pIg, such as pIgA or pIgM) from the basolateral region to the luminal surface at the mucosal barrier as part of mucosal surface defense against microorganisms. Following transport, a portion of the PiGR (or “pIgR”) called the secretory component (SC) is cleaved to release Ig, forming secreted Ig (“SIg”). SIg can modulate the microbial community and pathogens through several mechanisms via antigen-specific and non-specific binding: SIg and SC mediate the host’s innate protection via immune rejection, neutralization, and complement activation, and also aid adaptive immunity by modulating immune cell activation and function and by maintaining homeostasis. See, for example, H. Turula and CE Wobus (2018), Viruses 10(5):237. Therefore, in some embodiments, the chimeric receptor is the PiG receptor.

[0088] A chimeric antigen receptor (CAR) is an engineered molecule comprising an optional signal peptide, a target-binding domain, an optional hinge region, a transmembrane domain, an intracellular signal transduction domain, and an optional co-stimulatory domain. CARs are based on the structure of T-cell receptors, which are expressed on T cells and participate in cell-mediated immune responses. The target-binding domain is also referred to herein as the antigen-binding domain, and therefore the term "target" includes "antigen."

[0089] So-called "first-generation" CARs have a targeting domain and a CD3ξ signaling domain. So-called "second-generation" CARs further include co-stimulatory domains, such as CD28 or 4-1BB domains. So-called "third-generation" CARs contain multiple co-stimulatory domains. So-called "fourth-generation" CARs (also known as "TRUCKS") are engineered to release transgenic cytokines during CAR signaling.

[0090] A chimeric antigen receptor (“CAR”) comprises 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 containing a signal transduction domain. Optionally, a CAR may include any of the following: a CD3ζ signal transduction domain, an Fc receptor signal transduction domain, and / or a co-stimulatory (signal transduction) domain. That is, these optional elements may be included in addition to or in lieu of other optional elements. The target-binding domain is heterologous to at least one other domain. That is, the target-binding domain is not naturally present on T cell receptors.

[0091] The target-binding domain provides the binding specificity for the CAR. The signal peptide guides the polypeptide across the cell membrane. The target-binding domain can bind to the domain of an antibody that binds to the target antigen to form a so-called "universal CAR." The hinge region is a flexible connecting region, such as a natural or synthetic polypeptide, or any other type of molecule, providing structural flexibility and spacing for the side-attached polypeptide region. The transmembrane domain is a transmembrane protein domain, typically hydrophobic. The signal transduction domain, or signal transduction domain, transmits a signal into the cell via a signal transduction pathway upon binding. This signal transduction activates cellular activity. The co-stimulatory domain is an auxiliary signal transduction domain that further transmits the signal.

[0092] 1. Signal peptide Signal peptides can be any peptide that allows a polypeptide to cross the cell membrane. Signal peptides can be derived from receptors of the CD4, CD8, CD28, TLR, or immunoglobulin families.

[0093] An example signal peptide is: MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO:1) MALPVTALLLPLALLLHAAR (SEQ ID NO:26).

[0094] 2. Target-binding domain A. Structure The target-binding domain may include any polypeptide that incorporates target-binding functionality, such as an antibody as defined herein. In one embodiment, the target-binding domain may comprise a single-chain antibody (scFV). The scFV may be linked to the transmembrane domain via a hinge domain. The length, flexibility, and origin of the hinge domain provide variability in CAR design and, together with the transmembrane domain, can contribute to antigen interaction, immune synapse establishment, and influence the binding of the CAR to additional proteins required to deliver strong activation signals.

[0095] B. Target The chimeric antigen receptor disclosed herein includes a target-binding domain that binds to a target antigen (e.g., an autoimmune antigen), including but not limited to MBP, MOG, PLP, PDE4, VEGFR, S100-β, citrullinated vimentin (CV), TNF-like ligand 1A (TL1A), CD2, and CD23. (IgE receptor), IL-6R, IL-4Rα, IL-12R, IL-13Rα1, IL-13Rα2, IL-15R, IL-23R, CD19, CD20, CD22, CD3, CD4, CD5, IFN-γ, IgE, IL-1α, IL-1β, 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 v1, GLP2R, JAK1, C5, and TNF-α, or more of these. For example, in some embodiments, the CAR binds to a target antigen selected from the group consisting of CD19, CD20, CD22, and citrullinated vimentin (CV). Specifically, the CV target-binding domain can bind to one or more of (i) citrullinated vimentin, (ii) citrullinated fibrin, (iii) citrullinated fibrinogen, and (iv) their citrullinated peptides. In some embodiments, the target-binding domain can bind to citrullinated peptide fragments of (i)-(iii), wherein the peptide fragments are at least about 10 amino acids in length, for example, 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 tendinin C. In some embodiments, the target-binding domain can bind to two or more of the following: (i) citrullinated vimentin, (ii) citrullinated fibrin, (iii) citrullinated fibrinogen, and (iv) tendinin C or its citrullinated peptide fragments.

[0096] The target-binding domain may comprise sequences from the antibody's VH and VL domains. This includes a specific set of CDRs from the VH and VL domains. In some embodiments, the target-binding domain comprises a CDR from the VH domain of SEQ ID NO:67, SEQ ID NO:69, or SEQ ID NO:71. In some embodiments, the target-binding domain comprises a CDR from the VL domain of SEQ ID NO:68, SEQ ID NO:70, or SEQ ID NO:72. In some embodiments, the target-binding domain comprises CDRs from the VH and VL domains of SEQ ID NO:67 and SEQ ID NO:68, respectively. In some embodiments, the target-binding domain comprises CDRs from the VH and VL domains of SEQ ID NO:69 and SEQ ID NO:70, respectively. In some embodiments, the target-binding domain comprises CDRs from the VH and VL domains of SEQ ID NO:71 and SEQ ID NO:72, respectively. In some embodiments, the target-binding domain includes CDRs from the VH and VL domains of SEQ ID NO:67 and SEQ ID NO:70, respectively. In some embodiments, the target-binding domain includes CDRs from the VH and VL domains of SEQ ID NO:67 and SEQ ID NO:72, respectively. In some embodiments, the target-binding domain includes CDRs from the VH and VL domains of SEQ ID NO:69 and SEQ ID NO:68, respectively. In some embodiments, the target-binding domain includes CDRs from the VH and VL domains of SEQ ID NO:69 and SEQ ID NO:72, respectively. In some embodiments, the target-binding domain includes CDRs from the VH and VL domains of SEQ ID NO:71 and SEQ ID NO:68, respectively. In some embodiments, the target-binding domain includes CDRs from the VH and VL domains of SEQ ID NO:71 and SEQ ID NO:70, respectively.

[0097] In some embodiments, the target-binding domain includes complementarity-determining regions (CDRs) from the 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 includes VH-CDR1 containing the amino acid sequence of SEQ ID NO:34, VH-CDR2 containing the amino acid sequence of SEQ ID NO:35, and VH-CDR3 containing the amino acid sequence of SEQ ID NO:36, and the VL domain of the target-binding domain includes VL-CDR1 containing the amino acid sequence of SEQ ID NO:92, VL-CDR2 containing the amino acid sequence of SEQ ID NO:93, and VL-CDR3 containing the amino acid sequence of SEQ ID NO:94.

[0098] In some embodiments, the target-binding domain includes complementarity-determining regions (CDRs) of the VH and VL domains from SEQ ID NO:67 and SEQ ID NO:70, respectively. In some embodiments, the VH domain of the target-binding domain includes VH-CDR1 containing the amino acid sequence of SEQ ID NO:34, VH-CDR2 containing the amino acid sequence of SEQ ID NO:35, and VH-CDR3 containing the amino acid sequence of SEQ ID NO:36, and the VL domain of the target-binding domain includes VL-CDR1 containing the amino acid sequence of SEQ ID NO:37, VL-CDR2 containing the amino acid sequence of SEQ ID NO:38, and VL-CDR3 containing the amino acid sequence of SEQ ID NO:39.

[0099] In some embodiments, the target-binding domain includes complementarity-determining regions (CDRs) from the 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 includes VH-CDR1 containing the amino acid sequence of SEQ ID NO:40, VH-CDR2 containing the amino acid sequence of SEQ ID NO:41, and VH-CDR3 containing the amino acid sequence of SEQ ID NO:36, and the VL domain of the target-binding domain includes VL-CDR1 containing the amino acid sequence of SEQ ID NO:92, VL-CDR2 containing the amino acid sequence of SEQ ID NO:93, and VL-CDR3 containing the amino acid sequence of SEQ ID NO:94.

[0100] In some embodiments, the target-binding domain includes complementarity-determining regions (CDRs) of the VH and VL domains from SEQ ID NO:69 and SEQ ID NO:70, respectively. In some embodiments, the VH domain of the target-binding domain includes VH-CDR1 containing the amino acid sequence of SEQ ID NO:40, VH-CDR2 containing the amino acid sequence of SEQ ID NO:41, and VH-CDR3 containing the amino acid sequence of SEQ ID NO:36, and the VL domain of the target-binding domain includes VL-CDR1 containing the amino acid sequence of SEQ ID NO:37, VL-CDR2 containing the amino acid sequence of SEQ ID NO:38, and VL-CDR3 containing the amino acid sequence of SEQ ID NO:39.

[0101] In some embodiments, the target-binding domain includes complementarity-determining regions (CDRs) of the VH and VL domains from SEQ ID NO:71 and SEQ ID NO:72, respectively. In some embodiments, the VH domain of the target-binding domain includes VH-CDR1 containing the amino acid sequence of SEQ ID NO:42, VH-CDR2 containing the amino acid sequence of SEQ ID NO:43, and VH-CDR3 containing the amino acid sequence of SEQ ID NO:44, and the VL domain of the target-binding domain includes VL-CDR1 containing the amino acid sequence of SEQ ID NO:45, VL-CDR2 containing the amino acid sequence of SEQ ID NO:46, and VL-CDR3 containing the amino acid sequence of SEQ ID NO:47.

[0102] Table 2. Frame region (FR) and complementarity-determining region (CDR) of the heavy chain variable domain (VH). CV, citrullinated vimentin.

[0103] Table 3. Frame region (FR) and complementarity-determining region (CDR) of the light chain variable domain (VL). CV, citrullinated vimentin.

[0104] In some implementations, the target-binding domain comprises a VH sequence selected from the following: CV #1 VH HLHLQESGPGLVKPSETLSLTCTVSGGSINDTTYYWGWIRQPPGKGLEWIGSIYYRGNTHYNSSLRSRVTMSVDTSKNRFSLKVTSVTAADTAVYYCARLDPFDYWGRGTLVTVSS (SEQ ID NO:67); CV #2 / #4 VH QLQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLEWIGSIYYSGSTYYNPSLKSRVTISSVDTSKNQFSLKLSSVTAADTAVYYCARLDPFDYWGRGTLVTVSS (SEQ ID NO:69); CV #3 VH OR A sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% sequence identity with the aforementioned sequence, provided that the target-binding domain binds to the citrullinated antigen described herein.

[0105] The target binding region may contain VL sequences selected from the following: CV #1 / #4 VL SYVLTQPPSVSLAPGETATITCGGDDIENQNVNWYQQKSGQAPMLLIFFDTRRPSGIPERFSGSRSEDTANLTITRVEAGDDADYFCQVYDRKTDHQVFGPGTTVTVL (SEQ ID NO:68); CV #2 VL SYVLTQPPSVSVAPGKTARITCGGNNIGSKSVHWYQQKPGQAPVLVIYYDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHQVFGTGTKVTVL (SEQ ID NO:70); CV #3 VL OR A sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% sequence identity with the aforementioned sequence, provided that the target-binding domain binds to the citrullinated antigen described herein.

[0106] In another embodiment, the antigen-binding region comprises an scFV containing an amino acid sequence selected from the following: CV #1 scFv – VHVL – GGGSx3 connector HLHLQESGPGLVKPSETLSLTCTVSGGSINDTTYYWGWIRQPPGKGLEWIGSIYYRGNTHYNSSLRSRVTMSVDTSKNRFSLKVTSVTAADTAVYYCARLDPFDYWGRGTLVTVSS GGGGSGGGGSGGGGS SYVLTQPPSVSVAPGKTARITCGGNNIGSKSVHWYQQKPGQAPVLVIYYDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHQVFGTGTKVTVLR (SEQ ID NO:73) (GGGSx3 connector with underline); CV #2 scFv – VHVL – Whitlow 218 connector HLHLQESGPGLVKPSETLSLTCTVSGGSINDTTYYWGWIRQPPGKGLEWIGSIYYRGNTHYNSSLRSRVTMSVDTSKNRFSLKVTSVTAADTAVYYCARLDPFDYWGRGTLVTVSS GSTSGSGKPGSGEGSTKG SYVLTQPPSVSVAPGKTARITCGGNNIGSKSVHWYQQKPGQAPVLVIYYDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHQVFGTGTKVTVLR (SEQ ID NO:74) (Whitlow 218 connector with underline); CV #3 scFv – VHVL – AB pur connector HLHLQESGPGLVKPSETLSLTCTVSGGSINDTTYYWGWIRQPPGKGLEWIGSIYYRGNTHYNSSLRSRVTMSVDTSKNRFSLKVTSVTAADTAVYYCARLDPFDYWGRGTLVTVSS ASSGGSTSGSGKPGSGEGSSGSARSYVLTQPPSVSVAPGKTARITCGGNNIGSKSVHWYQQKPGQAPVLVIYYDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHQVFGTGTKVTVLR (SEQ ID NO:75) (AB pur connector with underline); CV #4 scFv – VHVL – AB pur connector EVKLIESGGGLVEPGRSLRLACTTSGFTFADYGLSWFRQGPGKGLEWVGFTGPKHLGETTECAPSVEDRCTISRDDSKSTVYLQMHRLQHEDTAVYFCVGPWFGDLLMWGQGTLVTVSSASSGGSTS GSGKPGSGEGSSGSARAIQMTQSPSSSLSASVGDRVSITCRATQDISTSLGWYHQRPGKAPRRLLIYGASKVQTGVPSRFSGNGSGTEFTLTISSLQPEDIGTYYCLQDDGFPFTVGQGTKLDIKRAAA (SEQ ID NO:76) (AB pur linker underlined); or A sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% sequence identity with the aforementioned sequence, provided that the target-binding domain binds to the citrullinated antigen described herein.

[0107] Therefore, an exemplary connector is: GGGGSGGGGSGGGGS (SEQ ID NO:77) GGGSx3 linker; GSTSGSGKPGSGEGSTKG (SEQ ID NO:78) Whitlow 218 connector; or ASSGGSTSGSGKPGSGEGSSGSAR (SEQ ID NO:79) AB pur connector.

[0108] Optionally, any of the aforementioned sequences may contain the set of CDRs described above.

[0109] 3. Hinge area In some embodiments, the hinge region of the disclosed CAR may be selected from the extracellular domains of CD8, CD4, or CD28, the Fc region of an IgG1 antibody, or the extracellular domain of any TLR receptor, as known to those skilled in the art and available in the GenBank database. In some embodiments, the hinge region is any suitable hinge region known in the art.

[0110] For example, the hinge area can contain the following sequence: IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO:27) (CD28); TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC(SEQ ID NO:28) (CD8); or A sequence that is identical to the aforementioned hinge region sequence in any of the following percentages: 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 99.5%.

[0111] 4. Transmembrane domain The transmembrane domain may contain a transmembrane domain of an immunoglobulin family receptor, such as CD8. The intracellular domain may be selected from any transmembrane molecule on a T cell. For example, the transmembrane (TM) domain of the disclosed CAR may contain a TM domain of CD2, CD3, CD16, CD32, CD64, CD28, CD247, 4-1BBL, CD4, or CD8.

[0112] For example, a transmembrane domain may contain sequences selected from the following: FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO:29); IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO:30); or A sequence that is identical to the aforementioned hinge region sequence in any of the following percentages: 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 99.5%.

[0113] 5. Signal transduction structural domain A. CD3Z signal transduction domain In some embodiments, the signal transduction domain comprises a CD3ζ signal transduction domain. The CD3ζ signal transduction domain of the disclosed CAR molecule may contain a CD3ζ amino acid sequence, such as a CD3ζ signal transduction domain.

[0114] For example, the CD3ζ signal transduction domain can have a sequence selected from the following: RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO:31); or A sequence that is identical to the aforementioned CD3ζ signal transduction domain sequence with at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% of the sequence.

[0115] For example, the CD3ζ signal transduction domain may contain amino acids 21-163, 31-142, 68-89 and / or 138-158 of the sequence shown in SEQ ID NO:31 or its functional variants (e.g., having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 10-20 amino acid substitutions, deletions or additions).

[0116] B. FC receptor signal transduction domain In some implementations, the signal transduction domain includes an Fc signaling domain. The Fc signaling domain can be any of the Fc-α, Fc-γ, Fc-ε, Fc-μ, and Fc-δ receptors. For example, the Fc receptor signaling domain can contain amino acids involved in interactions with Src (e.g., Fgr, Fyn, Hck, Lyn, Yes, and Src) and ZAP-70 family kinases, such as one or more ITAM domains (see, for example, 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, for example, derived from or substantially identical to any of the Fc-α, Fc-γ, Fc-ε, Fc-mu, and Fc-δ receptors.

[0117] Sequences can also be found in the following way:

[0118] 6. Co-stimulatory domains In addition to the signal transduction domains of the CD3ζ or Fc receptors, the CARs disclosed herein may include one or more co-stimulatory domains. These co-stimulatory domains may be derived from, for example, CD28, 4-1BB, CD2, CD27, CD30, OX40, CD40, PD-1, PD-L1, PD-L2, ICOS, LFA-1, CD7, LIGHT, NKG2C, B7-H3, CD83L, B7-1 (CD80), B7-2 (CD86), B7-H3, B7-H4, etc. The CAR construct may contain two or more co-stimulatory signal transduction domains (e.g., CD28 + 4-1BB).

[0119] The co-stimulatory domain can be located between the signal transduction domain and the transmembrane region.

[0120] In some implementations, the CD28 co-stimulatory domain may contain sequences selected from the following: RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO:32); or A sequence that is identical to the aforementioned sequence in any of the following percentages: 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 99.5%.

[0121] In some implementations, the 41BB co-stimulatory domain may contain sequences selected from the following: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO:33); or A sequence that is identical to the aforementioned sequence in any of the following percentages: 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 99.5%.

