Compositions and methods for engineering Treg cells for treating diabetes

Abstract

Compositions and methods are described for generating Treg cells for the treatment of diabetes. The recombinant Treg cells provided herein may be double-edited (i.e., edited at two different loci in the cell genome), with the first locus edited being the FOXP3 locus and the second locus being the TRAC locus. Recombinant engineering of the double-edited Treg cells provided herein may include selectively expanding the double-edited cells with a ligand that initiates and / or maintains IL-2 signaling in the double-edited cells. The double-edited Treg cells provided herein may be recombinantly engineered to stably express FoxP3 and exogenous TCR.

Description

[Technical Field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 292,125, filed December 21, 2021; U.S. Provisional Patent Application No. 63 / 363,918, filed April 29, 2022; U.S. Provisional Patent Application No. 63 / 364,285, filed May 6, 2022; U.S. Provisional Patent Application No. 63 / 378,928, filed October 10, 2022; and U.S. Provisional Patent Application No. 63 / 384,830, filed November 23, 2022, the contents of which are incorporated herein by reference in their entireties.

[0002] Reference to the electronic sequence listing The contents of the electronic sequence listing (G097170024WO-SEQ-NTJ.xml; size: 365,549 bytes; created: December 8, 2022) are incorporated herein by reference in their entirety. [Background technology]

[0003] Type 1 diabetes (T1D), also known as juvenile diabetes or insulin-dependent diabetes, is a chronic disease in which the pancreas produces little or no insulin. Cell therapy using regulatory T cells (Tregs) may be useful in treating various types of autoimmune diseases, including type 1 diabetes. Summary of the Invention [Means for solving the problem]

[0004] The present specification provides genetically engineered artificial regulatory T (EngTreg) cells for the treatment of type 1 diabetes (T1D), which contain two nucleic acids: a first nucleic acid inserted into the TRAC locus and a second nucleic acid inserted into the FOXP3 locus. This cell type accounts for 5% to 10% of diabetes cases worldwide, and there is no cure. Type 1 diabetes can occur at any age, but the average age of diagnosis is 8 years old. It is more prevalent in postpubertal males. The incidence of type 1 diabetes worldwide is increasing at a rate of 3% to 4% annually, and between 2001 and 2009, the number of type 1 diabetes patients aged 0 to 19 increased by approximately 20%. Type 1 diabetes is a chronic autoimmune disease caused by the destruction of insulin-producing beta cells by T lymphocytes. It is characterized by a variable presymptomatic period and ultimately leads to insulin deficiency accompanied by hyperglycemia. Inadequate control of hyperglycemia can lead to systemic, multi-organ damage that is often irreversible.

[0005] Lifelong administration of exogenous insulin is required to control hyperglycemia and its associated clinical signs and symptoms. Despite recent advances such as continuous blood glucose monitoring and automated insulin administration to maximize time in range (TIR), long-term euglycemic control remains a challenging therapeutic challenge. Poor blood glucose control can lead to significant sequelae and reduced quality of life due to microvascular and macrovascular complications. In addition to the complications of type 1 diabetes itself, patients and their families must contend with the risk of severe, potentially fatal hypoglycemic episodes caused by exogenous insulin therapy.

[0006] Although exogenous insulin is beneficial in managing type 1 diabetes, it does not cure the disease and requires daily blood glucose monitoring. The burden of blood glucose management, particularly in pediatric patients, can lead to family-related stress and significantly impact patients' quality of life. Even in patients whose insulin therapy is optimized, significant support is still required through various types of daily assessments to manage daily food intake, consider physical activity, align carbohydrates with insulin requirements, and monitor blood glucose levels. Therefore, managing blood glucose control while maintaining patients' quality of life remains a challenging task, especially in the pediatric patient population.

[0007] In newly diagnosed type 1 diabetes, subjects often experience a short-term remission shortly after initiating exogenous insulin therapy. Such remission is not reliable and occurs transiently in only approximately 50% of children and adolescents, with glycemic control relapsing within weeks to months. Without wishing to be bound by any theory, therapeutic intervention before the end of this remission period is expected to mitigate the progression of the autoimmune response against the pancreas while sparing the majority of functional islet cells, thereby reducing the need for exogenous insulin therapy. One such therapeutic intervention, contemplated herein, islet cell antigen-specific recombinant regulatory T cells (EngTregs), is designed to suppress autoimmune responses that adversely affect islet cell function.

[0008] The EngTregs described herein contain a recombinant TRAC locus, in which a heterologous promoter controls transcription of the first transmembrane protein portion of the chemical-induced signaling complex (CISC), which includes the FK506-binding protein 12 (FKBP) extracellular domain and the IL-2Rγ intracellular domain, and a recombinant FOXP3 locus, in which a heterologous promoter controls transcription of the second transmembrane protein portion of CISC, which includes the FKBP-rapamycin binding (FRB) domain and the IL-2Rβ intracellular domain, thereby facilitating IL-2 signaling in cells upon exposure to rapamycin and EngTreg proliferation in the presence of rapamycin. In some embodiments, the heterologous promoter controls transcription of the endogenous FOXP3 gene and the second transmembrane protein portion of CISC. Growth of these chemically induced, double-edited cells efficiently selects for cells in which both loci have been recombined to insert each CISC component, allowing for in vitro expansion. Specifically, the recombinant TRAC locus encodes a heterologous TCR β chain and a TCR α chain with a heterologous variable domain under the transcriptional control of the inserted promoter, allowing the edited cells to express a TCR specific for the IGRP peptide, a type 1 diabetes-associated antigen. Furthermore, the recombinant FOXP3 locus encodes a cytosolic FRB domain that binds intracellular rapamycin under the transcriptional control of the inserted promoter, preventing the undesirable effects of rapamycin exposure (e.g., mTOR inhibition) on CISC-mediated IL-2 signaling induction. Furthermore, the heterologous promoter of the recombinant FOXP3 locus is inserted downstream of the Treg-specific demethylation region (TSDR) of the FOXP3 locus, allowing the inserted promoter to regulate transcription of the endogenous FOXP3 coding sequence independently of TSDR methylation, which can occur in an inflammatory environment. The promoter inserted downstream of the TSDR allows us to bypass TSDR-mediated silencing of FOXP3 expression and maintain stable FOXP3 expression in cells even in inflammatory environments where FOXP3 expression is normally suppressed and Treg cells can transdifferentiate into inflammatory effector T cells.Thus, the dual-edited cells described herein are type 1 diabetes-associated antigen-specific Tregs, retain a stable suppressive phenotype in inflammatory environments (e.g., the inflamed pancreas), and can be expanded in a controllable manner in the presence of rapamycin.

[0009] Accordingly, some aspects of the present disclosure provide a method for producing a genetically modified cell, comprising: contacting a first nucleic acid and a second nucleic acid with a cell; (i) the first nucleic acid is (a) a first 5' homology arm having homology to a first nucleic acid sequence of the TRAC locus of the cellular genome; (b) the first promoter, which is the MND promoter; (c) a nucleotide sequence encoding a first chemically-induced signaling complex (CISC) component comprising: (1) an extracellular binding domain comprising the rapamycin-binding domain of FK506-binding protein 12 (FKBP); (2) a transmembrane domain of IL-2Rγ; and (3) an intracellular domain comprising the cytoplasmic domain of IL-2Rγ or a functional fragment thereof; (d) a nucleotide sequence encoding a TCRβ polypeptide or a functional fragment thereof; (e) a nucleotide sequence encoding at least a portion of a T cell receptor (TCR) comprising a TCRα polypeptide and a TCRβ polypeptide that binds to a type 1 diabetes (T1D)-associated antigen; the nucleotide sequence encoding at least a portion of a TCRα polypeptide comprising a variable region of TCRα and a junction region of TCRα; and (f) a first 3' homology arm having homology to a second nucleic acid sequence at the TRAC locus downstream of said first nucleic acid sequence at the TRAC locus; Including, (ii) the second nucleic acid is (a) a second 5' homologous arm having homology to a first nucleic acid sequence of a FOXP3 locus in the genome of the cell; (b) a second promoter that is the MND promoter; (c) a nucleotide sequence encoding a second CISC component comprising (1) an extracellular binding domain comprising the FKBP-rapamycin binding (FRB) domain of mTOR, (2) a transmembrane domain of IL-2Rβ, and (3) a cytoplasmic domain of IL-2Rβ or a functional fragment thereof; (d) a nucleotide sequence encoding a cytosolic FRB domain that binds rapamycin and does not contain a transmembrane domain; and (e) a second 3' homologous arm having homology to a second nucleic acid sequence at the FOXP3 locus downstream of the first nucleic acid sequence at the FOXP3 locus and downstream of a Treg-specific demethylation region (TSDR) at the FOXP3 locus; The present invention relates to a method, including:

[0010] In some embodiments, the first nucleic acid is a nucleotide sequence encoding a first 2A motif inserted in frame between the nucleotide sequence encoding the first CISC component and the nucleotide sequence encoding the TCR β polypeptide; a nucleotide sequence encoding a second 2A motif inserted in frame between the nucleotide sequence encoding the TCR β polypeptide and the nucleotide sequence encoding at least a portion of the TCR α polypeptide; Further includes:

[0011] In some embodiments, the nucleotide sequence encoding the first 2A motif has less than 90%, less than 80%, less than 70%, less than 60%, or less than 55% sequence identity with the nucleotide sequence encoding the second 2A motif.

[0012] In some embodiments, the first 2A motif is a T2A motif comprising the amino acid sequence of SEQ ID NO:222 and the second 2A motif is a P2A motif comprising the amino acid sequence of SEQ ID NO:226.

[0013] In some embodiments, the nucleotide sequence encoding the first 2A motif has at least 70% sequence identity to the nucleotide sequence of SEQ ID NO: 221, and the nucleotide sequence encoding the second 2A motif has at least 70% sequence identity to the nucleotide sequence of SEQ ID NO: 223.

[0014] In some embodiments, the second nucleic acid is a nucleotide sequence encoding a third 2A motif inserted in frame between the nucleotide sequence encoding the second CISC component and the nucleotide sequence encoding the cytosolic FRB domain polypeptide; a nucleotide sequence encoding a fourth 2A motif inserted in frame between the nucleotide sequence encoding the cytosolic FRB domain polypeptide and the nucleotide sequence encoding FoxP3 or a portion thereof; Further includes:

[0015] In some embodiments, the third 2A motif is a P2A motif comprising the amino acid sequence of SEQ ID NO:227, and the fourth 2A motif is a P2A motif comprising the amino acid sequence of SEQ ID NO:228.

[0016] In some embodiments, the nucleotide sequence encoding the third 2A motif has at least 70% sequence identity to the nucleotide sequence of SEQ ID NO: 224, and the nucleotide sequence encoding the fourth 2A motif has at least 70% sequence identity to the nucleotide sequence of SEQ ID NO: 225.

[0017] In some embodiments, the first CISC component further comprises a portion of the extracellular domain of IL-2Rγ.

[0018] In some embodiments, the second CISC component further comprises a portion of the extracellular domain of IL-2Rβ.

[0019] In some embodiments, the second CISC component comprises a threonine at a position corresponding to amino acid 2098 of wild-type mTOR having the amino acid sequence of SEQ ID NO:236.

[0020] In some embodiments, the first CISC component comprises an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or up to 100% sequence identity to the amino acid sequence of SEQ ID NO:66.

[0021] In some embodiments, the second CISC component comprises an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or up to 100% sequence identity to the amino acid sequence of SEQ ID NO:71.

[0022] In some embodiments, the first CISC component comprises the amino acid sequence of SEQ ID NO:66 and the second CISC component comprises the amino acid sequence of SEQ ID NO:71.

[0023] In some embodiments, the nucleotide sequence encoding at least a portion of the TCRα polypeptide is inserted in frame with an endogenous nucleotide sequence encoding at least a portion of the constant domain of the TCRα polypeptide, and the first MND promoter initiates transcription of the nucleotide sequence encoding the TCRα polypeptide comprising the variable region of the TCRα, the junction region of the TCRα, and the constant domain of the TCRα.

[0024] In some embodiments, the TCR beta polypeptide is (i) (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 4, (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 6; (ii) (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 14, (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 15, and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 16; or (iii) (a) CDR1 comprising the amino acid sequence of SEQ ID NO: 24, (b) CDR2 comprising the amino acid sequence of SEQ ID NO: 25, and (c) CDR3 comprising the amino acid sequence of SEQ ID NO: 26 Includes.

[0025] In some embodiments, the TCR alpha polypeptide is (i) (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 1, (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 3; (ii) (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 11, (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 12, and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 13; or (iii) (a) CDR1 comprising the amino acid sequence of SEQ ID NO: 21, (b) CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and (c) CDR3 comprising the amino acid sequence of SEQ ID NO: 23 Includes.

[0026] In some embodiments, the TCR alpha polypeptide comprises a variable domain comprising the amino acid sequence set forth in any of SEQ ID NOs: 7, 17, and 27.

[0027] In some embodiments, the TCR beta polypeptide comprises a variable domain comprising the amino acid sequence set forth in any of SEQ ID NOs: 8, 18, and 28.

[0028] In some embodiments, (i) the TCR alpha polypeptide comprises an alpha CDR1 having the amino acid sequence of SEQ ID NO: 1, an alpha CDR2 having the amino acid sequence of SEQ ID NO: 2, and an alpha CDR3 having the amino acid sequence of SEQ ID NO: 3, and the TCR beta polypeptide comprises a bCDR1 having the amino acid sequence of SEQ ID NO: 4, a bCDR2 having the amino acid sequence of SEQ ID NO: 5, and a bCDR3 having the amino acid sequence of SEQ ID NO: 6; (ii) the TCR alpha polypeptide comprises an alpha CDR1 having the amino acid sequence of SEQ ID NO: 11, an alpha CDR2 having the amino acid sequence of SEQ ID NO: 12, and an alpha CDR3 having the amino acid sequence of SEQ ID NO: 13, and the TCR beta polypeptide comprises a bCDR1 having the amino acid sequence of SEQ ID NO: 14, a bCDR2 having the amino acid sequence of SEQ ID NO: 15, and a bCDR3 having the amino acid sequence of SEQ ID NO: 16; or (iii) The TCR alpha polypeptide comprises an alpha CDR1 having the amino acid sequence of SEQ ID NO: 21, an alpha CDR2 having the amino acid sequence of SEQ ID NO: 22, and an alpha CDR3 having the amino acid sequence of SEQ ID NO: 23, and the TCR beta polypeptide comprises a bCDR1 having the amino acid sequence of SEQ ID NO: 24, a bCDR2 having the amino acid sequence of SEQ ID NO: 25, and a bCDR3 having the amino acid sequence of SEQ ID NO: 26.

[0029] In some embodiments, (i) the TCR alpha polypeptide comprises a variable domain comprising the amino acid sequence of SEQ ID NO:7, and the TCR beta polypeptide comprises a variable domain comprising the amino acid sequence of SEQ ID NO:8; (ii) the TCR alpha polypeptide comprises a variable domain comprising the amino acid sequence of SEQ ID NO: 17, and the TCR beta polypeptide comprises a variable domain comprising the amino acid sequence of SEQ ID NO: 18; or (iii) the TCR alpha polypeptide comprises a variable domain comprising the amino acid sequence of SEQ ID NO: 27, and the TCR beta polypeptide comprises a variable domain comprising the amino acid sequence of SEQ ID NO: 28. In some embodiments, (i) the TCR alpha polypeptide comprises the amino acid sequence of SEQ ID NO: 9 and the TCR beta polypeptide comprises the amino acid sequence of SEQ ID NO: 10; (ii) the TCR alpha polypeptide comprises the amino acid sequence of SEQ ID NO: 19, and the TCR beta polypeptide comprises the amino acid sequence of SEQ ID NO: 20; or (iii) the TCR alpha polypeptide comprises the amino acid sequence of SEQ ID NO: 29, and the TCR beta polypeptide comprises the amino acid sequence of SEQ ID NO: 30.

[0030] In some embodiments, inserting the second nucleic acid into the cell genome recombines the sequence of the first coding exon of the FOXP3 locus.

[0031] In some embodiments, insertion of the second nucleic acid into the cell genome does not alter the nucleotide sequence of the first coding exon of the FOXP3 locus.

[0032] In some embodiments, the method further comprises contacting the cell with a DNA endonuclease or a third nucleic acid encoding the DNA endonuclease.

[0033] In some embodiments, the third nucleic acid encoding the DNA endonuclease is RNA.

[0034] In some embodiments, the RNA encoding the DNA endonuclease is mRNA.

[0035] In some embodiments, the DNA endonuclease is an RNA-guided DNA endonuclease.

[0036] In some embodiments, the RNA-guided DNA endonuclease is a Cas endonuclease.

[0037] In some embodiments, the Cas endonuclease is a Cas9 endonuclease.

[0038] In some embodiments, the method further comprises contacting the cell with a TRAC locus-targeting guide RNA (gRNA) comprising a spacer sequence complementary to a sequence within the TRAC locus, or a fourth nucleic acid encoding the TRAC locus-targeting gRNA.

[0039] In some embodiments, the 5' homologous arm of the first nucleic acid comprises a sequence having at least 90% sequence identity to SEQ ID NO: 85, and the 3' homologous arm of the first nucleic acid comprises a sequence having at least 90% sequence identity to SEQ ID NO: 93.

[0040] In some embodiments, the 5' homologous arm of the first nucleic acid comprises a sequence having at least 90% sequence identity to SEQ ID NO: 96, and the 3' homologous arm of the first nucleic acid comprises a sequence having at least 90% sequence identity to SEQ ID NO: 105.

[0041] In some embodiments, the 5' homologous arm of the first nucleic acid comprises a sequence having at least 90% sequence identity to SEQ ID NO: 108, and the 3' homologous arm of the first nucleic acid comprises a sequence having at least 90% sequence identity to SEQ ID NO: 116.

[0042] In some embodiments, the 5' homologous arm of the first nucleic acid comprises a sequence having at least 90% sequence identity to SEQ ID NO: 119, and the 3' homologous arm of the first nucleic acid comprises a sequence having at least 90% sequence identity to SEQ ID NO: 127.

[0043] In some embodiments, the 5' homologous arm of the first nucleic acid comprises a sequence having at least 90% sequence identity to SEQ ID NO: 130, and the 3' homologous arm of the first nucleic acid comprises a sequence having at least 90% sequence identity to SEQ ID NO: 138.

[0044] In some embodiments, the method further comprises contacting the cell with a FOXP3 locus-targeting guide RNA (gRNA) comprising a spacer sequence complementary to a sequence within the FOXP3 locus, or a fourth nucleic acid encoding the FOXP3 locus-targeting gRNA.

[0045] In some embodiments, the 5' homologous arm of the second nucleic acid comprises a sequence having at least 90% sequence identity to SEQ ID NO: 141, and the 3' homologous arm of the second nucleic acid comprises a sequence having at least 90% sequence identity to SEQ ID NO: 149.

[0046] In some embodiments, the 5' homologous arm of the second nucleic acid comprises a sequence having at least 90% sequence identity to SEQ ID NO: 152, and the 3' homologous arm of the second nucleic acid comprises a sequence having at least 90% sequence identity to SEQ ID NO: 160.

[0047] In some embodiments, the 5' homologous arm of the second nucleic acid comprises a sequence having at least 90% sequence identity to SEQ ID NO: 163, and the 3' homologous arm of the second nucleic acid comprises a sequence having at least 90% sequence identity to SEQ ID NO: 171.

[0048] In some embodiments, the 5' homologous arm of the second nucleic acid comprises a sequence having at least 90% sequence identity to SEQ ID NO: 174, and the 3' homologous arm of the second nucleic acid comprises a sequence having at least 90% sequence identity to SEQ ID NO: 183.

[0049] In some embodiments, the 5' homologous arm of the second nucleic acid comprises a sequence having at least 90% sequence identity to SEQ ID NO: 186, and the 3' homologous arm of the second nucleic acid comprises a sequence having at least 90% sequence identity to SEQ ID NO: 194.

[0050] In some embodiments, the 5' homologous arm of the second nucleic acid comprises a sequence having at least 90% sequence identity to SEQ ID NO: 197, and the 3' homologous arm of the second nucleic acid comprises a sequence having at least 90% sequence identity to SEQ ID NO: 205.

[0051] In some embodiments, the 5' homologous arm of the second nucleic acid comprises a sequence having at least 90% sequence identity to SEQ ID NO: 208, and the 3' homologous arm of the second nucleic acid comprises a sequence having at least 90% sequence identity to SEQ ID NO: 217.

[0052] In some embodiments, the first nucleic acid is contained within a first vector.

[0053] In some embodiments, the first vector is an adeno-associated virus (AAV) vector.

[0054] In some embodiments, the first vector is an AAV vector derived from an AAV of the AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11 serotype.

[0055] In some embodiments, the second nucleic acid is contained within a second vector.

[0056] In some embodiments, the second vector is an adeno-associated virus (AAV) vector.

[0057] In some embodiments, the second vector is an AAV vector derived from an AAV of the AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11 serotype.

[0058] In some embodiments, the first nucleic acid comprises a nucleotide sequence between the first 5' homologous arm and the first 3' homologous arm that has at least 95% sequence identity to any of SEQ ID NOs: 94, 106, 117, 128, and 139.

[0059] In some embodiments, the second nucleic acid comprises a nucleotide sequence between the first 5' homologous arm and the first 3' homologous arm that has at least 95% sequence identity to any of SEQ ID NOs: 150, 161, 172, 184, 195, 206 and 218.

[0060] In some embodiments, the first nucleic acid comprises a nucleotide sequence having at least 95% sequence identity to any of SEQ ID NOs: 95, 107, 118, 129, and 140.

[0061] In some embodiments, the second nucleic acid comprises a nucleotide sequence having at least 95% sequence identity to any of SEQ ID NOs: 151, 162, 173, 185, 196, 207, and 219.

[0062] In some embodiments, the length of the one or more homology arms is between 100 and 2000 nucleotides in length.

[0063] In some embodiments, each of the homology arms is 300 to 700 nucleotides in length.

[0064] Some aspects of the present disclosure relate to genetically modified cells produced by the methods described herein.

[0065] Some aspects of the present disclosure provide a genetically modified cell comprising: a first nucleic acid inserted into a TRAC locus in the genome of a cell, and a second nucleic acid inserted into a FOXP3 locus in the genome of said cell; (i) the TRAC locus into which the first nucleic acid has been inserted is (a) The first promoter is the MND promoter; (b) an exogenous nucleotide sequence encoding a first chemical-inducible signaling complex (CISC) component comprising: (1) an extracellular binding domain comprising the rapamycin-binding domain of FK506-binding protein 12 (FKBP); (2) a transmembrane domain of IL-2Rγ; and (3) an intracellular domain comprising the cytoplasmic domain of IL-2Rγ or a functional fragment thereof; (c) an exogenous nucleotide sequence encoding an exogenous TCR β polypeptide or a functional fragment thereof; and (d) an exogenous nucleotide sequence encoding at least a portion of a T cell receptor (TCR) comprising a TCRα polypeptide and a TCRβ polypeptide that binds to a type 1 diabetes (T1D)-associated antigen, the TCRα polypeptide comprising a variable region of TCRα and a junction region of TCRα; Including, (ii) the FOXP3 locus into which the second nucleic acid has been inserted is (a) a second promoter, which is the MND promoter; (b) a nucleotide sequence encoding a second CISC component comprising (1) an extracellular binding domain comprising the FKBP-rapamycin binding (FRB) domain of mTOR, (2) a transmembrane domain of IL-2Rβ, and (3) a cytoplasmic domain of IL-2Rβ or a functional fragment thereof; and (c) a nucleotide sequence encoding a cytosolic FRB domain that binds rapamycin and does not contain a transmembrane domain; Including, The invention relates to a cell in which a second MND promoter has been inserted downstream of the Treg-specific demethylation region of the FOXP3 locus and initiates transcription of an endogenous nucleotide sequence encoding FoxP3 or a portion thereof.

[0066] In some embodiments, the first nucleic acid is a nucleotide sequence encoding a first 2A motif inserted in frame between the nucleotide sequence encoding the first CISC component and the nucleotide sequence encoding the TCR β polypeptide; a nucleotide sequence encoding a second 2A motif inserted in frame between the nucleotide sequence encoding the TCR β polypeptide and the nucleotide sequence encoding at least a portion of the TCR α polypeptide; Further includes:

[0067] In some embodiments, the nucleotide sequence encoding the first 2A motif has less than 90%, less than 80%, less than 70%, less than 60%, or less than 55% sequence identity with the nucleotide sequence encoding the second 2A motif.

[0068] In some embodiments, the first 2A motif is a T2A motif comprising the amino acid sequence of SEQ ID NO:222 and the second 2A motif is a P2A motif comprising the amino acid sequence of SEQ ID NO:226.

[0069] In some embodiments, the nucleotide sequence encoding the first 2A motif has at least 70% sequence identity to the nucleotide sequence of SEQ ID NO: 221, and the nucleotide sequence encoding the second 2A motif has at least 70% sequence identity to the nucleotide sequence of SEQ ID NO: 223.

[0070] In some embodiments, the second nucleic acid is a nucleotide sequence encoding a third 2A motif inserted in frame between the nucleotide sequence encoding the second CISC component and the nucleotide sequence encoding the cytosolic FRB domain polypeptide; a nucleotide sequence encoding a fourth 2A motif inserted in frame between the nucleotide sequence encoding the cytosolic FRB domain polypeptide and the nucleotide sequence encoding FoxP3 or a portion thereof; Further includes:

[0071] In some embodiments, the third 2A motif is a P2A motif comprising the amino acid sequence of SEQ ID NO:227, and the fourth 2A motif is a P2A motif comprising the amino acid sequence of SEQ ID NO:228.

[0072] In some embodiments, the nucleotide sequence encoding the third 2A motif has at least 70% sequence identity to the nucleotide sequence of SEQ ID NO: 224, and the nucleotide sequence encoding the fourth 2A motif has at least 70% sequence identity to the nucleotide sequence of SEQ ID NO: 225.

[0073] In some embodiments, the first CISC component further comprises a portion of the extracellular domain of IL-2Rγ.

[0074] In some embodiments, the second CISC component further comprises a portion of the extracellular domain of IL-2Rβ.

[0075] In some embodiments, the second CISC component comprises a threonine at a position corresponding to amino acid 2098 of wild-type mTOR having the amino acid sequence of SEQ ID NO:236.

[0076] In some embodiments, the first CISC component comprises an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or up to 100% sequence identity to the amino acid sequence of SEQ ID NO:66.

[0077] In some embodiments, the second CISC component comprises an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or up to 100% sequence identity to the amino acid sequence of SEQ ID NO:71.

[0078] In some embodiments, the first CISC component comprises the amino acid sequence of SEQ ID NO:66 and the second CISC component comprises the amino acid sequence of SEQ ID NO:71.

[0079] In some embodiments, the nucleotide sequence encoding at least a portion of the TCR alpha polypeptide is inserted in frame with an endogenous nucleotide sequence encoding at least a portion of a constant domain of the TCR alpha polypeptide; The first MND promoter initiates transcription of a nucleotide sequence encoding a TCRα polypeptide comprising the variable region of TCRα, the junction region of TCRα, and the constant domain of TCRα.

[0080] In some embodiments, the TCR beta polypeptide is (i) (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 4, (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 6; (ii) (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 14, (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 15, and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 16; or (iii) (a) CDR1 comprising the amino acid sequence of SEQ ID NO: 24, (b) CDR2 comprising the amino acid sequence of SEQ ID NO: 25, and (c) CDR3 comprising the amino acid sequence of SEQ ID NO: 26 Includes.

[0081] In some embodiments, the TCR alpha polypeptide is (i) (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 1, (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 3; (ii) (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 11, (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 12, and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 13; or (iii) (a) CDR1 comprising the amino acid sequence of SEQ ID NO: 21, (b) CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and (c) CDR3 comprising the amino acid sequence of SEQ ID NO: 23 Includes.

[0082] In some embodiments, the TCR alpha polypeptide comprises a variable domain comprising the amino acid sequence set forth in any of SEQ ID NOs: 7, 17, and 27.

[0083] In some embodiments, the TCR beta polypeptide comprises a variable domain comprising the amino acid sequence set forth in any of SEQ ID NOs: 8, 18, and 28.

[0084] In some embodiments, (i) the TCR alpha polypeptide comprises an alpha CDR1 having the amino acid sequence of SEQ ID NO: 1, an alpha CDR2 having the amino acid sequence of SEQ ID NO: 2, and an alpha CDR3 having the amino acid sequence of SEQ ID NO: 3, and the TCR beta polypeptide comprises a bCDR1 having the amino acid sequence of SEQ ID NO: 4, a bCDR2 having the amino acid sequence of SEQ ID NO: 5, and a bCDR3 having the amino acid sequence of SEQ ID NO: 6; (ii) the TCR alpha polypeptide comprises an alpha CDR1 having the amino acid sequence of SEQ ID NO: 11, an alpha CDR2 having the amino acid sequence of SEQ ID NO: 12, and an alpha CDR3 having the amino acid sequence of SEQ ID NO: 13, and the TCR beta polypeptide comprises a bCDR1 having the amino acid sequence of SEQ ID NO: 14, a bCDR2 having the amino acid sequence of SEQ ID NO: 15, and a bCDR3 having the amino acid sequence of SEQ ID NO: 16; or (iii) The TCR alpha polypeptide comprises an alpha CDR1 having the amino acid sequence of SEQ ID NO: 21, an alpha CDR2 having the amino acid sequence of SEQ ID NO: 22, and an alpha CDR3 having the amino acid sequence of SEQ ID NO: 23, and the TCR beta polypeptide comprises a bCDR1 having the amino acid sequence of SEQ ID NO: 24, a bCDR2 having the amino acid sequence of SEQ ID NO: 25, and a bCDR3 having the amino acid sequence of SEQ ID NO: 26.

[0085] In some embodiments, (i) the TCR alpha polypeptide comprises a variable domain comprising the amino acid sequence of SEQ ID NO:7, and the TCR beta polypeptide comprises a variable domain comprising the amino acid sequence of SEQ ID NO:8; (ii) the TCR alpha polypeptide comprises a variable domain comprising the amino acid sequence of SEQ ID NO: 17, and the TCR beta polypeptide comprises a variable domain comprising the amino acid sequence of SEQ ID NO: 18; or (iii) the TCR alpha polypeptide comprises a variable domain comprising the amino acid sequence of SEQ ID NO: 27, and the TCR beta polypeptide comprises a variable domain comprising the amino acid sequence of SEQ ID NO: 28.

