Antigen-specific artificial immunoregulatory t (AIRT) cells

Abstract

To provide stable antigen-specific immunoregulatory cells that can maintain antigen-specific immunosuppression in vitro and in vivo without exhibiting plasticity and are useful for administration to a subject in need of antigen-specific immunosuppression by adoptive transfer immunotherapy.SOLUTION: The present invention includes gene-edited artificial immunoregulatory T cells (airT cells), and the airT cells include FoxP3 gene products constitutively expressed at an expression level equal to or higher than that of natural regulatory T cells (Treg cells or suppressor T cells), and introduced T cell receptors (TCRs) (for example, TCRs artificially produced by gene-editing, transduction with viral vectors, transfection, or other genetic engineering methodologies). In some embodiments, it is preferred that the TCR is specific for an antigen associated with an autoimmune, allergic or other inflammatory disease.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 987,810, filed March 10, 2020, entitled "Antigen-Specific Artificial Immunoregulatory T (airT) Cells," and U.S. Provisional Patent Application No. 62 / 867670, filed June 27, 2019, entitled "Antigen-Specific Treg Therapy for Autoimmune Disease," which are expressly incorporated by reference in their entireties.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with government support under Contract No. U01AI101981 awarded by the National Institutes of Health (NIH) and Contract No. W81XWH-15-1-0003 awarded by the Department of Defense. The U.S. Government has certain rights in this invention.

[0003] Sequence Listing Reference This application has been filed with an electronic Sequence Listing, which is provided as an approximately 550 kb file created on June 23, 2020, under the file name SCRI252WOSEQLIST. The information set forth in this electronic Sequence Listing is incorporated herein by reference in its entirety.

[0004] Some embodiments provided herein include antigen-specific artificial immune regulatory T (airT) cells. The airT cells of the present invention include artificial immune system T lymphocytes that have been stably reprogrammed by gene editing to exhibit specific regulatory T cell (Treg) properties and engineered to express a desired functional T cell antigen receptor (TCR) or other antigen receptor (e.g., a chimeric antigen receptor (CAR)) by gene editing, viral vector transduction, transfection, or other genetic engineering methodologies. In some embodiments, the airT cells of the present invention can exert immunosuppressive activity in response to recognition of a specific antigen by their TCR. [Background technology]

[0005] 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.

[0006] 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).

[0007] 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).

[0008] 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).

[0009] Thus, there remains a need for stable antigen-specific immunoregulatory cells that can maintain antigen-specific immunosuppressive properties 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. The embodiments provided herein address this need and offer related advantages in addition to those discussed above. Summary of the Invention [Means for solving the problem]

[0010] Some embodiments of the methods and compositions provided herein comprise antigen-specific CD4+CD25+ artificial immunoregulatory T (airT) cells, (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; Contains airT cells.

[0011] In some embodiments, antigen-specific CD4+CD25+ artificial immunoregulatory T (airT) cells are obtained by artificially modifying the forkhead box protein 3 / winged helix transcription factor (FOXP3) gene in CD4+CD25- T cells and introducing at least one polynucleotide encoding an antigen-specific T cell receptor (TCR) polypeptide, The artificial modification provides airT cells that constitutively express the FOXP3 gene product at an expression level equal to or greater than that of natural regulatory T (Treg) cells.

[0012] In some embodiments, the FOXP3 gene is present in a FOXP3 locus that includes an intronic regulatory T cell (Treg)-specific demethylated region (TSDR) having multiple cytosine-guanine (CG) dinucleotides, each CG dinucleotide containing a methylated cytosine (C) nucleotide at a nucleotide position containing a demethylated C nucleotide in naturally occurring Treg cells. In some embodiments, at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the C nucleotides in the TSDR of naturally occurring Treg cells are methylated at the nucleotide positions containing a demethylated C nucleotide. 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 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.

[0013] In some embodiments, the artificial modification comprises insertion of a nucleic acid molecule comprising a constitutively active promoter into the native FOXP3 locus, the promoter positioned in the FOXP3 gene such that it can promote transcription of a nucleotide sequence encoding endogenous FOXP3 at the FOXP3 locus. In some embodiments, the inserted nucleic acid molecule 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, 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 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 the MND promoter. In some embodiments, the artificial modification comprises inserting into the native FOXP3 locus a nucleic acid molecule comprising a polynucleotide encoding exogenous FOXP3 and a constitutively active promoter operably linked thereto. 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 chemo-inducible 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 or second CISC component and capable of specifically binding to the CISC-inducing molecule.

[0014] 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.

[0015] In some embodiments, the artificial modification comprises 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 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 knocked out or inactivated at least one native TCR locus is the 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 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, 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 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 an exogenous FOXP3-encoding polynucleotide and a constitutively active promoter operably linked thereto has been inserted is within the T-cell receptor alpha chain (TRAC) locus.

[0016] 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.

[0017] 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.

[0018] 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.

[0019] 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 airT cell comprises 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 cell expresses 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 is 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 the activation and / or proliferation of effector T cells that recognize an antigen specifically recognized by the airT cell TCR comprising the TCR polypeptide encoded by the 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 airT cell TCR comprising the TCR polypeptide encoded by the at least one introduced polynucleotide; (iii) the production by the airT cells of one or more immunosuppressive cytokines, perforin / granzymes, or anti-inflammatory products, or the 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 the activation and / or proliferation of effector T cells that do not recognize the antigen specifically recognized by the airT cell TCR comprising the TCR polypeptide encoded by the at least one introduced polynucleotide. Contains one or more of the following:

[0020] In some embodiments, the TCR specifically recognizes an antigen associated with the pathogenesis of an autoimmune, allergic, or inflammatory disease. In some embodiments, (i) the autoimmune disease is selected from type 1 diabetes, multiple sclerosis, systemic lupus erythematosus, myasthenia gravis, rheumatoid arthritis, Crohn's disease, bullous pemphigoid, pemphigus vulgaris, autoimmune hepatitis, psoriasis, Sjogren's syndrome, and celiac disease; (ii) the allergic disease is selected from allergic asthma, pollen allergy, food allergy, drug hypersensitivity, and contact dermatitis; (iii) the inflammatory disease is selected from pancreatic islet cell transplantation, asthma, hepatitis, inflammatory bowel disease (IBD), ulcerative colitis, graft-versus-host disease (GVHD), transplant tolerance induction, transplant rejection, and sepsis. 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.

[0021] 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 any of Figures 139-140.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.

[0022] 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 the activation and / or proliferation of effector T cells that recognize an antigen specifically recognized by the airT cell TCR comprising the TCR polypeptide encoded by the 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 airT cell TCR comprising the TCR polypeptide encoded by the at least one introduced polynucleotide; (iii) the production by the airT cells of one or more immunosuppressive cytokines, perforin / granzymes, or anti-inflammatory products, or the 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 the activation and / or proliferation of effector T cells that do not recognize the antigen specifically recognized by the airT cell TCR comprising the TCR polypeptide encoded by the at least one introduced polynucleotide. Contains one or more of the following:

[0023] 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, which recognizes the IGRP peptide (residues 241 to 270) in an HLA DRB1*0404-restricted manner; or (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.

[0024] Some embodiments of the methods and compositions provided herein include any of the airT cells for use in treating, suppressing, or alleviating an autoimmune disease, an allergic disease, or an inflammatory disease, wherein the autoimmune disease is such as an autoimmune disease selected from type 1 diabetes, multiple sclerosis, systemic lupus erythematosus, myasthenia gravis, rheumatoid arthritis, Crohn's disease, bullous pemphigoid, pemphigus vulgaris, and autoimmune hepatitis; the allergic disease is such as an allergic disease selected from allergic asthma, pollen allergy, food allergy, drug hypersensitivity, and contact dermatitis; and the inflammatory disease is such as an inflammatory disease selected from pancreatic islet cell transplantation, asthma, hepatitis, inflammatory bowel disease (IBD), ulcerative colitis, graft-versus-host disease (GVHD), transplant tolerance induction, transplant rejection, and sepsis.

[0025] 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).

[0026] 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.

[0027] In some embodiments, the antigen comprises an epitope selected from the group consisting of Enol326, CILP297-1, Vim418, Agg520, and SLE3.

[0028] 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.

[0029] In some embodiments, the TCR polypeptide comprises a CD3α polypeptide having an amino acid sequence set forth in any of SEQ ID NOs: 1377 to 1390; and / or a CD3β polypeptide having an amino acid sequence set forth in any of SEQ ID NOs: 1377 to 1390.

[0030] Some embodiments of the methods and compositions provided herein include pharmaceutical compositions comprising any of the above airT cells and a pharmaceutically acceptable excipient.

[0031] Some embodiments of the methods and compositions provided herein include the use of any of the above airT cells as a medicament.

[0032] 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 or inactivate the native FOXP3 locus in CD4+ T cells and 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 within the native forkhead box protein 3 / winged helix transcription factor (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 said CD4+ T cells with at least one retroviral vector comprising a polynucleotide encoding said antigen-specific T cell receptor (TCR) polypeptide; and (ii) under conditions and for a time sufficient to knock out or inactivate the native T cell receptor alpha chain (TRAC) locus of said CD4+ T cells and 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; into the CD4+ T cells. is selected from.

[0033] 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 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, under conditions and for a time sufficient to knock out or inactivate the native T cell receptor alpha chain (TRAC) locus of said CD4+ T cells and insert all or a portion of a second TRAC locus donor template nucleic acid; (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:

[0034] In some embodiments, there is provided a method for producing antigen-specific artificial immunoregulatory T (airT) cells, comprising: under conditions and for a time sufficient to knock out or inactivate the native T cell receptor alpha chain (TRAC) locus in CD4+ T cells by homologous recombination repair and 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 inserted donor templates (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; The other of the inserted donor templates (the second donor template) further comprises a nucleic acid sequence encoding a second CISC component that is separate from the first CISC component and capable of specifically binding to the CISC-inducing molecule. 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 embodiments, (a) the DNA endonuclease is selected from CRISPR / Cas, TALEN, meganuclease, megaTAL, and 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 are mutually exclusively selected from IL2RB and IL2RG; (e) The third CISC component is FKBP; (f) the CISC-inducing molecule is rapamycin or an analog thereof.

[0035] Some embodiments of the methods and compositions provided herein include a method of producing any of the above-described antigen-specific artificial immunoregulatory T (airT) cells, comprising performing any of the above-described methods of producing antigen-specific artificial immunoregulatory T (airT) cells.

[0036] In some embodiments, a method of 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) 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, (i) the autoimmune disease is selected from type 1 diabetes, multiple sclerosis, systemic lupus erythematosus, myasthenia gravis, rheumatoid arthritis, Crohn's disease, bullous pemphigoid, pemphigus vulgaris, and autoimmune hepatitis; (ii) the allergic disease is selected from allergic asthma, pollen allergy, food allergy, drug hypersensitivity, and contact dermatitis; (iii) the inflammatory disease is selected from pancreatic islet cell transplantation, asthma, hepatitis, inflammatory bowel disease (IBD), ulcerative colitis, graft-versus-host disease (GVHD), transplant tolerance induction, transplant rejection, and sepsis. 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.

[0037] Some embodiments of the methods and compositions provided herein include a method of treating or alleviating a subject having a disorder, comprising administering to the subject any of the artificial immunoregulatory T (airT) cells.

[0038] In some embodiments, the disorder is selected from the group consisting of type 1 diabetes, multiple sclerosis, systemic lupus erythematosus (SLE), myasthenia gravis, rheumatoid arthritis (RA), Crohn's disease, bullous pemphigoid, pemphigus vulgaris, autoimmune hepatitis; allergic diseases such as an allergic disease selected from allergic asthma, pollen allergy, food allergy, drug hypersensitivity, and contact dermatitis; and inflammatory diseases such as an inflammatory disease selected from pancreatic islet cell transplantation, asthma, hepatitis, inflammatory bowel disease (IBD), ulcerative colitis, graft-versus-host disease (GVHD), transplant tolerance induction, transplant rejection, and sepsis. 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).

[0039] 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.

[0040] In some embodiments, the TCR polypeptide comprises a CD3α polypeptide having an amino acid sequence set forth in any of SEQ ID NOs: 1377 to 1390; and / or a CD3β polypeptide having an amino acid sequence set forth in any of SEQ ID NOs: 1377 to 1390. [Brief explanation of the drawings]

[0041] [Figure 1A-11] This involves using gene editing to generate airT cells from human CD4+ T cells.

[0042] [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) and after (bottom) 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 plots on the right side of the figure show the expression of CD25, CD127, Helios, CD45RO, ICOS, and CTLA-4, respectively, in cells gated on Foxp3+GFP+.

[0043] [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.

[0044] [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 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. edTreg / airT cells were generated by editing only Foxp3 in Tconv cells using Cas9 / Foxp3 gRNA and AAV-MND-LNGFR-Foxp3 ki. 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 in Figure 3D shows a comparison of the expression levels of Foxp3, CTLA-4, and ICOS in edTreg / airT cells (blue) and nTreg cells (red). The bottom table in Figure 3D shows MFI.

[0045] [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.

[0046] [Figure 5] Representative islet-specific TCR lentiviral constructs expressing rare islet-specific TCRs derived from subjects with type 1 diabetes (T1D). Figure 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. Figure B shows the structure of the islet-specific TCRs expressed by lentivirus. These TCR constructs contain human TCR variable regions derived from islet-specific TCRs and mouse TCR constant regions, improving pairing between human TCR chains upon transduction.

[0047] [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 antigen-specific TCRs (LV), and analyzed for mTCRβ expression. Panel B shows flow cytometry analysis of CD4+ T cells transduced with lentivirus encoding islet antigen-specific TCRs, labeled with Cell Trace Violet (CTV), and 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.

[0048] [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 the results of gating on LNGFR+ cells and analyzing the expression of CD25, CD127, CTLA-4, and ICOS.

[0049] [Figure 8]Regarding a typical antigen-specific suppression assay according to the present disclosure, Figure A shows a timeline of the process for generating edTreg cells expressing islet-specific TCRs. Using MACS LNGFR beads, edTreg cells expressing islet-specific TCRs (non-lentiviral TCRs; T1D4 TCR or T1D5-1 TCR) were enriched based on LNGFR expression. The resulting LNGFR+ cells were aliquoted and cryopreserved until further experiments. Figure 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 alone without edTreg cells. After 1 or 4 days of incubation, cells were stained and analyzed by flow cytometry to measure cytokine production and Teff cell proliferation.

[0050] [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 alone without edTreg cells. After 4 days of coculture, cells were stained and Teff cell proliferation was analyzed by CTV dilution. Flow cytometry plots show proliferation of Teff cells gated on CD3+CD4+CTV+EF670-LNGFR-.

[0051] [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 co-cultured 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 alone without edTreg cells. After 1 day of co-culture, cells were contacted with BFA for 4 hours, stained, and analyzed for cytokine production from Teff cells. Flow cytometry plots show TNF, IFNγ, or IL-17 production from T1D4 Teff cells gated on CD4+CTV+EF670-.

[0052] [Figure 12-17] FIG. 1 shows generation and characterization of human Foxp3-edited antigen-specific human CD4+ T cells.

[0053] [Figure 12] A typical scheme for generating antigen-specific human edTreg / airT cells from peripheral blood cells (top panel) and the phenotype of FOXP3-edited antigen-specific human CD4+ T cells (bottom panel) are shown. The bottom panel shows representative flow cytometry plots (left panel) and the percentage (right panel) of GFP expression in 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).

[0054] [Figure 13]Characterization of FOXP3-edited antigen-specific human CD4+ T cells is shown. Figure 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. Figure 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 healthy controls, antigen-presenting cells (APCs), and 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.

[0055] [Figure 14]We demonstrate that antigen-specific edTreg / airT cells were successfully generated by peptide stimulation followed by Foxp3 editing. Figure 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, then activated with CD3 / CD28 activation beads and subjected to gene editing. To enhance the proliferation of antigen-specific Treg cells, beads were added to cells sorted by flow cytometry. Figure 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.

[0056] [Figure 15]Antigen-specific suppression by Foxp3-edited Treg / airT cells. Figure 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. Figure 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. Figure 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. Figure 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 minus the proliferation rate (%).

[0057] [Figure 16] We demonstrate that peptide stimulation can expand islet-specific T cells with multiple specificities. Figure 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. On day 13, islet-specific T cell proliferation was analyzed by tetramer staining. Figure B shows flow cytometry plots of islet-specific T cells stained with each tetramer or tetramer pool, gated on CD4+ cells.

[0058] [Figure 17] We demonstrate 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.

[0059] [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.

[0060] [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.

[0061] [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.

[0062] [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.

[0063] [Figure 21] This study analyzed 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. Figure 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. Figure B shows a histogram showing the CD3 knockout rate.

[0064] [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.

[0065] [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.

[0066] [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.

[0067] [Figure 25]We demonstrate that double-editing of the TRAC locus in human CD4+ cells results in a double-positive cell population. Figure 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 . Figure B shows histograms showing the percentage of double-negative, GFP-single-positive, mCherry-single-positive, and GFP / mCherry-double-positive cells in the double-edited cells.

[0068] [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 transmembrane and intracellular domains. 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.

[0069] [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.

[0070] [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.

[0071] [Figure 29]Representative data demonstrating the enrichment of double-edited cells using rapalogs are shown. Figure 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 . Figure B shows histograms showing the percentage of double-negative, GFP-single-positive, mCherry-single-positive, and GFP / mCherry-double-positive cells in the double-edited cells.

[0072] [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.

[0073] [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.

[0074] [Figure 32] Figure 1 shows data from a FACS analysis of 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.

[0075] [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, double-edited cells containing both CISC components and therefore T1D4 TCR and Foxp3 can be selectively expanded.

[0076] [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.

[0077] [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 in blue.

[0078] [Figure 35]Figure 1 shows FACS analysis data for CD3 knockout in mouse CD4+ T cells. Panel A shows flow cytometry plots showing 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.

[0079] [Figure 36] This figure shows an outline of a typical experiment using AAV to perform double editing to evaluate knock-in of two alleles. Figure A shows a schematic diagram of each AAV construct used in this experiment. After gene editing, GFP / BFP expression is induced by the MND promoter. Figure 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.

[0080] [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.

[0081] [Figure 38-43] Regarding the function of airT cells under antigen-specific in vivo conditions.

[0082] [Figure 38]Schematic diagram of the experimental design to test whether MOG-specific edTreg / airT cells (shown in white) can suppress effector T cells (Teff) in a mouse model of experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis.

[0083] [Figure 39] This study demonstrates that mouse FOXP3 TALENs catalyze efficient FOXP3 disruption and initiate non-destructive donor template recombination. Figure A shows the binding sites of the FOXP3 TALEN pair in the human FOXP3 gene. Figure B shows the target binding sites of the mouse FOXP3 TALEN pair in the mouse FOXP3 gene. Figure 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.

[0084] [Figure 40] Generation of edTreg / airT cells from antigen-specific mouse CD4+ T cells. Figure 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. Figure B shows flow cytometry plots demonstrating GFP expression in antigen-specific mouse CD4+ T cells two days after gene editing. Figure C shows the average percentage of GFP+ cells across multiple experiments (n=10). Figure D shows flow cytometry plots demonstrating the expression of Treg markers associated with mouse edTreg / airT cells.

[0085] [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. Figure 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. Figure 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.

[0086] [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.

[0087] [Figure 43]These data demonstrate that antigen-specific edTreg / airT cells suppress Teff cell proliferation in vivo. Figure 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. Figure B shows a bar graph summarizing the average percentage of cells that incorporated EdU in various cell subsets. Figure 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.

[0088] [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.

[0089] [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.

[0090] [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. Center 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.

[0091] [Figure 46] This study investigated the phenotype of cells transferred into the NSG adoptive transfer mouse model. Figure A shows the experimental design, indicating the number and type of cells administered to each group of mice. Figure B shows flow cytometry plots demonstrating the phenotype of Teff, edTreg / airT, and nTreg cells injected into NSG mice.

[0092] [Figure 47]Antigen-specific T cell function in the adoptive transfer NSG mouse model. Figure 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. Figure B shows a graph showing 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.

[0093] [Figure 48-51] Regarding the construction of a mouse AAV donor template design for generating airT cell products containing a selectable marker (LNGFR).

[0094] [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.

[0095] [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).

[0096] [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).

[0097] [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.

[0098] [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). Figure 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. Figure D shows the percent suppression as a function of dilution of Treg or mock cells (top panel). Furthermore, histograms of proliferation dyes when Treg cells or mock cells were cultured with Teff cells at various ratios are shown (bottom). 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.Figure 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.

[0099] [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.

[0100] [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.

[0101] [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.

[0102] [Figure 55]Figure 1 shows the double-editing rate of the human TRAC locus in CD4+ T cells from donor R003657 edited with a CISC construct with or without rapalog selection. Figure 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). Figure 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). Figure 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.

[0103] [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. Figure 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. Figure 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). Figure 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.

[0104] [Figure 57]We demonstrate that dual editing of the TRAC locus in human CD4+ T cells can generate antigen-specific air T cells selectable with rapalogs. Figure 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. Figure B shows a timeline of the key steps in this study: dual editing of CD4+ T cells with AAV / RNP, expansion in the presence of rapalogs, 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 was analyzed by flow cytometry. The cells were then expanded in medium containing 50 ng / ml human IL-2 or 100 nM rapalog. Five and eight days after gene editing, the enrichment of double-positive cells expressing HA-FoxP3 and TCR was assessed by flow cytometry. Figure C shows the enrichment of double-edited cells using rapalogs. Left: Flow cytometry plots analyzing HA-FoxP3 and TCR expression in double-edited cells expanded for 8 days in the presence of IL-2 or rapalog. Right: 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.