[0122] B. Cell surface receptors In some embodiments, the expression cassette of the present invention comprises a nucleic acid encoding a cell surface receptor. The cell surface receptor can be used to identify cells expressing the expression cassette provided herein using standard methods in the art, such as, but not limited to, flow cytometry, Western blotting, immunohistochemistry and microscopy (e.g., fluorescence microscopy), radioligand binding assays, ELISA, etc.

[0123] In some embodiments, the cell surface receptor lacks a functional intracellular signal transduction domain or is unable to mediate intracellular signal transduction. In some embodiments, the cell surface receptor binds to its natural ligand (i.e., the wild-type ligand). In some embodiments, the cell surface receptor binds to a non-natural ligand, i.e., a ligand not naturally found in an organism containing cells containing 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 wild-type extracellular domain or a modified extracellular domain. In some embodiments, the modified extracellular domain contains one or more (e.g., at least about 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 the wild-type extracellular domain. In some embodiments, the modified extracellular domain binds to a non-natural ligand, thereby distinguishing cells that express only the wild-type cell surface receptor from cells that express both the wild-type cell surface receptor and an expression cassette containing nucleic acid encoding the modified cell surface receptor.

[0124] In some embodiments, the cell surface receptor includes an intracellular signal transduction domain. In some embodiments, the cell surface receptor does not include an intracellular signal transduction domain. In some embodiments, the cell surface receptor includes a truncated intracellular signal transduction domain. In some embodiments, the cell surface receptor includes an intracellular signal transduction domain having a length of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids, wherein the intracellular signal transduction domain has 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 wild-type cell surface receptor. In some embodiments, the cell surface receptor includes a modified intracellular signal transduction domain that contains one or more (e.g., any one of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) amino acid modifications (e.g., substitution, deletion or insertion) compared to the wild-type intracellular signal transduction domain, wherein the modified intracellular signal transduction domain is not capable of mediating intracellular signal transduction.

[0125] In some embodiments, the cell surface receptor is not endogenously expressed in cells transduced using any of the exogenous FOXP3 expression cassettes described herein. In some embodiments, the cell surface receptor is not endogenously expressed in T cells (e.g., Treg cells). In some embodiments, the cell surface receptor is not endogenously expressed in Treg cells. In some embodiments, the cell surface receptor 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 EGFR, HER2, CD2, and H-2K. k The group consists of IL-5Rα and its derivatives or mutants. In some embodiments, the cell surface receptor comprises an amino acid sequence selected from the group consisting of SEQ ID NO:17-19.

[0126] C. Double cis-reflex box In some embodiments, the expression cassette of the present invention comprises two nucleic acids encoding a unique polypeptide, wherein the nucleic acids can be separated by a separating element, such as by an IRES, a nucleic acid encoding a self-cleaving peptide (e.g., P2A peptide), or a nucleic acid encoding a peptide that induces ribosome jumping. Thus, post-translational, the expression cassette comprising the two nucleic acids produces two distinct polypeptides, such as a full-length protein or a derivative thereof, such as full-length FOXP3 and / or an alternative splice isoform lacking one or more exons (e.g., exon 2), and a cell surface receptor. For example, the expression cassette provided herein can produce, but is not limited to, one or more isoforms of the FOXP3 protein and cell surface receptors (e.g., EGFR, HER2, CD2, H-2K). k (or the cell membrane portion of IL-5Rα protein or its derivatives or mutants).

[0127] In some embodiments, this document provides an expression cassette containing nucleic acids 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 contains an intron sequence 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 isotype and a FOXP3 protein isotype lacking exon 2; and c) a nucleic acid encoding a cell surface receptor. In some embodiments, this document provides an expression cassette comprising nucleic acids 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 contains an intron sequence 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 isotype and an exon 2-deficient FOXP3 protein isotype; c) an IRES or a nucleic acid encoding a self-cleaving peptide or a peptide that induces ribosome jumping; 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 isotype, wherein the FOXP3 protein isotype is either a full-length FOXP3 protein or an exon 2-deficient FOXP3 protein isotype. In some embodiments, the nucleic acid encoding the FOXP3 protein encodes two FOXP3 protein isotypes: a full-length FOXP3 protein and a FOXP3 protein isotype lacking exon 2. In some embodiments, the nucleic acid encoding the FOXP3 protein, containing an intron sequence flanking the nucleic acid encoding exon 2, is alternatively spliced ​​to produce both a full-length FOXP3 protein isotype and a FOXP3 protein isotype lacking exon 2. In some embodiments, the nucleic acid encoding the FOXP3 protein is alternatively spliced ​​to produce both a full-length FOXP3 protein isotype and a FOXP3 protein isotype lacking exon 2, wherein the FOXP3 protein comprises the amino acid sequences SEQ ID NO: 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 NO: 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 FOXP3 does not contain intron sequences other than those flanking exon 2. In some embodiments, the nucleic acid encoding the FOXP3 protein is FOXP3 cDNA. In some embodiments, the cell surface receptor lacks a functional intracellular signal transduction domain or is unable to mediate intracellular signal transduction.In some implementations, the cell surface receptor is selected from EGFR, HER2, CD2, and H-2K. k The cassette comprises IL-5Rα and its derivatives or mutants. In some embodiments, the nucleic acid encoding the FOXP3 protein and the nucleic acid encoding the cell surface receptor are operatively linked to the same promoter. In some embodiments, the promoter is an inducible promoter or a constitutive promoter. In some embodiments, the promoter is a cell-specific promoter. In some embodiments, the promoter is specific to 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., a P2A peptide), or a nucleic acid encoding a peptide that induces ribosome jumping. In some embodiments, the expression cassette also contains 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 contains an intron sequence 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, wherein the FOXP3 protein contains the amino acid sequences of SEQ ID NO: 20 and 21; c) a nucleic acid encoding a self-cleaving 2A peptide (e.g., a P2A peptide), wherein the 2A peptide contains an amino acid sequence selected from the group consisting of SEQ ID NO: 2-10 and 89-91; and d) a nucleic acid encoding a cell surface receptor, wherein the cell surface receptor contains an amino acid sequence selected from the group consisting of SEQ ID NO: 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 contains an intron sequence 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 isotype and a FOXP3 protein isotype lacking exon 2, wherein the FOXP3 protein contains the amino acid sequences of SEQ ID NO: 22 and 23; c) a nucleic acid encoding a self-cleaving 2A peptide (e.g., a P2A peptide), wherein the 2A peptide contains an amino acid sequence selected from the group consisting of SEQ ID NO: 2-10 and 89-91; and d) a nucleic acid encoding a cell surface receptor, wherein the cell surface receptor contains an amino acid sequence selected from the group consisting of SEQ ID NO: 17-19.

[0128] In some embodiments, this document provides an expression cassette comprising nucleic acids encoding from 5' to 3': a) a promoter; b) a modified nucleic acid encoding the full-length FOXP3 protein; and c) a nucleic acid encoding a cell surface receptor. In some embodiments, this document provides an expression cassette comprising nucleic acids encoding from 5' to 3': a) a promoter; b) a modified nucleic acid encoding the full-length FOXP3 protein; c) an IRES or a nucleic acid encoding a self-cleaving peptide or a peptide that induces ribosome jumping; 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 isotype, wherein the FOXP3 protein isotype is the 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 attached 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 signal transduction domain or is unable to mediate intracellular signal transduction. In some embodiments, the cell surface receptor is selected from EGFR, HER2, CD2, and H-2K. k The cassette comprises IL-5Rα and its derivatives or mutants. In some embodiments, the nucleic acid encoding the FOXP3 protein and the nucleic acid encoding the cell surface receptor are operatively linked to the same promoter. In some embodiments, the promoter is an inducible promoter or a constitutive promoter. In some embodiments, the promoter is a cell-specific promoter. In some embodiments, the promoter is specific to 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., a P2A peptide), or a nucleic acid encoding a peptide that induces ribosome jumping. In some embodiments, the expression cassette also contains 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 implementations, the expression cassette contains nucleic acids encoding from 5' to 3': a) a promoter; b) a modified nucleic acid encoding a FOXP3 protein containing the amino acid 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.

[0129] In some embodiments, this document provides an expression cassette comprising nucleic acids encoding from 5' to 3': a) a promoter; b) a modified nucleic acid encoding a FOXP3 protein isotype lacking exon 2; and c) a nucleic acid encoding a cell surface receptor. In some embodiments, this document provides an expression cassette comprising nucleic acids encoding from 5' to 3': a) a promoter; b) a modified nucleic acid encoding a FOXP3 protein isotype lacking exon 2; c) an IRES or a nucleic acid encoding a self-cleaving peptide or a peptide that induces ribosome jumping; 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 isotype, wherein the FOXP3 protein isotype lacking exon 2. In some embodiments, the modified nucleic acid encoding the FOXP3 protein isotype 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 attached to the N-terminus of the FOXP3 protein. In some embodiments, the detection tag is attached 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 signal transduction domain or is unable to mediate intracellular signal transduction. In some embodiments, the cell surface receptor is selected from EGFR, HER2, CD2, and H-2K. k The cassette comprises IL-5Rα and its derivatives or mutants. In some embodiments, the nucleic acid encoding the FOXP3 protein and the nucleic acid encoding the cell surface receptor are operatively linked to the same promoter. In some embodiments, the promoter is an inducible promoter or a constitutive promoter. In some embodiments, the promoter is a cell-specific promoter. In some embodiments, the promoter is specific to 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., a P2A peptide), or a nucleic acid encoding a peptide that induces ribosome jumping. In some embodiments, the expression cassette also contains 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 implementations, the expression cassette contains nucleic acids encoding from 5' to 3': a) a promoter; b) a modified nucleic acid encoding a FOXP3 protein isotype lacking exon 2 containing 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.

[0130] In some embodiments, this document provides an expression cassette containing nucleic acids 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 contains an intron sequence 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 isotype and a FOXP3 protein isotype lacking exon 2. In some embodiments, this document provides an expression cassette comprising nucleic acids 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 induces ribosome jumping; and d) a modified nucleic acid encoding a FOXP3 protein, wherein the modified nucleic acid encoding the FOXP3 protein contains an intron sequence 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 isotype and an exon 2-deficient FOXP3 protein isotype. In some embodiments, the nucleic acid encoding the FOXP3 protein encodes only one FOXP3 protein isotype, wherein the FOXP3 protein isotype is either a full-length FOXP3 protein or an exon 2-deficient FOXP3 protein isotype. In some embodiments, the nucleic acid encoding the FOXP3 protein encodes two FOXP3 protein isotypes: a full-length FOXP3 protein and a FOXP3 protein isotype lacking exon 2. In some embodiments, the nucleic acid encoding the FOXP3 protein, containing an intron sequence flanking the nucleic acid encoding exon 2, is alternatively spliced ​​to produce both a full-length FOXP3 protein isotype and a FOXP3 protein isotype lacking exon 2. In some embodiments, the nucleic acid encoding the FOXP3 protein is alternatively spliced ​​to produce both a full-length FOXP3 protein isotype and a FOXP3 protein isotype lacking exon 2, wherein the FOXP3 protein comprises the amino acid sequences SEQ ID NO: 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 NO: 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 FOXP3 does not contain intron sequences other than those flanking exon 2. In some embodiments, the nucleic acid encoding the FOXP3 protein is FOXP3 cDNA.In some embodiments, the cell surface receptor lacks a functional intracellular signal transduction domain or is unable to mediate intracellular signal transduction. In some embodiments, the cell surface receptor is selected from EGFR, HER2, CD2, and H-2K. k The cassette comprises IL-5Rα and its derivatives or mutants. In some embodiments, the nucleic acid encoding the FOXP3 protein and the nucleic acid encoding the cell surface receptor are operatively linked to the same promoter. In some embodiments, the promoter is an inducible promoter or a constitutive promoter. In some embodiments, the promoter is a cell-specific promoter. In some embodiments, the promoter is specific to 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., a P2A peptide), or a nucleic acid encoding a peptide that induces ribosome jumping. In some embodiments, the expression cassette also contains 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 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 17-19; c) a nucleic acid encoding a self-cleaving 2A peptide (e.g., P2A peptide) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2-10 and 89-91; and d) a modified nucleic acid encoding a FOXP3 protein comprising an intron sequence flanking a modified nucleic acid encoding exon 2, wherein the nucleic acid encoding the FOXP3 protein is alternatively spliced ​​to produce a full-length FOXP3 protein isotype and a FOXP3 protein isotype lacking exon 2, wherein the FOXP3 protein comprises the amino acid sequences of SEQ ID NO: 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 NO: 17-19; c) a nucleic acid encoding a self-cleaving 2A peptide (e.g., P2A peptide) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 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 comprises an intron sequence 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 isotype and a FOXP3 protein isotype lacking exon 2, wherein the FOXP3 protein comprises the amino acid sequences of SEQ ID NO: 22 and 23.

[0131] In some embodiments, this document provides an expression cassette comprising nucleic acids encoding from 5' to 3': a) a promoter; b) a nucleic acid encoding a cell surface receptor; and c) a modified nucleic acid encoding the full-length FOXP3 protein. In some embodiments, this document provides an expression cassette comprising nucleic acids 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 induces ribosome jumping; and d) a modified nucleic acid encoding the full-length FOXP3 protein. In some embodiments, the nucleic acid encoding the FOXP3 protein encodes only one FOXP3 protein isotype, wherein the FOXP3 protein isotype 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 attached 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 signal transduction domain or is unable to mediate intracellular signal transduction. In some embodiments, the cell surface receptor is selected from EGFR, HER2, CD2, and H-2K. k The cassette comprises IL-5Rα and its derivatives or mutants. In some embodiments, the nucleic acid encoding the FOXP3 protein and the nucleic acid encoding the cell surface receptor are operatively linked to the same promoter. In some embodiments, the promoter is an inducible promoter or a constitutive promoter. In some embodiments, the promoter is a cell-specific promoter. In some embodiments, the promoter is specific to 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., a P2A peptide), or a nucleic acid encoding a peptide that induces ribosome jumping. In some embodiments, the expression cassette also contains 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 implementations, the expression cassette contains nucleic acids 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; and d) a modified nucleic acid encoding a full-length FOXP3 protein containing the amino acid sequence of SEQ ID NO: 20 or 22.

[0132] In some embodiments, this document provides an expression cassette comprising nucleic acids 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 isotype lacking exon 2. In some embodiments, this document provides an expression cassette comprising nucleic acids 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 induces ribosome jumping; and d) a modified nucleic acid encoding a FOXP3 protein isotype lacking exon 2. In some embodiments, the nucleic acid encoding the FOXP3 protein encodes only one FOXP3 protein isotype, wherein the FOXP3 protein isotype is a FOXP3 protein isotype lacking exon 2. In some embodiments, the modified nucleic acid encoding the FOXP3 protein isotype 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 the FOXP3 protein is FOXP3 cDNA. In some embodiments, the cell surface receptor lacks a functional intracellular signal transduction domain or is unable to mediate intracellular signal transduction. In some embodiments, the cell surface receptor is selected from EGFR, HER2, CD2, and H-2K. k The cassette comprises IL-5Rα and its derivatives or mutants. In some embodiments, the nucleic acid encoding the FOXP3 protein and the nucleic acid encoding the cell surface receptor are operatively linked to the same promoter. In some embodiments, the promoter is an inducible promoter or a constitutive promoter. In some embodiments, the promoter is a cell-specific promoter. In some embodiments, the promoter is specific to 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., a P2A peptide), or a nucleic acid encoding a peptide that induces ribosome jumping. In some embodiments, the expression cassette also contains 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 implementations, the expression cassette contains nucleic acids 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; and d) a modified nucleic acid encoding a FOXP3 protein isotype lacking exon 2 containing the amino acid sequence of SEQ ID NO: 21 or 23.

[0133] D. Tri-cistron / Multi-cistron box In some embodiments, the expression cassette of the present invention comprises three or more nucleic acids encoding unique polypeptides, wherein each nucleic acid can be separated by a separating element (e.g., an IRES, a nucleic acid encoding a self-cleaving peptide (e.g., P2A peptide), or a nucleic acid encoding a peptide that induces ribosome jumping). Thus, post-translational, the expression cassette comprising three or more nucleic acids produces three or more independent polypeptides, such as full-length proteins or derivatives thereof, such as full-length FOXP3 and / or alternatively spliced ​​isoforms lacking one or more exons (e.g., exon 2). For example, the expression cassette provided herein can produce, but is not limited to, one or more isoforms of the FOXP3 protein, cell surface receptors (e.g., EGFR, HER2, CD2, H-2K), etc. k (or the cell membrane portion of IL-5Rα protein or its derivatives or mutants) and chimeric receptors, such as chimeric antigen receptors (CARs), targeting, for example, the following CARs: MBP, MOG, PLP, PDE4, VEGFR, S100-β, citrullinated vimentin (CV), TNF-like ligand 1A (TL1A), CD2, CD23 (IgE receptor), IL-6R, IL-4Rα, IL-12R, IL-13Rα1, IL-13Rα2, IL-15R, IL-23R, CD19, CD20, CD22, CD3, CD4, CD5, IFN-γ, IgE, IL-1α, IL-1β, 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 v1, GLP2R, JAK1, C5, or TNF-α (such as citrullinated vimentin or CD19). Nucleic acids encoding additional proteins can be further incorporated into the expression cassette.

