Antigen-specific artificial immune control T (airT) cells

Abstract

Some embodiments of the methods and compositions provided herein relate to using a chemical-induced signaling complex (CISC) system to efficiently edit two or more loci in cells. Some embodiments include the use of such a system to edit the FOXP3 locus and the TRAC locus in Treg cells. Further embodiments relate to the use of gene-edited Treg cells to suppress the activation and / or proliferation of specific cell populations.

Description

[Technical Field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 274,375, filed November 1, 2021; U.S. Provisional Patent Application No. 63 / 179,068, filed April 23, 2021; U.S. Provisional Patent Application No. 63 / 155,486, filed March 2, 2021; and U.S. Provisional Patent Application No. 63 / 129,288, filed December 22, 2020, all of which are entitled "Antigen-Specific Artificial Immunoregulatory T (airT) Cells," the contents of which are expressly incorporated herein by reference in their entirety.

[0002] Sequence Listing Reference This application has been filed with an electronic Sequence Listing, which has been provided as a file of approximately 746,677 bytes created on December 20, 2021 under the file name SCRI351WOSEQLIST. The information set forth in this electronic Sequence Listing is incorporated herein by reference in its entirety.

[0003] Some embodiments of the methods and compositions provided herein relate to efficiently editing two or more gene loci in cells using a chemical-inducible signaling complex (CISC) system. Some embodiments include using such a system to edit the FOXP3 locus and the TRAC locus in Treg cells. Further embodiments relate to the use of gene-edited Treg cells to suppress the activation and / or proliferation of specific cell populations. [Background technology]

[0004] Autoimmune diseases, such as type 1 diabetes, multiple sclerosis, myocarditis, rheumatoid arthritis (RA), and systemic lupus erythematosus (SLE) (also known as "lupus"), are chronic conditions characterized by altered immunological self-tolerance, resulting in abnormal immune activation and end-organ pathology, often becoming life-threatening. Inappropriate and detrimental dysregulation of immune tolerance can also contribute to pathologies associated with allergy, asthma, transplant rejection, and / or graft-versus-host disease (GVHD). The role of specific thymus-derived, antigen-recognizing T lymphocytes known as regulatory T cells (Tregs) (also known as suppressor T cells) in maintaining immune tolerance and preventing autoimmunity is well documented, and various autoimmune diseases are characterized by dysfunction or dysregulation of the Treg compartment.

[0005] As a potential treatment for autoimmune diseases, adoptive transfer of functional Treg cells selected for their immunosuppressive potential into subjects with autoimmune diseases has been investigated in mouse models and early clinical trials. However, these Treg cells lack specificity for self-antigens and exhibit uncontrolled cellular plasticity (e.g., they can switch from an immunosuppressive negative regulator of the immune system to a pro-inflammatory effector-like phenotype), resulting in the loss of their immunosuppressive Treg activity. These two major limitations limit adoptive transfer of Treg cells from a single source to provide effective and sustained therapeutic benefits. The use of immunosuppressive Treg cells selected for antigen-specific responses to disease-associated self-antigens is believed to be a safer and more effective adoptive transfer therapy than the simple transfer of multispecific Treg cells. In this regard, these autoantigen-specific Treg cells are predicted to be specifically recruited to tissue sites where autoimmune activity is expressed, and importantly, to specifically exert immunosuppression in response to the autoantigens responsible for the pathogenesis of autoimmune diseases. Supporting this concept, studies using mouse models of autoimmune disease have shown that antigen-specific Treg cells are more effective than polyclonal Treg cells (Duggleby et al., 2018 Front. Immunol. 9:252; Tang et al., 2004 J Exp Med. 199(11):1455-1465; Tarbell et al., 2004 J Exp Med 199:1467-1477).

[0006] However, the therapeutic use of adoptively transferred antigen-specific Treg cells for the treatment of autoimmune diseases, and even polyclonal Treg cells for the same purpose, is limited by a number of issues, particularly the difficulty of isolating rare antigen-specific Treg cells in sufficient quantities from natural sources such as blood and lymph. Furthermore, natural Treg cells are present in small numbers in peripheral blood (e.g., only about 1-4% of peripheral blood mononuclear cells). Furthermore, it is difficult to expand Treg cell populations ex vivo to numbers suitable for therapeutic use while maintaining immunosuppressive function. Furthermore, the development of adoptive Treg transfer therapy is hindered by the fact that, after reinfusion of Treg cells, low-potency Treg cells survive and proliferate in the host that received the adoptively transferred Treg cells. Another issue is the plasticity of Treg cells, which can switch from an immunosuppressive negative regulator to a pro-inflammatory effector-like phenotype in inflammatory situations in vivo (Singer et al., 2014 Front. Immunol. 5:Art. 46; Trzonkowski et al., 2015 Sci. Translat. Med. 7(304):ps18 Romano et al., 2016 Transplant. Internatl. 30:745, McGovern et al., 2017 Front. Immunol. 8:Art. 1517).

[0007] Prior art methods have not provided large populations of stable Treg cells with desired antigen specificity and suppressive activity, such as specificity for antigens involved in the pathogenesis of diseases in which antigen-specific immunosuppression is thought to be beneficial (e.g., autoimmune diseases, allergies and / or other inflammatory diseases).

[0008] Therefore, there remains a need for stable antigen-specific immunoregulatory cells that can maintain antigen-specific immunosuppressive ability in vitro and in vivo without exhibiting plasticity and that are useful for administration to subjects in need of antigen-specific immunosuppression by adoptive transfer immunotherapy. Summary of the Invention [Means for solving the problem]

[0009] Some embodiments include: A chemical-inducible signaling complex (CISC) system for gene editing, comprising: a first polynucleotide encoding a nucleic acid encoding a first CISC component comprising a first extracellular binding domain, a transmembrane domain, and a first signaling domain, and a first promoter adjacent to the nucleic acid encoding the first CISC component; a second polynucleotide encoding a nucleic acid encoding a second CISC component comprising a second extracellular binding domain, a transmembrane domain, and a second signaling domain, and a second promoter; Including, The system includes a first CISC component and a second CISC component configured such that, when expressed in a cell, they can dimerize in the presence of rapamycin or a rapalog to form a CISC having signaling function. In some embodiments, the second promoter is adjacent to the nucleic acid encoding the second CISC component.

[0010] In some embodiments, the 3' end of the first promoter is comprised within 500 contiguous nucleotides from the 5' end of the nucleic acid encoding the first CISC component, hi some embodiments, the 3' end of the first promoter is comprised within 100 contiguous nucleotides from the 5' end of the nucleic acid encoding the first CISC component.

[0011] In some embodiments, the first polynucleotide is configured to integrate into a first target locus of a genome, and the second polynucleotide is configured to integrate into a second target locus of the genome. In some embodiments, the first target locus is selected from the TRAC locus and the FOXP3 locus, and the second target locus is selected from the TRAC locus and the FOXP3 locus.

[0012] In some embodiments, the first extracellular binding domain comprises an FK506 binding protein (FKBP)-rapamycin binding (FRB) domain and the second extracellular binding domain comprises an FKBP domain.

[0013] In some embodiments, the first signaling domain comprises an IL-2 receptor β subunit (IL2Rβ) domain or a functional derivative thereof, and the second signaling domain comprises an IL-2 receptor γ subunit (IL2Rβγ) domain or a functional derivative thereof. In some embodiments, the IL2Rβ domain comprises a truncated IL2Rβ domain.

[0014] In some embodiments, the first promoter and / or the second promoter comprises a constitutive promoter. In some embodiments, the first promoter and / or the second promoter comprises an MND promoter.

[0015] In some embodiments, the first polynucleotide is contained in a first vector and the second polynucleotide is contained in a second vector. In some embodiments, the first vector and / or the second vector comprises a viral vector. In some embodiments, the first vector and / or the second vector comprises a lentiviral vector, an adenoviral vector, or an adeno-associated viral (AAV) vector.

[0016] In some embodiments, the first polynucleotide and / or the second polynucleotide comprises a nucleic acid encoding a naked FRB domain, wherein the nucleic acid encoding the naked FRB domain lacks a nucleic acid encoding an endoplasmic reticulum localization signal peptide.

[0017] In some embodiments, the first polynucleotide and / or the second polynucleotide comprises a nucleic acid encoding a payload. In some embodiments, the first polynucleotide and / or the second polynucleotide comprises a nucleic acid encoding a self-cleaving polypeptide, wherein the nucleic acid encoding the self-cleaving polypeptide is located 5' to the nucleic acid encoding the payload. In some embodiments, the self-cleaving polypeptide is selected from the group consisting of P2A, T2A, E2A, and F2A. In some embodiments, the payload comprises a T cell receptor (TCR), a chimeric antigen receptor (CAR), or a functional fragment thereof. In some embodiments, the TCR or functional fragment thereof comprises a polypeptide sequence set forth in any of SEQ ID NOs: 1377-1390.

[0018] In some embodiments, the first polynucleotide and / or the second polynucleotide are configured to be integrated into the target genomic locus by homology directed repair (HDR) or non-homologous end joining (NHEJ).

[0019] Some embodiments further comprise a guide RNA (gRNA) and a DNA endonuclease, in some embodiments, the DNA endonuclease comprises Cas9 endonuclease.

[0020] In some embodiments, the rapalog is selected from the group consisting of everolimus, CCI-779, C20-methallylrapamycin, C16-(S)-3-methylindolerapamycin, C16-iRap, C16-(S)-7-methylindolerapamycin, AP21967, C16-(S)-butylsulfonamidorapamycin, AP23050, mycophenolate sodium, benidipine hydrochloride, AP1903, AP23573, and metabolites and derivatives thereof.

[0021] Some embodiments include cells comprising the system or the system described herein. Some embodiments herein provide cells (e.g., human cells) in which a locus encoding an endogenous TCR (e.g., the TRAC gene / locus) has been edited. In some embodiments, the TRAC gene of the cell is edited by promoter capture (e.g., the methods shown in Figures 54, 68, and 70). In some embodiments, the TRAC gene of the cell is edited by knocking in a promoter (e.g., the MND promoter) to the full-length sequence of the TCR (e.g., a pancreatic islet TCR). For an example of such a method, see Figure 67. In some embodiments, the TRAC gene of the cell is edited by hijacking the TRAC gene with a promoter (e.g., the MND promoter), as shown in Figure 164. In some embodiments, the cell provided herein is a human cell. In some embodiments, the cell is a lymphocyte (e.g., an NK1.1+ cell, a CD3+ cell, a CD4+ cell, or a CD8+ cell). In some embodiments, the cell is a T cell, a progenitor T cell, or a hematopoietic stem cell. In some embodiments, the cells are NK-T cells (e.g., FOXP3 - NK-T cells or FOXP3 +In some embodiments, the cells are regulatory B (Breg) cells (e.g., FOXP3- B cells or FOXP3+ B cells). In some embodiments, the cells are CD4+ T cells (e.g., FOXP3- CD4+ T cells or FOXP3+ CD4+ T cells) or CD8+ cells (e.g., FOXP3- CD8+ T cells or FOXP3+ CD8+ T cells). In some embodiments, the cells are CD25- T cells. In some embodiments, the cells are regulatory T (Treg) cells. Examples of Treg cells include, but are not limited to, Tr1 cells, Th3 cells, CD8+ CD28- cells, and Qa-1-restricted T cells. In some embodiments, the cells are type 1 regulatory T (Tr1) cells. In some embodiments, the Treg cells are FOXP3+ Treg cells. In some embodiments, the Treg cells express CTLA-4, LAG-3, CD25, CD39, neuropilin 1, galectin 1, and / or IL-2Rα on their surface. In some embodiments, the cells provided herein are recombinant cells. In some embodiments, the recombinant cells are cells in which one or more genes / locuses have been genetically engineered or edited (e.g., to stabilize expression of one or more genes). In some embodiments, the recombinant cells have been edited at the Foxp3 gene / locus, for example, by inserting a promoter (in some embodiments, inserting a promoter downstream of one or more regulatory elements, such as TSDR, and / or upstream of the first coding exon or first codon). In some embodiments, the cells are ex vivo cells. In some embodiments, the cells are in vivo cells. In some embodiments, the cells are human cells. In some embodiments, the cells described herein are cells isolated from a biological sample. A biological sample may be a sample obtained from a subject (e.g., a human subject) or a composition produced in a laboratory (e.g., by cell culture).The biological sample obtained from the subject may be a liquid sample (e.g., blood or a fraction thereof, bronchial lavage fluid, cerebrospinal fluid, or urine) or a solid sample (e.g., a tissue slice). In some embodiments, the cells are obtained from peripheral blood. In some embodiments, the cells are obtained from umbilical cord blood.

[0022] Some embodiments include a pharmaceutical composition comprising the cells and a pharmaceutically acceptable excipient.

[0023] Some embodiments provide a method of editing a cell, comprising: obtaining said system; introducing the first polynucleotide and the second polynucleotide into a cell to obtain a transduced cell; and Culturing the transduced cells. The method includes: Some embodiments further comprise contacting the transduced cells with rapamycin or a rapalog. In some embodiments, contacting the cells with rapamycin or a rapalog is performed ex vivo, e.g., for cell selection. In some embodiments, contacting the cells with rapamycin or a rapalog is performed in vivo, e.g., to activate the cells via the intracellular CISC machinery or to maintain cell activity. This results in a stable, functional suppressive phenotype (e.g., by maintaining expression of IL-2 or STAT5) or maintains a high number of active immunosuppressive cells (e.g., by stabilizing the suppressive phenotype). In some embodiments, contacting the cells with rapamycin or a rapalog in vivo maintains at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the administered immunosuppressive cells. In some embodiments, contacting cells in vivo with rapamycin or a rapalog can maintain the expression level of one or more cytokines at 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more of the level observed prior to administration. In some embodiments, cells comprising the CISC machinery described herein and the rapamycin or rapalog are administered to a subject simultaneously. In some embodiments, cells comprising the CISC machinery described herein and the rapamycin or rapalog are administered to a subject sequentially.

[0024] Some embodiments include a method of inhibiting proliferation of a cell population, the method comprising contacting the cell population comprising an endogenous T cell receptor (TCR) specific for a first epitope of an antigen with genetically engineered Treg cells (e.g., CD4+ Treg cells or CD8+ Treg cells) comprising an exogenous TCR specific for a second epitope of the antigen.

[0025] Some embodiments include a method of treating, alleviating, or suppressing a disease in a subject, comprising administering to the subject genetically modified T cell receptor (TCR) (e.g., CD4+ Treg cells or CD8+ Treg cells) comprising an exogenous T cell receptor specific for a second epitope of an antigen to suppress the proliferation of a cell population comprising an endogenous TCR specific for a first epitope of the antigen.

[0026] In some embodiments, the disease comprises an autoimmune disease. In some embodiments, the subject is a mammal. In some embodiments, the subject is human.

[0027] In some embodiments, the exogenous TCR has a higher avidity for the antigen than another TCR specific for the antigen.

[0028] In some embodiments, the exogenous TCR has a lower avidity for the antigen compared to another TCR specific for the antigen.

[0029] In some embodiments, the cell population comprises CD4+CD25- T cells. In some embodiments, the cell population comprises polyclonal T cells.

[0030] In some embodiments, the exogenous TCR is specific for a type 1 diabetes antigen. In some embodiments, the exogenous TCR is specific for a type 1 diabetes antigen selected from IGRP, GAD65, and PPI. In some embodiments, the exogenous TCR is selected from T1D2, T1D4, T1D5-1, T1D5-2, 4.13, GAD113, and PPI76. In some embodiments, the exogenous TCR comprises T1D5-2.

[0031] In some embodiments, the cell population is contacted with the genetically modified Treg cells in the presence of antigen-presenting cells and the antigen.

[0032] In some embodiments, the Treg cells are obtained by introducing into cells a vector comprising a nucleic acid encoding the exogenous TCR.

[0033] In some embodiments, the Treg cells are mammalian cells, hi some embodiments, the Treg cells are human cells.

[0034] Some embodiments of the methods and compositions provided herein involve the production of artificial cells (e.g., antigen-specific CD4+CD25+ immunoregulatory T (airT) cells; also referred to as recombinant regulatory T cell-like cells (EngTregs) or gene-edited regulatory T cell-like cells (edTregs)), which: (a) an artificially modified FOXP3 gene that constitutively expresses the forkhead box protein 3 / winged helix transcription factor (FOXP3) gene product at an expression level equal to or greater than that of natural regulatory T (Treg) cells; and (b) at least one introduced polynucleotide encoding an antigen-specific T cell receptor (TCR) polypeptide (e.g., a polynucleotide introduced into the TRAC locus / gene); Contains airT cells. Examples of such cells are shown in Figures 33 and 54. Figures 54, 67-68, 70 and 164 show examples of methods that may be used for transduction of TCR polypeptides.

[0035] In some embodiments, the present invention provides antigen-specific CD4+CD25+ artificial immunoregulatory T (airT) cells obtained by (i) artificially modifying the forkhead box protein 3 / winged helix transcription factor (FOXP3) gene in CD4+CD25- T cells and (ii) inserting at least one polynucleotide encoding an antigen-specific T cell receptor (TCR) polypeptide or a chimeric antigen receptor (CAR) polypeptide, wherein the artificial modification results in constitutive expression of the FOXP3 gene product at an expression level equal to or greater than that of natural regulatory T (Treg) cells. In some embodiments, the present invention provides antigen-specific artificial immunoregulatory T (airT) cells (e.g., CD4+ cells or CD8+ cells) obtained by (i) artificially modifying the forkhead box protein 3 / winged helix transcription factor (FOXP3) gene in CD25- T cells and (ii) inserting at least one polynucleotide encoding an antigen-specific T cell receptor (TCR) polypeptide or a chimeric antigen receptor (CAR) polypeptide, wherein the artificial modification results in constitutive expression of the FOXP3 gene product at an expression level equal to or greater than that of natural regulatory T (Treg) cells.

[0036] In some embodiments, the FOXP3 gene is present in a FOXP3 locus that includes a regulatory element capable of regulating FOXP3 expression in natural Treg cells. In some embodiments, the FOXP3 gene is present in a FOXP3 locus that includes an intronic regulatory T cell (Treg)-specific demethylation region (TSDR) having multiple cytosine-guanine (CG) dinucleotides, each CG dinucleotide including a methylated cytosine (C) nucleotide at a nucleotide position that includes a demethylated C nucleotide in natural Treg cells. In some embodiments, at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the C nucleotides in the TSDR of natural Treg cells are methylated at the nucleotide positions that include a demethylated C nucleotide. In some embodiments, the FOXP3 gene product is expressed at an amount sufficient for the airT cells to maintain a CD4+CD25+ phenotype in vitro for at least 21 days. In some embodiments, the FOXP3 gene product is expressed at an amount sufficient to cause the airT cells to maintain a CD4+CD25+ phenotype in vivo for at least 60 days after adoptive transfer of the airT cells into an immunocompatible mammalian host in need of antigen-specific immunosuppression. In some embodiments, the airT cells comprise a phenotype selected from (i) HeliosLo, (ii) CD152+, (iii) CD127-, and (iv) ICOS+. In some embodiments, the artificial modification comprises a knockout of the native FOXP3 locus.

[0037] In some embodiments, the artificial modification involves the insertion of a nucleic acid molecule containing a heterologous promoter into the native FOXP3 locus, where the heterologous promoter is positioned on the FOXP3 gene so as to promote transcription of the nucleotide sequence encoding endogenous FOXP3 at the FOXP3 locus. In some embodiments, the heterologous promoter is a constitutive promoter, promoting transcription of an operably linked sequence (e.g., the FOXP3 gene) with a consistent efficiency. The constitutive promoter may be a strong promoter that promotes transcription more efficiently than the endogenous promoter, or a weak promoter that promotes transcription less efficiently than either the strong promoter or the endogenous promoter. In some embodiments, the constitutive promoter is a strong promoter. In some embodiments, the constitutive promoter is a weak promoter. In some embodiments, the strong promoter is the MND promoter. In some embodiments, the constitutive promoter is the PGK promoter, the MND promoter, or the EF-1a promoter. In some embodiments, the heterologous promoter is an inducible promoter. An inducible promoter promotes transcription of an operably linked sequence in response to the presence of an activating signal or the absence of an inhibitory signal. In some embodiments, the inducible promoter is inducible by a drug or steroid.

[0038] In some embodiments, the inserted nucleic acid molecule further comprises a nucleic acid sequence encoding a first chemo-induced signaling complex (CISC) component capable of specifically binding to a CISC-inducing molecule. In some embodiments, the introduced polynucleotide encoding the TCR polypeptide further comprises a nucleic acid sequence encoding a second CISC component distinct from the first CISC component, capable of specifically binding to the CISC-inducing molecule. In some embodiments, the nucleic acid sequence encoding the first CISC component and the nucleic acid sequence encoding the second CISC component further comprise a nucleic acid sequence encoding a third CISC component distinct from the first and second CISC components, capable of specifically binding to the CISC-inducing molecule. In some embodiments, the third CISC component is soluble and does not comprise a transmembrane or extracellular domain. In some embodiments, the third soluble CISC component does not comprise a secreted peptide and is localized in the cytoplasm. In some embodiments, the nucleic acid molecule comprising the constitutively active promoter is inserted downstream of an intronic regulatory T cell (Treg)-specific demethylation region (TSDR) of the native FOXP3 locus. In some embodiments, the constitutively active promoter is an MND promoter. In some embodiments, the artificial modification comprises inserting into the native FOXP3 locus a nucleic acid molecule comprising a polynucleotide encoding an exogenous FOXP3 and a constitutively active promoter operably linked thereto. In some embodiments, the inserted nucleic acid molecule comprising a polynucleotide encoding an exogenous FOXP3 and a constitutively active promoter operably linked thereto further comprises a nucleic acid sequence encoding a first chemo-induced signaling complex (CISC) component capable of specifically binding to a CISC-inducing molecule. In some embodiments, the introduced polynucleotide encoding a gene (e.g., a FOXP3 polypeptide or a TCR polypeptide) further comprises a nucleic acid sequence encoding a second CISC component, separate from the first CISC component, capable of specifically binding to the CISC-inducing molecule.In some embodiments, at least one of the nucleic acid sequence encoding the first CISC component and the nucleic acid sequence encoding the second CISC component further comprises a nucleic acid sequence encoding a third CISC component that is distinct from the first CISC component and the second CISC component and capable of specifically binding to the CISC-inducing molecule. In some embodiments, the third CISC component is soluble and does not contain a transmembrane or extracellular domain. In some embodiments, the third soluble CISC component does not contain a secreted peptide and is localized in the cytoplasm. Examples of this are shown in Figures 33 and 54.

[0039] In some embodiments, the nucleic acid molecule comprising a polynucleotide encoding exogenous FOXP3 and an operably linked constitutively active promoter is inserted downstream of an intronic regulatory T cell (Treg)-specific demethylation region (TSDR) of the native FOXP3 locus. In some embodiments, the constitutively active promoter is an MND promoter.

[0040] In some embodiments, the artificial modification involves the insertion of a nucleic acid molecule comprising a polynucleotide encoding exogenous FOXP3 and a constitutively active promoter operably linked thereto at a chromosomal site other than the native FOXP3 locus. In some embodiments, at least one native T cell receptor (TCR) locus of the airT cell is knocked out or inactivated and replaced with the at least one introduced polynucleotide encoding an antigen-specific T cell receptor (TCR) polypeptide. In some embodiments, a promoter capture method is used in which an inserted TCR-encoding nucleic acid is driven by a native / natural / endogenous promoter, and this TCR-encoding nucleic acid may be followed by nucleic acids encoding one or more CISC components described herein (see, e.g., Figures 54, 68, and 70). In some embodiments, such an endogenous promoter capture method involves in-frame knock-in into exon 1 of the TRAC locus, thereby driving TCR expression via the endogenous TRAC promoter. In some embodiments, a TCR knock-in method is utilized to knock in a TCR-encoding nucleic acid and a promoter, which may be followed by nucleic acids encoding one or more of the CISC components described herein (see, e.g., Figure 67). In some embodiments, a native / natural / endogenous TCR gene or fragment thereof is hijacked by inserting a promoter upstream of the native / natural / endogenous TCR gene or fragment thereof, which may be upstream of a nucleic acid encoding one or more of the CISC components described herein (see, e.g., Figure 164). In some embodiments, editing of the TRAC gene / locus may be combined with inserting a promoter downstream of the TSDR of the Foxp3 gene / locus.

[0041] In some embodiments, one inserted nucleic acid encodes a first CISC component and the other nucleic acid encodes a second CISC component, such that cells into which both nucleic acids are inserted express both the first and second CISC components, whereas cells into which only one nucleic acid is inserted express only the first or second CISC component. In such embodiments, cells into which both nucleic acids are inserted can be selected by providing a CISC-inducing molecule that induces dimerization of the first and second CISC components to transduce a growth signal. If such inserted nucleic acid contains other genetic modifications, such as an exogenous TCR or CAR, or if such inserted nucleic acid contains a heterologous promoter that drives expression of the endogenous FOXP3 gene independently of regulation by endogenous regulatory elements, cells containing such genetic modifications can be selected based on expression of both CISC components. Thus, cells containing an inserted first nucleic acid molecule encoding a first CISC component and a TCR or CAR, and an inserted second nucleic acid molecule encoding a second CISC component and comprising a heterologous promoter operably linked to the endogenous FOXP3 gene, can be selected by providing a CISC-inducing molecule. Thus, by providing a CISC-inducing molecule, airT cells with a desired antigen specificity and expressing FOXP3 independently of endogenous regulatory mechanisms, such as repression by methylated TSDR, can be selected.

[0042] Any known gene editing method can be used to insert a nucleotide sequence or modify a genomic locus. Gene editing methods include, but are not limited to, gene editing using zinc finger nucleases (ZFNs), gene editing using transcription activator-like effector nucleases (TALENs), gene editing using meganucleases, gene editing using transposons, gene editing using serine integrases, gene editing using lentiviruses, gene editing using RNA-guided nucleases (RGNs), gene editing using CRISPR / Cas, gene editing using homologous recombination, and combinations thereof. ZFNs are restriction enzymes created by fusing a zinc finger DNA binding domain to a DNA cleavage domain. Therefore, ZFNs bind to specific DNA sequences based on their specificity and cleave DNA. TALENs are restriction enzymes containing a TAL effector domain that binds to specific DNA sequences and can be engineered to recognize the desired DNA sequence and the DNA cleavage domain of FokI endonuclease. Therefore, TALENs bind to specific DNA sequences based on their specificity, and cleave the DNA after binding to the target sequence. Meganucleases are targeted nucleases derived from homing endonucleases such as I-CreI and I-SceI, and bind to specific DNA sequences based on their specificity and cleave the DNA. Transposons are chromosomal elements that can transpose, and can excise transposons from one location on a chromosome and introduce them into another location. Transposases are responsible for integrating transposons into chromosomes. In transposon-based gene editing, a desired sequence is inserted into a transposon, and the transposon containing the desired sequence is introduced into cells together with transposase or a nucleic acid encoding the transposase. The transposon containing the desired sequence is inserted into a chromosome by the action of the transposase. See, for example, Ivics and Izsvak. Curr Gene Ther. 2006. 6(5):593-607.RNA-guided nuclease is a nuclease that binds to a conserved nucleotide sequence on the RNA guide, and this RNA guide has a targeting nucleotide sequence that is complementary to the target nucleotide sequence of the nucleic acid (e.g., chromosome) to be recombined.Therefore, the targeting nucleotide sequence guides the RNA-guided nuclease to the nucleic acid containing the target sequence, and when the RNA guide binds to the target sequence, the RNA-guided nuclease cuts the DNA.Examples of RNA-guided nucleases include, but are not limited to, those described in U.S. Patent No. 11,162,114 (this document is incorporated herein by reference in its entirety).Another example of RNA-guided nucleases is CRISPR-Cas-related nucleases.

[0043] In some embodiments, the at least one native TCR locus that has been knocked out or inactivated is a native TCR alpha chain (TRAC) locus. In some embodiments, the inserted nucleic acid molecule comprising a polynucleotide encoding exogenous FOXP3 and a constitutively active promoter operably linked thereto further comprises a nucleic acid sequence encoding a first chemically-induced signaling complex (CISC) component capable of specifically binding to a CISC-inducing molecule. In some embodiments, the introduced polynucleotide encoding the TCR polypeptide further comprises a nucleic acid sequence encoding a second CISC component distinct from the first CISC component and capable of specifically binding to the CISC-inducing molecule. In some embodiments, at least one of the nucleic acid sequence encoding the first CISC component and the nucleic acid sequence encoding the second CISC component further comprises a nucleic acid sequence encoding a third CISC component distinct from the first CISC component and the second CISC component and capable of specifically binding to the CISC-inducing molecule. In some embodiments, the third CISC component is soluble and does not comprise a transmembrane domain or an extracellular domain. In some embodiments, the third soluble CISC component does not comprise a secreted peptide and is localized in the cytoplasm. In some embodiments, the constitutively active promoter is an MND promoter. In some embodiments, the chromosomal site, other than the native FOXP3 locus, into which the nucleic acid molecule comprising a polynucleotide encoding exogenous FOXP3 and an operably linked constitutively active promoter has been inserted is within the T cell receptor alpha chain (TRAC) locus of the airT cell.

[0044] In some embodiments, at least one native T cell receptor (TCR) locus of the airT cell has been knocked out or inactivated and replaced with the at least one introduced polynucleotide encoding an antigen-specific T cell receptor (TCR) polypeptide. In some embodiments, the at least one knocked out native TCR locus is a native TCR alpha chain (TRAC) locus.

[0045] In some embodiments, the antigen-specific CD4+CD25+ artificial immune regulatory T (airT) cells are (a) an introduced nucleic acid sequence encoding an exogenous forkhead box protein 3 / winged helix transcription factor (FOXP3) gene product that is constitutively expressed at an expression level equal to or greater than that of natural regulatory T (Treg) cells; and (b) at least one introduced polynucleotide encoding an antigen-specific exogenous T cell receptor (TCR) polypeptide. Including, the introduced nucleic acid sequence encoding the exogenous FOXP3 gene product further comprises a nucleic acid sequence encoding a first chemical-induced signaling complex (CISC) component capable of specifically binding to a CISC-inducing molecule; the introduced nucleic acid sequence encoding the exogenous TCR gene product further comprises a nucleic acid sequence encoding a second CISC component separate from the first CISC component and capable of specifically binding to the CISC-inducing molecule; Provide airT cells.

[0046] In some embodiments, the antigen-specific CD4+CD25+ artificial immune regulatory T (airT) cells are (a) a FOXP3 locus that has been knocked out or inactivated by homologous recombination repair, and into which (i) a nucleic acid molecule comprising a constitutively active promoter capable of promoting transcription of a nucleotide sequence encoding endogenous FOXP3 of the native FOXP3 gene, or (ii) a nucleic acid molecule comprising a nucleotide sequence encoding an exogenous FOXP3 protein or a functional derivative thereof and a constitutively active promoter operably linked thereto, has been inserted, thereby constitutively expressing a FOXP3 gene product at an expression level equivalent to or greater than that of natural regulatory T (Treg) cells; and (b) a native T cell receptor alpha chain (TRAC) locus that has been knocked out by homologous recombination repair and inserted with at least one introduced polynucleotide encoding an antigen-specific exogenous T cell receptor (TCR) polypeptide; Including, the inserted nucleic acid molecule comprising the constitutively active promoter, or the inserted nucleic acid molecule comprising a nucleotide sequence encoding an exogenous FOXP3 protein or a functional derivative thereof and a constitutively active promoter operably linked thereto, further comprises a nucleic acid sequence encoding a first chemical-induced signaling complex (CISC) component capable of specifically binding to a CISC-inducing molecule; the introduced nucleic acid sequence encoding the exogenous TCR polypeptide further comprises a nucleic acid sequence encoding a second CISC component separate from the first CISC component and capable of specifically binding to the CISC-inducing molecule; Provide airT cells. The methods described herein, involving genetic manipulation of CD4+ cells, can also be applied to other cell types (e.g., CD8+ cells). In some embodiments, the methods provided herein involve editing CD3+ cells to produce edited CD3+ cells, such as CD4+ airT cells or CD8+ airT cells. In some embodiments, the methods involve editing CD4+ T cells to produce CD4+ airT cells. In some embodiments, the methods involve editing CD8+ T cells to produce CD8+ airT cells. In some embodiments, the methods involve editing NK1.1+ T cells to produce NK1.1+ airT cells. In some embodiments, the methods involve editing CD34+ hematopoietic stem cells (HSCs). In some embodiments, the methods involve editing induced pluripotent stem cells (iPSCs). The edited stem cells may be matured in vitro to produce airT cells or may be administered to a subject and developed in vivo to produce airT cells. The edited stem cells may be matured to obtain CD3+ airT cells, CD4+ airT cells, CD8+ airT cells, NK1.1+ airT cells, or a combination thereof.

[0047] In some embodiments, at least one of the nucleic acid sequence encoding the first CISC component and the nucleic acid sequence encoding the second CISC component further comprises a nucleic acid sequence encoding a third CISC component distinct from the first CISC component and the second CISC component, capable of specifically binding to the CISC-inducing molecule. In some embodiments, the third CISC component is soluble and does not comprise a transmembrane domain or an extracellular domain. In some embodiments, the third soluble CISC component does not comprise a secreted peptide and is localized in the cytoplasm. In some embodiments, the nucleic acid sequence encoding the first CISC component comprises a nucleic acid sequence homologous to a first locus in the genome of the cell, and the nucleic acid sequence encoding the second CISC component comprises a nucleic acid sequence homologous to a second locus in the genome of the cell, the first locus and the second locus being different loci. In some embodiments, the first locus is the FOXP3 locus, TRAC locus, AAVS1 locus, or ROSA26 locus; and the second locus is the FOXP3 locus, TRAC locus, AAVS1 locus, or ROSA26 locus, and the first and second loci are different loci. In some embodiments, the first and second loci are the same locus (e.g., upstream and downstream of the same locus), and the first and second loci are selected from the group consisting of the FOXP3 locus, TRAC locus, AAVS1 locus, and ROSA26 locus. In some embodiments, a nucleic acid comprising a nucleotide sequence encoding a first CISC component and a nucleic acid comprising a nucleotide sequence encoding a second CISC component are provided, and at least one of these nucleic acids may comprise a third CISC component. In some embodiments, one or more nucleic acids incorporated into a vector are provided. In some embodiments, a cell is provided that contains any one or more of the nucleic acid, the vector, and a genome modified by the insertion of the vector or the nucleic acid. In some embodiments, a composition is provided that includes the nucleic acid, the vector, or the genetically modified cell.