[0086] In some embodiments, (i) the TCR alpha polypeptide comprises the amino acid sequence of SEQ ID NO: 9 and the TCR beta polypeptide comprises the amino acid sequence of SEQ ID NO: 10; (ii) the TCR alpha polypeptide comprises the amino acid sequence of SEQ ID NO: 19, and the TCR beta polypeptide comprises the amino acid sequence of SEQ ID NO: 20; or (iii) the TCR alpha polypeptide comprises the amino acid sequence of SEQ ID NO: 29, and the TCR beta polypeptide comprises the amino acid sequence of SEQ ID NO: 30.

[0087] In some embodiments, inserting the second nucleic acid into the cell genome recombines the sequence of the first coding exon of the FOXP3 locus.

[0088] In some embodiments, insertion of the second nucleic acid into the cell genome does not alter the nucleotide sequence of the first coding exon of the FOXP3 locus.

[0089] In some embodiments, the genetically modified cells are CD3+ T cells, CD4+ T cells and / or CD8+ T cells.

[0090] In some embodiments, the genetically modified cells are CD4+ T cells.

[0091] In some embodiments, the genetically modified cells are Treg cells.

[0092] In some embodiments, the genetically modified cells are FoxP3+ Treg cells.

[0093] In some embodiments, the genetically modified cells are CTLA-4+, LAG-3+, CD25+, CD39+, CD27+, CD70+, GITR+, neuropilin 1+, galectin 1+ and / or IL-2Rα+.

[0094] Some aspects of the present disclosure relate to pharmaceutical compositions comprising the genetically modified cells described herein and a pharmaceutically acceptable excipient.

[0095] Some aspects of the present disclosure relate to methods comprising administering to a subject a pharmaceutical composition or a genetically modified cell described herein.

[0096] In some embodiments, the genetically modified cells are autologous cells to the subject.

[0097] In some embodiments, the genetically modified cells are cells from the same species as the subject.

[0098] In some embodiments, the subject has type 1 diabetes (T1D).

[0099] In some embodiments, the subject has been diagnosed with type 1 diabetes (T1D) within 6 months, 5 months, 4 months, 3 months, 3 months, 2 months, or 1 month prior to administering the cells.

[0100] In some embodiments, the subject has an insulin dose-adjusted hemoglobin A1c (IDAA1c) of 9.0 or less.

[0101] In some embodiments, the subject's IDAA1c is reduced from above 9.0 to 9.0 or less after the subject is diagnosed with type 1 diabetes (T1D).

[0102] In some embodiments, autoantibodies binding to an antigen selected from the group consisting of an islet cell antigen, insulin, glutamic acid decarboxylase, islet tyrosine phosphatase 2, and / or zinc transporter 8 have been detected in the subject within 6 months, 5 months, 4 months, 3 months, 3 months, 2 months, or 1 month prior to administering the cells.

[0103] In some embodiments, the subject has not been diagnosed with type 1 diabetes (T1D).

[0104] In some embodiments, the subject's hemoglobin A1c is between 5.7 and 6.4.

[0105] In some embodiments, the subject has a hemoglobin A1c of 6.5 or greater.

[0106] In some embodiments, the subject is 3 years of age or older and less than 6 years of age, and 8 pieces~6×10 8 A dose comprising said cells is administered.

[0107] In some embodiments, the dose is 2.4 x 10 8 pieces~3.6×10 8 The cell comprises:

[0108] In some embodiments, the dose is about 3×10 8 The cell comprises:

[0109] In some embodiments, the subject is 6 years of age or older and less than 12 years of age, and 8 pieces~1×10 9 A dose comprising said cells is administered.

[0110] In some embodiments, the dose is 4×10 8 pieces~6×10 8 The cell comprises:

[0111] In some embodiments, the dose is about 5×10 8 The cell comprises:

[0112] In some embodiments, the subject is 12 years of age or older and less than 18 years of age, and 8 pieces~2×10 9 A dose comprising said cells is administered.

[0113] In some embodiments, the dose is 8×10 8 pieces~1.2×10 9 The cell comprises:

[0114] In some embodiments, the dose is about 10 9 The cell comprises:

[0115] In some embodiments, the subject is 18 years of age or older and has a risk of developing 5×10 8 pieces~2×10 9 A dose comprising said cells is administered.

[0116] In some embodiments, the subject is under 46 years of age.

[0117] In some embodiments, the dose is 8×10 8 pieces~1.2×10 9 The cell comprises:

[0118] In some embodiments, the dose is about 10 9 The cell comprises:

[0119] In some embodiments, the pancreatic volume of the subject is estimated from the age of the subject; (a) If the estimated pancreatic volume is approximately 20 mL, 1 × 10 8 pieces~6×10 8 a dose comprising said cells is administered to said subject; (b) If the estimated pancreatic volume is approximately 35 mL, 2 × 10 8 pieces~1×10 9 a dose comprising said cells is administered to said subject; or (c) 5 × 10 if the estimated pancreatic volume is approximately 60 mL or more 8 pieces~2×10 9 A dose comprising said cells is administered to said subject.

[0120] In some embodiments, (a) If the estimated pancreatic volume is approximately 20 mL, 2.4 × 10 8 pieces~3.6×10 8 a dose comprising said cells is administered to said subject; (b) If the estimated pancreatic volume is approximately 35 mL, 4 × 10 8 pieces~6×10 8 a dose comprising said cells is administered to said subject; or (c) If the estimated pancreatic volume is approximately 60 mL or more, 8 × 10 8 pieces~1.2×10 9 A dose comprising said cells is administered to said subject.

[0121] In some embodiments, (a) If the estimated pancreatic volume is approximately 20 mL, approximately 3 × 10 8 a dose comprising said cells is administered to said subject; (b) If the estimated pancreatic volume is approximately 35 mL, approximately 5 × 10 8 a dose comprising said cells is administered to said subject; or (c) If the estimated pancreatic volume is 60 mL or more, approximately 10 9 A dose comprising said cells is administered to said subject.

[0122] In some embodiments, the subject's pancreatic volume is estimated from the subject's age, and the method further comprises measuring the subject's actual pancreatic volume; (a) If the estimated pancreatic volume is approximately 20 mL, the ratio of the subject's actual pancreatic volume to the estimated pancreatic volume is (1 × 10 8 pieces~6×10 8 a dose comprising 100 or more of the cells is administered to the subject; (b) If the estimated pancreatic volume is approximately 35 mL, then the ratio of (subject's actual pancreatic volume:estimated pancreatic volume) × (2 × 10 8 pieces~1×10 9 a dose comprising at least one of said cells is administered to said subject; or (c) If the estimated pancreatic volume is approximately 60 mL or greater, the ratio of the subject's actual pancreatic volume to the estimated pancreatic volume is calculated by multiplying the pancreatic volume by (5 × 108 pieces~2×10 9 A dose comprising 100 (100) of the cells is administered to the subject.

[0123] In some embodiments, (a) If the estimated pancreatic volume is approximately 20 mL, the ratio of the subject's actual pancreatic volume to the estimated pancreatic volume is (2.4 × 10 8 pieces~3.6×10 8 a dose comprising 100 or more of the cells is administered to the subject; (b) If the estimated pancreatic volume is approximately 35 mL, then the ratio of the subject's actual pancreatic volume to the estimated pancreatic volume is (4 × 10 8 pieces~6×10 8 a dose comprising at least one of said cells is administered to said subject; or (c) If the estimated pancreatic volume is approximately 60 mL or greater, the ratio of the subject's actual pancreatic volume to the estimated pancreatic volume is calculated by multiplying the pancreatic volume by 8 × 10. 8 pieces~1.2×10 9 A dose comprising 100 (100) of the cells is administered to the subject.

[0124] In some embodiments, (a) If the estimated pancreatic volume is approximately 20 mL, then the ratio of the subject's actual pancreatic volume to the estimated pancreatic volume is (approximately 3 × 10 8 a dose comprising 100 or more of the cells is administered to the subject; (b) If the estimated pancreatic volume is approximately 35 mL, then (ratio of subject's actual pancreatic volume: estimated pancreatic volume) × (approximately 5 × 10 8 a dose comprising at least one of said cells is administered to said subject; or (c) If the estimated pancreatic volume is approximately 60 mL or more, the ratio of the subject's actual pancreatic volume to the estimated pancreatic volume is calculated by multiplying the estimated pancreatic volume by approximately 10 9 A dose comprising 100 (100) of the cells is administered to the subject.

[0125] In some embodiments, the subject is a human.

[0126] Some embodiments of the present disclosure provide a system comprising a first nucleic acid and a second nucleic acid, (i) the first nucleic acid is (a) a first 5' homology arm having homology to a first nucleic acid sequence of the TRAC locus of the cellular genome; (b) the first promoter, which is the MND promoter; (c) a nucleotide sequence encoding a first chemically-induced signaling complex (CISC) component comprising: (1) an extracellular binding domain comprising the rapamycin-binding domain of FK506-binding protein 12 (FKBP); (2) a transmembrane domain of IL-2Rγ; and (3) an intracellular domain comprising the cytoplasmic domain of IL-2Rγ or a functional fragment thereof; (d) a nucleotide sequence encoding a TCRβ polypeptide or a functional fragment thereof; (e) a nucleotide sequence encoding at least a portion of a T cell receptor (TCR) comprising a TCRα polypeptide and a TCRβ polypeptide that binds to a type 1 diabetes (T1D)-associated antigen; the nucleotide sequence encoding at least a portion of a TCRα polypeptide comprising a variable region of TCRα and a junction region of TCRα; and (f) a first 3' homology arm having homology to a second nucleic acid sequence at the TRAC locus downstream of said first nucleic acid sequence at the TRAC locus; Including, (ii) the second nucleic acid is (a) a second 5' homologous arm having homology to a first nucleic acid sequence of a FOXP3 locus in the genome of the cell; (b) a second promoter that is the MND promoter; (c) a nucleotide sequence encoding a second CISC component comprising (1) an extracellular binding domain comprising the FKBP-rapamycin binding (FRB) domain of mTOR, (2) a transmembrane domain of IL-2Rβ, and (3) a cytoplasmic domain of IL-2Rβ or a functional fragment thereof; (d) a nucleotide sequence encoding a cytosolic FRB domain that binds rapamycin and does not contain a transmembrane domain; and (e) a second 3' homologous arm having homology to a second nucleic acid sequence at the FOXP3 locus downstream of the first nucleic acid sequence at the FOXP3 locus and downstream of a Treg-specific demethylation region (TSDR) at the FOXP3 locus; This relates to a system including:

[0127] In some embodiments, the first nucleic acid is a nucleotide sequence encoding a first 2A motif inserted in frame between the nucleotide sequence encoding the first CISC component and the nucleotide sequence encoding the TCR β polypeptide; a nucleotide sequence encoding a second 2A motif inserted in frame between the nucleotide sequence encoding the TCR β polypeptide and the nucleotide sequence encoding at least a portion of the TCR α polypeptide; Further includes:

[0128] In some embodiments, the nucleotide sequence encoding the first 2A motif has less than 90%, less than 80%, less than 70%, less than 60%, or less than 55% sequence identity with the nucleotide sequence encoding the second 2A motif.

[0129] In some embodiments, the first 2A motif is a T2A motif comprising the amino acid sequence of SEQ ID NO:222 and the second 2A motif is a P2A motif comprising the amino acid sequence of SEQ ID NO:226.

[0130] In some embodiments, the nucleotide sequence encoding the first 2A motif has at least 70% sequence identity to the nucleotide sequence of SEQ ID NO: 221, and the nucleotide sequence encoding the second 2A motif has at least 70% sequence identity to the nucleotide sequence of SEQ ID NO: 223.

[0131] In some embodiments, the second nucleic acid is a nucleotide sequence encoding a third 2A motif inserted in frame between the nucleotide sequence encoding the second CISC component and the nucleotide sequence encoding the cytosolic FRB domain polypeptide; a nucleotide sequence encoding a fourth 2A motif inserted in frame between the nucleotide sequence encoding the cytosolic FRB domain polypeptide and the nucleotide sequence encoding FoxP3 or a portion thereof; Further includes:

[0132] In some embodiments, the third 2A motif is a P2A motif comprising the amino acid sequence of SEQ ID NO:227, and the fourth 2A motif is a P2A motif comprising the amino acid sequence of SEQ ID NO:228.

[0133] In some embodiments, the nucleotide sequence encoding the third 2A motif has at least 70% sequence identity to the nucleotide sequence of SEQ ID NO: 224, and the nucleotide sequence encoding the fourth 2A motif has at least 70% sequence identity to the nucleotide sequence of SEQ ID NO: 225.

[0134] In some embodiments, the first CISC component further comprises a portion of the extracellular domain of IL-2Rγ.

[0135] In some embodiments, the second CISC component further comprises a portion of the extracellular domain of IL-2Rβ.

[0136] In some embodiments, the second CISC component comprises a threonine at a position corresponding to amino acid 2098 of wild-type mTOR having the amino acid sequence of SEQ ID NO:236.

[0137] In some embodiments, the first CISC component comprises an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or up to 100% sequence identity to the amino acid sequence of SEQ ID NO:66.

[0138] In some embodiments, the second CISC component comprises an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or up to 100% sequence identity to the amino acid sequence of SEQ ID NO:71.

[0139] In some embodiments, the first CISC component comprises the amino acid sequence of SEQ ID NO:66 and the second CISC component comprises the amino acid sequence of SEQ ID NO:71.

[0140] In some embodiments, the nucleotide sequence encoding at least a portion of the TCR alpha polypeptide is inserted in frame with a nucleotide sequence encoding at least a portion of a constant domain of the TCR alpha polypeptide in the 3' homology arm; The first MND promoter initiates transcription of a nucleotide sequence encoding a TCRα polypeptide comprising the variable region of TCRα, the junction region of TCRα, and the constant domain of TCRα.

[0141] In some embodiments, the TCR beta polypeptide is (i) (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 4, (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 6; (ii) (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 14, (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 15, and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 16; or (iii) (a) CDR1 comprising the amino acid sequence of SEQ ID NO: 24, (b) CDR2 comprising the amino acid sequence of SEQ ID NO: 25, and (c) CDR3 comprising the amino acid sequence of SEQ ID NO: 26 Includes.

[0142] In some embodiments, the TCR alpha polypeptide is (i) (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 1, (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 3; (ii) (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 11, (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 12, and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 13; or (iii) (a) CDR1 comprising the amino acid sequence of SEQ ID NO: 21, (b) CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and (c) CDR3 comprising the amino acid sequence of SEQ ID NO: 23 Includes.

[0143] In some embodiments, the TCR alpha polypeptide comprises a variable domain comprising the amino acid sequence set forth in any of SEQ ID NOs: 7, 17, and 27.

[0144] In some embodiments, the TCR beta polypeptide comprises a variable domain comprising the amino acid sequence set forth in any of SEQ ID NOs: 8, 18, and 28.

[0145] In some embodiments, (i) the TCR alpha polypeptide comprises an alpha CDR1 having the amino acid sequence of SEQ ID NO: 1, an alpha CDR2 having the amino acid sequence of SEQ ID NO: 2, and an alpha CDR3 having the amino acid sequence of SEQ ID NO: 3, and the TCR beta polypeptide comprises a bCDR1 having the amino acid sequence of SEQ ID NO: 4, a bCDR2 having the amino acid sequence of SEQ ID NO: 5, and a bCDR3 having the amino acid sequence of SEQ ID NO: 6; (ii) the TCR alpha polypeptide comprises an alpha CDR1 having the amino acid sequence of SEQ ID NO: 11, an alpha CDR2 having the amino acid sequence of SEQ ID NO: 12, and an alpha CDR3 having the amino acid sequence of SEQ ID NO: 13, and the TCR beta polypeptide comprises a bCDR1 having the amino acid sequence of SEQ ID NO: 14, a bCDR2 having the amino acid sequence of SEQ ID NO: 15, and a bCDR3 having the amino acid sequence of SEQ ID NO: 16; or (iii) The TCR alpha polypeptide comprises an alpha CDR1 having the amino acid sequence of SEQ ID NO: 21, an alpha CDR2 having the amino acid sequence of SEQ ID NO: 22, and an alpha CDR3 having the amino acid sequence of SEQ ID NO: 23, and the TCR beta polypeptide comprises a bCDR1 having the amino acid sequence of SEQ ID NO: 24, a bCDR2 having the amino acid sequence of SEQ ID NO: 25, and a bCDR3 having the amino acid sequence of SEQ ID NO: 26.

[0146] In some embodiments, (i) the TCR alpha polypeptide comprises a variable domain comprising the amino acid sequence of SEQ ID NO:7, and the TCR beta polypeptide comprises a variable domain comprising the amino acid sequence of SEQ ID NO:8; (ii) the TCR alpha polypeptide comprises a variable domain comprising the amino acid sequence of SEQ ID NO: 17, and the TCR beta polypeptide comprises a variable domain comprising the amino acid sequence of SEQ ID NO: 18; or (iii) the TCR alpha polypeptide comprises a variable domain comprising the amino acid sequence of SEQ ID NO: 27, and the TCR beta polypeptide comprises a variable domain comprising the amino acid sequence of SEQ ID NO: 28.

[0147] In some embodiments, (i) the TCR alpha polypeptide comprises the amino acid sequence of SEQ ID NO: 9 and the TCR beta polypeptide comprises the amino acid sequence of SEQ ID NO: 10; (ii) the TCR alpha polypeptide comprises the amino acid sequence of SEQ ID NO: 19, and the TCR beta polypeptide comprises the amino acid sequence of SEQ ID NO: 20; or (iii) the TCR alpha polypeptide comprises the amino acid sequence of SEQ ID NO: 29, and the TCR beta polypeptide comprises the amino acid sequence of SEQ ID NO: 30.

[0148] In some embodiments, inserting the second nucleic acid into the cell genome recombines the sequence of the first coding exon of the FOXP3 locus.

[0149] In some embodiments, insertion of the second nucleic acid into the cell genome does not alter the nucleotide sequence of the first coding exon of the FOXP3 locus.

[0150] In some embodiments, the system further comprises a DNA endonuclease or a third nucleic acid encoding the DNA endonuclease.

[0151] In some embodiments, the third nucleic acid encoding the DNA endonuclease is RNA.

[0152] In some embodiments, the RNA encoding the DNA endonuclease is mRNA.

[0153] In some embodiments, the DNA endonuclease is an RNA-guided DNA endonuclease.

[0154] In some embodiments, the RNA-guided DNA endonuclease is a Cas endonuclease.

[0155] In some embodiments, the Cas endonuclease is a Cas9 endonuclease.

[0156] In some embodiments, the system further comprises a TRAC locus-targeting guide RNA (gRNA) comprising a spacer sequence complementary to a sequence within the TRAC locus, or a fourth nucleic acid encoding the TRAC locus-targeting gRNA.

[0157] In some embodiments, the 5' homologous arm of the first nucleic acid comprises a sequence having at least 90% sequence identity to SEQ ID NO: 85, and the 3' homologous arm of the first nucleic acid comprises a sequence having at least 90% sequence identity to SEQ ID NO: 93.

[0158] In some embodiments, the 5' homologous arm of the first nucleic acid comprises a sequence having at least 90% sequence identity to SEQ ID NO: 96, and the 3' homologous arm of the first nucleic acid comprises a sequence having at least 90% sequence identity to SEQ ID NO: 105.

[0159] In some embodiments, the 5' homologous arm of the first nucleic acid comprises a sequence having at least 90% sequence identity to SEQ ID NO: 108, and the 3' homologous arm of the first nucleic acid comprises a sequence having at least 90% sequence identity to SEQ ID NO: 116.

[0160] In some embodiments, the 5' homologous arm of the first nucleic acid comprises a sequence having at least 90% sequence identity to SEQ ID NO: 119, and the 3' homologous arm of the first nucleic acid comprises a sequence having at least 90% sequence identity to SEQ ID NO: 127.

[0161] In some embodiments, the 5' homologous arm of the first nucleic acid comprises a sequence having at least 90% sequence identity to SEQ ID NO: 130, and the 3' homologous arm of the first nucleic acid comprises a sequence having at least 90% sequence identity to SEQ ID NO: 138.

[0162] In some embodiments, the system further comprises a FOXP3 locus-targeting guide RNA (gRNA) comprising a spacer sequence complementary to a sequence within the FOXP3 locus, or a fourth nucleic acid encoding the FOXP3 locus-targeting gRNA.

[0163] In some embodiments, the 5' homologous arm of the second nucleic acid comprises a sequence having at least 90% sequence identity to SEQ ID NO: 141, and the 3' homologous arm of the second nucleic acid comprises a sequence having at least 90% sequence identity to SEQ ID NO: 149.

[0164] In some embodiments, the 5' homologous arm of the second nucleic acid comprises a sequence having at least 90% sequence identity to SEQ ID NO: 152, and the 3' homologous arm of the second nucleic acid comprises a sequence having at least 90% sequence identity to SEQ ID NO: 160.

[0165] In some embodiments, the 5' homologous arm of the second nucleic acid comprises a sequence having at least 90% sequence identity to SEQ ID NO: 163, and the 3' homologous arm of the second nucleic acid comprises a sequence having at least 90% sequence identity to SEQ ID NO: 171.

[0166] In some embodiments, the 5' homologous arm of the second nucleic acid comprises a sequence having at least 90% sequence identity to SEQ ID NO: 174, and the 3' homologous arm of the second nucleic acid comprises a sequence having at least 90% sequence identity to SEQ ID NO: 183.

[0167] In some embodiments, the 5' homologous arm of the second nucleic acid comprises a sequence having at least 90% sequence identity to SEQ ID NO: 186, and the 3' homologous arm of the second nucleic acid comprises a sequence having at least 90% sequence identity to SEQ ID NO: 194.

[0168] In some embodiments, the 5' homologous arm of the second nucleic acid comprises a sequence having at least 90% sequence identity to SEQ ID NO: 197, and the 3' homologous arm of the second nucleic acid comprises a sequence having at least 90% sequence identity to SEQ ID NO: 205.

[0169] In some embodiments, the 5' homologous arm of the second nucleic acid comprises a sequence having at least 90% sequence identity to SEQ ID NO: 208, and the 3' homologous arm of the second nucleic acid comprises a sequence having at least 90% sequence identity to SEQ ID NO: 217.

[0170] In some embodiments, the first nucleic acid is contained within a first vector.

[0171] In some embodiments, the first vector is an adeno-associated virus (AAV) vector.

[0172] In some embodiments, the first vector is an AAV vector derived from an AAV of the AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11 serotype.

[0173] In some embodiments, the second nucleic acid is contained within a second vector.

[0174] In some embodiments, the second vector is an adeno-associated virus (AAV) vector.

[0175] In some embodiments, the second vector is an AAV vector derived from an AAV of the AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11 serotype.

[0176] In some embodiments, the first nucleic acid comprises a nucleotide sequence between the first 5' homologous arm and the first 3' homologous arm that has at least 95% sequence identity to any of SEQ ID NOs: 94, 106, 117, 128, and 139.

[0177] In some embodiments, the second nucleic acid comprises a nucleotide sequence between the first 5' homologous arm and the first 3' homologous arm that has at least 95% sequence identity to any of SEQ ID NOs: 150, 161, 172, 184, 195, 206 and 218.

[0178] In some embodiments, the first nucleic acid comprises a nucleotide sequence having at least 95% sequence identity to any of SEQ ID NOs: 95, 107, 118, 129, and 140.

[0179] In some embodiments, the second nucleic acid comprises a nucleotide sequence having at least 95% sequence identity to any of SEQ ID NOs: 151, 162, 173, 185, 196, 207, and 219.

[0180] In some embodiments, the length of the one or more homology arms is between 100 and 2000 nucleotides in length.

[0181] In some embodiments, each of the homology arms is 300 to 700 nucleotides in length. [Brief explanation of the drawings]

[0182] The following drawings form part of this specification and are included to further demonstrate certain aspects of the present disclosure. These aspects can be better understood by reference to one or more of the drawings in conjunction with the detailed description of specific embodiments set forth herein. The data shown in the drawings is not intended to limit the scope of the present disclosure in any way.

[0183] [Figure 1] For the treatment of diabetes, we present examples of polynucleotides used in Treg engineering to (i) insert fragments of MND, FKBP-IL2RG, and T1D2 TCR or T1D5-1 TCR via TRAC hijacking, and (ii) insert MND, FRB-ILRβ, and naked cytosolic FRB into the FOXP3 locus. This TRAC hijacking strategy involved knockout of the endogenous TCR but utilized the endogenous TRAC sequence. Cells with insertions at these two loci are referred to as "double-edited cells."

[0184] [Figure 2] The editing setup for Treg recombination using the polynucleotides shown in Figure 1 is shown, along with the CD4+ T cell donor, each AAV construct used, the starting cell number used for dual editing, and the name of the final product. A mock product was generated by electroporation without the addition of the AAV donor or nuclease.

[0185] [Figure 3]Figure 1 shows the initial editing rates in cells 3 days after insertion of the polynucleotides shown. CD4+ T cells from three donors (two type 1 diabetic subjects and one healthy donor) were double-edited using RNPs targeting the TRAC locus and RNPs targeting the FOXP3 locus, and delivered with either 1) VIN 10019-Genti 122 AAV T1D5-1+3362 AAV, or 2) VIN 10020-Genti 122 AAV T1D2+3362 AAV to generate hT1D5-1 and hT1D2 EngTregs, respectively. The graph demonstrates the high initial double-editing rates in this study. In each flow cytometry plot, double-edited cells are located in the upper right quadrant. Initial double-editing rates ranged from 10.1% to 18.9% as measured by FACS analysis for CD3+ / HA+ co-expression. In double-edited cells, successful introduction of an islet antigen-specific TCR (isTCR) at the TRAC locus restored CD3 expression, and HA staining demonstrated successful expression of HA-tagged endogenous FoxP3 after homologous recombination repair editing of the FOXP3 locus.

[0186] [Figure 4] An example protocol for engineered Treg cells, including expansion of dual-edited cells with rapamycin, is shown. Three days after gene editing, cells were seeded in 10 nM rapamycin and expanded for 12 days, with restimulation with anti-CD3 / CD28 beads on day 12. The total number of cells seeded ranged from 1.99 x 106 to 2.72 x 106 and is listed for each donor / TCR.

[0187] [Figure 5]Figure 1 shows the enrichment of double-edited cells 15 days after transfection with the indicated polynucleotides. The percentage of CD3+ / HA-FoxP3+ double-positive EngTregs at day 19 ranged from 81.1% to 89.2%, indicating that T1D2+ / FoxP3+ double-positive cells and T1D5-1+ / FoxP3+ double-positive cells were enriched in both donors with type 1 diabetes and healthy control donors. These results demonstrate that 1) as expected, T1D2+ / FoxP3+ double-positive EngTreg cells and T1D5-1+ / FoxP3+ double-positive EngTreg cells can be enriched with rapamycin, and 2) cells from donors with type 1 diabetes can be enriched in a similar manner to cells from healthy control donors.

[0188] [Figures 6A-6B] Figure 6 shows the suppression of activated Teff cells by recombinant Tregs generated by dual editing, which involved inserting the polynucleotides shown in Figure 1 to generate T1D2 TCR-expressing Tregs (Figure 6A) or T1D5-1 TCR-expressing Tregs (Figure 6B). Teff cells were activated with the IGRP305-324 cognate peptide or with anti-CD3 / CD28 in the presence of myeloid dendritic cells (mDCs) as antigen-presenting cells (APCs). Both T1D2-expressing and T1D5-1-expressing dual-edited EngTregs from each donor exhibited potent suppressive activity (>80% suppression) against Teff cells targeted with the same IGRP peptide, as summarized in the bar graph below.

[0189] [Figure 6C] To assess whether antigen-specific dual-edited EngTregs exert bystander suppression, we constructed and performed a polyclonal islet suppression assay using a pool of autologous Teff cells (derived from the same type 1 diabetic subject) activated in vitro with APCs (mDCs) pulsed with a pool of islet-specific peptides derived from four major islet antigens: IGRP, GAD65, PPI, and ZNT8.

[0190] We generated T1D2-expressing or TID5-1-expressing EngTregs by combining editing of the FOXP3 locus and the TRAC locus. We then compared these T1D2-expressing or TID5-1-expressing EngTregs with Tregs engineered by lentivirus (LV) delivery containing sequences encoding the same islet-specific TCRs (T1D2 or T1D5-1). LV-edited T1D2 / FoxP3+ and T1D5-1 / FoxP3+ cells differed from EngTregs dually edited at the TRAC and FOXP3 loci in several ways: (i) the islet-specific TCR expressed by LV-edited cells contained the mouse TCR β chain rather than the human TCR β chain; (ii) LV-edited cells expressed intact endogenous TDRs; and (iii) LV-edited cells did not express components of the chemo-inducible signaling complex (CISC), which mediates IL-2 signaling in the presence of rapamycin. The results showed that mouse LV-edited mT1D2+ / FoxP3+ and mT1D5-1+ / FoxP3+ cells suppressed the proliferation of mixed Teff cell populations stimulated with an islet-specific peptide pool at various Teff:DC ratios. The graph on the left shows the percentage of suppression, while the graph on the right shows the percentage of suppression normalized by the results of an anti-CD3 / CD28 bead assay. These findings support the notion that islet-specific EngTregs can exert bystander suppression of islet-specific autologous Teffs present in subjects with type 1 diabetes.

[0191] [Figures 7A-7B]We compared the suppressive function of dual-edited EngTregs (hTRAC and FOXP3) with that of Tregs generated by lentiviral vector-mediated insertion of the T1D2 TCR coding sequence or the T1D5-1 TCR coding sequence. Figure 7A shows data from a 30:1 Teff:DC ratio study. Using the assay described in Figure 6C, we analyzed bystander suppression by dual-HDR-edited hT1D2 / FoxP3 EngTregs and dual-HDR-edited hT1D5-1 / FoxP3 EngTregs. We measured the proliferation of islet antigen-specific Teff cells stimulated with a pancreatic islet-specific peptide pool (containing three IGRP peptides, five GAD65 peptides, one PPI peptide, and one ZNT8 peptide) at a Teff:Treg ratio of 1:1 or 1:0.5 (Treg1 / 2) in the presence or absence of hT1D2 / FoxP3 or hT1D5-1 / FoxP3 EngTregs. Both hT1D2 / FoxP3 and hT1D5-1 / FoxP3 double-edited EngTregs exhibited potent bystander suppressive activity. In this study, hT1D2 double-edited cells exhibited slightly greater suppressive activity than hT1D5-1 double-edited cells. Cells designated T1D2 and T1D5-1 (but not hT1D2 or hT1D5-1) represent LV-edited Tregs shown in Figure 6C, and these cells exhibited lower suppressive activity than the corresponding double-edited EngTregs expressing human TCRs (e.g., T1D2 vs. hT1D2). Figure 7B shows data obtained with various Teff:DC ratios. "T1D2" and "T1D5" represent Tregs engineered with a lentiviral vector encoding a mouse TCR without knockout of the endogenous TCR. These data demonstrate that polyclonal bystander suppression is reproducible at Teff:DC ratios of 5:1, 10:1, 20:1, and 30:1.Dual-edited hT1D2+ / FoxP3+ EngTregs and hT1D5-1+ / FoxP3+ EngTregs had higher suppressive activity compared with Tregs edited with LV to express mouse TCRs with the same specificity (e.g., hT1D5-1 vs. T1D5-1).