[0134] In some embodiments, this document provides an expression cassette containing nucleic acids 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 contains an intron sequence 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 isotype and a FOXP3 protein isotype lacking exon 2. In some embodiments, this document provides an expression cassette comprising nucleic acids 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 induces ribosome jumping; 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 induces ribosome jumping; and f) a modified nucleic acid encoding a FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein contains an intron sequence 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 isotype and an exon 2-deficient FOXP3 protein isotype. In some embodiments, the nucleic acid encoding the FOXP3 protein encodes only one FOXP3 isotype, wherein the FOXP3 protein isotype is either a full-length FOXP3 protein or an exon 2-deficient FOXP3 protein isotype. In some embodiments, the nucleic acid encoding the FOXP3 protein encodes two FOXP3 protein isotypes: a full-length FOXP3 protein and a FOXP3 protein isotype lacking exon 2. In some embodiments, the nucleic acid encoding the FOXP3 protein, containing an intron sequence flanking the nucleic acid encoding exon 2, is alternatively spliced ​​to produce both a full-length FOXP3 protein isotype and a FOXP3 protein isotype lacking exon 2. In some embodiments, the nucleic acid encoding the FOXP3 protein is alternatively spliced ​​to produce both a full-length FOXP3 protein isotype and a FOXP3 protein isotype lacking exon 2, wherein the FOXP3 protein comprises the amino acid sequences SEQ ID NO: 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 NO: 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 FOXP3 does not contain intron sequences other than those flanking exon 2.In some embodiments, the nucleic acid encoding the FOXP3 protein is FOXP3 cDNA. In some embodiments, the cell surface receptor lacks a functional intracellular signal transduction domain or is unable to mediate intracellular signal transduction. In some embodiments, the cell surface receptor is selected from EGFR, HER2, CD2, and H-2K. kThe group consists of IL-5Rα and its derivatives or mutants. In some embodiments, the chimeric receptor includes 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-β, citrullinated vimentin (CV), TNF-like ligand 1A (TL1A), CD2, CD23 (IgE receptor), IL-6R, IL-4Rα, IL-12R, IL-13Rα1, IL-13Rα2, IL-15R, IL-23R, CD19, CD20, CD22, CD3, CD4, CD5, IFN-γ, IgE, IL-1α, IL-1β, 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 v1, GLP2R, JAK1, C5, and TNF-α. 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 operatively linked to the same promoter. In some embodiments, the promoter is an inducible promoter or a constitutive promoter. In some embodiments, the promoter is a cell-specific promoter. In some embodiments, the promoter is specific to 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., a P2A peptide), or a nucleic acid encoding a peptide that induces ribosome jumping. In some embodiments, the expression cassette also contains 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 NO: 73-76); c) a nucleic acid encoding a self-cleaved 2A peptide (e.g., a P2A peptide), wherein the 2A peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 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 NO: 17-19; e) a nucleic acid encoding a self-cleaved 2A peptide (e.g., a P2A or T2A peptide), wherein the 2A peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 73-76. The amino acid sequence consisting of NO:2-10 and 89-91; and f) a modified nucleic acid encoding the FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein contains an intron sequence flanking the modified nucleic acid encoding exon 2, wherein the nucleic acid encoding the FOXP3 protein is alternatively spliced ​​to produce a full-length FOXP3 protein isotype and a FOXP3 protein isotype lacking exon 2, wherein the FOXP3 protein contains the amino acid sequences of SEQ ID NO: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 NO: 73-76); c) a nucleic acid encoding a self-cleaved 2A peptide (e.g., a P2A peptide), wherein the 2A peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 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 NO: 17-19; e) a nucleic acid encoding a self-cleaved 2A peptide (e.g., a P2A or T2A peptide), wherein the 2A peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 73-76. The amino acid sequence consisting of NO: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 contains an intron sequence flanking the modified nucleic acid encoding exon 2, wherein the nucleic acid encoding the FOXP3 protein is alternatively spliced ​​to produce a full-length FOXP3 protein isotype and a FOXP3 protein isotype lacking exon 2, wherein the FOXP3 protein contains the amino acid sequences of SEQ ID NO:22 and 23.

[0135] In some embodiments, this document provides an expression cassette comprising nucleic acids 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 the full-length FOXP3 protein. In some embodiments, this document provides an expression cassette comprising nucleic acids 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 ribosome-jumping peptide; 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 ribosome-jumping peptide; and f) a modified nucleic acid encoding the full-length FOXP3 protein. In some embodiments, the nucleic acid encoding the FOXP3 protein encodes only one FOXP3 isotype, wherein the FOXP3 protein isotype is the 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 signal transduction domain or is unable to mediate intracellular signal transduction. In some embodiments, the cell surface receptor is selected from EGFR, HER2, CD2, and H-2K. kThe group consists of IL-5Rα and its derivatives or mutants. In some embodiments, the chimeric receptor includes 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-β, citrullinated vimentin (CV), TNF-like ligand 1A (TL1A), CD2, CD23 (IgE receptor), IL-6R, IL-4Rα, IL-12R, IL-13Rα1, IL-13Rα2, IL-15R, IL-23R, CD19, CD20, CD22, CD3, CD4, CD5, IFN-γ, IgE, IL-1α, IL-1β, 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 v1, GLP2R, JAK1, C5, and TNF-α. 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 operatively linked to the same promoter. In some embodiments, the promoter is an inducible promoter or a constitutive promoter. In some embodiments, the promoter is a cell-specific promoter. In some embodiments, the promoter is specific to 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., a P2A peptide), or a nucleic acid encoding a peptide that induces ribosome jumping. In some embodiments, the expression cassette also contains 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 contains nucleic acids 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 containing the amino acid sequence of SEQ ID NO: 20 or 22.

[0136] In some embodiments, this document provides an expression cassette comprising nucleic acids 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 isotype lacking exon 2. In some embodiments, this document provides an expression cassette comprising nucleic acids 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 ribosome-jumping peptide; 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 ribosome-jumping peptide; and f) a modified nucleic acid encoding a FOXP3 protein isotype lacking exon 2. In some embodiments, the nucleic acid encoding the FOXP3 protein encodes only one FOXP3 isotype, wherein the FOXP3 protein isotype isotype lacking exon 2. In some embodiments, the modified nucleic acid encoding the FOXP3 protein isotype 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 the FOXP3 protein is FOXP3 cDNA. In some embodiments, the cell surface receptor lacks a functional intracellular signal transduction domain or is unable to mediate intracellular signal transduction. In some embodiments, the cell surface receptor is selected from EGFR, HER2, CD2, and H-2K. kThe group consists of IL-5Rα and its derivatives or mutants. In some embodiments, the chimeric receptor includes 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-β, citrullinated vimentin (CV), TNF-like ligand 1A (TL1A), CD2, CD23 (IgE receptor), IL-6R, IL-4Rα, IL-12R, IL-13Rα1, IL-13Rα2, IL-15R, IL-23R, CD19, CD20, CD22, CD3, CD4, CD5, IFN-γ, IgE, IL-1α, IL-1β, 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 v1, GLP2R, JAK1, C5, and TNF-α. 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 operatively linked to the same promoter. In some embodiments, the promoter is an inducible promoter or a constitutive promoter. In some embodiments, the promoter is a cell-specific promoter. In some embodiments, the promoter is specific to 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., a P2A peptide), or a nucleic acid encoding a peptide that induces ribosome jumping. In some embodiments, the expression cassette also contains 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 contains nucleic acids 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 FOXP3 protein isotype lacking exon 2 containing the amino acid sequence of SEQ ID NO: 21 or 23.

[0137] In some embodiments, this document provides an expression cassette containing nucleic acids 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 contains an intron sequence 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 isotype and a FOXP3 protein isotype lacking exon 2; and d) a nucleic acid encoding a cell surface receptor. In some embodiments, this document provides an expression cassette comprising nucleic acids 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 induces ribosome jumping; d) a modified nucleic acid encoding a FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein contains an intron sequence 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 an exon 2-deficient FOXP3 protein isoform; e) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that induces ribosome jumping; 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 either a full-length FOXP3 protein or an exon 2-deficient FOXP3 protein isoform. In some embodiments, the nucleic acid encoding the FOXP3 protein encodes two FOXP3 protein isotypes: a full-length FOXP3 protein and a FOXP3 protein isotype lacking exon 2. In some embodiments, the nucleic acid encoding the FOXP3 protein, containing an intron sequence flanking the nucleic acid encoding exon 2, is alternatively spliced ​​to produce both a full-length FOXP3 protein isotype and a FOXP3 protein isotype lacking exon 2. In some embodiments, the nucleic acid encoding the FOXP3 protein is alternatively spliced ​​to produce both a full-length FOXP3 protein isotype and a FOXP3 protein isotype lacking exon 2, wherein the FOXP3 protein comprises the amino acid sequences SEQ ID NO: 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 NO: 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 FOXP3 does not contain intron sequences other than those flanking exon 2.In some embodiments, the nucleic acid encoding the FOXP3 protein is FOXP3 cDNA. In some embodiments, the cell surface receptor lacks a functional intracellular signal transduction domain or is unable to mediate intracellular signal transduction. In some embodiments, the cell surface receptor is selected from EGFR, HER2, CD2, and H-2K. kThe group consists of IL-5Rα and its derivatives or mutants. In some embodiments, the chimeric receptor includes 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-β, citrullinated vimentin (CV), TNF-like ligand 1A (TL1A), CD2, CD23 (IgE receptor), IL-6R, IL-4Rα, IL-12R, IL-13Rα1, IL-13Rα2, IL-15R, IL-23R, CD19, CD20, CD22, CD3, CD4, CD5, IFN-γ, IgE, IL-1α, IL-1β, 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 v1, GLP2R, JAK1, C5, and TNF-α. 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 operatively linked to the same promoter. In some embodiments, the promoter is an inducible promoter or a constitutive promoter. In some embodiments, the promoter is a cell-specific promoter. In some embodiments, the promoter is specific to 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., a P2A peptide), or a nucleic acid encoding a peptide that induces ribosome jumping. In some embodiments, the expression cassette also contains 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 NO: 73-76); c) a nucleic acid encoding a self-cleaved 2A peptide (e.g., P2A peptide), wherein the 2A sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2-10 and 89-91; d) a modified nucleic acid encoding a FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein comprises an intron sequence 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 isotype and a FOXP3 protein isotype lacking exon 2, wherein the FOXP3 protein comprises an amino acid sequence of SEQ ID NO: 20 and 21; e) a nucleic acid encoding a self-cleaved 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 NO: 73-76. The amino acid sequence of the group consisting of NO:2-10 and 89-91; and f) the nucleic acid encoding a cell surface receptor comprising the amino acid sequence selected from the group consisting of SEQ ID NO: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 NO: 73-76); c) a nucleic acid encoding a self-cleaving 2A peptide (e.g., a P2A peptide), wherein the 2A sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 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 an intron sequence flanking the modified nucleic acid encoding exon 2, wherein the nucleic acid encoding the FOXP3 protein is alternatively spliced ​​to produce a full-length FOXP3 protein isotype and a FOXP3 protein isotype lacking exon 2, wherein the FOXP3 protein comprises SEQ ID NO: 73-76. The amino acid sequences of NO: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 NO: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 NO:17-19.

[0138] In some embodiments, this document provides an expression cassette comprising nucleic acids encoding, from 5' to 3': a) a promoter; b) a nucleic acid encoding a chimeric receptor; c) a modified nucleic acid encoding the full-length FOXP3 protein; and d) a nucleic acid encoding a cell surface receptor. In some embodiments, this document provides an expression cassette comprising nucleic acids 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 ribosome-jumping peptide; d) a modified nucleic acid encoding the full-length FOXP3 protein; e) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a ribosome-jumping peptide; and f) a nucleic acid encoding a cell surface receptor. In some embodiments, the nucleic acid encoding the FOXP3 protein encodes only one FOXP3 isotype, wherein the FOXP3 protein isotype is the 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 signal transduction domain or is unable to mediate intracellular signal transduction. In some embodiments, the cell surface receptor is selected from EGFR, HER2, CD2, and H-2K. kThe group consists of IL-5Rα and its derivatives or mutants. In some embodiments, the chimeric receptor includes 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-β, citrullinated vimentin (CV), TNF-like ligand 1A (TL1A), CD2, CD23 (IgE receptor), IL-6R, IL-4Rα, IL-12R, IL-13Rα1, IL-13Rα2, IL-15R, IL-23R, CD19, CD20, CD22, CD3, CD4, CD5, IFN-γ, IgE, IL-1α, IL-1β, 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 v1, GLP2R, JAK1, C5, and TNF-α. 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 operatively linked to the same promoter. In some embodiments, the promoter is an inducible promoter or a constitutive promoter. In some embodiments, the promoter is a cell-specific promoter. In some embodiments, the promoter is specific to 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., a P2A peptide), or a nucleic acid encoding a peptide that induces ribosome jumping. In some embodiments, the expression cassette also contains 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 contains nucleic acids 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 containing 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.

[0139] In some embodiments, this document provides an expression cassette containing nucleic acids 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 isotype lacking exon 2; and d) a nucleic acid encoding a cell surface receptor. In some embodiments, this document provides an expression cassette containing nucleic acids 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 induces ribosome jumping; d) a modified nucleic acid encoding a FOXP3 protein isotype lacking exon 2; e) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that induces ribosome jumping; and f) a nucleic acid encoding a cell surface receptor. In some embodiments, the nucleic acid encoding the FOXP3 protein encodes only one FOXP3 isotype, wherein the FOXP3 protein isotype isotype lacking exon 2. In some embodiments, the modified nucleic acid encoding the FOXP3 protein isotype 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 the FOXP3 protein is FOXP3 cDNA. In some embodiments, the cell surface receptor lacks a functional intracellular signal transduction domain or is unable to mediate intracellular signal transduction. In some embodiments, the cell surface receptor is selected from EGFR, HER2, CD2, and H-2K. kThe group consists of IL-5Rα and its derivatives or mutants. In some embodiments, the chimeric receptor includes 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-β, citrullinated vimentin (CV), TNF-like ligand 1A (TL1A), CD2, CD23 (IgE receptor), IL-6R, IL-4Rα, IL-12R, IL-13Rα1, IL-13Rα2, IL-15R, IL-23R, CD19, CD20, CD22, CD3, CD4, CD5, IFN-γ, IgE, IL-1α, IL-1β, 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 v1, GLP2R, JAK1, C5, and TNF-α. 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 operatively linked to the same promoter. In some embodiments, the promoter is an inducible promoter or a constitutive promoter. In some embodiments, the promoter is a cell-specific promoter. In some embodiments, the promoter is specific to 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., a P2A peptide), or a nucleic acid encoding a peptide that induces ribosome jumping. In some embodiments, the expression cassette also contains 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 contains nucleic acids 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 FOXP3 protein isotype lacking exon 2 containing 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.

[0140] In some embodiments, this document provides an expression cassette containing nucleic acids 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 contains an intron sequence 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 isotype and a FOXP3 protein isotype lacking exon 2; c) a nucleic acid encoding a chimeric receptor; and d) a nucleic acid encoding a cell surface receptor. In some embodiments, this document provides an expression cassette comprising nucleic acids 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 contains an intron sequence 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 an exon 2-deficient FOXP3 protein isoform; c) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that induces ribosome jumping; 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 induces ribosome jumping; 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 either a full-length FOXP3 protein or an exon 2-deficient FOXP3 protein isoform. In some embodiments, the nucleic acid encoding the FOXP3 protein encodes two FOXP3 protein isotypes: a full-length FOXP3 protein and a FOXP3 protein isotype lacking exon 2. In some embodiments, the nucleic acid encoding the FOXP3 protein, containing an intron sequence flanking the nucleic acid encoding exon 2, is alternatively spliced ​​to produce both a full-length FOXP3 protein isotype and a FOXP3 protein isotype lacking exon 2. In some embodiments, the nucleic acid encoding the FOXP3 protein is alternatively spliced ​​to produce both a full-length FOXP3 protein isotype and a FOXP3 protein isotype lacking exon 2, wherein the FOXP3 protein comprises the amino acid sequences SEQ ID NO: 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 NO: 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 FOXP3 does not contain intron sequences other than those flanking exon 2.In some embodiments, the nucleic acid encoding the FOXP3 protein is FOXP3 cDNA. In some embodiments, the cell surface receptor lacks a functional intracellular signal transduction domain or is unable to mediate intracellular signal transduction. In some embodiments, the cell surface receptor is selected from EGFR, HER2, CD2, and H-2K. kThe group consists of IL-5Rα and its derivatives or mutants. In some embodiments, the chimeric receptor includes 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-β, citrullinated vimentin (CV), TNF-like ligand 1A (TL1A), CD2, CD23 (IgE receptor), IL-6R, IL-4Rα, IL-12R, IL-13Rα1, IL-13Rα2, IL-15R, IL-23R, CD19, CD20, CD22, CD3, CD4, CD5, IFN-γ, IgE, IL-1α, IL-1β, 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 v1, GLP2R, JAK1, C5, and TNF-α. 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 operatively linked to the same promoter. In some embodiments, the promoter is an inducible promoter or a constitutive promoter. In some embodiments, the promoter is a cell-specific promoter. In some embodiments, the promoter is specific to 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., a P2A peptide), or a nucleic acid encoding a peptide that induces ribosome jumping. In some embodiments, the expression cassette also contains 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, wherein the nucleic acid encoding the FOXP3 protein contains an intron sequence 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 isotype and a FOXP3 protein isotype lacking exon 2, wherein the FOXP3 protein contains the amino acid sequences of SEQ ID NO: 20 and 21; c) a nucleic acid encoding a self-cleaving 2A peptide (e.g., a P2A peptide), wherein the 2A peptide contains an amino acid sequence selected from the group consisting of SEQ ID NO: 2-10 and 89-91; d) a chimeric receptor (e.g., a chimeric receptor containing an antigen-binding domain, the antigen-binding domain containing an amino acid sequence selected from SEQ ID NO: 20 and 21). Nucleic acid encoding the amino acid sequence of the group consisting of NO:73-76; e) nucleic acid encoding a self-cleaved 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 NO:2-10 and 89-91; and f) nucleic acid encoding a cell surface receptor comprising an amino acid sequence selected from the group consisting of SEQ ID NO: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 contains an intron sequence 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 isotype and a FOXP3 protein isotype lacking exon 2, wherein the FOXP3 protein contains the amino acid sequences of SEQ ID NO: 22 and 23; c) a nucleic acid encoding a self-cleaving 2A peptide (e.g., a P2A peptide), wherein the 2A peptide contains an amino acid sequence selected from the group consisting of SEQ ID NO: 2-10 and 89-91; d) a chimeric receptor (e.g., a chimeric receptor containing an antigen-binding domain, the antigen-binding domain containing an amino acid sequence selected from SEQ ID NO: 22 and 23). Nucleic acid encoding the amino acid sequence of the group consisting of NO:73-76; e) nucleic acid encoding a self-cleaved 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 NO:2-10 and 89-91; and f) nucleic acid encoding a cell surface receptor comprising an amino acid sequence selected from the group consisting of SEQ ID NO:17-19.