[0048] In some embodiments, a first polynucleotide encoding a first CISC component is inserted into a first locus (e.g., the Foxp3 locus), and a second polynucleotide encoding a second CISC component is inserted into a second locus (e.g., the TRAC locus). In some embodiments, the first polynucleotide is included in a first nucleic acid vector, and the second polynucleotide is included in a second nucleic acid vector. In some embodiments, the first polynucleotide and the second polynucleotide are included in a single nucleic acid vector. In some embodiments, the first polynucleotide further comprises one or more regulatory elements (e.g., a heterologous promoter such as MND) and / or a payload (e.g., a nucleic acid encoding a FOXP3 polypeptide). In some embodiments, the second polynucleotide further comprises one or more regulatory elements (e.g., a heterologous promoter such as MND) and / or a payload (e.g., a nucleic acid encoding a TCR or CAR). In some embodiments, a first CISC component encoded from a first locus comprises an extracellular domain comprising FKBP or a functional fragment thereof, and a second CISC component encoded from a first locus comprises an extracellular domain comprising FRB or a functional fragment thereof. In some embodiments, a first CISC component encoded from a first locus comprises an extracellular domain comprising FRB or a functional fragment thereof, and a second CISC component encoded from a first locus comprises an extracellular domain comprising FKBP or a functional fragment thereof. In some embodiments, a CISC component having an extracellular domain comprising FKBP or a functional fragment thereof further comprises an intracellular signaling domain of IL-2RB or a functional fragment thereof. In some embodiments, a CISC component having an extracellular domain comprising FKBP or a functional fragment thereof further comprises an intracellular signaling domain of IL-2RG or a functional fragment thereof. In some embodiments, a CISC component having an extracellular domain comprising FKBP or a functional fragment thereof further comprises an intracellular signaling domain of IL-2RB or a functional fragment thereof.In some embodiments, the CISC component having an extracellular domain comprising FRB or a functional fragment thereof further comprises the intracellular signaling domain of IL-2RB or a functional fragment thereof. In some embodiments, the first polynucleotide or the second polynucleotide further comprises a nucleic acid encoding a third CISC component that does not comprise the extracellular domain or the transmembrane domain but comprises FRB or a functional fragment thereof. The functional fragment of FKBP or FRB is a functional fragment that can bind to rapamycin or a rapalog.

[0049] In some embodiments, the airT cells comprise at least a first introduced polynucleotide encoding an antigen-specific TCR polypeptide and a second introduced polynucleotide encoding an antigen-specific TCR polypeptide, wherein the first polynucleotide encodes a TCR Vα polypeptide and the second polynucleotide encodes a TCR Vβ polypeptide, and the Vα polypeptide and the Vβ polypeptide constitute a functional TCR capable of specifically recognizing an antigen. In some embodiments, the airT cells express an antigen-specific T cell receptor (TCR) comprising the antigen-specific TCR polypeptide encoded by at least one introduced polynucleotide encoding the TCR polypeptide, and are capable of inducing antigen-specific immunosuppression in response to HLA-restricted stimulation by an antigen specifically recognized by the TCR polypeptide. In some embodiments, the antigen-specifically induced immunosuppression is (i) inhibiting one or both of the activation and proliferation of effector T cells that recognize an antigen specifically recognized by the TCR of said airT cell comprising said TCR polypeptide encoded by said at least one introduced polynucleotide; (ii) suppression of the expression of inflammatory cytokines or inflammatory mediators by effector T cells that recognize an antigen specifically recognized by the TCR of the airT cell comprising the TCR polypeptide encoded by the at least one introduced polynucleotide; (iii) production by the airT cells of one or more immunosuppressive cytokines, perforin / granzymes, or anti-inflammatory products; or induction in the airT cells of at least one of indoleamine-2,3-dioxygenase (IDO), competition for IL2 or adenosine, tryptophan catabolism, and expression of inhibitory receptors; and (iv) inhibiting one or both of the activation and proliferation of effector T cells that do not recognize the antigen specifically recognized by the TCR of the airT cell comprising the TCR polypeptide encoded by the at least one introduced polynucleotide. Contains one or more of the following:

[0050] In some embodiments, the TCR specifically recognizes an antigen involved in the pathogenesis of autoimmune diseases, allergic diseases, or inflammatory diseases. In some embodiments, the TCR specifically recognizes an antigen involved in the pathogenesis of autoimmune diseases (e.g., diabetes, such as type 1 diabetes, and primary biliary cholangitis), autoinflammatory diseases (e.g., acute respiratory distress syndrome (ARDS), stroke, and atherosclerotic cardiovascular disease), alloimmune diseases (e.g., graft-versus-host disease, solid organ transplantation, and immune-mediated recurrent pregnancy loss), and / or allergic diseases (e.g., asthma, drug hypersensitivity, and celiac disease). In some embodiments, the disease to be treated is cancer. Wang et al. (J Intern Med. 2015 Oct;278(4):369-95) has published a review article on autoimmune diseases (this review article is incorporated herein by reference). In some embodiments, (i) the autoimmune disease is selected from type 1 diabetes, multiple sclerosis, systemic lupus erythematosus, myasthenia gravis, rheumatoid arthritis, early-onset rheumatoid arthritis, ankylosing spondylitis, immune-mediated pregnancy loss, immune-mediated recurrent pregnancy loss, dermatomyositis, psoriatic arthritis, Crohn's disease, inflammatory bowel disease (IBD), ulcerative colitis, bullous pemphigoid, pemphigus vulgaris, autoimmune hepatitis, psoriasis, Sjogren's syndrome, and celiac disease; (ii) the allergic disease is selected from allergic asthma, atopic dermatitis, pollen allergy, food allergy, drug hypersensitivity, and contact dermatitis; (iii) the inflammatory disease is selected from pancreatic islet cell transplantation, asthma, steroid-resistant asthma, hepatitis, traumatic brain injury, primary sclerosing cholangitis, primary biliary cholangitis, polymyositis, stroke, Still's disease, acute respiratory distress syndrome, uveitis, inflammatory bowel disease (IBD), ulcerative colitis, graft-versus-host disease (GVHD), transplant tolerance induction, transplant rejection, and sepsis. In some embodiments, the inflammatory disease is primary biliary cholangitis. In some embodiments, the inflammatory disease is primary sclerosing cholangitis. In some embodiments, the inflammatory disease is autoimmune hepatitis. In some embodiments, the autoimmune disease is type 1 diabetes. In some embodiments, the inflammatory disease is pancreatic islet cell transplantation. In some embodiments, the inflammatory disease is transplant rejection. In some embodiments, the autoimmune disease is multiple sclerosis. In some embodiments, the inflammatory disease is inflammatory bowel disease. In some embodiments, the inflammatory disease is acute respiratory distress syndrome. In some embodiments, the inflammatory disease is stroke. In some embodiments, the inflammatory disease is graft-versus-host disease. In some embodiments, (i) the antigen associated with the pathogenesis of the autoimmune disease is selected from the autoantigens listed in any one of Figures 141 to 144; (ii) the antigen related to the etiology of the allergic disease is selected from the allergic antigens listed in any one of Figures 141 to 144; (iii) The antigen associated with the pathogenesis of the inflammatory disease is selected from the inflammation-associated antigens shown in any of Figures 141 to 144.

[0051] In some embodiments, the airT cells comprise at least one introduced polynucleotide sequence encoding a TCR polypeptide that specifically binds in a human HLA-restricted manner to an antigenic polypeptide epitope consisting of no more than 35, no more than 34, no more than 33, no more than 32, no more than 31, no more than 30, no more than 29, no more than 28, no more than 27, no more than 26, no more than 25, no more than 24, no more than 23, no more than 22, no more than 21, no more than 20, no more than 19, no more than 18, no more than 17, no more than 16, no more than 15, no more than 14, no more than 13, no more than 12, no more than 11, no more than 10, no more than 9, no more than 8, or no more than 7 consecutive amino acids of an amino acid sequence selected from the antigenic polypeptide sequences set forth in any of Figures 141-144, or at least one introduced polynucleotide sequence encoding a TCR polypeptide encoded by a nucleotide sequence set forth in Figure 139A or 140A.In some embodiments, the airT cells contain an introduced first polynucleotide sequence encoding a TCR Vα polypeptide of a TCR that specifically binds in a human HLA-restricted manner to an antigenic polypeptide epitope consisting of 35 or less, 34 or less, 33 or less, 32 or less, 31 or less, 30 or less, 29 or less, 28 or less, 27 or less, 26 or less, 25 or less, 24 or less, 23 or less, 22 or less, 21 or less, 20 or less, 19 or less, 18 or less, 17 or less, 16 or less, 15 or less, 14 or less, 13 or less, 12 or less, 11 or less, 10 or less, 9 or less, 8 or less, or 7 or less consecutive amino acids of an amino acid sequence selected from the antigenic polypeptide sequences of any of Figures 141 to 144 , and a TCR Vα polypeptide of the TCR. or an introduced first polynucleotide sequence encoding a TCR Vα polypeptide of a TCR comprising any of the TCR α polypeptide sequences set forth in any of Figures 136-140 and an introduced second polynucleotide sequence encoding a TCR Vβ polypeptide of the TCR; or an introduced first polynucleotide sequence encoding a TCR Vα polypeptide of a TCR encoded by a nucleotide sequence set forth in any of Figures 139-140 and an introduced second polynucleotide sequence encoding a TCR Vβ polypeptide of the TCR. In some embodiments, the airT cells comprise at least an introduced first polynucleotide sequence encoding a TCR Vα polypeptide of a TCR and an introduced second polynucleotide sequence encoding a TCR Vβ polypeptide of the TCR that specifically binds to an antigenic polypeptide in a human HLA-restricted manner, wherein the TCR Vα polypeptide and Vβ polypeptide comprise paired sequences selected from the paired TCR Vα polypeptide and TCR Vβ polypeptide sequences set forth in Figure 143.

[0052] In some embodiments, the biological Treg activity induced in the airT cells in response to MHC-restricted stimulation with an antigen recognized by a TCR polypeptide encoded by the at least one introduced polynucleotide is enhanced compared to the biological Treg activity of control airT cells in the absence of MHC-restricted stimulation with the same antigen; The biological Treg activity is (i) inhibiting one or both of the activation and proliferation of effector T cells that recognize an antigen specifically recognized by the TCR of said airT cell comprising said TCR polypeptide encoded by said at least one introduced polynucleotide; (ii) suppression of the expression of inflammatory cytokines or inflammatory mediators by effector T cells that recognize an antigen specifically recognized by the TCR of the airT cell comprising the TCR polypeptide encoded by the at least one introduced polynucleotide; (iii) production by the airT cells of one or more immunosuppressive cytokines, perforin / granzymes, or anti-inflammatory products; or induction in the airT cells of at least one of indoleamine-2,3-dioxygenase (IDO), competition for IL2 or adenosine, tryptophan catabolism, and expression of inhibitory receptors; and (iv) inhibiting one or both of the activation and proliferation of effector T cells that do not recognize the antigen specifically recognized by the TCR of the airT cell comprising the TCR polypeptide encoded by the at least one introduced polynucleotide. Contains one or more of the following:

[0053] In some embodiments, (1) The antigen associated with the pathogenesis of an autoimmune disease is an IGRP peptide (residues 241 to 270), the autoimmune disease is type 1 diabetes, and the TCR is TCR T1D4 that recognizes the IGRP peptide (residues 241 to 270) in an HLA DRB1*0404-restricted manner; (2) the antigen associated with the pathogenesis of an autoimmune disease is an IGRP peptide (residues 305 to 324), the autoimmune disease is type 1 diabetes, and the TCR is TCR T1D5, which recognizes the IGRP peptide (residues 305 to 324) in an HLA DRB1*0404-restricted manner; or (3) The antigen associated with the pathogenesis of an autoimmune disease is an IGRP peptide (residues 305 to 324), the autoimmune disease is type 1 diabetes, and the TCR is TCR T1D2, which recognizes the IGRP peptide (residues 305 to 324) in an HLA DRB1*0404-restricted manner.

[0054] Some embodiments of the methods and compositions provided herein include the airT cells, or airT cells as described elsewhere herein, for use in treating, inhibiting, or alleviating an autoimmune disease, an allergic disease, or an inflammatory disease, wherein the autoimmune disease, the allergic disease, or the inflammatory disease is, for example, type 1 diabetes, multiple sclerosis, systemic lupus erythematosus, myasthenia gravis, rheumatoid arthritis, early-onset rheumatoid arthritis, ankylosing spondylitis, immune-mediated pregnancy loss, immune-mediated recurrent pregnancy loss, dermatomyositis, psoriatic arthritis, Crohn's disease, inflammatory bowel disease (IBD), ulcerative colitis, In some embodiments, the airT cells are selected from the group consisting of inflammatory bowel disease, bullous pemphigoid, pemphigus vulgaris, autoimmune hepatitis, psoriasis, Sjogren's syndrome, celiac disease, allergic asthma, atopic dermatitis, pollen allergy, food allergy, drug hypersensitivity, contact dermatitis, pancreatic islet cell transplantation, asthma, steroid-resistant asthma, hepatitis, traumatic brain injury, primary sclerosing cholangitis, primary biliary cholangitis, polymyositis, stroke, Still's disease, acute respiratory distress syndrome, uveitis, inflammatory bowel disease (IBD), ulcerative colitis, graft-versus-host disease (GvHD), transplant tolerance induction, transplant rejection, and sepsis. Some embodiments of the methods and compositions provided herein comprise the airT cells, or airT cells as described elsewhere herein, for use in treating, suppressing, or alleviating autoimmune diseases (e.g., diabetes, such as type 1 diabetes, and primary biliary cholangitis), autoinflammatory diseases (e.g., acute respiratory distress syndrome (ARDS), stroke, and atherosclerotic cardiovascular disease), alloimmune diseases (e.g., graft-versus-host disease, solid organ transplantation, and immune-mediated recurrent pregnancy loss), and / or allergic diseases (e.g., asthma, drug hypersensitivity, and celiac disease). In some embodiments, the inflammatory disease is primary biliary cholangitis. In some embodiments, the inflammatory disease is primary sclerosing cholangitis. In some embodiments, the inflammatory disease is autoimmune hepatitis. In some embodiments, the autoimmune disease is type 1 diabetes. In some embodiments, the inflammatory disease is pancreatic islet cell transplantation. In some embodiments, the inflammatory disease is transplant rejection. In some embodiments, the autoimmune disease is multiple sclerosis.In some embodiments, the inflammatory disease is inflammatory bowel disease. In some embodiments, the inflammatory disease is acute respiratory distress syndrome. In some embodiments, the inflammatory disease is stroke. In some embodiments, the inflammatory disease is graft-versus-host disease. In some embodiments, the disease being treated is cancer. Wang et al. (J Intern Med. 2015 Oct;278(4):369-95) have published a review article on autoimmune diseases, which is incorporated herein by reference. In some embodiments, the TCR polypeptide binds to an antigen associated with a disorder selected from type 1 diabetes, multiple sclerosis, myocarditis, rheumatoid arthritis (RA), and systemic lupus erythematosus (SLE).

[0055] In some embodiments, the antigen is selected from the group consisting of vimentin, aggrecan, cartilage intermediate layer protein (CILP), preproinsulin, islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), and enolase.

[0056] In some embodiments, the antigen comprises an epitope selected from the group consisting of Enol326, CILP297-1, Vim418, Agg520, and SLE3.

[0057] In some embodiments, the antigen comprises an epitope having an amino acid sequence set forth in any of SEQ ID NOs: 1363-1376 and 1408-1415.

[0058] In some embodiments, the TCR polypeptide comprises a CD3α polypeptide having an amino acid sequence set forth in any of SEQ ID NOs: 1377-1390; and / or a CD3β polypeptide having an amino acid sequence set forth in any of SEQ ID NOs: 1377-1390.

[0059] Some embodiments of the methods and compositions provided herein include pharmaceutical compositions comprising the airT cells and a pharmaceutically acceptable excipient.

[0060] Some embodiments of the methods and compositions provided herein include the use of the airT cells as a pharmaceutical.

[0061] In some embodiments, there is provided a method for producing antigen-specific artificial immunoregulatory T (airT) cells, comprising: (a) knocking out or inactivating (e.g., by homology-directed repair or non-homologous end joining) a native forkhead box protein 3 / winged helix transcription factor (FOXP3) locus in a CD4+ T cell under conditions and for a time sufficient to insert all or a portion of a FOXP3 locus donor template nucleic acid; (1) a FOXP3 guide RNA (gRNA) comprising a spacer sequence complementary to a sequence in the native FOXP3 gene of a CD4+ T cell, or a nucleic acid encoding the FOXP3 gRNA; (2) a DNA endonuclease capable of forming a complex with the FOXP3 gRNA of (1), or a nucleic acid encoding the DNA endonuclease; and (3) a FOXP3 locus donor template selected from (i) a nucleic acid molecule comprising a constitutively active promoter capable of promoting transcription of a nucleotide sequence encoding endogenous FOXP3 of the FOXP3 gene, and (ii) a nucleic acid molecule comprising a nucleotide sequence encoding a FOXP3 protein or a functional derivative thereof and a constitutively active promoter operably linked thereto; into CD4+ T cells; and (b) simultaneously with (a), or consecutively with (a) in any order, transducing said CD4+ T cells with at least one polynucleotide encoding an antigen-specific T cell receptor (TCR) polypeptide. The present invention provides a method comprising: In some embodiments, step (b) comprises: (i) transducing the CD4+ T cells with at least one vector comprising a polynucleotide encoding the antigen-specific T cell receptor (TCR) polypeptide or chimeric antigen receptor (CAR) polypeptide; and (ii) knocking out or inactivating the native T cell receptor alpha chain (TRAC) locus of said CD4+ T cells (e.g., by homology-directed repair or non-homologous end joining) under conditions and for a time sufficient to insert all or a portion of a TRAC locus donor template nucleic acid; (1) a TRAC guide RNA (gRNA) comprising a spacer sequence complementary to a sequence within the native TRAC locus of the CD4+ T cell, or a nucleic acid encoding the TRAC gRNA; (2) a DNA endonuclease capable of forming a complex with the TRAC gRNA of (1), or a nucleic acid encoding the DNA endonuclease; and (3) a TRAC locus donor template comprising at least one polynucleotide encoding the antigen-specific T cell receptor (TCR) polypeptide or chimeric antigen receptor (CAR) polypeptide; into the CD4+ T cells. is selected from. In some embodiments, the FOXP3 locus donor template comprises a polynucleotide / nucleic acid encoding a first CISC component, and the TRAC locus donor template comprises a polynucleotide / nucleic acid encoding a second CISC component complementary to the first CISC component, wherein each of the first and second CISC components is capable of specific binding to a CISC-inducing molecule, and wherein the first and second CISC components dimerize in the presence of the CISC-inducing molecule. In another embodiment, the FOXP3 locus donor template encodes a second CISC component, and the TRAC locus donor template encodes a first CISC component, wherein the first and second CISC components specifically bind to the CISC-inducing molecule, thereby dimerizing the first and second CISC components in the presence of the CISC-inducing molecule. The first and second CISC components encoded by each donor template may be any of the first and second CISC components provided herein. In some embodiments, the FOXP3 locus donor template and / or the TRAC locus donor template contain a polynucleotide / nucleic acid encoding a third CISC component that is soluble in the cytoplasm and capable of specifically binding to a CISC-inducing molecule. Any gene editing method is contemplated herein, including, but not limited to, viral and non-viral methods capable of editing target genes (e.g., Cas9, ZFN, TALEN). Thus, the gene editing methods provided herein may utilize a nuclease that targets a locus or target locus on a nucleic acid sequence. In some embodiments, the nuclease is an RNA-guided nuclease (e.g., CRISPR / Cas nuclease), a meganuclease, a zinc finger nuclease, or a TALEN. In some embodiments, there is provided a method for producing antigen-specific artificial immunoregulatory T (airT) cells, comprising: (a) under conditions and for a time sufficient to knock out the native T cell receptor alpha chain (TRAC) locus of a CD4+ T cell (e.g., by homology-directed repair or non-homologous end joining) and insert all or a portion of a first TRAC locus donor template nucleic acid; (1) a first TRAC guide RNA (gRNA) comprising a first spacer sequence complementary to a first sequence within the native TRAC locus of a CD4+ T cell, or a nucleic acid encoding the first TRAC gRNA; (2) a first DNA endonuclease capable of forming a complex with the first TRAC gRNA of (1), or a nucleic acid encoding the first DNA endonuclease; and (3) a first TRAC locus donor template selected from (i) a nucleic acid molecule comprising a nucleotide sequence encoding a FOXP3 protein or a functional derivative thereof, and (ii) a nucleic acid molecule comprising a nucleotide sequence encoding a FOXP3 protein or a functional derivative thereof and a constitutively active promoter operably linked thereto; into CD4+ T cells; and (b) simultaneously with (a), or consecutively with (a) in any order, knocking out or inactivating (e.g., by homology-directed repair or non-homologous end joining) the native T-cell receptor alpha chain (TRAC) locus of said CD4+ T cell and inserting all or a portion of a second TRAC locus donor template nucleic acid under conditions and for a time sufficient to (1) a second TRAC guide RNA (gRNA) comprising a second spacer sequence that is separate from the first spacer sequence and complementary to a second sequence within the TRAC gene, or a nucleic acid encoding the second TRAC gRNA; (2) a second DNA endonuclease selected from the group consisting of the same DNA endonuclease as the first DNA endonuclease and a DNA endonuclease different from the first DNA endonuclease, capable of forming a complex with the second TRAC gRNA of (1), or a nucleic acid encoding the second DNA endonuclease; and (3) a second TRAC locus donor template comprising at least one polynucleotide encoding an antigen-specific T cell receptor (TCR) polypeptide; into the CD4+ T cells. The present invention provides a method comprising: In some embodiments, the FOXP3 locus donor template comprises a polynucleotide / nucleic acid encoding a first CISC component, and the TRAC locus donor template comprises a polynucleotide / nucleic acid encoding a second CISC component complementary to the first CISC component, wherein each of the first and second CISC components is capable of specific binding to a CISC-inducing molecule, and wherein the first and second CISC components dimerize in the presence of the CISC-inducing molecule. In another embodiment, the FOXP3 locus donor template encodes a second CISC component, and the TRAC locus donor template encodes a first CISC component, wherein the first and second CISC components specifically bind to the CISC-inducing molecule, thereby dimerizing the first and second CISC components in the presence of the CISC-inducing molecule. The first and second CISC components encoded by each donor template may be any of the first and second CISC components provided herein. In some embodiments, the FOXP3 locus donor template and / or the TRAC locus donor template contain a polynucleotide encoding a third CISC component that is soluble in the cytoplasm and capable of specifically binding to a CISC-inducing molecule. Any gene editing method is contemplated herein, including, but not limited to, viral and non-viral methods capable of editing a target gene (e.g., Cas9, ZFN, TALEN). Thus, the gene editing methods provided herein may utilize a nuclease that targets a locus or target locus on a nucleic acid sequence. In some embodiments, the nuclease is an RNA-guided nuclease (e.g., CRISPR / Cas nuclease), a meganuclease, a zinc finger nuclease, or a TALEN.

[0062] In some embodiments, there is provided a method for producing antigen-specific artificial immunoregulatory T (airT) cells, comprising: knocking out or inactivating the native T-cell receptor alpha chain (TRAC) locus in CD4+ T cells by homology-directed repair (HDR) or non-homologous end joining (NHEJ) under conditions and for a time sufficient to insert all or part of a TRAC locus donor template; (1) a TRAC guide RNA (gRNA) comprising a spacer sequence complementary to a sequence within the natural TRAC locus of a CD4+ T cell, or a nucleic acid encoding the TRAC gRNA; (2) a DNA endonuclease capable of forming a complex with the TRAC gRNA of (1), or a nucleic acid encoding the DNA endonuclease; and (3) a TRAC locus donor template comprising at least one polynucleotide encoding an antigen-specific T cell receptor (TCR) polypeptide; into CD4+ T cells. In some embodiments, one of the donor templates to be inserted (the first donor template) further comprises a nucleic acid sequence encoding a first CISC component capable of specifically binding to a CISC-inducing molecule, and the other donor template to be inserted (the second donor template) further comprises a nucleic acid sequence encoding a second CISC component, distinct from the first CISC component, capable of specifically binding to the CISC-inducing molecule. In some embodiments, the first donor template comprises a portion homologous to a first locus (e.g., the Foxp3 locus or the TRAC locus) in the genome of the cell, and the second donor template comprises a portion homologous to a second locus (e.g., the Foxp3 locus or the TRAC locus) in the genome of the cell, where the first and second loci can be different loci. The first and second loci can be any loci in the genome, for example, loci other than the Foxp3 locus or the TRAC locus. In some embodiments, at least one of the inserted first and second donor templates further comprises a nucleic acid sequence encoding a third CISC component that is separate from the first and second CISC components and capable of specifically binding to the CISC-inducing molecule. In some of the embodiments of the methods provided herein, (a) the DNA endonuclease is selected from an RNA-guided nuclease (RGN), a CRISPR / Cas nuclease, a TALEN, a meganuclease, a megaTAL, and a zinc finger nuclease; (b) the constitutively active promoter is MND and the insertion is by a mechanism selected from homology-directed repair and non-homologous end joining; (d) the first CISC component and the second CISC component comprise intracellular domains mutually exclusively selected from IL2RB and IL2RG and extracellular domains mutually exclusively selected from FKBP and FRB; (e) the third CISC component is an FKBP encoded by the first donor template or the second donor template; (f) the CISC-inducing molecule is rapamycin or an analog thereof; and / or (g) the first donor template and the second donor template are inserted into two separate loci, each locus independently selected from the group consisting of the FOXP3 locus, the TRAC locus, the AAVS1 locus, and the ROSA26 locus. Any gene editing method is contemplated herein, including but not limited to, for example, viral and non-viral methods (for example, Cas9, ZFN, TALEN) that can edit target gene.Therefore, the gene editing method provided herein can utilize nuclease that targets gene locus or target gene locus on nucleic acid sequence.In some embodiments, nuclease is Cas9, zinc finger nuclease or TALEN.

[0063] In some embodiments, there is provided a method for producing antigen-specific artificial immunoregulatory T (airT) cells, comprising: (a) knocking out or inactivating (e.g., by homology-directed repair or non-homologous end joining) a native forkhead box protein 3 / winged helix transcription factor (FOXP3) locus in a CD8+ T cell under conditions and for a time sufficient to insert all or a portion of a FOXP3 locus donor template nucleic acid; (1) a FOXP3 guide RNA (gRNA) comprising a spacer sequence complementary to a sequence in the native FOXP3 gene of a CD8+ T cell, or a nucleic acid encoding the FOXP3 gRNA; (2) a DNA endonuclease capable of forming a complex with the FOXP3 gRNA of (1), or a nucleic acid encoding the DNA endonuclease; and (3) a FOXP3 locus donor template selected from (i) a nucleic acid molecule comprising a constitutively active promoter capable of promoting transcription of a nucleotide sequence encoding endogenous FOXP3 of the FOXP3 gene, and (ii) a nucleic acid molecule comprising a nucleotide sequence encoding a FOXP3 protein or a functional derivative thereof and a constitutively active promoter operably linked thereto; into CD8+ T cells; and (b) simultaneously with (a), or consecutively with (a) in any order, transducing said CD8+ T cells with at least one polynucleotide encoding an antigen-specific T cell receptor (TCR) polypeptide. The present invention provides a method comprising: In some embodiments, step (b) comprises: (i) transducing the CD8+ T cells with at least one vector comprising a polynucleotide encoding the antigen-specific T cell receptor (TCR) polypeptide or chimeric antigen receptor (CAR) polypeptide; and (ii) knocking out or inactivating the native T cell receptor alpha chain (TRAC) locus of said CD8+ T cell (e.g., by homology-directed repair or non-homologous end joining) under conditions and for a time sufficient to insert all or a portion of a TRAC locus donor template nucleic acid; (1) a TRAC guide RNA (gRNA) comprising a spacer sequence complementary to a sequence within the native TRAC locus of the CD8+ T cell, or a nucleic acid encoding the TRAC gRNA; (2) a DNA endonuclease capable of forming a complex with the TRAC gRNA of (1), or a nucleic acid encoding the DNA endonuclease; and (3) a TRAC locus donor template comprising at least one polynucleotide / nucleic acid encoding said antigen-specific T cell receptor (TCR) polypeptide or chimeric antigen receptor (CAR) polypeptide; into the CD8+ T cells. is selected from. In some embodiments, the FOXP3 locus donor template comprises a polynucleotide / nucleic acid encoding a first CISC component, and the TRAC locus donor template comprises a polynucleotide / nucleic acid encoding a second CISC component complementary to the first CISC component, wherein each of the first and second CISC components is capable of specific binding to a CISC-inducing molecule, and wherein the first and second CISC components dimerize in the presence of the CISC-inducing molecule. In another embodiment, the FOXP3 locus donor template encodes a second CISC component, and the TRAC locus donor template encodes a first CISC component, wherein the first and second CISC components specifically bind to the CISC-inducing molecule, thereby dimerizing the first and second CISC components in the presence of the CISC-inducing molecule. The first and second CISC components encoded by each donor template may be any of the first and second CISC components provided herein. In some embodiments, the FOXP3 locus donor template and / or the TRAC locus donor template contain a polynucleotide / nucleic acid encoding a third CISC component that is soluble in the cytoplasm and capable of specifically binding to a CISC-inducing molecule. Any gene editing method is contemplated herein, including, but not limited to, viral and non-viral methods capable of editing target genes (e.g., Cas9, ZFN, TALEN). Thus, the gene editing methods provided herein may utilize a nuclease that targets a locus or target locus on a nucleic acid sequence. In some embodiments, the nuclease is an RNA-guided nuclease (e.g., CRISPR / Cas nuclease), a meganuclease, a zinc finger nuclease, or a TALEN. In some embodiments, there is provided a method for producing antigen-specific artificial immunoregulatory T (airT) cells, comprising: (a) under conditions and for a time sufficient to knock out the native T cell receptor alpha chain (TRAC) locus of a CD8+ T cell (e.g., by homology-directed repair or non-homologous end joining) and insert all or a portion of a first TRAC locus donor template nucleic acid; (1) a first TRAC guide RNA (gRNA) comprising a first spacer sequence complementary to a first sequence within the native TRAC locus of a CD8+ T cell, or a nucleic acid encoding the first TRAC gRNA; (2) a first DNA endonuclease capable of forming a complex with the first TRAC gRNA of (1), or a nucleic acid encoding the first DNA endonuclease; and (3) a first TRAC locus donor template selected from (i) a nucleic acid molecule comprising a nucleotide sequence encoding a FOXP3 protein or a functional derivative thereof, and (ii) a nucleic acid molecule comprising a nucleotide sequence encoding a FOXP3 protein or a functional derivative thereof and a constitutively active promoter operably linked thereto; into CD8+ T cells; and (b) simultaneously with (a), or consecutively with (a) in any order, knocking out or inactivating (e.g., by homology-directed repair or non-homologous end joining) the native T cell receptor alpha chain (TRAC) locus of said CD8+ T cell and inserting all or a portion of a second TRAC locus donor template nucleic acid under conditions and for a time sufficient to (1) a second TRAC guide RNA (gRNA) comprising a second spacer sequence that is separate from the first spacer sequence and complementary to a second sequence within the TRAC gene, or a nucleic acid encoding the second TRAC gRNA; (2) a second DNA endonuclease selected from the group consisting of the same DNA endonuclease as the first DNA endonuclease and a DNA endonuclease different from the first DNA endonuclease, capable of forming a complex with the second TRAC gRNA of (1), or a nucleic acid encoding the second DNA endonuclease; and (3) a second TRAC locus donor template comprising at least one polynucleotide / nucleic acid encoding an antigen-specific T cell receptor (TCR) polypeptide; into the CD8+ T cells. The present invention provides a method comprising: In some embodiments, the FOXP3 locus donor template comprises a polynucleotide / nucleic acid encoding a first CISC component, and the TRAC locus donor template comprises a polynucleotide / nucleic acid encoding a second CISC component complementary to the first CISC component, wherein each of the first and second CISC components is capable of specific binding to a CISC-inducing molecule, and wherein the first and second CISC components dimerize in the presence of the CISC-inducing molecule. In another embodiment, the FOXP3 locus donor template encodes a second CISC component, and the TRAC locus donor template encodes a first CISC component, wherein the first and second CISC components specifically bind to the CISC-inducing molecule, thereby dimerizing the first and second CISC components in the presence of the CISC-inducing molecule. The first and second CISC components encoded by each donor template may be any of the first and second CISC components provided herein. In some embodiments, the FOXP3 locus donor template and / or the TRAC locus donor template contain a polynucleotide / nucleic acid encoding a third CISC component that is soluble in the cytoplasm and capable of specifically binding to a CISC-inducing molecule. Any gene editing method is contemplated herein, including, but not limited to, viral and non-viral methods capable of editing target genes (e.g., Cas9, ZFN, TALEN). Thus, the gene editing methods provided herein may utilize a nuclease that targets a locus or target locus on a nucleic acid sequence. In some embodiments, the nuclease is an RNA-guided nuclease (e.g., CRISPR / Cas nuclease), a meganuclease, a zinc finger nuclease, or a TALEN.

[0064] In some embodiments, there is provided a method for producing antigen-specific artificial immunoregulatory T (airT) cells, comprising: knocking out or inactivating the native T-cell receptor alpha chain (TRAC) locus in CD8+ T cells by homology-directed repair (HDR) or non-homologous end joining (NHEJ) under conditions and for a time sufficient to insert all or part of a TRAC locus donor template; (1) a TRAC guide RNA (gRNA) comprising a spacer sequence complementary to a sequence within the natural TRAC locus of a CD8+ T cell, or a nucleic acid encoding the TRAC gRNA; (2) a DNA endonuclease capable of forming a complex with the TRAC gRNA of (1), or a nucleic acid encoding the DNA endonuclease; and (3) a TRAC locus donor template comprising at least one polynucleotide / nucleic acid encoding an antigen-specific T cell receptor (TCR) polypeptide; into CD8+ T cells. In some embodiments, one of the donor templates to be inserted (the first donor template) further comprises a nucleic acid sequence encoding a first chemo-induced signaling complex (CISC) component capable of specifically binding to a CISC-inducing molecule, and the other donor template to be inserted (the second donor template) further comprises a nucleic acid sequence encoding a second CISC component separate from the first CISC component capable of specifically binding to the CISC-inducing molecule. In some embodiments, the first donor template comprises a portion homologous to a first locus (e.g., the Foxp3 locus or the TRAC locus) in the genome of the cell, and the second donor template comprises a portion homologous to a second locus (e.g., the Foxp3 locus or the TRAC locus) in the genome of the cell, where the first locus and the second locus may be different loci. In some embodiments, at least one of the inserted first and second donor templates further comprises a nucleic acid sequence encoding a third CISC component that is separate from the first and second CISC components and capable of specifically binding to the CISC-inducing molecule. In some of the embodiments of the methods provided herein, (a) the DNA endonuclease is selected from an RNA-guided nuclease (RGN), a CRISPR / Cas nuclease, a TALEN, a meganuclease, a megaTAL, and a zinc finger nuclease; (b) the constitutively active promoter is MND and the insertion is by a mechanism selected from homology-directed repair and non-homologous end joining; (d) the first CISC component and the second CISC component comprise intracellular domains mutually exclusively selected from IL2RB and IL2RG and extracellular domains mutually exclusively selected from FKBP and FRB; (e) the third CISC component is an FKBP encoded by the first donor template or the second donor template; (f) the CISC-inducing molecule is rapamycin or an analog thereof; and / or (g) the first donor template and the second donor template are inserted into two separate loci, each locus independently selected from the group consisting of the FOXP3 locus, the TRAC locus, the AAVS1 locus, and the ROSA26 locus. Any gene editing method is contemplated herein, including but not limited to, for example, viral and non-viral methods (for example, Cas9, ZFN, TALEN) that can edit target gene.Therefore, the gene editing method provided herein can utilize nuclease that targets gene locus or target gene locus on nucleic acid sequence.In some embodiments, nuclease is Cas9, zinc finger nuclease or TALEN.