[0192] [Figures 8A-8C]The phenotype of dual-edited Tregs was shown. Antibodies were used to detect the expression of human T1D2 (anti-TCR Vβ13.6) and human T1D5-1 (anti-TCR Vβ7.2). Figure 8A shows the expression of TCRBb protein. Figure 8B shows the expression of TCR, FoxP3, and CD25. TCR Vβ13.6 staining was observed in both dual-edited cells targeting hT1D2 / FoxP3 and T1D2 cells edited with LV. TCR Vβ7.2 staining was observed in both dual-edited cells targeting hT1D5-1 / FoxP3 and T1D5-1 cells edited with LV. T1D2 TCR-expressing cells and hT1D2 TCR-expressing cells showed higher signals than T1D5-1 TCR-expressing cells or hT1D5-1 TCR-expressing cells, respectively, which may be due to differences in TCR expression levels or the efficacy of staining with the anti-TCR Vβ7.2 antibody. The expression of FoxP3 and CD25 was measured, demonstrating that hT1D2+ / FoxP3+ and hT1D5-1+ / FoxP3+ double-edited EngTreg cells exhibited a Treg phenotype (upper and lower right panels). Notably, both hT1D2+ / FoxP3+ and hT1D5-1+ / FoxP3+ double-edited EngTreg cells expressed higher levels of CD25 than cells edited with LV to express their respective mouse TCRs (e.g., hT1D2 double-edited vs. T1D2). Figure 8C shows the expression of CD39 and CD73 on hT1D2+ / FoxP3+ and hT1D5-1+ / FoxP3+ double-edited EngTreg cells. These two cell surface proteins are involved in adenosine generation, HLA-DR suppression, and Treg-mediated suppression of Teffs. Furthermore, T1D2 and T1D5-1 double-edited EngTreg cells expressed all three Treg markers evaluated (CD39, CD73, and HLA-DR) at higher frequencies than Treg cells edited with LV to express their corresponding mouse TCRs (e.g., hT1D2 double-edited vs. T1D2).

[0193] [Figure 9] 1 shows a graph of the initial double-editing rate for preparing T1D4 EngTreg cells.

[0194] [Figure 10] 1 shows the enrichment of dual-edited T1D4 EngTreg cells by rapamycin.

[0195] [Figure 11] Graph of editing rate in T1D4 EngTreg cells before and after enrichment with rapamycin is shown.

[0196] [Figure 12] Graphs showing the expression levels of FoxP3, CD25, and CTLA-4 in T1D4 EngTreg cells compared with mock-edited cells.

[0197] [Figure 13] A graph showing the relative production of TNF-α, IFN-γ, and IL-2 in T1D4 EngTreg cells compared with mock-edited cells.

[0198] [Figure 14] A graph showing the relative expression level of TGF-β in T1D4 EngTreg cells compared with mock-edited cells.

[0199] [Figure 15] A graph showing the relative suppression rate of T1D4 Teff cells by T1D4 EngTreg cells or mock-edited cells stimulated with anti-CD3 / CD28 or APC+IGRP241-260 is shown.

[0200] [Figure 16] Graphs showing the secretion of TNF-α, IFN-γ and IL-2 in T1D4 Teff cells co-cultured with T1D4 EngTreg cells or mock-edited cells are shown.

[0201] [Figure 17]The relative suppression rate of PPI-specific Teffs co-cultured with T1D4 EngTregs or mock-edited cells stimulated with a combination of PPI peptides alone and antigen-presenting cells, or a combination of both PPI peptides and IGRP peptides and antigen-presenting cells, is shown.

[0202] [Figure 18] Cytokine secretion of PPI-specific Teffs cultured with APCs loaded with PPI76-90 peptide, or with APCs loaded with PPI76-90 peptide and IGRP241-260 peptide, together with T1D4 EngTreg cells or mock-edited cells is shown.

[0203] [Figures 19A-19C] Figure 19 provides an overview of type 1 diabetes and the function of GNTI-122, a recombinant regulatory T cell therapy for the treatment of type 1 diabetes. Figure 19A illustrates the pathogenesis of type 1 diabetes, specifically the killing of insulin-producing β cells by T lymphocytes. Figure 19B illustrates the suppression of effector T cells by GNTI-122-recombinant Treg cells and the resulting protection of pancreatic islet cells. Figure 19C provides a schematic diagram of the development process of GNTI-122.

[0204] [Figure 20] 1 shows a method for producing GNTI-122 recombinant Tregs from autologous cells.

[0205] [Figure 21] Selective expansion of GNTI-122 cells during the manufacturing process is shown. The frequency of GNTI-122 cells is measured by flow cytometry. FACS analysis of GNTI-122 cells and mock-modified cells 3 days after editing (left) and upon cryopreservation (right) is shown.

[0206] [Figures 22A-22E]Figure 22A shows the effect of rapamycin stimulation on GNTI-122 Treg cells and mock-engineered cells. Figure 22B shows the median fluorescence intensity (MFI) of phosphorylated STAT5 (pSTAT5) in GNTI-122 or mock-engineered cells in response to various doses of rapamycin in culture. Repeated-measures ANOVA was performed for cell type, dose, and interaction (p<0.0001), followed by Sidak's multiple comparison test for each dose (*p<0.05, ***p<0.001). Error bars represent mean ± SEM (n=3 donors). For both mock-engineered and GNTI-122 cells, the live CD3+CD4+ cell population was gated. Figure 22C shows cell viability (measured as fold expansion) in culture in the presence of 10 mM rapamycin without TCR stimulation. Figure 22D shows cell viability (measured as fold proliferation) in culture in the presence of 10 mM rapamycin upon TCR stimulation with anti-CD3 / CD28 beads. Figure 22E shows fold proliferation upon TCR stimulation in the presence of rapamycin at concentrations ranging from 0 to 30 nM. Two-way ANOVA with Tukey's multiple comparison test was performed, and significance is indicated for the corresponding conditions on day 8 (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0207] [Figures 23A-23H]Figure 23 shows the expression of Treg-associated markers and suppression of T effector (Teff) cells by GNTI-122 and mock-modified cells. GNTI-122 cells and their corresponding mock controls were generated in parallel, thawed, and rested in culture medium for 3 days before staining. Mock-edited cells were gated on CD4+ cells, while GNTI-122 cells were gated on islet-specific T cell receptor (isTCR)+FoxP3+ cells. Figures 23A and 23B show representative data from a single donor; this phenotype was also reproduced in cells independently generated from six different donors. Figure 23C shows direct suppression of Teff cells expressing the same TCR as GNTI-122. Figure 23D shows bystander suppression of Teff cells expressing a different TCR specific for another type 1 diabetes-associated antigen, preproinsulin (PPI). Figure 23E shows the suppression of polyclonal Teff cell populations expressing TCRs specific for nine cognate peptides of type 1 diabetes-associated antigens. Figure 23F shows the editing efficiency of EngTregs generated from subjects with type 1 diabetes. Figure 23G shows the enrichment efficiency of EngTregs generated from subjects with type 1 diabetes. Figure 23H shows the phenotypic analysis of EngTregs generated from subjects with type 1 diabetes.

[0208] [Figures 24A-24B]Figure 24A shows the in vitro characteristics of GNTI-122 cells. Figure 24A shows cytokine production and expression of Treg activation markers by mock-recombinant cells, GNTI-122 cells alone, or GNTI-122 cells contacted with rapamycin after stimulation with PMA / ionomycin / monensin or anti-CD3 / CD28 beads. Relative MFI levels were normalized to mock cells. *** or **** indicates statistically significant differences by two-way analysis of variance. Data from a representative donor are shown; similar data were replicated in six independent donors. Figure 24B shows the suppression of Teff cells expressing the same isTCR by mock-recombinant cells or GNTI-122 cells. Mock-recombinant cells or GNTI-122 cells were cocultured with autologous isTCR+FoxP3- Teff cells and stimulated with monocyte-derived dendritic cells bearing the cognate peptide recognized by the isTCR. Percent suppression indicates Teff suppression as measured by flow cytometry analysis of Teff activation. *** or **** indicates statistically significant differences by two-way ANOVA. Data from a representative donor are shown; similar data were replicated in three independent donors.

[0209] [Figures 25A-25C] Experimental design and efficacy of murine recombinant Treg therapy in an adoptive transfer type 1 diabetes model are shown. Figure 25A shows the experimental timeline. Figures 25B-25C show the diabetes-free survival rate (Figure 25B) and blood glucose levels (Figure 25C) of NOD.Cg-Prkdcscid Il2rgtm1Wjl / SzJ (NSGTM) recipient mice 7 or 15 days after intravenous injection of splenocytes (T1D splenocytes) obtained from non-obese diabetic (NOD) mice followed by intravenous injection of murine BDC2.5 recombinant regulatory T cells (mEngTregs).

[0210] [Figures 26A-26B]Figure 26 shows the localization and suppressive function of mEngTregs in vivo. Mice were administered T1D splenocytes on day 0, and 14 days after T1D splenocyte administration, they were treated with or without mEngTregs. They were then euthanized on day 22, and mEngTreg cells and CD8+ Teff memory cells were quantified in the blood, bone marrow, liver, pancreas, and spleen. Figure 26A shows the quantification of mEngTregs (isTCR+FoxP3+). Figure 26B shows the quantification of CD8+ T effector memory (CD44+CD62L-) cells.

[0211] [Figures 27A-27C] These results demonstrate reduced islet inflammation and beta cell protection. Mice shown in Figures 25A-25C were euthanized 43 days after T1D splenocyte administration, and pancreatic histological analysis was performed. Figure 27A shows the severity of islet inflammation quantified by hematoxylin and eosin (H&E) staining. Figure 27B shows quantification of beta cell mass by insulin staining of the pancreas. Approximately 20 islets were quantified per mouse. Figure 27C shows representative H&E and insulin staining of pancreases isolated from mice treated with T1D splenocytes alone or T1D splenocytes plus mEngTregs 43 days after T1D splenocyte administration.

[0212] [Figure 28] 1 shows a mouse study in which mEngTregs were administered 7 days after administration of diabetogenic splenocytes.

[0213] [Figures 29A-29E]CD4+ T cells were edited to express one of various TCRs, and the phenotype of the edited cells was evaluated. Figure 29A shows an overview of CD4+ T cell editing, stimulation, and analysis. Figure 29B shows a representative gating strategy for assessing the expression of cell surface markers CD69, CD137, and CD154 after stimulation (day 8). Figure 29C shows the expression of cell surface markers (CD69, CD137, and CD154) after 20 hours of stimulation with HLA-DR-expressing K562 cells pulsed with the cognate peptides IGRP 305-324 or IGRP 241-260. Figure 29D shows a representative gating strategy for assessing TNF-α and IFN-γ production after stimulation (day 14). FIG. 29E shows the production of TNF-α and IFN-γ after 5 hours of stimulation in HLA-DR-expressing K562 cells pulsed with the cognate peptides IGRP 305-324 or IGRP 241-260.

[0214] [Figure 30A-30B] Figure 30 shows the dose response of T1D TCR-expressing CD4+ T cells to stimulation with IGRP 305-324 peptide. Cells were cultured for 20 hours in the presence of HLA-DR4-expressing K562 cells and analyzed by flow cytometry. Figure 30A shows the dose response measured by the intensity of cell surface expression of CD154. Figure 30B shows the dose response measured by the percentage of CD137-expressing cells. Dashed lines = 50% of the maximal response of each cell population (derived from the donor).

[0215] [Figures 31A-31D]Figures 31A and 31B show T1D2 tolerance to substitutions in the IGRP 305-324 peptide. Figures 31A and 31B show activation of T1D2 TCR-expressing CD4+ T cells in the presence of antigen-presenting cells pulsed with various alanine-substituted peptides, as measured by the intensity of CD154 expression (Figure 31A) or the percentage of CD137-expressing cells (Figure 31B). T cells were cultured for 20 hours in the presence of HLA-DR4-expressing K562 cells pulsed with various alanine-substituted peptides or the IGRP 305-324 peptide, and analyzed by flow cytometry. Figures 31C and 31D show activation of T1D2 TCR-expressing CD4+ T cells in the presence of antigen-presenting cells pulsed with various potentially off-target peptides derived from human-associated pathogens, as measured by the intensity of CD154 expression (Figure 31C) or the percentage of CD137-expressing cells (Figure 31D). "Control" indicates CD4+ T cells expressing the ZNT266 TCR.

[0216] [Figure 32]

[0023] Figure 1 outlines the study design for a Phase 1 / 2 trial to evaluate GNTI-122 cells in adult and pediatric subjects with recently diagnosed type 1 diabetes.

[0217] [Figure 33A] This figure shows the generation of islet-specific EngTregs by homologous recombination repair-mediated FOXP3 gene editing and lentiviral TCR transduction. A timeline of the major steps for generating and enriching islet-specific EngTregs from primary human CD4+ T cells is shown. On day 0, T cells were activated with CD3 / CD28 beads, and on day 1, they were transduced with a lentiviral vector (encoding an islet-specific TCR). On day 7, islet-specific TCR and Treg marker expression (mTCR, CD25, CD127, CTLA-4, and ICOS) was assessed by flow cytometry. On day 10, islet-specific EngTregs were enriched with LNGFR magnetic beads.

[0218] [Figure 33B] A schematic diagram of the FOXP3 locus is shown (top). Exons are boxed. An AAV6 donor template (bottom) was designed to insert the MND promoter, truncated LNGFR coding sequence, and P2A (2A) sequence. If gene editing is successful, the MND promoter drives expression of LNGFR and FOXP3.

[0219] [Figure 33C] Representative flow cytometry plots (day 7, 4 days after gene editing) are shown. The left panel shows co-expression of FOXP3 and LNGFR in gene-edited cells. From left to right, the panels show expression of mTCR, CD25, CD127, CTLA-4, and ICOS gated on LNGFR+FOXP3+ cells.

[0220] [Figure 33D] Representative flow cytometry plots (day 10, 7 days after gene editing) are shown, demonstrating the purity of LNGFR+ cells after enrichment with anti-LNGFR magnetic beads. During enrichment of LNGFR+ cells, LNGFR- T cells were also collected and used as a control for in vitro suppression assays.

[0221] [Figure 33E] This figure shows the expression of TCR in T cells transduced with islet TCRs and the antigen-specific proliferation of these T cells. A schematic diagram showing the structure of lentiviral islet-specific TCRs containing the variable regions of human islet-specific TCRs (huV-α and huV-β) and the constant regions of mouse TCRs (muV-α and muV-β) is shown.

[0222] [Figure 33F] This figure shows confirmation of islet-specific TCR expression in human CD4+ T cells transduced with islet-specific TCRs. CD4+ T cells were isolated, activated with CD3 / CD28 beads, and transduced with lentiviruses encoding islet-specific TCRs. Flow cytometry plots show the results of analyzing mTCR expression in CD4+ T cells 7 days after transduction using an antibody specific for the murine TCR constant region.

[0223] [Figure 33G] Proliferation of CD4+ T cells transduced with each islet TCR in the presence of cognate peptides recognized by each islet TCR and APCs is shown. CD4+ T cells transduced with each TCR were labeled with cell trace violet and then cocultured with the cognate peptide recognized by each TCR (or an irrelevant peptide) and APCs (irradiated PBMCs) for 4 days. Flow cytometry plots show cell proliferation measured as a function of CTV dilution.

[0224] [Figure 33H] This shows a comparison of the expression levels of mTCR in CD4 T cells transduced with the islet-specific TCR shown in Figure 33F.

[0225] [Figure 34A] This shows that islet-specific EngTregs suppress antigen-induced Teff proliferation. A schematic diagram showing direct suppression of Teffs by EngTregs with the same islet antigen specificity as Teffs is shown. Both EngTregs and Teffs shown in this figure express the T1D5-2 TCR specific for IGRP305-324.

[0226] [Figure 34B] Representative histograms show proliferation (measured by CTV dilution) of T1D5-2 Teffs cocultured with EF670-labeled EngTregs or control EngTregs in the presence of anti-CD3 / CD28 antibody-coated beads (top) or the cognate peptide (IGRP305-324) and APC (bottom). Each histogram was gated on EF670- cells.

[0227] [Figure 34C]The percentage of suppression of CD3 / CD28 bead-induced Teff proliferation by polyEngTregs, LNGFR-T cells, or islet-specific EngTregs bearing T1D5-2 TCR (left), PPI76 TCR (center), or GAD65 TCR (right) is shown.

[0228] [Figure 34D] Figures 34C and 34D show the percent inhibition of antigen-induced Teff proliferation by poly-EngTregs, LNGFR-T cells, or islet-specific EngTregs bearing T1D5-2 TCR (left), PPI76 TCR (center), or GAD65 TCR (right). The cognate peptides recognized by T1D5-2 TCR were IGRP305-324, PPI76 TCR (center), and GAD65 TCR (GAD65265-284). In Figures 34C and 34D, data are shown as the mean ± SD of three independent experiments using cells generated from three different healthy donors. P values ​​were calculated using a paired two-tailed Student's t-test (*P<0.05 and **P<0.01).

[0229] [Figure 34E] The main steps and timeline for the generation of islet-specific EngTregs and islet-specific Teffs and in vitro suppression assays are shown. CD4+ T cells were activated with CD3 / CD28 beads and then transduced with TCR to generate Teffs. Teffs were expanded and harvested on day 15. The procedure for generating EngTregs is shown in Figure 109A. Teffs were cocultured with EngTregs or LNGFR-T cells in the presence of APCs (irradiated autologous PBMCs) and various peptides, or in the presence of CD3 / CD28 beads, or Teffs were cultured without these T cells. Before coculture, Teffs were labeled with cell trace violet (CTV), and EngTregs and LNGFR-T cells were labeled with EF670. After 3 or 4 days of incubation, cells were harvested, stained, and analyzed by flow cytometry.

[0230] [Figure 34F] Representative histograms showing proliferation of T1D4 Teffs when T1D4 Teffs were co-cultured with poly-EngTregs or T1D4 EngTregs at various Treg:Teff ratios in the presence of CD3 / CD28 beads are shown.

[0231] [Figure 34G] Representative histograms showing proliferation of T1D4 Teffs in the presence of the cognate peptide (IGRP241-260) and APCs, performed in parallel with the CD3 / CD28 suppression assay shown in Figure 34F, are shown.

[0232] [Figure 34H] The percentage of suppression of CD3 / CD28 bead-induced Teff proliferation by polyEngTreg or T1D4 EngTreg is shown.

[0233] [Figure 34I] Figure 34 shows the rate of suppression of antigen-induced Teff proliferation by poly-EngTregs or T1D4 EngTregs. In Figures 34H and 34I, data are shown as the mean ± SD of five independent experiments using cells generated from four different healthy donors. P values ​​were calculated using a paired multiple t-test (***P < 0.005).

[0234] [Figure 35A] This shows that islet-specific EngTregs suppress antigen-induced cytokine production from Teffs. Representative flow cytometry plots show Teff cytokine production (TNF-α, IL-2, and IFN-γ) and Teff activation (CD25 expression) in an antigen-specific suppression assay. T1D5-2 Teffs were cultured alone or co-cultured with polyclonal EngTregs, LNGFR-T cells, or T1D5-2 EngTregs in the presence of IGRP305-324, the cognate peptide recognized by the T1D5-2 TCR, and APCs.

[0235] [Figure 35B] The percentage of suppression of antigen-induced TNFα (left), IL-2 (center), and IFNγ (right) production from T1D5-2 Teffs by polyEngTregs, LNGFR-T cells, or islet-specific T1D5-2 EngTregs is shown.

[0236] [Figure 35C] Figure 35B and Figure 35C show the suppression rate of antigen-induced CD25 expression from T1D5-2 Teff by poly-EngTregs, LNGFR-T cells, or islet-specific T1D5-2 EngTregs. In Figure 35B and Figure 35C, data are shown as the mean ± SD of four independent experiments using cells prepared from four different healthy donors. P values ​​were calculated using a paired two-tailed Student's t-test (*P<0.05, **P<0.01, and ***P<0.001).

[0237] [Figure 36A] This shows that islet-specific EngTregs suppress the proliferation of bystander Teffs. A schematic diagram showing bystander suppression of Teffs by EngTregs with different islet antigen specificities is shown. This diagram shows EngTregs expressing the T1D4 TCR specific for IGRP241-260 and Teffs expressing the T1D5-2 TCR specific for IGRP305-324.

[0238] [Figure 36B] Representative histograms show proliferation (measured by CTV dilution) of T1D5-2 Teffs cocultured with T1D5-2 EngTregs, T1D4 EngTregs, or poly-EngTregs in the presence of IGRP305-324 peptide alone and APC (top row) or a mixture of IGRP305-324 and IGRP241-260 peptides and APC (bottom row). EngTregs were labeled with EF670, and each histogram was gated on EF670- cells.

[0239] [Figure 36C] The figure shows the rate of inhibition of T1D5-2 Teff proliferation by polyEngTreg, T1D5-2 EngTreg, or T1D4 EngTreg in the presence of a mixture of IGRP305-324 peptide and IGRP241-260 peptide and APC.

[0240] [Figure 36D] Representative histograms show proliferation (measured by CTV dilution) of T1D5-2 Teffs cocultured with poly-EngTregs or GAD265 EngTregs in the presence of IGRP305-324 peptide alone and APC (top row) or a mixture of IGRP305-324 and GAD265-284 peptides and APC (bottom row). EngTregs were labeled with EF670, and each histogram was gated on EF670- cells.

[0241] [Figure 36E] The figure shows the rate of inhibition of T1D5-2 Teff proliferation by polyEngTreg or GAD265 EngTreg in the presence of a mixture of IGRP305-324 peptide and GAD265-284 peptide and APC.

[0242] [Figure 36F] The percentage of suppression of cytokine production from T1D5-2 Teff by polyEngTreg, T1D5-2 EngTreg, or T1D4 EngTreg in the presence of a mixture of IGRP305-324 and IGRP241-260 peptides and APC is shown.

[0243] [Figure 36G]Figures 36C, 36E, 36F, and 36G show the suppression rate of CD25 expression from T1D5-2 Teffs by poly-EngTregs, T1D5-2 EngTregs, or T1D4 EngTregs in the presence of a mixture of IGRP305-324 and IGRP241-260 peptides and APC. Data in Figures 36C, 36E, 36F, and 36G are shown as mean ± SD values ​​from three independent experiments using cells generated from three different healthy donors. P values ​​were calculated using a paired two-tailed Student's t-test (*P<0.05, P<0.01, and P<0.005). In all three experiments, LNGFR-T cells bearing T1D5-2 or T1D4 TCRs were used as negative controls. These negative control cells did not show significant suppression.

[0244] [Figure 36H] This shows that CD3 / CD28 bead-induced Teff proliferation was suppressed to a similar extent by different types of islet-specific EngTregs. Representative flow cytometry plots showing mTCR expression in FOXP3-edited cells (-) without TCR transduction, or in FOXP3-edited cells transduced with T1D4 or T1D5-2 TCRs. On day 7, edited cells were stained and gated for live, CD3+, CD4+, LNGFR+, and FOXP3+ cells.

[0245] [Figure 36I] Representative histograms showing proliferation of T1D5-2 Teffs in a CD3 / CD28 bead suppression assay performed in parallel with the bystander suppression assays shown in Figures 36B and 36C are shown. T1D5-2 Teffs were incubated with CD3 / CD28 beads in the absence (-) of Tregs or in the presence of polyclonal EngTregs, T1D5-2 EngTregs, or T1D4 EngTregs.

[0246] [Figure 36J]Figure 36I shows the percentage of inhibition of CD3 / CD28 bead-induced proliferation of T1D5-2 Teffs by polyEngTregs, T1D5-2 EngTregs, or T1D4 EngTregs.

[0247] [Figure 36K] Representative histograms showing proliferation of T1D5-2 Teffs in a CD3 / CD28 bead suppression assay performed in parallel with the bystander suppression assays shown in Figures 36D and 36E are shown. T1D5-2 Teffs were incubated with CD3 / CD28 beads in the absence (-) of Tregs or in the presence of poly-EngTregs or GAD265-EngTregs.

[0248] [Figure 36L] Figure 36K shows the percent inhibition of CD3 / CD28 bead-induced T1D5-2 Teff proliferation by poly-EngTregs or GAD265-EngTregs. In Figures 36J and 36L, data are shown as mean ± SD from three independent experiments using cells generated from three different healthy donors. P values ​​were calculated using a paired two-tailed Student's t-test.

[0249] [Figure 36M] Representative histograms showing the suppression of cytokine production from bystander Teffs by islet-specific EngTregs are shown. Representative histograms showing TNFα production from T1D5-2 Teffs in an antigen-specific bystander suppression assay are shown. The legends for each histogram are the same as in Figure 36M.

[0250] [Figure 36N] Representative histograms showing IL-2 production from T1D5-2 Teffs in an antigen-specific bystander suppression assay are shown. The legends for each histogram are the same as in Figure 36M.

[0251] [Figure 36O]Representative histograms showing IFNγ production from T1D5-2 Teffs in an antigen-specific bystander suppression assay are shown. The legends for each histogram are the same as in Figure 36M.

[0252] [Figure 36P] Representative histograms showing CD25 expression from T1D5-2 Teffs in an antigen-specific bystander suppression assay are shown. The legends for each histogram are the same as in Figure 36M. In Figures 36M-36P, T1D5-2 Teffs were cultured without Tregs or cocultured with poly-EngTregs, T1D5-2 EngTregs, or T1D4 EngTregs in the presence of IGRP305-324 peptide alone and APCs, or a mixture of IGRP305-324 and IGRP241-260 peptides and APCs.

[0253] [Figure 37A] This shows that islet-specific EngTregs suppress polyclonal islet-specific Teffs derived from PBMCs obtained from type 1 diabetic donors. The timeline and major steps of the production of islet-specific EngTregs, polyclonal islet-specific Teffs, and monocyte-derived DCs (mDCs) from PBMCs obtained from type 1 diabetic donors and the in vitro suppression assay are shown.

[0254] [Figure 37B] Representative histograms show proliferation (measured by CTV dilution) of polyclonal islet-specific Teffs co-cultured with T1D2 EngTregs, 4.13 EngTregs, LNGFR-T cells, or poly-EngTregs in the presence of CD3 / CD28 beads (top) or islet-specific antigens (nine islet-specific peptides and monocyte-derived DCs (mDCs)) (bottom). EngTregs were labeled with EF670, and each histogram was gated on EF670- cells.

[0255] [Figure 37C]Figure 1 shows the rate of inhibition of CD3 / CD28-induced polyclonal islet-specific Teff proliferation by T1D2 EngTregs, 4.13 EngTregs, LNGFR-T cells, or polyEngTregs.

[0256] [Figure 37D] Figure 1 shows the percent inhibition of antigen-induced polyclonal islet-specific Teff proliferation by T1D2 EngTregs, 4.13 EngTregs, LNGFR-T cells, or poly-EngTregs. Antigen stimulation was performed with a pool of nine islet-specific peptides in the presence of mDCs. Data are shown as the mean ± SD of three independent experiments using cells generated from three type 1 diabetic donors. P values ​​were calculated using a paired two-tailed Student's t-test (*P<0.05, **P<0.01, and ***P<0.0001). As a negative control, coculture in the presence of mDCs and DMSO also resulted in no significant Teff proliferation.

[0257] [Figure 37E] This paper presents the expansion of multispecific islet-specific T cells derived from PBMCs obtained from a type 1 diabetic donor. The timeline and key steps for peptide-stimulated expansion of islet-specific T cells are shown. CD4+CD25- T cells isolated from a type 1 diabetic donor were stimulated with nine HLA-DR0401-restricted islet peptides, including five GAD65-specific islet peptides, three IGRP-specific islet peptides, and one PPI-specific islet peptide, in combination with irradiated autologous APCs (CD4-CD25+). Tetramer staining was performed on days 12–14. T cells were cultured without IL-2 for up to day 7, after which IL-2 was added every 2–3 days for expansion.

[0258] [Figure 37F]Representative flow cytometry plots showing tetramer-positive T cells specific for individual antigenic peptides are shown. Negative staining was performed without tetramer. Cells were gated on CD4+ T cells, and percentages indicate the degree of tetramer staining relative to background.

[0259] [Figure 37G] CD4+ T cells from three type 1 diabetic donors were peptide-stimulated in vitro for 12–14 days in five separate experiments. The percentage of tetramer-positive CD4+ T cells was measured. The combined results are shown. Each bar represents the percentage of CD4+ T cells specific for each islet antigenic peptide. Each dot represents one experiment.

[0260] [Figure 37H] Islet-specific EngTregs are superior to tTregs in suppressing polyclonal islet-specific Teffs. Representative histograms show parallel measurements of polyclonal islet-specific Teff proliferation in the presence of anti-CD3 / CD28 antibody-coated beads (top) or in the presence of a pool of nine islet-specific peptides and mDCs (bottom). Polyclonal islet-specific Teffs were cultured without Tregs (-) or cocultured with T1D2 LNGFR-, T1D2 EngTregs, or tTregs. tTregs were selected for CD4+CD25+CD127- and cultured similarly to EngTregs. tTregs were activated with CD3 / CD28 beads for 2 days, expanded, and harvested on day 10. All cells used in suppression assays were autologous cells prepared from type 1 diabetic donors. As a negative control, co-culture with monocyte-derived DCs (mDCs) and DMSO was also performed, but no significant proliferation of Teffs was observed.

[0261] [Figure 37I] The percentage of inhibition of CD3 / CD28 bead-induced polyclonal islet-specific Teff proliferation by T1D2 LNGFR- cells, T1D2 EngTregs, or tTregs is shown.

[0262] [Figure 37J] The percentage of suppression of antigen-induced proliferation of polyclonal islet-specific Teffs by T1D2 LNGFR- cells, T1D2 EngTregs, or tTregs is shown.