[0141] In some embodiments, this document provides an expression cassette comprising nucleic acids encoding from 5' to 3': a) a promoter; b) a modified nucleic acid encoding the 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, this document provides an expression cassette comprising nucleic acids encoding from 5' to 3': a) a promoter; b) a modified nucleic acid encoding the full-length FOXP3 protein; c) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a ribosome-jumping peptide; 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 ribosome-jumping peptide; and f) a nucleic acid encoding a cell surface receptor. In some embodiments, the nucleic acid encoding the FOXP3 protein encodes only one FOXP3 isotype, wherein the FOXP3 protein isotype is the 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 signal transduction domain or is unable to mediate intracellular signal transduction. In some embodiments, the cell surface receptor is selected from EGFR, HER2, CD2, and H-2K. kThe group consists of IL-5Rα and its derivatives or mutants. In some embodiments, the chimeric receptor includes 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-β, citrullinated vimentin (CV), TNF-like ligand 1A (TL1A), CD2, CD23 (IgE receptor), IL-6R, IL-4Rα, IL-12R, IL-13Rα1, IL-13Rα2, IL-15R, IL-23R, CD19, CD20, CD22, CD3, CD4, CD5, IFN-γ, IgE, IL-1α, IL-1β, 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 v1, GLP2R, JAK1, C5, and TNF-α. 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 operatively linked to the same promoter. In some embodiments, the promoter is an inducible promoter or a constitutive promoter. In some embodiments, the promoter is a cell-specific promoter. In some embodiments, the promoter is specific to 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., a P2A peptide), or a nucleic acid encoding a peptide that induces ribosome jumping. In some embodiments, the expression cassette also contains 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 implementations, the expression cassette contains nucleic acids encoding from 5' to 3': a) a promoter; b) a modified nucleic acid encoding a full-length FOXP3 protein containing the amino acid sequence of SEQ ID NO: 20 or 22; 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.

[0142] In some embodiments, this document provides an expression cassette comprising nucleic acids encoding from 5' to 3': a) a promoter; b) a modified nucleic acid encoding a FOXP3 protein isotype lacking exon 2; c) a nucleic acid encoding a chimeric receptor; and d) a nucleic acid encoding a cell surface receptor. In some embodiments, this document provides an expression cassette comprising nucleic acids encoding from 5' to 3': a) a promoter; b) a modified nucleic acid encoding a FOXP3 protein isotype lacking exon 2; c) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a ribosome-jumping peptide; 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 ribosome-jumping peptide; and f) a nucleic acid encoding a cell surface receptor. In some embodiments, the nucleic acid encoding the FOXP3 protein encodes only one FOXP3 isotype, wherein the FOXP3 protein isotype isotype lacking exon 2. In some embodiments, the modified nucleic acid encoding the FOXP3 protein isotype 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 the FOXP3 protein is FOXP3 cDNA. In some embodiments, the cell surface receptor lacks a functional intracellular signal transduction domain or is unable to mediate intracellular signal transduction. In some embodiments, the cell surface receptor is selected from EGFR, HER2, CD2, and H-2K. kThe group consists of IL-5Rα and its derivatives or mutants. In some embodiments, the chimeric receptor includes 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-β, citrullinated vimentin (CV), TNF-like ligand 1A (TL1A), CD2, CD23 (IgE receptor), IL-6R, IL-4Rα, IL-12R, IL-13Rα1, IL-13Rα2, IL-15R, IL-23R, CD19, CD20, CD22, CD3, CD4, CD5, IFN-γ, IgE, IL-1α, IL-1β, 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 v1, GLP2R, JAK1, C5, and TNF-α. 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 operatively linked to the same promoter. In some embodiments, the promoter is an inducible promoter or a constitutive promoter. In some embodiments, the promoter is a cell-specific promoter. In some embodiments, the promoter is specific to 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., a P2A peptide), or a nucleic acid encoding a peptide that induces ribosome jumping. In some embodiments, the expression cassette also contains 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 contains nucleic acids encoding from 5' to 3': a) a promoter; b) a modified nucleic acid encoding a FOXP3 protein isotype lacking exon 2 containing 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.

[0143] E. Separating element In some embodiments, the expression cassettes provided herein comprise polycistronic expression systems. Separating elements are part of such polycistronic systems and allow the components of the expression cassette to be produced as independent proteins. Expression of gene products in polycistronic systems is co-regulated by the same promoter and terminator, thus minimizing differential expression and competition among transcription factors. Furthermore, controlling additional gene products via a single promoter and terminator reduces the required size of the transgenic expression system, improving cloning and transfection efficiency. Polycistronic expression can be achieved using various mechanisms, most commonly virus-derived sequences such as internal ribosome entry sites (IRES) sequences or 2A sequences.

[0144] In some embodiments, the components of the expression cassette are translated into individual polypeptides. In some embodiments, the components of the expression cassette are translated into individual polypeptides due to ribosome jumping. In some embodiments, the components of the expression cassette are translated into a single polypeptide, which is then further cleaved into individual polypeptides, i.e., undergoing proteolytic cleavage. In some embodiments, the presence of the separation element does not affect gene expression levels in cells (e.g., T cells, such as regulatory T (Treg) cells) that transduce and express any of the expression cassettes described herein.

[0145] Self-cleaving peptides were initially thought to function via proteolytic cleavage; however, 2A self-cleaving peptides specifically function by directly interfering with the translation process (by causing ribosomes to skip peptide bond formation). Therefore, nucleic acids encoding self-cleaving peptides can be used to isolate two or more components of any expression cassette described herein, while allowing the initiation of translation of two genes under the control of the same promoter and termination sequences.

[0146] The internal ribosome entry site (IRES) sequence is an internal ribosome entry site that allows the translation of two genes to be initiated under the control of the same promoter, resulting in the expression of two separate proteins. The IRES sequence allows ribosomes to initiate translation at the second open reading frame within the mRNA strand, and thus can be used to express multiple completely separate proteins. In some embodiments, the components of the expression cassette are separated from another component via the IRES.

[0147] In some embodiments, the expression cassette described herein further includes a cleavable adapter. In some embodiments, the cleavable adapter is a 2A peptide. In some embodiments, the expression cassette containing the FOXP3 protein includes a nucleic acid encoding a P2A adapter located at the junction of the FOXP3 protein and cell surface receptors (such as EGFR, HER2, CD2, H-2K). kBetween the nucleic acids of IL-5Rα or its derivatives or mutants. In some embodiments, the expression cassette containing the FOXP3 protein contains a nucleic acid encoding a P2A adapter, which is located between the nucleic acid encoding the FOXP3 protein and the chimeric receptor. In some embodiments, the expression cassette containing the FOXP3 protein contains a nucleic acid encoding a P2A adapter, which is located between the nucleic acid encoding the chimeric receptor and the cell surface receptor (such as EGFR, HER2, CD2, H-2K). k Between nucleic acids of IL-5Rα or its derivatives or mutants. In some embodiments, 2A-like sequences or “peptide-jumping” 2A sequences are derived from, for example, many different viruses, including, for example, those from *Echinochloa crus-galli*. These sequences are sometimes also referred to as “peptide-jumping sequences.” When this type of sequence is located within a cistron between two polypeptides to be separated, the ribosome appears to skip the peptide bond; in the case of the *Echinochloa crus-galli* sequence, the bond between the Gly and Pro amino acids at the C-terminus “PGP” is omitted. This can leave two or three polypeptides, such as 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 using this sequence, the 5' polypeptide encoding the 2A sequence can be terminated at the C-terminus with additional amino acids, including Gly residues and any upstream residues of the 2A sequence. The 3' polypeptide encoding the 2A sequence can be terminated at the N-terminus with additional amino acids, including Pro residues and any downstream residues following the 2A sequence. In some embodiments, the cleavable linker is a 2A peptide derived from porcine swine cisvirus-1 (P2A). In some implementations, the 2A co-translated sequence is a 2A-like sequence. In some implementations, the 2A co-translated sequence is T2A (Melilothorax virus 2A), F2A (Foot-and-mouth disease virus 2A), P2A (Swine disease virus-1 2A), BmCPV 2A (Polyhemotropic virus 2A), BmIFV 2A (…). silkworm The 2A co-translated sequence is either equine rhinitis virus 2A (E2A) or E2A (Equine rhinitis virus A 2A). In some embodiments, the 2A co-translated sequence is T2A-GSG, F2A-GSG, P2A-GSG, or E2A-GSG. In some embodiments, the 2A co-translated sequence is selected from the group consisting of T2A, P2A, and F2A. “Cleavable adapter” means that the adapter is cleaved by any means, including, for example, non-enzymatic (such as peptide jumping) or enzymatic (see, e.g., Donnelly, ML (2001), J. Gen. Virol. 82:1013-25, which is incorporated herein by reference in its entirety). In one particular embodiment, P2A comprises (or consists of) the sequences disclosed herein. In one particular embodiment, P2A comprises (or consists of) the sequences disclosed herein (e.g., the sequences disclosed in the examples below).

[0148] In some embodiments, the separating element is a 2A linker. In some embodiments, the 2A linker is selected from the group consisting of: P2A sequences, T2A sequences, E2A sequences, F2A sequences, their self-cleaving derivatives or mutants, and any combination thereof. In some embodiments, the 2A sequence is selected from the group consisting of SEQ ID NO:2-10 and 89-91. In some embodiments, the 2A linker is a P2A linker comprising the amino acid sequence of SEQ ID NO:2 (ATNFSLLKQAGDVEENPGP), SEQ ID NO:3 (SGATNFSLLKQAGDVEENPGP), or SEQ ID NO:4 (GSGATNFSLLKQAGDVEENPGP). In some embodiments, the 2A linker also comprises a GSG amino acid sequence at the amino terminus of the peptide; in other embodiments, the 2A linker comprises a GSGPR amino acid sequence (SEQ ID NO:24) at the amino terminus of the peptide. Therefore, the term “2A” can refer to the 2A sequence in the examples described herein, or it can also refer to the 2A sequence as listed herein, which also includes the 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.

[0149] Table 4. Exemplary 2A peptide sequences.

[0150] II. Cells This disclosure provides an adoptive cell immunotherapy composition comprising genetically modified cell preparations as described herein, such as genetically modified immune cells, such as lymphocytes. These cells are, for example, pluripotent cells, such as hematopoietic stem cells, progenitor or precursor cells of hematopoietic lineages, or immune cells (e.g., autologous or allogeneic T cells, natural killer (NK) cells, dendritic cells, 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, the pluripotent cells and multipotent cells are differentiated in vitro into the desired cell type prior to implantation into a patient.

[0151] In some alternatives, the genetically modified cell preparation is a T lymphocyte preparation. In some embodiments, the T lymphocyte preparation comprises CD4+ T cells having a chimeric receptor and an exogenous FOXP3 protein as described herein, the cells comprising an extracellular antibody variable domain, a spacer region, a transmembrane domain, and an intracellular signaling domain of a T cell receptor that are specific to ligands associated with a disease or condition. In some alternatives, the chimeric receptor-modified T cell population of this disclosure persists in vivo for at least about 3 days or longer. In some alternatives, each of these populations may be combined with each other or with other cell types to provide a composition.

[0152] In some alternatives, the genetically modified cell preparation is a Treg cell preparation. Tregs include both natural and induced allotypes. Natural Tregs (nTregs) are cells that 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 this disclosure. In some embodiments, CD4+ T cells are used to prepare Tregs expressing exogenous FOXP3 protein using the methods and compositions of this disclosure.

[0153] In some embodiments, the cells expressing the exogenous FOXP3 protein of this disclosure are not naturally occurring. In some embodiments, the cells are immune cells. In some embodiments, the cells are lymphocytes. In some embodiments, the cells are T cells. In some embodiments, the cells are regulatory T cells (Tregs). In some embodiments, the Tregs are compared with control Tregs lacking the exogenous FOXP3 protein. For example, in other embodiments, the Tregs are compared with control Tregs transduced with an empty vector lacking the FOXP3 protein and / or untransduced control Tregs.

[0154] Therefore, this document provides engineered regulatory T cells (Tregs) comprising an exogenous expression cassette containing a modified nucleic acid encoding a FOXP3 protein operatively linked to a promoter, wherein the nucleic acid encoding FOXP3 is modified to produce a full-length FOXP3 protein isotype and an alternative splicing isotype lacking one or more exons. In some embodiments, the nucleic acid encoding the FOXP3 protein contains an intron sequence flanking the nucleic acid encoding exon 2. In some embodiments, the nucleic acid encoding the FOXP3 protein containing the intron sequence flanking the nucleic acid encoding exon 2 is alternatively spliced ​​to produce a full-length FOXP3 protein isotype and a FOXP3 protein isotype 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 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 operatively 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., a P2A peptide), or a nucleic acid encoding a peptide that induces ribosome jumping. In some embodiments, the engineered Treg comprises an exogenous expression cassette that 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 implementations, the engineered Treg includes an exogenous expression cassette containing nucleic acids encoding: a) a promoter; b) a modified nucleic acid encoding a FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein contains an intron sequence flanking a nucleic acid encoding exon 2, wherein the FOXP3 protein contains the amino acid sequences of SEQ ID NO: 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 cell surface receptor.In some implementations, the engineered Treg includes an exogenous expression cassette containing nucleic acids encoding: a) a promoter; b) a modified nucleic acid encoding a FOXP3 protein linked to the detection tag, wherein the nucleic acid encoding the FOXP3 protein contains an intron sequence flanking the nucleic acid encoding exon 2, wherein the FOXP3 protein contains the amino acid sequences of SEQ ID NO: 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.

[0155] In some embodiments, this document provides engineered Tregs comprising exogenous expression cassettes containing nucleic acids 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 contains an intron sequence flanking a nucleic acid encoding exon 2; c) an IRES or a nucleic acid encoding a self-cleaving peptide or a peptide that induces ribosome jumping; and d) a nucleic acid encoding a cell surface receptor. In some embodiments, the nucleic acid encoding the FOXP3 protein containing an intron sequence flanking a nucleic acid encoding exon 2 is subjected to alternative splicing to produce a full-length FOXP3 protein isotype and an exon 2-deficient FOXP3 protein isotype. 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 FOXP3 does not contain intron sequences other than those flanking exon 2. In some embodiments, the nucleic acid encoding the FOXP3 protein is FOXP3 cDNA. In some embodiments, the cell surface receptor lacks a functional intracellular signal transduction domain or is unable to mediate intracellular signal transduction. In some embodiments, the cell surface receptor is selected from EGFR, HER2, CD2, and H-2K. kThe group consists of IL-5Rα and its derivatives or mutants. In some embodiments, the nucleic acid encoding the FOXP3 protein and the nucleic acid encoding the cell surface receptor are operatively linked to the same promoter. In some embodiments, the promoter is an inducible promoter or a constitutive promoter. In some embodiments, the promoter is a cell-specific promoter. In some embodiments, the promoter is specific to 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 each separated by an IRES, a nucleic acid encoding a self-cleaving peptide (e.g., a P2A peptide), or a nucleic acid encoding a peptide that induces ribosome jumping. In some embodiments, the engineered Treg comprises an exogenous expression cassette that also 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 engineered Treg comprises an exogenous expression cassette containing nucleic acids 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 contains an intron sequence flanking a nucleic acid encoding exon 2, wherein the FOXP3 protein contains the amino acid sequences of SEQ ID NO: 20 and 21; c) a nucleic acid encoding a self-cleaved P2A peptide; and d) a nucleic acid encoding a cell surface. In some embodiments, the engineered Treg comprises an exogenous expression cassette containing nucleic acids 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 contains an intron sequence flanking a nucleic acid encoding exon 2, wherein the FOXP3 protein contains the amino acid sequences of SEQ ID NO: 22 and 23; c) a nucleic acid encoding a self-cleaved P2A peptide; and d) a nucleic acid encoding a cell surface.

[0156] In some embodiments, this document provides engineered Tregs comprising exogenous expression cassettes containing nucleic acids 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 induces ribosome jumping; and d) a modified nucleic acid encoding a FOXP3 protein, wherein the modified nucleic acid encoding the FOXP3 protein contains an intron sequence flanking the nucleic acid encoding exon 2. In some embodiments, the nucleic acid encoding the FOXP3 protein containing the intron sequence flanking the nucleic acid encoding exon 2 is subjected to alternative splicing to produce a full-length FOXP3 protein isotype and an exon 2-deficient FOXP3 protein isotype. 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 FOXP3 does not contain intron sequences other than those flanking exon 2. In some embodiments, the nucleic acid encoding the FOXP3 protein is FOXP3 cDNA. In some embodiments, the cell surface receptor lacks a functional intracellular signal transduction domain or is unable to mediate intracellular signal transduction. In some embodiments, the cell surface receptor is selected from EGFR, HER2, CD2, and H-2K. kThe group consists of IL-5Rα and its derivatives or mutants. In some embodiments, the nucleic acid encoding the FOXP3 protein and the nucleic acid encoding the cell surface receptor are operatively linked to the same promoter. In some embodiments, the promoter is an inducible promoter or a constitutive promoter. In some embodiments, the promoter is a cell-specific promoter. In some embodiments, the promoter is specific to 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 each separated by an IRES, a nucleic acid encoding a self-cleaving peptide (e.g., a P2A peptide), or a nucleic acid encoding a peptide that induces ribosome jumping. In some embodiments, the engineered Treg comprises an exogenous expression cassette that also 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 engineered Treg comprises an exogenous expression cassette containing nucleic acids 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 contains an intron sequence flanking the nucleic acid encoding exon 2, wherein the FOXP3 protein contains the amino acid sequences of SEQ ID NO: 20 and 21. In some implementations, the engineered Treg comprises an exogenous expression cassette containing nucleic acids 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 contains an intron sequence flanking the nucleic acid encoding exon 2, wherein the FOXP3 protein contains the amino acid sequences of SEQ ID NO: 22 and 23.