[0065] In some embodiments, there is provided a method for producing antigen-specific artificial immunoregulatory T (airT) cells, comprising: (a) knocking out or inactivating (e.g., by homology-directed repair or non-homologous end joining) a native forkhead box protein 3 / winged helix transcription factor (FOXP3) locus in a CD3+ T cell under conditions and for a time sufficient to insert all or a portion of a FOXP3 locus donor template nucleic acid; (1) a FOXP3 guide RNA (gRNA) comprising a spacer sequence complementary to a sequence in the native FOXP3 gene of a CD3+ T cell, or a nucleic acid encoding the FOXP3 gRNA; (2) a DNA endonuclease capable of forming a complex with the FOXP3 gRNA of (1), or a nucleic acid encoding the DNA endonuclease; and (3) a FOXP3 locus donor template selected from (i) a nucleic acid molecule comprising a constitutively active promoter capable of promoting transcription of a nucleotide sequence encoding endogenous FOXP3 of the FOXP3 gene, and (ii) a nucleic acid molecule comprising a nucleotide sequence encoding a FOXP3 protein or a functional derivative thereof and a constitutively active promoter operably linked thereto; into CD3+ T cells; and (b) simultaneously with (a), or consecutively with (a) in any order, transducing said CD3+ T cells with at least one polynucleotide / nucleic acid encoding an antigen-specific T cell receptor (TCR) polypeptide. The present invention provides a method comprising: In some embodiments, step (b) comprises: (i) transducing said CD3+ T cells with at least one vector comprising a polynucleotide / nucleic acid encoding said antigen-specific T cell receptor (TCR) polypeptide or chimeric antigen receptor (CAR) polypeptide; and (ii) knocking out or inactivating the native T cell receptor alpha chain (TRAC) locus of said CD3+ T cells (e.g., by homology-directed repair or non-homologous end joining) under conditions and for a time sufficient to insert all or a portion of a TRAC locus donor template nucleic acid; (1) a TRAC guide RNA (gRNA) comprising a spacer sequence complementary to a sequence within the native TRAC locus of the CD3+ T cell, or a nucleic acid encoding the TRAC gRNA; (2) a DNA endonuclease capable of forming a complex with the TRAC gRNA of (1), or a nucleic acid encoding the DNA endonuclease; and (3) a TRAC locus donor template comprising at least one polynucleotide / nucleic acid encoding said antigen-specific T cell receptor (TCR) polypeptide or chimeric antigen receptor (CAR) polypeptide; into the CD3+ T cells. is selected from. In some embodiments, the FOXP3 locus donor template comprises a polynucleotide / nucleic acid encoding a first CISC component, and the TRAC locus donor template comprises a polynucleotide / nucleic acid encoding a second CISC component complementary to the first CISC component, wherein each of the first and second CISC components is capable of specific binding to a CISC-inducing molecule, and wherein the first and second CISC components dimerize in the presence of the CISC-inducing molecule. In another embodiment, the FOXP3 locus donor template encodes a second CISC component, and the TRAC locus donor template encodes a first CISC component, wherein the first and second CISC components specifically bind to the CISC-inducing molecule, thereby dimerizing the first and second CISC components in the presence of the CISC-inducing molecule. The first and second CISC components encoded by each donor template may be any of the first and second CISC components provided herein. In some embodiments, the FOXP3 locus donor template and / or the TRAC locus donor template contain a polynucleotide / nucleic acid encoding a third CISC component that is soluble in the cytoplasm and capable of specifically binding to a CISC-inducing molecule. Any gene editing method is contemplated herein, including, but not limited to, viral and non-viral methods capable of editing target genes (e.g., Cas9, ZFN, TALEN). Thus, the gene editing methods provided herein may utilize a nuclease that targets a locus or target locus on a nucleic acid sequence. In some embodiments, the nuclease is an RNA-guided nuclease (e.g., CRISPR / Cas nuclease), a meganuclease, a zinc finger nuclease, or a TALEN. In some embodiments, there is provided a method for producing antigen-specific artificial immunoregulatory T (airT) cells, comprising: (a) under conditions and for a time sufficient to knock out the native T cell receptor alpha chain (TRAC) locus of a CD3+ T cell (e.g., by homology-directed repair or non-homologous end joining) and insert all or a portion of a first TRAC locus donor template nucleic acid; (1) a first TRAC guide RNA (gRNA) comprising a first spacer sequence complementary to a first sequence within the native TRAC locus of a CD3+ T cell, or a nucleic acid encoding the first TRAC gRNA; (2) a first DNA endonuclease capable of forming a complex with the first TRAC gRNA of (1), or a nucleic acid encoding the first DNA endonuclease; and (3) a first TRAC locus donor template selected from (i) a nucleic acid molecule comprising a nucleotide sequence encoding a FOXP3 protein or a functional derivative thereof, and (ii) a nucleic acid molecule comprising a nucleotide sequence encoding a FOXP3 protein or a functional derivative thereof and a constitutively active promoter operably linked thereto; into CD3+ T cells; and (b) simultaneously with (a), or consecutively with (a) in any order, knocking out or inactivating (e.g., by homology-directed repair or non-homologous end joining) the native T cell receptor alpha chain (TRAC) locus of said CD3+ T cell and inserting all or a portion of a second TRAC locus donor template nucleic acid under conditions and for a time sufficient to (1) a second TRAC guide RNA (gRNA) comprising a second spacer sequence that is separate from the first spacer sequence and complementary to a second sequence within the TRAC gene, or a nucleic acid encoding the second TRAC gRNA; (2) a second DNA endonuclease selected from the group consisting of the same DNA endonuclease as the first DNA endonuclease and a DNA endonuclease different from the first DNA endonuclease, capable of forming a complex with the second TRAC gRNA of (1), or a nucleic acid encoding the second DNA endonuclease; and (3) a second TRAC locus donor template comprising at least one polynucleotide / nucleic acid encoding an antigen-specific T cell receptor (TCR) polypeptide; into the CD3+ T cells. The present invention provides a method comprising: In some embodiments, the FOXP3 locus donor template comprises a polynucleotide / nucleic acid encoding a first CISC component, and the TRAC locus donor template comprises a polynucleotide / nucleic acid encoding a second CISC component complementary to the first CISC component, wherein each of the first and second CISC components is capable of specific binding to a CISC-inducing molecule, and wherein the first and second CISC components dimerize in the presence of the CISC-inducing molecule. In another embodiment, the FOXP3 locus donor template encodes a second CISC component, and the TRAC locus donor template encodes a first CISC component, wherein the first and second CISC components specifically bind to the CISC-inducing molecule, thereby dimerizing the first and second CISC components in the presence of the CISC-inducing molecule. The first and second CISC components encoded by each donor template may be any of the first and second CISC components provided herein. In some embodiments, the FOXP3 locus donor template and / or the TRAC locus donor template contain a polynucleotide / nucleic acid encoding a third CISC component that is soluble in the cytoplasm and capable of specifically binding to a CISC-inducing molecule. Any gene editing method is contemplated herein, including, but not limited to, viral and non-viral methods capable of editing target genes (e.g., Cas9, ZFN, TALEN). Thus, the gene editing methods provided herein may utilize a nuclease that targets a locus or target locus on a nucleic acid sequence. In some embodiments, the nuclease is an RNA-guided nuclease (e.g., CRISPR / Cas nuclease), a meganuclease, a zinc finger nuclease, or a TALEN.

[0066] In some embodiments, there is provided a method for producing antigen-specific artificial immunoregulatory T (airT) cells, comprising: knocking out or inactivating the native T-cell receptor alpha chain (TRAC) locus in CD3+ T cells by homology-directed repair (HDR) or non-homologous end joining (NHEJ) under conditions and for a time sufficient to insert all or part of a TRAC locus donor template; (1) a TRAC guide RNA (gRNA) comprising a spacer sequence complementary to a sequence within the natural TRAC locus of a CD3+ T cell, or a nucleic acid encoding the TRAC gRNA; (2) a DNA endonuclease capable of forming a complex with the TRAC gRNA of (1), or a nucleic acid encoding the DNA endonuclease; and (3) a TRAC locus donor template comprising at least one polynucleotide / nucleic acid encoding an antigen-specific T cell receptor (TCR) polypeptide; into CD3+ T cells. In some embodiments, one of the donor templates to be inserted (the first donor template) further comprises a nucleic acid sequence encoding a first chemo-induced signaling complex (CISC) component capable of specifically binding to a CISC-inducing molecule, and the other donor template to be inserted (the second donor template) further comprises a nucleic acid sequence encoding a second CISC component separate from the first CISC component capable of specifically binding to the CISC-inducing molecule. In some embodiments, the first donor template comprises a portion homologous to a first locus (e.g., the Foxp3 locus or the TRAC locus) in the genome of the cell, and the second donor template comprises a portion homologous to a second locus (e.g., the Foxp3 locus or the TRAC locus) in the genome of the cell, where the first locus and the second locus may be different loci. In some embodiments, at least one of the inserted first and second donor templates further comprises a nucleic acid sequence encoding a third CISC component that is separate from the first and second CISC components and capable of specifically binding to the CISC-inducing molecule. In some of the embodiments of the methods provided herein, (a) the DNA endonuclease is selected from an RNA-guided nuclease (RGN), a CRISPR / Cas nuclease, a TALEN, a meganuclease, a megaTAL, and a zinc finger nuclease; (b) the constitutively active promoter is MND and the insertion is by a mechanism selected from homology-directed repair and non-homologous end joining; (d) the first CISC component and the second CISC component comprise intracellular domains mutually exclusively selected from IL2RB and IL2RG and extracellular domains mutually exclusively selected from FKBP and FRB; (e) the third CISC component is an FKBP encoded by the first donor template or the second donor template; (f) the CISC-inducing molecule is rapamycin or an analog thereof; and / or (g) the first donor template and the second donor template are inserted into two separate loci, each locus independently selected from the group consisting of the FOXP3 locus, the TRAC locus, the AAVS1 locus, and the ROSA26 locus. Any gene editing method is contemplated herein, including but not limited to, for example, viral and non-viral methods (for example, Cas9, ZFN, TALEN) that can edit target gene.Therefore, the gene editing method provided herein can utilize nuclease that targets gene locus or target gene locus on nucleic acid sequence.In some embodiments, nuclease is Cas9, zinc finger nuclease or TALEN.

[0067] Some embodiments of the methods and compositions provided herein include a method of producing the antigen-specific artificial immunoregulatory T (airT) cells, comprising performing the method of producing antigen-specific artificial immunoregulatory T (airT) cells.

[0068] In some embodiments, a method for treating, suppressing, or alleviating a disease requiring antigen-specific immunosuppression in a subject is provided, comprising administering to the subject a therapeutically effective amount of a plurality of the artificial immunoregulatory T (airT) cells expressing at least one T cell receptor (TCR) or chimeric antigen receptor (CAR) that specifically recognizes an antigen requiring antigen-specific immunosuppression. In some embodiments, the disease requiring antigen-specific immunosuppression is an autoimmune disease, an allergic disease, or an inflammatory disease. In some embodiments, the autoimmune disease is a disease in which an immune response targets normal cells, normal tissues, and / or normal organs, thereby causing an immune-related pathology. In some embodiments, the autoimmune disease is an immune-related pathology caused by dysregulation of the immune response. In some embodiments, (i) the autoimmune disease is selected from type 1 diabetes, multiple sclerosis, systemic lupus erythematosus, myasthenia gravis, rheumatoid arthritis, early-onset rheumatoid arthritis, ankylosing spondylitis, immune-mediated pregnancy loss, immune-mediated recurrent pregnancy loss, dermatomyositis, psoriatic arthritis, Crohn's disease, inflammatory bowel disease (IBD), ulcerative colitis, bullous pemphigoid, pemphigus vulgaris, autoimmune hepatitis, psoriasis, Sjogren's syndrome, and celiac disease; (ii) the allergic disease is selected from allergic asthma, atopic dermatitis, pollen allergy, food allergy, drug hypersensitivity, and contact dermatitis; (iii) the inflammatory disease is selected from pancreatic islet cell transplantation, asthma, steroid-resistant asthma, hepatitis, traumatic brain injury, primary sclerosing cholangitis, primary biliary cholangitis, polymyositis, stroke, Still's disease, acute respiratory distress syndrome, uveitis, inflammatory bowel disease (IBD), ulcerative colitis, graft-versus-host disease (GvHD), transplant tolerance induction, transplant rejection, and sepsis. In some embodiments, the inflammatory disease is primary biliary cholangitis. In some embodiments, the inflammatory disease is primary sclerosing cholangitis. In some embodiments, the inflammatory disease is autoimmune hepatitis. In some embodiments, the autoimmune disease is type 1 diabetes. In some embodiments, the inflammatory disease is pancreatic islet cell transplantation. In some embodiments, the inflammatory disease is transplant rejection. In some embodiments, the autoimmune disease is multiple sclerosis. In some embodiments, the inflammatory disease is inflammatory bowel disease. In some embodiments, the inflammatory disease is acute respiratory distress syndrome. In some embodiments, the inflammatory disease is stroke. In some embodiments, the inflammatory disease is graft-versus-host disease. In some embodiments, (i) the antigen associated with the pathogenesis of the autoimmune disease is selected from the autoantigens listed in any one of Figures 141 to 144; (ii) the antigen related to the etiology of the allergic disease is selected from the allergic antigens listed in any one of Figures 141 to 144; (iii) The antigen associated with the pathogenesis of the inflammatory disease is selected from the inflammation-associated antigens shown in any of Figures 141 to 144.

[0069] Some embodiments of the methods and compositions provided herein include a method of treating or alleviating a subject having a disorder, the method comprising administering the artificial immunoregulatory T (airT) cells to the subject. In some embodiments, the method of treating or alleviating further comprises administering a CISC-inducing molecule (e.g., rapamycin or a rapalog) to the subject prior to or after administration of the airT cells to promote proliferation, activation, and / or maintenance of the airT cells in vivo.

[0070] In some embodiments, the disorder is selected from the group consisting of autoimmune diseases (e.g., diabetes, such as type 1 diabetes, and primary biliary cholangitis), autoinflammatory diseases (e.g., acute respiratory distress syndrome (ARDS), stroke, and atherosclerotic cardiovascular disease), alloimmune diseases (e.g., graft-versus-host disease, solid organ transplantation, and immune-mediated recurrent pregnancy loss), and / or allergic diseases (e.g., asthma, drug hypersensitivity, and celiac disease). In some embodiments, the disease to be treated is cancer. Wang et al. (J Intern Med. 2015 Oct;278(4):369-95) have published a review article on autoimmune diseases (this review article is incorporated herein by reference). In some embodiments, the disorder is type 1 diabetes, multiple sclerosis, systemic lupus erythematosus, myasthenia gravis, rheumatoid arthritis, early-onset rheumatoid arthritis, ankylosing spondylitis, immune-mediated pregnancy loss, immune-mediated recurrent pregnancy loss, dermatomyositis, psoriatic arthritis, Crohn's disease, inflammatory bowel disease (IBD), ulcerative colitis, bullous pemphigoid, pemphigus vulgaris, autoimmune hepatitis, psoriasis, Sjogren's syndrome, celiac disease, allergic asthma. In some embodiments, the TCR polypeptide binds to an antigen associated with a disorder selected from the group consisting of type 1 diabetes, multiple sclerosis, myocarditis, rheumatoid arthritis (RA), and systemic lupus erythematosus (SLE).

[0071] In some embodiments, the TCR polypeptide binds to an antigen selected from the group consisting of vimentin, aggrecan, cartilage intermediate layer protein (CILP), preproinsulin, islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), and enolase. In some embodiments, the TCR polypeptide binds to an antigen comprising an epitope having an amino acid sequence set forth in any of SEQ ID NOs: 1363-1376 and 1408-1415. In some embodiments, the TCR polypeptide binds to an antigen carried by or derived from intestinal microorganisms. In some embodiments, the TCR polypeptide binds to an epitope of the bacterial protein OmpC. In some embodiments, the TCR polypeptide binds to an epitope of a type 1 diabetes antigen, such as GAD65, PPI, or ZNT8. In some embodiments, the TCR polypeptide binds to an epitope of a primary biliary cirrhosis (PBC) antigen, such as the E2 component of the pyruvate dehydrogenase complex (PDC-E2), hi some embodiments, the TCR polypeptide binds to a nuclear antigen, hi some embodiments, the TCR polypeptide binds to a mitochondrial antigen.

[0072] In some embodiments, the TCR polypeptide comprises a CD3α polypeptide having an amino acid sequence set forth in any of SEQ ID NOs: 1377-1390; and / or a CD3β polypeptide having an amino acid sequence set forth in any of SEQ ID NOs: 1377-1390. [Brief explanation of the drawings]

[0073] [Figure 1A-11] This involves using gene editing to generate airT cells from human CD4+ T cells.

[0074] [Figure 1]Figures 1A, 1B, and 1C show an exemplary scheme for converting CD4+ T cells into airT cells according to the present disclosure. Figure 1A shows a schematic diagram of the FOXP3 locus before (top panel) and after (bottom panel) gene editing using a FOXP3 TALEN or a FOXP3 guide RNA and CRISPR / Cas9. TALEN or CRISPR / Cas9 cleaves exon 1 of the FoxP3 locus, initiating site-specific double-stranded DNA breaks. AAV provides a donor template containing MND and GFP (for analyzing editing efficiency) and is inserted into the DNA cleavage site in exon 1. After homologous recombination repair, the MND promoter drives expression of the FoxP3 and GFP reporters. Figure 1B shows a timeline of the gene editing and cell analysis steps and the efficacy of generating airT cells from input conventional T cells (Tconv). Figure 1C shows a representative flow cytometry plot showing the correlation between Foxp3 and GFP on day 4 after gene editing. The three panels on the right side of the figure show the expression of CD25, CD127, Helios, CD45RO, ICOS, and CTLA-4 in Foxp3+GFP+ gated cells, respectively.

[0075] [Figure 2] Flow cytometry plots (bottom) showing GFP and Foxp3 expression at days 4 and 11 after gene editing according to the timeline shown at top. These data demonstrate efficient Foxp3 editing in CD4+ T cells, resulting in stable, high Foxp3 expression.

[0076] [Figure 3]Figures 3A, 3B, 3C, and 3D show data comparing airT cells with activated natural regulatory T (nTreg) cells. Figure 3A shows a timeline of the steps for generating recombinant regulatory T cell-like (edTreg) cells and activated nTreg cells for comparison. CD4+ cells were isolated from PBMCs using the MACS CD4+ Isolation Kit, and Tconv cells (CD25-CD127+) and Treg cells (CD25highCD127-) were sorted by flow cytometry. Sorted Tconv cells and Treg cells were activated with CD3 / CD28 activation beads for 48 hours, after which the beads were removed. Foxp3 alone in Tconv cells was edited using Cas9 / Foxp3 gRNA and AAV-MND-LNGFR-Foxp3 ki to generate edTreg / airT cells. nTreg cells were treated similarly, except that Foxp3 was not edited. On day 10, LNGFR+ cells were enriched from Foxp3-edited Tconv cells using MACS LNGFR beads. Suppression assays were performed using LNGFR+ edTreg cells and nTreg cells. Figure 3B shows a comparison of the efficacy of generating edTreg cells or nTreg cells from 1 x 107 PBMCs. On day 0, 1 x 107 PBMCs were obtained. Activation and expansion of Tconv and nTreg cells on day 0 resulted in a 10- to 30-fold increase in Tconv cells and a 1- to 2-fold increase in nTreg cells between days 0 and 10. The Treg cell yield on day 10 for edTreg cell generation was calculated based on the editing rate (10-30%). Figure 3C shows representative flow cytometry plots demonstrating the Treg phenotype of Foxp3-edited Tconv or nTreg cells on day 10. The top left panel shows the expression of LNGFR in edited Tconv cells, and the right panel shows the expression of Foxp3, Helios, CD25, CD127, ICOS, and CTLA-4 in edited Treg cells (gated on LNGFR+, upper panel) or nTreg cells (lower panel). The top panel of Figure 3D shows a comparison of the expression levels of Foxp3, CTLA-4, and ICOS in edTreg / airT cells and nTreg cells. The bottom table of Figure 3D shows MFI.

[0077] [Figure 4] Figures 4A and 4B show that airT cells have superior suppressive activity compared with nTreg cells in vitro. Figure 4A shows data from an in vitro suppression assay comparing the suppressive activity of edTreg / airT cells with nTreg cells against CD4+ Teff cells at the Treg / Teff ratios indicated in each graph. AirT cells and nTreg cells were labeled with EF670, and CD4+ Teff cells were labeled with Cell Trace Violet (CTV). AirT cells or nTreg cells were co-cultured with Teff cells at various ratios: 0:1 (Teff only), 1:1, 1:2, 1:4, 1:8, 1:16, or 1:32 (Treg:Teff). CD3 / CD28-activating beads were added at a ratio of 1:25 (beads:Teff cells) and the cells were incubated for 4 days. Cells were analyzed by flow cytometry. The dilution of CTV in Teff cells was measured as an indicator of proliferation. Figure 4B shows the suppression rate calculated using the following formula: (proliferation rate (%) in the presence of Teff cells alone + beads - proliferation rate (%) of Teff cells co-cultured with Treg cells) / (proliferation rate (%) in the presence of Teff cells alone + beads) × 100.

[0078] [Figure 5] Representative islet-specific TCR lentiviral constructs expressing rare islet-specific TCRs derived from subjects with type 1 diabetes (T1D). Panel A shows a table listing lentiviral vectors encoding GAD65- or IGRP-specific TCRs (4.13, T1D2, T1D4, T1D5-1, or T1D5-2), their epitope specificities, and the TCR α or β chains used. Panel B shows the structure of the lentivirally expressed islet-specific TCRs. These TCR constructs contain human TCR variable regions derived from islet-specific TCRs and mouse TCR constant regions, improving pairing of the human TCR chains upon transduction.

[0079] [Figure 6] To verify the expression of islet antigen-specific TCRs, we analyzed the expression of murine TCRβ and proliferation of islet antigen-specific T cells. Panel A shows flow cytometry plots of isolated CD4+ T cells activated with CD3 / CD28 beads and transduced with lentivirus encoding islet antigen-specific TCRs. These flow cytometry plots were gated on CD3 / CD28-activated CD4+ cells 9 days after transduction with lentivirus encoding islet specific TCRs (LV), and analyzed for mTCRβ expression. Panel B shows flow cytometry analysis of CD4+ T cells transduced with lentivirus encoding islet specific TCRs, labeled with Cell Trace Violet (CTV), cocultured with APCs (irradiated PBMCs) for 5 days in the presence of peptides recognized by the TCR or irrelevant peptides. These flow cytometry plots show the results of lentivirally transduced CTV-labeled CD4+ T cells cocultured with antigen-presenting cells (APCs; irradiated PBMCs) for 5 days in the presence of a peptide recognized by the TCR or an irrelevant peptide, and cell proliferation was analyzed by flow cytometry. CTV dilution was used as an indicator of proliferation.

[0080] [Figure 7] Generation of Foxp3-edited T cells with islet-specific TCRs. Panel A shows a timeline of the process for generating edTreg cells with islet-specific TCRs. Panel B shows representative flow cytometry plots showing mTCRβ and LNGFR / Foxp3 expression in CD4+ cells 7 days after transduction with T1D4 or T1D5-1 TCRs and Foxp3 editing. The right panel shows analysis of CD25, CD127, CTLA-4, and ICOS expression by gating on LNGFR+ cells.

[0081] [Figure 8]This figure shows a typical antigen-specific suppression assay according to the present disclosure. Panel A shows a timeline of the process for generating edTreg cells expressing islet-specific TCRs. edTreg cells expressing islet-specific TCRs (non-lentiviral TCRs; T1D4 TCR or T1D5-1 TCR) were enriched based on LNGFR expression using MACS LNGFR beads. The resulting LNGFR+ cells were aliquoted and cryopreserved until further experiments. Panel B shows an outline of the method for evaluating the antigen-specific suppression assay. CD4+ T cells transduced with islet-specific TCRs (T1D4 TCR or T1D5-1 TCR) were used as Teff cells. Teff cells and Treg cells were labeled with different reagents (e.g., CTV or EF670) and co-cultured with edTreg cells at a 1:1 or 1:2 ratio in the presence of APCs (irradiated autologous PBMCs) and various peptides, or Teff cells were cultured without edTregs. After 1 or 4 days of incubation, cells were stained and analyzed by flow cytometry to measure cytokine production and Teff cell proliferation.

[0082] [Figure 9-10] The graph shows the suppressive activity of edTreg / air T cells on Teff cell proliferation in the presence of the indicated peptides and APC. Teff cells were labeled with CTV, and Treg cells were labeled with EF670. T1D4-TCR-transduced CD4+ T cells (T1D4 Teff) were cocultured with T1D4-TCR-expressing edTreg cells (T1D4 edTreg) or T1D5-1-TCR-expressing edTreg cells (T1D5-1 edTreg) in the presence of various peptides (DMSO, IGRP 241, IGRP 305, or IGRP 241 + IGRP 305) and APC, or T1D4 Teff cells were cultured without edTreg. After 4 days of coculture, cells were stained to analyze Teff cell proliferation by dilution of CTV. Flow cytometry plots show Teff cell proliferation gated on CD3+CD4+CTV+EF670-LNGFR-.

[0083] [Figure 11] Figure 1 shows that edTreg / air T cells suppress cytokine production in Teff cells. Teff cells were labeled with CTV, and Treg cells were labeled with EF670. T1D4 Teff cells were cocultured with untransduced edTreg cells or T1D4-transduced edTreg / air T cells in the presence of APC and peptide (DMSO or IGRP 241), or T1D4 Teff cells were cultured without edTreg cells. After 1 day of coculture, cells were contacted with BFA for 4 hours and stained to analyze cytokine production from Teff cells. Flow cytometry plots show TNF, IFNγ, or IL-17 production from T1D4 Teff cells gated on CD4+CTV+EF670-.

[0084] [Figure 12-17] FIG. 1 shows generation and characterization of human Foxp3-edited antigen-specific human CD4+ T cells.

[0085] [Figure 12] A typical scheme for generating antigen-specific human edTreg / airT cells from peripheral blood cells (upper panel) and the phenotype of antigen-specific human CD4+ T cells after FOXP3 editing (lower panel) are shown. The lower panel shows a representative flow cytometry plot (left) and the percentage (right) of tetramer-positive (Tmr+; a mixture of influenza antigen peptide-MHC class II tetramer and tetanus toxin antigen peptide-MHC class II tetramer) human CD4+ T cells 4 days after gene editing (n=5).

[0086] [Figure 13]Characterization of FOXP3-edited antigen-specific human CD4+ T cells is shown. Panel A shows the phenotype of FOXP3-edited antigen-specific human CD4+ T cells. Flow cytometry data are summarized in a bar graph (n=5). The bar graph shows the expression of Treg markers and intracellular IL-2 production in Tmr+ed Treg cells, Tmr+ mock-edited cells, and thymus-derived Treg cells (tTregs) obtained from an unrelated donor. Data shown are representative of five independent experiments. P values ​​indicating statistical significance are indicated above each bar. Panel B shows that antigen-specific human edTreg / airT cells suppress Teff cell proliferation in vitro. Suppression assays were performed by co-culturing Tmr+ed Treg / airT cells or mock-edited Tmr+ cells with Teff cells obtained from a healthy control and antigen-presenting cells (APCs) in the presence of soluble anti-CD3 and anti-CD28 antibodies. The ratio of APC (irradiated CD4+ PBMCs) to Tmr+-ed Treg cells or mock-edited Tmr+ cells to Teff cells was 2:1:1. 18 hours before the end of the 4-day assay, 1 μCi of 3H was added, and cell proliferation was measured using a scintillation counter. Each bar graph represents the average of results from three experiments using three donors.

[0087] [Figure 14]This shows that antigen-specific edTreg / airT cells were successfully generated by peptide stimulation followed by Foxp3 editing. Panel A shows the timeline of the antigen-specific T cell expansion and gene editing processes. To expand T cells specific for MP peptide, HA peptide, or tetanus toxin antigen peptide (TT), cells were stimulated with a peptide pool for 9 days, followed by activation with CD3 / CD28 activation beads and gene editing. To enhance the expansion of antigen-specific Treg cells, beads were added to cells sorted by flow cytometry. Panel B shows flow cytometry plots showing GFP and Foxp3 expression 15 days after gene editing. GFP+Foxp3+ cells were CD25+CD127-, and tetramer staining revealed that approximately 60% of these cells were specific for MP peptide, HA peptide, or TT peptide.

[0088] [Figure 15]Antigen-specific suppression by Foxp3-edited Treg / airT cells is shown. Panel A shows a timeline of the process for generating antigen-specific edTreg / airT cells for use in suppression assays. 15 days after gene editing, GFP+ cells were selected by flow cytometry, activated with CD3 / CD28 beads, and expanded. After 7 days of incubation, the beads were removed and edTreg / airT cells were collected. Suppression assays were performed using edTreg / airT cells expanded for 11 days. Panel B shows an outline of the suppression assay design. CD4+CD25+ cells were isolated from autologous PBMCs, labeled with EF670, and used as Teff cells. Irradiated CD4-CD25+ cells were used as APCs. edTreg / airT cells were labeled with Cell Trace Violet (CTV). Teff cells were co-cultured with APCs and edTreg / airT cells in the presence of DMSO or a peptide pool (MP+HA+TT), or Teff cells and APCs without edTreg / airT cells. Panel C shows the results after 7 days of co-culture. Cells were stained and analyzed by flow cytometry. CD3+CD4+EF670+CTV- cells were gated as Teff cells. Panel D shows the proliferation rate measured by dilution of EF670 in Teff cells. The proliferation rate was normalized to 100% for EF670- cells, which accounted for 15% of the Teff cells in the co-culture of APCs and peptide pool. The inhibition rate was calculated as 100-proliferation rate (%).

[0089] [Figure 16]This demonstrates that islet-specific T cells with multiple specificities can be expanded by peptide stimulation. Panel A shows a typical timeline for generating islet antigen-specific edTreg / airT cells. Freshly isolated CD4+CD25- cells were stimulated with an islet-specific peptide pool and APCs (irradiated autologous CD4-CD25+ cells) for 14 days, and on day 13, islet-specific T cell proliferation was analyzed by tetramer staining. Panel B shows flow cytometry plots of islet-specific T cells stained with each tetramer or tetramer pool and gated on CD4+ cells.

[0090] [Figure 17] This figure shows the generation of islet-specific Treg cells with multiple specificities. Panel A shows islet-specific T cells stained with tetramers and sorted by flow cytometry on day 14. Sorted tetramer-positive cells were activated with CD3 / CD28 beads for 72 hours before Foxp3 editing. Cells were stained and analyzed 3 days after editing. Flow cytometry plots show Foxp3 and LNGFR expression in mock or Foxp3-edited cells (left) and CD25, CD127, and CD45RO expression in LNGFR+ gated cells (right). Panel B shows flow cytometry plots of cells stained with each tetramer or tetramer pool, demonstrating tetramer-positive cells among Foxp3-edited LNGFR+Foxp3+ cells.

[0091] [Figure 18-33] Dual editing of human CD4+ T cells by targeting two alleles results in inactivation of endogenous TCRs, resulting in artificial Treg cells expressing Foxp3 and antigen-specific TCRs.

[0092] [Figure 18]A schematic diagram of a typical CD4+ T cell that has been gene-edited to express an exogenous antigen-specific TCR and have a Treg phenotype without expressing an endogenous TCR is shown. In this scheme, the conversion of a normal CD4+ T cell to an antigen-specific Treg cell involves three genetic changes: 1) stable expression of the FOXP3 transcription factor in the CD4+ T cell to induce the Treg phenotype, 2) stable expression of a genetically rearranged T cell receptor specific to a given antigen (antigen-specific TCR) to induce Treg immunosuppressive activity, and 3) genetic deletion of the endogenous T cell receptor (TCR) to direct the immunosuppressive function only to the desired antigen.

[0093] [Figure 19] Typical AAV constructs used for CRISPR-mediated gene editing at the human and mouse TRAC loci are shown. This list shows the adeno-associated virus plasmid constructs generated for CRISPR-mediated homology-directed repair, which are constructed based on the associated gRNA. Each is numbered.

[0094] [Figure 20] A typical method for targeting the human TRAC locus using CRISPR for knockout / knockin is shown. More specifically, a schematic diagram of the human TRAC locus is shown, showing the relative positions of the four gRNA sequences tested (PC_TRAC_E1_gRNA1 to PC_TRAC_E1_gRNA4). Exon 1 of the TRAC locus is indicated by the bottommost bar and extends from approximately 1160 to over 1400. Common SNPs are shown near 1160 and 1400. The position of a previously reported positive control gRNA sequence (TCRa G4old) is shown near 1320.

[0095] [Figure 21]This study analyzes the suitability of each guide RNA (gRNA) for non-homologous end joining (NHEJ) to knock out CD3 in primary human CD4+ T cells. Data were obtained by FACS analysis. Panel A shows flow cytometry plots demonstrating CD3 expression 2 days after gene editing in mock-edited CD4+ T cells or CD4+ T cells in which the TCR locus was edited using each of the four guide RNAs. TCRa_G4old, which has previously been demonstrated to knock out CD3 expression, was used as a control. Panel B shows a histogram showing the CD3 knockout rate.

[0096] [Figure 22] The frequency of indels was investigated using ICE (Inference of CRISPR Edits). ICE (Inference of CRISPR Edits) was used to measure on-target site-specific activity, confirming that indels were specifically induced by gRNA_1 and gRNA_4 at the TRAC locus compared to predicted off-target sites.

[0097] [Figure 23] The results of ICE analysis of predicted off-target sites of gRNAs targeting the TRAC locus are shown. The frequency of indel induction at the top three predicted off-target sites (based on mismatch frequency and their locations) of TRAC gRNA 1 and TRAC gRNA 2 was examined by sequence deconvolution analysis using ICE.

[0098] [Figure 24]This figure shows an outline of a typical experiment using AAV to perform double editing to evaluate knock-in of two alleles. A. A schematic diagram of each AAV construct used in this experiment. After gene editing, GFP / BFP expression is induced by the MND promoter. B. A timeline of the experimental procedure. CD4+ T cells were stimulated with CD3 / CD28 beads for 3 days before gene editing. GFP and BFP expression in the cells was evaluated by flow cytometry 3 and 6 days after gene editing.

[0099] [Figure 25] This shows that double-editing of the TRAC locus in human CD4+ cells results in a double-positive cell population. Panel A shows flow cytometry plots demonstrating GFP and BFP expression in mock-edited cells and cells edited with a mixture of MND.GFP and MND.BFP (10% No. 3207 AAV virus + 10% No. 3208 AAV virus) two days after gene editing. The viral titer of No. 3207 was 3.3 x 10 12 , and the viral titer of No. 3208 was 2.53 x 10 12 . Panel B shows histograms showing the percentage of double-negative, GFP-only, mCherry-only, and GFP / mCherry-double-positive cells in the double-edited cells.