[0263] [Figure 38A] We demonstrate that islet-specific EngTregs inhibit APC maturation and suppress Teffs using both cell-contact-dependent and cell-contact-independent mechanisms. A schematic diagram of a transwell suppression assay using an upper and lower chamber separated by a permeable membrane is shown.

[0264] [Figure 38B] The percent proliferation inhibition of polyclonal islet-specific Teffs in the lower chamber (left panel) or upper chamber (right panel) as measured by CTV dilution is shown. As a positive control, polyclonal islet-specific Teffs were co-cultured with T1D2 EngTregs. Data are shown as mean ± SEM from three independent experiments using cells generated from three type 1 diabetic donors. **P<0.001, **P<0.01, *P<0.05 as determined by paired t-test.

[0265] [Figure 38C] A timeline of the DC maturation and APC modulation assay and its main steps are shown.

[0266] [Figure 38D] The normalized MFI of CD86 expression on mDCs is shown. HLA DR0401-restricted mature autologous mDCs were cocultured with T1D2 EngTreg or LNGFR-T cells for 2 days in the presence of IGRP305-324 peptide. The MFI of CD86 on DCs was normalized to the MFI measured in the DC-only condition. Data are shown as the mean ± SD of three independent experiments using cells generated from three different healthy donors. *P<0.05 as determined by paired t-test.

[0267] [Figure 38E] Representative histograms show proliferation of polyclonal islet-specific Teffs co-cultured with islet-specific antigens (10 antigens including IGRP305-324) and mDCs in the presence of T1D2 EngTregs and exogenous human IL2 (0.1 IU / ml). Prior to co-culture, Teffs were labeled with CTV and EngTregs with EF670, and proliferation was assessed by measuring the dilution of CTV.

[0268] [Figure 38F] Figure 38B shows the percent proliferation inhibition of Teffs shown in Figure 38E. The percent proliferation inhibition was calculated separately in the absence and presence of exogenous human IL2. Data are shown as the mean ± SEM of three independent experiments using cells generated from three type 1 diabetic donors. Ns: No significant difference as determined by paired t-test.

[0269] [Figure 38G] We demonstrate that islet-specific EngTregs exhibit contact-dependent and contact-independent bystander suppression. To investigate the mechanism of bystander suppression by islet-specific EngTregs, we generated polyclonal islet-specific Teffs. CD4+CD25- T cells isolated from type 1 diabetic donors were stimulated with nine HLA-DR0401-restricted islet peptides, including those specific for GAD65113-132, GAD265-284, GAD273-292, GAD305-324, IGRP17-36, IGRP241-260, PPI76-90, or ZNT8266-285, and irradiated autologous APCs (CD4-CD25+). Tetramer staining was performed on days 14 and 15. T1D2 TCRs specific for the IGRP305-324 peptide were excluded from Teff expansion. Representative flow cytometry plots showing tetramer-positive T cells specific for individual antigenic peptides are shown. Negative staining was performed without tetramer. Cells were gated on CD4+ T cells, and percentages indicate the degree of tetramer staining relative to background.

[0270] [Figure 38H] The figures show the combined results of measuring the percentage of tetramer-positive CD4+ T cells from three type 1 diabetic donors after 14-15 days of in vitro peptide stimulation. Each bar represents the percentage of CD4+ T cells specific for each islet antigenic peptide. Each dot represents a different type 1 diabetic donor.

[0271] [Figure 38I] Representative histograms show proliferation of polyclonal islet-specific Teff cells in the lower well (bottom row) or upper well (top row). mDCs loaded with a pool of islet-specific peptides (10 antigens including IGRP305-324) were seeded in the lower and upper wells. Polyclonal islet-specific Teff cells and / or T1D2 EngTreg cells were added to the lower and / or upper wells as indicated on the graph.

[0272] [Figure 38J] We demonstrate that islet-specific EngTregs suppress CD86 expression on dendritic cells. HLA-DR0401-restricted autologous monocytes were matured into DCs with GM-CSF / IL-4 and IFNγ / CL075. CTV-labeled islet TCR-expressing EngTregs or islet TCR-expressing LNGFR-T cells were cocultured with mature DCs in the presence of cognate peptides. After 2 days of incubation, cells were harvested, stained, and analyzed by flow cytometry.

[0273] [Figure 38K] Representative data showing the MFI of CD86 on DCs co-cultured with T1D2 EngTreg or LNGFR-T cells are shown.

[0274] [Figure 38L]Bar graphs showing the normalized expression levels of CD86 in DCs cocultured with T1D4 EngTreg or LNGFR-T cells in the presence of IGRP241-260 peptide (left graph) and the normalized expression levels of CD86 in DCs cocultured with PPI76 EngTreg or LNGFR-T cells in the presence of PPI76-90 peptide (right graph).

[0275] [Figure 38M] Expression of mTCR in FOXP3-edited cells transduced with no specific TCR (poly), T1D2 TCR, or 4.13 TCR is shown. On day 7, edited cells were stained and gated for live, CD3+, CD4+, and LNGFR+ cells. LNGFR+ cells (EngTregs) and LNGFR- T cells enriched using anti-LNGFR magnetic beads were used in the suppression assays shown in Figures 194A-194D.

[0276] [Figure 38N] Representative histograms show the results of parallel measurements of polyclonal islet-specific Teff proliferation in the presence of CD3 / CD28 beads (top panel) or mDCs and a pool of nine islet-specific peptides (bottom panel). Polyclonal islet-specific Teffs were cultured without Tregs (-) or co-cultured with T1D2 LNGFR- cells, T1D2 EngTregs, or tTregs. tTregs were selected for CD4+CD25+CD127- and cultured in the same manner as EngTregs. tTregs were activated with CD3 / CD28 beads for 2 days, expanded, and harvested on day 10. All cells used in suppression assays were autologous cells prepared from type 1 diabetic donors. As a negative control, co-culture with monocyte-derived DCs (mDCs) and DMSO also resulted in no significant Teff proliferation (data not shown).

[0277] [Figure 38O] The percentage of inhibition of CD3 / CD28 bead-induced polyclonal islet-specific Teff proliferation by T1D2 LNGFR- cells, T1D2 EngTregs, or tTregs is shown.

[0278] [Figure 38P] Figure 1 shows the percent inhibition of antigen-induced polyclonal islet-specific Teff proliferation by T1D2 LNGFR- cells, T1D2 EngTregs, or tTregs. The graph shows representative data from two independent experiments.

[0279] [Figure 39A] This graph shows the peptide dose response of T cells expressing T1D2 TCR, T1D4 TCR, or PPI76 TCR. CD4+ T cells transduced with T1D2 TCR, T1D4 TCR, or PPI76 TCR were cocultured with APCs for 4 days in the presence of various concentrations of the cognate peptides recognized by each TCR, i.e., IGRP305-324, IGRP241-260, and PPI76-90. Representative results from three independent experiments are shown.

[0280] [Figure 39B] Figure 1 shows the percent inhibition of antigen-induced polyclonal islet-specific Teff proliferation by T1D2 EngTregs, T1D4 EngTregs, or PPI76 EngTregs. Data are shown as the mean ± SEM of four independent experiments using cells generated from four different type 1 diabetic donors. P values ​​were calculated using a paired two-tailed Student's t-test (*P<0.05 and **P<0.01).

[0281] [Figure 39C]Graphs showing peptide dose responses of T cells expressing T1D2 TCR, T1D5-1 TCR, or T1D5-2 TCR are shown. CD4+ T cells transduced with T1D2 TCR, T1D5-1 TCR, or T1D5-2 TCR were cocultured with APCs for 4 days in the presence of various concentrations of the cognate peptide (IGRP305-324) recognized by each TCR. Representative results from three independent experiments are shown. For the T cell dose responses shown in Figures 39A and 39C, T cells were labeled with CTV before coculture, and cell proliferation was measured by dilution of the CTV.

[0282] [Figure 39D] Figure 39 shows the percent inhibition of antigen-induced polyclonal islet-specific Teff proliferation by T1D2 EngTregs, T1D5-1 EngTregs, or T1D5-2 EngTregs. Data are shown as the mean ± SEM of four independent experiments using cells generated from four different type 1 diabetes donors. P values ​​were calculated using a paired, two-tailed Student's t-test (*P<0.05). For the suppression assays shown in Figures 39B and 39D, data were normalized using the suppression activity obtained in a parallel suppression assay using CD3 / CD28 beads. The suppression activity was calculated as the percent inhibition / the lowest percent inhibition. Normalized antigen-specific suppression activity was calculated as the percent inhibition / the suppression activity in the antigen-specific assay.

[0283] [Figure 39E] Representative flow cytometry plots showing the expression of each mTCR in FOXP3-edited cells transduced with T1D2 TCR, T1D4 TCR, or PPI76 TCR are shown.

[0284] [Figure 39F] Figure 39B shows a comparison of mTCR expression levels. On day 7, edited cells were stained and gated for live, CD3+, CD4+, LNGFR+, and FOXP3+ cells. Enriched LNGFR+ cells (EngTregs) expressing T1D2 TCR, T1D4 TCR, or PPI76 TCR were used for suppression assays.

[0285] [Figure 39G] Representative histograms showing proliferation of polyclonal islet-specific Teffs in the presence of islet-specific antigens (10 islet-specific peptides + monocyte-derived DCs (mDCs)) and T1D2 EngTregs, T1D4 EngTregs, or PPI76 EngTregs are shown.

[0286] [Figure 39H] Representative flow cytometry plots showing the expression of each mTCR in FOXP3-edited cells transduced with T1D2 TCR, T1D5-1 TCR, or T1D5-2 TCR are shown.

[0287] [Figure 39I] Figure 39H shows a comparison of mTCR expression levels. On day 7, edited cells were stained and gated for live, CD3+, CD4+, LNGFR+, and FOXP3+ cells. Enriched LNGFR+ cells (EngTregs) expressing T1D2 TCR, T1D5-1 TCR, or T1D5-2 TCR were used for suppression assays.

[0288] [Figure 39J] Representative histograms show the proliferation of polyclonal islet-specific Teff cells in the presence of islet-specific antigens (10 islet-specific peptides + mDCs) and T1D2, T1D5-1, or T1D5-2 EngTregs. Polyclonal islet-specific Teff cells were labeled with CTV, and EngTreg cells were labeled with EF670. Cell proliferation was measured using the dilution of CTV.

[0289] [Figure 40A]Gene editing of BDC2.5 CD4+ T cells to generate mouse islet-specific EngTregs is shown. A schematic diagram of the MND LNGFR p2A knock-in donor template packaged in AAV5 for use in editing FOXP3 via homology-directed repair is shown. Exons are indicated by numbered boxes, and FOXP3 homology arms are also shown. If gene editing is successful, the MND promoter drives expression of the endogenous mouse FOXP3 protein and cell surface expression of LNGFR in cis configuration.

[0290] [Figure 40B] 1 shows a schematic diagram illustrating the experimental timeline for FOXP3 gene editing, cell analysis, and enrichment of edited LNGFR cells.

[0291] [Figure 40C] Representative flow cytometry plots (one of four independent experiments) showing LNGFR expression in mock-edited control cells (left), LNGFR+ cells edited with RNP and AAV donor template before column enrichment (center), or LNGFR+ cells after column enrichment (right) are shown.

[0292] [Figure 40D] Representative flow cytometry histograms (one of two independent experiments) showing the expression of Treg-associated markers in the indicated cell populations are shown.

[0293] [Figure 40E] A bar graph showing the MFI of Treg-associated markers in EngTreg or mock-edited cells is shown. Error bars indicate ±SD. P values ​​were calculated using an unpaired t-test to compare EngTreg and mock-edited cells.

[0294] [Figure 40F]Schematic representation of in vitro suppression assays performed with BDC2.5 CD4+ Teff cells and mock control cells, BDC2.5 tTreg cells or EngTreg cells.

[0295] [Figure 40G] Representative flow cytometry plot (one of three independent experiments) showing CTV-labeled BDC2.5 CD4+ Teffs co-cultured with the indicated cells 4 days after stimulation.

[0296] [Figure 40H] Figure 1 shows the percent inhibition of proliferation of BDC2.5 CD4+ Teffs co-cultured with the indicated Tregs at various Teff:Treg ratios. Percent inhibition = [100 - normalized percent inhibition]; normalized percent inhibition = 100 / (proliferation in Teff-only conditions) × (proliferation of Teffs in the presence of Tregs).

[0297] [Figure 41A] We demonstrate that polyclonal EngTregs cannot prevent the development of type 1 diabetes in vivo, whereas islet-specific EngTregs can. A schematic diagram showing the experimental timeline for conducting a mouse diabetes prevention study is shown.

[0298] [Figure 41B] This graph shows the diabetes-free survival rate of recipient NSG mice after co-transfer of islet-specific Teffs and the indicated cell populations. Data are the combined results of two independent experiments. ****, P ≤ 0.0001. P values ​​were calculated using the log-rank test (Mantel-Cox test) for comparisons of BDC2.5 tTreg or EngTreg groups with mock-edited controls.

[0299] [Figure 41C]The left panel shows representative flow cytometry plots of lymphocytes isolated from the pancreas of non-diabetic NSG recipient mice 49 days after injection of BDC2.5 CD4 Teffs. The top graph shows data from BDC2.5 tTreg recipient mice, while the bottom graph shows data from BDC2.5 EngTreg recipient mice. Pre-determined gates are indicated at the top of each flow cytometry plot. The right panel shows histograms of FOXP3 expression in each gate (color-coded) for each flow cytometry plot.

[0300] [Figure 41D] Representative flow cytometry plots showing LNGFR expression in CD4 T cells (polyclonal; top row) from NOD mice and CD4 T cells (islet-specific; bottom row) from NOD BDC2.5 mice gene-edited using the methods indicated in each plot (top of box).

[0301] [Figure 41E] This graph shows the diabetes-free survival rate in recipient NSG mice after transfer of mock-edited cells, polyclonal EngTreg cells, polyclonal tTreg cells, islet-specific EngTreg cells, or islet-specific tTreg cells co-transferred with islet-specific Teff cells. Data from two independent experiments are pooled. ****P ≤ 0.0001. P values ​​were calculated using the log-rank test (Mantel-Cox test) for comparisons of BDC2.5 tTregs or BDC2.5 EngTregs with polyclonal tTregs or polyclonal EngTregs, respectively. All flow cytometry plots are representative of results from at least two independent experiments.

[0302] [Figure 41F] 1 shows an experimental scheme for diabetes prevention research using diabetogenic NOD splenocytes.

[0303] [Figure 41G] This graph shows the diabetes-free survival rate of recipient NSG mice after injection of diabetogenic NOD Teffs with or without cotransfer of BDC2.5 EngTregs. Data from one experiment are shown. **, P ≤ 0.005. Comparison of BDC2.5 EngTregs vs. recipients of diabetogenic NOD Teffs alone was calculated using the log-rank test (Mantel-Cox test).

[0304] [Figure 41H] Representative histological images are shown, showing H&E staining (left panel), anti-CD3 staining (middle panel), and insulin staining (right panel) of a representative pancreatic islet. Results are shown for NSG mice treated with diabetogenic NOD splenocytes alone (top panel: mouse tissue removed at the time of euthanasia for diabetes) and NSG mice co-delivered with diabetogenic NOD splenocytes and BDC2.5 EngTregs (middle panel: mouse 6 survived until the end of the study without developing hyperglycemia), compared with age-matched untreated control NSG mice (mouse 22, bottom panel: removed at the end of the study). All images are at 20x magnification, and the marker in the image indicates 80 µm.

[0305] [Figure 41I]A summary of histological findings is shown below. Histological examinations were performed on two mice selected from each experimental treatment group indicated in the figure. L1 and L2 represent step-section specimens prepared from the same tissue block. The severity of lymphocytic insulitis was assessed for all islets in each H&E-stained section by determining the accumulation of lymphocytes within and / or around the islets. Individual islets in each pair of sections were then assigned a severity grade (normal to severe insulitis). The severity of insulitis is shown in the table, along with the area (mm²) of each islet per total area of ​​the pancreatic section (columns 3–6). Separate tissue sections from these specimens were also evaluated for the presence of insulin by immunohistochemistry (IHC). The total number of positively stained islets in each section is shown in the column labeled "Insulin-Positive Islets by IHC" (column 7). Because the area of ​​each section varied, the number of islets in each mouse and section was normalized by expressing the total number of islets assigned to each category as the number of islets per mm2 of pancreatic tissue section. Inflammation in the pancreatic interstitium was assessed using anti-CD3 stained sections and graded on a scale of 0 (normal) to 3+ (relative to other sections) (column 8). DETAILED DESCRIPTION OF THE INVENTION

[0306] Aspects of the present disclosure relate to methods and compositions for producing recombinant Treg cells that (i) have stable suppressive function, e.g., by stabilizing expression of FoxP3, (ii) have specificity for type 1 diabetes (T1D)-associated antigens, and (iii) exhibit IL-2-like signaling in the presence of rapamycin. This embodiment relates to the insertion of two nucleic acids into target loci in a cell's genome. The first nucleic acid inserted into the TRAC locus encodes, under the control of a strong constitutive promoter (e.g., the MND promoter), (a) a first component of a heterodimerizable protein complex that provides intracellular IL-2 signaling in the presence of rapamycin, comprising an extracellular FK506-binding protein 12 (FKBP) domain covalently linked to the transmembrane and cytoplasmic domains of IL-2Rγ; (b) a TCR β chain of a TCR specific for IGRP, a T1D-associated peptide; and (c) a constant region of endogenous TCR α in frame with the TCR β chain, such that an IGRP-specific TCR formed by the combination of the TCR α chain and the TCR β chain is expressed from the TRAC locus. The second nucleic acid inserted downstream of the Treg-specific demethylation region of the FOXP3 locus encodes, under the control of a strong constitutive promoter (e.g., the MND promoter), (a) a second component of the heterodimerizable protein complex for intracellular IL-2 signaling, comprising an extracellular FKBP-rapamycin-binding (FRB) domain covalently linked to the transmembrane and cytoplasmic domains of IL-2Rβ, (b) a soluble FRB domain for adsorbing intracellular rapamycin to limit its inhibition of mTOR, and (c) at least a portion of the endogenous first coding exon of FOXP3, thereby enabling FoxP3 expression to be regulated by the inserted promoter independently of TSDR regulation and the endogenous promoter. Thus, the dual-edited cells described herein are T1D-associated antigen-specific Tregs, retain a stable suppressive phenotype in inflammatory environments (e.g., the inflamed pancreas), and can be controllably expanded in the presence of rapamycin.

[0307] Methods for producing genetically modified cells Some embodiments of the present disclosure relate to methods for producing a genetically modified cell by introducing two nucleic acids into a cell, one of which is homologous to the TRAC locus of the cell and the other of which is homologous to the FOXP3 locus of the cell, thereby editing both loci by inserting each nucleic acid into each locus. The first nucleic acid targeting the TRAC locus comprises 5' and 3' homology arms that provide directionality for inserting the nucleic acid into the TRAC locus (e.g., by cleaving the DNA sequence at the TRAC locus with a nuclease followed by homology-directed repair (HDR)). The second nucleic acid targeting the FOXP3 locus comprises 5' and 3' homology arms that provide directionality for inserting the nucleic acid into the FOXP3 locus (e.g., by cleaving the DNA sequence at the FOXP3 locus with a nuclease followed by homology-directed repair (HDR)). By inserting each of the two nucleic acids into a separate locus in the cell, a double-edited cell (i.e., a cell in which each nucleic acid has been inserted into two different loci) is obtained.

[0308] In an embodiment of the methods described herein, the nucleic acid targeted for insertion into the TRAC locus is (i) a nucleotide sequence encoding a first chemically-induced signaling complex (CISC) component comprising: (a) an extracellular binding domain comprising, or derived from, FK506-binding protein 12 (FKBP); (b) a transmembrane domain comprising, or derived from, the transmembrane domain of IL-2Rγ; and (c) an intracellular signaling domain comprising, or derived from the cytoplasmic domain of IL-2Rγ; (ii) a nucleotide sequence encoding a full-length TCR β chain; and (iii) a nucleotide sequence encoding at least a portion of a TCR α chain The promoter is operably linked to the The nucleotide sequence encoding the heterologous TCR α is inserted in frame with an endogenous sequence encoding a portion of the endogenous TCR α (e.g., the constant domain of the TCR α), and the expressed mRNA is translated to produce a TCR α chain that associates with the heterologous TCR β chain to form a TCR. Because the antigen-binding region of the TCR α chain is encoded by the inserted nucleic acid, the specificity of the TCR is determined by the inserted nucleic acid. In the methods described herein, the TCR encoded by the inserted nucleic acid binds to a T1D-associated antigen. By inserting each sequence and a promoter into the TRAC locus, transcription of each sequence operably linked to the promoter is initiated (thereby promoting expression) by the promoter, and a T1D-associated antigen-specific TCR formed by the heterologous TCR β chain and the heterologous TCR α chain containing a portion of the heterologous sequence encoded by the inserted nucleic acid and an FKBP-IL2Rγ CISC component are expressed from the TRAC locus.

[0309] In an embodiment of the methods described herein, the nucleic acid targeted for insertion into the FOXP3 locus is (i) a nucleotide sequence encoding a first chemically-induced signaling complex (CISC) component comprising: (a) an extracellular binding domain comprising the FKBP-rapamycin binding (FRB) domain of mTOR or an extracellular binding domain derived from the FKBP-rapamycin binding (FRB) domain of mTOR; (b) a transmembrane domain comprising the transmembrane domain of IL-2Rβ or a transmembrane domain derived from the transmembrane domain of IL-2Rβ; and (c) an intracellular signaling domain comprising the cytoplasmic domain of IL-2Rβ or an intracellular signaling domain derived from the cytoplasmic domain of IL-2Rβ; (ii) a nucleotide sequence encoding a cytosolic FRB domain without a transmembrane domain; and (iii) a 3' homology arm having homology to a sequence downstream of the Treg-specific demethylation region in the FOXP3 locus (e.g., a 3' homology arm having homology to a sequence contained within 2,000 nucleotides upstream of exon 2, the first coding exon of the FOXP3 gene, or a sequence up to this 2,000 nucleotides); The promoter is operably linked to the By inserting each sequence and promoter downstream of the TSDR, which destabilizes FOXP3 expression under inflammatory conditions, the inserted promoter can initiate transcription of mRNA encoding FoxP3, independently of the endogenous FOXP3 promoter upstream of the TSDR. By inserting each sequence and promoter into the FOXP3 locus, transcription of each sequence operably linked to the promoter is initiated by this promoter, resulting in expression of the FRB-Il2Rβ CISC component, the cytosolic FRB component, and FoxP3 from the FOXP3 locus.

[0310] Cells dual-edited by inserting one of the two nucleic acids into the TRAC locus and the other into the FOXP3 locus contain (i) a first CISC component and a second CISC component that heterodimerize in the presence of rapamycin to transduce IL-2R signaling via dimerization of the cytoplasmic domain of IL-2Rβ and the intracellular domain of IL-2Rγ; (ii) a cytosolic FRB domain that binds to intracellular rapamycin and prevents its interaction with mTOR; (iii) FoxP3, which confers a stable Treg phenotype; and (iv) stably express a T1D-associated antigen-specific TCR. Thus, the methods described herein provide stable Treg cells with specificity for T1D-associated antigens, which can be induced to proliferate using rapamycin. Furthermore, because the nucleotide sequences encoding the first and second CISC components are separated into separate nucleic acids, rapamycin can be used to selectively induce the proliferation of cells expressing both CISC components (i.e., cells expressing a T1D antigen-specific TCR and FoxP3 due to the insertion of both nucleic acids). Thus, dual-edited cells can be readily selected and expanded in vitro to produce stable Treg cell populations specific for T1D-associated antigens for the treatment of type 1 diabetes. Furthermore, the in vivo engraftment and expansion of such stable Treg cells may be supported by administering rapamycin to a subject.

[0311] promoter Each nucleic acid for targeted insertion into a cell genome according to the methods described herein comprises a promoter operably linked to one or more nucleotide sequences on the respective nucleic acid. A promoter is "operably linked" to a sequence if it is capable of initiating transcription of the operably linked sequence (e.g., by recruiting RNA polymerase). The promoters of the first and second nucleic acids can be any promoter known in the art. In some embodiments, a heterologous promoter on the introduced nucleic acid is active and promotes RNA transcription even under inflammatory conditions. In some embodiments, the promoter is a constitutive promoter. A constitutive promoter can be a strong promoter that promotes transcription more efficiently than an endogenous promoter, or a weak promoter that promotes transcription less efficiently than a strong promoter or an endogenous promoter. In some embodiments, the constitutive promoter is a strong promoter. In some embodiments, the heterologous promoter is an inducible promoter. An inducible promoter promotes transcription of an operably linked sequence in response to the presence of an activating signal or the absence of an inhibitory signal. In some embodiments, the inducible promoter is inducible by a drug or steroid.

[0312] In some embodiments, the promoter of the first nucleic acid and the promoter of the second nucleic acid delivered to the cell are different promoters. In another embodiment, the first nucleic acid and the second nucleic acid comprise the same promoter. In some embodiments, both the first nucleic acid and the second nucleic acid comprise an MND promoter. In embodiments in which the first nucleic acid and the second nucleic acid comprise the same promoter, the promoter sequences of these nucleic acids may be the same. Alternatively, the promoter sequence of the first nucleic acid may comprise one or more mutations (e.g., insertions, deletions, substitutions) compared to the promoter sequence of the second nucleic acid. In some embodiments, the MND promoter of the first nucleic acid and / or the second nucleic acid has at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO: 220. In some embodiments, the MND promoter of the first nucleic acid and / or the second nucleic acid has at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 220. In some embodiments, the first nucleic acid and the second nucleic acid each comprise an MND promoter having the nucleic acid sequence of SEQ ID NO: 220.

[0313] In some embodiments, a stop codon is present upstream of or within the first five nucleotides of the promoter on the first nucleic acid for insertion into the TRAC locus. In some embodiments, a stop codon is present upstream of or within the first five nucleotides of the promoter on the second nucleic acid for insertion into the FOXP3 locus. The presence of a stop codon upstream of, within, or overlapping the first five nucleotides of the promoter is expected to terminate translation of mRNA that may be transcribed from upstream of the endogenous promoter of the recombined TRAC locus or FOXP3 locus, thereby suppressing expression of the inserted coding sequence (e.g., a sequence encoding a CISC component, a heterologous TCR β chain or TCR α chain, or FoxP3) under the control of the endogenous promoter. In some embodiments, the stop codon is in frame with one or more upstream start codons, such that mRNA produced by transcription from the upstream endogenous promoter is not translated beyond the stop codon.

[0314] Chemical-inducible signaling complex (CISC) In embodiments of the methods for producing genetically engineered cells described herein, each nucleic acid inserted into the cell genome comprises a nucleotide sequence encoding a component of a chemically-induced signaling complex (CISC), and each CISC component comprises an extracellular domain that binds rapamycin, a transmembrane domain, and an intracellular domain that comprises, or is derived from, the cytoplasmic domain of the interleukin-2 receptor (IL-2R). In some embodiments, the first nucleic acid (for insertion into the TRAC locus) encodes a first CISC component comprising: (i) an extracellular binding domain comprising an FK506-binding protein 12 (FKBP) domain; (ii) a transmembrane domain comprising or derived from the transmembrane domain of IL-2Rγ; and (iii) an intracellular domain comprising or derived from the cytoplasmic domain of IL-2Rγ; and the second nucleic acid (for insertion into the FOXP3 locus) encodes a first CISC component comprising: (i) an extracellular binding domain comprising an FKBP-rapamycin binding domain; (ii) a transmembrane domain comprising or derived from the transmembrane domain of IL-2Rβ; and (iii) an intracellular domain comprising or derived from the cytoplasmic domain of IL-2Rβ. A domain of a CISC component (e.g., the transmembrane domain of a first CISC component) is "derived from" a particular domain of an IL-2R polypeptide (e.g., IL-2Rγ) if it has at least 90% sequence identity with the wild-type (naturally occurring) amino acid sequence of that domain of that IL-2R polypeptide (e.g., the transmembrane domain of native IL-2Rγ).

[0315] By expressing CISC components in cells and manipulating the presence, absence, and / or concentration of rapamycin, IL-2 signaling can be selectively induced in the cells. In this manner, controllably inducing signaling allows, for example, IL-2 signaling events to induce cell proliferation, thereby selectively expanding cells expressing both CISC components. In some embodiments, in which two nucleic acids encoding different CISC components are introduced into cells, contacting cells containing only one CISC component with rapamycin will not induce dimerization or IL-2 signaling due to the absence of the other CISC component, allowing for such selective expansion to select for cells containing both nucleic acids.

[0316] Examples of intracellular signaling domains include, but are not limited to, the IL-2Rβ cytoplasmic domain and the IL-2Rγ cytoplasmic domain and functional derivatives thereof. In some embodiments, the intracellular signaling domain of a first CISC component comprises an IL-2Rγ domain or a functional derivative thereof, and the intracellular signaling domain of a second CISC component comprises an IL-2Rβ cytoplasmic domain or a functional derivative thereof. In some embodiments, dimerization of the first CISC component and the second CISC component induces phosphorylation of JAK1, JAK3, and / or STAT5 in cells. In some embodiments, dimerization of the first CISC component and the second CISC component induces cell proliferation.

[0317] Examples of transmembrane domains include, but are not limited to, the transmembrane domain of IL-2Rβ and the transmembrane domain of IL-2Rγ, and functional derivatives thereof. In some embodiments, the transmembrane domain of a CISC component is derived from the same protein as the protein from which the intracellular signaling domain of that CISC component is derived (e.g., a CISC component comprising the intracellular domain of IL-2Rβ comprises the transmembrane domain of IL-2Rβ). In some embodiments, one CISC component comprises the transmembrane domain of IL-2Rβ, and the other CISC component comprises the transmembrane domain of IL-2Rγ.

[0318] Examples of extracellular binding domains capable of binding to rapamycin include, but are not limited to, an FK506-binding protein (FKBP) domain and an FKBP-rapamycin binding (FRB) domain. The FKBP domain and the FRB domain can bind to rapamycin and form a heterodimer, as described below. In some embodiments, the extracellular binding domain of one CISC component comprises an FKBP domain, and the extracellular binding domain of the other CISC component comprises an FRB domain. In some embodiments, these CISC components form a heterodimer in the presence of rapamycin. In some embodiments, the FRB domain contains a threonine at the position corresponding to amino acid 2098 of wild-type mTOR having the amino acid sequence of SEQ ID NO: 236. This amino acid mutation increases the affinity of mTOR for compounds structurally related to rapamycin, but decreases the affinity of mTOR for rapamycin itself. Therefore, the presence of a threonine at this position maintains the binding ability of mTOR to rapamycin. The amino acid in a CISC component or FRB domain that "corresponds to" amino acid 2098 of wild-type mTOR may be determined by aligning a candidate sequence for a CISC component or FRB domain to SEQ ID NO:236 (e.g., by BLAST alignment algorithm or another alignment algorithm known in the art), and the amino acid that aligns to amino acid 2098 of SEQ ID NO:236 is the amino acid that "corresponds to" amino acid 2098 of SEQ ID NO:236.