[0105] [Figure 58]These data demonstrate that lowering serum concentration improves the overall and double-editing rates of the TRAC locus. Figure 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. Figure 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 . Figure C shows histograms showing the percentage of double-negative, FOXP3-HA-positive, T1D4-positive, and FOXP3 / T1D4 double-positive cells in double-edited cells.

[0106] [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. Figure 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 are shown only for the condition using recovery medium containing 2.5% FBS. Figure 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.

[0107] [Figure 60]We describe a method for testing a dual-editing method for editing two loci in human CD4+ T cells. Figure 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 using the dual-editing method. 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. Figure 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.

[0108] [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. Figure 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 and when the cells were recovered in 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 percentages of double-negative, GFP-positive, mCherry-positive, and GFP / mCherry double-positive cell populations in double-edited cells.

[0109] [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 shown 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.

[0110] [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.

[0111] [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.

[0112] [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.

[0113] [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.

[0114] [Figure 67]This figure shows a typical method for double editing of a single gene locus using the TRAC locus promoter. A schematic diagram of an AAV construct for gene editing using homologous recombination repair (HRR) designed for double editing within the TRAC locus is shown. The top diagram shows a schematic diagram of an AAV construct (No. 3240) for FOXP3 expression and introduction of split CISC. The bottom diagram shows a schematic diagram of an AAV construct (No. 3258) that is knocked in-frame into exon 1 of the TRAC locus to induce expression of T1D4 TCR and split CISC using the endogenous promoter of the TRAC locus.

[0115] [Figure 68] This figure shows that editing the TRAC locus using homologous recombination repair disrupts TCR expression and allows robust transgene expression driven by the endogenous TRAC locus enhancer and promoter. Figure 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, disrupting endogenous TCR expression while driving expression driven by the endogenous TRAC locus promoter. Figure 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. Figure C shows flow cytometric analysis of CD3 and mCherry expression in cells 7 days after gene editing. Flow cytometry plots revealed significant mCherry expression along with loss of CD3 in edited cells compared with mock-edited and AAV-only controls.

[0116] [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.

[0117] [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.

[0118] [Figure 71]Dual editing of human CD4+ T cells using a decoy-CISC (split-DISC) construct. Figure 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.

[0119] [Figure 72]We demonstrate that double editing of human CD4+ T cells with a split-DISC construct results in rapamycin-selectable cells. Double editing was performed as described in Figure 70. Panel A shows flow cytometry plots of 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 measuring the percentage of double-negative, GFP-positive, mCherry-positive, and GFP / mCherry double-positive cells in double-edited cells after enrichment with IL-2, rapalog (AP21967), or rapamycin, or no drug treatment.

[0120] [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.

[0121] [Fig. 74-94] Additional schematics and data regarding the generation and characterization of mouse airT cells are provided.

[0122] [Figure 74] The repair template used for editing mouse Foxp3 is shown. A schematic diagram of the additional AAV.GFP.KI and AAV.LNGFR.P2A constructs generated is shown. These constructs were tested for editing efficiency, FOXP3 expression, and suppression function in mouse T cells.

[0123] [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).

[0124] [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.

[0125] [Figure 77]Foxp3 expression levels in mouse air T cells using alternative homologous donor cassettes are shown. Panel A shows flow cytometry plots demonstrating LNGFR and GFP expression in spleen-derived T cells gene-edited using homologous recombination repair. 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 summarizing the FOXP3 expression data shown in Panel A. Panel C shows a bar graph showing the MFI of FOXP3 in edTreg / air T cells and nTreg cells generated using various alternative HDR donor constructs as shown in the graph. Highest FOXP3 expression was observed in donors containing the MND promoter.

[0126] [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. On day 4, cells were washed twice with PBS and stained with live / dead indicators, anti-CD4, anti-CD45, and anti-CD25 antibodies. The inhibitory effect of airT cells on Teff cell proliferation was analyzed by FACS (LSRII). B) Representative flow cytometry data showing the suppression of Teff cell proliferation in the presence of airT cells.

[0127] [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.

[0128] [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.

[0129] [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.

[0130] [Figure 82] Flow cytometry analysis of NOD BDC2.5+ mouse cells edited with LNGFR.P2A before and after column purification is shown. Figure A shows flow cytometry plots demonstrating LNGFR expression in mock-edited cells and cells edited with MND.LNGFR.P2A (No. 3189). Figure 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 purity greater than 90% and was used for in vitro and in vivo studies.

[0131] [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%.

[0132] [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.

[0133] [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.

[0134] [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).

[0135] [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 by homologous recombination repair in mouse T cells. 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 into primary mouse CD4+ T cells using R26 gRNA_1.

[0136] [Figure 88]This shows an overview of a gene editing experiment using homologous recombination repair at the mouse Rosa26 locus. Figure 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. Figure 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.

[0137] [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 showing viability, percentage of GFP-positive cells, and percentage of GFP-high expressing cells in the gene-edited cell population.

[0138] [Figure 90] This 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.

[0139] [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.

[0140] [Figure 92] This section describes the lentiviral CISC constructs used to transduce mouse CD4+ T cells and test their selective expansion with rapalogs. Figure 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). Figure 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 evaluated for mCherry expression by flow cytometry on days 2 and 5 post-transduction.

[0141] [Figure 93]We demonstrate that 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.

[0142] [Figure 94] We show that air T cells suppress the proliferation of CD8+ T cells and CD4+ T cells.

[0143] [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.

[0144] [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.

[0145] [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+.

[0146] [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.

[0147] [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.

[0148] [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.

[0149] [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.

[0150] [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.

[0151] [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.

[0152] [Figure 104] Figure 107 shows that antigen-specific GFP+ air T cells are detectable in the pancreas. See also Figure 107 and Figure 116.

[0153] [Figure 105] See also Figure 114 for the generation and enrichment of murine LNGFR+ air T cells used in in vivo suppression studies.

[0154] [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.

[0155] [Figure 107] This shows that antigen-specific GFP+ air T cells are detectable in the pancreas.

[0156] [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.

[0157] [Figure 109] Figure 1 shows that exposure to rapamycin in vivo enhances the persistence of CISC cells.

[0158] [Figure 110] 1 shows a schematic diagram of an exemplary gene-edited cell according to the present disclosure.

[0159] [Figure 111] Regarding the selection of gRNAs targeting the TRAC locus.

[0160] [Figure 112] A dual editing approach using split IL-2 CISC constructs targeting the TRAC locus.

[0161] [Figure 113] 1 shows selection of dual-edited cells by association of CISC components in vitro.

[0162] [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 a purity of 74.5% for in vivo studies.

[0163] [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. Figure 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). Figure B shows graphs demonstrating the development of diabetes in mice over a 49-day period. 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.

[0164] [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.

[0165] [Figure 117A] Flow cytometry plots are shown analyzing mTCRb expression gated on CD4+ cells 9 days after transduction.

[0166] [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.

[0167] [Figure 118B] Polyclonal and antigen-specific suppression assays using enolase-specific edTreg cells are shown.

[0168] [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.

[0169] [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.

[0170] [Figure 119B] Polyclonal and antigen-specific suppression assays using CILP-specific edTreg cells are shown.

[0171] [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.

[0172] [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.

[0173] [Figure 120B] Polyclonal and antigen-specific suppression assays using vimentin-specific edTreg cells are shown.

[0174] [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.

[0175] [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.

[0176] [Figure 121B] Polyclonal suppression assay using Agg520 or Vim418-specific edTreg cells or mock cells and Agg520 Teff cells is shown.

[0177] [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.

[0178] [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.

[0179] [Figure 121E] This 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.

[0180] [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.

[0181] [Figure 122B]Polyclonal suppression assay using CILP297-1 Teff cells with edTreg cells or mock cells specific for CILP297 or Vim418 is shown.

[0182] [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.

[0183] [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.

[0184] [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.

[0185] [Figure 123A] Flow cytometry plots showing mTCRb and LNGFR / Foxp3 expression in edited cells expressing SLE3-TCR at day 7.

[0186] [Figure 123B] Polyclonal and antigen-specific suppression assays using SLE-specific edTreg cells are shown.

[0187] [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.

[0188] [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.

[0189] [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.

[0190] [Figure 125B] Flow cytometry plots showing T1D4 and CD4 expression in mock-edited cells and cells edited with the virus mixture are shown.

[0191] [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.

[0192] [Figure 125D] A histogram comparing the CD3 knockout rate between FOXP3 / T1D4 double-edited and mock-edited cells is shown.

[0193] [Figure 126A] Flow cytometry plots showing viability and T1D4 and FOXP3 expression after treatment of dual-edited cells with 50 ng / mL IL-2 (top panel) or 100 nM rapalog (AP21967; bottom panel) for 7 days.

[0194] [Figure 126B]Flow cytometry plots comparing CTLA4 expression in T1D4 / FOXP3 double-positive and double-negative cell populations after 7 days of treatment with 50 ng / mL IL-2 (top panel) or 100 nM rapalog (AP21967; bottom panel).

[0195] [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).

[0196] [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.

[0197] [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.

[0198] [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.

[0199] [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.

[0200] [Figure 129B] Flow cytometry plots showing viability (top) and GFP and mCherry expression (bottom) after 7 days of expansion of 7.6 million edited cells in a gREX flask in the presence of AP21967, demonstrating a 32-fold expansion of double-positive cells. The total number of double-positive cells in the gRex flask was 11.1 million.

[0201] [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.

[0202] [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.

[0203] [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.

[0204] [Figure 132A] 1 shows the design of an in vitro suppression assay using mouse edTreg cells or mouse nTreg cells.

[0205] [Figure 132B] Representative flow cytometry data are shown demonstrating reduced proliferation of BDC2.5+Teff cells in the presence of BDC2.5+edTreg cells.

[0206] [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.

[0207] [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.

[0208] [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).

[0209] [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.

[0210] [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.

[0211] [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.

[0212] [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.

[0213] [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.

[0214] [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.

[0215] [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.

[0216] [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.

[0217] [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.

[0218] [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.

[0219] [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.

[0220] [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.

[0221] [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. DETAILED DESCRIPTION OF THE INVENTION

[0222] 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" cells or "gene-edited Treg" cells. Some embodiments include artificial T cells (e.g., T lymphocytes), which include CD4+CD25+ T cells having a forkhead box protein 3 / winged helix transcription factor (FOXP3) gene artificially modified to constitutively express 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.

[0223] In some embodiments, the airT cells of the 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 exaggerated immune response, to confer antigen-specific immunosuppression. Importantly, production of the airT cells of the invention avoids the time, cost, and inefficiencies associated with isolating relatively rare (1-4% of human PBMCs) natural Treg cells as starting material for gene editing, thereby offering the advantage of generating therapeutically effective amounts of the desired cells for adoptive immunotherapy.

[0224] 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 TCR comprising any of the TCR polypeptide sequences disclosed herein or any of the TCR polypeptides encoded by the TCR-encoding polynucleotide sequences disclosed herein, including the TCRs depicted in the figures.

[0225] 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.

[0226] 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 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 through 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, allergic, or other inflammatory diseases, without the risks associated with the plasticity of natural Treg cells (e.g., conversion to T effector behavior).

[0227] An advantage of the methods for producing airT cells disclosed herein is that, while typical production methods first involve isolating natural Treg cells, which are present at low frequencies in peripheral blood and account for only about 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 CD4+ T cells. While CD4+ T cells include a variety of T cells compared to cells with other cell surface markers, CD4+ T cells comprise about 25-60% of human PBMCs and therefore can be used as a relatively abundant starting material for gene editing using the various methods provided herein.

[0228] 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.

[0229] 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) inhibiting the activation and / or proliferation of effector T cells that recognize an antigen specifically recognized by an airT cell TCR comprising the TCR polypeptide encoded by the 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 an airT cell TCR comprising the TCR polypeptide encoded by the at least one introduced polynucleotide; (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, expression of inhibitory receptors by airT cells, and (iv) inhibiting the activation and / or proliferation of effector T cells that do not recognize the antigen specifically recognized by the air T cell TCR comprising the TCR polypeptide encoded by the at least one introduced polynucleotide. It may include one or more of: In some embodiments, such antigenic stimulation of airT cells is HLA-restricted.

[0230] 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.

[0231] To avoid such risks, in some embodiments provided herein, the FOXP3 gene is artificially modified using specific targeted gene editing rather than transferring the FOXP3 gene via a virus. Furthermore, gene editing may be performed to specifically target the TCR. In certain embodiments described herein, lentivirus-mediated gene delivery is used to introduce an autoimmune-associated TCR candidate into CD4 T cells, and the FOXP3 gene in these CD4 T cells is then edited to stably express FoxP3. In some related embodiments, this method is combined with a gene editing method to simultaneously delete (e.g., delete by inactivation (also referred to herein as "knockout")) the endogenous TCR gene.

[0232] 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 at a single locus are edited by performing two edits at a single locus (e.g., using one guide RNA and multiple AAV donor homology constructs).

[0233] 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 AAV donor homology cassettes specific for each locus).

[0234] These methods may be used to deliver the recombinant FOXP3 gene and the recombinant TCR gene 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 the inserted TCR gene and the Foxp3 gene, thereby enriching for air T cells.

[0235] 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 that is separate from the first CISC component and specifically binds to the CISC-inducing molecule, the second CISC component being present 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.

[0236] In certain embodiments, CD4+ T cells are obtained by enrichment from a biological sample, such as peripheral blood mononuclear cells (PBMCs), prior to undergoing a gene edit (e.g., dual edit) described herein. In certain embodiments, the CD4+ T cells obtained by enrichment are non-specifically activated (e.g., using solid-phase immobilized anti-CD3 and anti-CD28 antibodies) prior to undergoing a gene edit (e.g., dual edit) described herein.

[0237] In some embodiments, exposing dual-edited T cells described herein to a CISC-inducing molecule results in binding of the CISC-inducing molecule to the extracellular domain of the first CISC component and the extracellular domain of the second CISC component, forming a heterodimer of the first CISC component and the second CISC component, and activating a functional signaling complex formed by the first activation signaling intracellular domain and the second activation signaling intracellular domain. In this manner, expression of the first CISC component and the second CISC component in an airT cell population results in expression of FoxP3 and a heterologous TCR, allowing for the selective expansion of the airT cell population.

[0238] In some embodiments, in the airT cells of the present invention, the two gene edits that respectively generate a first CISC component co-expressed with the FoxP3 gene product and a second CISC component co-expressed with the TCR gene product can 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, a third CISC component that specifically binds to a CISC-inducing molecule can be co-expressed 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 molecule.

[0239] 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.

[0240] 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).

[0241] Phenotypic markers and suppressor function of FOXP3 / air T cells Editing the FOXP3 gene may involve artificial modification of the native FOXP3 locus and / or artificial modification of a chromosomal site other than the native FOXP3 locus. For example, gene editing may involve knocking in (e.g., inserting) a nucleic acid molecule comprising a polynucleotide encoding exogenous FOXP3 and an operably linked constitutive promoter into a chromosomal site other than the native FOXP3 locus, such as the T cell receptor alpha chain (TRAC) locus, the T cell receptor beta chain (TCRB) locus, the adeno-associated virus integration site 1 (AAVS1), or another locus. Thus, surprisingly, in certain embodiments, introducing an artificial FoxP3 gene sequence into a genomic site other than the native FOXP3 locus (e.g., the TRAC locus) provides airT cells that constitutively express a FOXP3 gene product at levels comparable to or greater than those of natural Treg cells, thereby constitutively ...

[0242] In certain embodiments, in the airT cells of the invention, the two gene edits that result in a FoxP3 gene product that is co-expressed with a first CISC component and a TCR gene product that is co-expressed with a second CISC component, respectively, can be designed to occur at separate alleles of the same locus (e.g., a dual-editing approach that edits two alleles) or at two different loci (e.g., a dual-editing approach that edits two loci). Surprisingly, in some embodiments, the airT cells disclosed herein are capable of expressing a FOXP3 gene product at an expression level sufficient to maintain a CD4+CD25+ phenotype for at least 21 days in vitro or for at least 60 days in vivo after adoptive transfer into an immunocompatible mammalian host in need of antigen-specific immunosuppression, and are capable of functionally expressing a TCR described herein that specifically recognizes an antigen associated with the pathogenesis of an autoimmune, allergic, or inflammatory disease, or a TCR that specifically recognizes an antigen described herein that is associated with the pathogenesis of an autoimmune, allergic, or inflammatory disease.

[0243] Thus, in certain embodiments, the CD4+CD25+ airT cells disclosed herein relate to genetically engineered cells obtained by artificially modifying the FOXP3 gene in CD4+CD25- T cells. In some embodiments, the airT cells of the present invention constitutively express the FOXP3 gene product at levels equivalent to or greater than those of natural regulatory T (Treg) cells due to the aforementioned artificial modification. In some embodiments, the airT cells of the present invention may further express the CD25 marker, CD152 marker, and / or ICOS marker on their cell surface at levels characteristic of immune regulatory cells, such as natural Treg cells. However, in some embodiments, the airT cells of the present invention may exhibit a HeliosLo phenotype on their cell surface, unlike natural Treg cells. For example, they may express the Helios marker on their cell surface at a level statistically significantly lower than that of natural Treg cells.

[0244] Detailed examples of gene editing methods for inducing FoxP3 expression in T cells are described elsewhere herein and in WO / 2018 / 080541 and WO / 2019 / 210078, both of which are expressly incorporated by reference in their entireties. Detailed examples of forced expression of FOXP3 using gene editing, including knocking in (inserting) a full-length codon-optimized FoxP3 cDNA into the FOXP3 locus or the AAVS1 locus, are described in WO / 2019 / 210042, which is expressly incorporated by reference in its entirety.

[0245] Briefly, WO / 2018 / 080541 describes CD4+ T cells stably expressing endogenous FoxP3 by knocking in (e.g., inserting) a constitutive promoter (EF1α promoter, PGK promoter, or MND promoter) using gene editing with Cas9, ZFN, or TALEN. FoxP3 expression may be achieved by targeted knock-in (insertion) of a polynucleotide comprising a sequence encoding the first-expressed FOXP3 exon and an operably linked regulatory sequence into the FOXP3 locus. The regulatory sequence may include a promoter, which in some embodiments may be the MND promoter, PGK promoter, or EF1α promoter, or another inducible promoter, another weak promoter, or another constitutive promoter. Exemplary gene-edited FOXP3+ cells may contain a FOXP3 gene in which the intronic regulatory T cell-specific demethylation region (TSDR) upstream of the promoter-integrated knock-in site is fully methylated.

[0246] WO / 2019 / 210078 describes forced expression of FoxP3 in CD4+ T cells to obtain cells with a Treg-like phenotype, a method for selecting cells with such a Treg-like phenotype to obtain a Treg-enriched preparation, and a method for in vitro expansion of a cell population with such a Treg-like phenotype. WO / 2019 / 210078 also describes compositions and methods for editing target genes in the FOXP3 locus, the AAVS1 locus, and / or the TCRα (TRAC) locus, which include guide RNA (gRNA) sequences specific to each of the FOXP3 locus, the AAVS1 locus, and / or the TCRα (TRAC) locus, and a donor template for gene editing via homologous recombination repair. WO / 2019 / 210078 also describes a CISC system in which dimerization of a first CISC component with a second CISC component via a chemical ligand induces an activation signal that promotes T cell proliferation, enabling selective expansion of gene-edited T cells. WO / 2019 / 210078 further describes a first CISC component and a second CISC component in which the CISC-inducing molecule is rapamycin or any of various rapamycin analogs, derivatives, and mimetics disclosed therein, and the activation signaling domains of the CISC components include functional portions of the cytoplasmic domains of the IL-2 receptor β subunit (IL2Rb (also referred to as IL2RP)) and the IL-2 receptor γ subunit (IL2Rg (also referred to as IL2Ry)) of the IL-2 receptor (IL2R).

[0247] Methods for assessing the phenotype and function of Treg cells, including cells induced to overexpress FoxP3, are known in the art (see, e.g., WO / 2018 / 080541; WO / 2019 / 210078; McMurchy et al., 2013 Meths. Mol. Biol. 946: 115-132; Thornton et al., 2019 Eur. J. Immunol. 49:398-412; Aarts-Riemens et al., 2008 Eur. J. Immunol. 38: 1381-1390; McGovern et al., 2017 Front. Immunol. 8: Art. 1517; all of which are expressly incorporated by reference in their entirety) and are described herein. These methods and related methodologies can also be used to characterize the airT cells of the invention described herein.

[0248] The airT cells disclosed herein differ from natural Treg cells in that a majority of cytosine (C) nucleotides at specific positions of cytosine-guanine (CG) dinucleotides in the intronic Treg-specific demethylated region (TSDR) of the FoxP3 locus are methylated. For example, in the airT cells of the present invention, at nucleotide positions containing demethylated C nucleotides in the TSDR region of natural Treg cells, at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of C nucleotides are methylated. Analysis of methylation in the TSDR region of the FoxP3 gene is known to be routinely performed in the art, and several methodologies exist (see, e.g., Salazar et al., 2017 Front. Immunol. 8:219; Ngalamika et al., 2014 Immunol. Invest. 44(2): 126-136; these references are expressly incorporated herein by reference in their entireties).