[0157] In some embodiments, this document provides engineered Tregs comprising exogenous expression cassettes containing nucleic acids 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 induces ribosome jumping; 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 induces ribosome jumping; and f) a modified nucleic acid encoding a FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein contains an intron sequence flanking the nucleic acid encoding exon 2. In some embodiments, the nucleic acid encoding the FOXP3 protein containing the intron sequence flanking the nucleic acid encoding exon 2 is subjected to alternative splicing to produce a full-length FOXP3 protein isotype and an exon 2-deficient FOXP3 protein isotype. In some embodiments, the FOXP3 protein is linked to a detection tag. In some embodiments, the detection tag is attached to the N-terminus of the FOXP3 protein. In some embodiments, the detection tag is attached to the C-terminus of the FOXP3 protein. In some embodiments, the nucleic acid encoding FOXP3 does not contain intron sequences other than those flanking exon 2. In some embodiments, the nucleic acid encoding the FOXP3 protein is FOXP3 cDNA. In some embodiments, the cell surface receptor lacks a functional intracellular signal transduction domain or is unable to mediate intracellular signal transduction. In some embodiments, the cell surface receptor is selected from EGFR, HER2, CD2, and H-2K. kThe group consists of IL-5Rα and its derivatives or mutants. In some embodiments, the chimeric receptor includes 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-β, citrullinated vimentin (CV), TNF-like ligand 1A (TL1A), CD2, CD23 (IgE receptor), IL-6R, IL-4Rα, IL-12R, IL-13Rα1, IL-13Rα2, IL-15R, IL-23R, CD19, CD20, CD22, CD3, CD4, CD5, IFN-γ, IgE, IL-1α, IL-1β, 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 v1, GLP2R, JAK1, C5, and TNF-α. 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 operatively linked to the same promoter. In some embodiments, the promoter is an inducible promoter or a constitutive promoter. In some embodiments, the promoter is a cell-specific promoter. In some embodiments, the promoter is specific to 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., a P2A peptide), or a nucleic acid encoding a peptide that induces ribosome jumping. In some embodiments, the engineered Treg comprises an exogenous expression cassette that also 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 engineered Treg comprises an exogenous expression cassette containing nucleic acids 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, wherein the nucleic acid encoding the FOXP3 protein contains an intron sequence flanking the nucleic acid encoding exon 2, wherein the FOXP3 protein contains the amino acid sequences of SEQ ID NO: 20 and 21.In some implementations, the engineered Treg includes an exogenous expression cassette containing nucleic acids 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 contains an intron sequence flanking the nucleic acid encoding exon 2, and wherein the FOXP3 protein contains the amino acid sequences of SEQ ID NO: 22 and 23.

[0158] In some embodiments, this document provides engineered Tregs comprising exogenous expression cassettes containing nucleic acids 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 induces ribosome jumping; d) a modified nucleic acid encoding a FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein contains an intron sequence 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 induces ribosome jumping; and f) a nucleic acid encoding a cell surface receptor. In some embodiments, the nucleic acid encoding the FOXP3 protein containing an intron sequence flanking the nucleic acid encoding exon 2 is alternatively spliced ​​to produce a full-length FOXP3 protein isotype and an exon 2-deficient FOXP3 protein isotype. In some embodiments, the FOXP3 protein is linked to a detection tag. In some embodiments, the detection tag is attached to the N-terminus of the FOXP3 protein. In some embodiments, the detection tag is attached to the C-terminus of the FOXP3 protein. In some embodiments, the nucleic acid encoding FOXP3 does not contain intron sequences other than those flanking exon 2. In some embodiments, the nucleic acid encoding the FOXP3 protein is FOXP3 cDNA. In some embodiments, the cell surface receptor lacks a functional intracellular signal transduction domain or is unable to mediate intracellular signal transduction. In some embodiments, the cell surface receptor is selected from EGFR, HER2, CD2, and H-2K. kThe group consists of IL-5Rα and its derivatives or mutants. In some embodiments, the chimeric receptor includes 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-β, citrullinated vimentin (CV), TNF-like ligand 1A (TL1A), CD2, CD23 (IgE receptor), IL-6R, IL-4Rα, IL-12R, IL-13Rα1, IL-13Rα2, IL-15R, IL-23R, CD19, CD20, CD22, CD3, CD4, CD5, IFN-γ, IgE, IL-1α, IL-1β, 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 v1, GLP2R, JAK1, C5, and TNF-α. 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 operatively linked to the same promoter. In some embodiments, the promoter is an inducible promoter or a constitutive promoter. In some embodiments, the promoter is a cell-specific promoter. In some embodiments, the promoter is specific to 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., a P2A peptide), or a nucleic acid encoding a peptide that induces ribosome jumping. In some embodiments, the engineered Treg comprises an exogenous expression cassette that also 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 implementations, the engineered Treg comprises an exogenous expression cassette containing nucleic acids 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 FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein contains an intron sequence flanking the nucleic acid encoding exon 2, wherein the FOXP3 protein contains the amino acid sequences of SEQ ID NO: 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 implementations, the engineered Treg includes an exogenous expression cassette containing nucleic acids 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 FOXP3 protein linked to a detection tag, wherein the nucleic acid encoding the FOXP3 protein contains an intron sequence flanking the nucleic acid encoding exon 2, wherein the FOXP3 protein contains the amino acid sequences of SEQ ID NO: 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.

[0159] In some embodiments, this document provides engineered Tregs comprising exogenous expression cassettes containing nucleic acids 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 contains an intron sequence flanking a 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 induces ribosome jumping; 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 induces ribosome jumping; and f) a nucleic acid encoding a cell surface receptor. In some embodiments, the nucleic acid encoding the FOXP3 protein containing an intron sequence flanking a nucleic acid encoding exon 2 is alternatively spliced ​​to produce a full-length FOXP3 protein isotype and an exon 2-deficient FOXP3 protein isotype. In some embodiments, the FOXP3 protein is linked to a detection tag. In some embodiments, the detection tag is attached to the N-terminus of the FOXP3 protein. In some embodiments, the detection tag is attached to the C-terminus of the FOXP3 protein. In some embodiments, the nucleic acid encoding FOXP3 does not contain intron sequences other than those flanking exon 2. In some embodiments, the nucleic acid encoding the FOXP3 protein is FOXP3 cDNA. In some embodiments, the cell surface receptor lacks a functional intracellular signal transduction domain or is unable to mediate intracellular signal transduction. In some embodiments, the cell surface receptor is selected from EGFR, HER2, CD2, and H-2K. kThe group consists of IL-5Rα and its derivatives or mutants. In some embodiments, the chimeric receptor includes 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-β, citrullinated vimentin (CV), TNF-like ligand 1A (TL1A), CD2, CD23 (IgE receptor), IL-6R, IL-4Rα, IL-12R, IL-13Rα1, IL-13Rα2, IL-15R, IL-23R, CD19, CD20, CD22, CD3, CD4, CD5, IFN-γ, IgE, IL-1α, IL-1β, 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 v1, GLP2R, JAK1, C5, and TNF-α. 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 operatively linked to the same promoter. In some embodiments, the promoter is an inducible promoter or a constitutive promoter. In some embodiments, the promoter is a cell-specific promoter. In some embodiments, the promoter is specific to 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., a P2A peptide), or a nucleic acid encoding a peptide that induces ribosome jumping. In some embodiments, the engineered Treg comprises an exogenous expression cassette that also 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 implementations, the engineered Treg includes an exogenous expression cassette containing nucleic acids 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 contains an intron sequence flanking a nucleic acid encoding exon 2, wherein the FOXP3 protein contains the amino acid sequences of SEQ ID NO: 20 and 21; 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.In some implementations, the engineered Treg includes an exogenous expression cassette containing nucleic acids encoding from 5' to 3': a) a promoter; b) a modified nucleic acid encoding a FOXP3 protein linked to the detection tag, wherein the nucleic acid encoding the FOXP3 protein contains an intron sequence flanking the nucleic acid encoding exon 2, wherein the FOXP3 protein contains the amino acid sequences of SEQ ID NO: 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.

[0160] In some embodiments, the Tregs expressing the exogenous expression cassette of this disclosure are not naturally occurring (not nTregs and / or not iTregs). In some embodiments, the cells express one or more markers characteristic of Tregs. Treg markers include high levels of CD25 (CD25+), low levels of CD127 (low CD127), or both high CD25 and low CD127. For example, the levels of CD25 and CD127 are compared to those of non-Treg CD4+ T cells. In some embodiments, Treg cells expressing the exogenous expression cassette have a high CD25, high CD4, and low CD127 phenotype.

[0161] In some implementations, Tregs express one or more proteins associated with the Treg phenotype. In some implementations, Tregs express FOXP3. In some implementations, Tregs express endogenous FOXP3. In some implementations, Tregs further express exogenous FOXP3. The expression level of FOXP3 can be assessed using conventional methods such as Western blotting, flow cytometry, or ELISA. The expression level can also be assessed by analyzing mRNA expression using techniques such as RT-qPCR. In some implementations, FOXP3 expression is increased compared to untransduced cells. In some implementations, FOXP3 expression is increased compared to cells without an exogenous expression cassette.

[0162] In some embodiments, FOXP3 expression is increased by 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. In some embodiments, after transduction with the exogenous expression cassette, FOXP3 expression is maintained at approximately the same level as on the day of peak FOXP3 expression.

[0163] In some implementations, the expression ratio of full-length FOXP3 protein in Tregs to the exon 2-deficient FOXP3 isoform (Δ2) is approximately 90:1, 80:1, 70:1, 60:1, 50:1, 40:1, 30:1, 25:1, 20:1, 15:1, 10:1, 9:1, 8:1, 7:1, and 6:1. 1. Approximately 5:1, Approximately 4:1, Approximately 3:1, Approximately 2:1, Approximately 1:1, Approximately 1:2, Approximately 1:3, Approximately 1:4, Approximately 1:5, Approximately 1:6, Approximately 1:7, Approximately 1:8, Approximately 1:9, Approximately 1:10, Approximately 1:15, Approximately 1:20, Approximately 1:25, Approximately 1:30, Approximately 1:40, Approximately 1:50, Approximately 1:60, Approximately 1:70, Approximately 1:80, or Approximately 1:90. In some implementations, the expression ratio of full-length FOXP3 protein to the exon 2-deficient FOXP3 isoform (Δ2) within the Treg containing the expression cassette is approximately 90:1, approximately 80:1, approximately 70:1, approximately 60:1, approximately 50:1, approximately 40:1, approximately 30:1, approximately 25:1, approximately 20:1, approximately 15:1, approximately 10:1, approximately 9:1, approximately 8:1, and approximately 7:1. The ratios are approximately 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:25, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, or 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.

[0164] In some implementations, the Treg is CD4 positive (CD4+). In some implementations, the Treg is CD4+ / CD25+. In some implementations, the Treg is CD4+ / CD127 low. In some implementations, the Treg is CD4+ / CD25+ / CD127 low.

[0165] Tregs can be characterized by CD25+ expression. In some implementations, Tregs are CD25+. In some implementations, Tregs are CD4+ / CD25+. In some implementations, Tregs are CD25+ / CD127 low. In some implementations, Tregs are CD4+ / CD25+ / CD127 low.

[0166] In some implementations, Tregs express low levels of CD127 (CD127 low). In some implementations, Tregs are CD4+ / CD127 low. In some implementations, Tregs are CD25 / CD127 low. In some implementations, Tregs transduced with the exogenous expression cassettes provided herein maintain a low CD127 state after transduction.

[0167] The Treg population can be further characterized by additional biomarkers. Such biomarkers can indicate Treg phenotypes and may be absent in Tregs, or may be present or absent in subsets of Tregs (e.g., activated Tregs, tissue-resident Tregs, senescent Tregs, and dysfunctional Tregs). For example, CD27 is a biomarker for highly repressive Tregs, while CD70 marks a significant loss of regulatory function in Tregs. Exemplary biomarkers may include, but are not limited to, CD27, CD70, CD71 (transferrin receptor, TfR), FOXP3, Helios, glucocorticoid-induced TNFR-related protein (GITR), latent-related peptide (LAP), glycoprotein A repeat dominant protein (GARP), and CD137 (4-1BB). In some embodiments, the genetically modified cell preparation is a Treg cell preparation (i.e., engineered Tregs) comprising Treg cells containing an expression cassette expressing exogenous FOXP3. In some embodiments, the engineered Tregs comprise the same set of biomarkers as wild-type Tregs. In some embodiments, the engineered Treg contains the same set of biomarkers as the control Treg lacking exogenous FOXP3. In some embodiments, the engineered Treg lacks the same set of biomarkers as the wild-type Treg. In some embodiments, the engineered Treg lacks the same set of biomarkers as the control Treg lacking the expression cassette of exogenous FOXP3. In some embodiments, additional biomarkers 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.

[0168] The Treg population can be further characterized by the presence or absence of specific cytokines and / or chemokines. For example, Treg cells typically do not express or express minimally IL-2. In some embodiments, Treg cells containing an expression cassette expressing exogenous FOXP3 further express TNF-α. In some embodiments, Treg cells containing an expression cassette expressing exogenous FOXP3 further express IFN-γ. In some embodiments, Treg cells containing an expression cassette expressing exogenous FOXP3 further express IL-10. In some embodiments, Treg cells containing an expression cassette expressing exogenous FOXP3 further express TGF-β. In some embodiments, Treg cells containing an expression cassette expressing exogenous FOXP3 further express IL-35. In some embodiments, Treg cells containing an expression cassette expressing exogenous FOXP3 do not express or express minimally IL-2. In some embodiments, Treg cells containing an expression cassette expressing exogenous FOXP3 do not express or express IL-17 (i.e., IL-17A) minimally. In some embodiments, Treg cells containing an expression cassette expressing exogenous FOXP3 do not express or express IL-4 minimally. In some embodiments, Treg cells containing an expression cassette expressing exogenous FOXP3 further express the same cytokines as wild-type Tregs. In some embodiments, Treg cells containing an expression cassette expressing exogenous FOXP3 further express the same cytokines as control Tregs lacking an expression cassette expressing exogenous FOXP3. In some embodiments, Treg cells containing an expression cassette expressing exogenous FOXP3 lack the same cytokine expression as wild-type Tregs. In some embodiments, Treg cells containing an expression cassette expressing exogenous FOXP3 lack the same cytokine expression as control Tregs lacking an expression cassette expressing exogenous FOXP3. In some embodiments, Treg cells containing an expression cassette expressing exogenous FOXP3 exhibit the same cytokine phenotype as wild-type Tregs. In some embodiments, Treg cells containing an expression cassette expressing exogenous FOXP3 exhibit the same cytokine phenotype as control Tregs lacking an expression cassette expressing exogenous FOXP3. In some embodiments, the cytokines are selected from the group consisting of TNF-α, IFN-γ, TGF-β, IL-10, IL-35, IL-17 (i.e., IL-17A), IL-2, IL-4, and any combination thereof.In some implementations, compared with control Tregs lacking an expression cassette expressing exogenous FOXP3, Treg cells containing an expression cassette expressing exogenous FOXP3 produce increased levels of IL-10 (e.g., at least about 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, or higher).

[0169] In some implementations, the Treg has an immunosuppressive phenotype. In one implementation, the Treg exerts an immunosuppressive effect in an individual suffering from an immune-related condition. In some implementations, the individual is human. In some implementations, the Treg is autologous to the individual.

[0170] In some embodiments, Treg suppresses, blocks, or inhibits graft-versus-host disease (GvHD) in an individual. In one embodiment, Treg suppresses, blocks, or inhibits immune-related illnesses in an individual. In some embodiments, Treg is administered before the onset of an immune-related illness. In some embodiments, Treg is administered after the onset of an immune-related illness. 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.

[0171] Methods for measuring biomarkers used to characterize Tregs will be readily apparent to those skilled in the art. For example, Tregs or T cell populations containing Tregs can be cultured using the methods described herein. In some embodiments, the method for measuring biomarkers includes measuring Treg biomarkers selected from the group consisting of CD4+, CD25+, low CD127, and FOXP3. In some embodiments, the method further includes measuring one or more additional biomarkers and comparing the presence / absence and / or level of such additional biomarkers to the presence / absence and / or level in wild-type Tregs and / or control Tregs that do not contain any of the exogenous FOXP3 expression cassettes described herein. In some embodiments, the additional biomarkers 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 biomarkers are Treg biomarkers. In some embodiments, additional biomarkers are Treg subgroup (e.g., activated Treg) biomarkers. In some embodiments, additional biomarkers are biomarkers of non-Treg cell populations (e.g., biomarkers of conventional T cells). After culture, Tregs can be collected and stained with antibodies that recognize and bind to target Treg biomarkers (such as FOXP3, CD25, and CD127), e.g., fluorophore-conjugated antibodies, and the expression levels of such Treg biomarkers can be analyzed using fluorescence activated flow cytometry (FACS) or fluorescence microscopy. Similarly, for intracellular production of cytokines (such as TNF-α, IFN-γ, and IL-10), Tregs can be cultured using the methods described herein with (e.g.) Golgi-Stop or Golgi-Plug, and then the Tregs can be collected, fixed (e.g., with paraformaldehyde), permeabilized, and stained. Treg cytokine production can also be assessed by ELISA or ELISpot to evaluate the levels of cytokines secreted by Tregs into the culture medium. Gene expression and / or cytokine expression of Treg markers can be measured by methods such as RT-qPCR and in situ hybridization.

[0172] III. Methods for generating TREGs overexpressing FOXP3 This document also provides a method for generating Tregs that overexpress FOXP3. For example, in some embodiments, a method is provided that includes transducing Tregs with any of the expression cassettes described herein. In other embodiments, a method is provided for generating Tregs that overexpress FOXP3 that includes transducing effector T cells (Teff) with any of the expression cassettes described herein.