[0319] The extracellular binding domain, transmembrane domain, and intracellular signaling domain of a CISC component described herein may each be connected to another domain of that CISC component via a linker. Linkers are known in the art. In some embodiments, the linker comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids (e.g., glycine), a number of amino acids (e.g., glycine), or a range between any two of these numbers. In some embodiments, the glycine spacer comprises at least three glycines. In some embodiments, the glycine spacer comprises a sequence set forth as GSG, GGGS (SEQ ID NO: 229), GGGSGGG (SEQ ID NO: 230), or GGG. In some embodiments, the glycine spacer comprises the amino acid sequence GSG.

[0320] The extracellular binding domain may be connected to the transmembrane domain via a hinge domain. "Hinge" refers to a domain that links the extracellular binding domain to the transmembrane domain and may thereby provide flexibility to the extracellular binding domain. In some embodiments, the hinge domain positions the extracellular binding domain in the vicinity of the cell membrane, minimizing the possibility of recognition by an antibody or binding fragment thereof. In some embodiments, the extracellular binding domain is located N-terminal to the hinge domain. In some embodiments, the hinge domain may be natural or synthetic.

[0321] In some embodiments, the first and second CISC components form a heterodimer in the presence of rapamycin. In some embodiments, the first and second CISC components form a heterodimer in the presence of a compound produced in vivo by metabolism of a rapalog. In some embodiments, the compound produced in vivo by metabolism of a rapalog is rapamycin. Examples of rapalogs include, but are not limited to, everolimus, CCI-779, C20-methallylrapamycin, C16-(S)-3-methylindolerapamycin, C16-iRap, C16-(S)-7-methylindolerapamycin, AP21967, C16-(S)-butylsulfonamidorapamycin, AP23050, mycophenolate sodium, benidipine hydrochloride, AP1903, and AP23573, as well as metabolites and derivatives thereof.

[0322] In some embodiments, the nucleic acid encoding the second CISC component (FRB-IL2Rβ) further comprises a nucleotide sequence encoding a third CISC component capable of binding rapamycin. Such a CISC component is useful, for example, for binding intracellular rapamycin, thereby preventing rapamycin bound to the CISC component from interacting with other intracellular molecules or structures (e.g., preventing rapamycin from interacting with mTOR). In some embodiments, the third CISC component is a soluble protein that does not contain a transmembrane domain. In some embodiments, the third CISC component comprises an intracellular FRB domain. In some embodiments, the third CISC component is a soluble protein that contains an FRB domain but not a transmembrane domain.

[0323] The nucleic acid encoding the first CISC component, the nucleic acid encoding the second CISC component, and / or the nucleic acid encoding the third CISC component may be contained in one or more vectors. In some embodiments, the nucleic acid encoding the first CISC component is contained in a vector separate from the vector containing the nucleic acid encoding the second CISC component. In some embodiments, the nucleic acid encoding the third CISC component is contained in the same vector as the vector containing the nucleic acid encoding the second CISC component. In some embodiments, the one or more vectors are viral vectors. In some embodiments, the one or more vectors are adeno-associated virus (AAV) vectors. In some embodiments, the one or more AAV vectors are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11 vectors. In some embodiments, the one or more AAV vectors are AAV5 vectors. In some embodiments, the one or more AAV vectors are AAV6 vectors.

[0324] In some embodiments, the CISC component comprises an amino acid sequence having at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or up to 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 66 or 71. In some embodiments, one or more CISC components further comprise a signal peptide. The signal peptide may be any signal peptide known in the art that transports the translated CISC component to the cell membrane. In some embodiments, both the first CISC component and the second CISC component comprise the signal peptide of LCN2. In some embodiments, both the first CISC component and the second CISC component comprise a signal peptide comprising the amino acid sequence of SEQ ID NO: 61.

[0325] In some embodiments, one CISC component comprises an amino acid sequence having at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or up to 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 66, and the other CISC component comprises an amino acid sequence having at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or up to 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 71. In some embodiments, each CISC component further comprises a signal peptide, which may have the same or different amino acid sequences. These signal peptides may be any signal peptide known in the art that transports the translated CISC component to the cell membrane.

[0326] In some embodiments, the third CISC component comprises an amino acid sequence having at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or up to 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 72. In some embodiments, the third CISC component consists of an amino acid sequence having at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or up to 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 72. In some embodiments, the third CISC component comprises the amino acid sequence of SEQ ID NO: 72. In some embodiments, the third CISC component consists of the amino acid sequence of SEQ ID NO: 72. In some embodiments, the third CISC component does not comprise a signal peptide. In some embodiments, the third CISC component does not include a transmembrane domain.

[0327] T cell receptor (TCR) In some embodiments of the methods described herein, the TRAC locus of a cell is edited by inserting a nucleic acid comprising a promoter operably linked to a nucleotide sequence encoding a full-length TCRβ protein and a nucleotide sequence encoding at least a portion of a TCRα protein, e.g., the TCRα variable region and TCRα junction (TRAJ) region that form the portion of the TCRα protein responsible for antigen specificity. In some embodiments, the nucleotide sequence encoding the TCRα variable and junction regions is inserted in frame with an endogenous nucleotide sequence encoding a portion of the TCRα constant domain, such that the inserted heterologous promoter initiates transcription of the sequence encoding the heterologous TCRβ protein and the sequence encoding the TCRα protein comprising the heterologous TRAV / TRAJ amino acid sequence and the endogenous TCRα constant domain. This embodiment utilizes the endogenous 3' regulatory region of the endogenous TRAC locus.

[0328] The genetically modified cells produced by the methods described herein express a T cell receptor specific for type 1 diabetes (T1D). 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, e.g., Janeway et al., Immunobiology: The Immune System in Health and Disease, 3rd Ed., Current Biology Publications, p. 433, 1997. TCRs can specifically bind to antigenic 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 the α, β, γ, and δ chains has a constant (C) domain and a highly variable (V) domain, each containing three complementarity-determining regions (CDRs) that are largely responsible for the TCR's recognition and binding to specific antigens. In certain embodiments, nucleic acids 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. See, e.g., Scholten et al., Clin Immunol. 2006. 119:135.

[0329] The extracellular domains of TCR chains (e.g., TCRα chain and TCRβ chain), like other antigen-binding members of the immunoglobulin superfamily (e.g., 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.

[0330] "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 Vα domain or Vβ domain of a TCR that binds to a specific antigen may be used to screen a library of complementarity-determining regions of Vα domains or Vβ domains, respectively, to isolate TCRs that bind to said specific antigen.

[0331] The terms "complementarity determining region" and "CDR" are used interchangeably with "hypervariable region" or "HVR" and are known in the art to refer to amino acid sequences within immunoglobulin (e.g., TCR) variable regions that confer 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, and αCDR3) in each TCR α chain variable region, and three CDRs (βCDR1, βCDR2, and βCDR3) in each TCR β chain variable region. In TCRs, the primary CDR responsible for recognizing peptide antigens bound to MHC is considered to be CDR3. Typically, CDR1 and CDR2 primarily interact with MHC, or only CDR1 and CDR2 interact with MHC.

[0332] CDR1 and CDR2 are encoded within the variable gene segments of the sequence encoding the variable domain 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 segments (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.

[0333] 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).

[0334] In some embodiments, the nucleic acids described herein encode at least a portion of a TCR β chain and a TCR α chain, which are expressed in combination to form a T1D2 TCR that binds to the IGRP(305-234) peptide. In another embodiment, the full-length TCR α chain and TCR β chain, portions of which are encoded by the nucleic acids described herein, form a T1D4 TCR that binds to the IGRP(241-260) peptide. In another embodiment, the full-length TCR α chain and TCR β chain, portions of which are encoded by the nucleic acids described herein, form a T1D5-1 TCR that binds to the IGRP(305-324) peptide. In some embodiments, the IGRP(305-324) peptide is recognized when bound to HLA-DRB1*0401. In some embodiments, the IGRP(241-260) peptide is recognized when bound to HLA-DRB1*0401.

[0335] In some embodiments, the TCR formed from (at least a portion of) a TCR β chain and a TCR α chain encoded by the nucleic acid described herein comprises a TCR α variable (Vα) domain having three complementarity determining regions (CDRs), αCDR1, αCDR2, and αCDR3, and a TCR β variable (Vβ) domain having three CDRs, βCDR1, βCDR2, and βCDR3. Representative amino acids of the CDRs of the TCRs described herein are shown in Table 1, and nucleotide sequences encoding the CDRs of the TCRs described herein are shown in Table 2. In some embodiments, (i) αCDR1 comprises SEQ ID NO: 1, (ii) αCDR2 comprises SEQ ID NO: 2, (iii) αCDR3 comprises SEQ ID NO: 3, (iv) βCDR1 comprises SEQ ID NO: 4, (v) βCDR2 comprises SEQ ID NO: 5, and (vi) βCDR3 comprises SEQ ID NO: 6. In some embodiments, (i) αCDR1 comprises SEQ ID NO: 11, (ii) αCDR2 comprises SEQ ID NO: 12, (iii) αCDR3 comprises SEQ ID NO: 13, (iv) βCDR1 comprises SEQ ID NO: 14, (v) βCDR2 comprises SEQ ID NO: 15, and (vi) βCDR3 comprises SEQ ID NO: 16. In some embodiments, (i) αCDR1 comprises SEQ ID NO: 21, (ii) αCDR2 comprises SEQ ID NO: 22, (iii) αCDR3 comprises SEQ ID NO: 23, (iv) βCDR1 comprises SEQ ID NO: 24, (v) βCDR2 comprises SEQ ID NO: 25, and (vi) βCDR3 comprises SEQ ID NO: 26. In another embodiment, each set consisting of αCDR1, αCDR2, αCDR3, βCDR1, βCDR2 and βCDR3 may have an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with each amino acid sequence shown in any of the above amino acid sequence combinations.

[0336] In some embodiments, Vα comprises SEQ ID NO: 7 and Vβ comprises SEQ ID NO: 8. In some embodiments, Vα comprises SEQ ID NO: 17 and Vβ comprises SEQ ID NO: 18. In some embodiments, Vα comprises SEQ ID NO: 27 and Vβ comprises SEQ ID NO: 28. In other embodiments, each pair of Vα and Vβ may have an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a respective amino acid sequence set forth in any of the above amino acid sequence combinations.

[0337] In some embodiments, the TCR alpha chain comprises SEQ ID NO: 9 and the TCR beta chain comprises SEQ ID NO: 10. In some embodiments, the TCR alpha chain comprises SEQ ID NO: 19 and the TCR beta chain comprises SEQ ID NO: 20. In some embodiments, the TCR alpha chain comprises SEQ ID NO: 29 and the TCR beta chain comprises SEQ ID NO: 30. In another embodiment, each pair of TCR alpha and TCR beta chains may have an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to each amino acid sequence set forth in any of the above amino acid sequence combinations.

[0338] Recombination at the FOXP3 locus In some embodiments of the methods described herein, the FOXP3 locus of a cell is edited by inserting a nucleic acid comprising a promoter operably linked to a nucleotide sequence encoding a portion of the endogenous FoxP3 protein. The inserted promoter is introduced into the genome downstream of the Treg-specific demethylation region (TSDR) of the FOXP3 locus. In unmodified cells, the TSDR epigenetically regulates FoxP3 expression, thereby suppressing FoxP3 production in cells exposed to inflammatory conditions. As a result, FoxP3 expression is lost and unmodified Treg cells are converted to a T effector (Teff) phenotype. By inserting a promoter downstream of the TSDR, TSDR-mediated regulation of FOXP3 expression is bypassed, allowing stable production of FoxP3 even under inflammatory conditions.

[0339] The heterologous promoter can be inserted anywhere downstream of the endogenous promoter of the FOXP3 coding sequence (e.g., downstream of the TSDR) and upstream of or within the first coding exon (known in the art as exon 2 because it is the second exon present in the pre-mRNA transcribed from the endogenous FOXP3 promoter, and known as the first coding exon because it is not exon 1 (the first exon in the pre-mRNA encoding FOXP3), which contains the start codon that initiates translation of wild-type FoxP3). In some embodiments, the heterologous promoter is 1 to 10,000 nucleotides downstream, 10 to 1,000 nucleotides downstream, 10 to 100 nucleotides downstream, 10 to 5,000 nucleotides downstream, 20 to 4,000 nucleotides downstream, 30 to 3,000 nucleotides downstream, 40 to 2,000 nucleotides downstream, 50 to 1,000 nucleotides downstream, 60 to 750 nucleotides downstream, 70 to 500 nucleotides downstream, 80 to 400 nucleotides downstream, 90 to 300 nucleotides downstream of the TSDR of the FOXP3 locus. The insertion may be 100 to 200 nucleotides downstream, 1 to 1,000 nucleotides downstream, 1,000 to 2,000 nucleotides downstream, 2,000 to 3,000 nucleotides downstream, 3,000 to 4,000 nucleotides downstream, 4,000 to 5,000 nucleotides downstream, 5,000 to 6,000 nucleotides downstream, 6,000 to 7,000 nucleotides downstream, 7,000 to 8,000 nucleotides downstream, 8,000 to 9,000 nucleotides downstream, or 9,000 to 10,000 nucleotides downstream.In some embodiments, the heterologous promoter is located between 1 and 10,000 nucleotides upstream, between 10 and 1,000 nucleotides upstream, between 100 nucleotides upstream, between 10 and 5,000 nucleotides upstream, between 20 and 4,000 nucleotides upstream, between 30 and 3,000 nucleotides upstream, between 40 and 2,000 nucleotides upstream, between 50 and 1,000 nucleotides upstream, between 60 and 750 nucleotides upstream, between 70 and 500 nucleotides upstream, between 80 and 400 nucleotides upstream, between 90 and 1000 nucleotides upstream, between 10 and 1500 nucleotides upstream, between 15 and 20 ... In some embodiments, the heterologous promoter is inserted between 100 and 200 nucleotides upstream, 1 and 1,000 nucleotides upstream, 1,000 and 2,000 nucleotides upstream, 2,000 and 3,000 nucleotides upstream, 3,000 and 4,000 nucleotides upstream, 4,000 and 5,000 nucleotides upstream, 5,000 and 6,000 nucleotides upstream, 6,000 and 7,000 nucleotides upstream, 7,000 and 8,000 nucleotides upstream, 8,000 and 9,000 nucleotides upstream, or 9,000 and 10,000 nucleotides upstream. In some embodiments, the heterologous promoter is inserted between the first coding exon to create a synthetic first coding exon that is distinct from the endogenous first coding exon but contains an initiation codon in-frame with the FOXP3 sequence encoding the downstream FOXP3 exon.

[0340] 2A motif and linker Some embodiments of the nucleic acids described herein that encode multiple polypeptides or portions thereof can include an intervening nucleotide sequence encoding a 2A motif. 2A motifs are known in the art and are useful for translating and facilitating the production of multiple polypeptides from a single nucleotide sequence. See, e.g., Kim et al., PLoS ONE. 2011. 6:e18556. In some embodiments, the 2A motif is translated, which results in self-cleavage of the polypeptide, releasing the separate polypeptides. In another embodiment, the presence of a nucleotide sequence encoding a 2A motif allows the ribosome to skip over the amino acids of the encoded 2A motif as it translates along the mRNA, thereby releasing a first polypeptide (e.g., a first FKBP-IL2Rγ CISC component) and initiating translation of a second polypeptide (e.g., a TCR β chain).

[0341] In some embodiments, in each pair of nucleic acid sequences, the nucleotide sequence encoding the 2A motif is positioned in frame between (i) the nucleotide sequence encoding the first (FKBP-IL2Rγ) CISC component, (ii) the nucleotide sequence encoding the TCR β chain, and (iii) the nucleotide sequence encoding the TCR α chain or a portion thereof. Thus, when a heterologous promoter (e.g., the MND promoter) initiates transcription of a single mRNA encoding each of the CISC components, the TCR β chain, and the TCR α chain, and containing an intervening 2A motif, the CISC components, the TCR β chain, and the TCR α chain are each produced as separate polypeptides. In some embodiments, in each pair of nucleic acid sequences, the nucleotide sequence encoding the 2A motif is positioned in frame between (i) the nucleotide sequence encoding the second (FKBP-IL2Rγ) CISC component, (ii) the nucleotide sequence encoding the cytosolic FRB domain, and (iii) the nucleotide sequence encoding FoxP3. Thus, when a heterologous promoter (e.g., the MND promoter) initiates transcription of a single mRNA encoding each of the CISC components, the cytosolic FRB domain, and FoxP3, and containing an intervening 2A motif, the CISC components, the cytosolic FRB domain, and FoxP3 are each produced as separate polypeptides.

[0342] In each pair of nucleic acid sequences, the 2A motif encoded by the nucleotide sequence located between the nucleotide sequences encoding two polypeptides (e.g., between the sequence encoding the FKBP-IL2Rγ CISC component and the sequence encoding the TCR β chain; and between the sequence encoding the TCR β chain and the sequence encoding a portion of the TCR α chain) may be any 2A motif known in the art. In some embodiments, the 2A motifs encoded between the nucleotide sequences encoding separate polypeptides in each pair of nucleic acid sequences may be independently selected from the group consisting of F2A, P2A, T2A, and E2A. In some embodiments, the first and second 2A motifs encoded on a single nucleic acid are different types of 2A motifs. Using different types of 2A motifs in a single inserted nucleic acid can reduce the probability of internal recombination. If internal recombination occurs, the nucleotide sequence between the recombined 2A motifs may be excised from the chromosome. In some embodiments, the nucleotide sequence encoding the first 2A motif on a single nucleic acid has 90% or less sequence identity with the nucleotide sequence encoding the second 2A motif on that nucleic acid. In some embodiments, the nucleotide sequence encoding a first 2A motif on a nucleic acid has 80% or less sequence identity with the nucleotide sequence encoding a second 2A motif on that nucleic acid. In some embodiments, the nucleotide sequence encoding a first 2A motif on a nucleic acid has 70% or less sequence identity with the nucleotide sequence encoding a second 2A motif on that nucleic acid. In some embodiments, the nucleotide sequence encoding a first 2A motif on a nucleic acid has 60% or less sequence identity with the nucleotide sequence encoding a second 2A motif on that nucleic acid. In some embodiments, the nucleotide sequence encoding a first 2A motif on a nucleic acid has 50% or less sequence identity with the nucleotide sequence encoding a second 2A motif on that nucleic acid. In some embodiments, the first 2A motif is a T2A motif and the second motif is a P2A motif.

[0343] In another embodiment, the first and second 2A motifs encoded by the nucleotide sequences on a single nucleic acid are the same type of 2A motif. In some embodiments, a single nucleic acid comprises a nucleotide sequence encoding a first P2A motif and a second nucleotide sequence encoding a second P2A motif, and the nucleotide sequence encoding the first P2A motif has at least 80% sequence identity with the nucleotide sequence encoding the second P2A motif. In some embodiments, the first nucleotide sequence encoding the first P2A motif and the second nucleotide sequence encoding the second P2A motif comprise the same nucleotide sequence.

[0344] In some embodiments, the nucleic acid for insertion into the TRAC locus (i) comprises a sequence encoding a T2A motif between the sequence encoding the first CISC component and the sequence encoding the TCR beta chain, and (ii) comprises a sequence encoding a P2A motif between the sequence encoding the TCR beta chain and the sequence encoding a portion of the heterologous TCR alpha chain.

[0345] In some embodiments, the nucleic acid for insertion into the FOXP3 locus comprises (i) a sequence encoding a P2A motif between the sequence encoding the second CISC component and the sequence encoding the cytosolic FRB domain, and (ii) a second sequence encoding a second P2A motif between the sequence encoding the cytosolic FRB domain and the sequence encoding FoxP3.

[0346] In some embodiments, the polypeptide (e.g., a CISC component and / or a TCR β chain) encoded by the nucleic acid for insertion into a cell genome comprises a C-terminal linker. Incorporation of a C-terminal linker may, for example, improve cleavage efficiency at the 2A motif and / or prevent amino acids of the encoded CISC component or TCR β chain from being excised upon cleavage of the 2A motif. In some embodiments, the encoded first CISC component comprises a C-terminal linker. In some embodiments, the encoded second CISC component comprises a C-terminal linker. In some embodiments, the encoded cytosolic FRB domain component comprises a C-terminal linker. In some embodiments, the encoded TCR β chain comprises a C-terminal linker.

[0347] The C-terminal linker of the encoded polypeptide can be any linker known in the art. In some embodiments, the C-terminal linker comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids (e.g., glycine), a number of amino acids (e.g., glycine), or a range of amino acids between any two of these numbers. In some embodiments, the C-terminal linker comprises at least three glycines. In some embodiments, the C-terminal linker comprises the sequence GSG, GGGS (SEQ ID NO: 229), GGGSGGG (SEQ ID NO: 230), or GGG. In some embodiments, the C-terminal linker comprises the amino acid sequence GSG. In some embodiments, the first CISC component, the second CISC component, the cytosolic FRB domain, and the TCR β chain all comprise C-terminal linkers having the amino acid sequence GSG.

[0348] vector The first nucleic acid for insertion into the TRAC locus and / or the second nucleic acid for insertion into the FOXP3 locus can be contained in one or more vectors.In some embodiments, the first nucleic acid for targeting the TRAC locus is contained in a first vector, and the nucleic acid for targeting the FOXP3 locus is contained in a second vector.In some cases, these vectors are packaged in a virus that can infect cells (for example, the vector is a viral vector).Typical viruses include adenoviruses, retroviruses, lentiviruses, adeno-associated viruses, and other viruses known in the art and disclosed herein.

[0349] The term "vector" is used to refer to any molecule (e.g., nucleic acid or plasmid) or molecular arrangement (e.g., virus) used to transfer coding information into a host cell. An "expression vector" refers to a vector suitable for introduction into a host cell and containing a nucleic acid sequence that induces and / or controls the expression of an introduced heterologous nucleic acid sequence. Expression includes, but is not limited to, processes such as transcription, translation, and RNA splicing (if introns are present). Examples of vectors include, but are not limited to, artificial chromosomes, minigenes, cosmids, plasmids, phagemids, and viral vectors. Examples of viral vectors include, but are not limited to, lentiviral vectors, retroviral vectors, herpesvirus vectors, adenoviral vectors, and adeno-associated viral vectors. In some embodiments, one or more vectors containing a nucleic acid for use in the methods provided herein are lentiviral vectors. In some embodiments, the one or more vectors are adenoviral vectors. In some embodiments, the one or more vectors are adeno-associated viral (AAV) vectors. In some embodiments, the one or more AAV vectors are AAV1 vectors, AAV2 vectors, AAV3 vectors, AAV4 vectors, AAV5 vectors, AAV6 vectors, AAV7 vectors, AAV8 vectors, AAV9 vectors, AAV10 vectors, or AAV11 vectors. In some embodiments, the vector comprising a nucleic acid for insertion into the TRAC locus is an AAV1 vector, AAV2 vectors, AAV3 vectors, AAV4 vectors, AAV5 vectors, AAV6 vectors, AAV7 vectors, AAV8 vectors, AAV9 vectors, AAV10 vectors, or AAV11 vectors. In some embodiments, the vector comprising a nucleic acid for insertion into the FOXP3 locus is an AAV1 vector, AAV2 vectors, AAV3 vectors, AAV4 vectors, AAV5 vectors, AAV6 vectors, AAV7 vectors, AAV8 vectors, AAV9 vectors, AAV10 vectors, or AAV11 vectors.

[0350] In some embodiments, the one or more AAV vectors are AAV5 vectors. In some embodiments, the one or more AAV vectors are AAV6 vectors. In some embodiments, the first nucleic acid and the second nucleic acid are each contained in a separate AAV5 vector. In some embodiments, the first nucleic acid and the second nucleic acid are each contained in a separate AAV6 vector.

[0351] In some embodiments, a nucleic acid for insertion into the TRAC locus comprises a nucleotide sequence between the 5' and 3' homology arms that has at least 90% sequence identity to any of SEQ ID NOs: 94, 106, 117, 128, and 139. In some embodiments, the nucleotide sequence has at least 95% sequence identity to any of SEQ ID NOs: 94, 106, 117, 128, and 139. In some embodiments, the nucleotide sequence comprises any of SEQ ID NOs: 94, 106, 117, 128, and 139. In some embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 94. In some embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 106. In some embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 117. In some embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 128. In some embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 139.

[0352] In some embodiments, a nucleic acid for insertion into the TRAC locus has at least 90% sequence identity to the nucleotide sequence set forth in any of SEQ ID NOs: 95, 107, 118, 129, and 140. In some embodiments, the nucleic acid has at least 95% sequence identity to the nucleotide sequence set forth in any of SEQ ID NOs: 95, 107, 118, 129, and 140. In some embodiments, the nucleic acid comprises the nucleotide sequence set forth in any of SEQ ID NOs: 95, 107, 118, 129, and 140. In some embodiments, the nucleic acid has at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 95. In some embodiments, the nucleic acid has at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 107. In some embodiments, the nucleic acid has at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 118. In some embodiments, the nucleic acid has at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 129. In some embodiments, the nucleic acid has at least 95% sequence identity to the nucleotide sequence of SEQ ID NO:140.

[0353] In some embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 95. In some embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 107. In some embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 118. In some embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 129. In some embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 140.

[0354] In some embodiments, a nucleic acid for insertion into the FOXP3 locus comprises a nucleotide sequence between the 5' homology arm and the 3' homology arm that has at least 90% sequence identity to any of SEQ ID NOs: 150, 161, 172, 184, 195, 206, and 218. In some embodiments, the nucleotide sequence has at least 95% sequence identity to any of SEQ ID NOs: 150, 161, 172, 184, 195, 206, and 218. In some embodiments, the nucleotide sequence comprises any of SEQ ID NOs: 150, 161, 172, 184, 195, 206, and 218. In some embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 150. In some embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 161. In some embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 172. In some embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 184. In some embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 195. In some embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 206. In some embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 218.

[0355] In some embodiments, a nucleic acid for insertion into the FOXP3 locus has at least 90% sequence identity to the nucleotide sequence set forth in any of SEQ ID NOs: 151, 162, 173, 185, 196, 207, and 219. In some embodiments, the nucleic acid has at least 95% sequence identity to the nucleotide sequence set forth in any of SEQ ID NOs: 151, 162, 173, 185, 196, 207, and 219. In some embodiments, the nucleic acid comprises the nucleotide sequence set forth in any of SEQ ID NOs: 151, 162, 173, 185, 196, 207, and 219. In some embodiments, the nucleic acid has at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 151. In some embodiments, the nucleic acid has at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 162. In some embodiments, the nucleic acid has at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 173. In some embodiments, the nucleic acid has at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 185. In some embodiments, the nucleic acid has at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 196. In some embodiments, the nucleic acid has at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 207. In some embodiments, the nucleic acid has at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 219.

[0356] In some embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 151. In some embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 162. In some embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 173. In some embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 185. In some embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 196. In some embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 207. In some embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 219.

[0357] Homologous arms The nucleic acids for insertion into the TRAC locus and the FOXP3 locus in the methods described herein each contain a 5' homology arm and a 3' homology arm, respectively, that target insertion of the nucleic acid into the TRAC locus or the FOXP3 locus by homology-directed repair after the introduction of a double-strand break. When considering the coding strand of the nucleic acid (i.e., the nucleic acid strand containing the reading frame encoding a polypeptide containing a CISC component, a TCR chain, and FoxP3), the 5' homology arm typically refers to a homology arm at the 5' end of the nucleic acid, and the 3' homology arm typically refers to another homology arm at the 3' end of the nucleic acid. The 5' homology arm is homologous to a first sequence at the target locus, and the 3' homology arm is homologous to a second sequence downstream of the first sequence at the target locus, thereby inserting the nucleic acid into the locus in a targeted manner. After insertion of the nucleic acid, the modified target locus contains a 5' homologous arm and a 3' homologous arm in place of the first and second sequences, and the sequence that was present between the homologous arms on the nucleic acid in place of the sequence that was present between the first and second sequences at the target locus. The 5' homologous arm and the 3' homologous arm may be the same length, may be of similar length (within a 100 bp difference in length), or may be of different lengths. In some embodiments, the length of one or both of the 5' homologous arm and the 3' homologous arm is 100 to 2,000 bp, 200 to 2,000 bp, 400 to 1,500 bp, or 500 to 1,000 bp. In some embodiments, the length of one or both of the 5' homologous arm and the 3' homologous arm is about 100 bp, about 200 bp, about 300 bp, about 400 bp, about 500 bp, about 600 bp, about 700 bp, about 800 bp, about 900 bp, about 1,000 bp, about 1,100 bp, about 1,200 bp, about 1,300 bp, about 1,400 bp, about 1,500 bp, about 1,600 bp, about 1,700 bp, about 1,800 bp, about 1,900 bp, or about 2,000 bp. In some embodiments, the length of both homologous arms is 100 to 2,000 nucleotides. In some embodiments, the length of both homologous arms is 300 to 1,000 nucleotides. In some embodiments, the length of both homologous arms is 300 to 700 nucleotides.In some embodiments, the length of both homologous arms is 300 to 500 nucleotides, in some embodiments, the length of both homologous arms is 500 to 700 nucleotides, in some embodiments, the length of both homologous arms is 700 to 1,000 nucleotides.