[0249] Despite these differences in epigenetic modifications in the TSDR region between the airT cells of the present invention and natural Treg cells, the airT cells of the present invention can induce immunosuppression in response to stimulation by specific TCR recognition of an antigen. Therefore, given the report by Wright et al. (2009 Proc. Nat. Acad. Sci. USA 106: 19078) that co-transfection of CD4+ cells with a viral vector encoding a FoxP3 construct and a viral vector encoding a TCR construct did not result in Treg-like cells exhibiting antigen-specific suppressive function, it was unexpected that the airT cells disclosed herein exhibit antigen-specific immunosuppressive properties. Therefore, without wishing to be bound by theory, it is believed that the airT cells disclosed herein possess unexpected advantages based, at least in part, on the methods for generating airT cells described herein, including artificial gene editing.

[0250] T cell receptor (TCR) The introduced polynucleotide encoding the exogenous TCR expressed in the airT cells of the invention may comprise an artificial modification of the native TCR locus (e.g., TRAC) and / or may comprise an artificial modification of a chromosomal site other than the native TCR locus, for example, by gene editing by knocking in (inserting) a nucleic acid molecule comprising a polynucleotide encoding the exogenous TCR into a chromosomal site other than the native TCR locus, such as the FOXP3 locus or the AAVS1 locus, or another locus.

[0251] In certain embodiments, in the airT cells of the present invention, the two gene edits that respectively result in a TCR gene product co-expressed with a first CISC component and a FoxP3 gene product co-expressed with a second CISC component 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).The amino acid sequences of exemplary TCRs that specifically recognize antigens associated with the pathogenesis of allergic, autoimmune, and / or inflammatory diseases and the nucleotide sequences encoding them are disclosed herein (see, e.g., Figures 136-144).

[0252] Gene editing As used herein, "knockout of a chromosomal gene" refers to the modification or inactivation of a gene in a host cell or the introduction of an inhibitor into a host cell to prevent (e.g., reduce, delay, suppress, or inhibit) the production of a functionally active endogenous polypeptide product by the host cell. Genetic modifications that result in the knockout or inactivation of a chromosomal gene include, for example, the introduction of nonsense mutations (including the formation of premature stop codons), missense mutations, gene deletions, or strand breaks, and the heterologous expression of inhibitory nucleic acid molecules that suppress the expression of endogenous genes in the host cell.

[0253] In certain embodiments, knocking out a chromosomal gene or knocking in (e.g., inserting) a chromosomal gene is achieved by editing the chromosome of a host cell. Chromosomal editing can be achieved, for example, using an endonuclease. As used herein, "endonuclease" refers to an enzyme that can catalyze the cleavage of a phosphodiester bond within a polynucleotide chain. In certain embodiments, cleaving a target gene with an endonuclease can inactivate or "knock out" the target gene. The endonuclease can be a natural endonuclease, a recombinant endonuclease, a recombinant endonuclease, or a fusion endonuclease. Examples of endonucleases used in gene editing include zinc finger nucleases (ZFNs), TALE nucleases (TALENs), CRISPR-Cas nucleases, meganucleases, and megaTALs.

[0254] The break in a nucleic acid strand caused by an endonuclease is usually a double-strand break (DSB), which is often repaired by two different mechanisms: homology-directed repair (HDR) via homologous recombination or non-homologous end joining (NHEJ) (NHEJ: Ghezraoui et al., 2014 Mol Cell 55(6):829-842; HDR: Jasin and Rothstein, 2013 Cold Spring Harb Perspect Biol 5(11):a012740, PMID 24097900). In homology-directed repair (HDR), or homologous recombination, a donor nucleic acid molecule may be used to "knock in" a donor gene, a donor nucleic acid molecule may be used to "knock out" a target gene, or a donor nucleic acid molecule may be used to knock in a donor gene or knock out a target gene, thereby inactivating the target gene. Non-homologous end joining (NHEJ) is an error-prone repair method, and often causes changes at the cut site of DNA sequence, such as at least one nucleotide substitution, deletion or addition.Non-homologous end joining can also be used to "knock out" target gene.When double-strand break occurs and donor template is present, homology recombination repair often occurs, and homology recombination repair is also preferred as the gene editing mechanism of certain embodiments described herein.

[0255] As used herein, "zinc finger nuclease (ZFN)" refers to a fusion protein in which a zinc finger DNA-binding domain is fused to a nonspecific DNA cleavage domain (e.g., Fok I endonuclease). Each zinc finger motif, approximately 30 amino acids long, binds to approximately three base pairs of DNA, and specific amino acid residues can be altered to change the triplet sequence specificity (see, e.g., Desjarlais et al., Proc. Natl. Acad. Sci. 90:2256-2260, 1993; Wolfe et al., J. Mol. Biol. 285:1917-1934, 1999). Linking multiple zinc finger motifs enables specific binding to a desired DNA sequence (e.g., a region approximately 9-18 base pairs long). This technology is based on the following: ZFN is involved in genome editing by catalyzing the formation of site-specific DNA double-strand breaks (DSBs) in genome, and then the transgene that comprises the flanking sequence that is homologous to the double-strand break site in genome is targeted and integrated into this break site by homology-directed repair (HDR).In another method, the double-strand break formed by ZFN can be repaired by non-homologous end joining (NHEJ) to knock out the target gene, and non-homologous end joining is an error-prone cell repair pathway, and causes the insertion or deletion of nucleotides at the break site.In certain embodiments, the knockout or inactivation of gene comprises the insertion, deletion, mutation, or a combination thereof that is introduced by using ZFN molecules.

[0256] As used herein, "transcription activator-like effector nuclease (TALEN)" refers to a fusion protein containing a TALE DNA-binding domain and a DNA cleavage domain (e.g., Fok I endonuclease). A "TALE DNA-binding domain" or "TALE" contains one or more TALE repeat domains / units, each of which typically has a highly conserved sequence of 33-35 amino acid residues, with the 12th and 13th amino acids being highly variable. This TALE repeat domain is responsible for TALE binding to target DNA sequences. The variable amino acid residues, called RVDs (Repeat Variable Diresidues), are involved in specific nucleotide recognition. The natural (canonical) codes for TALE DNA recognition are known: if positions 12 and 13 of the TALE are HD (histidine-aspartic acid), the TALE binds to cytosine (C); if positions 12 and 13 of the TALE are NG (asparagine-glycine), the TALE binds to T nucleotides; if positions 12 and 13 of the TALE are NI (asparagine-isoleucine), the TALE binds to A nucleotides; if positions 12 and 13 of the TALE are NN (asparagine-asparagine), the TALE binds to G or A nucleotides; and if positions 12 and 13 of the TALE are NG (asparagine-glycine), the TALE binds to T nucleotides. Non-canonical (atypical) RVDs are also known (see, e.g., U.S. Patent Publication No. 2011 / 0301073; the atypical RVDs described therein are incorporated by reference in their entirety). TALENs can be used to induce site-specific double-strand breaks (DSBs) in the T cell genome. Non-homologous end joining (NHEJ) involves joining the DNA at either end of the double-strand break, but with little or no overlapping sequences for annealing, introducing errors that knock out gene expression. Alternatively, homology-directed repair (HDR) can deliver a transgene to the double-strand break if the donor template containing the transgene is flanked by homologous sequences.In certain embodiments, the gene knockout comprises an insertion, deletion, mutation, or a combination thereof, introduced using a TALEN molecule.

[0257] As used herein, the term "clustered regularly interspaced short palindromic repeats / Cas (CRISPR / Cas)" nuclease system refers to a system that uses a CRISPR RNA (crRNA)-guided Cas nuclease to recognize and cleave DNA at a target site (known as a protospacer) in the genome through base pair complementarity when a conserved short protospacer-associated motif (PAM) is present immediately following the 3' end of the complementary target sequence. CRISPR / Cas systems are classified into three types (i.e., type I, type II, and type III) based on the sequence and structure of the Cas nuclease. Type I and type III crRNA-guided surveillance complexes require multiple Cas subunits. The most studied type II CRISPR / Cas system contains at least three components: an RNA-guided Cas9 nuclease, a crRNA, and a trans-activating crRNA (tracrRNA). The tracrRNA contains a duplex-forming region. The crRNA and tracrRNA form a duplex that can interact with the Cas9 nuclease. The Cas9 / crRNA:tracrRNA complex is guided to a specific site in the target DNA via Watson-Crick base pairing between the spacer on the crRNA and the protospacer on the target DNA upstream of the PAM. The Cas9 nuclease creates a double-strand break within the region defined by the crRNA spacer. Repair by non-homologous end joining (NHEJ) disrupts expression of the target locus by inserting and / or deleting the target locus. Alternatively, homology-directed repair (HDR) can introduce a transgene flanked by homologous sequences in a donor template into the double-strand break. The crRNA and tracrRNA can be used to generate a single-stranded guide RNA (also known as sgRNA or gRNA) (see, for example, Jinek et al., Science 337:816-21, 2012).Additionally, the region of the guide RNA complementary to the target site can be modified or programmed to target the desired sequence (Xie et al., PLOS One 9:e100448, 2014; U.S. Patent Application Publication No. 2014 / 0068797; U.S. Patent Application Publication No. 2014 / 0186843; U.S. Patent No. 8,697,359; and PCT Publication WO 2015 / 071474; all of which are incorporated herein by reference).

[0258] In certain embodiments, gene knockout or inactivation comprises insertion, deletion, mutation, or a combination thereof, introduced using a CRISPR / Cas nuclease system. U.S. Patent Publication No. 2016 / 033377 (which is expressly incorporated herein by reference in its entirety) teaches a method for enhancing gene editing using endonucleases, which method includes using AAV-expressed guide RNA in a CRISPR / Cas (e.g., Cas9) gene editing system. Exemplary gRNA sequences and methods using these sequences for knocking out endogenous genes encoding proteins in immune cells include those described in Ren et al., Clin. Cancer Res. 23(9):2255-2266 (2017), the gRNA, CAS9 DNA, vectors, and gene knockout techniques described therein are all expressly incorporated herein by reference in their entirety.

[0259] Herein, "meganuclease," also known as "homing endonuclease," refers to an endodeoxyribonuclease characterized by a long recognition site (a double-stranded DNA sequence consisting of approximately 12 to 40 base pairs). Based on their sequences and structural motifs, meganucleases can be classified into five families: LAGLIDADG, GIY-YIG, HNH, His-Cys box, and PD-(D / E)XK. Exemplary meganucleases include I-SceI, I-CeuI, PI-PspI, PI-Sce, I-SceIV, I-CsmI, I-PanI, I-SceII, I-PpoI, I-SceIII, I-CreI, I-TevI, I-TevII, and I-TevIII, and their recognition sequences are known (e.g., U.S. Pat. Nos. 5,420,032; 6,833,252; Belfort et al., Nucleic Acids Res. 25:3379-3388, 1997; Dujon et al., Gene 82:115-118, 1989; Perler et al., Nucleic Acids Res. 22:1125-1127, 1994; Jasin, Trends Genet. 12:224-228, 1998). 1996; Gimble et al., J. Mol. Biol. 263:163-180, 1996; Argast et al., J. Mol. Biol. 280:345-353, 1998).

[0260] In certain embodiments, natural meganucleases may be used to facilitate site-specific genomic modification of targets selected from genes encoding PD-1, LAG3, TIM3, CTLA4, TIGIT, FasL, HLA, and genes encoding components of the TCR. In another embodiment, recombinant meganucleases with novel binding specificities for target genes are used to perform site-specific genome modifications (e.g., Porteus et al., Nat. Biotechnol. 23:967-73, 2005; Sussman et al., J. Mol. Biol. 342:31-41, 2004; Epinat et al., Nucleic Acids Res. 31:2952-62, 2003; Chevalier et al., Molec. Cell 10:895-905, 2002; Ashworth et al., Nature 441:656-659, 2006; Paques et al., Curr. Gene Ther. 7:49-66, (See U.S. Patent Publication Nos. 2007; 2007 / 0117128; 2006 / 0206949; 2006 / 0153826; 2006 / 0078552; and 2004 / 0002092.) In a further embodiment, a homing endonuclease is attached to a TALEN DNA-binding domain module to create a fusion protein known as a megaTAL, which is used to knock out chromosomal genes. megaTALs can not only knock out or inactivate one or more target genes, but also, when used in conjunction with an exogenous donor template encoding a polypeptide of interest, can introduce heterologous or exogenous polynucleotides (knock-in).

[0261] Chromosomal gene knockout can be directly confirmed by DNA sequencing of host immune cells after the knockout procedure or use of a knockout agent. Chromosomal gene knockout can also be inferred from the absence of gene expression after knockout (e.g., the absence of mRNA or polypeptide product encoded by the knocked-out gene).

[0262] In certain embodiments, the knockout or inactivation of a chromosomal gene comprises knocking out or inactivating a gene for a TCR component selected from a TCR alpha variable region gene, a TCR beta variable region gene, a TCR constant region gene, and combinations thereof.

[0263] T cells and TCR The CD4+CD25+ air T cells of the present invention comprise an artificially modified FOXP3 gene as described herein and further comprise at least one introduced polynucleotide encoding an antigen-specific T cell receptor (TCR) polypeptide. In certain embodiments, the native TCR gene is preferably knocked out, e.g., the TCRα (TRAC) locus is preferably knocked out by targeted gene editing. As used herein, "knockout" may refer to the inactivation of a gene and / or gene product, e.g., by deleting a gene or a portion thereof, or by inserting a nucleic acid into a gene to disrupt transcription and / or translation of the gene and / or gene product. Furthermore, in certain embodiments, the introduced polynucleotide encoding the TCR is preferably knocked into a specific locus, such as the TRAC locus or another target locus, by gene editing.

[0264] Thus, in preferred embodiments, the airT cells of the present invention include artificial immunoregulatory T cells produced by selectively editing one or more specific genetic loci in T lymphocytes as described herein. The T cells are preferably mammalian, such as T cells obtained from humans, non-human primates (e.g., chimpanzees, rhesus monkeys, gorillas, etc.), rodents (e.g., mice, rats, etc.), lagomorphs (e.g., rabbits, hares, pikas, etc.), ungulates (e.g., cows, horses, pigs, sheep, etc.), or other mammals. In certain preferred embodiments, the T cells are human T cells.

[0265] T cells, or T lymphocytes, are immune system cells that mature in the thymus and produce T cell receptors (TCRs), which are cell surface receptors consisting of antigen-specific heterodimers, typically composed of αβ or γδ heterodimers. A given T cell clone typically expresses only a single clonal TCR, which recognizes a specific antigen epitope presented by a cognate antigen-presenting cell via determinants encoded by the major histocompatibility complex. T cells may be naive T cells ("TN"; T cells that have not been exposed to antigen; as described herein, they have increased expression of CD62L, CCR7, CD28, CD3, CD127, and CD45RA, and decreased or absent CD45RO expression, compared to TCMs), memory T cells (TM) (antigen-experienced, long-lived T cells) (including stem cell memory T cells), or effector cells (antigen-experienced, cytotoxic T cells). T cells can be further classified into two subsets: central memory T cells (T cells) (expressing CD62L, CCR7, CD28, CD95, CD45RO, and CD127) and effector memory T cells (T cells) (expressing CD45RO but with reduced expression of CD62L, CCR7, CD28, and CD45RA). Effector T cells (T cells) are antigen-experienced CD8+ cytotoxic T lymphocytes that express CD45RA and, compared with T cells, have reduced expression of CD62L, CCR7, and CD28 and are positive for granzymes and perforin. Helper T cells (T cells) are CD4+ cells that influence the activity of other immune cells by releasing cytokines. CD4+ T cells can activate or suppress adaptive immune responses, and the induction of these two functions depends on the presence of other cells and other signals.T cells can be collected using known techniques, such as by using antibodies that specifically recognize one or more T cell surface phenotypic markers, and various subpopulations or combinations thereof can be enriched or depleted by known techniques, such as affinity binding to antibodies, flow cytometry, fluorescence-activated cell sorting (FACS), or immunomagnetic bead selection. Other exemplary T cells include regulatory T cells (Tregs) (also known as suppressor T cells), such as CD4+CD25+ (Foxp3+) regulatory T cells and Treg17 cells, as well as Tr1 cells, Th3 cells, CD8+CD28- cells, and Qa-1-restricted T cells.

[0266] As used herein, "T cell receptor (TCR)" refers to a member of the immunoglobulin superfamily that has a variable binding domain, a constant domain, a transmembrane region, and a short cytoplasmic tail. See, for example, Janeway et al., Immunobiology: The Immune System in Health and Disease, 3rd Ed., Current Biology Publications, p. 433, 1997. TCRs can specifically bind to antigen peptides bound to receptors encoded by the major histocompatibility complex (MHC). TCRs are found on the surface of T cells but can also be released into the extracellular environment in a soluble form. They are typically composed of heterodimers with α and β chains (also known as TCRα and TCRβ) or γ and δ chains (also known as TCRγ and TCRδ), each of which has a characteristic constant (C) region and a highly diverse variable (V) region, which contains complementarity-determining regions (CDRs) that are largely responsible for TCR recognition and binding to specific antigens. In certain embodiments, polynucleotides encoding TCRs can be codon-optimized to improve expression in specific host cells, such as immune system cells, hematopoietic stem cells, T cells, primary T cells, T cell lines, NK cells, and natural killer T cells (Scholten et al., Clin. Immunol. 119:135, 2006).

[0267] Exemplary T cells capable of expressing a TCR encoded by heterologous genetic material introduced into a cell according to certain embodiments of the present disclosure include CD4+ T cells, CD8+ T cells, and related subpopulations thereof (e.g., naive T cells, central memory T cells, stem cell memory T cells, effector memory T cells). In a preferred embodiment, the exemplary T cells are CD4+ T cells into which genetic material encoding a TCR has been introduced by gene editing (e.g., by introducing a specifically targeted double-strand break in genomic DNA followed by homologous recombination repair), and the TCR specifically recognizes an antigen associated with the pathogenesis of an autoimmune disease, allergic disease, or inflammatory disease, e.g., a specific TCR structurally defined herein, or a TCR of the present disclosure that specifically recognizes a T cell epitope of a specific autoantigen, allergen, or inflammatory disease antigen.

[0268] The extracellular portions of TCR chains (e.g., α chains and β chains), like other antigen-binding members of the immunoglobulin superfamily (e.g., immunoglobulins (also called antibodies)), contain two immunoglobulin domains: an N-terminal variable domain (e.g., an α chain variable domain (Vα) and a β chain variable domain (Vβ); typically consisting of amino acids 1 to 116 according to Kabat numbering (Kabat et al., "Sequences of Proteins of Immunological Interest", U.S. Dept. Health and Human Services, Public Health Service National Institutes of Health, 1991, 5th ed.)) and one constant domain adjacent to the cell membrane (e.g., an α chain constant domain (Cα), typically consisting of amino acids 117 to 259 according to Kabat numbering; a β chain constant domain (Cβ), typically consisting of amino acids 117 to 295 according to Kabat numbering). Furthermore, like immunoglobulins, variable domains contain multiple complementarity-determining regions (CDRs) separated from each other by framework regions (FRs) (see, e.g., Jores et al., Proc. Nat'l Acad. Sci. USA 87:9138, 1990; Chothia et al., EMBO J. 7:3745, 1988; see also Lefranc et al., Dev. Comp. Immunol. 27:55, 2003). The TCRs used in the present disclosure may be derived from a variety of animals, including humans, non-human primates, mice, rats, rabbits, and other mammals.

[0269] "Variable region" or "variable domain" refers to the structural domain of an immunoglobulin superfamily binding protein (e.g., a TCR) that is involved in specific binding of the immunoglobulin superfamily binding protein (e.g., a TCR) to an antigen (e.g., the α or β chain of a TCR (or the γ and δ chains of a γδ TCR)). The variable domains of the α chain (Vα) and the β chain (Vβ) of naturally occurring TCRs typically have similar structures, with each variable domain containing four generally conserved framework regions (FR) and three CDRs. The Vα domain is encoded by two separate DNA segments, the variable gene segment and the joining gene segment (VJ), while the Vβ domain is encoded by three separate DNA segments, the variable gene segment, the diversity gene segment, and the joining gene segment (VDJ). In some cases, a single Vα or Vβ domain is sufficient to confer antigen-binding specificity. Furthermore, a library of complementarity-determining regions of Vα or Vβ domains of a TCR that binds to a specific antigen may be screened using the Vα or Vβ domain, respectively, to isolate TCRs that bind to the specific antigen.

[0270] The term "complementarity determining region" or "CDR" is used interchangeably with "hypervariable region" or "HVR" and is known in the art to refer to an amino acid sequence within an immunoglobulin (e.g., TCR) variable region that confers antigen specificity and / or binding affinity, with multiple CDRs separated from each other by framework regions in the primary amino acid sequence. Typically, there are three CDRs (αCDR1, αCDR2, αCDR3) in each TCR α chain variable region, and three CDRs (βCDR1, βCDR2, βCDR3) in each TCR β chain variable region. In TCRs, the primary CDR responsible for recognizing processed antigens is thought to be CDR3. Typically, CDR1 and CDR2 primarily interact with MHC, or only CDR1 and CDR2 interact with MHC.