[0173] In some embodiments, a method for generating a Treg overexpressing FOXP3 is provided, comprising transducing the Treg with an expression cassette containing a modified nucleic acid encoding a FOXP3 protein operatively linked to a promoter, wherein the nucleic acid encoding the FOXP3 protein is modified to produce a full-length FOXP3 protein isotype and an alternative splicing isotype lacking one or more exons. In some embodiments, the nucleic acid encoding the FOXP3 protein contains an intron sequence flanking a nucleic acid encoding exon 2. In some embodiments, the nucleic acid encoding the FOXP3 protein containing an intron sequence flanking a nucleic acid encoding exon 2 is alternatively spliced ​​to produce a full-length FOXP3 protein isotype and a FOXP3 protein isotype 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 protein is FOXP3 cDNA. In some embodiments, the exogenous expression cassette further comprises nucleic acids encoding a chimeric receptor and / or a cell surface receptor. In some embodiments, the nucleic acids encoding the FOXP3 protein and the nucleic acids encoding the chimeric receptor and / or the cell surface receptor are operatively linked to the same promoter. In some embodiments, the nucleic acids encoding the FOXP3 protein and the nucleic acids encoding the chimeric receptor and / or the cell surface receptor are each separated by an IRES, a nucleic acid encoding a self-cleaving peptide (e.g., a P2A peptide), or a nucleic acid encoding a peptide that induces ribosome jumping. Therefore, in some embodiments, a method for overexpressing FOXP3 in a Treg is provided, comprising transducing the Treg with an exogenous expression cassette comprising nucleic acids encoding: (I) a promoter; (II) a nucleic acid encoding the FOXP3 protein; (III) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that induces ribosome jumping; (IV) a nucleic acid encoding a cell surface receptor; or (I) a promoter; (II) a nucleic acid encoding the FOXP3 protein; (III) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that induces ribosome jumping; (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 induces ribosome jumping; and (VI) a nucleic acid encoding a cell surface receptor, wherein the order of the FOXP3 protein, the chimeric receptor, and / or the cell surface receptor from 5' to 3' is interchangeable, and wherein the components are separated from each other by a self-cleaving peptide (e.g., P2A). In some implementations, the method also includes measuring Treg markers selected from a group consisting of CD4+, CD25+, CD127 low, and FOXP3.In some embodiments, the method further includes measuring one or more additional biomarkers and comparing the presence / absence and level of the one or more additional biomarkers to the presence / absence and level in wild-type Tregs and / or control Tregs that do not contain any of the expression cassettes expressing exogenous FOXP3 described herein. For example, a specific biomarker may indicate a Treg population, while other biomarkers may be absent in Tregs (e.g., instead indicating an effector T cell population), and the combination of the presence of a specific Treg biomarker and the absence of a specific non-Treg biomarker can be used to assess the cellular phenotype of Tregs overexpressing FOXP3 as described herein. In some embodiments, Tregs maintain the expression of at least one Treg biomarker selected from the group consisting of CD4+, CD25+, low CD127, and FOXP3. In some embodiments, Tregs maintain the expression of FOXP3 and one or more Treg biomarkers selected from the group consisting of CD4+, CD25+, and low CD127. In some embodiments, the Treg expresses additional biomarkers at similar protein and / or gene expression levels to wild-type Tregs or control Tregs, wherein the control Treg does not contain any of the expression cassettes for exogenous FOXP3 described herein. In some embodiments, the additional biomarkers 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 biomarkers are Treg biomarkers. In some embodiments, the additional biomarkers are Treg subgroup (e.g., activated Treg) biomarkers. In some embodiments, the additional biomarkers are biomarkers for non-Treg cell populations (e.g., biomarkers for conventional T cells). In some embodiments, the Treg maintains the Treg functional phenotype, characterized by one or more Treg activities selected from the group consisting of: suppressing effector T cell activity, producing inhibitory cytokines (e.g., IL-10), suppressing effector T cell cytokines (e.g., IL-2, IFNγ, TNFα), and reducing the rate of effector T cell division. In some embodiments, the Treg expresses one or more cytokines similar to those expressed by wild-type Tregs or control Tregs, wherein the control Treg does not contain any of the expression cassettes described herein that express exogenous FOXP3. In some embodiments, the Treg does not express one or more cytokines similar to those expressed by wild-type Tregs or control Tregs, wherein the control Treg does not contain any of the expression cassettes described herein that express exogenous FOXP3.In some embodiments, the one or more cytokines are selected from the group consisting of TNF-α, IFN-γ, TGF-β, IL-10, IL-35, IL-17 (i.e., IL-17A), IL-2, IL-4, and any combination thereof. In some embodiments, the Treg produces IL-10 at an increased level (e.g., at least about 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, or higher) compared to wild-type Tregs or control Tregs lacking the expression cassette of exogenous FOXP3.

[0174] In some embodiments, a method for generating Tregs is provided, comprising transducing effector T cells (Teffs) with an expression cassette containing a modified nucleic acid encoding a FOXP3 protein operatively linked to a promoter, wherein the nucleic acid encoding the FOXP3 protein is modified to produce a full-length FOXP3 protein isotype and an alternative splice isotype lacking one or more exons, wherein expression of the FOXP3 protein leads to Teff differentiation or conversion into Tregs. In some embodiments, the nucleic acid encoding the FOXP3 protein contains an intron sequence flanking a nucleic acid encoding exon 2. In some embodiments, the nucleic acid encoding the FOXP3 protein containing an intron sequence flanking a nucleic acid encoding exon 2 is alternatively spliced ​​to produce a full-length FOXP3 protein isotype and a FOXP3 protein isotype 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 nucleic acids encoding a chimeric receptor and / or a cell surface receptor. In some embodiments, the nucleic acids encoding the FOXP3 protein and the nucleic acids encoding the chimeric receptor and / or the cell surface receptor are operatively linked to the same promoter. In some embodiments, the nucleic acids encoding the FOXP3 protein and the nucleic acids encoding the chimeric receptor and / or the cell surface receptor are each separated by an IRES, a nucleic acid encoding a self-cleaving peptide (e.g., a P2A peptide), or a nucleic acid encoding a peptide that induces ribosome jumping. Therefore, in some embodiments, a method for generating Tregs is provided, which includes transducing effector T cells (Teffs) 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 self-cleaving peptide or a peptide that induces ribosome jumping; (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). The nucleic acid may encode a self-cleaving peptide or a peptide that induces ribosome jumping; (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 induces ribosome jumping; and (VI) a nucleic acid encoding a cell surface receptor; wherein the order of the FOXP3 protein, the chimeric receptor, and / or the cell surface receptor from 5' to 3' is interchangeable, and wherein the components are separated from each other by a self-cleaving peptide (e.g., P2A); and wherein expression of the FOXP3 protein leads to Teff differentiation or conversion to Treg. In some embodiments, the nucleic acid encoding the FOXP3 protein is FOXP3 cDNA.In some embodiments, the method further includes measuring Treg biomarkers selected from the group consisting of CD4+, CD25+, low CD127, and FOXP3. In some embodiments, the method further includes measuring one or more additional biomarkers and comparing the presence / absence and level of these additional biomarkers with the presence / absence and level in wild-type Tregs and / or control Tregs that do not contain any of the exogenous FOXP3 expression cassettes described herein. In some embodiments, Teffs differentiate or transform into Tregs after transduction with an exogenous expression cassette, wherein the differentiated or transformed Tregs maintain the expression of at least one Treg biomarker selected from the group consisting of CD4+, CD25+, low CD127, and FOXP3. In some embodiments, the differentiated or transformed Tregs maintain the expression of FOXP3 and one or more Treg biomarkers selected from the group consisting of CD4+, CD25+, and low CD127. In some embodiments, differentiated or transformed Tregs express additional biomarkers at similar protein and / or gene expression levels to wild-type Tregs or control Tregs that do not contain any of the expression cassettes for exogenous FOXP3 described herein. In some embodiments, the additional biomarkers 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 biomarkers are Treg biomarkers. In some embodiments, the additional biomarkers are Treg subgroup (e.g., activated Treg) biomarkers. In some embodiments, the additional biomarkers are biomarkers for non-Treg cell populations (e.g., effector T cell biomarkers). In some embodiments, the presence of one or more additional biomarkers indicates differentiated or transformed Tregs compared to the presence of one or more additional biomarkers in wild-type Tregs. In some embodiments, the absence of one or more additional markers indicates a differentiated or transformed Treg, compared to the absence of one or more additional markers in wild-type Tregs. In some embodiments, the differentiated or transformed Treg maintains a Treg functional phenotype characterized by one or more Treg activities selected from the group consisting of: suppressing effector T cell activity, producing suppressive cytokines (e.g., IL-10), suppressing effector T cell cytokines (e.g., IL-2, IFNγ, TNFα), and reducing the rate of effector T cell division. In some embodiments, the differentiated or transformed Treg expresses one or more cytokines similar to those expressed by wild-type Tregs or control Tregs that do not contain any of the expression cassettes for exogenous FOXP3 described herein.In some embodiments, the differentiated or transformed Treg does not express one or more similar cytokines not expressed by wild-type Tregs or control Tregs, which do not contain any of the expression cassettes for exogenous FOXP3 described herein. In some embodiments, the one or more cytokines are selected from the group consisting of TNF-α, IFN-γ, TGF-β, IL-10, IL-35, IL-17 (i.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 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, or higher) of IL-10 compared to wild-type Tregs or control Tregs lacking an expression cassette for exogenous FOXP3.

[0175] IV. Preparation Method The expression cassette described herein can be prepared and transduced into host cells (e.g., Treg cells). Sequences encoding the expression cassette can be synthesized. After obtaining the 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 live host cells stably expressing the expression cassette of the present invention.

[0176] In some embodiments, the expression cassette may be DNA. In some embodiments, the expression cassette is inserted into a vector, such as an expression vector, viral vector, or cloning vector. To express the expression cassette, the vector may be introduced into a host cell to allow the expression cassette to be expressed within the host cell. The expression vector may contain a variety of elements for controlling expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, optional markers, and signal sequences. These elements may be appropriately selected by those skilled in the art. For example, a promoter sequence may be selected to promote transcription of the expression cassette in the vector. Suitable promoter sequences include, but are not limited to, MND, T7 promoter, T3 promoter, SP6 promoter, β-actin promoter, EF1a promoter, CMV promoter, and SV40 promoter. An enhancer sequence may be selected to enhance transcription of the expression cassette. Optional markers may be included to allow selection of host cells containing the vector from those host cells lacking the vector; for example, optional markers may be transmembrane genes (such as EGFR, HER2, CD2, H-2K) that can be identified by flow cytometry and FACS analysis. k IL-5Rα or any of its derivatives or mutants).

[0177] In some embodiments, the expression cassette (or the vector containing the expression cassette, such as an expression vector, viral vector, or clonal vector) expresses the FOXP3 protein, such as the full-length FOXP3 protein and / or an alternative splice isotype lacking one or more exons (e.g., exon 2). In some embodiments, the expression cassette (or the vector containing the expression cassette, such as an expression vector, viral vector, or clonal 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 signal transduction receptor. The chimeric receptor may target an antigen of interest (e.g.,Targets in the site of inflammation. Antigens may include, but are not limited to, AFP (alpha-fetoprotein), αvβ6 or another integrin, BCMA, B7-H3, B7-H6, CA9 (carbonic anhydrase 9), CCL-1 (CC 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), blocking protein, c-MET, DLL3 (delta-like protein 3), DLL4, ENPP3 (exonucleotide pyrophosphatase / phosphodiesterase family member 3), EpCAM, EPG-2. (Epidermal glycoprotein 2), EPG-40, liver glycoside B2, EPHa2 (liver glycoside receptor A2), ERBB dimer, estrogen receptor, ETBR (endothelin B receptor), FAP-α (fibroblast activation protein α), fetal AchR (fetal acetylcholine receptor), FBP (folate-binding protein), FCRL5, FR-α (folate receptor α), GCC (guanylate cyclase C), GD2, GD3, GPC2 (phosphatidylinositol proteoglycan-2), GPC3, gp100 (glycoprotein 100), GPNMB (glycoprotein NMB), GPRC5D (G protein-coupled receptor 5D), HER2, HER3, HER4, hepatitis B surface antigen, HLA-A1 (human leukocyte antigen A1), HLA-A2 (human leukocyte antigen A2), HMW-MAA (human high molecular weight melanoma-associated antigen), IGF1R (Insulin-like growth factor 1 receptor), Igκ, Igλ, IL-22Rα (IL-22 receptor α), IL-13Rα2 (IL-13 receptor α2), KDR (kinase insertion domain receptor), LI cell adhesion molecule (LI-CAM), Liv-1, LRRC8A (leucine-rich repeat protein family member 8A), Lewis Y, melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, MART-1 (melan A), murine cytomegalovirus (MCMV), MCSP (melanoma-associated chondroitin sulfate proteoglycan), mesothelin, mucin 1 (MUC1), MUC16, MHC / peptide complex (e.g.,HLA-A (a peptide complex derived from AFP, KRAS, NY-ESO, MAGE-A, and WT1), NCAM (neural cell adhesion molecule), cohesin-4, NKG2D (natural killer group 2 member D) ligand, NY-ESO, carcinoembryonic antigen, PD-1, PD-L1, PRAME (preferred expression antigen of melanoma), progesterone receptor, PSA (prostate-specific antigen), PSCA (prostate stem cell antigen), PSMA (prostate-specific membrane antigen), ROR1, ROR2, SIRPα (signal regulator protein α), SLIT, SLITRK6 (NTRK-like protein 6), STEAP1 (prostate six-transmembrane epithelial antigen 1), susceptin, 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 implementations, the target antigen is selected from the group consisting of: carboxypeptidase H, chromogranin A, glutamate decarboxylase, Imogen-38, insulin, proinsulin, insulinoma antigen-2 and 2β, islet-specific glucose-6-phosphatase catalytic subunit-associated protein (IGRP), α-enolase, aquaporin-4, β-repressor protein, myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG), protein lipoprotein (PLP), phosphodiesterase 4 (PDE4), vascular endothelial growth factor receptor (VEGFR), S100-β, citrullinated vimentin (CV), collagen II, human chondroitin 39, La antigen, phospholipid-β-2 glycoprotein I complex, Sm antigen of U-1 small ribonucleoprotein complex, tumor necrosis factor (TNF)-like ligand 1A (TL1A), complement C1s, Cam-3001, CD125, CD154, CD2, CD23 (IgE receptor), IL-6 receptor (IL-6R), IL-4Rα, IL-12R, IL-13Rα1, IL-13Rα2, IL-15R, IL-23R, rhuMAb β7, NKG2D, NKp30, AOC3 (VAP-1), CCL11 (eosinophil chemokine-1), CD19, CD20, CD22, CD3, CD4, CD5, IFN-γ, IgE, IgE Fc region, IL-1 (i.e.,IL-1α and / or IL-1β), IL-12, IL-23, IL-15, IL-17, IL-17A, IL-22, IL-6, IL-13, IL-33, TSLP, integrin α4β7, LFA-1 (CD11a), OX-40, OX-40L, p53, CD38, CD40L, CD28, ICOS, RIPK1, BTK, Betv1, GLP2R, JAK1, C5, and TNF-α.

[0178] In some embodiments, the host cell (e.g., Treg cells) contains the vector described above. The vector can be introduced into the cell using any suitable method known in the art, including, but not limited to, DEAE-dextran-mediated delivery, calcium phosphate precipitation, cationic lipid-mediated delivery, liposome-mediated transfection, electroporation, gene gun methods, receptor-mediated gene delivery, and delivery mediated by polylysine, histones, chitosan, and peptides. Standard methods for transducing cells to express the vector of interest are well known in the art. In some embodiments, the host cell contains a vector containing an expression cassette encoding the FOXP3 protein.

[0179] In some embodiments, this application provides a method for expressing any of the expression cassettes described herein, comprising culturing isolated host cells containing a vector. The isolated host cells are cultured under conditions that allow expression of the expression cassette inserted into the vector. Suitable conditions for expressing the expression cassette may include, but are not limited to, a suitable culture medium, a suitable density of host cells in the culture medium, the presence of essential nutrients, the presence of supplemental factors, suitable temperature and humidity, and the absence of microbial contaminants. Those skilled in the art can appropriately select suitable conditions for the purpose of expression.

[0180] carrier In some respects, this disclosure provides expression cassettes encoding the FOXP3 protein. Such expression cassettes can be inserted into suitable vectors (e.g., viral vectors or non-viral plasmid vectors) for introduction into host cells of interest (e.g., Tregs).

[0181] As used herein, the terms “recombinant” or “non-natural” refer to an organism, microorganism, cell, nucleic acid molecule, or vector that includes at least one genetic alteration or has been modified by introducing an exogenous expression cassette, wherein such alteration or modification is introduced through genetic engineering. Genetic alterations include, for example, the introduction of an expressible expression cassette encoding one or more proteins, fusion proteins, or enzymes, or the addition, deletion, substitution, or other functional disruption of other expression cassettes in the cellular genetic material. Additional modifications include, for example, non-coding regulatory regions, wherein the modification alters the expression of a gene or operon. In some embodiments, by introducing an expression cassette as described herein, cells obtained from a subject (such as regulatory T cells (Tregs) or effector T cells) can be converted into non-natural or recombinant regulatory T cells (Tregs) (e.g., non-natural or recombinant regulatory T cells (Tregs)).

[0182] The vector encoding the core virus is referred to herein as a "viral vector." A large number of available viral vectors are suitable for use with the compositions of this disclosure, including those identified for human gene therapy applications (see Pfeifer and Verma (2001), Ann. Rev. Genomics Hum. Genet. 2:177, which is incorporated herein by reference in its entirety). Suitable viral vectors include RNA virus-based vectors, such as retrovirus-derived vectors, for example, vectors derived from Maronie mouse leukemia virus (MLV), and more complex retrovirus-derived vectors, such as lentivirus-derived vectors. HIV-1-derived vectors fall into this category. Other examples include lentivirus vectors derived from HIV-2, FIV, equine infectious anemia virus, SIV, and Medivesna virus (sheep lentivirus). Methods using retroviral and lentiviral vectors and packaging cells to transduce mammalian host regulatory T cells (Tregs) with viral particles containing chimeric antigen receptor transgenes are known in the art and have previously been described, for example, in U.S. Patent No. 8,119,772; Walchli et al. (2011), PLoS One 6:327930; Zhao et al. (2005), J. Immunol. 174:4415; Engels et al. (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.

[0183] In some embodiments, a viral vector is used to introduce any of the expression cassettes described herein. The viral vector may be a retroviral vector or a lentiviral vector. The viral vector may also contain a nucleic acid sequence encoding a cell surface receptor as a marker of successful cell transduction. Transduction markers for the viral vector are known in the art and include selectable markers that can confer 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 a particular embodiment, the viral vector may also contain a genetic marker for transduction, comprising green fluorescent protein or a truncated human EGFR (huEGFRt; see Wang et al. (2011), Blood 118:1255). When the viral vector genome contains multiple nucleic acid sequences to be expressed as individual transcripts in host cells (e.g., T cells, such as Tregs), the viral vector may also contain additional sequences between two (or more) transcripts that allow bicistronic or polycistronic expression. Examples of such sequences for viral vectors include internal ribosome entry sites (IRES), furin cleavage sites, viral 2A peptides, or any combination thereof.

[0184] Other vectors can also be used for expression cassette delivery, including DNA viral vectors, such as adenovirus-based vectors and adeno-associated virus (AAV)-based vectors; and vectors derived from herpes simplex virus (HSV), including amplicon vectors, replication-defective HSV, and attenuated HSV (Krisky et al. (1998), Gene Ther. 5: 1517).

[0185] Other vectors recently developed for gene therapy purposes can also be used with the compositions and methods disclosed herein. Such vectors include those derived from baculoviruses and alpha viruses (Jolly, DJ (1999) Emerging Viral Vectors. pp. 209-40 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).