[0358] The homologous arm of a nucleic acid for insertion into a targeted genomic locus may be selected based on homologous sequences located upstream and / or downstream of the target site cleaved by a nuclease at the target locus. For example, in some embodiments in which a nucleic acid is inserted by cleavage at a specific position (cleavage site) of the target locus followed by homologous recombination repair, the 5' homologous arm of the nucleic acid for insertion is homologous to a sequence upstream of the cleavage site, and the 3' homologous arm of the nucleic acid for insertion is homologous to a sequence downstream of the cleavage site. In some embodiments, the 5' homologous arm is homologous to a 100-2,000 nucleotide sequence terminating 25-5,000 nucleotides, 50-3,000 nucleotides, 75-2,000 nucleotides, 100-1,000 nucleotides, or 150-500 nucleotides upstream of the cleavage site. In some embodiments, the 5' homologous arm has homology to a 100-2,000 nucleotide sequence terminating 25-5,000 nucleotides, 50-3,000 nucleotides, 75-2,000 nucleotides, 100-1,000 nucleotides, or 150-500 nucleotides upstream of the PAM sequence cleaved by the RNA-guided nuclease. In some embodiments, the 5' homologous arm has homology to a 100-2,000 nucleotide sequence terminating 25-5,000 nucleotides, 50-3,000 nucleotides, 75-2,000 nucleotides, 100-1,000 nucleotides, or 150-500 nucleotides upstream of the genomic sequence complementary to the spacer sequence of the gRNA. In some embodiments, the 5' homologous arm has homology to a 100-2,000 nucleotide sequence terminating 150-500 nucleotides upstream of the cleavage site. In some embodiments, the 5' homologous arm has homology to a 100-2,000 nucleotide sequence that terminates 150-500 nucleotides upstream of the PAM sequence cleaved by the RNA-guided nuclease. In some embodiments, the 5' homologous arm has homology to a 100-2,000 nucleotide sequence that terminates 150-500 nucleotides upstream of the genomic sequence complementary to the spacer sequence of the gRNA.

[0359] In some embodiments, the 3' homologous arm has homology to a 100-2,000 nucleotide sequence that terminates 25-5,000 nucleotides, 50-3,000 nucleotides, 75-2,000 nucleotides, 100-1,000 nucleotides, or 150-500 nucleotides upstream of the cleavage site. In some embodiments, the 3' homologous arm has homology to a 100-2,000 nucleotide sequence that terminates 25-5,000 nucleotides, 50-3,000 nucleotides, 75-2,000 nucleotides, 100-1,000 nucleotides, or 150-500 nucleotides upstream of the PAM sequence cleaved by the RNA-guided nuclease. In some embodiments, the 3' homology arm has homology to a 100-2,000 nucleotide sequence that terminates 25-5,000 nucleotides, 50-3,000 nucleotides, 75-2,000 nucleotides, 100-1,000 nucleotides, or 150-500 nucleotides upstream of the genomic sequence complementary to the spacer sequence of the gRNA. In some embodiments, the 3' homology arm has homology to a 100-2,000 nucleotide sequence that terminates 150-500 nucleotides upstream of the cleavage site. In some embodiments, the 3' homology arm has homology to a 100-2,000 nucleotide sequence that terminates 150-500 nucleotides upstream of the PAM sequence that is cleaved by the RNA-guided nuclease. In some embodiments, the 3' homology arm has homology to a 100-2,000 nucleotide long sequence that terminates 150-500 nucleotides upstream of the genomic sequence complementary to the spacer sequence of the gRNA.

[0360] In some embodiments, when the method of the present invention includes a gRNA containing a spacer sequence, the 5' and 3' homologous arms of the nucleic acid for insertion into the genome do not contain sequences complementary to this spacer sequence. In such embodiments, the absence of a complementary sequence on the donor template can reduce the likelihood that the gRNA will bind to and cleave the donor template, and cleavage of the donor template by such a gRNA can reduce the efficiency of insertion into the genome. In some embodiments, the donor template does not contain a sequence complementary to the spacer sequence. In embodiments in which a different type of nuclease is used that does not require a gRNA for targeted cleavage, the donor template does not contain a sequence that is cleaved by the nuclease.

[0361] In some embodiments, a nucleic acid for insertion into the TRAC locus comprises a 5' homology arm having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 85 and a 3' homology arm having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 93. In some embodiments, the 5' homology arm has at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 85, and the 3' homology arm has at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 93. In some embodiments, the 5' homology arm comprises the nucleotide sequence of SEQ ID NO: 85, and the 3' homology arm comprises the nucleotide sequence of SEQ ID NO: 93.

[0362] In some embodiments, a nucleic acid for insertion into the TRAC locus comprises a 5' homology arm having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 96 and a 3' homology arm having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 105. In some embodiments, the 5' homology arm has at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 96, and the 3' homology arm has at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 105. In some embodiments, the 5' homology arm comprises the nucleotide sequence of SEQ ID NO: 96, and the 3' homology arm comprises the nucleotide sequence of SEQ ID NO: 105.

[0363] In some embodiments, a nucleic acid for insertion into the TRAC locus comprises a 5' homology arm having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 108 and a 3' homology arm having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 116. In some embodiments, the 5' homology arm has at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 108, and the 3' homology arm has at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 116. In some embodiments, the 5' homology arm comprises the nucleotide sequence of SEQ ID NO: 108, and the 3' homology arm comprises the nucleotide sequence of SEQ ID NO: 116.

[0364] In some embodiments, a nucleic acid for insertion into the TRAC locus comprises a 5' homology arm having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 119 and a 3' homology arm having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 127. In some embodiments, the 5' homology arm has at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 119, and the 3' homology arm has at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 127. In some embodiments, the 5' homology arm comprises the nucleotide sequence of SEQ ID NO: 119, and the 3' homology arm comprises the nucleotide sequence of SEQ ID NO: 127.

[0365] In some embodiments, a nucleic acid for insertion into the TRAC locus comprises a 5' homology arm having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 130 and a 3' homology arm having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 138. In some embodiments, the 5' homology arm has at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 130, and the 3' homology arm has at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 138. In some embodiments, the 5' homology arm comprises the nucleotide sequence of SEQ ID NO: 130, and the 3' homology arm comprises the nucleotide sequence of SEQ ID NO: 138.

[0366] In some embodiments, a nucleic acid for insertion into the FOXP3 locus comprises a 5' homology arm having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 141 and a 3' homology arm having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 149. In some embodiments, the 5' homology arm has at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 141, and the 3' homology arm has at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 149. In some embodiments, the 5' homology arm comprises the nucleotide sequence of SEQ ID NO: 141, and the 3' homology arm comprises the nucleotide sequence of SEQ ID NO: 149.

[0367] In some embodiments, a nucleic acid for insertion into the FOXP3 locus comprises a 5' homology arm having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 152 and a 3' homology arm having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 160. In some embodiments, the 5' homology arm has at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 152, and the 3' homology arm has at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 160. In some embodiments, the 5' homology arm comprises the nucleotide sequence of SEQ ID NO: 152, and the 3' homology arm comprises the nucleotide sequence of SEQ ID NO: 160.

[0368] In some embodiments, a nucleic acid for insertion into the FOXP3 locus comprises a 5' homology arm having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 163 and a 3' homology arm having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 171. In some embodiments, the 5' homology arm has at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 163, and the 3' homology arm has at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 171. In some embodiments, the 5' homology arm comprises the nucleotide sequence of SEQ ID NO: 163, and the 3' homology arm comprises the nucleotide sequence of SEQ ID NO: 171.

[0369] In some embodiments, a nucleic acid for insertion into the FOXP3 locus comprises a 5' homology arm having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 174 and a 3' homology arm having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 183. In some embodiments, the 5' homology arm has at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 174, and the 3' homology arm has at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 183. In some embodiments, the 5' homology arm comprises the nucleotide sequence of SEQ ID NO: 174, and the 3' homology arm comprises the nucleotide sequence of SEQ ID NO: 183.

[0370] In some embodiments, a nucleic acid for insertion into the FOXP3 locus comprises a 5' homology arm having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 186 and a 3' homology arm having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 194. In some embodiments, the 5' homology arm has at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 186, and the 3' homology arm has at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 194. In some embodiments, the 5' homology arm comprises the nucleotide sequence of SEQ ID NO: 186, and the 3' homology arm comprises the nucleotide sequence of SEQ ID NO: 194.

[0371] In some embodiments, a nucleic acid for insertion into the FOXP3 locus comprises a 5' homology arm having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 197 and a 3' homology arm having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 205. In some embodiments, the 5' homology arm has at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 197 and the 3' homology arm has at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 205. In some embodiments, the 5' homology arm comprises the nucleotide sequence of SEQ ID NO: 197 and the 3' homology arm comprises the nucleotide sequence of SEQ ID NO: 205.

[0372] In some embodiments, a nucleic acid for insertion into the FOXP3 locus comprises a 5' homology arm having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 208 and a 3' homology arm having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 217. In some embodiments, the 5' homology arm has at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 208, and the 3' homology arm has at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 217. In some embodiments, the 5' homology arm comprises the nucleotide sequence of SEQ ID NO: 208, and the 3' homology arm comprises the nucleotide sequence of SEQ ID NO: 217.

[0373] genetically modified cells Some embodiments of the present disclosure relate to genetically engineered cells containing two nucleic acids, each introduced into a separate locus in the cellular genome. The cells are double-edited by inserting one nucleic acid into the TRAC locus and the other nucleic acid into the FOXP3 locus (i.e., cells in which each nucleic acid is inserted into two different loci). The exact insertion locations vary depending on the homologous arms on the nucleic acids targeting each locus. The first nucleic acid targeting the TRAC locus contains 5' and 3' homologous arms that provide directionality for inserting the nucleic acid into the TRAC locus (e.g., by cleaving the DNA sequence at the TRAC locus with a nuclease followed by homology-directed repair (HDR)). The second nucleic acid targeting the FOXP3 locus contains 5' and 3' homologous arms that provide directionality for inserting the nucleic acid into the FOXP3 locus (e.g., by cleaving the DNA sequence at the FOXP3 locus with a nuclease followed by homology-directed repair (HDR)). By inserting each of the two nucleic acids into a separate locus in the cell, a double-edited cell (i.e., a cell in which each nucleic acid has been inserted into two different loci) is obtained.

[0374] In the cell embodiments described herein, the recombinant TRAC locus includes: (i) a nucleotide sequence encoding a first chemically-induced signaling complex (CISC) component comprising: (a) an extracellular binding domain comprising, or derived from, FK506-binding protein 12 (FKBP); (b) a transmembrane domain comprising, or derived from, the transmembrane domain of IL-2Rγ; and (c) an intracellular signaling domain comprising, or derived from the cytoplasmic domain of IL-2Rγ; (ii) a nucleotide sequence encoding a full-length TCR β chain; and (iii) a nucleotide sequence encoding at least a portion of a heterologous TCR α chain. A promoter operably linked to the The nucleotide sequence encoding the heterologous TCR α chain is inserted in frame with an endogenous sequence encoding a portion of the endogenous TCR α (e.g., the constant domain of the TCR α), allowing the expressed mRNA to be translated to produce a TCR α chain that associates with the heterologous TCR β chain to form a TCR. Because the antigen-binding region of the TCR α chain is encoded by the inserted nucleic acid, the specificity of the TCR is determined by the inserted nucleic acid. In the cells described herein, the TCR encoded by the inserted nucleic acid binds to a T1D-associated antigen. Thus, transcription of each sequence operably linked to the inserted promoter in the recombinant TRAC locus is initiated by the promoter, and a T1D-associated antigen-specific TCR formed by the heterologous TCR β chain and the heterologous TCR α chain containing a portion of the heterologous sequence encoded by the inserted nucleic acid, and an FKBP-IL2Rγ CISC component, are expressed from the recombinant TRAC locus.

[0375] In the cell embodiments described herein, the recombinant FOXP3 locus comprises: (i) a nucleotide sequence encoding a first chemically-induced signaling complex (CISC) component comprising: (a) an extracellular binding domain comprising the FKBP-rapamycin binding (FRB) domain of mTOR or an extracellular binding domain derived from the FKBP-rapamycin binding (FRB) domain of mTOR; (b) a transmembrane domain comprising the transmembrane domain of IL-2Rβ or a transmembrane domain derived from the transmembrane domain of IL-2Rβ; and (c) an intracellular signaling domain comprising the cytoplasmic domain of IL-2Rβ or an intracellular signaling domain derived from the cytoplasmic domain of IL-2Rβ; (ii) a nucleotide sequence encoding a cytosolic FRB domain without a transmembrane domain; and (iii) a nucleotide sequence encoding FoxP3 A promoter operably linked to the The promoter is inserted downstream of the Treg-specific demethylation region of the FOXP3 locus (e.g., using a homology arm homologous to a sequence within 2,000 nucleotides upstream of exon 2, the first coding exon of the FOXP3 gene, or to a sequence up to this 2,000 nucleotide sequence). By inserting a promoter downstream of the TSDR, which destabilizes FOXP3 expression under inflammatory conditions, the inserted promoter can initiate transcription of mRNA encoding FoxP3 independently of the endogenous FOXP3 promoter upstream of the TSDR. Thus, in the recombinant FOXP3 locus, transcription of each sequence operably linked to the inserted promoter is initiated by this promoter, resulting in expression of the FRB-Il2Rβ CISC component, the cytosolic FRB component, and FoxP3 from the FOXP3 locus.

[0376] As described in the previous section, the double-edited cells obtained by recombining the TRAC and FOXP3 loci contain (i) a first CISC component and a second CISC component that form a heterodimer in the presence of rapamycin to transduce IL-2R signaling through dimerization of the cytoplasmic domain of IL-2Rβ and the intracellular domain of IL-2Rγ; (ii) a cytosolic FRB domain that binds to intracellular rapamycin and prevents its interaction with mTOR; (iii) FoxP3, which confers a stable Treg phenotype; and (iv) stably express a T1D-associated antigen-specific TCR. Thus, the cells described herein are stable Treg cells with specificity for T1D-associated antigens, and these Treg cells can be induced to proliferate using rapamycin. Furthermore, because the nucleotide sequences encoding the first and second CISC components are inserted at separate loci, rapamycin can be used to selectively induce the proliferation of cells expressing both CISC components (i.e., cells expressing a T1D antigen-specific TCR and FoxP3 due to the recombination of both loci). Thus, dual-edited cells can be readily selected and expanded in vitro to produce stable Treg cell populations specific for T1D-associated antigens for the treatment of type 1 diabetes. Furthermore, the in vivo engraftment and expansion of such stable Treg cells may be supported by administering rapamycin to a subject.

[0377] promoter Any nucleic acid inserted into the genome of a genetically modified cell described herein includes a promoter operably linked to one or more nucleotide sequences inserted into the FOXP3 locus or the TRAC locus. A promoter is "operably linked" to a sequence if it is capable of initiating transcription of the operably linked sequence (e.g., by recruiting RNA polymerase). The promoters inserted into the modified TRAC and FOXP3 loci can be any promoter known in the art. In some embodiments, the inserted heterologous promoter is active and promotes RNA transcription, even under inflammatory conditions. In some embodiments, the promoter is a constitutive promoter. A constitutive promoter can be a strong promoter that promotes transcription more efficiently than an endogenous promoter, or a weak promoter that promotes transcription less efficiently than a strong promoter or an endogenous promoter. In some embodiments, the constitutive promoter is a strong promoter. In some embodiments, the heterologous promoter is an inducible promoter. An inducible promoter promotes transcription of an operably linked sequence in response to the presence of an activating signal or the absence of an inhibitory signal. In some embodiments, the inducible promoter is inducible by a drug or steroid.

[0378] In some embodiments, the promoters inserted into the TRAC locus and the FOXP3 locus of the cell are different promoters. In another embodiment, the TRAC locus and the FOXP3 locus comprise the same promoter. In some embodiments, both the TRAC locus and the FOXP3 locus comprise an MND promoter. In embodiments where the TRAC locus and the FOXP3 locus comprise the same promoter, the promoter sequences of the TRAC locus and the FOXP3 locus may be the same. Alternatively, the promoter sequence of the recombined TRAC locus may contain one or more mutations (e.g., insertions, deletions, substitutions) compared to the promoter sequence of the FOXP3 locus. In some embodiments, the MND promoter of the TRAC locus and / or the FOXP3 locus has at least 90% sequence identity with the nucleotide sequence of SEQ ID NO: 220. In some embodiments, the MND promoter of the TRAC locus and / or the FOXP3 locus has at least 95% sequence identity with the nucleotide sequence of SEQ ID NO: 220. In some embodiments, the TRAC locus and the FOXP3 locus each comprise an MND promoter having the nucleotide sequence of SEQ ID NO:220.

[0379] In some embodiments, a stop codon is present upstream of the first five nucleotides of the promoter inserted into the TRAC locus, or within any of those five nucleotides. In some embodiments, a stop codon is present upstream of the first five nucleotides of the promoter inserted into the FOXP3 locus, or within any of those five nucleotides. The presence of a stop codon upstream of, within, or overlapping the first five nucleotides of the promoter is expected to terminate translation of mRNA that may be transcribed from upstream of the endogenous promoter of the recombined TRAC locus or FOXP3 locus, thereby suppressing expression of the inserted coding sequence (e.g., a sequence encoding a CISC component, a heterologous TCR β chain or TCR α chain, or FoxP3) under the control of the endogenous promoter. In some embodiments, the stop codon is in frame with one or more upstream start codons, such that mRNA produced by transcription from the upstream endogenous promoter is not translated beyond the stop codon.

[0380] Chemical-inducible signaling complex (CISC) Embodiments of the genetically engineered cells described herein comprise, at each of the TRAC and FOXP3 loci of their genome, nucleotide sequences encoding components of a chemically-induced signaling complex (CISC), each CISC component comprising an extracellular domain that binds rapamycin, a transmembrane domain, and an intracellular domain that includes, or is derived from, the cytoplasmic domain of the interleukin-2 receptor (IL-2R). In some embodiments, the TRAC locus encodes a first CISC component comprising: (i) an extracellular binding domain comprising an FK506-binding protein 12 (FKBP) domain; (ii) a transmembrane domain comprising, or derived from, the transmembrane domain of IL-2Rγ; and (iii) an intracellular domain comprising, or derived from, the cytoplasmic domain of IL-2Rγ; and the FOXP3 locus encodes a first CISC component comprising: (i) an extracellular binding domain comprising an FKBP-rapamycin binding domain; (ii) a transmembrane domain comprising, or derived from, the transmembrane domain of IL-2Rβ; and (iii) an intracellular domain comprising, or derived from the cytoplasmic domain of IL-2Rβ. A domain of a CISC component (e.g., the transmembrane domain of a first CISC component) is "derived from" a particular domain of an IL-2R polypeptide (e.g., IL-2Rγ) if it has at least 90% sequence identity with the wild-type (naturally occurring) amino acid sequence of that domain of that IL-2R polypeptide (e.g., the transmembrane domain of native IL-2Rγ).

[0381] By expressing CISC components in cells and manipulating the presence and / or concentration of rapamycin, IL-2 signaling can be selectively induced in the cells. In this manner, controllably inducing signaling allows, for example, IL-2 signaling events to induce cell proliferation, thereby selectively expanding cells expressing both CISC components. In some embodiments in which two loci are recombined, nucleotides encoding different CISC components are inserted at each locus, so that contacting cells containing only one CISC component with rapamycin does not induce dimerization or IL-2 signaling due to the absence of the other CISC component, allowing for such selective expansion to select for cells in which both loci have been recombined.

[0382] Examples of intracellular signaling domains include, but are not limited to, the IL-2Rβ cytoplasmic domain and the IL-2Rγ cytoplasmic domain and functional derivatives thereof. In some embodiments, the intracellular signaling domain of a first CISC component comprises an IL-2Rγ domain or a functional derivative thereof, and the intracellular signaling domain of a second CISC component comprises an IL-2Rβ cytoplasmic domain or a functional derivative thereof. In some embodiments, dimerization of the first CISC component and the second CISC component induces phosphorylation of JAK1, JAK3, and / or STAT5 in cells. In some embodiments, dimerization of the first CISC component and the second CISC component induces cell proliferation.

[0383] Examples of transmembrane domains include, but are not limited to, the transmembrane domain of IL-2Rβ and the transmembrane domain of IL-2Rγ, and functional derivatives thereof. In some embodiments, the transmembrane domain of a CISC component is derived from the same protein as the protein from which the intracellular signaling domain of that CISC component is derived (e.g., a CISC component comprising the intracellular domain of IL-2Rβ comprises the transmembrane domain of IL-2Rβ). In some embodiments, one CISC component comprises the transmembrane domain of IL-2Rβ, and the other CISC component comprises the transmembrane domain of IL-2Rγ.

[0384] Examples of extracellular binding domains capable of binding to rapamycin include, but are not limited to, an FK506-binding protein (FKBP) domain and an FKBP-rapamycin binding (FRB) domain. The FKBP domain and the FRB domain can bind to rapamycin and form a heterodimer, as described below. In some embodiments, the extracellular binding domain of one CISC component comprises an FKBP domain, and the extracellular binding domain of the other CISC component comprises an FRB domain. In some embodiments, these CISC components form a heterodimer in the presence of rapamycin. In some embodiments, the FRB domain contains a threonine at the position corresponding to amino acid 2098 of wild-type mTOR having the amino acid sequence of SEQ ID NO: 236. This amino acid mutation increases the affinity of mTOR for compounds structurally related to rapamycin, but decreases the affinity of mTOR for rapamycin itself. Therefore, the presence of a threonine at this position maintains the binding ability of mTOR to rapamycin. The amino acid in a CISC component or FRB domain that "corresponds to" amino acid 2098 of wild-type mTOR may be determined by aligning a candidate sequence for a CISC component or FRB domain to SEQ ID NO:236 (e.g., by BLAST alignment algorithm or another alignment algorithm known in the art), and the amino acid that aligns to amino acid 2098 of SEQ ID NO:236 is the amino acid that "corresponds to" amino acid 2098 of SEQ ID NO:236.

[0385] The extracellular binding domain, transmembrane domain, and intracellular signaling domain of a CISC component described herein may each be connected to another domain of that CISC component via a linker. Linkers are known in the art. In some embodiments, the linker comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids (e.g., glycine), a number of amino acids (e.g., glycine), or a range between any two of these numbers. In some embodiments, the glycine spacer comprises at least three glycines. In some embodiments, the glycine spacer comprises a sequence set forth as GSG, GGGS (SEQ ID NO: 229), GGGSGGG (SEQ ID NO: 230), or GGG. In some embodiments, the glycine spacer comprises the amino acid sequence GSG.

[0386] The extracellular binding domain may be connected to the transmembrane domain via a hinge domain. "Hinge" refers to a domain that links the extracellular binding domain to the transmembrane domain and may thereby provide flexibility to the extracellular binding domain. In some embodiments, the hinge domain positions the extracellular binding domain in the vicinity of the cell membrane, minimizing the possibility of recognition by an antibody or binding fragment thereof. In some embodiments, the extracellular binding domain is located N-terminal to the hinge domain. In some embodiments, the hinge domain may be natural or synthetic.

[0387] In some embodiments, the first and second CISC components form a heterodimer in the presence of rapamycin. In some embodiments, the first and second CISC components form a heterodimer in the presence of a compound produced in vivo by metabolism of a rapalog. In some embodiments, the compound produced in vivo by metabolism of a rapalog is rapamycin. Examples of rapalogs include, but are not limited to, everolimus, CCI-779, C20-methallylrapamycin, C16-(S)-3-methylindolerapamycin, C16-iRap, C16-(S)-7-methylindolerapamycin, AP21967, C16-(S)-butylsulfonamidorapamycin, AP23050, mycophenolate sodium, benidipine hydrochloride, AP1903, and AP23573, as well as metabolites and derivatives thereof.

[0388] In some embodiments, the FOXP3 locus further comprises a nucleotide sequence encoding a third CISC component that binds rapamycin. Such a CISC component may be useful, for example, to bind intracellular rapamycin, thereby preventing rapamycin bound to the CISC component from interacting with other intracellular molecules or structures (e.g., preventing rapamycin from interacting with mTOR). In some embodiments, the third CISC component is a soluble protein that does not contain a transmembrane domain. In some embodiments, the third CISC component comprises an intracellular FRB domain. In some embodiments, the third CISC component is a soluble protein that contains an FRB domain but does not contain a transmembrane domain.

[0389] In some embodiments, the CISC component comprises an amino acid sequence having at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or up to 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 66 or 71. In some embodiments, one or more CISC components further comprise a signal peptide. The signal peptide may be any signal peptide known in the art that transports the translated CISC component to the cell membrane. In some embodiments, both the first CISC component and the second CISC component comprise the signal peptide of LCN2. In some embodiments, both the first CISC component and the second CISC component comprise a signal peptide comprising the amino acid sequence of SEQ ID NO: 73.

[0390] In some embodiments, one CISC component comprises an amino acid sequence having at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or up to 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 66, and the other CISC component comprises an amino acid sequence having at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or up to 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 71. In some embodiments, each CISC component further comprises a signal peptide, which may have the same or different amino acid sequences. These signal peptides may be any signal peptide known in the art that transports the translated CISC component to the cell membrane.

[0391] In some embodiments, the third CISC component comprises an amino acid sequence having at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or up to 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 72. In some embodiments, the third CISC component consists of an amino acid sequence having at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or up to 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 72. In some embodiments, the third CISC component comprises the amino acid sequence of SEQ ID NO: 72. In some embodiments, the third CISC component consists of the amino acid sequence of SEQ ID NO: 72. In some embodiments, the third CISC component does not comprise a signal peptide. In some embodiments, the third CISC component does not include a transmembrane domain.

[0392] T cell receptor (TCR) In some embodiments of the cells described herein, the TRAC locus has been edited by inserting a nucleic acid comprising a promoter operably linked to a nucleotide sequence encoding a full-length TCRβ protein and a nucleotide sequence encoding at least a portion of a TCRα protein, e.g., the TCRα variable region and TCRα junction (TRAJ) region that form the portion of the TCRα protein responsible for antigen specificity. In some embodiments, the inserted nucleotide sequence encoding the TCRα variable and junction regions is in-frame with an endogenous nucleotide sequence encoding a portion of the TCRα constant domain, such that the inserted heterologous promoter initiates transcription of the sequence encoding the heterologous TCRβ protein and the sequence encoding the TCRα protein comprising the heterologous TRAV / TRAJ amino acid sequence and the endogenous TCRα constant domain. This embodiment utilizes the endogenous 3' regulatory region of the endogenous TRAC locus.

[0393] The genetically engineered cells described herein express a T cell receptor specific for type 1 diabetes (T1D). The T cell receptor expressed by the genetically engineered cells described herein is described in the section entitled "Methods for Producing Genetically Engineered Cells" and the subheading "T Cell Receptor (TCR)." In certain embodiments, the sequence encoding the TCR in the cell genome can be codon-optimized to improve expression in certain host cells, such as, for example, immune system cells, hematopoietic stem cells, T cells, primary T cells, T cell lines, NK cells, and natural killer T cells. See, e.g., Scholten et al., Clin Immunol. 2006. 119:135.

[0394] In some embodiments, the recombinant TRAC locus of the genetically engineered cells described herein encodes at least a portion of a TCR β chain and a TCR α chain, which are expressed in combination to form a T1D2 TCR that binds to the IGRP(305-234) peptide. In another embodiment, the full-length TCR α chain and TCR β chain, partially encoded by the recombinant TRAC locus described herein, form a T1D4 TCR that binds to the IGRP(241-260) peptide. In another embodiment, the full-length TCR α chain and TCR β chain, partially encoded by the inserted nucleotide sequence described herein, form a T1D5-1 TCR that binds to the IGRP(305-324) peptide. In some embodiments, the IGRP(305-324) peptide is recognized when bound to HLA-DRB1*0401. In some embodiments, the IGRP(241-260) peptide is recognized when bound to HLA-DRB1*0401.

[0395] In some embodiments, the TCR formed from (at least a portion of) the TCR β chain and TCR α chain encoded by the recombinant TRAC locus of the genetically engineered cell described herein comprises a TCR α variable (Vα) domain having three complementarity determining regions (CDRs), αCDR1, αCDR2, and αCDR3, and a TCR β variable (Vβ) domain having three CDRs, βCDR1, βCDR2, and βCDR3. Representative amino acids of the CDRs of the TCRs described herein are set forth in Table 1, and nucleotide sequences encoding the CDRs of the TCRs described herein are set forth in Table 2. In some embodiments, (i) αCDR1 comprises SEQ ID NO: 1, (ii) αCDR2 comprises SEQ ID NO: 2, (iii) αCDR3 comprises SEQ ID NO: 3, (iv) βCDR1 comprises SEQ ID NO: 4, (v) βCDR2 comprises SEQ ID NO: 5, and (vi) βCDR3 comprises SEQ ID NO: 6. In some embodiments, (i) αCDR1 comprises SEQ ID NO: 11, (ii) αCDR2 comprises SEQ ID NO: 12, (iii) αCDR3 comprises SEQ ID NO: 13, (iv) βCDR1 comprises SEQ ID NO: 14, (v) βCDR2 comprises SEQ ID NO: 15, and (vi) βCDR3 comprises SEQ ID NO: 16. In some embodiments, (i) αCDR1 comprises SEQ ID NO: 21, (ii) αCDR2 comprises SEQ ID NO: 22, (iii) αCDR3 comprises SEQ ID NO: 23, (iv) βCDR1 comprises SEQ ID NO: 24, (v) βCDR2 comprises SEQ ID NO: 25, and (vi) βCDR3 comprises SEQ ID NO: 26. In another embodiment, each set consisting of αCDR1, αCDR2, αCDR3, βCDR1, βCDR2 and βCDR3 may have an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with each amino acid sequence shown in any of the above amino acid sequence combinations.

[0396] In some embodiments, Vα comprises SEQ ID NO: 7 and Vβ comprises SEQ ID NO: 8. In some embodiments, Vα comprises SEQ ID NO: 17 and Vβ comprises SEQ ID NO: 18. In some embodiments, Vα comprises SEQ ID NO: 27 and Vβ comprises SEQ ID NO: 28. In other embodiments, each pair of Vα and Vβ may have an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a respective amino acid sequence set forth in any of the above amino acid sequence combinations.

[0397] In some embodiments, the TCR alpha chain comprises SEQ ID NO: 9 and the TCR beta chain comprises SEQ ID NO: 10. In some embodiments, the TCR alpha chain comprises SEQ ID NO: 19 and the TCR beta chain comprises SEQ ID NO: 20. In some embodiments, the TCR alpha chain comprises SEQ ID NO: 29 and the TCR beta chain comprises SEQ ID NO: 30. In another embodiment, each pair of TCR alpha and TCR beta chains may have an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to each amino acid sequence set forth in any of the above amino acid sequence combinations.

[0398] Recombination at the FOXP3 locus In some embodiments of the genetically engineered cells described herein, the FOXP3 locus contains an inserted promoter operably linked to a nucleotide sequence encoding at least a portion of the endogenous FoxP3 protein. The inserted promoter is introduced into the genome downstream of the Treg-specific demethylation region (TSDR) of the FOXP3 locus. In unmodified cells, the TSDR epigenetically regulates FoxP3 expression, thereby suppressing FoxP3 production in cells exposed to inflammatory conditions, resulting in loss of FoxP3 expression and conversion of unmodified Treg cells to a T effector (Teff) phenotype. Inserting a promoter downstream of the TSDR bypasses TSDR-mediated regulation of FOXP3 expression, allowing stable production of FoxP3 even under inflammatory conditions.