[0100] [Figure 26]A schematic diagram showing a typical split IL-2 CISC-containing homology-directed repair (HDR) knock-in construct for the selection of double-edited cells is shown. In the construct shown, the CISC (chemically induced signaling complex) is split into two separate constructs, with each CISC component coexpressed with a different reporter (in this case, GFP or mCherry). Each construct contains one half of a rapamycin-binding complex (either the FKBP or FRB domain) linked to a chimeric endoplasmic reticulum targeting domain, which is fused to one half of the IL-2R signaling complex (IL-2RB or IL-2RG), consisting of a transmembrane and intracellular domain. By delivering cDNA encoding each CISC component coexpressed with a GFP or mCherry tag into primary human CD4+ T cells, double-edited cells containing both CISC components and therefore GFP and BFP can be selectively grown.

[0101] [Figure 27] A typical timeline for double-editing CD4+ T cells with AAV, expansion in the presence of a rapalog, and analysis of enriched cells is shown. Cells were stimulated with CD3 / CD28 beads for 3 days before gene editing. Two days after gene editing, cells were analyzed for GFP and mCherry expression by flow cytometry, and then expanded in medium containing 50 ng / ml human IL-2 or 100 nM of a rapalog. Enrichment of GFP+mCherry+ double-positive cells was assessed by flow cytometry 6, 8, and 10 days after gene editing.

[0102] [Figure 28]FACS analysis was performed to examine the initial double-editing rate. Panel A shows flow cytometry plots demonstrating GFP and mCherry expression in mock-edited cells, cells edited with MND.GFP.FRB.IL-2RB (No. 3207 AAV 20%), cells edited with MND.mCherry.FKBP.IL-2RB (No. 3208 AAV 20%), and cells edited with a mixture of MND.GFP.FRB.IL-2RB and MND.mCherry.FKBP.IL-2RB (No. 3207 10% + No. 3208 10%). The viral titer of No. 3207 was 3.3 x 10 12 , and the viral titer of No. 3208 was 2.53 x 10 12 . Panel B shows histograms showing the percentage of double-negative, GFP-positive, mCherry-positive, and GFP / mCherry double-positive cells in the double-edited cells.

[0103] [Figure 29] Representative data showing enrichment of double-edited cells using rapalogs are shown. Panel A shows flow cytometry plots demonstrating GFP and BFP expression in mock-edited cells and cells edited with a mixture of MND.GFP and MND.BFP (10% No. 3207 AAV virus + 10% No. 3208 AAV virus) 2 days after gene editing. The viral titer of No. 3207 was 3.3 x 10 12 , and the viral titer of No. 3208 was 2.53 x 10 12 . Panel B shows histograms showing the percentage of double-negative, GFP-only, mCherry-only, and GFP / mCherry-double-positive cells in the double-edited cells.

[0104] [Figure 30] Histograms showing the percentage of double-negative, GFP-only, and mCherry-only cells after exposure to IL-2 or a rapalog are shown. These data demonstrate that treatment with a rapalog does not significantly alter the percentage of the single-positive and unedited cell populations.

[0105] [Figure 31] Figure 1 shows data from FACS analysis of initial double-editing rates using two different donors. Panel A shows a timeline of the gene editing and analysis steps. Panel B shows histograms showing the percentage of double-negative, GFP-positive, mCherry-positive, and GFP / mCherry double-positive cells in double-edited cells from each donor. Donor R003657 was a 28-year-old Caucasian male, and donor R003471 was a 29-year-old Caucasian male.

[0106] [Figure 32] FACS analysis data are shown for cells from donor R003471 that were bi-allele-edited and enriched in the presence of a rapalog. Panel A shows a flow cytometry plot demonstrating GFP and mCherry expression after 5 days of enrichment in the presence of a rapalog. Panel B shows a histogram showing the percentage of GFP / mCherry double-positive cells after growth in the presence of IL-2 or a rapalog.

[0107] [Figure 33] A schematic diagram showing a typical split CISC construct for inserting TCR and Foxp3 and enriching for double-edited cells is shown. CISC is split into two separate constructs, and each CISC component is coexpressed with an antigen-specific TCR (the exemplary T1D4 TCR shown in the schematic) or Foxp3. Each construct contains one half of a rapamycin-binding complex (either the FKBP or FRB domain) linked to a chimeric endoplasmic reticulum targeting domain, which is fused to one half of the IL-2R signaling complex (IL-2RB or IL-2RG), consisting of a transmembrane and intracellular domain. By delivering cDNA encoding each CISC component coexpressed with T1D4 TCR / Foxp3 into primary human CD4+ T cells, we were able to selectively expand double-edited cells containing both CISC components and therefore also containing T1D4 TCR and Foxp3.

[0108] [Figure 34-37] This paper relates to the production of reagents for evaluating the function of antigen-specific airT cells in in vivo models of autoimmune diseases.

[0109] [Figure 34] A schematic diagram of the mouse TRAC locus is shown, showing the relative positions of the three novel gRNA sequences tested (PC_mmTrac_E1_gRNA1 to PC_mmTrac_E1_gRNA3). Exon 1 of the TRAC locus is shown.

[0110] [Figure 35] Figure 1 shows FACS analysis data for CD3 knockout in mouse CD4+ T cells. Panel A shows flow cytometry plots demonstrating mouse CD3 expression 2 days after gene editing in mock-edited CD4+ T cells or CD4+ T cells in which the TCR locus was edited using each of the three guides. Panel B shows histograms showing the mCD3 knockout rate using each guide RNA.

[0111] [Figure 36] A typical experiment using AAV for double editing to evaluate knock-in of two alleles is shown. Panel A shows a schematic diagram of each AAV construct used in this experiment. After gene editing, GFP / BFP expression is driven by the MND promoter. B shows a timeline of the experimental procedure. Mouse CD4+ T cells were stimulated with CD3 / CD28 beads for 3 days before gene editing. GFP and BFP expression in the cells was assessed by flow cytometry 3 and 5 days after gene editing.

[0112] [Figure 37]FACS analysis data for single- and double-editing rates at the mouse TCRα locus are shown. Flow cytometry plots show GFP and BFP expression 3 days after gene editing in mock-edited cells, cells edited with MND.GFP (No. 3211 10%), cells edited with MND.BFP (No. 3212 10%), and cells edited with a mixture of MND.GFP and MND.BFP (No. 3207 5% + No. 3208 5%). The mixture-edited cells had a combined percentage of 1.97% GFP / BFP double-positive cells.

[0113] [Figure 38-43] Regarding the function of airT cells under antigen-specific in vivo conditions.

[0114] [Figure 38] Schematic diagram of the experimental design to test whether MOG-specific edTreg / air T cells (shown in white) can suppress effector T cells (Teff) (shown in gray) in a mouse model of experimental autoimmune encephalomyelitis, a model of multiple sclerosis.

[0115] [Figure 39] This study demonstrates that mouse FOXP3 TALENs catalyze efficient FOXP3 disruption and initiate non-destructive donor template recombination. Panel A shows the binding site of the FOXP3 TALEN pair in the human FOXP3 gene. Panel B shows the target binding site of the mouse FOXP3 TALEN pair in the mouse FOXP3 gene. Panel C shows the frequency of indels at the FOXP3 TALEN cleavage site in human CD4+ T cells (left) and mouse CD4+ T cells (right) 5–7 days after transfection with control mRNA (encoding blue fluorescent protein) or mRNA encoding TALENs specific for human FOXP3 or mouse FoxP3. Each graph shows the average frequency of indels determined by colony sequencing of PCR-amplified amplicons surrounding the target site in gDNA. Sequences were determined from 20–40 colonies per experiment.

[0116] [Figure 40] Generation of edTreg / airT cells from antigen-specific mouse CD4+ T cells. Panel A shows a schematic diagram of the FOXP3 locus after gene editing using mouse FOXP3 TALEN and an AAV donor template for mouse FOXP3 MND-GFP knock-in (ki). Upon gene editing, expression of the chimeric GFP-FoxP3 protein is driven by the MND promoter. Panel B shows flow cytometry plots demonstrating GFP expression in antigen-specific mouse CD4+ T cells two days after gene editing. Panel C shows the average percentage of GFP+ cells across multiple experiments (n=10). Panel D shows flow cytometry plots demonstrating the expression of Treg markers associated with mouse edTreg / airT cells.

[0117] [Figure 41] The results of a comparative evaluation of the function of antigen-specific edTreg / airT cells and polyclonal edTreg / airT cells in a mouse model of multiple sclerosis are shown. Panel A shows flow cytometry plots demonstrating GFP expression in MOG-specific and polyclonal mouse CD4+ T cells after FACS sorting two days after gene editing. Panel B shows a schematic diagram illustrating the in vivo experimental design and timeline using a mouse model of experimental autoimmune encephalomyelitis (EAE). 2D2 (MOG-specific) Teff cells (30,000 cells) were delivered into RAG1- / - recipient mice, and simultaneously transferred with edTreg / airT cells (30,000 cells) generated from 2D2 or C57Bl / 6 mice. Alternatively, 2D2 (MOG-specific) Teff cells (30,000 cells) alone were delivered into RAG1- / - recipient mice without edTreg / airT cell transfer. Both mouse strains were on a C57Bl / 6 genetic background. The analysis was performed on the seventh day.

[0118] [Figure 42]Data show that antigen-specific edTreg / airT cells delayed Teff cell proliferation, activation, and cytokine production. The immunophenotype of T cells obtained from the inguinal and axillary lymph nodes of recipient mice 7 days after cell transfer was assessed by flow cytometry. CD45+ cells were detected using a panCD45 antibody (an antibody that recognizes all CD45 isoforms and both CD45.1 and CD45.2 alloantigens). (A) Total number of CD45+CD4+ cells, (B) total number of other T cell subsets indicated on the graph, and (C) proliferation of GFP+ cells are shown. Data are from three independent experiments. Each bar graph shows the mean ± SD. p values ​​indicating statistical significance are indicated above each bar graph.

[0119] [Figure 43] These data demonstrate that antigen-specific edTreg / airT cells suppress Teff cell proliferation in vivo. Panel A shows flow cytometry plots in which actively dividing cells were labeled by administering the thymidine analog 5-ethynyl-2'-deoxyuridine (EdU) to selected mice 2 hours before sacrifice. EdU incorporation by T cells was measured by intracellular labeling with an anti-EdU antibody and flow cytometry. Each flow cytometry plot was obtained by analyzing T cells isolated from lymph nodes 7 days after cell transfer. Panel B shows a bar graph summarizing the average percentage of cells that incorporated EdU in various cell subsets. Panel C shows a bar graph summarizing the percentage of GFP+ lymphocytes. Each flow cytometry plot shows representative results from at least three independent experiments. Each bar graph shows the mean ± SD. p values ​​indicating statistical significance are indicated above each bar graph.

[0120] [Figure 44-47]This study investigated antigen-specific T cell function in an adoptive transfer NSG mouse model of type 1 diabetes. NSG mice were infused with antigen-specific (BDC) recombinant edTreg / airT cells or polyclonal (NOD) recombinant edTreg / airT cells, or antigen-specific nTreg cells, followed by antigen-specific Teff cells. Mice were monitored for diabetes for up to 90 days after infusion. The graph shows the percentage of mice that developed diabetes after administration of mock-edited, Foxp3-edited, or nTreg cells from NOD or BDC2.5 mice with effector cells, as indicated.

[0121] [Figure 44] Editing of Foxp3 in CD4+ T cells from antigen-specific NOD mice. Panel A shows the cleavage efficiency of CAS9 / CRISPR RNP using various guide RNAs in BDC2.5 NOD mice. Panel B shows the repair template delivered by AAV5. Upon gene editing, expression of a chimeric GFP-Foxp3 protein is driven by the MND promoter. Panel C shows flow cytometry plots showing GFP expression in mock-edited and GFP-Foxp3-edited antigen-specific mouse CD4+ T cells 2 days after editing.

[0122] [Figure 45] Phenotype of FOXP3-edited antigen-specific NOD CD4+ T cells. Left panel: Flow cytometry plots showing GFP and Foxp3 expression in edited cells. Middle panel: Flow cytometry plots showing IL-2, IFN-γ, and IL-4 expression in antigen-specific murine NOD CD4+ T cells edited with the GFP-Foxp3 construct (top plot) and mock-edited antigen-specific murine NOD CD4+ T cells (bottom plot). Right panel: Histograms showing the percentage of cells positive for IL-2, IFN-γ, and IL-4 4 days after editing.

[0123] [Figure 46] This study examines the phenotype of cells transferred into an adoptive transfer NSG mouse model. Panel A shows the experimental design, indicating the number and type of cells administered to each group of mice. Panel B shows flow cytometry plots demonstrating the phenotype of Teff, edTreg / airT, and nTreg cells injected into NSG mice.

[0124] [Figure 47] Antigen-specific T cell function in the adoptive transfer NSG mouse model. Panel A shows the experimental design. NSG mice were infused with antigen-specific (BDC)-modified edTreg / airT cells or polyclonal (NOD)-modified edTreg / airT cells, or antigen-specific nTreg cells, followed by antigen-specific Teff cells. After infusion, mice were monitored for diabetes for up to 90 days. Panel B shows a graph depicting the percentage of mice that developed diabetes after administration of the indicated mock-edited, Foxp3-edited, or nTreg cells from NOD or BDC2.5 mice in combination with effector cells. Antigen-specific edTreg / airT cells provided significantly greater protection against type 1 diabetes compared with mock-edited T cells, polyclonal edTreg / airT cells, and polyclonal nTreg cells.

[0125] [Figure 48-51] Regarding the construction of a mouse AAV donor template design for generating airT cell products containing a selectable marker (LNGFR).

[0126] [Figure 48] A typical repair template used for editing mouse Foxp3 is shown. Stable expression of Foxp3 via the AAV promoter-LNGF.P2A knock-in construct was tested in mouse T cells.

[0127] [Figure 49]Figure 1 shows the phenotype of murine edTreg / airT cells using different homologous donor cassettes. Flow cytometry plots show the expression of LNGFR, FOXP3, CD25, and CTLA-4 in mock-edited cells or cells edited with MND.LNGFR.P2A KI (No. 3189) or PGK.LNGFR.P2A KI (No. 3227).

[0128] [Figure 50] Data showing the editing rate and LNGFR expression in gene-edited mouse Treg / airT cells are shown. Flow cytometry plots show LNGFR and GFP expression in mock-edited cells or cells edited with MND-GFPki (No. 1331) or MND.LNGFR.P2A.KI (No. 3189).

[0129] [Figure 51] Figure 1 shows data from enrichment of gene-edited LNGFR+ T cells from B6 mice using an anti-LNGFR column. Flow cytometry plots show LNGFR expression in cells before purification on a Miltenyi anti-LNGFR column, in the flow-through fraction, and in cells eluted from the column.

[0130] [Figure 52]This figure shows a comparison of FOXP3-edited human CD4+ T cells and human CD4+ T cells transduced with FOXP3 lentivirus (LV). Panel A shows a schematic diagram of the LV construct. The MND promoter drives expression of a transcript encoding the same GFP-FOXP3 fusion protein contained in air T cells. This transcript contains a WPRE and poly(A) signal for efficient nuclear transport and mRNA stability. The bottom panel shows representative flow cytometry plots showing FOXP3 and GFP expression in mock-edited T cells or FACS-sorted tTreg cells (CD4+CD25++CD127-), air T cells, or LV Treg cells (CD4+GFP+). All of these cells were expanded in vitro with CD3 / CD28 bead stimulation for over 14 days. Panel B shows the mean (±SD) viral copy number in FACS-sorted cells transduced with LV (left panel; n=6). Scatter plots (right) show the MFI of the GFP+ population in each sample (n = 5; P values ​​are determined by two-tailed Student's t-test). Panel C shows bar graphs depicting the mean percentage (top graph) and MFI (bottom graph) of each cell analyzed by flow cytometry staining for each protein indicated in the graph. Live singlet cells were further gated by CD4+GFP+ (LV Tregs and edTregs), CD4+FOXP3+ (tTregs), or CD4+ (mock). For markers that showed a clear bimodal distribution, only the MFI of the positive population was calculated. Error bars indicate ±SD. A conventional two-way ANOVA was performed, with P values ​​corrected by Tukey's multiple comparison test. P values ​​in black indicate comparisons with mock-edited cells, and P values ​​in red indicate comparisons with the group indicated by the dashed line. Panel D shows the percent suppression as a function of dilution of Treg or mock cells (top panel). Additionally, histograms of proliferation dyes when Treg cells or mock cells were cultured with Teff cells at various ratios are shown (bottom panel). Suppression rate (%) = [(percentage of dividing cells in the absence of Treg cells (%) - percentage of dividing cells in the presence of Treg cells (%)) / percentage of dividing cells in the absence of Treg cells (%)] × 100.Panel E shows plots of time-dependent data points and simple linear regression of the percentage of GFP+ cells in cultures after FACS purification. Air T cells (n=4) and LV Treg cells (n=6); data from six experiments are shown. Dashed lines indicate 95% confidence intervals. P values ​​were determined using an F-test.

[0131] [Figure 53-73] FIG. 1 shows another schematic and data for an exemplary dual-editing method according to the present disclosure to generate antigen-specific airT cells in which the endogenous TCR has been knocked out and which can be drug-selected.

[0132] [Figure 53] A schematic diagram of a double-editing method designed to a) eliminate endogenous TCR expression and b) generate selectable antigen-specific air T cells is shown. The IL-2 CISC / DISC is split into two halves (FKBP-IL2RG and FRB-IL2RB), and one halves is combined with an expression cassette for FOXP3, while the other halves are combined with an islet antigen-specific TCR candidate (T1D4) and delivered to the same locus (Method 1) or to two separate loci (Method 2). Endogenous TCRs can be deleted by targeting the TRAC locus in CD4+ T cells. Method 2 can achieve a higher initial double-editing rate, but requires two nuclease target sites, resulting in two double-strand breaks in the host cell genome, each of which undergoes homologous recombination repair. Method 1 utilizes a single nuclease target site to create a single double-strand break.

[0133] [Figure 54]A schematic diagram of the AAV HDR donor constructs used for dual editing of human T cells is shown. The first seven constructs are split IL-2 CISC repair templates carrying GFP, mCherry, HA-tagged FOXP3, or T1D4 driven by the MND promoter. Each split CISC component contains one half of a complex (either the FKBP or FRB domain) that binds and heterodimerizes with rapamycin, linked to a chimeric endoplasmic reticulum targeting domain. The FKBP or FRB domain is fused to one half of the IL-2R signaling complex (IL-2RB or IL-2RG), which consists of a transmembrane and intracellular domain. Each repair template was flanked by 300-bp homology arms compatible with gRNAs targeting the TRAC locus (gRNA_4) or the FOXP3 locus (gRNA_T9) (constructs 3207, 3208, 3240, 3243, 3251, 3252, and 3273). The next four constructs (constructs 3253, 3258, 3292, and 0001) were used to in-frame knock-in a promoterless TCR cassette containing CISC components, targeting the first exon of the TRAC locus (gRNA_1). The final two constructs (3280 and 3262) are split DISC repair templates containing CISC components and cDNA encoding a free FRB domain that sequesters rapamycin in the cytoplasm (thus eliminating or reducing the negative effects of rapamycin on gene-edited cells). These two constructs also contain mCherry or FOXP3 driven by the MND promoter.

[0134] [Figure 55]The double-editing rate of the human TRAC locus in CD4+ T cells from donor R003657 edited with a CISC construct was shown with or without rapalog selection. Panel A shows a timeline of the gene editing, enrichment, and analysis steps for CD4+ T cells from donor R003657 using AAV No. 3207 and AAV No. 3208 (via co-delivery of RNP and AAV). Panel B shows flow cytometry plots comparing the percentage of GFP / mCherry double-positive cells initially in mock-edited and double-edited samples, and after 7 days of enrichment in the presence of IL-2 or a rapalog (AP21967). Panel C shows histograms comparing the percentage of double-negative, GFP-positive, mCherry-positive, and GFP / mCherry double-positive cells in double-edited cells enriched with IL-2 and rapalogs.

[0135] [Figure 56] The double-editing rate of the TRAC locus in CD4+ T cells from donor R003471 edited with a CISC construct and selected with a rapalog is shown. Panel A shows a timeline of the gene editing, enrichment, and analysis steps for CD4+ T cells from donor R003471 using AAV No. 3207 and AAV No. 3208. Panel B shows flow cytometry plots comparing the initial percentage of GFP / mCherry double-positive cells between mock-edited and double-edited samples, as well as the percentage of GFP / mCherry double-positive cells after 7 days of enrichment in the presence of IL-2 or a rapalog (AP21967). Panel C shows histograms comparing the percentage of double-negative, GFP-positive, mCherry-positive, and GFP / mCherry double-positive cells in double-edited cells enriched with IL-2 and those enriched with a rapalog.

[0136] [Figure 57]Dual editing of the TRAC locus in human CD4+ T cells generates antigen-specific air T cells that can be selected with rapalogs. Panel A shows a schematic diagram of the AAV HDR donor construct used to deliver the "split" IL-2 CISC components for the selection of dual-edited cells. The CISC components (IL2RG and IL2RB) were separately integrated into two constructs and coexpressed with HA-FoxP3 cDNA (AAV No. 3240) or the islet-specific TCR T1D4 (AAV No. 3243). Each repair template is flanked by identical homologous arms that are not cleaved by gRNA targeting the TRAC locus. By integrating a single copy of each construct, only edited CD4+ T cells are predicted to be selectively expandable by treatment with rapalogs. Panel B shows a timeline of the key steps in this study: double-editing CD4+ T cells with AAV / RNP, expansion in the presence of a rapalog, and analysis of enriched cells. Cells were stimulated with CD3 / CD28 beads for 3 days before gene editing. Two days after gene editing, HA-FoxP3 and TCR expression in the cells were analyzed by flow cytometry. The cells were then expanded in medium containing 50 ng / ml human IL-2 or 100 nM of a rapalog. Five and eight days after gene editing, enrichment of double-positive cells positive for HA-FoxP3 and TCR was assessed by flow cytometry. Panel C shows enrichment of double-edited cells using a rapalog. Left panel: Flow cytometry plots analyzing HA-FoxP3 and TCR expression in double-edited cells expanded for 8 days in the presence of IL-2 or a rapalog. Right panel: Quantification of the percentage of HA-FoxP3 / TCR double positive cells expanded for 5 or 8 days in the presence of IL-2 or rapalog.

[0137] [Figure 58]Data show that lowering serum concentration improves the total and double-editing rates of the TRAC locus. Panel A shows a timeline illustrating the double-editing process of CD4+ T cells using AAV and the expansion process with rapalogs. Gene editing of human CD4+ T cells was performed using TRAC gRNA_4 and AAV No. 3243 and AAV No. 3240 constructs (double editing at one locus). Immediately after electroporation to deliver RNPs, cells were seeded in medium containing 20%, 2.5%, 1%, or 0% FBS (recovery medium) and infected with AAV. Approximately 16 hours later, the medium was replaced with 20% FBS-containing medium, and editing rates were measured by FACS analysis on day 3. Cells recovered in medium containing 2.5% FBS were expanded for an additional 7 days in the presence of IL-2 or rapalogs. Panel B shows flow cytometry plots demonstrating T1D4 and FOXP3 expression in mock-edited cells, single-edited cells, and cells edited with an AAV mixture (10% AAV No. 3243 and 10% AAV No. 3240) 3 days after gene editing. The viral titer of AAV No. 3243 (pAAV.MND.T1D4.FRB.IL2RB) was 4.2 x 10 11 , and the viral titer of AAV No. 3240 (pAAV.MND.FOXP3-HA.FKBP.IL2RG) was 1.3 x 10 12 . Panel C shows histograms showing the percentage of double-negative, FOXP3-HA-positive, T1D4-positive, and FOXP3 / T1D4 double-positive cells in double-edited cells.

[0138] [Figure 59]This figure shows a comparison of IL-2 enrichment and rapalog enrichment of double-edited cell populations. Dual editing of the TRAC locus was performed using the method described in Figure 5. Panel A shows flow cytometry plots comparing T1D4 and FOXP3 expression in mock-edited and FOXP3 / T1D4 (No. 3240 / No. 3243) double-edited cells treated with 50 ng / mL IL-2 or 100 nM rapalog (AP21967) for 7 days. Data represent only the condition using recovery medium containing 2.5% FBS. Panel B shows histograms showing the percentage of double-negative, FOXP3-HA-positive, T1D4-positive, and FOXP3 / T1D4 double-positive cells in the enriched double-edited cells.

[0139] [Figure 60]This paper describes a method for testing a dual-editing method for editing two loci in human CD4+ T cells. Panel A shows a schematic diagram of an AAV HDR donor construct designed to deliver a split IL-2 construct for selection of dual-edited cells. The CISC component is split between the two constructs and coexpressed with mCherry (No. 3207) or GFP (No. 3251). One repair template is flanked by homologous arms compatible with a gRNA targeting the TRAC locus, and the other repair template is flanked by homologous arms compatible with a gRNA targeting the FOXP3 locus. Only CD4+ T cells that have been edited with both expression cassettes (at the appropriate loci) are predicted to be selectively expanded under rapalog treatment. Panel B shows a timeline illustrating the AAV-based dual-editing and rapalog-based expansion steps for CD4+ T cells. Human CD4+ T cells were gene-edited using human TRAC gRNA_4 and human FOXP3 gRNA_T9 with AAV constructs No. 3251 (MND.mCherry.FKBP.IL2RG) and No. 3207 (MND.GFP.FRB.IL2RB) (a dual-locus editing approach). Immediately after electroporation, cells were seeded in medium containing 20% ​​or 2.5% FBS (recovery medium). Approximately 16 hours later, the medium was replaced with 20% FBS-containing medium, and editing rates were measured by FACS analysis on day 3. Cells recovered in 2.5% FBS-containing medium were cultured for an additional 7 days in the presence of IL-2 or a rapalog to monitor enrichment.

[0140] [Figure 61]This figure shows that recovery in medium containing 2.5% FBS improves the double-editing rate, measured 3 days after gene editing. A double-editing method was performed to edit two loci using the method described in Figure 59. Panel A shows flow cytometry plots comparing GFP and mCherry expression in mock-edited and double-edited cells 3 days after gene editing, when the cells were recovered in recovery medium containing 20% ​​FBS versus recovery medium containing 2.5% FBS. The viral titer of No. 3251 (pAAV.MND.mCherry.FKBP.IL2RG) was 6.55 x 10 10 , and the viral titer of No. 3207 (pAAV.MND.GFP.FRB.IL2RB) was 2.50 x 10 12 . The amount of each AAV virus used in each editing reaction was 10% of the medium volume. Panel B shows histograms showing the percentage of double-negative, GFP-positive, mCherry-positive, and GFP / mCherry double-positive cell populations in double-edited cells.

[0141] [Figure 62] This shows that double-edited cells with edited two loci are highly enriched by selection with rapalog treatment. A double-editing method for editing two loci was performed using the method described in Figure 59. Panel A shows flow cytometry plots comparing GFP and mCherry expression in mock-edited cells and GFP / mCherry (No. 3207 / No. 3251)-edited cells (edited in medium containing 2.5% serum) treated with 50 ng / mL IL-2 for 10 days and 100 nM rapalog (AP21967) for 10 days. Panel B shows histograms comparing the percentages of double-negative, GFP-positive, mCherry-positive, and GFP / mCherry double-positive cells in the double-edited cell population treated with IL-2 for 10 days and rapalog for 10 days.

[0142] [Figure 63]This paper describes a dual-editing method for editing two gene loci in human CD4+ T cells. This figure shows a timeline of the AAV-based CD4+ T cell dual-editing process and the rapalog-based expansion process, along with the editing conditions. Human CD4+ T cells were edited using human TRAC gRNA_4 and human FOXP3 gRNA_T9 with AAV No. 3251 (MND.mCherry.FKBP.IL2RG) and AAV No. 3207 (MND.GFP.FRB.IL2RB) (a dual-editing method for editing two gene loci). Editing conditions were varied as described in the table by using various viral stock ratios in the presence or absence of HDR enhancers or DMSO. Immediately after electroporation, cells were seeded in medium containing 2.5% FBS (recovery medium). Approximately 16 hours later, the medium was replaced with medium containing 20% ​​FBS, and editing rates were measured by FACS analysis on day 3. Cells recovered in 2.5% FBS-containing medium were cultured for an additional 10 days in the presence of IL-2 or a rapalog to monitor enrichment.

[0143] [Figure 64] This graph shows that using an optimized amount (10% by volume) of AAV HDR donor improves the double-editing rate. Gene editing was performed using the method summarized in Figure 62. The graph shows the percentages of double-negative, GFP-positive, mCherry-positive, and GFP / mCherry double-positive cell populations in double-edited cells 3 days after editing with various amounts of AAV No. 3207 and AAV No. 3251 in the presence of 30 μM HDR enhancer or DMSO.

[0144] [Figure 65]This figure shows data demonstrating that selection with rapalogs can highly enrich for dual-edited CD4+ T cells with edited loci. Optimal results were obtained using a medium containing 2.5% FBS and an optimized amount (10% by volume) of AAV donor. Gene editing was performed using the method summarized in Figure 62. The cells shown in Figure 10 (gene-edited with an optimized amount (10%) of virus in a medium containing 2.5% serum, with or without HDR enhancers) were contacted with IL-2 or a rapalog for 10 days, and a graph showing the percentage of double-negative cells, GFP-positive cells, mCherry-positive cells, and GFP / mCherry double-positive cells in the edited cell population is shown.

[0145] [Figure 66] A schematic diagram of a typical split-CISC construct for inserting an islet-specific TCR and FOXP3 using a dual-editing method to edit two gene loci and enrich for dual-edited cells is shown. The IL-2 CISC (chemically induced signaling complex) was split into two separate constructs and co-expressed with either the T1D4 TCR (No. 3243) or FOXP3 (No. 3252). Each construct contains one half of a complex that binds to rapamycin and heterodimerizes (either the FKBP domain or the FRB domain) linked to a chimeric endoplasmic reticulum targeting domain. The FKBP or FRB domain is fused to one half of the IL-2R signaling complex (IL-2RB or IL-2RG), consisting of a transmembrane and intracellular domain. By delivering cDNA encoding each CISC component co-expressed with T1D4 TCR or FOXP3 into primary human CD4 + T cells, we were able to propagate only dual-edited cells that contain both CISC components and therefore express T1D4 TCR and FOXP3.

[0146] [Figure 67]This figure shows a typical method for dual editing of a single gene locus using the TRAC locus promoter. A schematic diagram of an AAV construct for homologous recombination repair-based gene editing designed for dual editing within the TRAC locus is shown. The upper panel shows a schematic diagram of an AAV construct (No. 3240) for FOXP3 expression and introduction of split CISC. The lower panel shows a schematic diagram of an AAV construct (No. 3258) for in-frame knock-in into exon 1 of the TRAC locus to induce expression of T1D4 TCR and split CISC using the endogenous promoter of the TRAC locus.

[0147] [Figure 68] This figure shows that editing of the TRAC locus using homologous recombination repair disrupts TCR expression and allows robust transgene expression driven by the endogenous TRAC locus enhancer and promoter. Panel A shows the editing strategy for in-frame integration of an mCherry-split CISC cassette into the endogenous TRAC locus. Using gRNA targeting exon 1 of the TRAC locus, we integrated a construct (No. 3253) containing a fluorescent dye marker (mCherry) followed by a P2A element, FRB-IL2RB CISC, in-frame, disrupting endogenous TCR expression while driving expression driven by the endogenous TRAC locus promoter. Panel B shows a timeline illustrating the steps in gene editing of CD4+ T cells using AAV construct No. 3253. Cells were stimulated with CD3 / CD28 beads for 3 days before gene editing. Panel C shows flow cytometry analysis of CD3 and mCherry expression in cells 7 days after gene editing. Flow cytometry plots show significant mCherry expression along with loss of CD3 in edited cells compared to mock-edited and AAV-only controls.

[0148] [Figure 69]This figure shows a comparison of mCherry expression driven by the endogenous promoter of the TRAC locus and mCherry expression driven by the MND promoter. Gene editing was performed as in Figure 67 using a different HDR donor (No. 3253 vs. No. 3208), and the relative expression activity from the endogenous promoter of the TRAC locus and the relative expression activity from the MND promoter were assessed and compared. Flow cytometry plots showed that the level of mCherry expression driven by the endogenous promoter (using P2A.mCherry.FRB.IL2RB (No. 3253)) was lower than the level of mCherry expression driven by the MND promoter (using MND.mCherry.FKBP.IL2RG (No. 3208)). The bottom panel shows data from a repeat experiment using donor No. 3253.

[0149] [Figure 70] Another exemplary dual-editing strategy is shown, utilizing constructs targeted to the TRAC and / or FOXP3 loci and knocked in in-frame to utilize the endogenous promoter of the TRAC locus. A schematic diagram of an exemplary AAV donor construct for generating IL-2 DISC-selectable antigen-specific airT cells is shown for testing dual-editing strategies for one or two loci. The T1D4 TCR is shown as an exemplary TCR; alternative TCRs can be replaced based on disease target or other relevant features for therapeutic use. The same applies to the IL-2 DISC construct.

[0150] [Figure 71]Dual editing of human CD4+ T cells using a decoy-CISC (split-DISC) construct. Panel A shows a schematic diagram of the HDR knock-in construct (No. 3280) containing a split IL-2 DISC for selection of dual-edited cells with rapamycin. To generate the decoy-CISC (split-DISC), a free FRB domain was added to the MND.mCherry.FKBP.IL2RG construct to sequester rapamycin in the cytoplasm, resulting in MND.mCherry.FKBP.IL2RG.FRB (No. 3280). Each repair template (No. 3280 and No. 3207 (not shown)) is flanked by identical homology arms compatible with a gRNA targeting the TRAC locus. CD4+ T cells edited by integration of a single copy of each construct are expected to be selectively expandable by treatment with a rapalog or rapamycin. Panel B shows a timeline illustrating the double-editing of CD4+ T cells using AAV No. 3280 and AAV No. 3207, expansion in the presence of a rapalog / rapamycin, and analysis of enriched cells. Cells were stimulated with CD3 / CD28 beads for 3 days before gene editing. Two days after gene editing, cells were analyzed by flow cytometry for GFP and mCherry expression, and then expanded in medium containing 50 ng / ml human IL-2, 100 nM of a rapalog, or 10 nM of rapamycin. Panel C shows a flow cytometry plot showing the percentage of GFP / mCherry double-positive cells 3 days after gene editing.

[0151] [Figure 72]Dual editing of human CD4+ T cells with a split-DISC construct results in rapamycin-selectable cells. Dual editing was performed as described in Figure 70. Panel A shows flow cytometry plots depicting the percentage of GFP / mCherry double-positive cells after 8 days of culture in the presence of 50 ng / mL human IL-2, 100 nM rapalog (AP21967), 10 nM rapamycin, or no drug treatment. Panel B shows histograms depicting the percentage of double-negative, GFP-positive, mCherry-positive, and GFP / mCherry double-positive cells in dual-edited cells after enrichment with IL-2, rapalog (AP21967), or rapamycin, or no drug treatment.