[0271] CDR1 and CDR2 are encoded within the variable gene segments of the sequence encoding the variable region of the TCR, and CDR3 is encoded by the region spanning from the variable segment of Vα to the joining segment (VJ) or the region spanning from the variable segment of Vβ to the diversity and joining segment (VDJ). Therefore, when a variable gene segment of Vα or a variable gene segment of Vβ is identified, the sequences of their corresponding CDR1 and CDR2 can be deduced, for example, according to the numbering scheme described herein. CDR3 is typically significantly more diverse than CDR1 or CDR2 due to the addition and / or deletion of nucleotides during the recombination process.

[0272] TCR variable domain sequences can be aligned using a numbering scheme (e.g., Kabat, Chothia, EU, IMGT, Enhanced Chothia, and Aho) to annotate identical residue positions and compare different molecules, for example, using the ANARCI software tool (2016, Bioinformatics 15:298-300). Utilizing a numbering scheme allows for standardized definition of the framework regions and CDRs of TCR variable domains. In certain embodiments, the CDRs of the present disclosure are identified according to the IMGT numbering scheme (Lefranc et al., Dev. Comp. Immunol. 27:55, 2003; imgt.org / IMGTindex / V-QUEST.php).

[0273] As used herein, "CD4" refers to a glycoprotein that functions as an immunoglobulin coreceptor, assisting in the communication between TCRs and antigen-presenting cells (see Campbell & Reece, Biology 909 (Benjamin Cummings, Sixth Ed., 2002)). CD4 is found on the surface of immune cells, such as helper T cells, monocytes, macrophages, and dendritic cells. It contains four immunoglobulin domains (D1-D4), which are expressed on the cell surface. During antigen presentation, CD4 is recruited along with the TCR complex, and CD4 and the TCR complex bind to different regions of the MHC class II molecule (CD4 binds to MHCIIβ2, while the TCR complex binds to MHCIIα1 / β1). Without wishing to be bound by theory, it is believed that the location of CD4 adjacent to the TCR complex allows CD4-associated kinase molecules to phosphorylate immunoreceptor tyrosine-based activation motifs (ITAMs) present in the cytoplasmic domain of CD3. This activity is thought to promote immune responses by amplifying signals generated by activated TCRs to generate and recruit various types of immune cells, including helper T cells.

[0274] In certain embodiments, TCRs are found on the surface of T cells (i.e., T lymphocytes) and associate with the CD3 complex. "CD3" is a multiprotein complex consisting of six chains (see Abbas and Lichtman, 2003; Janeway et al., pp. 172 and 178, 1999) and is involved in antigen signal transduction in T cells. In mammals, the CD3 complex comprises a homodimer of a CD3γ chain, a CD3δ chain, two CD3ε chains, and a CD3ζ chain. The CD3γ chain, CD3β chain, and CD3ε chain are associated with each other as cell surface proteins of the immunoglobulin superfamily containing a single immunoglobulin domain. The transmembrane regions of the CD3γ chain, CD3β chain, and CD3ε chain are negatively charged, which is thought to enable these CD3γ chain, CD3β chain, and CD3ε chain to associate with the positively charged regions of the T cell receptor chains. The cytoplasmic tails of the CD3γ, CD3β, and CD3ε chains each contain one conserved motif known as an immunoreceptor tyrosine-based activation motif (ITAM), while each CD3ζ chain has three of these motifs. Without wishing to be bound by any particular theory, ITAMs are thought to be important for the signal transduction ability of the TCR complex. The CD3 used in the present disclosure may be obtained from various types of animals, such as humans, non-human primates, mice, rats, and other mammals.

[0275] As used herein, the term "TCR complex" refers to a complex formed by the association of CD3 and TCR. For example, the TCR complex may be composed of a CD3γ chain, a CD3β chain, two CD3ε chains, a homodimer of a CD3ζ chain, a TCRα chain, and a TCRβ chain. Alternatively, the TCR complex may be composed of a CD3γ chain, a CD3β chain, two CD3ε chains, a homodimer of a CD3ζ chain, a TCRγ chain, and a TCRβ chain.

[0276] As used herein, "a component of a TCR complex" refers to a TCR chain (i.e., TCRα, TCRβ, TCRγ, or TCRδ), a CD3 chain (i.e., CD3γ, CD3δ, CD3ε, or CD3ζ), or a complex formed by two or more TCR chains or CD3 chains (e.g., a complex consisting of TCRα and TCRβ, a complex consisting of TCRγ and TCRδ, a complex consisting of CD3ε and CD3δ, a complex consisting of CD3γ and CD3ε, or a TCR subcomplex consisting of TCRα, TCRβ, CD3γ, CD3δ, and two CD3ε chains).

[0277] As used herein, "chimeric antigen receptor (CAR)" refers to an artificial fusion protein in which two or more naturally occurring amino acid sequences, domains, or motifs are linked in a manner not found in nature or in a host cell, which fusion protein can function as a receptor when expressed on the surface of a cell, such as a T cell. A CAR may comprise an extracellular portion comprising an antigen-binding domain (e.g., an extracellular portion obtained from or derived from an immunoglobulin or immunoglobulin-like molecule; e.g., a TCR antigen-binding domain obtained from or derived from a TCR specific for an autoantigen, an allergen, or an antigen associated with an inflammatory disease; an scFv obtained from or derived from an antibody; an antigen-binding domain obtained from or derived from a killer immune receptor of an NK cell, etc.) linked to one or more intracellular signaling domains (which may include one or more costimulatory domains) and a transmembrane domain (see, e.g., Sadelain et al., Cancer Discov., 3(4):388 (2013); Harris and Kranz, Trends Pharmacol. Sci., 37(3):220 (2016), Stone et al., Cancer Immunol. Immunother., 63(11):1163 (2014), and Walseng et al., Scientific Reports 7:10713 (2017); the CAR constructs and methods for producing them described in these documents are incorporated herein by reference.

[0278] Many of the polypeptides encoded by polynucleotide sequences may contain a "signal peptide" (also known as a leader sequence, leader peptide, or transit peptide). A signal peptide directs a newly synthesized polypeptide to the appropriate location inside or outside a cell. A signal peptide may be removed from a polypeptide during biosynthesis, or may be removed from the polypeptide after the polypeptide has been localized to the appropriate location inside the cell or after the polypeptide has been secreted outside the cell. A polypeptide having a signal peptide is referred to herein as a "preprotein," and a polypeptide from which the signal peptide has been removed is referred to herein as a "mature" protein or polypeptide.

[0279] A "linker" refers to an amino acid sequence that links two proteins, polypeptides, peptides, domains, regions, or motifs, and may provide a spacer function that accommodates the interaction of the two linked domains so that the resulting polypeptide retains specific binding affinity to a target molecule (e.g., scTCR) or signaling activity (e.g., TCR complex). In certain embodiments, the linker is composed of approximately 2 to 35 amino acids, e.g., approximately 4 to 20 amino acids, approximately 8 to 15 amino acids, or approximately 15 to 25 amino acids. Exemplary linkers include glycine-serine linkers known in the art.

[0280] Exemplary TCR V region sequences of TCRs that specifically recognize antigens associated with the autoimmune, allergic, and inflammatory diseases described herein, and polynucleotide sequences encoding the same, are disclosed herein, including those described in the Examples and Figures. Furthermore, the present specification, including the Examples and Figures, discloses polypeptide sequences comprising antigenic epitopes of antigens recognized by TCRs associated with the autoimmune, allergic, and inflammatory diseases described herein.

[0281] An "antigen" typically refers to an immunogenic molecule that elicits an immune response. This immune response may involve the production of antibodies that specifically bind to the antigen, the activation of specific immunocompetent cells (e.g., T cells, such as helper T cells, effector T cells, and Treg cells), or both. While antigens are often thought of as "non-self" structures that the host's immune system recognizes as foreign and responds to, in this disclosure, "antigen" is not limited to such non-self antigens and, in certain embodiments, also includes autoantigens (also referred to as "self" molecules, "self" cells, "self" organs, or "self" tissue structures) to which the host's immune system responds inappropriately in autoimmune diseases. Antigens (immunogenic molecules) may be, for example, peptides, glycopeptides, polypeptides, glycopolypeptides, polynucleotides, polysaccharides, lipids, etc. It is readily understood that antigens can be artificially synthesized, produced by recombinant technology, or obtained from biological samples. Exemplary biological samples that may contain one or more antigens include tissue samples, cells, body fluids, biopsies, primary cultures, or combinations thereof. Antigens can be produced by cells that have been modified or genetically engineered to express the antigen, or can be produced by cells that endogenously express the immunogenic mutation or polymorphism (e.g., without human modification or genetic engineering).

[0282] In certain embodiments, the T cells provided herein may be used as host cells that may be modified to include one or more heterologous polynucleotides comprising regulatory sequences (e.g., promoters, enhancers, etc.), a nucleic acid sequence encoding a desired TCR, and / or a nucleic acid sequence encoding all or a portion of a FoxP3 transcription factor, as described herein. Methods for transfecting / transducing T cells with a desired nucleic acid have been previously reported (e.g., U.S. Patent Application Publication No. 2004 / 0087025), and it has also been reported that adoptive transfer can be performed using T cells with a desired target specificity (e.g., Schmitt et al., Hum. Gen. 20:1240, 2009; Dossett et al., Mol. Ther. 17:742, 2009; Till et al., Blood 112:2261, 2008; Wang et al., Hum. Gene Ther. 18:712, 2007; Kuball et al., Blood 109:2331, 2007; U.S. Patent Publication No. 2011 / 0243972; U.S. Patent Publication No. 2011 / 0189141; Leen et al., Ann. Rev. Immunol. 25:243, 2007), and based on the teachings herein, it is contemplated that such methodologies may be used in conjunction with the embodiments disclosed herein. Particularly preferred embodiments relate to artificially modifying the genome of T cells by editing targeted genes as described herein.

[0283] Transfection or transduction of cells, such as T cells, or administration of polynucleotides or compositions according to the methods of the present invention can be carried out using any suitable method. Known methods for delivering polynucleotides to host cells include, for example, the use of cationic polymers, lipid-like molecules, or certain commercially available products (e.g., IN-VIVO-JET PEI). Other methods include ex vivo transduction, injection, electroporation, DEAE-dextran, ultrasound loading, liposome-mediated transfection, receptor-mediated transduction, biolistics, transposon-mediated transfer, and the like. Methods for transfecting or transducing host cells further include the use of vectors described herein and known in the art.

[0284] Nucleic acids may include DNA or RNA, which may be wholly or partially synthetic. Reference to a nucleotide sequence described herein encompasses DNA molecules having the sequence described herein, and, unless otherwise specified, also encompasses RNA molecules having the sequence described herein in which T is replaced with U. As used herein, an "isolated polynucleotide" refers to a genomic polynucleotide, a cDNA polynucleotide, a synthetic polynucleotide, or a combination thereof, and, depending on its origin, an isolated polynucleotide is (1) separated from all or part of a polynucleotide with which the isolated polynucleotide can be found in nature, (2) linked to a polynucleotide with which it is not linked in nature, or (3) present as part of a larger sequence not found in nature.

[0285] "Operably linked" means that the components to which the term is applied are in a relationship that allows them, under appropriate conditions, to perform their specific functions. For example, a transcriptional regulatory sequence "operably linked" to a protein coding sequence is ligated to the protein coding sequence such that expression of the protein coding sequence is achieved under conditions compatible with the transcriptional activity of the transcriptional regulatory sequence.

[0286] As used herein, the term "regulatory sequence" refers to a polynucleotide sequence that can affect the expression, processing, or subcellular localization of a coding sequence by being ligated or operably linked to the coding sequence. The characteristics of such regulatory sequences may depend on the host organism. In certain embodiments, prokaryotic transcriptional regulatory sequences include promoters, ribosomal binding sites, and transcription termination sequences. In other specific embodiments, eukaryotic transcriptional regulatory sequences include promoters containing one or more sites for recognition of transcription factors, transcriptional enhancer sequences, transcription termination sequences, or polyadenylation sequences. In certain embodiments, a "regulatory sequence" may include a leader sequence and / or a fusion partner sequence.

[0287] In certain embodiments, the airT cells of the present invention may be gene-edited to express a FoxP3 gene product encoded by a nucleotide sequence encoding FoxP3 operably linked to a constitutive promoter. Here, "constitutive expression of the FoxP3 gene product" refers to an expression level of FOXP3 equal to or greater than that of natural regulatory T (Treg) cells. In certain preferred embodiments, the constitutive promoter is the MND promoter, and in certain preferred embodiments, the MND promoter is knocked into the native FOXP3 locus by homology-directed repair-mediated gene editing. In certain embodiments, the constitutively active promoter is knocked into the native FOXP3 locus downstream of an intronic regulatory T cell (Treg)-specific demethylation region (TSDR). In certain embodiments, a nucleic acid molecule comprising a polynucleotide encoding exogenous FOXP3 and an operably linked constitutive promoter thereto is knocked into the native FOXP3 locus by homology-directed repair-mediated gene editing. In certain embodiments, a nucleic acid molecule comprising a polynucleotide encoding exogenous FOXP3 and an operably linked constitutive promoter is knocked into a chromosomal site other than the native FOXP3 locus (e.g., the TRAC locus, the AAVS1 locus, or another locus) by homologous recombination repair-mediated gene editing. Thus, in these and related embodiments, the present disclosure teaches for the first time that artificial gene editing such that expression of the FOXP3 gene is regulated by a constitutively active promoter (in a particularly preferred embodiment, a constitutively active MND promoter) provides unexpected advantages in the production of recombinant artificial immunoregulatory T (airT) cells of the invention.

[0288] The term "polynucleotide" as used herein refers to a single- or double-stranded nucleic acid polymer. In certain embodiments, the nucleotides contained in this polynucleotide may be ribonucleotides, deoxyribonucleotides, or modified forms of ribonucleotides or deoxyribonucleotides. Modifications of ribonucleotides or deoxyribonucleotides include base modifications such as bromouridine; ribose modifications such as arabinoside and 2',3'-dideoxyribose; and internucleotide linkage modifications such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate, and phosphoramidate. More specifically, "polynucleotide" includes DNA in single- or double-stranded form.

[0289] "Natural nucleotides" include deoxyribonucleotides and ribonucleotides. "Modified nucleotides" include nucleotides with modified or substituted sugar groups, etc. "Oligonucleotide linkages" include oligonucleotide linkages such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate, phosphoramidate, etc. For example, LaPlanche et al., 1986, Nucl. Acids Res., 14:9081; Stec et al., 1984, J. Am. Chem. Soc., 106:6077; Stein et al., 1988, Nucl. Acids Res., 16:3209; Zon et al., 1991, Anti-Cancer Drug Design, 6:539; Zon et al., 1991, OLIGONUCLEOTIDES AND ANALOGUES: A PRACTICAL APPROACH, pp. 87-108 (F. Eckstein, Ed.), Oxford University Press, Oxford England; Stec et al., US Pat. No. 5,151,510; Uhlmann and Peyman, 1990, Chemical Reviews, 90:543, the disclosures of which are expressly incorporated herein by reference in their entireties. The oligonucleotide may include a detectable label that allows for detection of the oligonucleotide or its hybridization.

[0290] The term "vector" is used to refer to any molecule (e.g., nucleic acid, plasmid, or virus) used to transfer coding information into a host cell. An "expression vector" refers to a vector suitable for transformation of a host cell and contains nucleic acid sequences that direct and / or regulate the expression of an inserted heterologous nucleic acid sequence. Expression includes, but is not limited to, processes such as transcription, translation, and RNA splicing (if introns are present).

[0291] As will be readily understood by those skilled in the art, polynucleotides may include genomic sequences, plasmid-encoded extragenomic sequences, and small artificial gene segments that express or are engineered to express proteins, polypeptides, peptides, etc. Such segments may be isolated from nature or synthetically engineered by those skilled in the art.

[0292] Furthermore, as will be readily understood by those skilled in the art, polynucleotides may be single-stranded (coding or antisense) or double-stranded, and may be DNA molecules (genomic, cDNA, or synthetic) or RNA molecules. RNA molecules may include HnRNA molecules (which contain introns and correspond one-to-one to DNA molecules) and mRNA molecules (which do not contain introns). Additional coding or non-coding sequences may, but are not necessarily, present within a polynucleotide according to the present disclosure, and polynucleotides may, but are not necessarily, attached to other molecules and / or carrier materials. Polynucleotides may include naturally occurring sequences or sequences that encode variants or derivatives of naturally occurring sequences.

[0293] In another related embodiment, a polynucleotide variant may have substantial sequence identity with a polynucleotide sequence encoding an immunomodulatory polypeptide described herein. For example, a polynucleotide may have at least 70% sequence identity, preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity, or a range of sequence identity between any two of these percentages, as measured using methods described herein (e.g., BLAST analysis using standard parameters, as described below), with a reference polynucleotide sequence (such as a sequence encoding an antibody described herein). Those skilled in the art will readily understand that the identity of proteins encoded by two nucleotide sequences can be determined by appropriately adjusting each parameter, taking into account codon degeneracy, amino acid similarity, reading frame position, etc.

[0294] Typically, a variant of a polynucleotide will contain one or more substitutions, additions, deletions, and / or insertions, and preferably a variant of a polypeptide encoded by a variant of a polynucleotide and capable of specifically binding to and interacting with another molecule, derived from a given polypeptide, will contain one or more such substitutions, additions, deletions, and / or insertions that do not substantially reduce its binding affinity compared to the polypeptide encoded by the polynucleotide sequences specifically set forth herein.

[0295] In another specific related embodiment, a polynucleotide fragment may comprise, or consist essentially of, a contiguous sequence of varying lengths identical to or complementary to a sequence encoding a polypeptide described herein, for example, at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, or more of the sequences encoding a polypeptide disclosed herein or variants thereof. , 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 200, 300, 400, 500, 1000 or more consecutive nucleotides, or at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 200, 300, 400, 500, 1000 or more consecutive nucleotides contained in a sequence encoding a polypeptide or variant thereof disclosed herein. 10 pieces, about 11 pieces, about 12 pieces, about 13 pieces, about 14 pieces, about 15 pieces, about 16 pieces, about 17 pieces, about 18 pieces, about 19 pieces, about 20 pieces, about 21 pieces, about 22 pieces, about 23 pieces, about 24 pieces, about 25 pieces, about 26 pieces, about 27 pieces, about 28 pieces, about 29 pieces, about 30 pieces, about 31 pieces, about 32 pieces, about 33 pieces, about 34 pieces, about 35 pieces, about 36 pieces, about 37 pieces, about 38 pieces, about 39 pieces, about 40 pieces, about 45 pieces, about 50 pieces, about 55 pieces, about 60 pieces, about 65 pieces, about 70 pieces, about 75 pieces, about 80 pieces, Polynucleotides containing about 85, about 90, about 95, about 100, about 110, about 120, about 130, about 140, about 150, about 200, about 300, about 400, about 500, about 1000 or more contiguous nucleotides, or polynucleotides containing lengths of contiguous nucleotides of any intermediate length therebetween, or polynucleotides consisting essentially of such numbers of contiguous nucleotides, are provided.As used herein, the term "intermediate length" refers to a length between the numerical values ​​described herein, such as 50, 51, 52, 53, etc.; 100, 101, 102, 103, etc.; 150, 151, 152, 153, etc.; any integer between 200 and 500; or 500 to 1,000, etc. It will be readily understood that the polynucleotide sequences described herein may be extended by adding nucleotides not found in the native sequence to one or both termini. This additional sequence may consist of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides at one or both termini of a polynucleotide encoding a polypeptide described herein.

[0296] In another embodiment, a polynucleotide is provided that can hybridize to a polynucleotide sequence encoding a polypeptide or variant thereof provided herein, a fragment thereof, or a complementary sequence thereof under moderately to highly stringent conditions. Hybridization techniques are well known in the field of molecular biology. As an example, moderately stringent conditions suitable for testing hybridization of a polynucleotide provided herein with another polynucleotide include a prewash step in a solution containing 5xSSC, 0.5% SDS, and 1.0 mM EDTA (pH 8.0); overnight hybridization in 5xSSC at 50-60°C; and two wash steps using 2xSSC containing 0.1% SDS, 0.5xSSC, and 0.2xSSC at 65°C for 20 minutes each. Those skilled in the art will appreciate that stringent hybridization conditions can be easily modified, for example, by changing the salt content of the hybridization solution and / or the temperature at which hybridization is performed. For example, in another embodiment, suitable highly stringent hybridization conditions are the same as those described above, except that the hybridization temperature is increased to, for example, 60-65°C or 65-70°C.

[0297] The polynucleotides described herein, or fragments thereof, may be combined with other DNA sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, and other coding segments, regardless of the length of the coding sequence itself, and their total length may vary considerably. Therefore, it is contemplated that nucleic acid fragments of almost any length can be used, with the total length preferably being limited by the ease of preparation and the recombinant DNA protocol used. For example, polynucleotide segments of lengths of 10,000 base pairs, 5,000 base pairs, 3,000 base pairs, 2,000 base pairs, 1,000 base pairs, 500 base pairs, 200 base pairs, 100 base pairs, 50 base pairs, etc., or about 10,000 base pairs, about 5,000 base pairs, about 3,000 base pairs, about 2,000 base pairs, about 1,000 base pairs, about 500 base pairs, about 200 base pairs, about 100 base pairs, about 50 base pairs, etc. (including all intermediate lengths) are contemplated as useful.