[0399] The heterologous promoter can be inserted anywhere downstream of the endogenous promoter of the FOXP3 coding sequence (e.g., downstream of the TSDR) and upstream of or within the first coding exon (known in the art as exon 2 because it is the second exon present in the pre-mRNA transcribed from the endogenous FOXP3 promoter, and known as the first coding exon because it is not exon 1 (the first exon in the pre-mRNA encoding FOXP3), which contains the start codon that initiates translation of wild-type FoxP3). In some embodiments, the heterologous promoter is 1 to 10,000 nucleotides downstream, 10 to 1,000 nucleotides downstream, 10 to 100 nucleotides downstream, 10 to 5,000 nucleotides downstream, 20 to 4,000 nucleotides downstream, 30 to 3,000 nucleotides downstream, 40 to 2,000 nucleotides downstream, 50 to 1,000 nucleotides downstream, 60 to 750 nucleotides downstream, 70 to 500 nucleotides downstream, 80 to 400 nucleotides downstream, 90 to 300 nucleotides downstream of the TSDR of the FOXP3 locus. The insertion may be 100 to 200 nucleotides downstream, 1 to 1,000 nucleotides downstream, 1,000 to 2,000 nucleotides downstream, 2,000 to 3,000 nucleotides downstream, 3,000 to 4,000 nucleotides downstream, 4,000 to 5,000 nucleotides downstream, 5,000 to 6,000 nucleotides downstream, 6,000 to 7,000 nucleotides downstream, 7,000 to 8,000 nucleotides downstream, 8,000 to 9,000 nucleotides downstream, or 9,000 to 10,000 nucleotides downstream.In some embodiments, the heterologous promoter is located between 1 and 10,000 nucleotides upstream, between 10 and 1,000 nucleotides upstream, between 100 nucleotides upstream, between 10 and 5,000 nucleotides upstream, between 20 and 4,000 nucleotides upstream, between 30 and 3,000 nucleotides upstream, between 40 and 2,000 nucleotides upstream, between 50 and 1,000 nucleotides upstream, between 60 and 750 nucleotides upstream, between 70 and 500 nucleotides upstream, between 80 and 400 nucleotides upstream, between 90 and 1000 nucleotides upstream, between 10 and 1500 nucleotides upstream, between 15 and 20 ... In some embodiments, the heterologous promoter is inserted between 100 and 200 nucleotides upstream, 1 and 1,000 nucleotides upstream, 1,000 and 2,000 nucleotides upstream, 2,000 and 3,000 nucleotides upstream, 3,000 and 4,000 nucleotides upstream, 4,000 and 5,000 nucleotides upstream, 5,000 and 6,000 nucleotides upstream, 6,000 and 7,000 nucleotides upstream, 7,000 and 8,000 nucleotides upstream, 8,000 and 9,000 nucleotides upstream, or 9,000 and 10,000 nucleotides upstream. In some embodiments, the heterologous promoter is inserted between the first coding exon to create a synthetic first coding exon that is distinct from the endogenous first coding exon but contains an initiation codon in-frame with the FOXP3 sequence encoding the downstream FOXP3 exon.

[0400] 2A motif and linker Some embodiments of the recombinant TRAC locus and / or recombinant FOXP3 locus encoding multiple polypeptides or portions thereof in the genetically engineered cells described herein may include an intervening nucleotide sequence encoding a 2A motif. 2A motifs are known in the art and are useful for translating and facilitating the production of multiple polypeptides from a single nucleotide sequence. See, e.g., Kim et al., PLoS ONE. 2011. 6:e18556. In some embodiments, the 2A motif is translated, which results in self-cleavage of the polypeptide, releasing separate polypeptides. In another embodiment, the presence of a nucleotide sequence encoding a 2A motif allows the ribosome to skip over the amino acids of the encoded 2A motif as it translates along the mRNA, thereby releasing a first polypeptide (e.g., a first FKBP-IL2Rγ CISC component) and initiating translation of a second polypeptide (e.g., a TCR β chain).

[0401] In some embodiments, in each pair of nucleic acid sequences, the nucleotide sequence encoding the 2A motif is positioned in frame between (i) the nucleotide sequence encoding the first (FKBP-IL2Rγ) CISC component, (ii) the nucleotide sequence encoding the TCR β chain, and (iii) the nucleotide sequence encoding the TCR α chain or a portion thereof. Thus, when a heterologous promoter (e.g., the MND promoter) initiates transcription of a single mRNA encoding each of the CISC components, the TCR β chain, and the TCR α chain, and containing an intervening 2A motif, the CISC components, the TCR β chain, and the TCR α chain are each produced as separate polypeptides. In some embodiments, in each pair of nucleic acid sequences, the nucleotide sequence encoding the 2A motif is positioned in frame between (i) the nucleotide sequence encoding the second (FKBP-IL2Rγ) CISC component, (ii) the nucleotide sequence encoding the cytosolic FRB domain, and (iii) the nucleotide sequence encoding FoxP3. Thus, when a heterologous promoter (e.g., the MND promoter) initiates transcription of a single mRNA encoding each of the CISC components, the cytosolic FRB domain, and FoxP3, and containing an intervening 2A motif, the CISC components, the cytosolic FRB domain, and FoxP3 are each produced as separate polypeptides.

[0402] In each pair of nucleic acid sequences, the 2A motif encoded by the nucleotide sequence located between the nucleotide sequences encoding two polypeptides (e.g., between the sequence encoding the FKBP-IL2Rγ CISC component and the sequence encoding the TCR β chain; and between the sequence encoding the TCR β chain and the sequence encoding a portion of the TCR α chain) may be any 2A motif known in the art. In some embodiments, in each pair of nucleic acid sequences, the 2A motifs encoded between the nucleotide sequences encoding separate polypeptides may be independently selected from the group consisting of F2A, P2A, T2A, and E2A. In some embodiments, the first and second 2A motifs encoded in one recombinant TRAC locus and / or one recombinant FOXP3 locus are different types of 2A motifs. Using different types of 2A motifs in one recombinant TRAC locus and / or one recombinant FOXP3 locus can reduce the probability of internal recombination. When internal recombination occurs, the nucleotide sequence between the recombined 2A motifs may be excised from the chromosome. In some embodiments, the nucleotide sequence encoding a first 2A motif at a recombinant TRAC locus and / or a recombinant FOXP3 locus has 90% or less sequence identity to the nucleotide sequence encoding a second 2A motif at that recombinant TRAC locus and / or the recombinant FOXP3 locus. In some embodiments, the nucleotide sequence encoding a first 2A motif at a recombinant TRAC locus and / or a recombinant FOXP3 locus has 80% or less sequence identity to the nucleotide sequence encoding a second 2A motif at that recombinant TRAC locus and / or the recombinant FOXP3 locus. In some embodiments, the nucleotide sequence encoding a first 2A motif at a recombinant TRAC locus and / or a recombinant FOXP3 locus has 70% or less sequence identity to the nucleotide sequence encoding a second 2A motif at that recombinant TRAC locus and / or the recombinant FOXP3 locus.In some embodiments, the nucleotide sequence encoding a first 2A motif at a recombinant TRAC locus and / or a recombinant FOXP3 locus has 60% or less sequence identity with the nucleotide sequence encoding a second 2A motif at that recombinant TRAC locus and / or that recombinant FOXP3 locus. In some embodiments, the nucleotide sequence encoding a first 2A motif at a recombinant TRAC locus and / or a recombinant FOXP3 locus has 50% or less sequence identity with the nucleotide sequence encoding a second 2A motif at that recombinant TRAC locus and / or that recombinant FOXP3 locus. In some embodiments, the first 2A motif is a T2A motif and the second motif is a P2A motif.

[0403] In another embodiment, the first and second 2A motifs encoded by the nucleotide sequences in one recombinant TRAC locus and / or one recombinant FOXP3 locus are the same type of 2A motif. In some embodiments, one recombinant TRAC locus and / or one recombinant FOXP3 locus comprises a nucleotide sequence encoding a first P2A motif and a second nucleotide sequence encoding a second P2A motif, and the nucleotide sequence encoding the first P2A motif has at least 80% sequence identity with the nucleotide sequence encoding the second P2A motif. In some embodiments, the first nucleotide sequence encoding the first P2A motif and the second nucleotide sequence encoding the second P2A motif comprise the same nucleotide sequence.

[0404] In some embodiments, the recombinant TRAC locus (i) comprises a sequence encoding a T2A motif between the sequence encoding the first CISC component and the sequence encoding the TCR β chain, and (ii) comprises a sequence encoding a P2A motif between the sequence encoding the TCR β chain and the sequence encoding a portion of the heterologous TCR α chain.

[0405] In some embodiments, the recombinant FOXP3 locus comprises (i) a sequence encoding a P2A motif between the sequence encoding the second CISC component and the sequence encoding the cytosolic FRB domain, and (ii) a second sequence encoding a second P2A motif between the sequence encoding the cytosolic FRB domain and the sequence encoding FoxP3.

[0406] In some embodiments, the polypeptide (e.g., a CISC component and / or a TCR β chain) encoded by the nucleotide sequence inserted into the recombinant TRAC locus or recombinant FOXP3 locus comprises a C-terminal linker. Incorporation of a C-terminal linker may, for example, improve cleavage efficiency at the 2A motif and / or prevent amino acids of the encoded CISC component or TCR β chain from being excised upon cleavage of the 2A motif. In some embodiments, the encoded first CISC component comprises a C-terminal linker. In some embodiments, the encoded second CISC component comprises a C-terminal linker. In some embodiments, the encoded cytosolic FRB domain component comprises a C-terminal linker. In some embodiments, the encoded TCR β chain comprises a C-terminal linker.

[0407] The C-terminal linker of the encoded polypeptide can be any linker known in the art. In some embodiments, the C-terminal linker comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids (e.g., glycine), a number of amino acids (e.g., glycine), or a range of amino acids between any two of these numbers. In some embodiments, the C-terminal linker comprises at least three glycines. In some embodiments, the C-terminal linker comprises the sequence GSG, GGGS (SEQ ID NO: 229), GGGSGGG (SEQ ID NO: 230), or GGG. In some embodiments, the C-terminal linker comprises the amino acid sequence GSG. In some embodiments, the first CISC component, the second CISC component, the cytosolic FRB domain, and the TCR β chain all comprise C-terminal linkers having the amino acid sequence GSG.

[0408] In some embodiments, the recombinant TRAC locus comprises a nucleotide sequence having at least 90% sequence identity to any of SEQ ID NOs: 94, 106, 117, 128, and 139. In some embodiments, the nucleotide sequence has at least 95% sequence identity to any of SEQ ID NOs: 94, 106, 117, 128, and 139. In some embodiments, the nucleotide sequence comprises any of SEQ ID NOs: 94, 106, 117, 128, and 139. In some embodiments, the recombinant TRAC locus comprises the nucleotide sequence of SEQ ID NO: 94. In some embodiments, the recombinant TRAC locus comprises the nucleotide sequence of SEQ ID NO: 106. In some embodiments, the recombinant TRAC locus comprises the nucleotide sequence of SEQ ID NO: 117. In some embodiments, the recombinant TRAC locus comprises the nucleotide sequence of SEQ ID NO: 128. In some embodiments, the recombinant TRAC locus comprises the nucleotide sequence of SEQ ID NO: 139.

[0409] In some embodiments, the recombinant FOXP3 locus comprises a nucleotide sequence having at least 90% sequence identity to any of SEQ ID NOs: 150, 161, 172, 184, 195, 206, and 218. In some embodiments, the nucleotide sequence has at least 95% sequence identity to any of SEQ ID NOs: 150, 161, 172, 184, 195, 206, and 218. In some embodiments, the nucleotide sequence comprises any of SEQ ID NOs: 150, 161, 172, 184, 195, 206, and 218. In some embodiments, the recombinant FOXP3 locus comprises the nucleotide sequence of SEQ ID NO: 150. In some embodiments, the recombinant FOXP3 locus comprises the nucleotide sequence of SEQ ID NO: 161. In some embodiments, the recombinant FOXP3 locus comprises the nucleotide sequence of SEQ ID NO: 172. In some embodiments, the recombinant FOXP3 locus comprises the nucleotide sequence of SEQ ID NO: 184. In some embodiments, the recombinant FOXP3 locus comprises the nucleotide sequence of SEQ ID NO: 195. In some embodiments, the recombinant FOXP3 locus comprises the nucleotide sequence of SEQ ID NO: 206. In some embodiments, the recombinant FOXP3 locus comprises the nucleotide sequence of SEQ ID NO: 218.

[0410] System for producing genetically modified cells Some embodiments of the present disclosure relate to a system for producing a genetically engineered cell comprising two nucleic acids, one of which is homologous to the TRAC locus of the cell and the other of which is homologous to the FOXP3 locus of the cell, such that both loci may be edited by inserting the respective nucleic acids into each locus. The first nucleic acid targeting the TRAC locus comprises 5' and 3' homologous arms that provide directionality for inserting the nucleic acid into the TRAC locus (e.g., by cleaving the DNA sequence of the TRAC locus with a nuclease followed by homology-directed repair (HDR)). The second nucleic acid targeting the FOXP3 locus comprises 5' and 3' homologous arms that provide directionality for inserting the nucleic acid into the FOXP3 locus (e.g., by cleaving the DNA sequence of the FOXP3 locus with a nuclease followed by homology-directed repair (HDR)). By inserting each of the two nucleic acids into a separate locus in the cell, a double-edited cell (i.e., a cell in which each nucleic acid has been inserted into two different loci) is obtained.

[0411] In an embodiment of the system described herein, the nucleic acid targeted for insertion into the TRAC locus is (i) a nucleotide sequence encoding a first chemically-induced signaling complex (CISC) component comprising: (a) an extracellular binding domain comprising, or derived from, FK506-binding protein 12 (FKBP); (b) a transmembrane domain comprising, or derived from, the transmembrane domain of IL-2Rγ; and (c) an intracellular signaling domain comprising, or derived from the cytoplasmic domain of IL-2Rγ; (ii) a nucleotide sequence encoding a full-length TCR β chain; and (iii) a nucleotide sequence encoding at least a portion of a TCR α chain The promoter is operably linked to the The nucleotide sequence encoding the heterologous TCR α is inserted in frame with an endogenous sequence encoding a portion of the endogenous TCR α (e.g., the constant domain of the TCR α), resulting in translation of the expressed mRNA to generate a TCR α chain that associates with the heterologous TCR β chain to form a TCR. Because the antigen-binding region of the TCR α chain is encoded by the inserted nucleic acid, the specificity of the TCR is determined by the inserted nucleic acid. In the system described herein, the TCR encoded by the inserted nucleic acid binds to a T1D-associated antigen. By inserting each sequence and a promoter into the TRAC locus, transcription of each sequence operably linked to the promoter is initiated (thereby promoting expression) by the promoter, and a T1D-associated antigen-specific TCR formed by the heterologous TCR β chain and the heterologous TCR α chain containing a portion of the heterologous sequence encoded by the inserted nucleic acid, and an FKBP-IL2Rγ CISC component, are expressed from the TRAC locus.

[0412] In an embodiment of the system described herein, the nucleic acid targeted for insertion into the FOXP3 locus is: (i) a nucleotide sequence encoding a first chemically-induced signaling complex (CISC) component comprising: (a) an extracellular binding domain comprising the FKBP-rapamycin binding (FRB) domain of mTOR or an extracellular binding domain derived from the FKBP-rapamycin binding (FRB) domain of mTOR; (b) a transmembrane domain comprising the transmembrane domain of IL-2Rβ or a transmembrane domain derived from the transmembrane domain of IL-2Rβ; and (c) an intracellular signaling domain comprising the cytoplasmic domain of IL-2Rβ or an intracellular signaling domain derived from the cytoplasmic domain of IL-2Rβ; (ii) a nucleotide sequence encoding a cytosolic FRB domain without a transmembrane domain; and (iii) a 3' homology arm having homology to a sequence downstream of the Treg-specific demethylation region in the FOXP3 locus (e.g., a 3' homology arm having homology to a sequence contained within 2,000 nucleotides upstream of exon 2, the first coding exon of the FOXP3 gene, or a sequence up to this 2,000 nucleotides); The promoter is operably linked to the By inserting each sequence and promoter downstream of the TSDR, which destabilizes FOXP3 expression under inflammatory conditions, the inserted promoter can initiate transcription of mRNA encoding FoxP3, independently of the endogenous FOXP3 promoter upstream of the TSDR. By inserting each sequence and promoter into the FOXP3 locus, transcription of each sequence operably linked to the promoter is initiated by this promoter, resulting in expression of the FRB-Il2Rβ CISC component, the cytosolic FRB component, and FoxP3 from the FOXP3 locus.

[0413] Cells dual-edited by inserting one of the two nucleic acids into the TRAC locus and the other into the FOXP3 locus contain (i) a first CISC component and a second CISC component that heterodimerize in the presence of rapamycin to transduce IL-2R signaling via dimerization of the cytoplasmic domain of IL-2Rβ and the intracellular domain of IL-2Rγ; (ii) a cytosolic FRB domain that binds to intracellular rapamycin and prevents its interaction with mTOR; (iii) FoxP3, which confers a stable Treg phenotype; and (iv) stably express a T1D-associated antigen-specific TCR. Thus, the system described herein provides stable Treg cells specific for T1D-associated antigens, which can be induced to proliferate using rapamycin. Furthermore, because the nucleotide sequences encoding the first and second CISC components are separated into separate nucleic acids, rapamycin can be used to selectively induce the proliferation of cells expressing both CISC components (i.e., cells expressing a T1D antigen-specific TCR and FoxP3 due to the insertion of both nucleic acids). Thus, dual-edited cells can be readily selected and expanded in vitro to produce stable Treg cell populations specific for T1D-associated antigens for the treatment of type 1 diabetes. Furthermore, the in vivo engraftment and expansion of such stable Treg cells may be supported by administering rapamycin to a subject.

[0414] promoter Each nucleic acid for targeted insertion into a cell genome using the systems described herein includes a promoter operably linked to one or more nucleotide sequences on the respective nucleic acid. A promoter is "operably linked" to a sequence if it is capable of initiating transcription of the operably linked sequence (e.g., by recruiting RNA polymerase). The promoters of the first and second nucleic acids can be any promoter known in the art. In some embodiments, a heterologous promoter on the introduced nucleic acid is active and promotes RNA transcription, even under inflammatory conditions. In some embodiments, the promoter is a constitutive promoter. A constitutive promoter can be a strong promoter that promotes transcription more efficiently than an endogenous promoter, or a weak promoter that promotes transcription less efficiently than a strong promoter or an endogenous promoter. In some embodiments, the constitutive promoter is a strong promoter. In some embodiments, the heterologous promoter is an inducible promoter. An inducible promoter promotes transcription of an operably linked sequence in response to the presence of an activating signal or the absence of an inhibitory signal. In some embodiments, the inducible promoter is inducible by a drug or steroid.

[0415] In some embodiments, the promoter of the first nucleic acid and the promoter of the second nucleic acid for insertion into the cell genome are different promoters. In another embodiment, the first nucleic acid and the second nucleic acid comprise the same promoter. In some embodiments, both the first nucleic acid and the second nucleic acid comprise an MND promoter. In embodiments in which the first nucleic acid and the second nucleic acid comprise the same promoter, the promoter sequences of these nucleic acids may be the same. Alternatively, the promoter sequence of the first nucleic acid may comprise one or more mutations (e.g., insertions, deletions, substitutions) compared to the promoter sequence of the second nucleic acid. In some embodiments, the MND promoter of the first nucleic acid and / or the second nucleic acid has at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO: 220. In some embodiments, the MND promoter of the first nucleic acid and / or the second nucleic acid has at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 220. In some embodiments, the first nucleic acid and the second nucleic acid each comprise an MND promoter having the nucleic acid sequence of SEQ ID NO: 220.

[0416] In some embodiments, a stop codon is present upstream of or within the first five nucleotides of the promoter on the first nucleic acid for insertion into the TRAC locus. In some embodiments, a stop codon is present upstream of or within the first five nucleotides of the promoter on the second nucleic acid for insertion into the FOXP3 locus. The presence of a stop codon upstream of, within, or overlapping the first five nucleotides of the promoter is expected to terminate translation of mRNA that may be transcribed from upstream of the endogenous promoter of the recombined TRAC locus or FOXP3 locus, thereby suppressing expression of the inserted coding sequence (e.g., a sequence encoding a CISC component, a heterologous TCR β chain or TCR α chain, or FoxP3) under the control of the endogenous promoter. In some embodiments, the stop codon is in frame with one or more upstream start codons, such that mRNA produced by transcription from the upstream endogenous promoter is not translated beyond the stop codon.

[0417] Chemical-inducible signaling complex (CISC) In embodiments of the system for producing genetically engineered cells described herein, each nucleic acid for insertion into the genome of a cell comprises a nucleotide sequence encoding a component of a chemically-induced signaling complex (CISC), each CISC component comprising an extracellular domain that binds rapamycin, a transmembrane domain, and an intracellular domain that comprises, or is derived from, the cytoplasmic domain of the interleukin-2 receptor (IL-2R). In some embodiments, the first nucleic acid (for insertion into the TRAC locus) encodes a first CISC component comprising: (i) an extracellular binding domain comprising an FK506-binding protein 12 (FKBP) domain; (ii) a transmembrane domain comprising or derived from the transmembrane domain of IL-2Rγ; and (iii) an intracellular domain comprising or derived from the cytoplasmic domain of IL-2Rγ; and the second nucleic acid (for insertion into the FOXP3 locus) encodes a first CISC component comprising: (i) an extracellular binding domain comprising an FKBP-rapamycin binding domain; (ii) a transmembrane domain comprising or derived from the transmembrane domain of IL-2Rβ; and (iii) an intracellular domain comprising or derived from the cytoplasmic domain of IL-2Rβ. A domain of a CISC component (e.g., the transmembrane domain of a first CISC component) is "derived from" a particular domain of an IL-2R polypeptide (e.g., IL-2Rγ) if it has at least 90% sequence identity with the wild-type (naturally occurring) amino acid sequence of that domain of that IL-2R polypeptide (e.g., the transmembrane domain of native IL-2Rγ).

[0418] By expressing CISC components in cells and manipulating the presence, absence, and / or concentration of rapamycin, IL-2 signaling can be selectively induced in the cells. In this manner, controllably inducing signaling allows, for example, IL-2 signaling events to induce cell proliferation, thereby selectively expanding cells expressing both CISC components. In some embodiments, in which two nucleic acids encoding different CISC components are introduced into cells, contacting cells containing only one CISC component with rapamycin will not induce dimerization or IL-2 signaling due to the absence of the other CISC component, allowing for such selective expansion to select for cells containing both nucleic acids.

[0419] Examples of intracellular signaling domains include, but are not limited to, the IL-2Rβ cytoplasmic domain and the IL-2Rγ cytoplasmic domain and functional derivatives thereof. In some embodiments, the intracellular signaling domain of a first CISC component comprises an IL-2Rγ domain or a functional derivative thereof, and the intracellular signaling domain of a second CISC component comprises an IL-2Rβ cytoplasmic domain or a functional derivative thereof. In some embodiments, dimerization of the first CISC component and the second CISC component induces phosphorylation of JAK1, JAK3, and / or STAT5 in cells. In some embodiments, dimerization of the first CISC component and the second CISC component induces cell proliferation.

[0420] Examples of transmembrane domains include, but are not limited to, the transmembrane domain of IL-2Rβ and the transmembrane domain of IL-2Rγ, and functional derivatives thereof. In some embodiments, the transmembrane domain of a CISC component is derived from the same protein as the protein from which the intracellular signaling domain of that CISC component is derived (e.g., a CISC component comprising the intracellular domain of IL-2Rβ comprises the transmembrane domain of IL-2Rβ). In some embodiments, one CISC component comprises the transmembrane domain of IL-2Rβ, and the other CISC component comprises the transmembrane domain of IL-2Rγ.

[0421] Examples of extracellular binding domains capable of binding to rapamycin include, but are not limited to, an FK506-binding protein (FKBP) domain and an FKBP-rapamycin binding (FRB) domain. The FKBP and FRB domains can bind to rapamycin or a rapalog, as described below, to form heterodimers. In some embodiments, the extracellular binding domain of one CISC component comprises an FKBP domain, and the extracellular binding domain of the other CISC component comprises an FRB domain. In some embodiments, these CISC components form heterodimers in the presence of rapamycin. In some embodiments, the FRB domain contains a threonine at the position corresponding to amino acid 2098 of wild-type mTOR having the amino acid sequence of SEQ ID NO: 236. This amino acid mutation increases the affinity of mTOR for compounds structurally related to rapamycin, but decreases the affinity of mTOR for rapamycin itself. Thus, the presence of a threonine at this position maintains the binding ability of mTOR to rapamycin. The amino acid in a CISC component or FRB domain that "corresponds to" amino acid 2098 of wild-type mTOR may be determined by aligning a candidate sequence for a CISC component or FRB domain to SEQ ID NO:236 (e.g., by BLAST alignment algorithm or another alignment algorithm known in the art), and the amino acid that aligns to amino acid 2098 of SEQ ID NO:236 is the amino acid that "corresponds to" amino acid 2098 of SEQ ID NO:236.

[0422] The extracellular binding domain, transmembrane domain, and intracellular signaling domain of a CISC component described herein may each be connected to another domain of that CISC component via a linker. Linkers are known in the art. In some embodiments, the linker comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids (e.g., glycine), a number of amino acids (e.g., glycine), or a range between any two of these numbers. In some embodiments, the glycine spacer comprises at least three glycines. In some embodiments, the glycine spacer comprises a sequence set forth as GSG, GGGS (SEQ ID NO: 229), GGGSGGG (SEQ ID NO: 230), or GGG. In some embodiments, the glycine spacer comprises the amino acid sequence GSG.

[0423] The extracellular binding domain may be connected to the transmembrane domain via a hinge domain. "Hinge" refers to a domain that links the extracellular binding domain to the transmembrane domain and may thereby provide flexibility to the extracellular binding domain. In some embodiments, the hinge domain positions the extracellular binding domain in the vicinity of the cell membrane, minimizing the possibility of recognition by an antibody or binding fragment thereof. In some embodiments, the extracellular binding domain is located N-terminal to the hinge domain. In some embodiments, the hinge domain may be natural or synthetic.

[0424] In some embodiments, the first and second CISC components form a heterodimer in the presence of rapamycin. In some embodiments, the first and second CISC components form a heterodimer in the presence of a compound produced in vivo by metabolism of a rapalog. In some embodiments, the compound produced in vivo by metabolism of a rapalog is rapamycin. Examples of rapalogs include, but are not limited to, everolimus, CCI-779, C20-methallylrapamycin, C16-(S)-3-methylindolerapamycin, C16-iRap, C16-(S)-7-methylindolerapamycin, AP21967, C16-(S)-butylsulfonamidorapamycin, AP23050, mycophenolate sodium, benidipine hydrochloride, AP1903, and AP23573, as well as metabolites and derivatives thereof.

[0425] In some embodiments, the nucleic acid encoding the second CISC component (FRB-IL2Rβ) further comprises a nucleotide sequence encoding a third CISC component capable of binding rapamycin. Such a CISC component is useful, for example, for binding intracellular rapamycin, thereby preventing rapamycin bound to the CISC component from interacting with other intracellular molecules or structures (e.g., preventing rapamycin from interacting with mTOR). In some embodiments, the third CISC component is a soluble protein that does not contain a transmembrane domain. In some embodiments, the third CISC component comprises an intracellular FRB domain. In some embodiments, the third CISC component is a soluble protein that contains an FRB domain but not a transmembrane domain.

[0426] The nucleic acid encoding the first CISC component, the nucleic acid encoding the second CISC component, and / or the nucleic acid encoding the third CISC component may be contained in one or more vectors. In some embodiments, the nucleic acid encoding the first CISC component is contained in a vector separate from the vector containing the nucleic acid encoding the second CISC component. In some embodiments, the nucleic acid encoding the third CISC component is contained in the same vector as the vector containing the nucleic acid encoding the second CISC component. In some embodiments, the one or more vectors are viral vectors. In some embodiments, the one or more vectors are adeno-associated virus (AAV) vectors. In some embodiments, the one or more AAV vectors are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11 vectors. In some embodiments, the one or more AAV vectors are AAV5 vectors. In some embodiments, the one or more AAV vectors are AAV6 vectors.

[0427] In some embodiments, the CISC component comprises an amino acid sequence having at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or up to 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 66 or 71. In some embodiments, one or more CISC components further comprise a signal peptide. The signal peptide may be any signal peptide known in the art that transports the translated CISC component to the cell membrane. In some embodiments, both the first CISC component and the second CISC component comprise the signal peptide of LCN2. In some embodiments, both the first CISC component and the second CISC component comprise a signal peptide comprising the amino acid sequence of SEQ ID NO: 61.

[0428] In some embodiments, one CISC component comprises an amino acid sequence having at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or up to 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 66, and the other CISC component comprises an amino acid sequence having at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or up to 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 71. In some embodiments, each CISC component further comprises a signal peptide, which may have the same or different amino acid sequences. These signal peptides may be any signal peptide known in the art that transports the translated CISC component to the cell membrane.

[0429] In some embodiments, the third CISC component comprises an amino acid sequence having at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or up to 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 72. In some embodiments, the third CISC component consists of an amino acid sequence having at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or up to 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 72. In some embodiments, the third CISC component comprises the amino acid sequence of SEQ ID NO: 72. In some embodiments, the third CISC component consists of the amino acid sequence of SEQ ID NO: 72. In some embodiments, the third CISC component does not comprise a signal peptide. In some embodiments, the third CISC component does not include a transmembrane domain.

[0430] T cell receptor (TCR) In some embodiments of the systems described herein, the TRAC locus of a cell is edited by inserting a nucleic acid comprising a promoter operably linked to a nucleotide sequence encoding a full-length TCRβ protein and a nucleotide sequence encoding at least a portion of a TCRα protein, e.g., the TCRα variable region and TCRα junction (TRAJ) region that form the portion of the TCRα protein responsible for antigen specificity. In some embodiments, the nucleotide sequence encoding the TCRα variable and junction regions is inserted in frame with an endogenous nucleotide sequence encoding a portion of the TCRα constant domain, such that the inserted heterologous promoter initiates transcription of the sequence encoding the heterologous TCRβ protein and the sequence encoding the TCRα protein comprising the heterologous TRAV / TRAJ amino acid sequence and the endogenous TCRα constant domain. This embodiment utilizes the endogenous 3' regulatory region of the endogenous TRAC locus.