[0152] [Figure 73] Representative constructs for in vivo testing of dual-edited (split-DISC) Treg cells are shown. A schematic diagram of the FOXP3 split-IL-2 DISC HDR knock-in construct (No. 3262) used in combination with the T1D4 CISC construct (No. 3243) for rapamycin selection of dual-edited cells is shown. The CISC component is split between the two constructs and co-expressed with HA-FoxP3 or T1D4 TCR. This FOXP3 CISC construct also contains the FRB domain, which is predicted to protect mTOR signaling in the presence of rapamycin (FOXP3 DISC construct). Each repair template is flanked by identical homology arms compatible with a gRNA targeting the TRAC locus. CD4+ T cells edited by integration of a single copy of each construct are expected to be selectively expanded in both rapalog and rapamycin treatment.

[0153] [Fig. 74-94] Additional schematics and data regarding the generation and characterization of mouse airT cells are provided.

[0154] [Figure 74] The repair template used for editing mouse Foxp3 is shown. Schematic diagrams of the additional AAV.GFP.KI and AAV.LNGFR.P2A constructs generated are shown. These constructs were tested for editing efficiency, FOXP3 expression, and suppression function in mouse T cells.

[0155] [Figure 75] FIG. 1 shows a schematic illustrating the method used to generate mouse airT cells using the MND.GFP.KI HDR donor construct (or another HDR donor construct).

[0156] [Figure 76] We demonstrate the generation and enrichment of murine airT cells expressing endogenous Foxp3 using alternative promoters. Flow cytometry plots are shown for mock-edited cells, MND.GFP.KI (No. 1331)-edited cells, MND.LNGFR.P2A (No. 3189 or No. 3261)-edited cells, PGK.LNGFR.P2A (No. 3227)-edited cells, and EF-1a-LNGFR.P2A (No. 3229)-edited cells, before and after FACS sorting for enrichment of LNGFR. The top plot shows the initial editing rate, and the bottom plot shows enrichment after FACS sorting. These data demonstrate the success of generating airT cells using each candidate donor construct.

[0157] [Figure 77]Panel A shows flow cytometry plots demonstrating LNGFR and GFP expression in spleen-derived T cells gene-edited using homologous recombination repair (HDR). Each plot represents unmodified control cells from C57BL / 6 mice; mock-edited T cells, MND.GFP.KI (No. 1331), UCOE-containing MND.GFP.KI (No. 3213), and PGK.GFP.KI (No. 3209) T cells from C57BL / 6 mice. Panel B shows a histogram of the FOXP3 expression data shown in Panel A. Panel C shows a bar graph showing the MFI of FOXP3 in edTreg / airT cells and nTreg cells generated using various alternative HDR donor constructs as indicated. Highest FOXP3 expression was observed in donors containing the MND promoter.

[0158] [Figure 78]The design and results of an in vitro suppression assay using mouse tTreg cells or mouse airT cells are shown. A) For in vitro suppression assays, airT cells were enriched by FACS sorting two days after gene editing and resuspended in RPMI medium containing 10% FBS. nTreg cells (CD4+CD25+), Teff cells (CD4+CD25-), and antigen-presenting cells (APCs) (CD4-CD25-) were isolated from a mixture of spleen and lymph node cells from 8-10 week-old C56BL / 6 mice by column enrichment. 5 x 106 enriched Teff cells were resuspended in 2 ml of PBS, labeled with Cell Trace Violet (CTV) by incubation at 37°C for 15 minutes, washed in medium, resuspended in medium, and then added to the suppression assay. Assays were set up in U-bottom 96-well tissue culture plates by co-culture of 1.25 x 105 irradiated (2500 rad) APCs, 0.25 x 105 Teff cells, and counted nTreg or airT cells in the presence of 1 mg / ml anti-CD3 in a total volume of 300 μl of medium in a 37°C CO2 incubator for 4 days. On day 4, cells were washed twice with PBS and stained with live / dead indicators, anti-CD4, anti-CD45, and anti-CD25 antibodies to analyze the inhibitory effect of airT cells on Teff cell proliferation by FACS (LSRII). B) Representative flow cytometry data showing the suppression of Teff cell proliferation in the presence of airT cells.

[0159] [Figure 79]Figure 1 shows the results of testing the suppressive function of murine air T cells in vitro. Flow cytometry plots show Cell Trace Violet-labeled CD4+ T cells from C57BL / 6 mice in the presence or absence of mock-edited T cells, MND.GFP.KI (No. 1331)-edited T cells, MND.LNGFR.P2A (No. 3261)-edited T cells, or nTreg cells. These data demonstrate that murine air T cells (generated using MND.GFP.KI HDR donors or MND.LNGFR.P2A HDR donors) and nTreg cells both exhibited potent suppressive function in vitro, and the suppressive function was comparable.

[0160] [Figure 80] In vitro suppressive function of murine airT cells carrying various alternative promoters is shown. Flow cytometry plots show Cell Trace Violet-labeled CD4+ T cells from C57BL / 6 mice in the presence or absence of mock-edited T cells, MND.GFP.KI (No. 1331)-edited T cells, MND.LNGFR.P2A (No. 3261)-edited T cells, PGK.LNGFR.P2A (No. 3227)-edited T cells, EF-1α.LNGFR.P2A (No. 3229)-edited T cells, or nTreg cells. MND promoter-driven murine airT cells exhibit suppressive function comparable to nTreg cells. In contrast, PGK promoter-driven or EF-1α promoter-driven airT cells exhibit limited or no suppressive function.

[0161] [Figure 81] Figure 1 shows the experimental design comparing LNGFR+ edited cells enriched by FACS sorting with LNGFR+ edited cells enriched by column purification in an NSG adoptive transfer model. The number of recipient NSG host animals in the five experimental groups, as well as the origin and number of adoptively transferred control, airT, or nTreg cells, are shown in the table.

[0162] [Figure 82] Flow cytometry analysis of NOD BDC2.5+ mouse cells edited with LNGFR.P2A before and after column purification is shown. Panel A shows flow cytometry plots demonstrating LNGFR expression in mock-edited cells and cells edited with MND.LNGFR.P2A (No. 3189). Panel B shows flow cytometry plots demonstrating LNGFR expression in MND.LNGFR.P2A (No. 3189)-edited cells enriched by FACS sorting. FACS sorting consistently enriched edTreg cell products with greater than 90% purity, which were used for in vitro and in vivo studies.

[0163] [Figure 83] Flow cytometry analysis of gene-edited mouse cells before and after column enrichment is shown. Flow cytometry plots of cells edited with MND-LNGFR.P2A (No. 3189) before and after column enrichment are shown. In this enrichment example, 72 x 106 cells, with an initial editing efficiency of approximately 7%, were applied to the anti-LNGFR column, resulting in 2 x 106 edTreg cells with a purity of >84%.

[0164] [Figure 84]Experimental design and results are shown to evaluate the function of islet antigen-specific airT cells in the NSG adoptive transfer model. A comparison of FACS-sorted and column-enriched airT cells is shown. Islet antigen-specific (BDC) airT cells (generated using HDR donors (3261 or 3389)) or antigen-specific nTreg cells were adoptively transferred into 8-10-week-old adult NSG recipient mice by delivery into the retroorbital plexus (RO), followed by injection of antigen-specific Teff cells. Mice were monitored for the development of diabetes for up to 60 days. A graph shows the percentage of mice that developed diabetes after administration of the indicated mock-edited cells, MND.LNGFR.P2A-edited cells (FACS-sorted or column-enriched), or nTreg cells and effector cells from NOD BDC2.5 mice. Column-enriched antigen-specific MND.LNGFR.P2A airT cells reduced the development of diabetes in NSG mice and exhibited similar functionality to FACS-sorted airT cells. High doses of column-enriched MND.LNGFR.P2A airT cells or nTreg cells completely protected recipient mice from developing diabetes.

[0165] [Figure 85]This figure shows the in vivo function of airT cells generated using various promoters compared in the NSG adoptive transfer model. Engineered antigen (BDC)-specific airT cells (airT cells generated using the MND promoter (donor construct 1331) or the PGK promoter (donor construct 3209)) or antigen-specific nTreg cells were adoptively transferred into NSG recipient mice followed by injection of antigen-specific Teff cells. Mice were monitored for the development of diabetes for up to 60 days. This figure shows the percentage of mice that developed diabetes after administration of the indicated mock-edited, MND.GFP.KI (No. 1331)-edited, or PGK.GFP.KI (No. 3209)-edited airT cells or nTreg cells (5 × 104 each) from NOD BDC2.5 mice in combination with effector cells (5 × 104). Antigen-specific airT cells carrying the MND promoter prevented the development of diabetes in all recipient mice. nTreg cells prevented diabetes in 4 of 5 recipient mice, whereas antigen-specific airT cells engineered with the PGK promoter conferred little or no protection. These data directly demonstrate that protection from type 1 diabetes is specific to airT cells generated using the MND promoter to drive Foxp3 expression and support the use of this construct in human clinical trials for type 1 diabetes or other immune diseases.

[0166] [Figure 86]We demonstrate that islet antigen-specific MND.GFP.KI airT cells maintain viability and a stable phenotype in the target organ (pancreas) in vivo for at least 60 days. Flow cytometry plots show FOXP3 and GFP expression in MND.GFP.KI (No. 1331) NOD BDC2.5 airT cells harvested from the pancreas 60 days after adoptive transfer into NSG mice. Data are from two mice. Recipient mice demonstrate expansion of iTreg cells (FOXP3+GFP- CD4 T cells) derived from the transferred Teff cell population or endogenous Treg cells (presumably secondary to a beneficial bystander effect of airT cell delivery).

[0167] [Figure 87] This figure shows the design and results of targeting the mouse Rosa26 locus using CRISPR for knock-in / knock-out. Panel A shows the selection of the Rosa26 locus as a model safe-harbor site for integration in mouse T cells by homologous recombination repair. The locations of two novel gRNAs (gRNA_1 and gRNA_2) within the mouse Rosa26 locus are shown. The gRNAs reported by Pesch et al. and Wu et al. contain previously reported gRNAs within this locus. Panel B shows on-target site-specific activity measured by Inference of CRISPR Edits (ICE), demonstrating that specific indels were induced at the Rosa26 locus by delivering Cas9-RNP to primary mouse CD4+ T cells using R26 gRNA_1.

[0168] [Figure 88]This shows an overview of a gene editing experiment using homologous recombination repair at the mouse Rosa26 locus. Panel A shows a schematic diagram of the AAV construct No. 3245 used in this experiment. Gene editing of mouse T cells using homologous recombination repair induces GFP expression via the MND promoter. Panel B shows a timeline of the experimental procedure. Mouse C57BL / 6J CD4+ T cells were isolated and stimulated with CD3 / CD28 beads for 3 days before gene editing. GFP expression in the cells was assessed by flow cytometry on days 3 and 8 after gene editing.

[0169] [Figure 89] These data demonstrate homologous recombination repair-mediated gene editing within the Rosa26 locus in mouse CD4+ T cells. CD4+ T cells were gene-edited according to the method outlined in Figure 88 and assessed by flow cytometry on day 3. Panel A shows flow cytometry plots demonstrating GFP expression in mock-edited cells, cells edited with AAV No. 3245 alone, and cells edited with AAV No. 3245 and RNP 3 days after gene editing. Panel B shows histograms demonstrating viability, percentage of GFP-positive cells, and percentage of GFP-high expressing cells in the gene-edited cell population.

[0170] [Figure 90] Figure 8 shows that stable GFP expression is maintained in mouse T cells edited at the Rosa26 locus using homologous recombination repair. CD4+ T cells were gene-edited according to the method outlined in Figure 88 and assessed by flow cytometry on day 8. Panel A shows flow cytometry plots demonstrating GFP expression in mock-edited cells, cells edited with AAV No. 3245 alone, and cells edited with AAV No. 3245 and RNP 8 days after gene editing. Panel B shows a histogram showing the percentage of GFP-positive cells in the gene-edited cell population.

[0171] [Figure 91]A schematic diagram of the AAV HDR donor constructs for expressing mouse Foxp3 and P2A-linked LNGFR in the Rosa26 locus of mouse T cells is shown. These repair templates are flanked by 300-bp homology arms compatible with the R26_gRNA_1 cleavage site and contain different promoters (MND or PGK) driving the expression of mFOXP3 and LNGFR. Additionally, a cassette containing a 4x CDK phosphorylation site mutant of Foxp3 is included, which is predicted to improve Foxp3 stability.

[0172] [Figure 92] Lentiviral CISC constructs used to transduce mouse CD4+ T cells and test their selective expansion with rapalogs. Panel A shows a schematic diagram of lentiviral construct No. 1272. This construct was developed to demonstrate the concept that human CISC components can be used to enrich mouse T cells in the presence of rapalogs. Upon transduction of mouse T cells, the MND promoter drives expression of mCherry linked to IL-2 CISC components (FKBP-IL2RG and FRB-IL2RB). Panel B shows a timeline of the experimental procedure. Mouse C57BL / 6J CD4+ T cells were stimulated with CD3 / CD28 beads for 3 days before transduction. Cells were assessed for mCherry expression by flow cytometry on days 2 and 5 post-transduction.

[0173] [Figure 93]Lentivirally transduced mouse CD4+ T cells containing CISCs are highly enriched in the presence of a rapalog. Panel A shows flow cytometry plots of mCherry expression 2 days after mock transduction or lentiviral transduction (No. 1272) of mouse CD4+ T cells. Panel B shows flow cytometry plots of mCherry expression in mock mouse cells treated with IL-2, IL-7, and IL-15, or in lentiviral transduced mouse cells treated with IL-2, IL-7, and IL-15, rapalog alone, or rapalog plus bead stimulation.

[0174] [Figure 94] We show that air T cells suppress the proliferation of CD8+ T cells and CD4+ T cells.

[0175] [Figure 95] A schematic diagram of the method for generating antigen-specific airT cells by stimulating with a model antigen peptide (MP) and expressing FoxP3 by gene editing is shown.

[0176] [Figure 96] Antigen-specific suppression by air T cells specific for a model antigen peptide (MP) is shown. Briefly described below. Teff: T cells on day 23 stimulated with MP peptide (right) or HA peptide (left). Treg: T cells on day 23, gene-edited with CRISPR / Cas9 and AAV containing Foxp3-MND-LNGFRki and specific for MP peptide (right) or HA peptide (left). APC: Irradiated CD4-CD25+ autologous cells, incubated for 6 days in the presence of DMSO or 5 μg / ml HA peptide.

[0177] [Figure 97]We demonstrate that air T cells exert suppressive activity against Teff cell proliferation. Briefly described below. Both assays were incubated for 3 days. For bead-stimulated suppression assays, Teff cells were co-cultured with Treg cells (untransduced Treg cells, T1D5-1 air T cells, or T1D5-1 mock cells). For antigen-specific suppression assays, T1D5-1 Teff cells were co-cultured with Treg cells. Teff cells were gated on CD4+CD11c-CTV+EF670-mTCRb+.

[0178] [Figure 98] We demonstrate that airT cells suppress cytokine production by Teff cells. Briefly, T1D4 Teff cells and Treg cells (T1D4 mock cells or T1D4 airT cells) (day 10) were incubated for 3 days in the presence of 1 μg / ml of peptide.

[0179] [Figure 99] We demonstrate that air T cells exert antigen-specific and bystander suppressive effects on Teff cells. Briefly, 1.25 × 104 Teff cells, 2.5 × 104 Treg cells, and 1 × 105 APCs were cocultured in the presence of 5 μg / ml of peptide.

[0180] [Figure 100] We demonstrate that air T cells exert antigen-specific and bystander suppressive effects on Teff cells. Briefly, 1.25 × 104 Teff cells, 2.5 × 104 Treg cells, and 1 × 105 APCs were cocultured in the presence of 5 μg / ml of peptide.

[0181] [Figure 101]The bystander suppressive effect on cytokine production by Teff cells is shown below. Briefly, T1D5-2 Teff cells and Treg cells (T1D4 mock cells or T1D4 edTreg cells on day 10) were incubated for 3 days in the presence of 1 μg / ml of peptide.

[0182] [Figure 102] A proliferation assay to confirm the dose response of TCR is shown below. The mTCR expression data was obtained 8 days after transduction. The proliferation assay was performed using day 11 cells, incubated for 4 days.

[0183] [Figure 103] To verify the expression of TCR specific to islet antigens, mTCRβ expression assay and proliferation assay were performed. Briefly described below. T cells: Cell Trace Violet-labeled cells 9 days after transduction. APC: Irradiated CD4-CD25+ cells. Incubated for 5 days.

[0184] [Figure 104] Figure 107 shows that antigen-specific GFP+ air T cells are detectable in the pancreas. See also Figure 107 and Figure 116.

[0185] [Figure 105] See also Figure 114 for the generation and enrichment of murine LNGFR+ air T cells used in in vivo suppression studies.

[0186] [Figure 106] Figure 115, Figure 134 and Figure 135 show that antigen-specific MND.LNGFR.P2A-air T cells completely prevented diabetes in NSG mice.

[0187] [Figure 107] This shows that antigen-specific GFP+ air T cells are detectable in the pancreas.

[0188] [Figure 108] 1 shows a schematic diagram of an exemplary IL-2 CISC according to the present disclosure and data related to this IL-2 CISC.

[0189] [Figure 109] Figure 1 shows that exposure to rapamycin in vivo enhances the persistence of CISC cells.

[0190] [Figure 110] 1 shows a schematic diagram of an exemplary gene-edited cell according to the present disclosure.

[0191] [Figure 111] Regarding the selection of gRNAs targeting the TRAC locus.

[0192] [Figure 112] A dual editing approach using split IL-2 CISC constructs targeting the TRAC locus.

[0193] [Figure 113] 1 shows selection of dual-edited cells by association of CISC components in vitro.

[0194] [Figure 114] Flow cytometry analysis of NOD BDC2.5+ mouse cells edited with LNGFR.P2A before and after column purification is shown. Panel A shows flow cytometry plots demonstrating specific LNGFR expression in cells edited with MND.LNGFR.P2A (No. 3261), but not in mock cells. Panel B shows flow cytometry plots demonstrating LNGFR expression in the flow-through fraction (FT) and in the eluted sample after concentration following column purification of cells edited with MND.LNGFR.P2A (No. 3261). Column concentration yielded an airT cell product with 74.5% purity for in vivo studies.

[0195] [Figure 115] This figure shows the evaluation of islet antigen-specific airT cell function in the NSG adoptive transfer model. Islet antigen-specific (BDC) airT cells (generated using HDR donor 3261) or antigen-specific nTreg cells (50,000 each) were adoptively transferred into 8-10 week-old adult NSG recipient mice by delivery into the retro-orbital plexus (RO), followed by injection of 50,000 antigen-specific Teff cells. Panel A shows flow cytometry plots demonstrating the CD4 and CD25 expression profile of nTreg cells and LNGFR expression in cells edited with MND.LNGFR.P2A (No. 3261). Panel B shows graphs demonstrating the development of diabetes in mice monitored for up to 49 days. The graph shows the percentage of mice that developed diabetes after administration of the indicated mock-edited cells, MND.LNGFR.P2A-edited cells (column-enriched), or nTreg cells plus effector cells derived from NOD BDC2.5 mice. Column-enriched antigen-specific MND.LNGFR.P2A-air T cells completely prevented diabetes in NSG mice.

[0196] [Figure 116] This shows that islet antigen-specific MND.GFP.KI airT cells maintain viability and a stable phenotype in the target organ (pancreas) in vivo for at least 49 days. Flow cytometry plots show LNGFR and FOXP3 expression in NOD BDC2.5 airT cells harvested from the pancreas 49 days after adoptive transfer into NSG mice.

[0197] [Figure 117A] Flow cytometry plots are shown analyzing mTCRb expression gated on CD4+ cells 9 days after transduction.

[0198] [Figure 117B]Flow cytometry plots showing the results of CD4+ T cells transduced with a TCR specific for a rheumatoid arthritis antigen, labeled with CTV, and co-cultured with APCs (irradiated PBMCs) for 3 days in the presence of a peptide recognized by the TCR or DMSO.

[0199] [Figure 118B] Polyclonal and antigen-specific suppression assays using enolase-specific edTreg cells are shown.

[0200] [Figure 118C] This graph shows the results of calculating the rate of Teff cell proliferation inhibition by no Treg cells, untended Treg cells, Enol-ed Treg cells, or mock cells in the presence of anti-CD3 / CD28 (black) or APC and enolase peptide (gray) from the proliferation rates shown in Figure 118B.

[0201] [Figure 119A] Flow cytometry plots analyzing mTCRb expression in non-transduced edTreg cells or CILP297-1 edTreg cells by gating on LNGFR+Foxp3+ on edited cells that were not transduced with lentivirus or transduced with lentivirus encoding CILP297-1-TCR.

[0202] [Figure 119B] Polyclonal and antigen-specific suppression assays using CILP-specific edTreg cells are shown.

[0203] [Figure 119C]A graph showing the results of calculating the rate of inhibition of CILP Teff cell proliferation by no Treg cells, untended Treg cells, CILP edTreg cells, or mock cells in the presence of anti-CD3 / CD28 (black) or APC and CILP peptide (gray) from the proliferation rates shown in Figure 119B is shown.

[0204] [Figure 120A] Flow cytometry plots are shown analyzing mTCRb expression in non-transduced edTreg cells or Vim418 edTreg cells by gating on LNGFR+Foxp3+ on edited cells that were not transduced with lentivirus or transduced with lentivirus encoding Vim418-TCR.

[0205] [Figure 120B] Polyclonal and antigen-specific suppression assays using vimentin-specific edTreg cells are shown.

[0206] [Figure 120C] This graph shows the results of calculating the rate of inhibition of Vim Teff cell proliferation by no Treg cells, untd ed Treg cells, Vim ed Treg cells, or mock cells in the presence of anti-CD3 / CD28 (black) or APC and vimentin peptide (gray) from the proliferation rates shown in Figure 120B.

[0207] [Figure 121A] Flow cytometry plots are shown analyzing mTCRb expression in untransduced edTreg cells, Agg520 edTreg cells, or Vim418 edTreg cells by gating on LNGFR+Foxp3+ on edited cells not transduced with lentivirus, edited cells transduced with lentivirus encoding Agg520-TCR, or edited cells transduced with lentivirus encoding Vim418-TCR.

[0208] [Figure 121B] Polyclonal suppression assay using Agg520 or Vim418-specific edTreg cells or mock cells and Agg520 Teff cells is shown.

[0209] [Figure 121C] A graph showing the results of calculating the rate of inhibition of Agg520 Teff cell proliferation by no Treg cells, untd edTreg cells, Agg edTreg cells, Agg mock cells, Vim edTreg cells, or Vim mock cells from the proliferation rates shown in Figure 121B is shown.

[0210] [Figure 121D] Antigen-specific suppression assays and bystander suppression assays using Agg520- or Vim418-specific edTreg cells or mock cells and Agg520 Teff cells are shown.

[0211] [Figure 121E] This figure shows a graph illustrating the results of calculating the rate of inhibition of Agg520 Teff cell proliferation by the absence of Treg cells, edTreg cells, or mock cells from the proliferation rates shown in Figure 121D.

[0212] [Figure 122A] Flow cytometry plots are shown analyzing mTCRb expression in untransduced edTreg cells, CILP297-1 edTreg cells, or Vim418 edTreg cells by gating on LNGFR+Foxp3+ on edited cells not transduced with lentivirus, edited cells transduced with lentivirus encoding CILP297-1-TCR, or edited cells transduced with lentivirus encoding Vim418-TCR.

[0213] [Figure 122B]Polyclonal suppression assay using CILP297-1 Teff cells with edTreg cells or mock cells specific for CILP297 or Vim418 is shown.

[0214] [Figure 122C] A graph showing the results of calculating the rate of inhibition of CILP Teff cell proliferation by no Treg cells, untd edTreg cells, CILP edTreg cells, CILP mock cells, Vim edTreg cells, or Vim mock cells from the proliferation rates shown in Figure 122B is shown.

[0215] [Figure 122D] Antigen-specific suppression assays and bystander suppression assays using CILP297-1 Teff cells with CILP297- or Vim418-specific edTreg cells or mock cells are shown.

[0216] [Figure 122E] This figure shows a graph illustrating the results of calculating the rate of inhibition of CILP Teff cell proliferation by the absence of Treg cells, edTreg cells, or mock cells from the proliferation rates shown in Figure 122D.

[0217] [Figure 123A] Flow cytometry plots showing mTCRb and LNGFR / Foxp3 expression in edited cells expressing SLE3-TCR at day 7.

[0218] [Figure 123B] Polyclonal and antigen-specific suppression assays using SLE-specific edTreg cells are shown.

[0219] [Figure 124A] A schematic diagram of the AAV HDR donor constructs designed to deliver each component of split CISC to the TRAC locus using a dual-locus editing approach is shown.

[0220] [Figure 124B] A timeline of the key steps in this study, double editing of CD4+ T cells with AAV and expansion in the presence of rapalogs, is shown.

[0221] [Figure 125A] Flow cytometry plots showing the expression of T1D4 and FOXP3 in mock-edited, single-edited, and double-edited cells (using 10% by volume of AAV No. 3243 and AAV No. 3240, respectively) 3 days after gene editing.

[0222] [Figure 125B] Flow cytometry plots showing T1D4 and CD4 expression in mock-edited cells and cells edited with the virus mixture are shown.

[0223] [Figure 125C] Histograms showing the percentage of double-negative cells, FOXP3-HA-positive cells, T1D4-positive cells, and FOXP3 / T1D4 double-positive cells in double-edited cells are shown.

[0224] [Figure 125D] A histogram comparing the CD3 knockout rate between FOXP3 / T1D4 double-edited and mock-edited cells is shown.

[0225] [Figure 126A] Flow cytometry plots showing viability and T1D4 and FOXP3 expression after treatment of dual-edited cells with 50 ng / mL IL-2 (upper panel) or 100 nM rapalog (AP21967; lower panel) for 7 days are shown.

[0226] [Figure 126B]Flow cytometry plots comparing CTLA4 expression in T1D4 / FOXP3 double-positive and double-negative cell populations after treatment with 50 ng / mL IL-2 (upper panel) or 100 nM rapalog (AP21967; lower panel) for 7 days are shown.

[0227] [Figure 127A] Flow cytometry plots comparing viability (right plots) and T1D4 and FOXP3 expression (left plots) in double-edited cells treated with 50 ng / mL IL-2 (upper plots) and double-edited cells treated with 100 nM AP21967 and then recovered in medium containing IL-2 (lower plots).

[0228] [Figure 127B] A graph showing the fold enrichment over 10 days of T1D4 / FOXP3 double positive cells treated with 50 ng / mL IL-2 or 100 nM rapalog (AP21967) and allowed to recover in recovery medium containing IL-2 for the last 3 days.

[0229] [Figure 128A] A schematic diagram of the HDR knock-in construct (No. 3280) containing a split IL-2 DISC for selection of double-edited cells with rapamycin or a rapalog is shown.

[0230] [Figure 128B] A timeline of the key steps in this study, double editing of CD4+ T cells using AAV No. 3280 and AAV No. 3207, expansion in the presence of a rapalog / rapamycin, and analysis of the enriched cells, is shown.

[0231] [Figure 129A]Flow cytometry plots showing mCherry and GFP expression in double-edited cells (AAV donor No. 3280 and AAV donor No. 3207 were added at 10% of the medium volume) 4 days after gene editing. The viral titer of No. 3280 was 3.30 x 1012, and the viral titer of No. 3207 was 3.1 x 1010. The initial double-positive rate across 4 million double-edited cells was 4.47%. A total of 7.6 million cells were seeded into gRex flasks, of which 340,000 were double-positive.

[0232] [Figure 129B] Flow cytometry plots showing viability (top panel) and GFP and mCherry expression (bottom panel) after 7 days of expansion in the presence of AP21967 in a gREX flask containing 7.6 million edited cells, demonstrating a 32-fold expansion of double-positive cells. The total number of double-positive cells in the gRex flask was 11.1 million.

[0233] [Figure 130A] A timeline of the steps for dual editing of CD4+ T cells with AAV No. 3280 and AAV No. 3207, expansion in the presence of rapalogs, and analysis of enriched cells is shown.

[0234] [Figure 130B] Flow cytometry plots showing mCherry and GFP expression in double-edited cells (AAV No. 3280 and AAV No. 3207 were each added at 10% concentration). The viral titer of No. 3280 (MND.mCherry.FKBP.IL2RG.FRB) was 3.30 x 1012, and the viral titer of No. 3207 (pAAV.MND.GFP.FRB.IL2RB) was 3.1 x 1010. A total of 10 million cells were gene-edited, resulting in an initial double-positive rate of 2.37%. 9.1 million cells were seeded into gRex flasks, of which 216,000 were double-positive.

[0235] [Figure 131] Flow cytometry plots showing viability and GFP and mCherry expression after 7 days of expansion of gene-edited cells in gREX flasks in the presence of AP21967. After 7 days of expansion, the total number of double-positive cells in the gRex flask reached 9.7 million, representing a ~45-fold increase from the initial 216,000 double-positive cells seeded.

[0236] [Figure 132A] 1 shows the design of an in vitro suppression assay using mouse edTreg cells or mouse nTreg cells.

[0237] [Figure 132B] Representative flow cytometry data are shown demonstrating reduced proliferation of BDC2.5+Teff cells in the presence of BDC2.5+edTreg cells.

[0238] [Figure 133] Flow cytometry plots show Cell Trace Violet-labeled CD4+ T cells from NOD BDC2.5+ mice in the presence or absence of mock, MND.LNGFR.p2A (No. 3261)-edited Treg, or nTreg cells. Islet antigen-specific TCR+ murine edTreg cells (generated using a MND.LNGFR p2A (No. 3261) HDR donor) and murine tTreg cells demonstrated antigen-specific suppressive function in vitro. 50,000 Teff cells + anti-CD3 (1 μg / ml) + 200,000 irradiated (2500 rad) APCs were used. Analysis was performed on day 4. CTV = Cell Trace Violet. Data shown are from an experiment performed at a 1:1 Treg:Teff ratio.

[0239] [Figure 134](A) Graph showing the percentage of mice that developed diabetes after administration of the indicated mock-edited, MND.LNGFR.P2A-edited, or nTreg cells from NOD BDC2.5 mice in combination with effector cells. MND.LNGFR p2A-edTreg cells, like nTreg cells, completely prevented the onset of diabetes, whereas mock-edited control cells had no effect on diabetes development.

[0240] [Figure 135] Figure 1 shows the percentage of mice that developed diabetes after administration of mock-edited, MND.LNGFR.P2A-edited, or nTreg cells from NOD BDC2.5 mice with effector cells in a repeat experiment. Column-enriched antigen-specific LNGFR p2A edTreg cells completely prevented diabetes in NSG mice (day 33).

[0241] [Figure 136] Figures 136A, 136B and 136C show tables listing the amino acid sequences of the CDR3 and J regions of the α and β chains of TCRs that specifically recognize antigens associated with the pathogenesis of autoimmune, allergic or inflammatory diseases.

[0242] [Figure 137] The table shows a list of amino acid sequences of the CDR3 and J regions of the α and β chains of TCRs that specifically recognize antigens involved in the pathogenesis of autoimmune, allergic, or inflammatory diseases.

[0243] [Figure 138] 1 shows a table listing the amino acid sequences of the J regions of TCRs that specifically recognize antigens involved in the pathogenesis of autoimmune, allergic, or inflammatory diseases.

[0244] [Figure 139A]A table listing nucleotide sequences encoding the V regions of the α and β chains of TCRs that specifically recognize antigens involved in the pathogenesis of autoimmune, allergic, or inflammatory diseases is shown.

[0245] [Figure 139B] 1 shows a table listing the amino acid sequences of the J regions of TCRs that specifically recognize antigens involved in the pathogenesis of autoimmune, allergic, or inflammatory diseases.

[0246] [Figure 140A] A table listing nucleotide sequences encoding the V regions of the α and β chains of TCRs that specifically recognize antigens involved in the pathogenesis of autoimmune, allergic, or inflammatory diseases is shown.

[0247] [Figure 140B] 1 shows a table listing the amino acid sequences of the J regions of the α and β chains of TCRs that specifically recognize antigens involved in the pathogenesis of autoimmune, allergic, or inflammatory diseases.

[0248] [Figure 141] 1 shows a table listing the amino acid sequences of antigenic epitopes recognized by TCRs specific to the CYP2D6 antigen associated with type 2 autoimmune hepatitis.

[0249] [Figure 142] 1 shows a table listing the amino acid sequences of antigenic epitopes recognized by TCRs specific to the BP230 antigen or BP180 antigen associated with bullous pemphigoid.

[0250] [Figure 143A] This table shows a list of amino acid sequences of polypeptide antigens that are involved in the pathogenesis of autoimmune, allergic, and / or inflammatory diseases and contain antigenic epitopes recognized by specific TCRs, as well as the amino acid sequences of the CDR3 regions of the α and β chains of TCRs that specifically recognize these antigens.

[0251] [Figure 143B] This table shows a list of amino acid sequences of polypeptide antigens that are involved in the pathogenesis of autoimmune, allergic, and / or inflammatory diseases and contain antigenic epitopes recognized by specific TCRs, as well as the amino acid sequences of the CDR3 regions of the α and β chains of TCRs that specifically recognize these antigens.

[0252] [Figure 144] This table shows a list of amino acid sequences of polypeptide antigens that are involved in the pathogenesis of autoimmune, allergic, or inflammatory diseases and contain antigenic epitopes recognized by specific TCRs, as well as the amino acid sequences of the CDR3 regions of the α and β chains of TCRs that specifically recognize these antigens.

[0253] [Figure 145] A table listing certain nucleic acid sequences useful in the embodiments provided herein, including guide RNAs (gRNAs) and AAV vectors containing sequences for editing FOXP3, is provided.

[0254] [Figure 146] (Panel A) Schematic map of the FOXP3 knock-in construct (3324) containing elements encoding the FKBP-IL2RG and FRB polypeptides. (Panel B) Schematic map of the TRAC-targeting HDR construct (3243) containing elements encoding the T1D4 and FRB-IL2B polypeptides. (Panel C) Schematic map of the TRAC-targeting HDR construct (3333) containing elements encoding the T1D4 and FRB-IL2Bmin polypeptides containing a truncated intracellular IL-2 receptor β signaling domain.

[0255] [Figure 147] FACS analysis of CD4+ T cells transduced with a combination of 3333 and 3324 constructs (A), a combination of 3243 and 3324 constructs (B), or a control (C) is shown.

[0256] [Figure 148] (Panel A) CD4+ T cells transduced with the combination of the 3333 and 3324 constructs (top row) or the combination of the 3243 and 3324 constructs (bottom row) were treated with 100 nM AP21967 (rapalog) for 7 days and then subjected to FACS analysis. Comparison of cell viability and cell size (FSC-A), T1D4 expression levels versus CD3 expression levels, or T1D4 expression levels versus HA-tagged FOXP3 expression levels are shown. (Panel B) Graph showing the fold enrichment of double-positive cells in transduced CD4+ T cells.