[0186] In some embodiments, hematopoietic progenitor cells or embryonic stem cells are modified to include the expression cassette of this disclosure. The hematopoietic progenitor cells may comprise induced pluripotent stem cells, which may be derived from or derived from fetal liver tissue, bone marrow, umbilical cord blood, or peripheral blood. The hematopoietic progenitor cells may be derived from humans, mice, rats, or other mammals. In some embodiments, the host cells transfected to express the expression cassette of this disclosure are functional Tregs or effector T cells.

[0187] V. Methods of Cell Amplification and Culture In some embodiments, the cells transfected with the expression cassette of this disclosure are eukaryotic cells. In some embodiments, the cells transfected with the expression cassette of this disclosure are human cells. In some embodiments, the cells transfected with the expression cassette of this disclosure are immune cells. In some embodiments, the cells transfected with the expression cassette of this disclosure are T cells. In some embodiments, the cells transfected with the expression cassette of this disclosure are functional regulatory T cells (Tregs).

[0188] In some embodiments, Tregs are isolated from peripheral blood mononuclear cells (PBMCs). In some embodiments, Tregs are isolated from PBMCs using density gradient centrifugation. In some embodiments, Tregs are enriched by positive selection. In some embodiments, Tregs are enriched by positive selection targeting CD25+ cells. In some embodiments, Tregs are enriched by positive selection targeting CD4+CD25+CD127 low-dose cells. In some embodiments, enrichment occurs via FACS. In some embodiments, Tregs are stimulated with anti-CD3 and / or anti-CD28 antibodies on day 0 after positive selection. In some embodiments, Tregs are restimulated with anti-CD3 and / or anti-CD28 antibodies on day 9 of in vitro culture after positive selection.

[0189] One or more growth factor cytokines can be added to an in vitro culture, wherein the one or more growth factor cytokines promote the proliferation of T cells (such as Tregs, including Tregs containing any expression cassettes of this disclosure, and / or effector T cells expressing expression cassettes of this disclosure). The cytokines can be human or non-human cytokines. Exemplary growth factor cytokines that can be used to promote the proliferation of regulatory T cells (Tregs) include IL-2, IL-4, IL-7, IL-9, IL-15, TGFβ, etc. In some embodiments, the cytokines are added to the culture medium prior to selection. In some embodiments, the cytokines are added to the culture medium after cell isolation. In some embodiments, the cytokines are added to the cell culture medium approximately every 12 to 60 hours (e.g., approximately 24 to approximately 48 hours). In some embodiments, the cytokines are added to the culture medium during the duration of culture. In some embodiments, Tregs are cultured in a culture medium containing two, three, four, five, or more cytokines.

[0190] In some embodiments, cells expressing any expression cassette of this disclosure are cultured for a sufficient time to induce proliferation or differentiation. Cells are maintained in culture for approximately 3 to 5 days, approximately 4 to 10 days, approximately 5 to 20 days, approximately 10 to 23 days, approximately 15 to 30 days, or approximately 23 to 30 days. It should be understood that the appropriate amount of time required to maintain cells to achieve the desired result (i.e., the desired cell composition or proliferation level) can be achieved. For example, to produce a cell composition primarily containing Tregs, cells may be maintained in culture for approximately 30 days.

[0191] In some embodiments, the method further includes detecting one or more Treg biomarkers provided herein. In some embodiments, the method further includes detecting FOXP3 levels and / or FOXP3.

[0192] In some embodiments, the inhibitory activity of a Treg containing any expression cassette of this disclosure is increased compared to a Treg lacking an expression cassette. In some embodiments, the production of inhibitory cytokines is increased by a Treg containing any expression cassette of this disclosure compared to a Treg lacking an expression cassette. In some embodiments, the production of cytotoxic and pro-inflammatory cytokines by cytotoxic T cells and / or effector T cells is reduced by a Treg containing any expression cassette of this disclosure. In some embodiments, the activity of cytotoxic T cells and / or effector T cells is reduced by a Treg containing any expression cassette of this disclosure.

[0193] VI. Pharmaceutical compositions, products and reagent kits This application also provides pharmaceutical compositions comprising cells containing the recombinant cytokine receptors described herein, such as T cells like Treg cells.

[0194] The pharmaceutical composition is suitable for a variety of administration modes described herein, including, for example, systemic or local administration. In some embodiments, the pharmaceutical composition is formulated for intravenous administration.

[0195] Pharmaceutical compositions intended for internal administration are typically formulated as sterile, substantially isotonic compositions and fully compliant with all Good Manufacturing Practices (GMP) requirements of the U.S. Food and Drug Administration. Sterility is readily achieved through filtration via sterile filter membranes. In some embodiments, the composition is pathogen-free. For injection, the pharmaceutical composition may be in the form of a liquid solution, such as in physiologically compatible buffer solutions like Hank's solution or Ringer's solution.

[0196] In some embodiments, the pharmaceutical composition is suitable for administration to humans. In some embodiments, the pharmaceutical composition is suitable for administration to rodents (e.g., mice, rats) or non-human primates (e.g., cynomolgus monkeys). In some embodiments, the pharmaceutical composition is cryopreserved.

[0197] This application also provides a medicine box that includes the compositions described herein (such as pharmaceutical compositions), and may further include one or more instructions regarding methods of using the composition, uses such as those described herein. The medicine box described herein may also include other materials desirable from a commercial and user perspective, including additional buffers, diluents, filters, needles, syringes, and packaging inserts with instructions for performing any of the methods described herein.

[0198] VII. Treatment Methods In some embodiments, this document provides a method for treating an immune-related condition, comprising administering an engineered Treg containing an exogenous expression cassette comprising a modified nucleic acid encoding a FOXP3 protein operatively linked to a promoter, wherein the nucleic acid encoding the FOXP3 protein is modified to produce a full-length FOXP3 protein isotype and an alternative splice isotype lacking one or more exons. In some embodiments, the nucleic acid encoding the FOXP3 protein comprises an intron sequence flanking a nucleic acid encoding exon 2. In some embodiments, the nucleic acid encoding the FOXP3 protein containing an intron sequence flanking a nucleic acid encoding exon 2 is alternatively spliced ​​to produce a full-length FOXP3 protein isotype and a FOXP3 protein isotype 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 operatively linked to the same promoter. In some implementations, 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 IRES, the nucleic acid encoding a self-cleaving peptide (e.g., the P2A peptide), or the nucleic acid encoding a peptide that induces ribosome jumping. Therefore, in some embodiments, a method for treating an immune-related condition is provided, comprising administering a Treg containing 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-cleaving peptide or a peptide that induces ribosome jumping; (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 induces ribosome jumping; (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 induces ribosome jumping; and (VI) a nucleic acid encoding a cell surface receptor; wherein the order of the FOXP3 protein, the chimeric receptor, and / or the 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 the 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 embodiments, the cell is a Treg.

[0199] In some embodiments, this document provides a method for treating an immune-related condition, comprising administering an engineered Treg containing an exogenous expression cassette comprising a modified nucleic acid encoding a FOXP3 protein and a nucleic acid encoding a cell surface receptor, wherein the nucleic acid encoding the FOXP3 protein contains an intron sequence flanking a nucleic acid encoding exon 2. In some embodiments, the nucleic acid encoding the FOXP3 protein containing the intron sequence flanking an exon 2 nucleic acid is subjected to alternative splicing to produce a full-length FOXP3 protein isotype and an exon 2-deficient FOXP3 protein isotype. In some embodiments, the nucleic acid encoding the FOXP3 protein and the nucleic acid encoding the cell surface receptor are operatively 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 nucleic acids encoding IRES, self-cleaving peptides (e.g., P2A peptides), or peptides that induce ribosome jumping. Therefore, in some embodiments, a method for treating an immune-related condition is provided, comprising administering a Treg containing 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 contains an intron sequence flanking a nucleic acid encoding exon 2; (III) an IRES or a nucleic acid encoding a self-cleaving peptide or a peptide that induces ribosome jumping; and (IV) a nucleic acid encoding a cell surface receptor. In some embodiments, the nucleic acid encoding the 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 embodiments, the cell is a Treg.

[0200] In some embodiments, this document provides a method for treating an immune-related condition, comprising administering an engineered Treg containing an exogenous expression cassette comprising 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 contains an intron sequence flanking a nucleic acid encoding exon 2. In some embodiments, the nucleic acid encoding the FOXP3 protein containing the intron sequence flanking an exon 2 nucleic acid is subjected to alternative splicing to produce a full-length FOXP3 protein isotype and an exon 2-deficient FOXP3 protein isotype. In some embodiments, the nucleic acid encoding the FOXP3 protein and the nucleic acid encoding the cell surface receptor are operatively 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 nucleic acids encoding IRES, self-cleaving peptides (e.g., P2A peptides), or peptides that induce ribosome jumping. Therefore, in some embodiments, a method for treating an immune-related condition is provided, comprising administering a Treg containing 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 induces ribosome jumping; and (IV) a modified nucleic acid encoding a FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein contains an intron sequence flanking a nucleic acid encoding exon 2. In some embodiments, the nucleic acid encoding the 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 embodiments, the cell is a Treg.

[0201] In some embodiments, this document provides a method for treating an immune-related condition, comprising administering an engineered Treg containing 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 contains an intron sequence flanking a nucleic acid encoding exon 2. In some embodiments, the nucleic acid encoding the FOXP3 protein containing the intron sequence flanking an exon 2 nucleic acid is subjected to alternative splicing to produce a full-length FOXP3 protein isotype and an exon 2-deficient FOXP3 protein isotype. 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 operatively 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., a P2A peptide), or a nucleic acid encoding a peptide that induces ribosome jumping. Therefore, in some embodiments, a method for treating an immune-related condition is provided, comprising administering a Treg containing 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 induces ribosome jumping; (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 induces ribosome jumping; and (VI) a modified nucleic acid encoding a FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein contains an intron sequence flanking the nucleic acid encoding exon 2. In some embodiments, the nucleic acid encoding the 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 embodiments, the cell is a Treg.

[0202] In some embodiments, this document provides a method for treating an immune-related condition, comprising administering an engineered Treg containing an exogenous expression cassette comprising a nucleic acid encoding a chimeric receptor, a modified nucleic acid encoding a FOXP3 protein, and a nucleic acid encoding a cell surface receptor, wherein the nucleic acid encoding the FOXP3 protein contains an intron sequence flanking a nucleic acid encoding exon 2. In some embodiments, the nucleic acid encoding the FOXP3 protein containing the intron sequence flanking an exon 2 nucleic acid is subjected to alternative splicing to produce a full-length FOXP3 protein isotype and an exon 2-deficient FOXP3 protein isotype. 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 operatively 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., a P2A peptide), or a nucleic acid encoding a peptide that induces ribosome jumping. Therefore, in some embodiments, a method for treating an immune-related condition is provided, comprising administering a Treg containing 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 induces ribosome jumping; (IV) a modified nucleic acid encoding a FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein contains an intron sequence flanking a nucleic acid encoding exon 2; (V) an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that induces ribosome jumping; and (VI) a nucleic acid encoding a cell surface receptor. In some embodiments, the nucleic acid encoding the 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 embodiments, the cell is a Treg.

[0203] In some embodiments, this document provides a method for treating an immune-related condition, comprising administering an engineered Treg containing an exogenous expression cassette comprising a modified nucleic acid encoding a FOXP3 protein, a nucleic acid encoding a chimeric receptor, and a nucleic acid encoding a cell surface receptor, wherein the nucleic acid encoding the FOXP3 protein contains an intron sequence flanking a nucleic acid encoding exon 2. In some embodiments, the nucleic acid encoding the FOXP3 protein containing the intron sequence flanking an exon 2 nucleic acid is subjected to alternative splicing to produce a full-length FOXP3 protein isotype and an exon 2-deficient FOXP3 protein isotype. 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 operatively 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., a P2A peptide), or a nucleic acid encoding a peptide that induces ribosome jumping. Therefore, in some embodiments, a method for treating an immune-related condition is provided, comprising administering a Treg containing 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 contains an intron sequence flanking a 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 induces ribosome jumping; (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 induces ribosome jumping; and (VI) a nucleic acid encoding a cell surface receptor. In some embodiments, the nucleic acid encoding the 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 embodiments, the cell is a Treg.

[0204] In some embodiments, this document provides a method for treating an immune-related condition, comprising administering an engineered Treg containing an exogenous expression cassette comprising 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 contains an intron sequence flanking a nucleic acid encoding exon 2. In some embodiments, the nucleic acid encoding the FOXP3 protein containing the intron sequence flanking an exon 2 nucleic acid is subjected to alternative splicing to produce a full-length FOXP3 protein isotype and an exon 2-deficient FOXP3 protein isotype. In some embodiments, the nucleic acid encoding the FOXP3 protein and the nucleic acid encoding the cell surface receptor are operatively 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 nucleic acids encoding IRES, self-cleaving peptides (e.g., P2A peptides), or peptides that induce ribosome jumping. Therefore, in some embodiments, a method for treating an immune-related condition is provided, comprising administering a Treg containing 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 induces ribosome jumping; and (IV) a modified nucleic acid encoding a FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein contains an intron sequence flanking a nucleic acid encoding exon 2. In some embodiments, the nucleic acid encoding the 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 embodiments, the cell is a Treg.

[0205] In some embodiments, this document provides a method for treating an immune-related condition, comprising administering an engineered Treg containing an exogenous expression cassette comprising nucleic acids encoding a FOXP3 protein and nucleic acids encoding a cell surface receptor, wherein the nucleic acid encoding the FOXP3 protein does not contain a nucleic acid sequence encoding exon 2. In some embodiments, the nucleic acid encoding the FOXP3 protein and the nucleic acid encoding the cell surface receptor are operatively 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., a P2A peptide), or a nucleic acid encoding a peptide that induces ribosome jumping. Therefore, in some embodiments, a method for treating an immune-related condition is provided, comprising administering a Treg containing 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 contain a nucleic acid sequence encoding exon 2; (III) an IRES or a nucleic acid encoding a self-cleaving peptide or a peptide that induces ribosome jumping; and (IV) a nucleic acid encoding a cell surface receptor. In some embodiments, the nucleic acid encoding the 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 embodiments, the cell is a Treg.

[0206] In some embodiments, a method of treating an immune-related condition includes administering a composition containing Tregs. In some embodiments, the method includes transducing Tregs with a vector encoding any 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 the FOXP3 protein is FOXP3 cDNA. In some embodiments, the cells are immune cells. In some embodiments, the cells are lymphocytes. In some embodiments, the cells are T cells. In some embodiments, the cells are Tregs.

[0207] In some embodiments, the expression cassettes of this disclosure are expressed in T cells (e.g., Tregs). In some embodiments, one or more expression cassettes are expressed in T cells (e.g., Tregs). In some embodiments, the T cells are regulatory T cells (Tregs). In some embodiments, the Tregs are CD4+, CD25+, and CD127-low. In some embodiments, the Tregs express endogenous FOXP3. In some embodiments, the expression cassettes are expressed in Treg cells.

[0208] The immune disorders, diseases, disorders, and reactions or responses treated by the methods and compositions according to the present invention refer to diseases in which the immune system contributes to the pathogenesis or may be part of the treatment. These reactions include, but are not limited to: cancer, inflammation, autoimmune disorders, disorders, or diseases, and persistent and progressive immune responses to infectious non-self antigens from bacteria, viruses (e.g., HCV), fungi, or parasites that invade and persist within mammals and humans. Such disorders and disorders include allergies and / or asthma. Allergies and asthma may be due to sensitization by exogenous or non-self antigens such as pollen, animal dander, and food proteins. The source of the provoking exogenous antigen may be plants, fungi, molds, or other environmental contaminants.

[0209] Autoimmunity is defined as a persistent and progressive immune response to non-infectious self-antigens, distinct from infectious non-self-antigens from bacteria, viruses, fungi, or parasites that invade and persist within mammals and humans. Autoimmune diseases include: graft-versus-host disease, autoimmune polyendocrine syndrome, type 1 diabetes mellitus (TIDM), autoimmune gastritis, autoimmune uveitis, autoimmune vasculitis, colitis, thyroiditis, Addison's disease, agammaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, anti-GBM / anti-TBM nephritis, antiphospholipid syndrome, autoimmune hepatitis, autoimmune inner ear disease, axonal and neuronal neuropathy, and Behçet's syndrome. Diseases including bullous pemphigoid, Castleman's disease, celiac disease, Chagas disease, acute inflammatory demyelinating polyneuropathy (CIDP), chronic relapsing multifocal osteomyelitis, Churg-Strauss, cicatricial pemphigoid / benign mucositis pemphigus, Cogan syndrome, cold agglutinin disease, congenital heart block, Coxsackievirus myocarditis, CREST syndrome, Crohn's disease, herpetic dermatitis, dermatomyositis, Dermatomyositis, Dervian disease, discoid lupus, Dressler syndrome, endometriosis, eosinophilic esophagitis, eosinophilic fasciitis, erythema nodosum, idiopathic mixed cryoprecipitate globulinemia, Evans syndrome, fibromyalgia, fibrosing alveolitis, giant cell arteritis (temporal arteritis), giant cell myocarditis, glomerulonephritis, Goodpasture syndrome, granulomatous polyangiitis, Graves' disease. Guillian-Barre syndrome, Hashimoto's thyroiditis, hemolytic anemia, Sergey-Henri purpura, herpes gestationis or pemphigoid of pregnancy, hypogammaglobulinemia, IgA nephropathy, IgG4-related sclerosis, inclusion body myositis, interstitial cystitis, juvenile arthritis, juvenile diabetes mellitus (type 1 diabetes), juvenile myositis, Kawasaki disease, Lambert-Eaton syndrome, leukocytic clotting vasculitis, lichen planus, lichen sclerosus, woody conjunctivitis, linear IgA disease, lupus, Lyme disease, chronic Meniere's disease, microscopic polyangiitis, mixed connective tissue disease, Moren's ulcer, Mucha-Habermann disease, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neuromyelitis optica, neutropenia, ocular cicatricial pemphigoid, optic neuritis, relapsing rheumatism, PANDAS (Pediatric autoimmune neuropsychiatric disorders with streptococcal infection), paraneoplastic cerebellar degeneration, paroxysmal nocturnal hemoglobinuria, ParryRomber syndrome, pars plana cyclitis (intermediate uveitis), Parsonnage-Turner syndrome, pemphigus, peripheral neuropathy, perivenous encephalomyelitis, pernicious anemia, POEMS syndrome, polyarteritis nodosa, polymyalgia rheumatica, polymyositis, post-myocardial infarction syndrome, post-cardiotomy syndrome, primary biliary cirrhosis, primary sclerosing cholangitis, progesterone dermatitis, psoriasis, psoriatic arthritis, pure red cell aplasia, pyoderma gangrene, Raynaud's phenomenon, reactive arthritis, reflex sympathetic dystrophy, Reiter syndrome, recurrent pyoderma Polychondritis, restless legs 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 syndrome, sympathetic ophthalmia, aortitis, temporal arteritis / giant cell arteritis, thrombocytopenic purpura, Tolosa-Hunt syndrome, transverse myelitis, ulcerative colitis, undifferentiated connective tissue disease, uveitis, vasculitis, vitiligo, and Wegener's granulomatosis (granulomatous polyangiitis). As used herein, "autoantigen" or "self-antigen" refers to a naturally occurring antigen or epitope in mammals that generates immunity in the diseases of said mammals. One aspect of this application provides a method for treating immune-related disorders.