[0298] In comparing polynucleotide sequences, two sequences are said to be "identical" if the nucleotide sequences of the two sequences are identical when aligned for maximum matching, as described below. Comparison of two sequences is typically performed by comparing sequences within a comparison window to identify local regions of sequence homology and then comparing these regions. As used herein, a "comparison window" refers to a segment of at least 20 or at least about 20 (usually 30-75 or 40-50) contiguous positions within which the two sequences are compared at the same number of contiguous positions after optimal alignment of the reference sequence and the specific sequence.

[0299] Optimal alignment of sequences for comparison may be performed using the Megalign program in the Lasergene suite of bioinformatics software (DNASTAR, Madison, Wis.) with default parameters.This program has been used for several alignment schemes described in various publications, including Dayhoff, MO (1978) A model of evolutionary change in proteins - Matrices for detecting distant relationships, in Dayhoff, MO (ed.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington DC, Vol. 5, Suppl. 3, pp. 345-358; Hein J., Unified Approach to Alignment and Phylogenes, pp. 626-645 (1990); Enzymology vol. 183, Academic Press, Inc., San Diego, CA; Higgins, DG and Sharp, PM, CABIOS 5:151-153 (1989); Myers, EW and Muller W., CABIOS 4:11-17 (1988); Robinson, ED, Comb. Theor 11:105 (1971); Santou, N. Nes, M., Mol. Biol. Evol. 4:406-425 (1987); Sneath, PHA and Sokal, RR, Numerical Taxonomy - the Principles and Practice of Numerical Taxonomy, Freeman Press, San Francisco, CA (1973); Wilbur, WJ and Lipman, DJ, Proc. Natl. Acad., Sci. USA 80:726-730 (1983).

[0300] Alternatively, optimal alignment of sequences for comparison may be achieved by the local identity algorithm described in Smith and Waterman, Add. APL. Math 2:482 (1981), the identity alignment algorithm described in Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), the similarity search method described in Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85: 2444 (1988), computer implementations of these algorithms (GAP, BESTFIT, BLAST, FASTA, and TFASTA, available in the Wisconsin Genetics Software Package from Genetics Computer Group (GCG), Inc., 575 Science Drive, Madison, Wis.), or by thorough search.

[0301] Preferred algorithms suitable for determining sequence identity and sequence homology include the BLAST algorithm described in Altschul et al., Nucl. Acids Res. 25:3389-3402 (1977) and the BLAST 2.0 algorithm described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). For example, BLAST or BLAST 2.0 can be used with the parameters described herein to determine sequence identity between two or more polynucleotides. Software for performing BLAST analyses is publicly available from the National Center for Biotechnology Information. As an illustrative example, a cumulative score can be calculated using the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0) for nucleotide sequences. Extension of hit words in both directions is stopped when the cumulative alignment score begins to drop by the value of X from the maximum, when the accumulation of one or more negatively scoring residues causes the cumulative score to fall below zero, or when the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for comparing nucleotide sequences) uses default parameters: word length (W) = 11, expectation (E) = 10, alignment using the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)), (B) = 50, expectation (E) = 10, M = 5, N = -4, and comparison of both strands.

[0302] In certain embodiments, "sequence identity (%)" is determined by comparing two sequences in an optimized alignment within a comparison window containing at least 20 positions, and a portion of the polynucleotide sequence within the comparison window may contain additions or deletions (i.e., gaps) of 20% or less (typically 5-15% or 10-12%) compared to the reference sequence (which does not contain additions or deletions) in the optimized alignment of these two sequences. The sequence identity (%) is calculated by determining the number of positions where identical nucleic acid bases exist in both sequences, calculating the number of positions where the nucleic acid bases match, dividing the number of matched positions by the total number of positions in the reference sequence (i.e., the size of the comparison window), and multiplying the result by 100.

[0303] As those skilled in the art will appreciate, due to the degeneracy of the genetic code, there are many different nucleotide sequences encoding the FoxP3 peptides, TCR peptides, or antigenic peptides described herein, or the antibodies described herein that specifically bind to such peptides. Some of these polynucleotides have minimal sequence identity to the natural or original polynucleotide sequences encoding the FoxP3 polypeptides, TCR polypeptides, or antigenic polypeptides described herein. However, polynucleotides that vary due to differences in codon usage are also expressly contemplated in this disclosure. In certain embodiments, codon-optimized sequences for expression in mammals are specifically contemplated.

[0304] Thus, in another embodiment of the present invention, mutagenesis techniques, such as site-directed mutagenesis, may be used to generate variants and / or derivatives of the FoxP3 polypeptides, TCR polypeptides, or antigenic polypeptides described herein. Mutagenesis techniques allow specific modifications to be introduced into the polypeptide sequence by introducing mutations into the original polynucleotide encoding these polypeptides. Such techniques provide a straightforward method for generating and testing sequence variants that take into account one or more of the above considerations, for example, by introducing one or more nucleotide sequence changes into the polynucleotide.

[0305] In site-directed mutagenesis, variants can be generated by using a specific oligonucleotide sequence encoding the desired mutated DNA sequence and a sufficient number of flanking nucleotides, providing primer sequences of sufficient size and sequence complexity to extend the primers from both ends of the deletion junction to form a stable duplex. Mutations introduced into a selected polynucleotide sequence may improve, alter, reduce, modify, or otherwise change the properties of the polynucleotide itself and / or may alter the properties, activity, composition, stability, or primary sequence of the polypeptide encoded by the polynucleotide.

[0306] In certain embodiments, the inventors contemplate introducing mutations into a polynucleotide sequence encoding a FoxP3 polypeptide, TCR polypeptide, or antigenic polypeptide or variant thereof disclosed herein to alter one or more properties of the polypeptide encoded by the polynucleotide sequence, such as altering the binding affinity of the polypeptide or variant thereof for a ligand recognized by the polypeptide or variant (e.g., in the case of a TCR or antigenic peptide) or the immunosuppressive effect (e.g., in the case of FoxP3). Site-directed mutagenesis techniques are well known in the art and are widely used to generate variant polypeptides and polynucleotides. For example, site-directed mutagenesis is often used to modify specific portions of DNA molecules. In such embodiments, primers, such as 14-25 nucleotides in length, are typically used to modify approximately 5-10 residues or 5-10 residues on either side of the junction of the sequences.

[0307] As those skilled in the art can well understand, site-directed mutagenesis techniques often use phage vectors in single-stranded or double-stranded form.Standard vectors useful for site-directed mutagenesis include vectors such as M13 phage.These phages are readily available commercially, and their use is generally well known to those skilled in the art.Double-stranded plasmids are also routinely used in site-directed mutagenesis, which eliminates the step of transferring the gene of interest from the plasmid to the phage.

[0308] The site-directed mutagenesis described herein typically begins with obtaining a single-stranded vector containing a DNA sequence encoding the desired peptide, or melting a double-stranded vector containing a DNA sequence encoding the desired peptide to separate it into two strands. An oligonucleotide primer bearing the desired mutated sequence is then prepared, typically synthetically. This primer is then annealed to the single-stranded vector, and the mutation-bearing strand is synthesized by DNA polymerization using an enzyme such as the Klenow fragment of E. coli polymerase I. A heteroduplex is thus formed, with one strand encoding the unmutated original sequence and the second strand bearing the desired mutation. This heteroduplex vector is then used to transform appropriate cells (e.g., E. coli cells), and clones containing recombinant vectors bearing the mutated sequence are selected.

[0309] The preparation of sequence variants of selected DNA segments encoding peptides using site-directed mutagenesis provides a means for producing potentially useful peptide species, but is not limited to this. Another method by which sequence variants of peptides and the DNA sequences encoding them can be obtained is by treating or contacting a recombinant vector encoding the desired peptide sequence with a mutagen such as hydroxylamine. Specific details regarding these methods and protocols are taught in Maloy et al., 1994; Segal, 1976; Prokop and Bajpai, 1991; Kuby, 1994, and Maniatis et al., 1982 (all of which are incorporated herein by reference for this purpose).

[0310] As used herein, "oligonucleotide-directed mutagenesis" refers to a template-dependent method and vector-mediated propagation in which the concentration of a particular nucleic acid molecule is increased above its initial concentration or the level of a detectable signal (e.g., amplification) is increased. As used herein, "oligonucleotide-directed mutagenesis" refers to a method involving template-dependent extension of a primer molecule. "Template-dependent methods" refer to nucleic acid synthesis of RNA or DNA molecules, characterized in that the sequence of the newly synthesized nucleic acid strand is determined by the well-known rule of complementary base pairing (see, e.g., Watson (1987)). Furthermore, vector-based methodologies generally involve introducing a nucleic acid fragment into a DNA or RNA vector, amplifying the resulting vector clone, and recovering the amplified nucleic acid fragment. An example of such a methodology is described in U.S. Pat. No. 4,237,224, which is expressly incorporated herein by reference in its entirety.

[0311] Another method for producing polypeptide variants may utilize recursive sequence recombination, as described in U.S. Patent No. 5,837,458, which is expressly incorporated herein by reference in its entirety. This method allows for the "evolution" of individual polynucleotide variants through repeated recombination and screening or selection, e.g., with improved binding affinity. Certain embodiments further provide constructs in the form of plasmids, vectors, transcription cassettes, or expression cassettes, comprising at least one polynucleotide described herein.

[0312] It will be readily understood that the practice of certain embodiments of the present invention will employ, unless otherwise indicated, conventional methods in virology, immunology, microbiology, molecular biology and recombinant DNA technology that are within the skill of the art, most of which are described below for illustrative purposes, and such techniques are fully described in the literature. For example, Current Protocols in Molecular Biology or Current Protocols in Immunology, John Wiley & Sons, New York, NY (2009); Ausubel et al., Short Protocols in Molecular Biology, 3rd ed., Wiley & Sons, 1995; Sambrook and Russell, Molecular Cloning: A Laboratory Manual (3rd Edition, 2001); Maniatis et al. Molecular Cloning: A Laboratory Manual (1982); DNA Cloning: A Practical Approach, vol. I & II (D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed., 1984); Nucleic Acid Hybridization (B. Hames & S. Higgins, eds., 1985); Transcription and Translation (B. Hames & S. Higgins, eds., 1984); Animal Cell Culture (R. Freshney, ed., 1986); See Perbal, A Practical Guide to Molecular Cloning (1984), both of which are incorporated herein by reference in their entireties.

[0313] Standard techniques may be used for DNA recombination, oligonucleotide synthesis, tissue culture, and transformation (e.g., electroporation, lipofection, etc.). Enzymatic reactions and purification techniques may be performed according to manufacturer's instructions, by methods common in the art, or as described herein. These and related techniques and procedures may generally be performed according to conventional methods well known in the art or as described in the various general or more detailed references cited or discussed herein. Unless specific definitions are provided, the nomenclature, laboratory methods, and techniques used in connection with molecular biology, analytical chemistry, organic synthetic chemistry, and medicinal chemistry described herein are those commonly used and known in the art. Standard techniques may also be used for recombinant technology, molecular biological synthesis, microbiological synthesis, chemical synthesis, chemical analysis, pharmaceutical preparation, formulation, and delivery, and patient treatment.

[0314] Autoimmune, allergic and inflammatory diseases and antigens associated therewith As noted above, the airT cells of the present invention may be used to treat and / or alleviate certain autoimmune, allergic, and inflammatory diseases, the clinical signs and symptoms, and diagnostic criteria of which are known in the art. Examples of diseases that may benefit from administration of the airT cells of the present invention to human patients or other mammalian hosts in need of antigen-specific immunosuppression, i.e., diseases in individuals in which clinically inappropriate pro-inflammatory mediators (e.g., cytokines, lymphokines, hormones, etc.) and / or localized or systemic increased inflammatory cells may be present, include, but are not limited to, type 1 diabetes, multiple sclerosis, systemic lupus erythematosus, myasthenia gravis, rheumatoid arthritis, Crohn's disease, inflammatory bowel disease, bullous pemphigoid, pemphigus vulgaris, autoimmune hepatitis, asthma, allergies (e.g., specific hypersensitivity to food allergens, plant allergens, animal allergens, environmental allergens, drug allergens, chemical allergens, or other allergens), transplant tolerance induction (e.g., pancreatic islet cell transplantation), and graft-versus-host disease (GVHD) following stem cell (e.g., hematopoietic stem cell) transplantation.

[0315] Some of the antigens associated with these diseases, particularly those for which epitopes recognized by TCRs exist, are known and are shown in the figures, which also include the TCR V-region sequences of TCRs designated based on their ability to recognize antigens of the present disclosure associated with autoimmune, allergic, or inflammatory diseases.

[0316] Methods for identifying and characterizing antigens related to the pathogenesis of autoimmune disease, allergic disease or inflammatory disease (including determining autoreactive T cell epitopes) are known in the art.For example, a typical method for identifying islet autoantigenic polypeptides, including fragments of islet autoantigenic polypeptides recognized by T cells, from subjects suffering from type 1 diabetes (T1D) has been reported by Cerosaletti et al. (2017 J. Immunol. 199:323; this document is expressly incorporated herein by reference in its entirety).Other polypeptide antigens related to the pathogenesis of autoimmune disease, allergic disease or inflammatory disease (including determined autoreactive T cell epitopes) are disclosed herein, including those shown in the drawings.

[0317] Cerosaletti et al. (2017) also describes a typical methodology for determining the structure of T cell receptors (TCRs) that recognize antigens associated with the pathogenesis of autoimmune, allergic, or inflammatory diseases, but is not limited to this methodology. Structural features of various TCRs specific to various antigens associated with the pathogenesis of autoimmune, allergic, or inflammatory diseases, such as the entire or partial amino acid sequences of the variable region (Vα) of the TCR α chain and / or the variable region (Vβ) of the TCR β chain, and polynucleotide sequences encoding them, are disclosed herein, including those depicted in the drawings.

[0318] Composition and Method of Use Accordingly, certain embodiments disclosed herein contemplate administering the airT cells described herein as adoptively transferred immunotherapy cells to provide antigen-specific immunosuppression for diseases in which excessive and / or clinically deleterious antigen-specific immune activity is present. For example, by way of example only, certain embodiments contemplate immunotherapy protocols that include adoptively transferring the airT cells disclosed herein into a subject (e.g., a patient with an autoimmune, allergic, or other inflammatory disease). Adoptive transfer protocols using unselected or selected T cells are known in the art (e.g., Schmitt et al., 2009 Hum. Gen. 20:1240; Dossett et al., 2009 Mol. Ther. 17:742; Till et al., 2008 Blood 112:2261; Wang et al., 2007 Hum. Gene Ther. 18:712; Kuball et al., 2007 Blood 109:2331; U.S. Patent Publication No. 2011 / 0243972; U.S. Patent Publication No. 2011 / 0189141; Leen et al., 2007 Ann. Rev. Immunol. 25:243; (See U.S. Patent Publication Nos. 2011 / 0052530, 2010 / 0310534; Ho et al., 2006 J. Imm. Meth. 310:40; Ho et al., 2003 Canc. Cell 3:431), these adoptive transfer protocols may be modified in accordance with the teachings herein and used to transfer cell populations containing the desired airT cells generated as described herein.

[0319] The airT cells of the present invention can be administered using any of the generally accepted methods for administering drugs with similar utilities. The airT cells of the present invention can be formulated into pharmaceutical compositions by mixing with suitable physiologically acceptable carriers, diluents, or additives, such as aqueous liquids that may contain appropriate salts, buffers, and / or stabilizers. The airT cells can be administered by various routes, including intravenous, intrahepatic, intraperitoneal, intragastric, intraarticular, intrathecal, and other routes, and in a preferred embodiment, by intravenous infusion.

[0320] Preferred methods of administration will depend on the characteristics of the condition to be treated or prevented, and in certain embodiments, on the type of harmful or clinically undesirable condition whose extent, severity, likelihood of occurrence, and / or duration may be reduced (e.g., statistically significantly reduced compared to an appropriate control condition, such as an untreated control) by a particular method provided herein. Efficacy is considered to be a detectable reduction, suppression, or delay of such a condition, e.g., a reduction, suppression, or delay of local or systemic autoimmune, allergic, or other harmful inflammatory activity, following administration of the airT cells of the invention. Those skilled in the art related to the present invention will be familiar with various diagnostic, surgical, and other clinical criteria that can be adapted to assess the efficacy of administering the immunoregulatory airT cell compositions described herein by adoptive transfer. See, e.g., Humar et al., Atlas of Organ Transplantation, 2006, Springer; Kuo et al., Comprehensive Atlas of Transplantation, 2004 Lippincott, Williams & Wilkins; Gruessner et al., Living Donor Organ Transplantation, 2007 McGraw-Hill Professional; Antin et al., Manual of Stem Cell and Bone Marrow Transplantation, 2009 Cambridge University Press; Wingard et al. (Ed.), Hematopoietic Stem Cell Transplantation: A Handbook for Clinicians, 2009 American Association of Blood Banks.

[0321] Thus, in some embodiments, the airT cells of the present invention may express an antigen-specific T cell receptor (TCR) comprising an antigen-specific TCR polypeptide encoded by at least one introduced polynucleotide encoding the antigen-specific TCR polypeptide, and may induce antigen-specific immunosuppression in response to HLA-restricted stimulation by an antigen specifically recognized by the TCR polypeptide. The presence or absence of immunosuppression may be determined by various criteria familiar to those skilled in the art. See, for example, Sakaguchi et al., 2020 Ann. Rev. Immunol. 38:541 (which is expressly incorporated herein by reference in its entirety).

[0322] For example, several mechanisms have been reported that contribute to the suppressive phenotype of Treg cells, including the CTLA-4 immune checkpoint; expression of immunosuppressive cytokines such as IL-10 and TGF-β; cytotoxicity against target cells via the perforin / granzyme pathway; induction of indoleamine-2,3-dioxygenase (IDO) and tryptophan catabolism in target cells; adenosine consumption through expression of CD73; and effector T (T) cells against IL-2, based on their constitutive expression of CD25 (a subunit of the high-affinity receptor for IL-2). eff(Immune Dysregulation Leading to Chronic Autoimmunity.) See, for example, Verbsky, JW, and Chatila, TA (2014). Chapter 23 - Immune Dysregulation Leading to Chronic Autoimmunity. in Stiehm's Immune Deficiencies, K. E. Sullivan, and E. R. Stiehm, eds., (Amsterdam: Academic Press), pp. 497-516; Campbell et al. 2020 Cell Metab. 31(1):18-25; Dominguez-Villar et al., 2018 Nat. Immunol. 19:665-673; Sakaguchi et al., 2008 Cell 133(5):775-787.

[0323] Thus, in some embodiments, antigen-specifically induced immunosuppression is (i) inhibiting the activation and / or proliferation of effector T cells that recognize an antigen specifically recognized by an airT cell TCR comprising the TCR polypeptide encoded by the 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 an airT cell TCR comprising the TCR polypeptide encoded by the at least one introduced polynucleotide; (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, expression of inhibitory receptors by airT cells, and (iv) inhibiting the activation and / or proliferation of effector T cells that do not recognize the antigen specifically recognized by the air T cell TCR comprising the TCR polypeptide encoded by the at least one introduced polynucleotide. It may include one or more of:

[0324] In certain instances, adoptive transfer immunotherapy using airT cells of the present invention (which may be administered in conjunction with at least one other therapeutic agent) may be administered for 1 to 14 days during a 30-day treatment period. In certain instances, adoptive transfer immunotherapy using airT cells of the present invention (which may be administered in conjunction with at least one other therapeutic agent) may be administered for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days during a 60-day treatment period. Alternative protocols may be appropriate for individual subjects. When administered as described above, an appropriate dose is an amount of compound that allows a detectable change or alleviation of symptoms, or that causes a statistically significant decrease of at least one indicator of autoimmune activity, allergic immune activity, or other inflammatory immune activity by at least 10-50% compared to the baseline state (e.g., untreated state), and the change or alleviation of symptoms or the indicator can be monitored by measuring the amount of specific blood components, for example, by measuring the number of detectable immune cells and / or other inflammatory cells in the blood and / or the amount of detectable soluble inflammatory mediators, such as pro-inflammatory cytokines.

[0325] Appropriate dosages and treatment regimens typically provide sufficient amounts of the airT cells of the present invention to provide therapeutic and / or prophylactic benefit. Response to such administration can be monitored by observing improved clinical outcomes (e.g., more frequent remission, complete or partial remission, or longer disease-free survival) in treated subjects compared with untreated subjects. A reduction (e.g., a statistically significant reduction compared with relevant controls) in pre-existing immune responses to antigens associated with the autoimmune, allergic, or other inflammatory diseases described herein typically correlates with improved clinical outcomes. Such immune responses may typically be assessed by analyzing standard leukocyte and / or lymphocyte surface markers, cytokine expression, cell proliferation, cytotoxicity, or released cytokines; such analyses are routine in the art and may be performed using samples taken from the subject before and after treatment.

[0326] For example, the airT cells of the invention may be administered in an amount sufficient to clinically meaningfully reduce the symptoms of an autoimmune disease (e.g., sufficient to clinically significantly reduce the symptoms of an autoimmune disease, preferably sufficient to detectably reduce the symptoms of an autoimmune disease with statistical significance compared to an appropriate control disease), including, but not limited to, type 1 diabetes, rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), multiple sclerosis, inflammatory bowel disease (IBD), psoriatic arthritis, Crohn's disease, ulcerative colitis, seronegative spondyloarthropathy, Behcet's disease, vasculitis, and other autoimmune diseases.