[0257] [Figure 149] (Top) Schematic map of another TRAC-targeting HDR construct in which the μCISC component is placed adjacent to the MND promoter. (Bottom) Schematic map of another TRAC-targeting HDR construct in which the TCR (T1D4) component is placed adjacent to the MND promoter.

[0258] [Figure 150A] (Panel A) Graph showing the fold enrichment of double-positive cells when CD4+ T cells transduced with a combination of 3324 and 3323 constructs were treated with 50 ng / mL IL-2, 100 nM AP21967, or 10 nM rapamycin for 7 days. (Panel B) Graph showing the fold enrichment of double-positive cells when CD4+ T cells transduced with a combination of 3324 and 3333 constructs were treated with 50 ng / mL IL-2, 100 nM AP21967, or 10 nM rapamycin for 7 days.

[0259] [Figure 150B]A schematic map showing several constructs (3323 and 3354) in which the sequence encoding truncated FRB-IL2RB is positioned close to the MND promoter, and a construct (3234) in which the sequence encoding truncated FRB-IL2RB is positioned distal to the MND promoter by inserting a sequence encoding a T1D4 TCR polypeptide between the sequence encoding truncated FRB-IL2RB and the MND promoter.

[0260] [Figure 150C] Figure 150B shows FACS analysis of cells transduced with various constructs, including the combination of FOX3P targeting constructs (3324) and 3323, FOX3P targeting constructs (3324) and 3354, and FOX3P targeting constructs (3324) and 3243. The enrichment ability of these different TRAC targeting constructs, different from those in the previous examples, was compared over an 8-day period using 100 nM AP21967 (a rapalog) or 10 nM rapamycin.

[0261] [Figure 150D] A comparison of absolute and fold enrichment graphs is shown for the various double-edited groups shown in Figures 150B and 150C.

[0262] [Figure 151] (Panel A) Schematic map of CISC constructs containing the MND promoter (1272) or the EF1a promoter (3312). (Panel B) FACS analysis comparing mCherry signal and cell size in CD4+ T cells transduced with the 1272 or 3312 constructs.

[0263] [Figure 152](Panel A) FACS analysis comparing viability and cell size (FSC-A) and mCherry and cell size after 7 days of culture of CD4+ T cells transduced with the 1272 or 3312 construct in the presence of 100 nM AP21967 (rapalog). (Panel B) Graph showing fold enrichment of mCherry double-positive cells over 7 days after treatment of CD4+ T cells transduced with the 1272 or 3312 construct with 100 nM AP21967 (rapalog).

[0264] [Figure 153] (Panel A) Steps and timeline for expanding islet-specific T cells by peptide stimulation. (Panel B) Representative FACS analysis of tetramer-positive T cells specific for individual antigenic peptides. (Panel C) Bar graph showing the mean percentage of the tetramer-positive population among CD4+ T cells measured after 12-14 days of peptide stimulation in vitro. Each bar represents the percentage of CD4+ T cells specific for each islet antigenic peptide, and each dot represents a different experiment.

[0265] [Fig. 154] (Panel A) Timeline of steps for generating islet-specific edTregs, polyclonal islet-specific Teffs, and monocyte-derived DCs (mDCs) from PBMCs and performing an in vitro suppression assay. (Panel B) Histogram showing the proliferation of polyclonal islet-specific Teffs in an antigen-specific suppression assay. (Panel C) Bar graph showing the percentage of polyclonal islet-specific Teff proliferation suppressed by polyclonal edTregs, T1D2 mock, T1D2 edTregs, 4.13 mock, or 4.13 edTregs in the presence of a pool of nine islet-specific peptides and mDCs. The percentage of proliferation suppression was calculated using the following formula: (% proliferation in the absence of Tregs - % proliferation in the presence of edTregs) / % proliferation in the absence of Tregs × 100. **P<0.001 and *P<0.05 were calculated by paired t-test.

[0266] [Figure 155] (Panel A) A graph showing the proliferation of T cells expressing T1D2 TCR, T1D5-1 TCR, or T1D5-2 TCR. (Panel B) A table listing the peptide specificities and avidities of T1D2 TCR, T1D5-1 TCR, and T1D5-2 TCR. (Panel C) A histogram showing the proliferation of T1D5-2 Teffs in an antigen-specific suppression assay using T1D2 edTregs or T1D5-1 edTregs. (Panel D) A bar graph showing the rate of suppression of T1D5-2 Teff proliferation by T1D2 edTregs or T1D5-1 edTregs in an antigen-specific suppression assay. (Panel E) A histogram showing the proliferation of polyclonal islet-specific Teffs in an antigen-specific suppression assay. (Panel F) Bar graph showing the percent inhibition of polyclonal islet-specific Teff proliferation by T1D2 edTregs or T1D5-1 edTregs in the presence of a pool of nine islet-specific peptides and mDCs.

[0267] [Figure 156A](Panel A) Graph showing proliferation of T cells expressing islet-specific TCRs. (Panel B) Table summarizing the peptide specificity and avidity of islet-specific TCRs. (Panel C) Histogram (left) showing proliferation of polyclonal islet-specific Teffs in an antigen-specific suppression assay using T1D2 edTregs or 4.13 edTregs, and bar graph (right) showing the percentage of suppression of polyclonal islet-specific Teff proliferation by T1D2 edTregs or 4.13 edTregs in the presence of a pool of nine islet-specific peptides and mDCs. These results were obtained from three independent experiments using cells generated from three type 1 diabetic donors. Statistical significance was determined by paired t-test. (Panel D) Histograms (left) show the proliferation of polyclonal islet-specific Teffs in an antigen-specific suppression assay using T1D2 edTregs, GAD113 edTregs, or PPI edTregs, and bar graphs (right) show the percent inhibition of polyclonal islet-specific Teff proliferation by T1D2 edTregs, GAD113 edTregs, or PPI edTregs. (Panel E) Histograms (left) show the proliferation of polyclonal islet-specific Teffs in an antigen-specific suppression assay using T1D2 edTregs, T1D4 edTregs, or T1D5-2 edTregs, and bar graphs (right) show the percent inhibition of polyclonal islet-specific Teff proliferation by T1D2 edTregs, T1D4 edTregs, or T1D5-2 edTregs.

[0268] [Figure 156B] (Panel A) A graph showing the proliferation of T cells expressing islet-specific TCRs. (Panel B) A table summarizing the peptide specificity and avidity of islet-specific TCRs.

[0269] [Figure 157] (Panel A) Schematic diagram showing the generation of edTreg cells expressing islet-specific TCR. (Panel B) Summary of the methods used to evaluate antigen-specific suppression assay.

[0270] [Figure 158] T1D5-1 TCR-transduced CD4+ T cells (T1D5-1 Teff) were labeled with CTV and co-cultured with EF670-labeled untransduced edTregs (untd edTregs), EF670-labeled T1D5-1 TCR-expressing edTregs (T1D5-1 edTregs), or EF670-labeled T1D5-1 mock cells in the presence of CD3 / CD28 activation beads. Histograms show FACS analysis of Teff cells. The top histogram shows Teff proliferation in response to CD3 / CD28 bead activation, and the bottom histogram shows antigen-specific Teff proliferation.

[0271] [Figure 159] (Left panel) Histogram showing proliferation of Teffs gated on CD3+CD4+CTV+EF670-LNGFR-. (Right panel) Graph showing proliferation rate of T1D4 Teff cells cultured without edTregs or co-cultured with T1D4 edTregs or T1D5-1 edTregs in the presence of APC and IGRP 241+IGRP 305 peptide.

[0272] [Figure 160] FACS analysis (left panel) and graph (right panel) of TNF, IL-2 or IFN-γ production and CD25 expression in T1D4 Teff cells gated on CD4+CTV+EF670- are shown.

[0273] [Figure 161] FACS analysis (top row) and graphs (bottom row) of TNF, IL-2, or IFN-γ production and CD25 expression in bystander T1D5-2 Teff cells gated on CD4+CTV+EF670-.

[0274] [Figure 162](Panel A) Histograms showing Teff proliferation in CTV-labeled T1D5-2 or T1D4 Teffs cocultured with EF670-labeled T1D5-1 or T1D5-2 edTregs, or without edTregs, in the presence of IGRP 305 peptide, IGRP 241 peptide, or IGRP 241 + IGRP 305 peptide and APC. (Panel B) Graphs showing the proliferation rates of T1D5-1 Teffs (top) and T1D4 Teffs (bottom). (Panel C) Graphs showing the production rates of TNF and IL-2 by T1D5-2 Teffs (left) and T1D4 Teffs (right).

[0275] [Figure 163] (Panel A) Histograms showing Teff proliferation in response to CD3 / CD28 bead activation. (Panel B) Histograms showing islet-specific T cell proliferation in the presence of a pool of nine islet-specific peptides or DMSO and monocyte-derived DCs when islet-specific T cells were cultured for 4 days without Tregs or co-cultured with untransduced (untd)edTregs, T1D2 edTregs, or T1D2 mock cells for 4 days.

[0276] [Fig. 164]Figure 164A shows a schematic map illustrating (Panel A) a full-length CISC TRAC hijack construct (3354) containing elements encoding the FRB-IL2RB AA237-551 polypeptide, elements encoding the full-length TCRb polypeptide, and elements encoding the TRAV / TRAJ polypeptide; (Panel B) a CISC component swap TRAC hijack construct (3363) containing elements encoding the FKBP-IL2RG polypeptide, elements encoding the full-length TCRb polypeptide, and elements encoding the TRAV / TRAJ polypeptide; and (Panel C) a CISC component swap FOXP3 construct (3362) containing elements encoding the FRB-IL2RB AA237-551 polypeptide and elements encoding the FRB polypeptide. Figure 164B shows the gene editing efficiency of T cells when endogenous TCR expression was disrupted using the 3363 and 3362 constructs, exogenous TCRb polypeptides and exogenous TRAV / TRAJ polypeptides were introduced, stable FOXP3 expression was promoted, and FKBP-IL2RG polypeptide, FRB-IL2RB polypeptide, and FRB polypeptide were expressed. Figure 164C shows the time course of the frequency of double-positive cells expressing FOXP3 and exogenous TCRb after incubation with rapamycin.

[0277] [Figure 165] Schematic maps showing (Panel A) the T1D4 full-length CDS CISC order-swap construct (3364) containing elements encoding the FKBP-IL2RG polypeptide and elements encoding the T1D4 polypeptide; and (Panel B) the A2-CAR CISC construct containing elements encoding the FRB-IL2RB AA237-551 polypeptide and elements encoding the A2-CAR polypeptide.

[0278] [Figure 166]Figure 166A shows how ex vivo expanded natural autologous Treg cells or umbilical cord blood (UCB)-derived allogeneic Treg cells can be induced and delivered to a subject as Treg therapy. Figure 166B shows the introduction of a FOXP3 expression cassette driven by the MND promoter at the FOXP3 locus via homologous recombination repair.

[0279] [Figure 167] This figure shows the optimization of the expansion of cord blood-derived EngTreg cells generated by homologous recombination repair-mediated gene editing. Panel A shows the experimental timeline for gene editing of CD4+ cells, followed by expansion and confirmation of gene editing of the resulting EngTreg cells expressing FOXP3 cDNA and the LNGFR marker. Panel B shows the expansion of cord blood-derived CD4+ T cells (n = 6) and PBMC-derived CD4+ T cells (n = 5) from Days 0 to 4 shown in Panel A. Cells were stimulated with CD3 / CD28 Dynabeads for 3 days, then removed and cultured for an additional 16 hours. Significance was determined by unpaired t-test. Panel C shows a comparison of the editing efficiency of cord blood-derived cells (n = 6) and PBMC-derived cells (n = 5). After RNP delivery using a Lonza 4D nucleofector, 20% v / v AAV was added to the cell culture. Two days after gene editing, homologous recombination repair efficiency was assessed by flow cytometry. Gene-edited cells are shown as the percentage of live singlet CD4 cells staining positive for LNGFR. Significance was determined by unpaired t-test. Panel D shows the expansion of cord blood-derived EngTregs and peripheral blood-derived EngTregs enriched with LNGFR and cultured for 7 days in the G-rex device. Panel E shows the effect of IL-2 concentration and culture device on the fold expansion of cord blood-derived EngTregs enriched with LNGFR and cultured for 7 days. Panel F shows the viability of cord blood-derived EngTregs cultured in different devices.

[0280] [Figure 168]These data demonstrate that cord blood-derived EngTregs exhibit a Treg phenotype and reduced production of proinflammatory cytokines. Panel A shows representative histograms depicting selected markers expressed in cord blood-derived EngTregs after 7 days of expansion. Panel B shows cytokine production from cord blood-derived LNGFR+ EngTregs (n=5) compared to mock-edited cells. After resting, cells were stimulated with PMA and ionomycin in the presence of Golgi-stop for 5 hours, and intracellular cytokine production was analyzed by flow cytometry.

[0281] [Figure 169] These results demonstrate that cord blood-derived EngTregs suppress allogeneic T cells and protect against graft-versus-host disease (GvHD). Panel A shows the timeline and study design of an in vivo study comparing the suppression of allogeneic Teffs by PBMC-derived EngTregs with that by cord blood-derived EngTregs in NSG mice. Panel B shows the survival curve for the duration of this in vivo study. A 20% weight loss was set as the humane study endpoint. Panel C shows the change in weight of each mouse from D0 to the study endpoint.

[0282] [Figure 170]Figure 170A shows the mechanism by which CISC activity is regulated by the presence or absence of rapamycin. Figure 170B shows the CISC AAV donor template introduced upstream of the endogenous FOXP3 gene. Figure 170C shows the relative MFI values ​​of the indicated Treg markers in umbilical cord blood-derived EngTregs compared to mock-edited cord blood-derived CD4+ cells as a baseline. Figure 170D shows cytokine production from umbilical cord blood-derived CISC-expressing EngTregs expanded in tissue culture plates compared to mock-edited cells. Cells were treated with PMA, ionomycin, and Golgi-stop for 5 hours, followed by intracellular staining for the indicated cytokines. Figure 170E shows flow cytometry plots demonstrating that umbilical cord blood-derived EngTregs expressing CISC express FOXP3 and P2A, indicating that initial translation produces each CISC component separated by the P2A self-cleavage motif. Figure 170F shows the percentage of cord blood-derived CD4+ cells that expressed CISC at day 3, demonstrates expansion upon exposure to rapamycin, and shows editing efficiency at day 17. Figure 170G shows the fold expansion upon treatment of cord blood-derived mock-edited EngTregs or cord blood-derived CISC-expressing EngTregs with rapamycin.

[0283] [Figure 171] 1 shows the timeline and procedures for scaling up the production of cord blood-derived EngTregs.

[0284] [Fig. 172]Figure 172A shows the immunophenotype of cord blood-derived LNGFR+ EngTregs, with representative histograms depicting selected markers expressed in cord blood-derived EngTregs obtained from two donors after 7 days of expansion. Figure 172B shows bar graphs comparing cytokine production from cord blood-derived LNGFR+ EngTregs obtained from two donors with mock-edited cells. After resting the cells, they were stimulated with PMA and ionomycin in the presence of Golgi-stop for 5 hours, and intracellular cytokine production was analyzed by flow cytometry.

[0285] [Figure 173] Figure 173A shows an in vivo evaluation of the suppressive capacity of cord blood-derived EngTregs against allogeneic effector T cells (Teffs) in a xenogeneic GvHD mouse model. The figure shows the timeline and study design for an in vivo study comparing the suppression of allogeneic Teffs by PBMC-derived EngTregs with that by cord blood-derived EngTregs in NSG mice. Figure 173B shows survival curves for the duration of this in vivo study. A 20% weight loss was set as the humane study endpoint. This graph represents the combined data for EngTregs obtained from two cord blood donors. Figure 173C shows the change in body weight for each mouse from DO to the study endpoint. This graph represents the combined data for EngTregs obtained from two donors.

[0286] [Fig. 174] Survival curves for NSG mice are shown, combining results from xenogeneic GvHD studies. Data shown represent the combined results of two cord blood-derived cell preparations versus each of three peripheral blood-derived allogeneic CD4 Teffs. Peripheral blood-derived autologous EngTregs and mock-edited cells served as controls. P values ​​were calculated using the log-rank test (Mantel-Cox test).

[0287] [Figure 175]Figure 175A shows that gene editing of CD8+ T cells can induce FOXP3 expression and convert them to Treg characteristics. A schematic diagram of the process for generating EngTregs expressing MND-driven IL-2 CISC by editing the FOXP3 gene in T cells is shown. T cells were electroporated with a Cas9-containing RNP complex and a FOXP3-specific gRNA, followed by AAV transduction. Homologous recombination repair resulted in alleles expressing MND-driven IL-2 CISC and endogenous FOXP3. Figure 175B shows the main steps and timeline for generating IL-2 CISC EngTregs. CD3+ T cells were isolated from healthy donors, gene-edited, and then enriched and expanded. Figure 175C shows a bar graph showing gene editing efficiency analyzed 2 days after gene editing. The percentage of FOXP3+ cells was analyzed in the CD4+ and CD8+ subsets of live singlets. Figure 175D shows the percentage of FOXP3+ cells analyzed after the enrichment and expansion process on day 16 shown in panel B. Figure 175E shows histograms of the indicated Treg markers in the CD4+ and CD8+ subsets in the resulting cell product. Figure 175F (left panel) shows the results of flow cytometry analysis of the intracellular expression of the indicated cytokines in CD8+ mock-edited cells (top row) or CD8+ EngTregs (bottom row) in response to stimulation with PMA and ionomycin. Figure 175F (right panel) shows a graph showing the relative positivity of the cytokines compared to mock-edited cells. Figure 175G shows cell trace violet (CTV)-labeled CD4+ (left graph) or CD8+ (right graph) autologous responder cells were treated with CD3 / CD28 T cell activation beads at a bead to responder cell ratio of 10:1, and then cocultured with CD4+ or CD8+ CISC-EngTregs or their corresponding mock-edited cells at the ratios indicated. After 96 hours of incubation, proliferation of CD4+ or CD8+ responder cells was analyzed by flow cytometry.Inhibition rate (%) = [(proliferation rate (%) of responder cells without addition of suppressor cells) - (proliferation rate (%) of responder cells with addition of suppressor cells)] / (proliferation rate (%) of responder cells without addition of suppressor cells)] × 100.

[0288] [Figure 176] FACS analysis of CD8+ T cells and CD8+ CISC EngTregs generated from purified CD8+ cells is shown. FACS analysis of CD8+ CISC EngTregs shows editing efficiency 2 days after gene editing.

[0289] [Figure 177] Figure 177A shows CD8 CISC EngTregs after rapamycin enrichment and expansion, showing the time course of the percentage of FOXP3+ cells. Figure 177B shows FACS analysis.

[0290] [Figure 178] 1 shows FACS analysis of the immunophenotype of CD8 CISC EngTregs.

[0291] [Figure 179] 1 shows FACS analysis of cytokine production by CD8 CISC EngTregs.

[0292] [Figure 180A] 1 shows the timeline for manufacturing A2CAR EngTreg.

[0293] [Fig. 180B-D] Figure 180B shows the LV.A2CAR.P2A.LNGFR construct (LV3350) and the AAV3195 construct. Figure 180C shows a table summarizing groups 1-9 for generating various cells and a schematic diagram of the 3362 and 3407 constructs. Figure 180D shows FACS analysis of the recombinant cells.

[0294] [Figure 181]Gene editing in CD3 cells. Homologous recombination repair was detected by FACS analysis 3 days after gene editing.

[0295] [Figure 182] Gene editing in CD3 cells is shown. Homologous recombination repair was detected by FACS analysis in CD4 and CD8 subsets.

[0296] [Figure 183] Gene editing in CD8 cells is shown. Homologous recombination repair was analyzed by FACS analysis.

[0297] [Figure 184] Figure 184A shows dual editing of TRAC / FOXP3 using split CISC and A2CAR, showing constructs 3362 and 3407. Figure 185B shows FACS analysis of cells recombined with the two constructs shown in Figure 184A.

[0298] [Figure 185] Figure 184A shows FACS analysis of FOXP3 double-edited cells using split CISC and A2CAR, which contain the 3362 and 3407 constructs shown in Figure 184A.

[0299] [Figure 186] Figure 186A shows the pRRL_MND.A2CAR.PA2.LNGFR construct for TCR null editing of LVA2CAR.CISC EngTreg. Figure 186B shows FACS analysis of cells recombined with the construct shown in Figure 186A.

[0300] [Figure 187] 1 shows FACS analysis of enrichment of A2CAR+ cells by affinity selection for LNGFR.

[0301] [Figure 188]An example of an in vivo study, its timeline, and experimental groups is shown. This study will be performed similarly to the in vivo CD4 A2CAR study disclosed herein. Mice will be irradiated and then co-injected with EngTregs and PBMCs the following day. EngTregs and PBMCs will be mixed immediately prior to injection. If CD3-derived cells are used, CD4 and CD8 cells will be isolated prior to injection. The ratio of EngTregs to PBMCs in the mouse groups will be 1:1, 2:1, or 4:1. It is proposed to use R003791 and R003798 as donors for A2+ PBMCs in the first and second experiments, respectively, and to use the same donors for the CD4 A2CAR LNGFR study.

[0302] [Figure 189] A table showing the sequences of various constructs encoding A2 CAR is provided.

[0303] [Figure 190A] This figure shows the generation of islet-specific EngTregs by homologous recombination repair-mediated FOXP3 gene editing and lentiviral TCR transduction. A timeline of the major steps for generating and enriching islet-specific EngTregs from primary human CD4+ T cells is shown. On day 0, T cells were activated with CD3 / CD28 beads, and on day 1, they were transduced with a lentiviral vector (encoding an islet-specific TCR). On day 7, islet-specific TCR and Treg marker expression (mTCR, CD25, CD127, CTLA-4, and ICOS) was assessed by flow cytometry. On day 10, islet-specific EngTregs were enriched with LNGFR magnetic beads.

[0304] [Figure 190B] A schematic diagram of the FOXP3 locus is shown (top). Exons are boxed. An AAV6 donor template (bottom) was designed to accommodate the insertion of the MND promoter, truncated LNGFR coding sequence, and P2A (2A) sequence. If gene editing is successful, the MND promoter will drive expression of LNGFR and FOXP3.

[0305] [Figure 190C] Representative flow cytometry plots (day 7, 4 days after gene editing) are shown. The left panel shows co-expression of FOXP3 and LNGFR in gene-edited cells. From left to right, the panels show expression of mTCR, CD25, CD127, CTLA-4, and ICOS gated on LNGFR+FOXP3+ cells.

[0306] [Figure 190D] Representative flow cytometry plots (day 10, 7 days after gene editing) are shown, demonstrating the purity of LNGFR+ cells after enrichment with anti-LNGFR magnetic beads. During enrichment of LNGFR+ cells, LNGFR- T cells were also collected and used as a control for in vitro suppression assays.

[0307] [Figure 190E] This figure shows the expression of TCR in T cells transduced with islet TCRs and the antigen-specific proliferation of these T cells. A schematic diagram showing the structure of lentiviral islet-specific TCRs containing the variable regions of human islet-specific TCRs (huV-α and huV-β) and the constant regions of mouse TCRs (muV-α and muV-β) is shown.

[0308] [Figure 190F] This figure shows confirmation of islet-specific TCR expression in human CD4+ T cells transduced with islet-specific TCRs. CD4+ T cells were isolated, activated with CD3 / CD28 beads, and transduced with lentiviruses encoding islet-specific TCRs. Flow cytometry plots show the results of analyzing mTCR expression in CD4+ T cells 7 days after transduction using an antibody specific for the murine TCR constant region.

[0309] [Figure 190G]The proliferation of CD4+ T cells transduced with each islet TCR in the presence of peptides recognized by each islet TCR and APCs is shown. CD4+ T cells transduced with each TCR were labeled with cell trace violet and then co-cultured with peptides recognized by each TCR (or an irrelevant peptide) and APCs (irradiated PBMCs) for 4 days. Flow cytometry plots show cell proliferation measured as a function of CTV dilution.

[0310] [Figure 191A] This shows that islet-specific EngTregs suppress antigen-induced Teff proliferation. A schematic diagram showing direct suppression of Teffs by EngTregs with the same islet antigen specificity as Teffs is shown. Both EngTregs and Teffs shown in this figure express the T1D5-2 TCR specific for IGRP305-324.

[0311] [Figure 191B] Representative histograms show proliferation (measured by CTV dilution) of T1D5-2 Teffs cocultured with EF670-labeled EngTregs or control EngTregs in the presence of anti-CD3 / CD28 antibody-coated beads (top) or a TCR-recognized peptide (IGRP305-324) and APC (bottom). Each histogram was gated on EF670- cells.

[0312] [Figure 191C] The percentage of suppression of CD3 / CD28 bead-induced Teff proliferation by polyEngTregs, LNGFR-T cells, or islet-specific EngTregs bearing T1D5-2 TCR (left), PPI76 TCR (center), or GAD65 TCR (right) is shown.

[0313] [Figure 191D]Figures 191C and 191D show the rate of inhibition of antigen-induced Teff proliferation by poly-EngTregs, LNGFR-T cells, or islet-specific EngTregs bearing T1D5-2 TCR (left), PPI76 TCR (center), or GAD65 TCR (right). The peptide recognized by T1D5-2 TCR was IGRP305-324, the peptide recognized by PPI76 TCR was PPI76-90, and the peptide recognized by GAD65 TCR was GAD65265-284. In Figures 191C and 191D, data are shown as the mean ± SD of three independent experiments using cells generated from three healthy donors. P values ​​were calculated using a paired two-tailed Student's t-test (*P<0.05 and **P<0.01).

[0314] [Figure 191E] The main steps and timeline for the generation of islet-specific EngTregs and islet-specific Teffs and in vitro suppression assays are shown. CD4+ T cells were activated with CD3 / CD28 beads and then transduced with TCR to generate Teffs. Teffs were expanded and harvested on day 15. The procedure for generating EngTregs is shown in Figure 190A. Teffs were cocultured with EngTregs or LNGFR-T cells in the presence of APCs (irradiated autologous PBMCs) and various peptides, or in the presence of CD3 / CD28 beads, or without these T cells. Before coculture, Teffs were labeled with cell trace violet (CTV), and EngTregs and LNGFR-T cells were labeled with EF670. After 3 or 4 days of incubation, cells were harvested, stained, and analyzed by flow cytometry.

[0315] [Figure 192A]This shows that islet-specific EngTregs suppress antigen-induced cytokine production from Teffs. Representative flow cytometry plots showing cytokine production from Teffs (TNF-α, IL-2, and IFN-γ) and Teff activation (CD25 expression) in an antigen-specific suppression assay are shown. T1D5-2 Teffs were cultured alone or co-cultured with polyclonal EngTregs, LNGFR-T cells, or T1D5-2 EngTregs in the presence of IGRP305-324, a peptide recognized by the T1D5-2 TCR, and APC.

[0316] [Figure 192B] The percentage of suppression of antigen-induced TNFα (left), IL-2 (center), and IFNγ (right) production from T1D5-2 Teffs by polyEngTregs, LNGFR-T cells, or islet-specific T1D5-2 EngTregs is shown.

[0317] [Figure 192C] Figure 192B and Figure 192C show the suppression rate of antigen-induced CD25 expression from T1D5-2 Teffs by poly-EngTregs, LNGFR-T cells, or islet-specific T1D5-2 EngTregs. In Figure 192B and Figure 192C, data are shown as the mean ± SD of four independent experiments using cells prepared from four healthy donors. P values ​​were calculated using a paired two-tailed Student's t-test (*P<0.05, **P<0.01, and ***P<0.001).

[0318] [Figure 193A] This shows that islet-specific EngTregs suppress the proliferation of bystander Teffs. A schematic diagram showing the bystander suppressive effect of EngTregs with different islet antigen specificities on Teffs is shown. This diagram shows EngTregs expressing the T1D4 TCR specific for IGRP241-260 and Teffs expressing the T1D5-2 TCR specific for IGRP305-324.

[0319] [Figure 193B] Representative histograms show proliferation (measured by CTV dilution) of T1D5-2 Teffs cocultured with T1D5-2 EngTregs, T1D4 EngTregs, or poly-EngTregs in the presence of IGRP305-324 peptide alone and APC (top row) or a mixture of IGRP305-324 and IGRP241-260 peptides and APC (bottom row). EngTregs were labeled with EF670, and each histogram was gated on EF670- cells.

[0320] [Figure 193C] The figure shows the rate of inhibition of T1D5-2 Teff proliferation by polyEngTreg, T1D5-2 EngTreg, or T1D4 EngTreg in the presence of a mixture of IGRP305-324 peptide and IGRP241-260 peptide and APC.

[0321] [Figure 193D] Representative histograms show proliferation (measured by CTV dilution) of T1D5-2 Teffs cocultured with poly-EngTregs or GAD265 EngTregs in the presence of IGRP305-324 peptide alone and APC (top row) or a mixture of IGRP305-324 and GAD265-284 peptides and APC (bottom row). EngTregs were labeled with EF670, and each histogram was gated on EF670- cells.

[0322] [Figure 193E] The figure shows the rate of inhibition of T1D5-2 Teff proliferation by polyEngTreg or GAD265 EngTreg in the presence of a mixture of IGRP305-324 peptide and GAD265-284 peptide and APC.

[0323] [Figure 193F]The figure shows the rate of suppression of cytokine production from T1D5-2 Teff by polyEngTreg, T1D5-2 EngTreg, or T1D4 EngTreg in the presence of a mixture of IGRP305-324 peptide and IGRP241-260 peptide and APC.

[0324] [Figure 193G] Figures 193C, 193E, 193F, and 193G show the suppression rate of CD25 expression from T1D5-2 Teffs by poly-EngTregs, T1D5-2 EngTregs, or T1D4 EngTregs in the presence of a mixture of IGRP305-324 and IGRP241-260 peptides and APCs. Data are shown as mean ± SD from three independent experiments using cells generated from three healthy donors. P values ​​were calculated using a paired two-tailed Student's t-test (*P<0.05, P<0.01, and P<0.005). In all three experiments, LNGFR-T cells bearing T1D5-2 or T1D4 TCRs were used as negative controls. These negative control cells did not show significant suppression.

[0325] [Figure 193H] This shows that CD3 / CD28 bead-induced Teff proliferation was suppressed to a similar extent by different types of islet-specific EngTregs. Representative flow cytometry plots showing mTCR expression in FOXP3-edited cells (-) without TCR transduction, or in FOXP3-edited cells transduced with T1D4 or T1D5-2 TCRs. On day 7, gene-edited cells were stained and gated for live, CD3+, CD4+, LNGFR+, and FOXP3+ cells.

[0326] [Figure 193I]Representative histograms showing proliferation of T1D5-2 Teffs in a CD3 / CD28 bead suppression assay performed in parallel with the bystander suppression assays shown in Figures 193B and 193C are shown. T1D5-2 Teffs were incubated with CD3 / CD28 beads in the absence (-) of Tregs or in the presence of polyclonal EngTregs, T1D5-2 EngTregs, or T1D4 EngTregs.

[0327] [Figure 193J] Figure 19 shows the percentage of inhibition of CD3 / CD28 bead-induced proliferation of T1D5-2 Teffs by polyEngTregs, T1D5-2 EngTregs, or T1D4 EngTregs in the experiment shown in Figure 3I.

[0328] [Figure 193K] Representative histograms showing proliferation of T1D5-2 Teffs in a CD3 / CD28 bead suppression assay performed in parallel with the bystander suppression assay shown in Figures 193D and 193E are shown. T1D5-2 Teffs were incubated with CD3 / CD28 beads in the absence (-) of Tregs or in the presence of poly-EngTregs or GAD265-EngTregs.

[0329] [Figure 193L] Figure 193K shows the percent inhibition of CD3 / CD28 bead-induced T1D5-2 Teff proliferation by poly-EngTregs or GAD265-EngTregs. Figures 193J and 193L show data as mean ± SD from three independent experiments using cells generated from three healthy donors. P values ​​were calculated using a paired two-tailed Student's t-test.

[0330] [Figure 193M]Representative histograms showing the suppression of cytokine production from bystander Teffs by islet-specific EngTregs are shown. Representative histograms showing TNFα production from T1D5-2 Teffs in an antigen-specific bystander suppression assay are shown. The legends for each histogram are the same as in Figure 193M.

[0331] [Figure 193N] Representative histograms showing IL-2 production from T1D5-2 Teffs in an antigen-specific bystander suppression assay are shown. The legends for each histogram are the same as in Figure 193M.

[0332] [Figure 193O] Representative histograms showing IFNγ production from T1D5-2 Teffs in an antigen-specific bystander suppression assay are shown. The legends for each histogram are the same as in Figure 193M.

[0333] [Figure 193P] Representative histograms showing CD25 expression from T1D5-2 Teffs in an antigen-specific bystander suppression assay are shown. The legends for each histogram are the same as in Figure 193M. In Figures 193M-193P, T1D5-2 Teffs were cultured without Tregs or cocultured with poly-EngTregs, T1D5-2 EngTregs, or T1D4 EngTregs in the presence of IGRP305-324 peptide alone and APCs, or a mixture of IGRP305-324 and IGRP241-260 peptides and APCs.

[0334] [Figure 194A] This demonstrates that islet-specific EngTregs suppress polyclonal islet-specific Teffs derived from PBMCs obtained from type 1 diabetic donors. This figure shows the main steps and their timeline for producing islet-specific EngTregs, polyclonal islet-specific Teffs, and monocyte-derived DCs (mDCs) from PBMCs obtained from type 1 diabetic donors and performing in vitro suppression assays.

[0335] [Figure 194B] Representative histograms show proliferation (measured by CTV dilution) of polyclonal islet-specific Teffs co-cultured with T1D2 EngTregs, 4.13 EngTregs, LNGFR-T cells, or poly-EngTregs in the presence of CD3 / CD28 beads (top) or islet-specific antigens (nine islet-specific peptides and monocyte-derived DCs (mDCs)) (bottom). EngTregs were labeled with EF670, and each histogram was gated on EF670- cells.

[0336] [Figure 194C] Figure 1 shows the rate of inhibition of CD3 / CD28-induced polyclonal islet-specific Teff proliferation by T1D2 EngTregs, 4.13 EngTregs, LNGFR-T cells, or polyEngTregs.

[0337] [Figure 194D] Figure 1 shows the percent inhibition of antigen-induced polyclonal islet-specific Teff proliferation by T1D2 EngTregs, 4.13 EngTregs, LNGFR-T cells, or poly-EngTregs. Antigen stimulation was performed with a pool of nine islet-specific peptides in the presence of mDCs. Data are shown as the mean ± SD of three independent experiments using cells generated from three type 1 diabetic donors. P values ​​were calculated using a paired two-tailed Student's t-test (*P<0.05, **P<0.01, and ***P<0.0001). As a negative control, coculture in the presence of mDCs and DMSO also resulted in no significant Teff proliferation.