[0210] Cells can be “allogeneic cells” (allectic), which are cells isolated from one individual (donor) and infused into another; while “autologous cells” (autologous approach) refer to cells 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 Treg cells. In some embodiments, Treg cells are isolated from human peripheral blood mononuclear cells (PBMCs). In some embodiments, Treg cells are modified in vitro with a vector encoding one or more expression cassettes. In some embodiments, Treg cells are modified with a vector containing an expression cassette. In some embodiments, Treg cells are modified to express exogenous FOXP3. In some embodiments, Treg cells are CD4+, CD25+, and CD127 low. In some embodiments, Treg cells express endogenous FOXP3. In some embodiments, Treg cells are administered to the same individual to treat an immune-related disorder.

[0211] In some implementations, methods of treating immune-related conditions include administering Treg to individuals in need. In some implementations, the Treg is CD4+, CD25+, and CD127-low. In some implementations, the Treg expresses endogenous FOXP3.

[0212] In some embodiments, a method of treating immune-related illness includes administering Tregs that are autologous to the individual. In some embodiments, the individual is a human. In some embodiments, a method of treating immune-related illness includes administering Tregs isolated from human peripheral blood mononuclear cells (PBMCs). In some embodiments, a method of treating immune-related illness includes administering Treg cells modified with a vector containing an expression cassette. In some embodiments, a method of treating immune-related illness includes administering Tregs in vitro modified with a vector containing one or more expression cassettes. In some embodiments, a method of treating immune-related illness includes administering Tregs modified to express exogenous FOXP3. In some embodiments, a method of treating immune-related illness includes administering Tregs modified to overexpress FOXP3. In some embodiments, a method of treating immune-related illness includes administering Tregs with low CD4+, CD25+, and CD127 expression. In some embodiments, a method of treating immune-related illness includes administering Tregs expressing endogenous FOXP3.

[0213] In some embodiments, a method of treating immune-related conditions includes administering a Treg containing an expression cassette of the present discl...

Claims

1. An engineered regulatory T cell (Treg) comprising an exogenous expression cassette containing a modified nucleic acid encoding a FOXP3 protein operatively linked to a promoter, wherein the nucleic acid encoding the FOXP3 protein is modified to produce a full-length FOXP3 protein isotype and an alternative splicing isotype lacking one or more exons.

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

3. The engineered Treg of claim 2, wherein the nucleic acid encoding the FOXP3 protein is subjected to alternative splicing to produce a full-length FOXP3 protein isotype and a FOXP3 protein isotype lacking exon 2.

4. The engineered Treg according to 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 operatively 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 jumping between the nucleic acid encoding the FOXP3 protein and the nucleic acid encoding the chimeric receptor.

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

9. The engineered Treg according to 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 at the 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 IRES, or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosomal jumping 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 IRES, or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosomal jumping between the nucleic acid encoding the cell surface receptor and the nucleic acid encoding the chimeric receptor.

13. The engineered Treg as described in any one of claims 1-3 and 9-12, wherein the exogenous expression cassette from 5' to 3' comprises: a) The promoter; b) The modified nucleic acid encoding the FOXP3 protein optionally linked to the detection tag, wherein the nucleic acid encoding the FOXP3 protein contains an intron sequence located flanking the nucleic acid encoding exon 2; c) IRES or nucleic acids encoding self-cleaving peptides or peptides that induce ribosome jumping; and d) The nucleic acid encoding the cell surface receptor.

14. The engineered Treg cell according to any one of claims 1-3 and 9-12, comprising from the 5' to the 3': a) The promoter; b) The nucleic acid encoding the cell surface receptor; c) Internal ribosome entry sites (IRES), or nucleic acids encoding self-cleaving peptides or peptides that induce ribosome jumping; and d) The modified nucleic acid encoding the FOXP3 protein, wherein the modified nucleic acid encoding the FOXP3 protein contains an intron sequence located flanking the nucleic acid encoding exon 2.

15. The engineered Treg as described in any one of claims 1-12, wherein the exogenous expression cassette from 5' to 3' comprises: a) The promoter; b) The nucleic acid encoding the chimeric receptor; c) Internal ribosome entry site (IRES), or nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome jumping; d) The nucleic acid encoding the cell surface receptor; e) Internal ribosome entry sites (IRES), or nucleic acids encoding self-cleaving peptides or peptides that induce ribosome jumping; and f) The modified nucleic acid encoding the FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein contains an intron sequence located flanking the nucleic acid encoding exon 2.

16. The engineered Treg as described in any one of claims 1-12, wherein the exogenous expression cassette from 5' to 3' comprises: a) The promoter; b) The nucleic acid encoding the chimeric receptor; c) Internal ribosome entry site (IRES), or nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome jumping; d) The modified nucleic acid encoding the FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein contains an intron sequence located flanking the nucleic acid encoding exon 2; e) Internal ribosome entry sites (IRES), or nucleic acids encoding self-cleaving peptides or peptides that induce ribosome jumping; 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 from 5' to 3' comprises: a) The promoter; b) The modified nucleic acid encoding the FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein contains an intron sequence located flanking the nucleic acid encoding exon 2; c) Internal ribosome entry site (IRES), or nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome jumping; d) The nucleic acid encoding the chimeric receptor; e) Internal ribosome entry sites (IRES), or nucleic acids encoding self-cleaving peptides or peptides that induce ribosome jumping; and f) The nucleic acid encoding the cell surface receptor.

18. An engineered Treg comprising an exogenous expression cassette, said exogenous expression cassette comprising, from 5' to 3': a) Promoter; b) Nucleic acid encoding the chimeric receptor; c) Internal ribosome entry site (IRES), or nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome jumping; d) The nucleic acid encoding the FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein is FOXP3 cDNA; e) Internal ribosome entry sites (IRES), or nucleic acids encoding self-cleaving peptides or peptides that induce ribosome jumping; and f) Nucleic acids encoding cell surface receptors.

19. An engineered Treg comprising an exogenous expression cassette, said exogenous expression cassette comprising, from 5' to 3': a) Promoter; b) A nucleic acid encoding the FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein is FOXP3 cDNA; c) IRES or nucleic acids encoding self-cleaving peptides or peptides that induce ribosome jumping; and d) Nucleic acids encoding cell surface receptors.

20. An engineered Treg comprising an exogenous expression cassette, the exogenous expression cassette comprising, from 5' to 3': a) Promoter; b) Nucleic acids encoding cell surface receptors; c) Internal ribosome entry sites (IRES), or nucleic acids encoding self-cleaving peptides or peptides that induce ribosome jumping; and d) A nucleic acid encoding the FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein is FOXP3 cDNA.

21. An engineered Treg comprising an exogenous expression cassette, said exogenous expression cassette comprising, from 5' to 3': a) Promoter; b) A nucleic acid encoding the FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein is FOXP3 cDNA; c) Internal ribosome entry site (IRES), or nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome jumping; d) Nucleic acids encoding chimeric receptors; e) Internal ribosome entry sites (IRES), or nucleic acids encoding self-cleaving peptides or peptides that induce ribosome jumping; and f) Nucleic acids encoding cell surface receptors.

22. An engineered Treg comprising an exogenous expression cassette, said exogenous expression cassette comprising, from 5' to 3': a) Promoter; b) Nucleic acid encoding the chimeric receptor; c) Internal ribosome entry site (IRES), or nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome jumping; d) Nucleic acids encoding cell surface receptors; e) Internal ribosome entry sites (IRES), or nucleic acids encoding self-cleaving peptides or peptides that induce ribosome jumping; and f) A nucleic acid encoding the FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein is FOXP3 cDNA.

23. An engineered Treg comprising an exogenous expression cassette, said exogenous expression cassette comprising, from 5' to 3': a) Promoter; b) A nucleic acid encoding the FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein does not contain a nucleic acid sequence encoding exon 2; c) IRES or nucleic acids encoding self-cleaving peptides or peptides that induce ribosome jumping; and d) Nucleic acids encoding cell surface receptors.

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

25. The engineered Treg according to any one of claims 9-24, wherein the cell surface receptor lacks a functional intracellular signal transduction domain or is unable to mediate intracellular signal transduction.

26. The engineered Treg according to any one of claims 9-25, wherein the cell surface receptor is selected from EGFR, HER2, CD2, and H-2K. k The group consists of IL-5Rα and its derivatives or mutants.

27. The engineered Treg according to 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 according to 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, S100-β, citrullinated vimentin (CV), TNF-like ligand 1A (TL1A), CD2, CD23. (IgE receptor), IL-6R, IL-4Rα, IL-12R, IL-13Rα1, IL-13Rα2, IL-15R, IL-23R, CD19, CD20, CD22, CD3, CD4, CD5, IFN-γ, IgE, IL-1α, IL-1β, 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-α.

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 P2A, T2A, E2A and F2A sequences.

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

31. The engineered Treg according to any one of claims 1-30, wherein the promoter is a cell-specific promoter.

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

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

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

35. The engineered Treg according to 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 according to any one of claims 1-37, wherein the exogenous expression cassette is located in the chromosome of the Treg.

39. The engineered Treg of any one of claims 1-38, wherein the engineered Treg expresses a higher level of FOXP3 protein compared to a Treg lacking the exogenous expression cassette.

40. An expression cassette comprising 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 isotype and an alternative splicing isotype lacking one or more exons.

41. The expression cassette of claim 40, wherein the nucleic acid encoding the FOXP3 protein comprises an intron sequence located 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 operatively 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 jumping between the nucleic acid encoding the FOXP3 protein and the nucleic acid encoding the chimeric receptor.

45. The expression cassette according to 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 IRES, or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosomal jumping 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 IRES, or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome jumping between the nucleic acid encoding the cell surface receptor and the nucleic acid encoding the chimeric receptor.

48. The expression box as claimed in 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 the detection tag, wherein the nucleic acid encoding the FOXP3 protein contains an intron sequence located flanking the nucleic acid encoding exon 2; c) IRES or nucleic acids encoding self-cleaving peptides or peptides that induce ribosome jumping; and d) The nucleic acid encoding the cell surface receptor.

49. The expression box as claimed in 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) Internal ribosome entry sites (IRES), or nucleic acids encoding self-cleaving peptides or peptides that induce ribosome jumping; and d) The modified nucleic acid encoding the FOXP3 protein, wherein the modified nucleic acid encoding the FOXP3 protein contains an intron sequence located flanking the nucleic acid encoding exon 2.

50. The expression box as claimed in any one of claims 40-47, wherein from 5' to 3' it comprises: a) The promoter; b) The nucleic acid encoding the chimeric receptor; c) Internal ribosome entry site (IRES), or nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome jumping; d) The nucleic acid encoding the cell surface receptor; e) Internal ribosome entry sites (IRES), or nucleic acids encoding self-cleaving peptides or peptides that induce ribosome jumping; and f) The modified nucleic acid encoding the FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein contains an intron sequence located flanking the nucleic acid encoding exon 2.

51. The expression box as claimed in any one of claims 40-47, wherein from 5' to 3' it comprises: a) The promoter; b) The nucleic acid encoding the chimeric receptor; c) Internal ribosome entry site (IRES), or nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome jumping; d) The modified nucleic acid encoding the FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein contains an intron sequence located flanking the nucleic acid encoding exon 2; e) Internal ribosome entry sites (IRES), or nucleic acids encoding self-cleaving peptides or peptides that induce ribosome jumping; and f) The nucleic acid encoding the cell surface receptor.

52. The expression box as claimed in any one of claims 40-47, wherein from 5' to 3' it comprises: a) The promoter; b) The modified nucleic acid encoding the FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein contains an intron sequence located flanking the nucleic acid encoding exon 2; c) Internal ribosome entry site (IRES), or nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome jumping; d) The nucleic acid encoding the chimeric receptor; e) Internal ribosome entry sites (IRES), or nucleic acids encoding self-cleaving peptides or peptides that induce ribosome jumping; and f) The nucleic acid encoding the cell surface receptor.

53. An expression box comprising, from 5' to 3': a) Promoter; b) Nucleic acid encoding the chimeric receptor; c) Internal ribosome entry site (IRES), or nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome jumping; d) A nucleic acid encoding the FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein is FOXP3 cDNA; e) Internal ribosome entry sites (IRES), or nucleic acids encoding self-cleaving peptides or peptides that induce ribosome jumping; and f) Nucleic acids encoding cell surface receptors.

54. An expression box comprising, from 5' to 3': a) Promoter; b) A nucleic acid encoding the FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein is FOXP3 cDNA; c) IRES or nucleic acids encoding self-cleaving peptides or peptides that induce ribosome jumping; and d) Nucleic acids encoding cell surface receptors.

55. An expression box comprising, from 5' to 3': a) Promoter; b) Nucleic acids encoding cell surface receptors; c) Internal ribosome entry sites (IRES), or nucleic acids encoding self-cleaving peptides or peptides that induce ribosome jumping; and d) A nucleic acid encoding the FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein is FOXP3 cDNA.

56. An expression box comprising, from 5' to 3': a) Promoter; b) A nucleic acid encoding the FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein is FOXP3 cDNA; c) Internal ribosome entry site (IRES), or nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome jumping; d) Nucleic acids encoding chimeric receptors; e) Internal ribosome entry sites (IRES), or nucleic acids encoding self-cleaving peptides or peptides that induce ribosome jumping; and f) Nucleic acids encoding cell surface receptors.

57. An expression box comprising, from 5' to 3': a) Promoter; b) Nucleic acid encoding the chimeric receptor; c) Internal ribosome entry site (IRES), or nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome jumping; d) Nucleic acids encoding cell surface receptors; e) Internal ribosome entry sites (IRES), or nucleic acids encoding self-cleaving peptides or peptides that induce ribosome jumping; and f) A nucleic acid encoding the FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein is FOXP3 cDNA.

58. An expression box comprising, from 5' to 3': a) Promoter; b) A nucleic acid encoding the FOXP3 protein, wherein the nucleic acid encoding the FOXP3 protein does not contain a nucleic acid sequence encoding exon 2; c) IRES or nucleic acids encoding self-cleaving peptides or peptides that induce ribosome jumping; and d) Nucleic acids encoding cell surface receptors.

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

60. The expression cassette of any one of claims 45-59, wherein the cell surface receptor lacks a functional intracellular signal transduction domain or is unable to mediate intracellular signal transduction, optionally wherein the cell surface receptor is selected from EGFR, HER2, CD2, and H-2K. k The group consists of IL-5Rα and its derivatives or mutants.

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-β, citrullinated vimentin (CV), TNF-like ligand 1A (TL1A), CD2, CD23. (IgE receptor), IL-6R, IL-4Rα, IL-12R, IL-13Rα1, IL-13Rα2, IL-15R, IL-23R, CD19, CD20, CD22, CD3, CD4, CD5, IFN-γ, IgE, IL-1α, IL-1β, 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-α.

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 P2A, T2A, E2A and F2A sequences.

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

64. The expression cassette according to any one of claims 40-63, wherein the promoter is a cell-specific promoter, optionally wherein the promoter is specific to 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 the chromosome of the Treg.

67. A method for increasing the persistence of Tregs and / or cytokine signaling, comprising: The expression box as described in any one of claims 40-66 is provided to the Treg. Compared to Tregs that do not contain the expression cassette, the persistence and / or cytokine signaling of the Tregs are increased.

68. The method of claim 67, wherein the expression box is provided to the Treg by transducing the Treg with a carrier containing the expression box.

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

70. A method for generating Tregs, comprising transducing effector T cells (Teffs) with an expression cassette as described in any one of claims 40-66.

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

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

73. The method of claim 70 or claim 71, further comprising measuring the level of one or more biomarkers in the engineered Treg and comparing the level of the one or more biomarkers with that of a wild-type Treg or a control Treg lacking exogenous FOXP3 protein.

74. The engineered Treg as described in any one of claims 1-39, wherein: a) The engineered Treg expression is expressed by one or more markers of wild-type Treg or control Treg lacking exogenous FOXP3 protein; and / or b) The engineered Treg does not express one or more biomarkers that are not expressed by wild-type Treg or by control Treg lacking exogenous FOXP3 protein.

75. The engineered Treg of claim 74, wherein one or more markers are expressed at protein and / or gene levels similar to those in wild-type Tregs or control Tregs lacking exogenous FOXP3 protein.

76. The method of claim 73 or the engineered Treg of claim 74 or claim 75, wherein one or more biomarkers include CD27, CD70, CD71, Helios, glucocorticoid-induced TNFR-related protein (GITR), latent-related peptide (LAP), glycoprotein A repeat dominant protein (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 of the one or more cytokines produced by a wild-type Treg or a control Treg lacking exogenous FOXP3 protein.

78. The method of claim 77, wherein the one or more cytokines comprise TNF-α, IFN-γ, IL-10, IL-4, IL-2, IL-17A, any functional derivative thereof, and any combination thereof.

79. The method of claim 78, wherein the one or more cytokines comprise at least IL-10, and wherein, under non-inflammatory conditions, the levels of IL-10 protein and / or gene expression in the engineered Treg are substantially the same or increased compared to the wild-type Treg or the control Treg lacking exogenous FOXP3 protein.

80. The engineered Treg according to any one of claims 1-39, 72, and 74-76, wherein the engineered Treg expresses a full-length FOXP3 protein at a similar ratio to that of a wild-type Treg or a control Treg lacking exogenous FOXP3 protein, and is isotyped with alternatively spliced ​​FOXP3 lacking one or more exons.

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