[0327] Thus, in some embodiments, following adoptive transfer of airT cells expressing a TCR that specifically recognizes an antigenic epitope associated with an autoantigen associated with type 1 diabetes (T1D) into a patient with type 1 diabetes, a reduction in one or more relevant clinical criteria known in the art used to assess type 1 diabetes may be identified. Exemplary antigens (usually autoantigens) associated with type 1 diabetes and the structures of TCRs that specifically recognize such antigens are described herein.

[0328] Common criteria for stage 2 type 1 diabetes may include the detection of two or more islet-specific autoantibodies in a patient and evidence of abnormal glucose metabolism in an oral glucose tolerance test. In some embodiments, abnormal glucose metabolism may be defined as a fasting blood glucose level of 110-125 mg / dL (6.1-6.9 mmol / L), a 2-hour postprandial plasma glucose level of 140 mg / dL (7.8 mmol / L) or greater but less than 200 mg / dL (11.1 mmol / L), or a 30-, 60-, or 90-minute postprandial plasma glucose level greater than 200 mg / dL. In some embodiments, clinical type 1 diabetes may be defined as the presence of diabetic symptoms (e.g., increased thirst, frequent urination, and / or unexplained weight loss compared to normal subjects known to be not at risk for or known not to have type 1 diabetes) and a blood glucose level of 200 mg / dL or greater, a fasting blood glucose level of 126 mg / dL or greater, a 2-hour oral glucose tolerance test (OGTT) of 200 mg / dL or greater, or a hemoglobin A1c level of 6.5% or greater (see, e.g., Khokhar et al., 2017 Clin. Diabetes 35(3):133).

[0329] The reduction of rheumatoid arthritis symptoms may be, for example, but not limited to, the reduction of any one or more of the following: fatigue; loss of appetite; low-grade fever; swollen lymph nodes; weakness; swollen joints; joint pain; morning stiffness; hot, brittle or stiff joints after only an hour of disuse; bilateral joint pain (may affect knuckles (excluding fingertips), wrists, elbows, shoulders, hips, knees, ankles, toes, jaw joints and neck); reduced range of motion of affected joints; pleurisy; burning eyes; itchy eyes; eye discharge; subcutaneous nodules; and numbness, tingling or burning in hands and feet.The diagnostic criteria and clinical monitoring of rheumatoid arthritis patients are well known to those skilled in the art.For example, see Hochberg et al., Rheumatology, 2010 Mosby; Firestein et al., Textbook of Rheumatology, 2008 Saunders. The diagnostic criteria and clinical monitoring of patients with rheumatoid arthritis and / or other autoimmune diseases are also well known to those skilled in the art.See, for example, Petrov, Autoimmune Disorders: Symptoms, Diagnosis and Treatment, 2011 Nova Biomedical Books; Mackay et al. (Eds.), The Autoimmune Diseases-Fourth Edition, 2006 Academic Press; Brenner (Ed.), Autoimmune Diseases: Symptoms, Diagnosis and Treatment, 2011 Nova Science Pub. Inc.

[0330] Standard techniques may be used for recombinant DNA, peptide and oligonucleotide synthesis, immunoassays, tissue culture, and transformation (e.g., electroporation, lipofection, etc.). Enzymatic reactions and purification techniques may be performed according to manufacturer's instructions, as is common in the art, or as described herein. These and related techniques and manipulations may generally be performed according to conventional methods well known in the art, or as described in the various general or more detailed references on microbiological, molecular biological, biochemical, molecular genetic, cell biological, virological, or immunological techniques cited or discussed herein. For example, Sambrook, et al., Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Current Protocols in Molecular Biology (John Wiley and Sons, updated July 2008); Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Glover, DNA Cloning: A Practical Approach, vol. I & II (IRL Press, Oxford Univ. Press USA, 1985); Current Protocols in Immunology (Edited by: John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M.Shevach, Warren Strober 2001 John Wiley & Sons, NY, NY); Real-Time PCR: Current Technology and Applications, Edited by Julie Logan, Kirstin Edwards and Nick Saunders, 2009, Caister Academic Press, Norfolk, UK; Anand, Techniques for the Analysis of Complex Genomes, (Academic Press, New York, 1992); Guthrie and Fink, Guide to Yeast Genetics and Molecular Biology (Academic Press, New York, 1991); Oligonucleotide Synthesis (N. Gait, Ed., 1984); Nucleic Acid Hybridization (B. Hames & S. Higgins, Eds., 1985); Transcription and Translation (B. Hames & S. Higgins, Eds., 1984); Animal Cell Culture (R. Freshney, Ed., 1986); Perbal, A Practical Guide to Molecular Cloning (1984); Next-Generation Genome Sequencing (Janitz, 2008 Wiley-VCH); PCR Protocols (Methods in Molecular Biology) (Park, Ed., 3rd Edition, 2010 Humana Press); Immobilized Cells And Enzymes (IRL Press, 1986); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H.Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Harlow and Lane, Antibodies, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1998); Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and CC Blackwell, eds., 1986); Roitt, Essential Immunology, 6th Edition (Blackwell Scientific Publications, Oxford, 1988); Embryonic Stem Cells: Methods and Protocols (Methods in Molecular Biology) (Kurstad Turksen, Ed., 2002); Embryonic Stem Cell Protocols: Volume I: Isolation and Characterization (Methods in Molecular Biology) (Kurstad Turksen, Ed., 2006); Embryonic Stem Cell Protocols: Volume II: Differentiation Models (Methods in Molecular Biology) (Kurstad Turksen, Ed., 2006); Human Embryonic Stem Cell Protocols (Methods in Molecular Biology) (Kursad Turksen Ed.Hematopoietic Stem Cell Protocols (Methods in Molecular Medicine) (Christopher A. Klug, and Craig T. Jordan Eds., 2001); See Hematopoietic Stem Cell Protocols (Methods in Molecular Biology) (Kevin D. Bunting Ed., 2008) Neural Stem Cells: Methods and Protocols (Methods in Molecular Biology) (Leslie P. Weiner Ed., 2008). .

[0331] Unless specifically defined, the nomenclatures, laboratory methods, and techniques used in connection with molecular biology, analytical chemistry, synthetic organic chemistry, and medicinal chemistry described herein are those commonly used and known in the art, and standard techniques may be used for recombinant technology, molecular biological synthesis, microbiological synthesis, chemical synthesis, chemical analysis, pharmaceutical preparation, formulation, and delivery, and patient treatment.

[0332] Each embodiment described herein applies mutatis mutandis to each of the other embodiments unless otherwise stated.

[0333] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless expressly stated otherwise. Also, in this specification, unless expressly stated otherwise, the term "comprise," and its variations "comprises" or "comprising," are understood to include any component or element, or group of components or elements, described herein, but not to exclude any component or element, or group of components or elements other than those described herein. Each embodiment described herein applies mutatis mutandis to each of the other embodiments, unless expressly stated otherwise. [Example]

[0334] Example 1 - Generation of airT cells We developed a platform to convert normal human CD4 T cells into Treg-like cells (edTreg; airT) by editing the Foxp3 gene (Figure 1A, 1B, 1C, 2). This platform involved generating antigen-specific edTreg cells by lentiviral transduction of TCR genes.

[0335] Antigen-specific T cells were identified by activating PBMCs with a peptide pool and assessing CD154 expression. This method utilized single-cell RNA-seq analysis to identify TCR clonotypes that were upregulated in subjects with type 1 diabetes and generate full-length sequences for each TCR (Cerosaletti et al. 2017 J. Immunol. PMID: 28566371). Based on the islet-specific TCR sequences identified in this study, we generated several lentiviral TCR constructs for TCR gene transfer. These TCR constructs expressed human TCR variable regions derived from the islet-specific TCR and constant regions from a mouse TCR, resulting in improved pairing of the human TCR chains upon transduction (Figure 5). Expression of the islet-specific TCR was verified by T cell proliferation assays using peptides recognized by the TCR (or irrelevant peptides) and antigen-presenting cells (APCs). T cells transduced with an islet-specific TCR proliferated only in response to peptides recognized by the TCR and APCs (FIG. 6).

[0336] To generate antigen-specific airT cells, we edited the Foxp3 locus in CD4 T cells transduced with an islet-specific TCR, and successfully generated airT cells expressing the islet-specific TCR. The airT cells expressing the islet-specific TCR exhibited a Treg phenotype: CD25+, CD127-, CTLA4+, and ICOS+ (Figure 7). Notably, the airT cells expressing the islet-specific TCR exhibited antigen-specific and bystander suppressive functions in in vitro suppression assays (Figures 7-11). Furthermore, the airT cells were able to inhibit T eff The production of inflammatory cytokines such as TNF, IFN-γ, IL-17, and IL-2 by the cells was specifically suppressed by the airT antigen (Fig. 11).

[0337] We investigated the Treg phenotype, generation efficiency, and suppressive capacity of airT cells by comparing them with expanded nTreg cells. While airT cells could be expanded to three times the number of PBMCs added, nTreg cells were only obtained at 1–4% of the number of cells added, even after 10 days of expansion. Furthermore, airT cells exhibited a phenotype similar to nTreg cells, but expressed higher levels of Foxp3, CTLA-4, and ICOS than nTreg cells (Figure 3).

[0338] Notably, the suppressive activity of airT cells against the proliferation of effector T cells in vitro was equal to or greater than that of expanded nTreg cells (Figure 4).

[0339] Example 2 - Acquisition of Treg phenotype and in vitro immunosuppressive properties by FOXP3-edited antigen-specific human T cells As an alternative approach to generating FOXP3-edited antigen-specific CD4+ T cells, we developed a method for isolating, gene-editing, and expanding antigen-specific effector T cells from healthy subjects or patients with autoimmune diseases. To test whether FOXP3 editing is possible while maintaining proliferation of antigen-specific human T cells, CD4+ T cells obtained from a human donor with the HLA DRB1*0401 allele were expanded in the presence of influenza antigen (flu) and tetanus toxin antigen, and then gene-edited. After gene editing, the cells were expanded for an additional 4–7 days in the presence of an antigen cocktail consisting of influenza antigen and tetanus toxin antigen. The average editing rate (GFP+) at this time was 28 ± 2.1% (Figure 12). Cells were labeled with a mixture of PE-labeled influenza antigen peptide-MHC-II tetramer and PE-labeled tetanus toxin antigen peptide-MHC-II tetramer, and antigen-specific cells were purified by FACS. The resulting tetramer-positive airT (Tmr+airT) cells recapitulated the immunophenotype of activated tTreg cells, characterized by canonical regulatory T cell markers. More specifically, these tetramer-positive airT cells, unlike Tmr+mock cells analyzed in parallel, exhibited upregulated expression of FOXP3, CD25, CTLA4, and Helios, and suppressed IL-2 production (Fig. 13A). Furthermore, unlike Tmr+mock cells, Tmr+airT cells were able to express polyclonally activated autologous CD4+T cells in vitro. eff These results demonstrate that CD4+ T cells obtained from human peripheral blood can be enriched for target antigen-specific CD4+ T cells by tetramer-based flow cytometry sorting and can be modified using gene editing to confer tTreg-like phenotypes and suppressive properties while retaining antigen specificity (Figure 13B).

[0340] We further developed this method to enrich for antigen-specific T cells by stimulating them with model antigens (MP, HA, and TT). After approximately two weeks of expansion, the cells were stained with tetramers to identify antigen-specific T cells, and the FoxP3 locus was edited (Figure 14). We used this method to generate antigen-specific Treg cells, and these air T cells exhibited antigen-specific suppressive activity in vitro (Figure 15). Furthermore, we enriched for islet-specific T cells by stimulating them with a pool of islet-specific peptides, and then isolated islet-specific T cells with multiple specificities by tetramer staining. In this experiment, we also obtained islet-specific air T cells by editing the Foxp3 gene in these cells (Figures 16 and 17).

[0341] Example 3 - Generating double-edited human CD4+ T cells by gene editing of two alleles using homologous recombination repair Figures 18, 19 and 110 summarize the experimental methods used to demonstrate whether two separate expression cassettes can be introduced into the human TRAC locus (Figure 18) and the constructs used in these experiments (Figure 19).

[0342] Using a CRISPR-based approach, we tested the efficacy of four novel gRNAs targeting the first exon of the human TRAC locus for inducing complete TCR knockout (Figure 20). Forty-eight hours after RNP delivery, CD3 expression was assessed by flow cytometry, demonstrating that gRNA_1 knocked out 96.8% of CD3, while gRNA_4 knocked out 84.7% of CD3 (Figure 21). Furthermore, on-target site-specific activity was measured using Inference of CRISPR Edits (ICE), confirming that gRNA_1 and gRNA_4 specifically induced indels at the TRAC locus compared with predicted off-target sites (Figure 22). Next, to test the specificity of these novel guides, we evaluated the top three off-target sites (predicted based on bioinformatics analysis of the most similar sequences in the human genome) for each gRNA. These off-target sites were directly analyzed by amplifying each off-target site from nuclease-transfected human T cells. The amplicons were sequenced and analyzed using the ICE program. The observed cleavage activity at each off-target site candidate was 0%. In contrast, at the on-target site in the same assay, the cleavage activity at the targeted TRAC site by gRNA_1 was 78% and by gRNA_4 was 66% (Figure 23). This result indicated that these new donor templates were highly specific for the TRAC locus.

[0343] Next, we investigated the dual-editing ability of each gRNA in human CD4+ T cells using constructs that allow easy tracking of successfully edited cells. We engineered MND-GFP and MND-BFP cassettes flanked by identical 300-bp homology arms compatible with gRNA_1 or gRNA_4 (Figure 24A) targeting the TRAC locus and tested whether these expression cassettes could be used to edit two alleles of the TRAC locus to generate T cells stably expressing both GFP and BFP. A timeline of cell expansion, gene editing, and analysis is shown in Figure 24B. FACS analysis revealed that 20.3% of BFP / GFP double-positive cells were obtained with gRNA_1, and 10.6% of BFP / GFP double-positive cells were obtained with gRNA_4, confirming that both repair cassettes were integrated after the creation of a single double-strand break (Figure 25).

[0344] In some cases, selective expansion of recombinant cells in vitro may be useful to obtain sufficient numbers of cells for therapeutic use. To selectively expand dual-edited cells, we generated a homology-directed repair (HDR) knock-in construct containing a split IL-2 chemo-inducible signaling complex (CISC) for enrichment and selection. We have previously reported a method for enriching gene-edited CD4+ T cells using the IL-2 CISC component in the presence of rapamycin or AP21967 (a rapalog), a rapamycin homolog capable of inducing heterodimerization. See, for example, Figure 108. In this method, the FRB-IL2RB and FKBP-IL2RG components are inserted into the same cassette to allow selection for single integration events.

[0345] To conduct this study, the FRB-IL2RB and FKBP-IL2RG constructs were split into two separate cassettes. One cassette contained GFP and the other contained mCherry, allowing for the selection of two independent integrations. These constructs are shown in Figure 26, and the timeline and editing conditions for this experiment are shown in Figure 27. The initial double-editing rate with these constructs was 1.44% as a percentage of GFP / mCherry double-positive cells (Figure 28). This was likely due to the large size of the homologous recombination repair template, but the use of rapalogs significantly enriched for double-edited cells. Treatment with 100 nM rapalog increased the percentage of GFP / mCherry double-positive cells from 1.4% to 9% over 8 days (Figure 29). Importantly, the proportions of GFP-, mCherry-, and double-negative cells remained unchanged in the presence of the rapalog, suggesting that dual expression of FRB-IL2RB and FKBP-IL2RG results in proliferation of GFP / mCherry double-positive cells only in the presence of functional IL-2 CISC protein. As expected, no proliferation was observed in the presence of IL-2 (Figure 30).

[0346] We tested the reproducibility between experiments and the variability between donors (Figure 31). We edited genes in cells from the same donor as in the previous experiment (shown in Figure 29) and compared them with cells from another Caucasian male donor of the same age. The double-editing rate in cells from donor R003657 was 1.1%, which was similar to the results obtained in the previous experiment (Figure 29). The bi-allele editing rate in the second donor (R003471) was 6.4%. Overall, the editing rate varied between donors, but the proportions of GFP-positive, mCherry-positive, and double-positive cells were similar, suggesting that the variability in editing rates may be due to how well the donor's cells were edited. Importantly, double-edited cells from both donors were enriched in the presence of the rapalog, with donor R003657 yielding 13.8% GFP / mCherry double-positive cells and donor R003471 yielding 28.5% GFP / mCherry double-positive cells (Figure 32).

[0347] These results suggest that the double-editing method, which incorporates split IL-2 CISCs by homologous recombination repair-mediated gene editing, can provide a means to efficiently select and enrich for double-edited cells and to obtain the necessary numbers of edited cells for therapeutic use.

[0348] Based on our results demonstrating successful bi-allele editing using the MND-eGFP-FRB-IL2RB and MND-mCherry-FKBP-IL2RG cassettes, and the ability to enrich for double-edited cells using rapalogs, we generated constructs capable of delivering FoxP3 in combination with the IL-2 CISC component and a pancreatic islet antigen-specific TCR (T1D4) in combination with the IL-2 CISC component, and used these to generate antigen-specific Foxp3+ air T cells (Figure 33).

[0349] Example 4: Targeting two alleles to generate double-edited mouse CD4+ T cells To evaluate the efficacy of antigen-specific FoxP3 air T cells in animal models of diabetes or other autoimmune diseases, we constructed a similar tool to edit the mouse Trac locus. Three novel gRNA target sequences within the first exon of the mouse Trac locus were selected and tested for CD3 knockout in mouse (C57 / B6) CD4+ primary T cells (Figure 34). As shown in Figure 35, mCD3 expression was measured by flow cytometry analysis two days after transfection, and mTrac_gRNA_2 demonstrated the highest knockout rate at 87.8%. As with the constructs for human cells, we generated MND-GFP and MND-BFP constructs to allow for convenient tracking of gene-edited cells. The constructs used in this experiment and the timeline for this experiment are shown in Figure 36. As with the double-edited human cells with TRAC locus editing, the double-editing efficiency in mouse cells was relatively low (1.97%) (Figure 37).

[0350] Example 5: Function of air T cells in an antigen-specific mouse model of multiple sclerosis To investigate the function of air T cells in an antigen-specific in vivo condition, T cells for gene editing were selected from myelin oligodendrocyte glycoprotein peptide fragment 35-55 (MOG)-specific TCR transgenic mice (C57Bl / 6-Tg(Tcra2D2, Tcrb2D2)1Kuch mice (abbreviated as 2D2 mice)). When challenged with MOG, 2D2 transgenic mice develop experimental autoimmune encephalomyelitis (EAE), which can be used as a mouse model of multiple sclerosis. EAE in 2D2 mice is not regulated by endogenous 2D2 tTreg cells present in the central nervous system (CNS), which is likely a pathogenic T cell. eff This is thought to be due to the production of large amounts of inflammatory cytokines by antigen-specific 2D2 airT cells. Adoptive transfer of antigen-specific 2D2 airT cells stimulates T cells in the periphery before these activated effector T cells migrate to the central nervous system. effThis may be able to suppress cell proliferation (Figure 38). To test this hypothesis, we designed TALEN and AAV donor templates to closely mimic the GFP knock-in editing method used to generate GFP+ human airT cells. We designed an improved procedure for stimulating mouse T cells and electroporating mRNA, and transfected them with mRNA encoding a TALEN pair specific for the first coding exon of mouse Foxp3. Seven to nine days after transduction, colony sequencing of PCR-amplified gDNA revealed that approximately 80% of alleles contained indels (Figure 39). This result indicated efficient excision of the target site. We cloned an AAV donor template in which the human Foxp3 homology arm sequence used in the previous homology-directed repair experiment was replaced with the mouse Foxp3 homology arm sequence. The homology arm was adjacent to, but did not overlap with, the mouse TALEN binding site. By slightly modifying the conditions used for gene editing of human CD4+ T cells, including the use of AAV5 capsids for donor template delivery, gene editing was performed using mCD4+ T cells isolated from the spleen and lymph nodes (LN) of 2D2 mice. Approximately 25-30% editing rates (GFP+ cells) were consistently achieved. The resulting GFP+ cells had a FOXP3+CD25+CTLA-4+ phenotype (Figure 40). To compare with airT cells generated from 2D2 mice, CD4+ T cells were isolated from littermate mice (C57BL / 6) that did not have the 2D2 phenotype and gene-edited to generate airT cells containing polyclonal TCR pools with various specificities. Next, 3.0 × 10 4 CD4+T eff Cells, 3.0 x 10 4These cells were adoptively transferred into lymphopenic Rag1- / - mice along with mock or air T cells. Recipient mice were challenged with the MOG35-55 peptide in adjuvant, followed by pertussis toxin administration to disrupt the blood-brain barrier. Figure 41 shows the experimental timeline, including cell transfer, immunization, and cell analysis. Recipient mice in this model develop symptoms of EAE (tail droop and complete ptosis, followed by hind limb weakness and paralysis) approximately 7–10 days after cell transfer. To assess effector T cell priming, recipient mice were euthanized 7 days after cell transfer, and inguinal and axillary lymph nodes were harvested. The percentage of CD45+CD4+ T cells in lymph nodes from recipients of antigen-specific 2D2 air T cells was half that of recipients of mock-edited cells and 1.7-fold lower than that of recipients of polyclonal C57Bl / 6 air T cells. The absolute number of CD4+CD45+ T cells was significantly reduced in both airT cell groups compared to mock controls, with a 49-fold reduction in the absolute number of CD4+CD45+ T cells in recipients of 2D2 airT cells and an 18-fold reduction in the absolute number of CD4+CD45+ T cells in recipients of C57Bl / 6 airT cells. In both groups, the majority of CD45+CD4+ cells were GFP-, and mice receiving 2D2 edTreg cells had fewer GFP- T cells expressing the inflammatory markers CD25 or IFN-γ (Figure 42). The observed effects were due to the T effTo confirm whether this was due to reduced cell proliferation, we injected some mice with the thymidine analog 5-ethynyl-2'-deoxyuridine (EdU) 2 hours before sacrifice and detected EdU incorporation into gDNA by flow cytometry using the "Click" reaction (Figure 43). In the group treated with 2D2 airT cells, the overall percentage of GFP- cells that incorporated EdU was reduced by 22% compared to the group treated with mock-edited cells and by 18% compared to the group treated with polyclonal airT cells. Importantly, GFP+ cells in the group treated with 2D2 airT cells also incorporated EdU (approximately 25%) and, to a lesser extent, GFP+ cells in the group treated with C57Bl / 6 airT cells (approximately 10%). This result was consistent with the in vivo proliferation capacity of tTregs in response to autoantigen stimulation. These findings suggest that murine airT cells are a pathogenic T cell in vivo. eff It was shown that priming of cells was suppressed, and antigen-specific airT cells showed stronger activity and proliferation than polyclonal airT cells.