[0338] [Figure 194E]This paper presents the expansion of multispecific islet-specific T cells derived from PBMCs obtained from a type 1 diabetic donor, showing the main steps and timeline for peptide-stimulated expansion of islet-specific T cells. CD4+CD25- T cells isolated from a type 1 diabetic donor were stimulated with nine HLA-DR0401-restricted islet peptides, including five GAD65-specific islet peptides, three IGRP-specific islet peptides, and one PPI-specific islet peptide, in combination with irradiated autologous APCs (CD4-CD25+). Tetramer staining was performed on days 12–14. T cells were cultured without IL-2 for up to day 7, after which IL-2 was added every 2–3 days for expansion.

[0339] [Figure 194F] Representative flow cytometry plots showing tetramer-positive T cells specific for individual antigenic peptides are shown. Negative staining was performed without tetramer. Cells were gated on CD4+ T cells, and percentages indicate the degree of tetramer staining relative to background.

[0340] [Figure 194G] CD4+ T cells from three type 1 diabetic donors were peptide-stimulated in vitro for 12–14 days in five separate experiments. The percentage of tetramer-positive CD4+ T cells was measured. The combined results are shown. Each bar represents the percentage of CD4+ T cells specific for each islet antigenic peptide. Each dot represents one experiment.

[0341] [Figure 194H]This shows that islet-specific EngTregs are superior to tTregs in suppressing polyclonal islet-specific Teffs. Representative histograms show parallel measurements of polyclonal islet-specific Teff proliferation in the presence of anti-CD3 / CD28 antibody-coated beads (top panel) or in the presence of a pool of nine islet-specific peptides and mDCs (bottom panel). Polyclonal islet-specific Teffs were cultured without Tregs (-) or co-cultured with T1D2 LNGFR-, T1D2 EngTregs, or tTregs. tTregs were selected for CD4+CD25+CD127- and cultured similarly to EngTregs. tTregs were activated with CD3 / CD28 beads for 2 days, expanded, and harvested on day 10. All cells used in the suppression assay were autologous cells prepared from type 1 diabetic donors. As a negative control, co-culture with monocyte-derived DCs (mDCs) and DMSO also resulted in no significant Teff proliferation.

[0342] [Figure 194I] The percentage of inhibition of CD3 / CD28 bead-induced polyclonal islet-specific Teff proliferation by T1D2 LNGFR- cells, T1D2 EngTregs, or tTregs is shown.

[0343] [Figure 194J] The percentage of suppression of antigen-induced proliferation of polyclonal islet-specific Teffs by T1D2 LNGFR- cells, T1D2 EngTregs, or tTregs is shown.

[0344] [Figure 195A] We demonstrate that islet-specific EngTregs inhibit APC maturation and suppress Teffs using both cell-contact-dependent and cell-contact-independent mechanisms. A schematic diagram of a transwell suppression assay using an upper and lower chamber separated by a permeable membrane is shown.

[0345] [Figure 195B]The percent proliferation inhibition of polyclonal islet-specific Teffs in the lower chamber (left panel) or upper chamber (right panel) as measured by CTV dilution is shown. As a positive control, polyclonal islet-specific Teffs were co-cultured with T1D2 EngTregs. Data are shown as mean ± SEM from three independent experiments using cells generated from three type 1 diabetic donors. ***P<0.001, **P<0.01, *P<0.05, indicating significant differences calculated by paired t-test.

[0346] [Figure 195C] The main steps and timeline of the DC maturation and APC modulation assays are shown.

[0347] [Figure 195D] The normalized MFI of CD86 expression on mDCs is shown. HLA DR0401-restricted mature autologous mDCs were cocultured with T1D2 EngTregs or LNGFR-T cells for 2 days in the presence of IGRP305-324 peptide. The MFI of CD86 on DCs was normalized by the MFI measured in the DC-only condition. Data are shown as the mean ± SD of three independent experiments using cells generated from three healthy donors. *P<0.05 indicates a significant difference calculated by paired t-test.

[0348] [Figure 195E] Representative histograms show proliferation of polyclonal islet-specific Teffs co-cultured with islet-specific antigens (10 antigens including IGRP305-324) and mDCs in the presence of T1D2 EngTregs and exogenous human IL2 (0.1 IU / ml). Prior to co-culture, Teffs were labeled with CTV and EngTregs with EF670, and proliferation was assessed by measuring the dilution of CTV.

[0349] [Figure 195F]Figure 19 shows the percent proliferation inhibition of Teffs shown in Figure 5E. The percent proliferation inhibition was calculated separately in the absence and presence of exogenous human IL2. Data are shown as the mean ± SEM of three independent experiments using cells generated from three type 1 diabetic donors. Ns indicates no significant difference as calculated by paired t-test.

[0350] [Figure 195G] We demonstrate that islet-specific EngTregs exhibit contact-dependent and contact-independent bystander suppression. To investigate the mechanism of bystander suppression by islet-specific EngTregs, we generated polyclonal islet-specific Teffs. CD4+CD25- T cells isolated from type 1 diabetic donors were stimulated with nine HLA-DR0401-restricted islet peptides, including those specific for GAD65113-132, GAD265-284, GAD273-292, GAD305-324, IGRP17-36, IGRP241-260, PPI76-90, or ZNT8266-285, and irradiated autologous APCs (CD4-CD25+). Tetramer staining was performed on days 14 and 15. T1D2 TCRs specific for the IGRP305-324 peptide were excluded from Teff expansion. Representative flow cytometry plots showing tetramer-positive T cells specific for individual antigenic peptides are shown. Negative staining was performed without tetramer. Cells were gated on CD4+ T cells, and percentages indicate the degree of tetramer staining relative to background.

[0351] [Figure 195H] The figures show the combined results of measuring the percentage of tetramer-positive CD4+ T cells from three type 1 diabetic donors after 14-15 days of in vitro peptide stimulation. Each bar represents the percentage of CD4+ T cells specific for each islet antigenic peptide. Each dot represents a different type 1 diabetic donor.

[0352] [Figure 195I]Representative histograms show proliferation of polyclonal islet-specific Teff cells in the lower well (bottom row) or upper well (top row). mDCs loaded with a pool of islet-specific peptides (10 antigens including IGRP305-324) were seeded in the lower and upper wells. Polyclonal islet-specific Teff cells and / or T1D2 EngTreg cells were added to the lower and / or upper wells as indicated on the graph.

[0353] [Figure 195J] We demonstrate that islet-specific EngTregs suppress CD86 expression on dendritic cells. HLA-DR0401-restricted autologous monocytes were matured into DCs with GM-CSF / IL-4 and IFNγ / CL075. CTV-labeled islet TCR-expressing EngTregs or islet TCR-expressing LNGFR-T cells were cocultured with mature DCs in the presence of a peptide recognized by the islet TCR. After 2 days of incubation, cells were harvested, stained, and analyzed by flow cytometry.

[0354] [Figure 195K] Representative data showing the MFI of CD86 on DCs co-cultured with T1D2 EngTreg or LNGFR-T cells are shown.

[0355] [Figure 195L] Bar graphs showing the corrected expression levels of CD86 in DCs cocultured with T1D4 EngTreg or LNGFR-T cells in the presence of IGRP241-260 peptide (left graph) and the corrected expression levels of CD86 in DCs cocultured with PPI76 EngTreg or LNGFR-T cells in the presence of PPI76-90 peptide (right graph).

[0356] [Figure 196A]This shows that islet-specific EngTregs with low functional avidity exhibit superior suppression of polyclonal islet-specific Teffs derived from PBMCs obtained from type 1 diabetic donors. This graph shows the peptide dose-response of T cells expressing the T1D2 TCR, T1D5-1 TCR, or T1D5-2 TCR. CD4+ T cells transduced with the T1D2 TCR, T1D5-1 TCR, or T1D5-2 TCR were cocultured with APCs for 4 days in the presence of various concentrations of the peptide (IGRP305-324) recognized by each TCR. Prior to coculture, T cells were labeled with CTV, and cell proliferation was measured by dilution of the CTV.

[0357] [Figure 196B] Representative histograms show the proliferation of polyclonal islet-specific Teff cells in the presence of islet-specific antigens (10 islet-specific peptides + monocyte-derived DCs (mDCs)) and T1D2 EngTregs, T1D5-1 EngTregs, or T1D5-2 EngTregs. Polyclonal islet-specific Teff cells were labeled with CTV, and EngTreg cells were labeled with EF670. Cell proliferation was measured using the dilution of CTV.

[0358] [Figure 196C] Figure 1 shows the percent inhibition of antigen-induced proliferation of polyclonal islet-specific Teffs by T1D2 EngTregs, T1D5-1 EngTregs, or T1D5-2 EngTregs. Data were corrected using the inhibitory activity obtained in a parallel suppression assay using CD3 / CD28 beads. The inhibitory activity was calculated as the percent inhibition / the lowest percent inhibition. The corrected antigen-specific inhibitory activity was calculated as the percent inhibition / the inhibitory activity in the antigen-specific assay.

[0359] [Figure 196D]We demonstrate the generation of polyclonal islet-specific Teffs from PBMCs obtained from a type 1 diabetic donor. CD4+CD25- T cells isolated from the donor were stimulated with 10 HLA-DR0401-restricted islet peptides, including those specific for GAD65113-132, GAD265-284, GAD273-292, GAD305-324, IGRP17-36, IGRP241-260, PPI76-90, or ZNT8266-285, along with irradiated autologous APCs (CD4-CD25+). Tetramer staining was performed on days 14 and 15. Representative flow cytometry plots show tetramer-positive T cells specific for individual antigenic peptides. Negative staining was performed without tetramer. Cells were gated on CD4+ T cells, and the percentages indicate the degree of tetramer staining relative to background.

[0360] [Figure 196E] The percentage of tetramer-positive CD4+ T cells was measured in five separate experiments in which CD4+ T cells from four type 1 diabetic donors were peptide-stimulated in vitro for 14–15 days. The combined results are shown. Each bar represents the percentage of CD4+ T cells specific for each islet antigenic peptide. Each dot represents a different T1D donor.

[0361] [Figure 196F] 196B and 196C show normalized antigen-specific suppression of polyclonal islet-specific Teffs. Representative histograms showing proliferation of polyclonal islet-specific Teffs in the presence of anti-CD3 / CD28 antibody-coated beads or in the presence of a pool of 9 islet-specific peptides and mDCs (bottom panel) are shown. This experiment was performed in parallel with the proliferation suppression experiment performed in the presence of a pool of 10 islet-specific peptides and mDCs (Figures 196B and 196C).

[0362] [Figure 196G]Figure 196F shows the rate of inhibition of proliferation of polyclonal islet-specific Teff induced by T1D2 EngTreg, T1D5-1 EngTreg, or T1D5-2 EngTreg.

[0363] [Figure 197A] Gene editing of BDC2.5 CD4+ T cells to generate mouse islet-specific EngTregs is shown. A schematic diagram of the MND LNGFR p2A knock-in donor template packaged in AAV5 for use in editing FOXP3 via homology-directed repair is shown. Exons are indicated by numbered boxes, and FOXP3 homology arms are also shown. If gene editing is successful, the MND promoter drives expression of the endogenous mouse FOXP3 protein and cell surface expression of LNGFR in cis configuration.

[0364] [Figure 197B] 1 shows a schematic diagram illustrating the experimental timeline for FOXP3 gene editing, cell analysis, and enrichment of edited LNGFR cells.

[0365] [Figure 197C] Representative flow cytometry plots (one of four independent experiments) showing LNGFR expression in mock-edited control cells (left), LNGFR+ cells edited with RNP and AAV donor template before column enrichment (center), or LNGFR+ cells after column enrichment (right) are shown.

[0366] [Figure 197D] Representative flow cytometry histograms (one of two independent experiments) showing the expression of Treg-associated markers in the indicated cell populations are shown.

[0367] [Figure 197E]A bar graph showing the MFI of Treg-associated markers in EngTreg or mock-edited cells is shown. Error bars indicate ±SD. P values ​​were calculated using an unpaired t-test to compare EngTreg and mock-edited cells.

[0368] [Figure 197F] Schematic representation of in vitro suppression assays performed with BDC2.5 CD4+ Teff cells and mock control cells, BDC2.5 tTreg cells or EngTreg cells.

[0369] [Figure 197G] Representative flow cytometry plot (one of three independent experiments) showing CTV-labeled BDC2.5 CD4+ Teffs co-cultured with the indicated cells 4 days after stimulation.

[0370] [Figure 197H] Figure 1 shows the percent inhibition of proliferation of BDC2.5 CD4+ Teffs co-cultured with the indicated Tregs at various Teff:Treg ratios. Percent inhibition = [100 - corrected percent inhibition]; corrected percent inhibition = 100 / Teff proliferation alone × Teff proliferation in the presence of Tregs.

[0371] [Figure 198A] We demonstrate that polyclonal EngTregs cannot prevent the development of type 1 diabetes in vivo, whereas islet-specific EngTregs can. A schematic diagram showing the experimental timeline for conducting a mouse diabetes prevention study is shown.

[0372] [Figure 198B]This graph shows the diabetes-free survival rate of recipient NSG mice after co-transfer of islet-specific Teffs and the indicated cell populations. Data are the combined results of two independent experiments. ****, P ≤ 0.0001. P values ​​were calculated using the log-rank test (Mantel-Cox test) for comparisons of BDC2.5 tTreg or EngTreg groups with mock-edited controls.

[0373] [Figure 198C] The left panel shows representative flow cytometry plots of lymphocytes isolated from the pancreas of non-diabetic NSG recipient mice 49 days after injection of BDC2.5 CD4 Teffs. The top graph shows data from BDC2.5 tTreg recipient mice, while the bottom graph shows data from BDC2.5 EngTreg recipient mice. Pre-determined gates are indicated at the top of each flow cytometry plot. The right panel shows histograms of FOXP3 expression in each gate (color-coded) for each flow cytometry plot.

[0374] [Figure 198D] Representative flow cytometry plots showing LNGFR expression in CD4 T cells (polyclonal; top row) from NOD mice and CD4 T cells (islet-specific; bottom row) from NOD BDC2.5 mice gene-edited using the methods indicated in each plot (top of box).

[0375] [Figure 198E]This graph shows the diabetes-free survival rate in recipient NSG mice after transfer of mock-edited cells, polyclonal EngTreg cells, polyclonal tTreg cells, islet-specific EngTreg cells, or islet-specific tTreg cells co-transferred with islet-specific Teff cells. Data from two independent experiments are pooled. ****P ≤ 0.0001. P values ​​were calculated using the log-rank test (Mantel-Cox test) for comparisons of BDC2.5 tTregs or BDC2.5 EngTregs with polyclonal tTregs or polyclonal EngTregs, respectively. All flow cytometry plots are representative of results from at least two independent experiments. DETAILED DESCRIPTION OF THE INVENTION

[0376] Provided herein are compositions and methods for editing two or more genomic loci in a cell by editing the TRAC locus of the cellular genome using a chemically-inducible signaling complex (CISC) system (including promoter capture, TCR / CAR knock-in, and TRAC gene promoter hijacking). Also provided herein are compositions and methods for suppressing effector T cell proliferation using genetically engineered / modified airT cells, and methods for treating autoimmune, allergic, and / or inflammatory diseases in a subject using genetically engineered / modified airT cells. Furthermore, provided herein are methods for designing TCRs with specific properties, e.g., specific avidity.

[0377] Editing two or more gene loci in cells using the chemically inducible signaling complex (CISC) system Some embodiments of the methods and compositions provided herein relate to efficiently editing two or more gene loci in a cell using a chemical-inducible signaling complex (CISC) system. In some embodiments, the CISC complex includes a first CISC component comprising a first extracellular binding domain, a transmembrane domain, and a first signaling domain, and a second CISC component comprising a second extracellular binding domain, a transmembrane domain, and a second signaling domain, wherein the first CISC component and the second CISC component are capable of binding to a CISC-inducing molecule (e.g., a small molecule such as rapamycin or an analog thereof) (e.g., via the first extracellular binding domain and the second extracellular binding domain). In some embodiments, the CISC complex system includes a CISC complex that further includes a third CISC component in addition to the first CISC component and the second CISC component, wherein the third CISC component is distinct from both the first and second CISC components and is capable of specifically binding to the CISC-inducing molecule. In some embodiments, the third CISC component is soluble and does not contain a transmembrane or extracellular domain. In some embodiments, the third soluble CISC component does not contain a secreted peptide and is localized in the cytoplasm. In some embodiments, the first extracellular binding domain comprises FKBP and the second extracellular binding domain comprises FRB. In some embodiments, the third CISC component comprises a soluble FRB or a cytosolic FRB. Co-expression of two (e.g., first and second) transmembrane CISC components that can dimerize in the presence of a CISC-inducing molecule (e.g., rapamycin or a rapalog) provides a molecule that dimerizes, which is useful for regulating signal transduction. However, the inducing molecule may have undesirable effects on cellular metabolism. For example, upon entering cells, rapamycin binds to free FKBP protein to form an FKBP-rapamycin complex, which may stimulate the target of rapamycin (mTOR) to suppress mRNA translation and cell proliferation.However, when a third CISC component, such as soluble FRB, is expressed in the cytoplasm, this third CISC component can function as a decoy receptor for rapamycin or other CISC-inducing molecules, preventing or reducing the undesirable effects of the CISC-inducing molecules on cell physiology. CISC components and methods useful in embodiments of the cells and methods provided herein are described in WO2019 / 210057, which is incorporated herein by reference in its entirety.

[0378] In some embodiments, the first polynucleotide comprises a nucleic acid encoding a first CISC component provided herein. In some embodiments, the second polynucleotide comprises a nucleic acid encoding a second CISC component provided herein. In some embodiments, the third polynucleotide comprises a nucleic acid encoding a third CISC component provided herein. In some embodiments, the first polynucleotide comprising the nucleic acid encoding the first CISC component further comprises a first regulatory element (e.g., a first promoter). In some embodiments, the second polynucleotide comprising the nucleic acid encoding the second CISC component further comprises a second regulatory element (e.g., a second promoter). In some embodiments, the first polynucleotide comprising the nucleic acid encoding the third CISC component further comprises a third regulatory element (e.g., a third promoter). In some embodiments, the polynucleotide comprising the nucleic acid encoding the first CISC component, the second CISC component, and / or the third CISC component does not comprise a regulatory element, is dependent on, or is operably linked to, one or more regulatory elements in the genome into which it is inserted. In some embodiments, a combination of a first CISC component and a second CISC component, a combination of a first CISC component and a third CISC component, a combination of a second CISC component and a third CISC component, or a combination of a first CISC component, a second CISC component and a third CISC component can be inserted into a single locus. In some embodiments, a combination of a first CISC component and a second CISC component, a combination of a first CISC component and a third CISC component, a combination of a second CISC component and a third CISC component, or a combination of a first CISC component, a second CISC component and a third CISC component can be present on a single polynucleotide.In some embodiments, the first polynucleotide and the second polynucleotide are the same, and both comprise a nucleic acid encoding both the first CISC component and a nucleic acid encoding the second CISC component. In some embodiments, the first polynucleotide and the third polynucleotide are the same, and both comprise a nucleic acid encoding both the first CISC component and a nucleic acid encoding the third CISC component. In some embodiments, the second polynucleotide and the third polynucleotide are the same, and both comprise a nucleic acid encoding both the second CISC component and a nucleic acid encoding the third CISC component. In some embodiments, the polynucleotide comprising nucleic acids encoding the first CISC component and the second CISC component further comprises a nucleic acid encoding a third CISC component provided herein. In some embodiments, any two or three of the first CISC component, the second CISC component, and the third CISC component share a common regulatory element or are operably linked to a single regulatory element.

[0379] In some embodiments, the first CISC component and the second CISC component are configured such that when the first CISC component and the second CISC component are expressed in a cell, they can dimerize in the presence of an inducer molecule (e.g., rapamycin or a rapalog) to form a CISC having signaling function.

[0380] In some embodiments, a method for editing two or more loci (e.g., a first locus and a second locus) in a cell includes providing to the cell a first polynucleotide comprising a nucleic acid encoding a first CISC component (and optionally a third CISC component) provided herein for insertion into the first locus; and / or a second polynucleotide comprising a nucleic acid encoding a second CISC component (and optionally a third CISC component) provided herein for insertion into the second locus. In some embodiments, the first CISC component and the second CISC component (and optionally a third CISC component) are contained in a single polynucleotide and inserted into the same locus (e.g., the Foxp3 gene / locus or the TRAC gene / locus). Specific loci may be targeted by designing an endonuclease that cleaves at a specific position and inserting sequences (e.g., homology arms) that flank one or more nucleic acids encoding the CISC components. It is understood that the methods disclosed herein can be used to edit three or more loci by inserting any one or more of the first, second, and third CISC components provided herein.

[0381] In some embodiments, the method of editing one or more loci of a gene / genome in a cell comprises: (a)(i) a first nucleic acid comprising a polynucleotide encoding a first CISC component comprising a first extracellular binding domain, a first transmembrane domain, and a first intracellular signaling domain; (ii) a second nucleic acid comprising a second polynucleotide encoding a second CISC component comprising a second extracellular binding domain, a transmembrane domain, and a second intracellular signaling domain; and (iii) a first endonuclease capable of cleaving a first nucleotide sequence within a first locus, or a nucleic acid encoding the first endonuclease; (iv) a second endonuclease capable of cleaving a second nucleotide sequence within the second locus, or a nucleic acid encoding the second endonuclease. to integrate a first polynucleotide or fragment thereof into the first locus and insert a second polynucleotide or fragment thereof into the second locus; and (b) contacting the cells with a dimerizing agent (e.g., a ligand) that binds to a first extracellular binding domain of a first CISC component and a second extracellular binding domain of a second CISC component. Including, In cells expressing both the first and second CISC components, the first and second intracellular signaling domains dimerize and a signal is transmitted via interaction between the first and second intracellular signaling domains. In some embodiments, the first polynucleotide further comprises a homologous arm that targets a first locus. In some embodiments, the second polynucleotide further comprises a homologous arm that targets a second locus. In some embodiments, the first CISC component comprises an FKBP extracellular domain, a transmembrane domain, and an IL2RB intracellular signaling domain, and the second CISC component comprises an FRB extracellular domain, a transmembrane domain, and an IL2RG intracellular signaling domain, and the first and second CISC components dimerize in the presence of rapamycin or a rapalog, thereby rendering cells responsive to rapamycin in a manner similar to stimulation with IL-2. In some embodiments, a first CISC component comprises an FKBP extracellular domain, a transmembrane domain, and an IL2RG intracellular signaling domain, and a second CISC component comprises an FRB extracellular domain, a transmembrane domain, and an IL2RB intracellular signaling domain; the first and second CISC components dimerize in the presence of rapamycin or a rapalog, thereby conferring responsiveness to rapamycin to cells in a manner analogous to stimulation with IL-2. In some embodiments, the transmembrane domain of a CISC component comprises a transmembrane domain that corresponds to the transmembrane domain of the intracellular signaling domain of the CISC component (e.g., a CISC component comprising an IL2RB intracellular domain comprises a transmembrane domain derived from IL2RB). In some embodiments, both the first and second CISC components comprise transmembrane domains derived from the same protein (e.g., IL2RB). In some embodiments, the transmembrane domain of a CISC component has a transmembrane domain derived from a protein different from the intracellular signaling domain of the CISC component (e.g., a CISC component comprising an IL2RB intracellular signaling domain comprises a transmembrane domain derived from a protein other than the full-length IL2RB protein).

[0382] The extracellular domain of a CISC component can be any domain that can dimerize upon binding to an agent. Such agents can be small molecules (e.g., rapamycin), proteins (e.g., lysozyme), or other agents. The extracellular domain can also be a domain that can bind to a small molecule or protein (e.g., an antibody binding domain or fragment thereof) and dimerize upon binding to such a ligand.

[0383] In some embodiments, the first locus is the FOXP3 locus, and the second locus is the TRAC locus. In another embodiment, the first locus is the TRAC locus, and the second locus is the FOXP3 locus. In some embodiments, the first locus and the second locus are independently selected from the FOXP3 locus, the TRAC locus, the AAVS1 locus, and the ROSA26 locus. In some embodiments, both nucleic acid molecules are inserted into different alleles of the same locus, and the locus is selected from the FOXP3 locus, the TRAC locus, the AAVS1 locus, and the ROSA26 locus.

[0384] In some embodiments, the method further comprises separating cells that bind to the ligand at a higher concentration than other cells. In some embodiments, the first polynucleotide further comprises one or more regulatory elements and / or a first payload. In some embodiments, the first polynucleotide encodes the first CISC component and the regulatory element, but does not encode a payload. The payload described herein is a protein or RNA encoded by a nucleic acid. In some embodiments, the second polynucleotide further comprises one or more regulatory elements and / or a nucleic acid encoding a second payload. In some embodiments, (i) a first polynucleotide comprises an MND promoter operably linked to a nucleic acid encoding (a) a first CISC component comprising an FRB extracellular domain, a transmembrane domain, and an IL2RB intracellular signaling domain, and (b) a third CISC component comprising a soluble FRB domain, wherein the MND promoter is inserted upstream of a first coding exon or first codon of an endogenous FOXP3 gene in a cell and downstream (e.g., 10 to 10,000 bp downstream) of a TSDR of the endogenous FOXP3 locus; (ii) The second polynucleotide comprises an MND promoter operably linked to a nucleic acid encoding (a) a second CISC component comprising an FKBP extracellular domain, a transmembrane domain, and an IL2RG intracellular domain, (b) an exogenous TCRβ, and (c) an exogenous TRAV amino acid sequence and an exogenous TRAJ amino acid sequence, wherein the second nucleic acid is inserted upstream of the nucleic acid encoding the amino acid sequence of the TCRα constant region within the TRAC locus, and the inserted MND promoter controls transcription of the exogenous TCRβ, the exogenous TCRα comprising the exogenous TRAV amino acid sequence and the exogenous TRAJ amino acid sequence, and the endogenous TCRα amino acid sequence. In some embodiments, a first endonuclease and / or a second endonuclease, or a nucleic acid encoding the same, and one or more gRNAs targeting a first locus and / or a second locus are provided. In some embodiments, the endonuclease is an RNA-guided nuclease. In some embodiments, the RNA-guided nuclease is a CRISPR / Cas nuclease. In some embodiments, the CRISPR / Cas nuclease is a type I Cas nuclease. In some embodiments, the CRISPR / Cas nuclease is a type II Cas nuclease. In some embodiments, the CRISPR / Cas nuclease is a type III Cas nuclease. In some embodiments, the CRISPR / Cas nuclease is a type V Cas nuclease. In some embodiments, the CRISPR / Cas nuclease is selected from the group consisting of Cas9, SaCas9, CjCas9, xCas9, C2C1, Cas13a / C2c2, C2c3, Cas13b, Cpf1, and variants thereof.

[0385] In some embodiments, the first polynucleotide and / or second polynucleotide described herein further comprises a payload or nucleic acid encoding a polypeptide or a functional fragment thereof. Examples of payloads or nucleic acids encoding a polypeptide or a functional fragment thereof include, but are not limited to, TCR, CAR, Foxp3, or functional fragments thereof. In some embodiments, the first polynucleotide and / or second polynucleotide comprises one or more regulatory elements (e.g., one or more promoters). In some embodiments, the first polynucleotide and / or second polynucleotide does not comprise a payload or nucleic acid encoding a polypeptide or a functional fragment thereof, but does comprise one or more regulatory elements (e.g., one or more promoters) (provided that the nucleic acid encoding the polypeptide does not constitute a targeting sequence or homology arm used to target a specific locus within the genome). For example, the targeting sequence or homology arm may be a Foxp3 targeting arm comprising a nucleic acid encoding an exon of Foxp3, but the Foxp3 targeting arm is not required for the coding region of the FOXP3 gene or a functional fragment thereof. In some embodiments, the first polynucleotide and / or second polynucleotide described herein further comprises a payload or nucleic acid encoding a polypeptide or functional fragment thereof and one or more regulatory elements. For example, the first polynucleotide may comprise a nucleic acid encoding a first CISC component (and optionally a third CISC component described herein) and a promoter (e.g., an MND promoter) for insertion into a first locus (e.g., the Foxp3 locus), and the second polynucleotide may comprise a nucleic acid encoding a second CISC component (and optionally a third CISC component described herein) and a nucleic acid encoding a polypeptide (e.g., a TCR or CAR) or a functional fragment thereof for insertion into a second locus (e.g., the TRAC locus). In some embodiments, two or more payloads are delivered to one or more loci.For example, a nucleic acid encoding Foxp3, or a nucleic acid not encoding Foxp3 but comprising only regulatory elements (e.g., a promoter), may be inserted into the Foxp3 locus, and a nucleic acid encoding a TCR or CAR may be inserted into the TRAC locus. Note that any gene or locus may be used in the methods disclosed herein, which involve inserting one or more polynucleotides comprising nucleic acids encoding any one or more of a first CISC component, a second CISC component, and a third CISC component.

[0386] In some embodiments, the FOXP3 locus / gene and the TRAC locus / gene are loci / genes in a cell (e.g., a T cell). In some embodiments, the T cell is a Treg cell (e.g., Foxp+, Tr1, Th3, CD4+, CD8+, CD8+CD28-, or Qa-1-restricted) and is edited using the CISC components provided herein. In some embodiments, the cells provided herein are recombinant cells. In some embodiments, the recombinant cell is a cell in which one or more genes / locuses have been genetically engineered or edited (e.g., to stabilize expression of one or more genes). In some embodiments, the recombinant cell comprises an edited Foxp3 gene / locus, such as by inserting a promoter (in some embodiments, the promoter is inserted downstream (e.g., 10-10,000 bp downstream) of one or more regulatory elements such as TSDR and / or upstream of the first coding exon or first codon).

[0387] Methods for editing the TRAC locus in the genome of a cell Various methods for editing the TRAC locus in cells are provided herein. In some embodiments, editing of the TRAC locus is performed to introduce a nucleic acid encoding an exogenous antigen-specific receptor, such as a TCR or CAR, and a nucleic acid encoding a first or second CISC component described herein. In some embodiments, a promoter capture method is used in which an inserted nucleic acid encoding a TCR is controlled by a native / natural / endogenous promoter, and this TCR-encoding nucleic acid may be followed by nucleic acids encoding one or more CISC components described herein (see, for example, Figures 54, 68, and 70). In some embodiments, such an endogenous promoter capture method involves in-frame knock-in into exon 1 of the TRAC locus, thereby inducing TCR expression dependent on the endogenous TRAC promoter. In some embodiments, a TCR knock-in method is used in which a nucleic acid encoding a TCR and a promoter are knocked in, and this TCR-encoding nucleic acid may be followed by nucleic acids encoding one or more CISC components described herein (see, for example, Figure 67). Embodiments of the methods that involve inserting a nucleic acid encoding a TCR or a TCR can be modified so that a nucleic acid encoding a CAR or a CAR is inserted. In some embodiments, a native / natural / endogenous TCR gene or fragment thereof is hijacked by inserting a promoter upstream of the native / natural / endogenous TCR gene or fragment thereof, which may be upstream of a nucleic acid encoding one or more CISC components described herein (see, e.g., Figure 164).

[0388] Promoter capture method In some embodiments, the TRAC locus of a cell is edited by promoter capture (e.g., the methods shown in Figures 54, 68, and 70). In some embodiments of the methods provided herein, the cell is edited by inserting a nucleic acid molecule comprising a nucleic acid encoding an exogenous TCR or CAR into the TRAC locus. The nucleic acid encoding the exogenous TCR or CAR is inserted downstream (e.g., 10 to 10,000 bp downstream) of the endogenous TRAC promoter and in frame with the TRAC coding sequence, thereby operably linking the inserted nucleic acid to the endogenous TRAC promoter and driving expression of the exogenous TCR or CAR via the endogenous TRAC promoter. Thus, in some embodiments, the exogenous TCR or CAR is expressed at an expression level comparable to that of the endogenous TCR prior to genetic modification. In some embodiments, the nucleic acid molecule comprising a nucleic acid encoding an exogenous TCR or CAR further comprises a nucleic acid encoding a first or second CISC component described herein. In some embodiments, the nucleic acid molecule comprising a nucleic acid encoding an exogenous TCR or CAR further comprises a nucleic acid encoding a third CISC component described herein.

[0389] TCR / CAR knock-in method In some embodiments, the TRAC locus in a cell is edited by knocking in a full-length TCR (e.g., a pancreatic islet TCR) or CAR and a promoter (e.g., an MND promoter) operably linked to a polynucleotide / nucleic acid encoding the TCR or CAR, terminating transcription with a stop codon and polyadenylation signal, thereby expressing the TCR or CAR independently of endogenous TRAC regulatory elements. For an example of such a method, see Figure 67. In some embodiments, the cell is edited by inserting a nucleic acid molecule comprising a heterologous promoter operably linked to a polynucleotide / nucleic acid encoding a TCR or CAR into the TRAC locus, disrupting the endogenous TRAC gene while simultaneously driving expression of the inserted TCR or CAR via the heterologous promoter. In some embodiments, the nucleic acid molecule comprising the polynucleotide / nucleic acid encoding an exogenous TCR or CAR further comprises a polynucleotide / nucleic acid encoding a first CISC component or a second CISC component described herein. In some embodiments, the polynucleotide molecule comprising the nucleic acid encoding an exogenous TCR or CAR further comprises a nucleic acid encoding a third CISC component described herein.

[0390] Promoter-mediated hijacking of the TRAC gene In some embodiments, the TRAC gene of a cell is edited by hijacking the TRAC locus with a promoter (e.g., the MND promoter), as shown in Figure 164. In some embodiments, the cell is edited by (a) inserting a polynucleotide molecule comprising a promoter operably linked to a nucleic acid encoding a full-length TCRβ protein and a nucleic acid encoding the TCRα variable region (TRAV) and TCR joining region (TRAJ). The coding sequence for the TRAV region and the coding sequence for the TRAJ region are inserted in frame with the coding sequence encoding the TCRα constant region, such that transcription of the TCRα protein comprising the heterologous TRAV / TRAJ amino acid sequence and the endogenous TCRα constant region amino acid sequence, and transcription of the heterologous TCRβ protein, are controlled by the inserted heterologous promoter. In this embodiment, the endogenous 3' regulatory region from the endogenous TRAC gene is utilized. In some embodiments, the polynucleotide molecule comprising a nucleic acid encoding an exogenous TCR or CAR further comprises a nucleic acid encoding a first CISC component or a second CISC component described herein. In some embodiments, the polypeptide molecule comprising a nucleic acid encoding an exogenous TCR or CAR further comprises a nucleic acid encoding a third CISC component described herein.

[0391] Provided herein are airT cells, e.g., cells having one, two, or more loci edited by inserting nucleic acid encoding at least two CISC components (e.g., at least a first CISC component and a second CISC component) and optionally a third CISC component.

[0392] Some embodiments include the use of such systems to edit the FOXP3 and TRAC loci in Treg cells. Further embodiments relate to the use of gene-edited Treg cells to suppress the activation and / or proliferation of specific cell populations. Such suppression can be achieved in vitro, ex vivo, or in vivo (e.g., by administering gene-edited Treg cells to a subject with or without a ligand (e.g., rapamycin) to activate and maintain the suppressive phenotype of the Treg cells).