[0351] Example 6: Function of air T cells in an antigen-specific mouse model of type 1 diabetes To investigate the efficacy of antigen-specific air T cells in an in vivo model of autoimmune type 1 diabetes, we used the BDC2.5NOD-NSG adoptive transfer model to determine whether Foxp3-edited antigen-specific T cells could delay or reverse the onset of type 1 diabetes. NOD mice (NOD / ShiLtJ strain) were used as a polygenic model of autoimmune type 1 diabetes. The development of diabetes in this model is characterized by moderate glucosuria and non-fasting plasma glucose levels exceeding 250 mg / dL. These diabetic mice exhibit hypoinsulinemia and hyperglucagonemia, indicating selective destruction of pancreatic islet beta cells. This mouse model is currently the most widely used polyclonal autoimmune animal model for studying spontaneous type 1 diabetes. BDC2.5 NOD mice [NOD.Cg-Tg(TcraBDC2.5,TcrbBDC2.5)1Doi / DoiJ] harbor rearranged TCRα and TCRβ genes derived from the cytotoxic CD4+ T cell clone BDC-2.5. Mature T cells from these mice express only the BDC2.5 TCR. These mice, on a NOD background, possess this TCR transgene and exhibit a reduced incidence of diabetes compared with control NOD / ShiLtJ mice. However, transfer of CD4+CD25- BDC-2.5 T cells into immunodeficient recipient mice rapidly develops significant diabetes. Previous studies have shown that nTregs derived from antigen-specific BDC2.5 NOD mice can prevent and reverse autoimmune diabetes in NOD mice more effectively than nTregs derived from polyclonal WT NOD mice. Based on these findings, in this study, we used these animal models to determine whether Foxp3-edited antigen-specific T cells could delay or reverse the onset of autoimmune type 1 diabetes compared with nTregs derived from wild-type NOD mice. First, we tested whether air T cells could be generated in NOD mice. Previous studies have demonstrated that air T cells can be generated from murine CD4+ T cells derived from B6 mice by editing the Foxp3 locus using homologous recombination repair (WO 2018 / 080541 and U.S. Patent Publication No. 2019 / 0247443, both of which are incorporated herein by reference in their entireties).In this study, we extended these studies by performing non-homologous end joining (NHEJ) and homology-directed repair (HDR) editing in CD4+ T cells from NOD mice using the same AAV donor template, demonstrating comparable editing efficiencies (Figure 44). Importantly, Foxp3-edited BDC2.5 NOD mouse CD4+ T cells have a Treg phenotype with increased Foxp3 expression and decreased expression of inflammatory cytokines compared to mock cells (Figure 45).

[0352] In adoptively transferred NSG mouse models, antigen-specific airT cells may be more effective than non-antigen-specific airT cells in delaying or reversing the onset of autoimmune type 1 diabetes. Because nTreg cells have previously been reported to reduce the onset of type 1 diabetes, we also included nTreg cells in this study. The experimental design and the transferred T eff The phenotypes of airT cells, airT cells, and nTreg cells are shown in Figure 46. AirT cells, like nTreg cells, were significantly different from mice receiving mock airT cells and mice receiving T cells. eff The incidence of diabetes was reduced compared to mice receiving only BDC airT cells (Figure 47). Importantly, administration of BDC airT cells resulted in a statistically significant reduction in the incidence of diabetes compared to polyclonal NOD airT cells. This finding indicates that antigen-specific airT cells are more effective at preventing the onset of diabetes than polyclonal airT cells. Of note, previous studies have shown that N-terminal GFP-FOXP3 fusion proteins function as hypomorphic forms and can actually accelerate autoimmune diabetes in immunocompetent NOD mice. Consistent with this notion, we also observed significant suppressive effects with airT cells expressing GFP-FOXP3 fusion proteins, although antigen-specific nTreg cells performed better than antigen-specific airT cells. Studies using FOXP3-expressing airT cells without the N-terminal GFP fusion protein (including airT cells expressing the clinically relevant LNGFR selectable marker in cis configuration; see below) are expected to provide improved protection.

[0353] These findings clearly demonstrate that antigen-specific air T cells, among BDC2.5 NOD T cells transferred into the adoptive transfer NSG mouse model, have a protective function against the onset of diabetes compared with polyclonal air T cells.

[0354] Example 7: Construction of an AAV donor template design for generating airT cell products containing the LNGFR selection marker The ability to enrich mouse cells after gene editing is important when attempting to obtain sufficient numbers of edited cells for in vivo experiments without flow cytometric sorting. To achieve this goal, we developed a cis-configured LNGFR selectable marker for use in purifying mouse edTreg cell products. Figure 48 shows the design of the repair templates used for editing mouse Foxp3. Each template contains a knock-in (KI) of LNGF.P2A but uses a different promoter. Furthermore, we tested the presence or absence of a 0.7 kb UCOE under the control of the MND promoter. When AAV5 No. 1331 (MND-GFP), AAV5 No. 3189 (MND-LNGFR), or AAV5 No. 3227 (PGK-LNGFR) and RNP were transfected into B6 CD4+ T cells, the GFP and LNGFR knock-in editing efficiencies were very similar (Figure 49, Figure 50). Furthermore, magnetic field separation using anti-LNGFR microbeads demonstrated an 8.7-fold enrichment of LNGFR+ cells (Figure 51). These data suggest that LNGFR can be successfully used as a method for selecting and enriching mouse CD4+ T cells.

[0355] Example 8 - Methods Editing Foxp3 CD4+ T cells were isolated from PBMCs using the MACS CD4+ T Cell Isolation Kit and activated with CD3 / CD28 activation beads (beads:cells = 1:1) and IL-2, IL-7, and IL-15. After 48 hours of activation, the beads were removed and the cells were rested for 16–24 hours. For Cas9 / CRISPR Foxp3 editing, cells were electroporated with an RNP complex consisting of Cas9 and guide RNA, followed by transduction with an AAV containing the template (AAV FOXP3 exon1.MND-LNGFRki). For TALEN nuclease-based Foxp3 editing, cells were electroporated with TALEN RNA targeting FOXP3, followed by transduction with an AAV containing the template (AAV FOXP3 exon1.MND-GFPki). After gene editing, cells were expanded in medium containing IL-2.

[0356] Generation of air T cells with islet-specific TCR CD4+ T cells were isolated from PBMCs and activated with CD3 / CD28-activated beads and IL-2, IL-7, and IL-15. After 24 hours of activation, they were transduced with lentiviral vectors encoding TCRs specific for GAD65 or IGRP (4.13, T1D2, T1D4, T1D5-1, or T1D5-2) at an MOI of 10 using protamine sulfate. After a total incubation of 48 hours from the initial activation, the beads were removed. Cells were allowed to rest for 16–24 hours before gene editing. For Foxp3 editing, cells were electroporated with an RNP complex consisting of Cas9 and guide RNA, followed by transfection with an AAV FOXP3 exon 1.MND-LNGFRki template. After gene editing, cells were expanded in medium containing IL-2, and editing and TCR transduction rates were measured 3–4 days after gene editing. The resulting airT cells were enriched based on LNGFR expression using MACSelect LNGFR beads, aliquoted, and cryopreserved until use in experiments.

[0357] Generation of antigen-specific airT cells To generate T cells specific for influenza antigen (Flu) or tetanus toxin antigen (TT), CD4+CD25- cells were isolated from PBMCs and co-cultured with irradiated autologous CD4-CD25+ cells (antigen-presenting cells) in the presence of IL-2 with MP, HA, and TT peptides. After two rounds of stimulation with these peptides and APCs for 9 days, CD4+ T cells were activated with CD3 / CD28 beads and Foxp3 editing was performed using TALEN and the AAV FOXP3 exon 1.MND-GFPki template. Three days after gene editing, GFP+ cells were selected by flow cytometry and expanded with CD3 / CD28 beads. After 7 days of expansion, CD3 / CD28 beads were removed, and the cells were incubated for an additional 4 days before harvesting and performing suppression assays.

[0358] To generate islet-specific T cells by peptide stimulation, CD4+CD25- cells were isolated from PBMCs and co-cultured with APCs and a pool of islet-specific antigens (a total of nine peptides selected from IGRP, GAD65, and PPI). After 2 weeks of expansion, cells were harvested, stained with tetramers specific for the nine islet peptides, and sorted by flow cytometry. Selected islet-specific CD4+ T cells were activated with CD3 / CD28 activation beads and Foxp3 was edited using Cas9 / CRISPR and the AAV FOXP3 exon 1.MND-LNGFRki template. Three days after gene editing, cells were stained with each tetramer and analyzed by flow cytometry.

[0359] Specific nucleic acid sequences useful in the embodiments provided herein are listed in the table shown in FIG.

[0360] Example 9 - Comparison of lentivirally delivered FOXP3-expressing T cells with air T cells We have previously shown that lentiviral delivery of a FOXP3 cDNA expression cassette into normal T cells confers a Treg-like phenotype and suppressive properties in vitro and in vivo (Allan et al. Mol Ther 16:194-202 (2008); Passerini et al. Sci Transl Med 5:215ra174 (2013)). To compare with the gene-editing method disclosed herein, we generated a lentiviral (LV) construct delivering a cDNA encoding the same GFP-FOXP3 fusion protein produced by airT cells (Figure 52A). Both gene-editing and viral transduction methods yielded similar percentages of GFP+FOXP3+ cells (Figure 52A). LV-treated cells (LV Tregs) harbored an average of 3.0 copies of lentivirus per genome of GFP+ cells. The airT cells in this experiment contained only a single targeted insertion per gene-edited T cell from a male donor. Despite the difference in inserted copy number, the MFI of GFP+ cells was consistently lower in LV Treg cells than in air T cells (Fig. 52B), consistent with more efficient expression from the genome than from cDNA or with the locus where LV is integrated being poorly transcribed. eff The air T cells and LV Treg cells were converted from a regulatory T phenotype to a tTreg phenotype, with upregulation of CD25, CTLA-4, and Helios, and downregulation of IL-2, TNF-α, and IFN-γ (Fig. 52C). With the exception of FOXP3 expression, the percentage of cells expressing regulatory T cell markers and the mean expression levels per cell (assessed by MFI) were similar between air T cells and LV Treg cells. Furthermore, in vitro polyclonal T effAirT cells and LV Treg cells had similar suppressive abilities against CD4 T cells (Figure 52D). However, importantly, FACS-purified LV Treg cells showed reduced GFP expression compared to airT cells over the 5-week culture period (Figure 52E; the initial percentage of GFP+ cells was >99% for both cell types). This finding indicates that homology-directed repair-mediated gene editing can more effectively maintain high FOXP3 expression than LV delivery using the same promoter construct. These findings were unexpected based on previous reports of LV-mediated FOXP3 delivery and support the notion that homology-directed repair-mediated editing of the FOXP3 locus provides a more stable platform for sustained FOXP3 expression in CD4 T cells.

[0361] Example 10 - Dual Editing Method Figure 53 provides an overview of the homology-directed repair gene editing method we developed to generate antigen-specific airT cells using a gene editing strategy based solely on homology-directed repair. This novel method eliminates the need for LVs for TCR delivery and is designed to generate airT cells that a) lack endogenous TCR expression, b) express an islet-specific T1D TCR (or another disease-associated antigen-specific TCR), and c) can be enriched in vitro and in vivo using a novel CISC / DISC IL-2 platform. As shown in Figure 53, we have developed methods that can achieve these goals by targeting one locus (TRAC) or two loci (TRAC and FOXP3). Examples of successful application of these two strategies are presented below.

[0362] Figure 54 shows a schematic overview of the AAV HDR donor constructs used in the studies described below to generate antigen-specific edTreg cells using a dual editing approach at one or two loci, along with their numbers. These AAV HDR donor constructs include those with and without IL2-CISC / DISC selectability.

[0363] Dual editing of human CD4+ T cells - an example of how to edit a single locus Figures 55 and 56 show inter-experiment reproducibility and inter-donor variability. Cells from two donors were edited using AAV No. 3207 (MND.GFP.FRB-IL2RG) and AAV No. 3208 (MND.mCherry.FKBP-IL2RG), which were used in previous experiments, and the data from these replicate experiments were compared with the data from the first experiment. In these experiments, both homologous recombination repair templates targeted a single sgRNA cut site within the first exon of the TRAC locus. The double-editing rate (integration of both GFP and mCherry from the split CISC cassette into a single cell) in cells from donor R003657 was 2.75% (Figure 55), which was similar to the results observed in the two datasets from the previous experiment (double-editing rates in cells from donor R003657 in the previous experiment were 1.44% and 1.1%). The double-editing rate in cells from the second donor (R003471) was 6.78% (Figure 56), similar to the 6.4% double-editing rate observed in the first dataset. Both donors were Caucasian males of the same age. Overall, the editing rates varied between donors, but within each donor, the editing rates were similar across experiments, suggesting that this variation in editing rates was due to the ability to successfully edit genes in the donor's cells and not due to variability between different experiments on the same donor. Importantly, using edited T cells from both human donors, we were able to enrich for double-edited cells in the presence of a rapamycin analog (rapalog, AP21967) capable of inducing heterodimerization with similar efficiency to that observed in the previous experiments. In this study, 7 days of enrichment with rapalogs resulted in 44.7% enrichment of GFP / mCherry double-positive cells from donor R003657 and 46.1% enrichment of GFP / mCherry double-positive cells from donor R003471 (Figure 55, Figure 56). As expected, enrichment was not observed in the presence of IL-2.

[0364] These studies demonstrate that the homology-directed double-editing approach to integrate split IL-2 CISC provides an efficient means for selecting and enriching for double-edited cells, and is reproducible across donors and replicates. Based on our successful double-editing using the MND.GFP.FRB.IL2RB cassette (No. 3207) and the MND.mCherry.FKBP.IL2RG cassette (No. 3208), and our enrichment of double-edited cells using rapalogs, we developed two constructs: one capable of delivering HA-tagged FOXP3 in combination with the IL-2 CISC construct (No. 3240) and the other capable of delivering an islet antigen-specific TCR (T1D4) in combination with the IL-2 CISC construct (No. 3243), and used these to generate antigen-specific FOXP3+ed Treg cells (Figure 57).

[0365] We tested the initial editing rate and proliferation of T1D4-positive human edTreg cells expressing FOXP3 using the MND.T1D4.FRB.IL2RB construct (No. 3243), in which GFP was replaced with the T1D4 TCR, and the MND.HA.FOXP3.FKBP.IL2RG construct (No. 3240), in which mCherry was replaced with HA-tagged FOXP3. These constructs are shown in Figure 57(A), and the timeline and editing conditions for this experiment are shown in Figure 57(B). Using the CISC construct containing GFP and the CISC construct containing mCherry, the percentage of GFP / mCherry double-positive cells was 2.75% (Figure 55) and 6.37% (Figure 56), respectively. This was compared to the low percentage of FOXP3 / T1D4 double-positive cells (0.65%) (Figure 57). Thus, despite the low initial editing rate, we were able to significantly enrich for FOXP3 / T1D4 double-positive cells. Treatment with the rapalog for 8 days enriched FOXP3 / T1D4 double-positive cells from 0.65% to 11%, whereas treatment with IL-2 only enriched them to 1.1% (Figure 57(C)). The initial editing rate with these constructs was lower than that with the GFP-containing split CISC construct (No. 3207) and the mCherry-containing split CISC construct (No. 3208), likely due to the larger size of the homologous recombination repair template containing the T1D4 TCR. The size of MND.T1D4.FRB.IL2RB (No. 3243) was 4.3 kb, significantly larger than the 2.7 kb size of MND.mCherry.FKBP.IL2RB (No. 3208).

[0366] To obtain sufficient numbers of edited cells for therapeutic use, it is believed that improving the editing rate and / or enrichment of FOXP3 / TCR double-positive cells is crucial. To improve the initial editing rate, we varied serum concentrations during the gene editing step. Figures 58 and 59 show the results of a double-editing experiment using the AAV constructs MND.HA.FOXP3.FKBP.IL2RG (No. 3240) and MND.T1D4.FRB.IL2RB (No. 3243), comparing four serum concentrations during the gene editing step of human CD4+ T cells. Analysis by FACS showed that the initial editing rate improved from 1.8% with 20% FBS to 3.96%–4.75% with low serum concentrations or no serum (Figure 58). Importantly, when cells recovered in medium containing 2.5% FBS were enriched by treatment with IL-2 for 7 days, the percentage of double-positive cells was 1.97%, whereas when cells were enriched by treatment with rapalogs for 7 days, the percentage of double-positive cells increased from 1.8% to 17.6%, a nearly 10-fold enrichment of double-positive cells (Figure 59).

[0367] In summary, the results shown in Figures 55-59 demonstrate that dual editing using homologous recombination repair can be efficiently performed at the TRAC locus, resulting in antigen-specific airT cells that can be enriched using the IL-2 CISC platform.

[0368] Dual editing of human CD4+ T cells - an example of how to edit two loci As an alternative dual-editing approach to generate antigen-specific airT cell products containing an IL-2 CISC component, we developed a construct targeting the TRAC locus and another targeting the FOXP3 locus, and performed dual editing. As shown in the schematic diagram in Figure 53, instead of using two constructs targeting the TRAC locus, we used one construct targeting the TRAC locus and one targeting the FOXP3 locus. This approach may improve the dual-editing rate and may be compatible with the multiple methods developed in previous experiments for sustained FOXP3 expression. To test this approach, we developed a construct that allows for easy tracking of successfully edited cells. We tested whether we could generate double-edited cells stably expressing both mCherry and GFP linked to IL-2 CISCs by using the MND.mCherry.FKBP.IL2RG (No. 3251) cassette with FOXP3 homology arms and the MND.GFP.FKB.IL2RB (No. 3207) cassette with TRAC homology arms. The constructs used and a timeline for gene editing, cell expansion, and analysis are shown in Figure 60. Previous experiments suggesting that lower serum concentrations result in higher double-editing rates for single-locus editing (Figure 58), we compared high-serum and low-serum editing conditions in this experiment. FACS analysis showed that the dual-locus editing strategy was successful under both serum concentrations (Figure 61). Similar to single-locus editing, the use of 2.5% serum in the gene editing step significantly improved the double-editing rate on day 3 (4.99% editing rate using 20% ​​serum, compared with 11.0% editing rate using 2.5% serum). Cells edited in 2.5% serum were then expanded for 10 days in the presence of IL2 or a rapalog (AP21967). Figure 62 shows that mCherry / GFP double-positive cells were highly enriched in the presence of the rapalog, reaching a total of 59%.

[0369] To test reproducibility between experiments and further improve the initial editing rate, we investigated various additional editing conditions. Figure 63 shows a timeline of gene editing, cell expansion, and analysis, along with an overview of each editing condition. In this experiment, a medium containing 2.5% serum was used during the gene editing stage for all conditions. The percentage of virus (volume %) was varied for each reaction, and editing results were compared using AAV No. 3207 and AAV No. 3251 in the presence and absence of a homology-directed repair enhancer (HDR-E). Figure 64 shows a histogram summarizing flow cytometry data for each condition 3 days after editing. The editing rate in 2.5% serum in this experiment was comparable to that in the previous experiment shown in Figure 61 (15.4% GFP / mCherry double-positive cells in this experiment, compared with 11% GFP / mCherry double-positive cells in the previous experiment). Furthermore, enrichment of the double-edited cell population with rapalogs resulted in 54% GFP / mCherry double-positive cells (Figure 65), which was comparable to previous data, demonstrating reproducibility between experiments. While the presence of HDR-E did not affect the initial editing rate, the proportion of virus in the reaction did affect the editing outcome (Figure 64). The results indicated that a 10% concentration of each virus relative to the media volume was optimal compared to a 15% concentration of each virus relative to the media volume or a combined concentration of both viruses at 30%.

[0370] These studies demonstrated that the dual-editing method disclosed herein, which edits two loci, can be used to introduce a split IL-2 CISC cassette and efficiently enrich for dual-edited cells with rapalogs. Given the success of this method for generating and enriching dual-edited cells, we designed and cloned constructs targeting the FOXP3 locus to express FOXP3 with an IL-2 CISC component and the TRAC locus to express an islet antigen-specific TCR (T1D4) with an IL-2 CISC component. These and a variety of other HDR donors can be used to generate antigen-specific FOXP3 airT cells (Figure 66).

[0371] In-frame knock-in at the TRAC locus as a dual editing strategy As a further modification or improvement of our dual editing method, we established a method that targets the first exon of the TRAC locus to knock in a promoterless TCR cassette containing the IL-2 CISC component in frame (Figure 67). This editing method utilizes the promoter / enhancer activity of the endogenous TRAC locus to drive antigen-specific TCR expression. The advantages of this method include the elimination of endogenous TCR expression (and potentially the potential for inappropriate pairing with the delivered TCR component), concomitantly driving autoantigen-specific TCR expression at near-endogenous levels. To establish this method, we tested gene editing and exogenous gene expression using a proof-of-concept construct (No. 3253) containing mCherry IL-2 CISC (Figure 68). The percentage of CD3-deleted mCherry-positive cells was 63.8% (the percentage of CD3-expressing cells was 99.9% when transduced with AAV alone, but decreased to 3.01% when edited with AAV (No. 3253) containing P2A.mCherry.FRB.IL2RB). After gene editing, mCherry expression was easily detected by flow cytometry. As expected, the MFI of mCherry expression in cells edited with AAV (No. 3253) containing P2A.mCherry.FRB.IL2RB was lower than that in T cells edited at the same locus using the MND promoter expression cassette (MND.mCherry.FKBP.IL2RG (No. 3208)) (Figure 69). Thus, this homologous recombination repair method exploits the promoter / enhancer activity of the endogenous TRAC locus to drive transgene expression.

[0372] In follow-up studies, two HDR donor cassettes will be used to perform dual editing of the TRAC locus (and utilize the endogenous promoter) and / or dual editing of both the TRAC and FOXP3 loci. Each HDR donor will deliver split components of IL-2 CISC to allow enrichment for dual-edited cells, deliver an antigen-specific TCR (under the control of the TRAC promoter), and express FOXP3 via expression from a cDNA or by targeting endogenous FOXP3 expression. The dual-editing approach will be tested in combination with a split CISC mCherry construct (P2A.mCherry.FRB.IL2RB (No. 3253)) that utilizes the endogenous promoter of the TRAC locus and MND.GFP.FKBP.IL2RG (No. 3273) for editing the FOXP3 locus. Because expressing the two components of the split IL-2 CISC from two different promoters may affect overall CISC function, we also test a dual-editing approach to a single locus by using P2A.mCherry.FRB.IL2RB (No. 3253) and P2A.GFP.FKBP.IL2RG (No. 3292) to drive expression of each component of the IL-2 CISC from the endogenous promoter of the TRAC locus (Figure 70).

[0373] Given our successful editing of two alleles (using one or both promoterless mCherry / GFP constructs and enriching for double-edited cells with rapalogs), we create constructs that can deliver FOXP3 and an antigen-specific TCR (T1D4) in combination with an IL-2 CISC component as an alternative approach to generating antigen-specific FOXP3+ airT cells (see Figure 70). We compare airT cells generated using these methods to cells in which the antigen-specific TCR is derived from an exogenous MND promoter.

[0374] Dual editing using the Decoy-CISC (Split-DISC) construct Although CISC-expressing cells expand efficiently in the presence of a rapalog (AP21967) capable of inducing heterodimerization, the FDA-approved drug rapamycin may be preferred for clinical applications. It is well documented that intracellular binding of rapamycin to its target, mTOR, exerts an inhibitory effect on T cell proliferation. To address this issue, constructs and methods utilizing a naked "decoy" FRB domain expressed within CISC-expressing cells are disclosed in WO2019210057 (incorporated herein by reference in its entirety). Constructs expressing a naked FRB domain together with CISC are referred to as "decoy-CISC" or "DISC."

[0375] To confirm whether DISC functions in dual editing methods, a CISC-containing construct was modified to include an additional naked FRB domain at the 3' end of the CISC receptor (Figures 70 and 71). Constructs containing the additional FRB domain along with the CISC component are referred to as "decoy-CISC" or "DISC." When used in dual editing methods, this construct is referred to as "split-DISC." When split-DISC is used, the naked FRB domain competes with the endogenous FRB domain of mTOR for binding to rapamycin, thereby allowing signaling by the split-CISC component to be induced via rapamycin without inhibiting cell proliferation.

[0376] As shown in Figure 71, dual editing of T cells with an mCherry CISC construct containing an additional FRB domain (mCherry DISC, No. 3280) and a GFP CISC construct (No. 3207) resulted in 7.07% GFP / mCherry double-positive cells. As predicted from this experiment with the DISC construct, GFP / mCherry double-positive cells were enriched to a similar extent by treatment with rapamycin or a rapalog (AP21967) (79.5% and 86.4%, respectively) (Figure 72).

[0377] Experiments will be performed to examine the in vivo enrichment / engraftment of cells edited with a GFP / mCherry split DISC construct in NSG mice treated with rapamycin. GFP / mCherry double-edited cells demonstrate improved in vivo engraftment / enrichment. This study will be extended to assess the engraftment and expansion of islet-specific airT cells in vivo by using a FOXP3-containing construct and a T1D4-containing construct. The DISC construct shown in Figure 73 (MND.FOXP3.FKBP.IL2RG.FRB (No. 3262)) will be combined with the previously generated MND.T1D4.FRB.IL2RB construct (No. 3243) to perform dual editing of the TRAC locus and generate islet-specific airT cells that can be enriched in vivo using rapamycin.

[0378] Example 11 - Functional activity of antigen-specific murine air T cells in vitro and in vivo In vitro characterization of murine AirT cell products: We conducted a study designed to identify advantageous HDR donor template designs for generating airT cell products with a suppressive Treg-like phenotype from murine CD4+ T cells. The questions we sought to answer were: a) which of the promoter / enhancer constructs tested would provide the best performing airT cells in vitro and ultimately in vivo; and b) whether the clinically relevant LNGFR selection marker in cis configuration could be used to enrich airT cells in a manner suitable for GMP manufacturing. Figures 74-80 show the results of (1) experiments evaluating the use of a clinically relevant cis-configured LNGFR selectable marker as a method to enrich for mouse cells after gene editing, (2) experiments testing a variety of promoter candidates (in addition to the MND promoter), (3) experiments testing two Foxp3 homology arms of different sizes in a donor template, and (4) experiments comparing donor templates containing UCOE elements with donor templates lacking UCOE elements (as a means of limiting silencing of the promoter introduced within the Foxp3 locus).

[0379] We first evaluated the effect of extending the homology arms of the MND.LNGFR.P2A donor template of the Foxp3 gene from 0.6 kb to 1.0 kb on the editing efficiency of CD4+ T cells derived from C57BL / 6 mice (Figure 76). Results from this study showed that MND.LNGFR.P2A (No. 3261), which contains 1.0 kb homology arms, had slightly higher editing efficiency than MND.LNGFR.P2A (No. 3189), which contains 0.6 kb homology arms. This extension of the homology arms improved editing efficiency by approximately 10%, and this improvement in editing efficiency was reproducible between experiments. Therefore, in subsequent studies, we selected AAV No. 3261 as the preferred target donor template for generating murine MND.LNGFR.P2A airT cells. We also tested the editing efficiency and post-enrichment purity of C57BL / 6 edTreg cells using AAV donor templates containing alternative promoters (Figure 76). The results showed that the overall editing rate and purity of enriched LNGFR+ cells were comparable between AAV donor templates containing the MND promoter, the PGK promoter, and the EF-1α promoter.Furthermore, the purity of enriched LNGFR+ and GFP+ cells was also comparable, demonstrating the feasibility of using LNGFR as a method for selecting and enriching mouse CD4+ T cells.

[0380] Although editing efficiency and purity of edited cells were comparable across various AAV donor templates with different promoters, it is important to note that FOXP3 expression levels varied depending on the promoter. Figure 77 shows the results of evaluating GFP and FOXP3 expression in CD4+ T cells from C57BL / 6 mice after mock editing, editing with MND.GFP.KI (No. 1331), or editing with PGK.GFP.KI (No. 3209). The results showed that the MFI of FOXP3 in cells edited with MND.GFP.KI (No. 1331) was significantly higher than that in cells edited with PGK.GFP.KI (No. 3209). FOXP3 expression levels in CD4+ cells edited with PGK.GFP.KI (No. 3209) were similar to those observed in splenic nTreg cells. These studies demonstrated that the overall expression level of FOXP3 in airT cell products can be controlled by introducing various promoters into the endogenous Foxp3 locus. This suggests that the generation of airT cell products can be flexible and that airT cells with diverse functional properties can be obtained. The relationship between FOXP3 expression level and its function in vitro or in vivo was further investigated in the studies shown in Figures 79 and 85 (see below). Furthermore, we tested whether the ubiquitous chromatin opening element (UCOE) can stabilize FOXP3 expression (Figure 77). UCOE can limit adverse effects on promoter elements by suppressing silencing. These studies demonstrated that FOXP3 was stable regardless of the presence or absence of UCOE, and that the inclusion of the UCOE element did not adversely affect the relative expression level of FOXP3 (comparison of MND.GFP.KI (No. 1331) and MND.GFP.KI (No. 3213) with UCOE). These results suggest that donors with suppressed silencing due to the inclusion of UCOE function effectively. Furthermore, the inclusion of UCOE may protect expression in vivo or over time, potentially improving the duration of functional activity, suggesting that UCOE may be useful in airT cell products.

[0381] Further studies were performed to (a) assess the functional activity of LNGFR-expressing airT cells in vitro and (b) investigate whether the promoter driving the expression of endogenous FOXP3 had any effect on the functional activity of Treg cells. MND.GFP.KI-edited and MND.LNGFR.P2A-edited airT cells were sorted by flow cytometry, and Treg activity was determined in the presence and absence of these airT cells using an in vitro suppression assay as outlined in Figure 78. eff The proliferation of these airT cells was analyzed. The efficacy of these airT cells was compared with the activity of purified murine nTreg cells. The results, shown in Figure 79, demonstrate that both murine airT cells (generated using either the MND.GFP.KI HDR donor or the MND.LNGFR.P2A HDR donor) and nTreg cells exhibited potent and comparable suppressive function in vitro. Furthermore, these findings demonstrate that airT cells expressing the LNGFR selectable marker (those edited with MND.LNGFR.P2A (No. 3261)) can be successfully used to select and enhance murine CD4+ cells without loss of functional activity, making them useful for modeling the functional activity of clinically relevant human airT cell products utilizing this selectable marker in vitro or in vivo.

[0382] Using the same in vitro suppression assay, we investigated whether FOXP3 expression levels (as assessed in Figure 77) correlated with promoter functional activity by examining the functional activity of promoters with varying strengths. Figure 80 shows a comparison of CD4+ T cells from C57BL / 6 mice edited with the MND promoter-based LNGFR construct (MND.LNGFR.P2A), the PGK promoter-based LNGFR construct (PGK.LNGFR.P2A), or the EF-1α promoter-based LNGFR construct (EF-1α.LNGFR.P2A), CD4+ T cells from C57BL / 6 mice edited with MND.GFP.KI, and nTreg cells from C57BL / 6 mice. The results of this experiment demonstrated that murine air T cells carrying the MND promoter exhibited suppressive function comparable to that of nTreg cells. In contrast to the MND promoter-containing construct, airT cells using the PGK promoter exhibited only partial suppressive function in vitro, and airT cells using the EF-1α promoter exhibited no suppressive function. Interestingly, although FOXP3 expression levels in PGK.GFP.KI-edited cells were comparable to those in nTreg cells (Figure 77), the suppressive activity of nTreg cells in vitro was significantly higher than that of PGK.LNGFR.P2A-edited cells. These findings suggest, surprisingly, that a threshold level of FOXP3 expression in edited CD4+ T cells may be required for proper reprogramming and effective functional activity in vitro. The results described below also clearly demonstrate that the MND promoter was effective in alleviating diabetes in vivo.

[0383] Functional characterization of edTreg cell products in vivo: The experimental results summarized above suggest that murine airT cells containing the clinically relevant LNGFR selectable marker in cis-configuration retained functional activity in vitro and that the MND promoter possessed superior repressive activity to other promoters in vitro. Extending these studies, we evaluated islet-specific airT cell products in an NSG adoptive transfer diabetes model in which the transfer of islet-specific NOD (murine) CD4+ T cells into adult NSG recipient mice rapidly developed diabetes.

[0384] Using this model, we evaluated whether islet-specific MND.LNGFR.P2A airT cells derived from NOD BDC2.5 mice could delay or prevent the onset of diabetes. The in vitro experiments shown in Figures 76-80 utilized cells enriched by flow cytometry sorting, but this sorting process is time-consuming and expensive. Furthermore, FACS sorting can significantly affect the engraftment and / or survival of adoptively transferred cells in vivo. To efficiently enrich for in vivo-usable gene-edited mouse airT cells, we compared the purification and functional activity of airT cells purified using various methods. Specifically, we compared (1) enrichment of LNGFR+ cells by cell sorting using a flow cytometer with (2) enrichment of LNGFR+ cells using LNGFR column separation. Figures 82-83 show flow cytometry plots before and after purification using FACS sorting or column enrichment. Although FACS-sorted purification yielded a somewhat purer LNGFR+ cell population, column enrichment yielded an approximately 84% pure LNGFR+ cell population, significantly saving time and materials. Importantly, both column-enriched and FACS-sorted LNGFR+ airT cells delayed or prevented the onset of diabetes (Figure 84).

[0385] The combined data shown in Figures 82-84 demonstrate that both LNGFR column isolation and FACS sorting yield highly purified islet-specific edited cells. Both cell products overexpressed LNGFR / FOXP3 and mitigated or prevented diabetes in vivo, demonstrating a Treg-like phenotype.

[0386] Furthermore, as shown in Figure 85, the functional activity of islet-specific airT cell products in the NSG adoptive transfer model varied depending on the promoter used to drive endogenous FOXP3. While both MND.GFP.KI-edited and nTreg cells were able to delay or prevent the onset of diabetes, PGK.GFP.KI-edited airT cells failed to do so. This result was consistent with the in vitro suppression data shown in Figure 80, suggesting that promoter choice plays a role in achieving optimized function. Importantly, consistent with the observed protective effect against diabetes, islet-specific airT cells were recruited to the pancreas and maintained viability in the NSG model while stably expressing FOXP3 (Figure 86).

[0387] These data suggest that mouse air T cells can be used for in vitro and in vivo studies. eff Consistent with findings in human T cells, mouse T eff By using the MND promoter in islet-specific mouse airT cells, we were able to effectively convert them into airT cells that highly express FOXP3 and exhibit potent suppressive activity in vitro comparable to that of nTreg cells. Importantly, the MND promoter-driven islet-specific mouse airT cells and nTreg cells (1) exhibited similarly potent suppressive function in vitro and (2) transformed islet-specific T cells in recipient mice. effFurthermore, these data demonstrate that (3) airT cells expressing the LNGFR selectable marker can be enriched in vitro and function in vivo without loss of functional activity, and (4) airT cells with the MND promoter outperform airT cells generated using alternative promoters, such as PGK or EF1α, suggesting that promoter choice plays a role in improving function.

[0388] The NSG adoptive transfer diabetes model described herein allows for the analysis of lymph node trafficking, cell proliferation, activation state, and T eff We were able to rapidly assess important functional characteristics of mouse airT cells, such as their ability to restrict early cell activation. Using this method, we compared the functional activity of enriched antigen-specific LNGFR airT cells in a mouse model of type 1 diabetes on an immunocompetent NOD background.

[0389] Editing the Rosa26 locus to generate gene-edited mouse T cells To expand the toolset for evaluating the efficacy of antigen-specific FOXP3 airT cells in animal models of diabetes or other autoimmune diseases, we designed and tested gRNAs targeting the mouse Rosa26 locus. The Rosa26 locus is a well-characterized safe harbor locus and has been used in previous studies to stably express transgenes in mouse models. We selected two novel gRNA target sequences within the intronic region of the Rosa26 locus, adjacent to previously reported gRNA target sites, and delivered RNPs to primary mouse CD4+ T cells. We then measured on-target site-specific activity using Inference of CRISPR Edits (ICE). ICE analysis confirmed the induction of indels specific to R26_gRNA_1 at the Rosa26 locus (Figure 87).

[0390] Next, we tested the editing ability of each gRNA in mouse T cells using constructs that appeared to allow easy tracking of successfully edited cells. We engineered an MND-GFP cassette flanked by identical 300-base-pair Rosa26 homology arms compatible with R26_gRNA_1 (No. 3245). We then used this MND-GFP cassette to edit the Rosa26 locus, generating T cells stably expressing GFP. A timeline of cell expansion, gene editing, and analysis is shown in Figure 88. FACS analysis showed that 3 days after gene editing, the percentage of highly GFP-expressing cells was 0.02% when AAV No. 3245 was transfected alone, whereas the percentage of highly GFP-expressing cells was 11.4% when AAV No. 3245 and RNP were transfected, confirming integration of the MND-GFP repair cassette into the Rosa26 locus (Figure 89). FACS analysis performed 8 days after gene editing showed that a similar percentage of GFP+ cells (10.8%) was obtained, indicating stable GFP expression (Figure 90).

[0391] Given our success in editing the Rosa26 locus in mouse T cells using the MND-GFP cassette, we constructed a repair template containing the mFoxp3 coding sequence with an LNGFR marker (for purification) and a potential alternative promoter (other than the MND promoter) to generate another construct capable of stably expressing FOXP3 at this safe-harbor locus in mouse cells (Figure 91). These constructs will be used to investigate dual editing in mouse cells, with the goal of generating antigen-specific FOXP3-expressing mouse air T cells for use in mouse models of autoimmune disease. We will test FOXP3 mutant variants predicted to have improved stability, including a 4xCDK phosphorylation mutant in which a series of four target residues phosphorylated by cyclin-dependent kinases are substituted with alanine to prevent proteolysis-associated phosphorylation.

[0392] These data demonstrate that the safe harbor site Rosa26 locus can be used for gene editing of mouse T cells using homologous recombination repair. This finding enables dual-editing studies in mouse T cells and parallel studies in human T cells, facilitating the generation of preclinical animal models of antigen-specific air T cells.

[0393] Development of a tool for expanding mouse cells using CISC elements A key feature of the antigen-specific human airT cell platform is the ability to expand airT cells in vitro and in vivo, for example, by utilizing the IL-2-CISC system. To evaluate the function of airT cells containing the IL-2-CISC cassette in immunocompetent animal disease models, we performed experiments to investigate whether a human IL-2R sequence containing a CISC / DISC cassette could promote selective expansion of mouse cells in vitro and in vivo in the presence of a rapalog / rapamycin. Experimental data using a lentiviral construct (No. 1272) containing a human IL-2 CISC element in cis with an mCherry reporter driven by the MND promoter demonstrated this concept. Figure 92 shows a schematic diagram of the lentiviral cassette and a timeline for T cell transduction, expansion, and analysis. In this study, transduced cells were cultured 2 days after transduction in either (a) the presence of IL-2, IL-7, and IL-15, (b) the presence of a rapalog alone, or (c) the presence of a rapalog and boost with CD3 / CD28 beads. Figure 93 shows that 8.85% of the transduced cells expressed mCherry and were further enriched by treatment with a rapalog for 3 days. The greatest enrichment (46.1%) was observed in transduced T cells treated simultaneously with a rapalog and boost with CD3 / CD28 beads.

[0394] These data demonstrate that IL-2 CISC technology can be used to enrich for murine CD4+ T cells and that human CISCs are functional in the murine system. These findings demonstrate the feasibility of studies investigating the enrichment and function of antigen-specific murine air T cells using split CISC or split DISC methods in nonclinical animal models.

[0395] Example 12 - Generation and testing of antigen-specific edTreg cells Rheumatoid arthritis antigen-specific TCR identified from rheumatoid arthritis patients Rheumatoid arthritis antigen-specific TCRs were identified from T cell clones isolated from rheumatoid arthritis patients. Based on the sequences of these TCRs, several lentiviral TCR constructs for TCR gene transfer were constructed. Table 1 shows the lentiviral constructs encoding rheumatoid arthritis antigen-specific TCRs constructed in this study, their epitope specificity, and HLA restriction. The targeted T cell epitope sequences contained citrulline modifications. TCRs recognizing citrullinated vimentin, citrullinated aggrecan, citrullinated CILP, or citrullinated enolase were identified from T cell clones previously isolated from rheumatoid arthritis patients. [Table 1]

[0396] CD4+ T cells were isolated, activated with CD3 / CD28 beads, and transduced with lentivirus encoding a rheumatoid arthritis antigen-specific TCR. Flow cytometry plots show the expression of mTCRb gated on CD4+ cells 9 days after transduction (Figure 117A). CD4+ T cells transduced with a TCR specific for a rheumatoid arthritis antigen were labeled with CTV and cocultured with APCs (irradiated PBMCs) for 3 days in the presence of a peptide recognized by the TCR or DMSO. Flow cytome...

Claims

[Claim 1] The invention described herein.

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