[0393] Some embodiments of the methods and compositions provided herein relate to antigen-specific artificial immunoregulatory T (airT) cells. The airT cells of the present invention are also referred to as "edTreg," "gene-edited Treg," "EngTreg," or "recombinant Treg" cells. AirT cells may be Tr1 cells, e.g., cells that express IL-10. Some embodiments include artificial T cells (e.g., T lymphocytes). Some embodiments include CD4+CD25+ T cells that have an artificially modified forkhead box protein 3 / winged helix transcription factor (FOXP3) gene that constitutively expresses the FOXP3 gene product at an expression level equal to or greater than that of natural regulatory T (Treg) cells, and at least one introduced polynucleotide encoding an antigen-specific T cell receptor (TCR) polypeptide.

[0394] Some embodiments provided herein relate to the efficient editing of two or more genetic loci in cells using a chemically inducible signaling complex (CISC) system, where the components of the CISC system are expressed at increased levels relative to another CISC system with different orientations and / or different elements. In some embodiments, the gene editing systems described herein include one or more (e.g., two) constructs, each comprising a promoter and one or more elements (e.g., a polynucleotide / nucleic acid encoding a first CISC component and a polynucleotide / nucleic acid encoding a TCR), configured to optimize expression of all CISC components. This configuration is based, at least in part, on the discovery that the orientation of split CISCs (where the first and second CISC components are contained in separate cassettes, each targeting a different genetic locus) is critical to their function. Unexpectedly, it has been found that the function of split CISC depends on the expression of various components of the CISC system and / or also depends on the arrangement of each component within a construct / cassette (e.g., a homology-directed repair cassette). Thus, in some embodiments, one or more constructs for the gene editing system described herein comprise a promoter (e.g., an MND promoter) that drives high expression of a CISC component compared to a promoter (e.g., an EF1α) that drives low expression of a CISC component. In some embodiments, each construct targets a different locus.

[0395] Suppression of Teff cell proliferation using genetically engineered / modified airT cells and the bystander effect Some embodiments provided herein relate to methods of suppressing the proliferation of Teff cells using genetically modified / altered Treg cells, some of which may involve a bystander effect, in which Treg cells contain a TCR specific for a first epitope of an antigen and Teff cells contain a TCR specific for a second epitope of the antigen.

[0396] Certain features useful for certain embodiments provided herein are disclosed in International Patent Application No. PCT / US2020 / 039445, filed June 24, 2020, entitled "Antigen-Specific Artificial Immunoregulatory T (airT) Cells"; U.S. Provisional Application No. 62 / 987,810, filed March 10, 2020, entitled "Antigen-Specific Artificial Immunoregulatory T (airT) Cells"; and U.S. Provisional Application No. 62 / 867670, filed June 27, 2019, entitled "Antigen-Specific Treg Therapy for Autoimmune Diseases" (all of which are expressly incorporated by reference in their entirety). Another particular feature useful for certain embodiments provided herein is disclosed in International Patent Application No. PCT / US2017 / 065746, filed December 12, 2017, entitled "Method for Activating Chemically Induced Signaling Complexes Expressed in Recombinant Cells In Vitro and In Vivo by Exogenous Drugs"; and International Patent Application No. PCT / US2019 / 029118, filed April 25, 2019, entitled "Rapamycin-Resistant Cells," both of which are expressly incorporated herein by reference in their entireties.

[0397] In some embodiments, the airT cells of the present invention can be induced by a specific antigen recognized by the TCR, such as a self-antigen, an allergen, or another antigen associated with the pathogenesis of inflammatory diseases characterized by an excessive immune response, to confer antigen-specific immunosuppression. Importantly, production of the airT cells of the present invention avoids the time, cost, and inefficiencies associated with isolating relatively rare natural Treg cells (1-4% of human PBMCs) as starting material for gene editing, thereby providing the advantage of generating therapeutically effective amounts of desired cells for adoptive immunotherapy. The airT cells of the present application can be generated using cells other than T cells, such as stem cells. In some embodiments, the airT cells are produced by gene editing induced pluripotent stem cells (iPSCs). In some embodiments, the airT cells are produced by gene editing CD34+ hematopoietic stem cells (HSCs). In some embodiments, stem cells are first differentiated into T cells, and then the T cells are transformed into airT cells.

[0398] In some embodiments, the airT cells of the invention express a functional TCR that specifically recognizes an antigen associated with the pathogenesis of an autoimmune disease, an allergic disease, an inflammatory disease, a solid organ transplant, or graft-versus-host disease, including, for example, a TCR comprising any of the TCR polypeptide sequences disclosed herein or any of the TCR polypeptides encoded by a TCR-encoding nucleic acid sequence disclosed herein, including the TCRs depicted in the figures.

[0399] In some embodiments, the airT cells of the present invention express a functional TCR that specifically recognizes an antigen associated with the pathogenesis of an autoimmune, allergic, or inflammatory disease, including, for example, a polypeptide autoantigen, allergen, and / or inflammation-associated antigen that comprises the amino acid sequence of a polypeptide antigen disclosed herein, or a polypeptide antigen that immunologically cross-reacts with a polypeptide autoantigen, allergen, and / or inflammation-associated antigen that comprises the amino acid sequence of a polypeptide antigen disclosed herein, including antigens shown in the figures.

[0400] Certain embodiments disclosed herein relate to a gene editing method for generating airT cells of the invention, which involves (i) stably expressing FoxP3 through targeted FoxP3 gene editing by introducing a promoter (e.g., a constitutive promoter) into cells that do not express FoxP3, thereby inducing FoxP3 expression at levels equivalent to or greater than those of natural regulatory T (Treg) cells and maintaining a stable FoxP3-controlled immune regulatory (immunosuppressive) program; and (ii) further gene editing the same cells to stably express an exogenous TCR by introducing a specific nucleotide sequence of the present disclosure encoding a TCR that recognizes an antigen associated with the pathogenesis of an autoimmune, allergic, or inflammatory disease. This stable expression of FoxP3 via targeted FoxP3 gene editing and stable expression of an exogenous TCR have a surprisingly advantageous functional relationship, allowing for the selection and expansion of recombinant T cells characterized by stable immune suppression coupled with expression of a desired TCR. Without wishing to be bound by any particular theory, it is believed that some of the airT cell embodiments disclosed herein, by artificially stably expressing FoxP3, may provide safe and effective adoptively transferred immunotherapy cells for indications where antigen-specific immunosuppression is desired, such as autoimmune diseases, allergic diseases, graft-versus-host disease (GVHD) or other inflammatory diseases, without the risks associated with the plasticity of natural Treg cells (e.g., conversion to T effector behavior).

[0401] The methods for producing airT cells disclosed herein have the advantage that, while typical production methods begin with the isolation of natural Treg cells, which are present at low frequencies in peripheral blood and account for only approximately 1-4% of human peripheral blood mononuclear cells, as described above, some embodiments of the present invention omit this isolation step. Instead, as described herein, airT cells of the present invention can be produced by isolating T cells, such as CD4+ T cells or CD8+ T cells. While CD4+ T cells and CD8+ T cells represent a greater variety of T cells than cells with other cell surface markers, CD4+ T cells and CD8+ T cells comprise approximately 25-60% of human PBMCs and therefore can be used as relatively abundant starting materials for gene editing using the various methods provided herein.

[0402] In certain embodiments, compositions comprising antigen-specific immunoregulatory T (airT) cells of the invention and methods for producing the airT cells of the invention can be used to treat and / or alleviate certain autoimmune, allergic, and / or inflammatory diseases (e.g., adoptive transfer immunotherapy), and in such uses, the stable survival and maintenance of antigen-specific immunoregulatory function of the airT cells offers unprecedented advantages.

[0403] In certain embodiments, it has been unexpectedly found that the airT cells described herein are capable of inducing an antigen-specific immunosuppressive response when stimulated with an antigen associated with the pathogenesis of an autoimmune, allergic, or inflammatory disease (e.g., any of the antigens disclosed herein). Such antigen-specific induced immunosuppression can be (i) suppression of either or both of the activation and proliferation of effector T cells that recognize an antigen specifically recognized by the TCR of an airT cell comprising said TCR polypeptide encoded by said at least one introduced polynucleotide / nucleic acid; (ii) suppression of the expression of inflammatory cytokines or inflammatory mediators by effector T cells that recognize an antigen specifically recognized by the TCR of airT cells comprising said TCR polypeptide encoded by said at least one introduced polynucleotide / nucleic acid; (iii) production of one or more immunosuppressive cytokines or anti-inflammatory products by airT cells, e.g., release of immunosuppressive cytokines or perforin / granzymes, induction of indoleamine-2,3-dioxygenase (IDO), competition for IL2 or adenosine, catabolism of tryptophan, or expression of inhibitory receptors by airT cells, or the establishment of one or more inhibitory mechanisms; (iv) suppression of either or both of the activation and proliferation of effector T cells that do not recognize the antigen specifically recognized by the TCR of airT cells comprising the TCR polypeptide encoded by the at least one introduced polynucleotide / nucleic acid; and (v) suppression of the expression of inflammatory cytokines or inflammatory mediators by effector T cells that do not recognize the antigen specifically recognized by the TCR of said airT cells comprising said TCR polypeptide encoded by said at least one introduced polynucleotide / nucleic acid, or (vi). It may include one or more of: In some embodiments, such antigenic stimulation of airT cells is HLA-restricted.

[0404] In some embodiments, the generation of airT cells of the present invention stably expressing FoxP3 described herein overcomes the drawbacks of conventional methodologies for expressing a FOXP3 transgene via retroviral or lentiviral gene transfer. Cell populations virally transduced with FoxP3 using these conventional methodologies are genetically heterogeneous, resulting from random integration of the FOXP3 transgene at various genomic sites with varying stability and expression levels. While such transduced cell populations at least transiently exhibit Treg characteristics, such as phenotypic markers and cytokine expression profiles, they are susceptible to associated genotoxicity and susceptibility to silencing by regulatory elements localized at the viral integration site.

[0405] To avoid such risks, in some embodiments provided herein, the FOXP3 gene is artificially modified using specific targeted gene editing rather than virally mediated FOXP3 gene transfer. Furthermore, gene editing may be specifically targeted to the TCR. In certain embodiments described herein, lentivirus-mediated gene delivery is used to introduce an autoimmune-associated TCR candidate (or CAR candidate) into T cells, such as CD4 T cells or CD8 T cells, and the FOXP3 gene in these T cells is then edited to stably express FoxP3. In some related embodiments, this method is used in conjunction with one of the TRAC locus editing methods disclosed herein, e.g., in conjunction with a gene editing method that simultaneously deletes (e.g., deletes by inactivation (also referred to herein as "knockout")) the endogenous TCR gene.

[0406] As an alternative to lentiviral delivery of TCRs, some embodiments described herein relate to simultaneous gene editing of separate alleles at the same locus, for example, dual editing methods in which two alleles are edited at one locus by making two edits at one locus (e.g., using one guide RNA and multiple AAV donor homology constructs). In some embodiments, the method for gene editing two alleles at a locus comprises: i) inserting a first donor template into a first allele of a locus; and ii) inserting a second donor template into a second allele of the same locus. Includes. Each donor template may be inserted into an allele of a locus by a gene editing method provided herein. In some embodiments, the method includes providing a nucleic acid encoding an RNA-guided nuclease or an RNA-guided endonuclease and a guide RNA that guides the RNA-guided nuclease to cleave a nucleotide sequence within the locus. In this way, the RNA-guided nuclease cleaves the nucleotide sequences of the two alleles within the locus, and each donor template is inserted into a separate allele within the locus by homologous recombination repair. Because the two donor templates compete for one double-strand break, one copy of each donor template is integrated, resulting in a predetermined subpopulation of cells in which two alleles have been edited. In some embodiments, the method for gene editing two alleles within a locus includes providing cells with a nuclease (e.g., meganuclease, TALEN, or ZFN) that cleaves the nucleotide sequences of the two alleles within a locus. For either single allele modification or double allele modification, a combination of homology recombination repair template and locus (such as the TRAC locus in primary T cells) that shows a very high homology recombination repair rate can be used, and a low- or high-efficiency endonuclease can be selected. Alternatively, gRNA can be selected to more efficiently edit one allele. Any method can be used to evaluate single allele modification or double allele modification, for example, sequence analysis can be used.

[0407] Alternatively, some embodiments described herein relate to gene editing two different loci simultaneously, e.g., dual editing methods in which two loci are edited by performing separate gene edits at each of the two loci (e.g., using two different guide RNAs and multiple AAV donor homology cassettes specific for each locus). In some embodiments, the method of gene editing two different loci comprises: i) inserting a first donor template into a first locus; and ii) inserting the second donor template into a second locus different from the first locus. Includes. Each donor template may be inserted into each locus by the gene editing method provided herein. In some embodiments, the method includes providing a nucleic acid encoding an RNA-guided nuclease or an RNA-guided endonuclease, a first guide RNA for guiding the RNA-guided nuclease to cleave a nucleotide sequence within a first locus, and a second guide RNA for guiding the RNA-guided nuclease to cleave a nucleotide sequence within a second locus. In this manner, after each guide RNA guides the RNA-guided nuclease to each locus, the RNA-guided nuclease cleaves the two loci, and each donor template is inserted into each locus by homologous recombination repair. In some embodiments, a method for gene editing two different loci includes providing a cell with a first nuclease (e.g., a meganuclease, TALEN, or ZFN) that cleaves a nucleotide sequence within a first locus and a second nuclease (e.g., a meganuclease, TALEN, or ZFN) that cleaves a nucleotide sequence within a second locus. As previously mentioned, the insertion of the first donor template into the first locus and the insertion of the second donor template into the second locus can be performed by: (i) a first polypeptide comprising a nucleic acid encoding a first CISC component comprising a first extracellular binding domain, a first transmembrane domain, and a first intracellular signaling domain; (ii) a second polypeptide comprising a second nucleic acid encoding a second CISC component comprising a second extracellular binding domain, a transmembrane domain, and a second intracellular signaling domain; and (iii) a first endonuclease capable of cleaving a first nucleotide sequence within a first locus, or a nucleic acid encoding the first endonuclease; (iv) a second endonuclease capable of cleaving a second nucleotide sequence within the second locus, or a nucleic acid encoding the second endonuclease. to integrate the first polynucleotide or fragment thereof into the first locus and insert the second polynucleotide or fragment thereof into the second locus. This can be done by:

[0408] These methods may be used to deliver a recombinant FOXP3 gene and a recombinant TCR gene (or another payload) to one specific locus or to two different specific loci. Additionally, as described herein, in some embodiments, the method may further include incorporating a split chemo-inducible signaling complex (split CISC) component to selectively expand only T cells that express both FOXP3 and the inserted TCR, thereby enriching for air T cells. Provided herein is a method for selectively expanding cells, comprising: (i) inserting (a) a first polynucleotide molecule comprising a nucleic acid encoding a first CISC component and (b) a second polynucleotide molecule comprising a nucleic acid encoding a second CISC component into one or more cells in the cell population; and (ii) contacting the cells with a CISC-inducing molecule to promote proliferation and / or activation of cells expressing both the first CISC component and the second CISC component. Including, Methods are provided, wherein a first CISC component and a second CISC component are capable of specifically binding to and dimerizing in the presence of said CISC-inducing molecule. In some embodiments, the first polynucleotide molecule or the second polynucleotide molecule comprises a nucleic acid encoding a third CISC component that is soluble in the cytoplasm and capable of specifically binding to the CISC-inducing molecule, and expression of this third CISC component can block or attenuate the effect of the CISC-inducing molecule on the endogenous signaling pathway. Any transfection or genome modification method can be used to insert the first nucleic acid or nucleic acid molecule and the second nucleic acid or nucleic acid molecule. As previously mentioned, in some embodiments, the insertion of the first donor template into the first locus and the insertion of the second donor template into the second locus is performed by: (i) a first nucleic acid comprising a nucleic acid encoding a first CISC component comprising a first extracellular binding domain, a first transmembrane domain, and a first intracellular signaling domain; (ii) a second nucleic acid comprising a second nucleic acid encoding a second CISC component comprising a second extracellular binding domain, a transmembrane domain, and a second intracellular signaling domain; and (iii) a first endonuclease capable of cleaving a first nucleotide sequence within a first locus, or a nucleic acid encoding the first endonuclease; (iv) a second endonuclease capable of cleaving a second nucleotide sequence within the second locus, or a nucleic acid encoding the second endonuclease. to integrate the first polynucleotide or fragment thereof into the first locus and insert the second polynucleotide or fragment thereof into the second locus. This can be done by:

[0409] Chemical-inducible signaling complex (CISC) As described herein, some embodiments utilize a split chemical-induced signaling complex (CISC)-based method. In this method, (i) a FoxP3 gene product constitutively expressed by editing the FoxP3 gene and (ii) a heterologous TCR gene product introduced by introducing an edited heterologous TCR gene are expressed in the same cell, resulting in gene-edited airT cells, which may then be selectively expanded. Expression of the FoxP3 gene product constitutively expressed by editing the FoxP3 gene is associated with cell surface expression of a first CISC component that specifically binds to a CISC-inducing molecule. The first CISC component exists as a transmembrane fusion protein having an extracellular domain that binds to the first CISC-inducing molecule, a transmembrane domain, and a first activation signaling intracellular domain. Expression of the heterologous TCR gene product by introduction of the edited heterologous TCR gene is associated with cell surface expression of a second CISC component, separate from the first CISC component, that specifically binds to the CISC-inducing molecule, where the second CISC component exists as a transmembrane fusion protein having an extracellular domain that binds to the second CISC-inducing molecule, a transmembrane domain, and a second activation signaling intracellular domain that is separate from the first activation signaling intracellular domain. In some embodiments, the first CISC component comprises an FKBP extracellular domain and an IL2RB intracellular domain, and the second CISC component comprises an FRB extracellular domain and an IL2RG intracellular domain. In some embodiments, the first CISC component comprises an FKBP extracellular domain and an IL2RG intracellular domain, and the second CISC component comprises an FRB extracellular domain and an IL2RB intracellular domain. In some embodiments, one or both of the first CISC component and the second CISC component comprise a hinge domain disposed between the extracellular domain and the transmembrane domain. In some embodiments, the first CISC component and the second CISC component each comprise a hinge domain disposed between the extracellular domain and the transmembrane domain.

[0410] In certain embodiments, CD3+ T cells, CD4+ T cells, or CD8+ T cells are obtained by enrichment from a biological sample, such as peripheral blood mononuclear cells (PBMCs), prior to undergoing gene editing (e.g., dual editing) as described herein. In certain embodiments, the CD3+ T cells, CD4+ T cells, or CD8+ T cells obtained by enrichment are non-specifically activated (e.g., using anti-CD3 and anti-CD28 antibodies immobilized on a solid phase) prior to undergoing gene editing (e.g., dual editing) as described herein.

[0411] In some embodiments, exposure of dual-edited T cells described herein to a CISC-inducing molecule results in binding of the CISC-inducing molecule to the extracellular domains of the first and second CISC components, forming a heterodimer of the first and second CISC components and activating a functional signaling complex formed by the first and second activation signaling intracellular domains. In this manner, expression of the first and second CISC components in an airT cell population results in expression of the first and second CISC components and a payload (e.g., FoxP3 and a heterologous TCR), allowing the airT cell population to be selectively expanded in culture conditions that provide the CISC-inducing molecule but not endogenous signaling substances.

[0412] In some embodiments, in the airT cells of the invention, the two gene edits that respectively generate a first CISC component that is co-expressed with a FoxP3 gene product and a second CISC component that is co-expressed with a TCR gene product may be designed to occur at separate alleles of the same locus (e.g., a dual-editing method that edits two alleles) or at two different loci (e.g., a dual-editing method that edits two loci). In some embodiments, the method for gene editing two alleles at a locus comprises: i) inserting a first donor template into a first allele of a locus; and ii) inserting a second donor template into a second allele of the same locus. Includes. Each donor template may be inserted into an allele of a locus by a gene editing method provided herein. In some embodiments, the method includes providing a nucleic acid encoding an RNA-guided nuclease or an RNA-guided endonuclease and a guide RNA that guides the RNA-guided nuclease to cleave a nucleotide sequence within the locus. In this way, the RNA-guided nuclease cleaves the nucleotide sequences of the two alleles within the locus, and each donor template is inserted into a separate allele within the locus by homology-directed repair. In some embodiments, the method for gene editing two alleles within the same locus includes providing a cell with a nuclease (e.g., meganuclease, TALEN, or ZFN) that cleaves the nucleotide sequences of the two alleles within the locus.

[0413] In some embodiments, the method of gene editing two different loci comprises: i) inserting a first donor template into a first locus; and ii) inserting the second donor template into a second locus different from the first locus. Includes. Each donor template may be inserted into each locus by the gene editing method provided herein. In some embodiments, the method includes providing a nucleic acid encoding an RNA-guided nuclease or an RNA-guided endonuclease, a first guide RNA for guiding the RNA-guided nuclease to cleave a nucleotide sequence within a first locus, and a second guide RNA for guiding the RNA-guided nuclease to cleave a nucleotide sequence within a second locus. In this manner, after each guide RNA guides the RNA-guided nuclease to each locus, the RNA-guided nuclease cleaves the two loci, and each donor template is inserted into each locus by homologous recombination repair. In some embodiments, a method for gene editing two different loci includes providing a cell with a first nuclease (e.g., a meganuclease, TALEN, or ZFN) that cleaves a nucleotide sequence within a first locus and a second nuclease (e.g., a meganuclease, TALEN, or ZFN) that cleaves a nucleotide sequence within a second locus.

[0414] In some embodiments, a locus includes a nucleic acid sequence encoding a gene. In some embodiments, a locus includes a nucleic acid including one or more exons encoding a gene and one or more nucleotides between the one or more exons. In some embodiments, a locus includes a nucleic acid including one or more regulatory elements and a nucleic acid sequence encoding a gene. In some embodiments, a gene in a genome includes multiple loci. In some embodiments, the multiple loci include multiple nucleic acid sequences that do not share a common nucleotide. In some embodiments, the multiple loci include multiple nucleic acid sequences, and no nucleotides belong to more than one locus. In some embodiments, a third CISC component that specifically binds to a CISC-inducing molecule may be coexpressed with the FoxP3 gene product or the TCR gene product. When expressed, this third CISC component remains intracellularly and acts as a decoy that binds to CISC-inducing molecules that may enter the cell, preventing toxicity caused by the CISC-inducing molecules.

[0415] Details of the CISC system, including the structures of the first, second, and third CISC components and the CISC-inducing molecule, are described elsewhere herein and in WO / 2018 / 111834 and WO / 2019 / 210078 (both of which are expressly incorporated herein by reference in their entirety). Briefly, WO / 2018 / 111834 describes compositions and methods for genetically editing host cells by knocking in (inserting) a genetic construct encoding a chemically inducible signaling complex (CISC), a fusion protein capable of dimerization via a ligand. By expressing the two subunits of this fusion protein in cells and then exposing the host cells to a chemical ligand, the dimerization of these subunits can be induced to form a CISC, which then induces the transmission of a cell activation signal. Thus, by utilizing the CISC system, cells into which two CISC components have been genetically engineered can be selected and expanded (e.g., activated to induce proliferation). WO / 2019 / 210078 also describes a gene editing composition and a gene editing method for introducing a nucleic acid sequence encoding a first CISC subunit component and a nucleic acid sequence encoding a second CISC subunit component into a host cell as part of a single-targeted gene editing of the FOXP3 locus, TRAC locus, or AAVS1 locus. Induction of CISC subunit dimerization via a chemical ligand induces biological signaling, allowing for the selection and expansion of gene-edited cells. In some related embodiments, a nucleic acid encoding a third CISC subunit component may be expressed in the host cell. When expressed in the cell, this third CISC subunit remains within the cell and can function as a decoy to suppress the harmful effects of a CISC ligand that has entered the cell.

[0416] Each of the exemplary first and second CISC subunit components may include a functional intracellular signaling domain of the IL2 receptor beta subunit (IL2RB) or a functional intracellular signaling domain of the IL2 receptor gamma subunit (IL2RG). An exemplary third CISC component may include a rapamycin-binding functional domain of FK506-binding protein (FKBP). An exemplary third CISC component may include an FKBP-rapamycin-binding protein (FRB) that binds to the complex of FKBP and rapamycin.

[0417] Polynucleotides containing nucleic acids encoding CISC components Some embodiments of the methods provided herein include inserting into the genome of a cell (i) a first polynucleotide molecule comprising a first nucleic acid encoding a first CISC component and (ii) a second polynucleotide molecule comprising a second nucleic acid encoding a second CISC component. In some embodiments, the first polynucleotide molecule comprises a nucleic acid encoding a first CISC component comprising an FKBP extracellular domain, a transmembrane domain, and an IL2RB intracellular signaling domain, and the second polynucleotide molecule comprises a second nucleic acid encoding a second CISC component comprising an FRB extracellular domain, a transmembrane domain, and an IL2RB intracellular signaling domain. In some embodiments, the first polynucleotide molecule comprises a nucleic acid encoding a first CISC component comprising an FKBP extracellular domain, a transmembrane domain, and an IL2RG intracellular signaling domain, and the second polynucleotide molecule comprises a second nucleic acid encoding a second CISC component comprising an FRB extracellular domain, a transmembrane domain, and an IL2RB intracellular signaling domain. In some embodiments, the nucleic acid encoding the first CISC component or the nucleic acid encodi...

Claims

1. A recombinant regulatory T (Treg) cell, comprising: (i) a first polynucleotide comprising a first promoter operably linked to a nucleotide sequence encoding a first chemical-inducible signaling complex (CISC) component; (ii) a second polynucleotide comprising a second promoter operably linked to a nucleotide sequence encoding a second CISC component; the first polynucleotide is in the genome of the cell, and the first CISC component comprises: (a) a first extracellular binding domain; (b) a first transmembrane domain; and (c) comprising a first cytoplasmic domain; the second polynucleotide is at the TRAC locus of the genome, and the second CISC element comprises: (a) a second extracellular binding domain; (b) a second transmembrane domain; and (c) comprising a second cytoplasmic domain; and wherein the first CISC component and the second CISC component dimerize in the presence of a ligand, thereby inducing signaling events and proliferation of the recombinant Treg cell; the first promoter is operably linked to a nucleotide sequence encoding FOXP3; (iii)(a)(1) the second promoter is operably linked to a nucleotide sequence encoding a chimeric antigen receptor (CAR); or (iii)(a)(2) the second promoter is operably linked to (A) a nucleotide sequence encoding a T cell receptor (TCR) α protein and (B) a nucleotide sequence encoding a TCR β protein; or (iii) (b) the second promoter is operably linked to (A) a nucleotide sequence encoding a TCR β protein and (B) a first portion of a TCR α protein, the first portion of the TCR α protein comprising a TCR α variable region and a TCR α joining region, the nucleotide sequence encoding the first portion of the TCR α protein is in frame with an endogenous nucleotide sequence encoding a second portion of the TCR α protein, the second portion comprising at least a portion of a TCR α constant region. Recombinant Treg cells.

2. The recombinant Treg cell of claim 1, wherein the first promoter is a constitutive promoter and the second promoter is a constitutive promoter.

3. The recombinant Treg cell of claim 1 or 2, wherein the first promoter is an MND promoter and the second promoter is an MND promoter.

4. The recombinant Treg cell of any one of claims 1 to 3, wherein the second promoter is operably linked to a nucleotide sequence encoding the CAR.

5. The recombinant Treg cell of any one of claims 1 to 3, wherein the second promoter is operably linked to (A) a nucleotide sequence encoding the TCR α protein and (B) a nucleotide sequence encoding the TCR β protein.

6. 4. The recombinant Treg cell of any one of claims 1 to 3, wherein the second promoter is operably linked to (A) a nucleotide sequence encoding the TCR β protein and (B) the first portion of the TCR α protein, the first portion of the TCR α protein comprising the TCR α variable region and the TCR α joining region, and wherein the nucleotide sequence encoding the first portion of the TCR α protein is in frame with an endogenous nucleotide sequence encoding the second portion of the TCR α protein.

7. The recombinant Treg cell according to any one of claims 1 to 6, which is a CD8+ Treg cell.

8. The recombinant Treg cell according to any one of claims 1 to 6, which is a CD4+ Treg cell.

9. (i)(a) the first CISC component comprises, in order from N-terminus to C-terminus, an FK506 binding protein (FKBP) domain, an IL-2 receptor gamma (IL-2Rγ) transmembrane domain, and an IL-2Rγ cytoplasmic domain; and (i)(b) the second CISC component comprises, in order from N-terminus to C-terminus, an FKBP-rapamycin binding (FRB) domain, an IL-2 receptor beta (IL-2Rβ) transmembrane domain, and an IL-2Rβ cytoplasmic domain; or (ii)(a) the first CISC component comprises, in order from N-terminus to C-terminus, an FRB domain, an IL-2Rβ transmembrane domain and an IL-2Rβ cytoplasmic domain; and (ii)(b) the second CISC component comprises, in order from N-terminus to C-terminus, an FKBP domain, an IL-2Rγ transmembrane domain and an IL-2Rγ cytoplasmic domain. The recombinant Treg cell according to any one of claims 1 to 8.

10. The recombinant Treg cell of any one of claims 1 to 9, wherein the ligand is rapamycin.

11. 11. The recombinant Treg cell of any one of claims 1 to 10, wherein the first promoter is operably linked to a first coding exon of an endogenous FOXP3 gene in the Treg cell and downstream of a Treg-specific demethylated region (TSDR) of the endogenous FOXP3 gene.

12. The recombinant Treg cell of any one of claims 1 to 10, wherein the nucleotide sequence encoding FOXP3 is a FOXP3 cDNA sequence.

13. A method for producing recombinant regulatory T (Treg) cells, comprising: The method comprises contacting a cell with (i) a first polynucleotide comprising a first promoter operably linked to a nucleotide sequence encoding a first CISC component; and (ii) a second polynucleotide comprising a second promoter operably linked to a nucleotide sequence encoding a second CISC component, the first CISC component comprising: (a) a first extracellular binding domain; (b) a first transmembrane domain; and (c) comprising a first cytoplasmic domain; the second CISC component comprising: (a) a second extracellular binding domain; (b) a second transmembrane domain; and (c) comprising a second cytoplasmic domain; and wherein the first CISC component and the second CISC component dimerize in the presence of a ligand to induce signaling events and proliferation of the cell; the first polynucleotide is inserted into a FOXP3 locus in the genome of the cell, and after insertion, at the FOXP3 locus, the first promoter is operably linked to a nucleotide sequence encoding FOXP3; the second polynucleotide is inserted into the TRAC locus of the genome, and after the insertion, at the TRAC locus: (iii)(a)(1) the second promoter is operably linked to a nucleotide sequence encoding a chimeric antigen receptor (CAR); or (iii)(a)(2) the second promoter is operably linked to (A) a nucleotide sequence encoding a T cell receptor (TCR) α protein and (B) a nucleotide sequence encoding a TCR β protein; or (iii) (b) the second promoter is operably linked to (A) a nucleotide sequence encoding a TCR β protein and (B) a first portion of a TCR α protein, the first portion of the TCR α protein comprising a TCR α variable region and a TCR α joining region, the nucleotide sequence encoding the first portion of the TCR α protein is in frame with an endogenous nucleotide sequence encoding a second portion of the TCR α protein, the second portion comprising at least a portion of a TCR α constant region. method.

14. 14. The method of claim 13, wherein the first promoter is a constitutive promoter and the second promoter is a constitutive promoter.

15. 15. The method of claim 13 or 14, wherein the first promoter is an MND promoter and the second promoter is an MND promoter.

16. The method of any one of claims 13 to 15, wherein the second promoter is operably linked to a nucleotide sequence encoding the CAR.

17. The method of any one of claims 13 to 15, wherein the second promoter is operably linked to (A) a nucleotide sequence encoding the TCR α protein and (B) a nucleotide sequence encoding the TCR β protein.

18. 16. The method of any one of claims 13 to 15, wherein the second promoter is operably linked to (A) a nucleotide sequence encoding the TCRβ protein and (B) the first portion of the TCRα protein, the first portion of the TCRα protein comprising the TCRα variable region and the TCRα joining region, and the nucleotide sequence encoding the first portion of the TCRα protein is in frame with an endogenous nucleotide sequence encoding the second portion of the TCRα protein.

19. The method of any one of claims 13 to 18, wherein the cell is a CD8+ cell.

20. The method of any one of claims 13 to 18, wherein the cell is a CD4+ cell.

21. The method according to any one of claims 13 to 18, wherein the cells are hematopoietic stem cells or progenitor stem cells.

22. (i)(a) the first CISC component comprises, in order from N-terminus to C-terminus, an FK506 binding protein (FKBP) domain, an IL-2 receptor gamma (IL-2Rγ) transmembrane domain, and an IL-2Rγ cytoplasmic domain; and (i)(b) the second CISC component comprises, in order from N-terminus to C-terminus, an FKBP-rapamycin binding (FRB) domain, an IL-2 receptor beta (IL-2Rβ) transmembrane domain, and an IL-2Rβ cytoplasmic domain; or (ii)(a) the first CISC component comprises, in order from N-terminus to C-terminus, an FRB domain, an IL-2Rβ transmembrane domain and an IL-2Rβ cytoplasmic domain; and (ii)(b) the second CISC component comprises, in order from N-terminus to C-terminus, an FKBP domain, an IL-2Rγ transmembrane domain and an IL-2Rγ cytoplasmic domain. The method according to any one of claims 13 to 21.

23. The method of any one of claims 13 to 22, wherein the ligand is rapamycin.

24. 24. The method of any one of claims 13 to 23, wherein the first promoter is inserted so as to be operably linked to a first coding exon of an endogenous FOXP3 gene in the Treg cell and is downstream of a Treg-specific demethylation region (TSDR) of the endogenous FOXP3 gene.

25. The method of any one of claims 13 to 23, wherein the nucleotide sequence encoding FOXP3 is a FOXP3 cDNA sequence.

26. The cells, (i) a first DNA endonuclease that targets the FOXP3 locus, or a nucleic acid encoding the first DNA endonuclease; (ii) a second DNA endonuclease that targets the TRAC locus, or a nucleic acid encoding the second DNA endonuclease. The method of any one of claims 13 to 25, further comprising contacting

27. The cells, (i) RNA-guided DNA endonucleases; (ii) a first guide RNA (gRNA) comprising a spacer sequence that targets the FOXP3 locus, or a nucleic acid encoding the first gRNA; and (iii) a second gRNA comprising a spacer sequence targeting the TRAC locus, or a nucleic acid encoding the second gRNA The method of any one of claims 13 to 25, further comprising contacting

28. The method of any one of claims 13 to 27, further comprising contacting said cell with said ligand.

29. A recombinant Treg cell produced by the method according to any one of claims 13 to 28.

30. 30. The recombinant Treg cell of any one of claims 1 to 12 and 29 for use in a method for treating, alleviating or suppressing a disease in a subject in need thereof.

31. 31. The recombinant Treg cell for use according to claim 30, wherein said ligand is rapamycin and said method comprises administering rapamycin to said subject.

32. 32. The recombinant Treg cell for use according to claim 30 or 31, wherein the disease is selected from the group consisting of an autoimmune disease, an inflammatory disease, an alloimmune disease and an allergic disease.

33. A recombinant Treg cell for use according to any one of claims 30 to 32, which is an autologous cell of the subject.

34. A recombinant Treg cell for use according to any one of claims 30 to 32, which is an allogeneic cell of the subject.