[0431] The genetically engineered cells produced by the systems described herein express a T cell receptor specific for type 1 diabetes (T1D). The T cell receptors expressed by the genetically engineered cells described herein are described in the section entitled "Methods for Producing Genetically Engineered Cells" and the subheading "T Cell Receptors (TCRs)." In certain embodiments, the nucleic acid encoding the TCR is codon-optimized for improved expression in certain host cells, such as, for example, immune system cells, hematopoietic stem cells, T cells, primary T cells, T cell lines, NK cells, and natural killer T cells. See, e.g., Scholten et al., Clin Immunol. 2006. 119:135.

[0432] In some embodiments, the nucleic acids described herein encode at least a portion of a TCR β chain and a TCR α chain, which are expressed in combination to form a T1D2 TCR that binds to the IGRP(305-234) peptide. In another embodiment, the full-length TCR α chain and TCR β chain, portions of which are encoded by the nucleic acids described herein, form a T1D4 TCR that binds to the IGRP(241-260) peptide. In another embodiment, the full-length TCR α chain and TCR β chain, portions of which are encoded by the nucleic acids described herein, form a T1D5-1 TCR that binds to the IGRP(305-324) peptide. In some embodiments, the IGRP(305-324) peptide is recognized when bound to HLA-DRB1*0401. In some embodiments, the IGRP(241-260) peptide is recognized when bound to HLA-DRB1*0401.

[0433] In some embodiments, the TCR formed from (at least a portion of) a TCR β chain and a TCR α chain encoded by the nucleic acid described herein comprises a TCR α variable (Vα) domain having three complementarity determining regions (CDRs), αCDR1, αCDR2, and αCDR3, and a TCR β variable (Vβ) domain having three CDRs, βCDR1, βCDR2, and βCDR3. Representative amino acids of the CDRs of the TCRs described herein are shown in Table 1, and nucleotide sequences encoding the CDRs of the TCRs described herein are shown in Table 2. In some embodiments, (i) αCDR1 comprises SEQ ID NO: 1, (ii) αCDR2 comprises SEQ ID NO: 2, (iii) αCDR3 comprises SEQ ID NO: 3, (iv) βCDR1 comprises SEQ ID NO: 4, (v) βCDR2 comprises SEQ ID NO: 5, and (vi) βCDR3 comprises SEQ ID NO: 6. In some embodiments, (i) αCDR1 comprises SEQ ID NO: 11, (ii) αCDR2 comprises SEQ ID NO: 12, (iii) αCDR3 comprises SEQ ID NO: 13, (iv) βCDR1 comprises SEQ ID NO: 14, (v) βCDR2 comprises SEQ ID NO: 15, and (vi) βCDR3 comprises SEQ ID NO: 16. In some embodiments, (i) αCDR1 comprises SEQ ID NO: 21, (ii) αCDR2 comprises SEQ ID NO: 22, (iii) αCDR3 comprises SEQ ID NO: 23, (iv) βCDR1 comprises SEQ ID NO: 24, (v) βCDR2 comprises SEQ ID NO: 25, and (vi) βCDR3 comprises SEQ ID NO: 26. In another embodiment, each set consisting of αCDR1, αCDR2, αCDR3, βCDR1, βCDR2 and βCDR3 may have an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with each amino acid sequence shown in any of the above amino acid sequence combinations.

[0434] In some embodiments, Vα comprises SEQ ID NO: 7 and Vβ comprises SEQ ID NO: 8. In some embodiments, Vα comprises SEQ ID NO: 17 and Vβ comprises SEQ ID NO: 18. In some embodiments, Vα comprises SEQ ID NO: 27 and Vβ comprises SEQ ID NO: 28. In other embodiments, each pair of Vα and Vβ may have an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a respective amino acid sequence set forth in any of the above amino acid sequence combinations.

[0435] In some embodiments, the TCR alpha chain comprises SEQ ID NO: 9 and the TCR beta chain comprises SEQ ID NO: 10. In some embodiments, the TCR alpha chain comprises SEQ ID NO: 19 and the TCR beta chain comprises SEQ ID NO: 20. In some embodiments, the TCR alpha chain comprises SEQ ID NO: 29 and the TCR beta chain comprises SEQ ID NO: 30. In another embodiment, each pair of TCR alpha and TCR beta chains may have an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to each amino acid sequence set forth in any of the above amino acid sequence combinations.

[0436] Recombination at the FOXP3 locus In some embodiments of the systems described herein, the nucleic acid for targeted insertion into the FOXP3 locus contains a promoter, which is operably linked to a nucleotide sequence encoding a portion of the endogenous FoxP3 protein after insertion of the nucleic acid. The inserted promoter is introduced into the genome downstream of the Treg-specific demethylation region (TSDR) of the FOXP3 locus. In non-recombined cells, the TSDR epigenetically regulates FoxP3 expression, thereby suppressing FoxP3 production in cells exposed to inflammatory conditions, resulting in the loss of FoxP3 expression and the conversion of non-recombined Treg cells to a T effector (Teff) phenotype. Inserting a promoter downstream of the TSDR bypasses TSDR-mediated regulation of FOXP3 expression, allowing stable production of FoxP3 even under inflammatory conditions.

[0437] The heterologous promoter can be inserted anywhere downstream of the endogenous promoter of the FOXP3 coding sequence (e.g., downstream of the TSDR) and upstream of or within the first coding exon (known in the art as exon 2 because it is the second exon present in the pre-mRNA transcribed from the endogenous FOXP3 promoter, and known as the first coding exon because it is not exon 1 (the first exon in the pre-mRNA encoding FOXP3), which contains the start codon that initiates translation of wild-type FoxP3). In some embodiments, the heterologous promoter is 1 to 10,000 nucleotides downstream, 10 to 1,000 nucleotides downstream, 10 to 100 nucleotides downstream, 10 to 5,000 nucleotides downstream, 20 to 4,000 nucleotides downstream, 30 to 3,000 nucleotides downstream, 40 to 2,000 nucleotides downstream, 50 to 1,000 nucleotides downstream, 60 to 750 nucleotides downstream, 70 to 500 nucleotides downstream, 80 to 400 nucleotides downstream, 90 to 300 nucleotides downstream of the TSDR of the FOXP3 locus. The insertion may be 100 to 200 nucleotides downstream, 1 to 1,000 nucleotides downstream, 1,000 to 2,000 nucleotides downstream, 2,000 to 3,000 nucleotides downstream, 3,000 to 4,000 nucleotides downstream, 4,000 to 5,000 nucleotides downstream, 5,000 to 6,000 nucleotides downstream, 6,000 to 7,000 nucleotides downstream, 7,000 to 8,000 nucleotides downstream, 8,000 to 9,000 nucleotides downstream, or 9,000 to 10,000 nucleotides downstream.In some embodiments, the heterologous promoter is located between 1 and 10,000 nucleotides upstream, between 10 and 1,000 nucleotides upstream, between 100 nucleotides upstream, between 10 and 5,000 nucleotides upstream, between 20 and 4,000 nucleotides upstream, between 30 and 3,000 nucleotides upstream, between 40 and 2,000 nucleotides upstream, between 50 and 1,000 nucleotides upstream, between 60 and 750 nucleotides upstream, between 70 and 500 nucleotides upstream, between 80 and 400 nucleotides upstream, between 90 and 1000 nucleotides upstream, between 10 and 1500 nucleotides upstream, between 15 and 20 ... In some embodiments, the heterologous promoter is inserted between 100 and 200 nucleotides upstream, 1 and 1,000 nucleotides upstream, 1,000 and 2,000 nucleotides upstream, 2,000 and 3,000 nucleotides upstream, 3,000 and 4,000 nucleotides upstream, 4,000 and 5,000 nucleotides upstream, 5,000 and 6,000 nucleotides upstream, 6,000 and 7,000 nucleotides upstream, 7,000 and 8,000 nucleotides upstream, 8,000 and 9,000 nucleotides upstream, or 9,000 and 10,000 nucleotides upstream. In some embodiments, the heterologous promoter is inserted between the first coding exon to create a synthetic first coding exon that is distinct from the endogenous first coding exon but contains an initiation codon in-frame with the FOXP3 sequence encoding the downstream FOXP3 exon.

[0438] 2A motif and linker Some embodiments of the nucleic acids described herein that encode multiple polypeptides or portions thereof can include an intervening nucleotide sequence encoding a 2A motif. 2A motifs are known in the art and are useful for translating and facilitating the production of multiple polypeptides from a single nucleotide sequence. See, e.g., Kim et al., PLoS ONE. 2011. 6:e18556. In some embodiments, the 2A motif is translated, which results in self-cleavage of the polypeptide, releasing the separate polypeptides. In another embodiment, the presence of a nucleotide sequence encoding a 2A motif allows the ribosome to skip over the amino acids of the encoded 2A motif as it translates along the mRNA, thereby releasing a first polypeptide (e.g., a first FKBP-IL2Rγ CISC component) and initiating translation of a second polypeptide (e.g., a TCR β chain).

[0439] In some embodiments, in each pair of nucleic acid sequences, the nucleotide sequence encoding the 2A motif is positioned in frame between (i) the nucleotide sequence encoding the first (FKBP-IL2Rγ) CISC component, (ii) the nucleotide sequence encoding the TCR β chain, and (iii) the nucleotide sequence encoding the TCR α chain or a portion thereof. Thus, when a heterologous promoter (e.g., the MND promoter) initiates transcription of a single mRNA encoding each of the CISC components, the TCR β chain, and the TCR α chain, and containing an intervening 2A motif, the CISC components, the TCR β chain, and the TCR α chain are each produced as separate polypeptides. In some embodiments, in each pair of nucleic acid sequences, the nucleotide sequence encoding the 2A motif is positioned in frame between (i) the nucleotide sequence encoding the second (FKBP-IL2Rγ) CISC component, (ii) the nucleotide sequence encoding the cytosolic FRB domain, and (iii) the nucleotide sequence encoding FoxP3. Thus, when a heterologous promoter (e.g., the MND promoter) initiates transcription of a single mRNA encoding each of the CISC components, the cytosolic FRB domain, and FoxP3, and containing an intervening 2A motif, the CISC components, the cytosolic FRB domain, and FoxP3 are each produced as separate polypeptides.

[0440] In each pair of nucleic acid sequences, the 2A motif encoded by the nucleotide sequence located between the nucleotide sequences encoding two polypeptides (e.g., between the sequence encoding the FKBP-IL2Rγ CISC component and the sequence encoding the TCR β chain; and between the sequence encoding the TCR β chain and the sequence encoding a portion of the TCR α chain) may be any 2A motif known in the art. In some embodiments, the 2A motifs encoded between the nucleotide sequences encoding separate polypeptides in each pair of nucleic acid sequences may be independently selected from the group consisting of F2A, P2A, T2A, and E2A. In some embodiments, the first and second 2A motifs encoded on a single nucleic acid are different types of 2A motifs. Using different types of 2A motifs in a single inserted nucleic acid can reduce the probability of internal recombination. If internal recombination occurs, the nucleotide sequence between the recombined 2A motifs may be excised from the chromosome. In some embodiments, the nucleotide sequence encoding the first 2A motif on a single nucleic acid has 90% or less sequence identity with the nucleotide sequence encoding the second 2A motif on that nucleic acid. In some embodiments, the nucleotide sequence encoding a first 2A motif on a nucleic acid has 80% or less sequence identity with the nucleotide sequence encoding a second 2A motif on that nucleic acid. In some embodiments, the nucleotide sequence encoding a first 2A motif on a nucleic acid has 70% or less sequence identity with the nucleotide sequence encoding a second 2A motif on that nucleic acid. In some embodiments, the nucleotide sequence encoding a first 2A motif on a nucleic acid has 60% or less sequence identity with the nucleotide sequence encoding a second 2A motif on that nucleic acid. In some embodiments, the nucleotide sequence encoding a first 2A motif on a nucleic acid has 50% or less sequence identity with the nucleotide sequence encoding a second 2A motif on that nucleic acid. In some embodiments, the first 2A motif is a T2A motif and the second motif is a P2A motif.

[0441] In another embodiment, the first and second 2A motifs encoded by the nucleotide sequences on a single nucleic acid are the same type of 2A motif. In some embodiments, a single nucleic acid comprises a nucleotide sequence encoding a first P2A motif and a second nucleotide sequence encoding a second P2A motif, and the nucleotide sequence encoding the first P2A motif has at least 80% sequence identity with the nucleotide sequence encoding the second P2A motif. In some embodiments, the first nucleotide sequence encoding the first P2A motif and the second nucleotide sequence encoding the second P2A motif comprise the same nucleotide sequence.

[0442] In some embodiments, the nucleic acid for insertion into the TRAC locus (i) comprises a sequence encoding a T2A motif between the sequence encoding the first CISC component and the sequence encoding the TCR beta chain, and (ii) comprises a sequence encoding a P2A motif between the sequence encoding the TCR beta chain and the sequence encoding a portion of the heterologous TCR alpha chain.

[0443] In some embodiments, the nucleic acid for insertion into the FOXP3 locus comprises (i) a sequence encoding a P2A motif between the sequence encoding the second CISC component and the sequence encoding the cytosolic FRB domain, and (ii) a second sequence encoding a second P2A motif between the sequence encoding the cytosolic FRB domain and the sequence encoding FoxP3.

[0444] In some embodiments, the polypeptide (e.g., a CISC component and / or a TCR β chain) encoded by the nucleic acid for insertion into a cell genome comprises a C-terminal linker. Incorporation of a C-terminal linker may, for example, improve cleavage efficiency at the 2A motif and / or prevent amino acids of the encoded CISC component or TCR β chain from being excised upon cleavage of the 2A motif. In some embodiments, the encoded first CISC component comprises a C-terminal linker. In some embodiments, the encoded second CISC component comprises a C-terminal linker. In some embodiments, the encoded cytosolic FRB domain component comprises a C-terminal linker. In some embodiments, the encoded TCR β chain comprises a C-terminal linker.

[0445] The C-terminal linker of the encoded polypeptide can be any linker known in the art. In some embodiments, the C-terminal linker comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids (e.g., glycine), a number of amino acids (e.g., glycine), or a range of amino acids between any two of these numbers. In some embodiments, the C-terminal linker comprises at least three glycines. In some embodiments, the C-terminal linker comprises the sequence GSG, GGGS (SEQ ID NO: 229), GGGSGGG (SEQ ID NO: 230), or GGG. In some embodiments, the C-terminal linker comprises the amino acid sequence GSG. In some embodiments, the first CISC component, the second CISC component, the cytosolic FRB domain, and the TCR β chain all comprise C-terminal linkers having the amino acid sequence GSG.

[0446] vector The first nucleic acid for insertion into the TRAC locus and / or the second nucleic acid for insertion into the FOXP3 locus can be contained in one or more vectors.In some embodiments, the first nucleic acid for targeting the TRAC locus is contained in a first vector, and the nucleic acid for targeting the FOXP3 locus is contained in a second vector.In some cases, these vectors are packaged in a virus that can infect cells (for example, the vector is a viral vector).Typical viruses include adenoviruses, retroviruses, lentiviruses, adeno-associated viruses, and other viruses known in the art and disclosed herein.

[0447] The term "vector" is used to refer to any molecule (e.g., nucleic acid or plasmid) or molecular arrangement (e.g., virus) used to transfer coding information into a host cell. An "expression vector" refers to a vector suitable for introduction into a host cell and containing a nucleic acid sequence that induces and / or controls the expression of an introduced heterologous nucleic acid sequence. Expression includes, but is not limited to, processes such as transcription, translation, and RNA splicing (if introns are present). Examples of vectors include, but are not limited to, artificial chromosomes, minigenes, cosmids, plasmids, phagemids, and viral vectors. Examples of viral vectors include, but are not limited to, lentiviral vectors, retroviral vectors, herpesvirus vectors, adenoviral vectors, and adeno-associated viral vectors. In some embodiments, one or more vectors containing a nucleic acid for use in the systems provided herein are lentiviral vectors. In some embodiments, the one or more vectors are adenoviral vectors. In some embodiments, the one or more vectors are adeno-associated viral (AAV) vectors. In some embodiments, the one or more AAV vectors are AAV1 vectors, AAV2 vectors, AAV3 vectors, AAV4 vectors, AAV5 vectors, AAV6 vectors, AAV7 vectors, AAV8 vectors, AAV9 vectors, AAV10 vectors, or AAV11 vectors. In some embodiments, the vector comprising a nucleic acid for insertion into the TRAC locus is an AAV1 vector, AAV2 vectors, AAV3 vectors, AAV4 vectors, AAV5 vectors, AAV6 vectors, AAV7 vectors, AAV8 vectors, AAV9 vectors, AAV10 vectors, or AAV11 vectors. In some embodiments, the vector comprising a nucleic acid for insertion into the FOXP3 locus is an AAV1 vector, AAV2 vectors, AAV3 vectors, AAV4 vectors, AAV5 vectors, AAV6 vectors, AAV7 vectors, AAV8 vectors, AAV9 vectors, AAV10 vectors, or AAV11 vectors.

[0448] In some embodiments, the one or more AAV vectors are AAV5 vectors. In some embodiments, the one or more AAV vectors are AAV6 vectors. In some embodiments, the first nucleic acid and the second nucleic acid are each contained in a separate AAV5 vector. In some embodiments, the first nucleic acid and the second nucleic acid are each contained in a separate AAV6 vector.

[0449] In some embodiments, a nucleic acid for insertion into the TRAC locus comprises a nucleotide sequence between the 5' and 3' homology arms that has at least 90% sequence identity to any of SEQ ID NOs: 94, 106, 117, 128, and 139. In some embodiments, the nucleotide sequence has at least 95% sequence identity to any of SEQ ID NOs: 94, 106, 117, 128, and 139. In some embodiments, the nucleotide sequence comprises any of SEQ ID NOs: 94, 106, 117, 128, and 139. In some embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 94. In some embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 106. In some embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 117. In some embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 128. In some embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 139.

[0450] In some embodiments, a nucleic acid for insertion into the TRAC locus has at least 90% sequence identity to the nucleotide sequence set forth in any of SEQ ID NOs: 95, 107, 118, 129, and 140. In some embodiments, the nucleic acid has at least 95% sequence identity to the nucleotide sequence set forth in any of SEQ ID NOs: 95, 107, 118, 129, and 140. In some embodiments, the nucleic acid comprises the nucleotide sequence set forth in any of SEQ ID NOs: 95, 107, 118, 129, and 140. In some embodiments, the nucleic acid has at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 95. In some embodiments, the nucleic acid has at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 107. In some embodiments, the nucleic acid has at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 118. In some embodiments, the nucleic acid has at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 129. In some embodiments, the nucleic acid has at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 140. In some embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 95. In some embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 107. In some embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 118. In some embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 129. In some embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 140.

[0451] In some embodiments, a nucleic acid for insertion into the FOXP3 locus comprises a nucleotide sequence between the 5' homology arm and the 3' homology arm that has at least 90% sequence identity to any of SEQ ID NOs: 150, 161, 172, 184, 195, 206, and 218. In some embodiments, the nucleotide sequence has at least 95% sequence identity to any of SEQ ID NOs: 150, 161, 172, 184, 195, 206, and 218. In some embodiments, the nucleotide sequence comprises any of SEQ ID NOs: 150, 161, 172, 184, 195, 206, and 218. In some embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 150. In some embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 161. In some embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 172. In some embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 184. In some embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 195. In some embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 206. In some embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 218.

[0452] In some embodiments, a nucleic acid for insertion into the FOXP3 locus has at least 90% sequence identity to the nucleotide sequence set forth in any of SEQ ID NOs: 151, 162, 173, 185, 196, 207, and 219. In some embodiments, the nucleic acid has at least 95% sequence identity to the nucleotide sequence set forth in any of SEQ ID NOs: 151, 162, 173, 185, 196, 207, and 219. In some embodiments, the nucleic acid comprises the nucleotide sequence set forth in any of SEQ ID NOs: 151, 162, 173, 185, 196, 207, and 219. In some embodiments, the nucleic acid has at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 151. In some embodiments, the nucleic acid has at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 162. In some embodiments, the nucleic acid has at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 173. In some embodiments, the nucleic acid has at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 185. In some embodiments, the nucleic acid has at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 196. In some embodiments, the nucleic acid has at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 207. In some embodiments, the nucleic acid has at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 219. In some embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 151. In some embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 162. In some embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 173. In some embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 185. In some embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 196. In some embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 207. In some embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 219.

[0453] Homologous armsNucleic acids for insertion into the TRAC locus and the FOXP3 locus using the systems described herein each contain a 5' homology arm and a 3' homology arm, respectively, that target insertion of the nucleic acid into the TRAC locus or the FOXP3 locus by homology-directed repair after the introduction of a double-strand break. When considering the coding strand of a nucleic acid (i.e., the nucleic acid strand containing the reading frame encoding a polypeptide containing a CISC component, a TCR chain, and FoxP3), the 5' homology arm typically refers to a homology arm at the 5' end of the nucleic acid, and the 3' homology arm typically refers to another homology arm at the 3' end of the nucleic acid. The 5' homology arm is homologous to a first sequence at the target locus, and the 3' homology arm is homologous to a second sequence downstream of the first sequence at the target locus, thereby inserting the nucleic acid into the locus in a targeted manner. After insertion of the nucleic acid, the modified target locus contains a 5' homologous arm and a 3' homologous arm in place of the first and second sequences, and the sequence that was present between the homologous arms on the nucleic acid is replaced by the sequence that was present between the first and second sequences at the target locus. The 5' homologous arm and the 3' homologous arm may be the same length, may be of similar length (within a 100 bp difference in length), or may be of different lengths. In some embodiments, the length of one or both of the 5' homologous arm and the 3' homologous arm is 100 to 2,000 bp, 400 to 1,500 bp, or 500 to 1,000 bp. In some embodiments, the length of one or both of the 5' homologous arm and the 3' homologous arm is about 100 bp, about 200 bp, about 300 bp, about 400 bp, about 500 bp, about 600 bp, about 700 bp, about 800 bp, about 900 bp, about 1,000 bp, about 1,100 bp, about 1,200 bp, about 1,300 bp, about 1,400 bp, about 1,500 bp, about 1,600 bp, about 1,700 bp, about 1,800 bp, about 1,900 bp, or about 2,000 bp. In some embodiments, the length of both homologous arms is 100 to 2,000 nucleotides. In some embodiments, the length of both homologous arms is 300 to 1,000 nucleotides. In some embodiments, the length of both homologous arms is 300 to 700 nucleotides.In some embodiments, the length of both homologous arms is 300 to 500 nucleotides, in some embodiments, the length of both homologous arms is 500 to 700 nucleotides, in some embodiments, the length of both homologous arms is 700 to 1,000 nucleotides.

[0454] The homologous arm of a nucleic acid for insertion into a targeted genomic locus may be selected based on homologous sequences located upstream and / or downstream of the target site cleaved by a nuclease at the target locus. For example, in some embodiments in which a nucleic acid is inserted by cleavage at a specific position (cleavage site) of the target locus followed by homologous recombination repair, the 5' homologous arm of the nucleic acid for insertion is homologous to a sequence upstream of the cleavage site, and the 3' homologous arm of the nucleic acid for insertion is homologous to a sequence downstream of the cleavage site. In some embodiments, the 5' homologous arm is homologous to a 100-2,000 nucleotide sequence terminating 25-5,000 nucleotides, 50-3,000 nucleotides, 75-2,000 nucleotides, 100-1,000 nucleotides, or 150-500 nucleotides upstream of the cleavage site. In some embodiments, the 5' homologous arm has homology to a 100-2,000 nucleotide sequence terminating 25-5,000 nucleotides, 50-3,000 nucleotides, 75-2,000 nucleotides, 100-1,000 nucleotides, or 150-500 nucleotides upstream of the PAM sequence cleaved by the RNA-guided nuclease. In some embodiments, the 5' homologous arm has homology to a 100-2,000 nucleotide sequence terminating 25-5,000 nucleotides, 50-3,000 nucleotides, 75-2,000 nucleotides, 100-1,000 nucleotides, or 150-500 nucleotides upstream of the genomic sequence complementary to the spacer sequence of the gRNA. In some embodiments, the 5' homologous arm has homology to a 100-2,000 nucleotide sequence terminating 150-500 nucleotides upstream of the cleavage site. In some embodiments, the 5' homologous arm has homology to a 100-2,000 nucleotide sequence that terminates 150-500 nucleotides upstream of the PAM sequence cleaved by the RNA-guided nuclease. In some embodiments, the 5' homologous arm has homology to a 100-2,000 nucleotide sequence that terminates 150-500 nucleotides upstream of the genomic sequence complementary to the spacer sequence of the gRNA.

[0455] In some embodiments, the 3' homologous arm has homology to a 100-2,000 nucleotide sequence that terminates 25-5,000 nucleotides, 50-3,000 nucleotides, 75-2,000 nucleotides, 100-1,000 nucleotides, or 150-500 nucleotides upstream of the cleavage site. In some embodiments, the 3' homologous arm has homology to a 100-2,000 nucleotide sequence that terminates 25-5,000 nucleotides, 50-3,000 nucleotides, 75-2,000 nucleotides, 100-1,000 nucleotides, or 150-500 nucleotides upstream of the PAM sequence cleaved by the RNA-guided nuclease. In some embodiments, the 3' homology arm has homology to a 100-2,000 nucleotide sequence that terminates 25-5,000 nucleotides, 50-3,000 nucleotides, 75-2,000 nucleotides, 100-1,000 nucleotides, or 150-500 nucleotides upstream of the genomic sequence complementary to the spacer sequence of the gRNA. In some embodiments, the 3' homology arm has homology to a 100-2,000 nucleotide sequence that terminates 150-500 nucleotides upstream of the cleavage site. In some embodiments, the 3' homology arm has homology to a 100-2,000 nucleotide sequence that terminates 150-500 nucleotides upstream of the PAM sequence that is cleaved by the RNA-guided nuclease. In some embodiments, the 3' homology arm has homology to a 100-2,000 nucleotide long sequence that terminates 150-500 nucleotides upstream of the genomic sequence complementary to the spacer sequence of the gRNA.

[0456] In some embodiments, when the system of the present invention includes a gRNA containing a spacer sequence, the 5' and 3' homologous arms of the nucleic acid for insertion into the genome do not contain sequences complementary to this spacer sequence. In such embodiments, the absence of a complementary sequence on the donor template can reduce the likelihood that the gRNA will bind to and cleave the donor template, and cleavage of the donor template by such a gRNA can reduce the efficiency of insertion into the genome. In some embodiments, the donor template does not contain a sequence complementary to the spacer sequence. In embodiments in which a different type of nuclease is used that does not require a gRNA for targeted cleavage, the donor template does not contain a sequence that can be cleaved by the nuclease.

[0457] In some embodiments, a nucleic acid for insertion into the TRAC locus comprises a 5' homology arm having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 85 and a 3' homology arm having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 93. In some embodiments, the 5' homology arm has at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 85, and the 3' homology arm has at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 93. In some embodiments, the 5' homology arm comprises the nucleotide sequence of SEQ ID NO: 85, and the 3' homology arm comprises the nucleotide sequence of SEQ ID NO: 93.

[0458] In some embodiments, a nucleic acid for insertion into the TRAC locus comprises a 5' homology arm having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 96 and a 3' homology arm having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 105. In some embodiments, the 5' homology arm has at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 96, and the 3' homology arm has at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 105. In some embodiments, the 5' homology arm comprises the nucleotide sequence of SEQ ID NO: 96, and the 3' homology arm comprises the nucleotide sequence of SEQ ID NO: 105.

[0459] In some embodiments, a nucleic acid for insertion into the TRAC locus comprises a 5' homology arm having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 108 and a 3' homology arm having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 116. In some embodiments, the 5' homology arm has at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 108, and the 3' homology arm has at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 116. In some embodiments, the 5' homology arm comprises the nucleotide sequence of SEQ ID NO: 108, and the 3' homology arm comprises the nucleotide sequence of SEQ ID NO: 116.

[0460] In some embodiments, a nucleic acid for insertion into the TRAC locus comprises a 5' homology arm having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 119 and a 3' homology arm having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 127. In some embodiments, the 5' homology arm has at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 119, and the 3' homology arm has at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 127. In some embodiments, the 5' homology arm comprises the nucleotide sequence of SEQ ID NO: 119, and the 3' homology arm comprises the nucleotide sequence of SEQ ID NO: 127.

[0461] In some embodiments, a nucleic acid for insertion into the TRAC locus comprises a 5' homology arm having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 130 and a 3' homology arm having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 138. In some embodiments, the 5' homology arm has at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 130, and the 3' homology arm has at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 138. In some embodiments, the 5' homology arm comprises the nucleotide sequence of SEQ ID NO: 130, and the 3' homology arm comprises the nucleotide sequence of SEQ ID NO: 138.

[0462] In some embodiments, a nucleic acid for insertion into the FOXP3 locus comprises a 5' homology arm having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 141 and a 3' homology arm having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 149. In some embodiments, the 5' homology arm has at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 141, and the 3' homology arm has at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 149. In some embodiments, the 5' homology arm comprises the nucleotide sequence of SEQ ID NO: 141, and the 3' homology arm comprises the nucleotide sequence of SEQ ID NO: 149.

[0463] In some embodiments, a nucleic acid for insertion into the FOXP3 locus comprises a 5' homology arm having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 152 and a 3' homology arm having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 160. In some embodiments, the 5' homology arm has at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 152, and the 3' homology arm has at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 160. In some embodiments, the 5' homology arm comprises the nucleotide sequence of SEQ ID NO: 152, and the 3' homology arm comprises the nucleotide sequence of SEQ ID NO: 160.

[0464] In some embodiments, a nucleic acid for insertion into the FOXP3 locus comprises a 5' homology arm having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 163 and a 3' homology arm having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 171. In some embodiments, the 5' homology arm has at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 163, and the 3' homology arm has at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 171. In some embodiments, the 5' homology arm comprises the nucleotide sequence of SEQ ID NO: 163, and the 3' homology arm comprises the nucleotide sequence of SEQ ID NO: 171.

[0465] In some embodiments, a nucleic acid for insertion into the FOXP3 locus comprises a 5' homology arm having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 174 and a 3' homology arm having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 183. In some embodiments, the 5' homology arm has at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 174, and the 3' homology arm has at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 183. In some embodiments, the 5' homology arm comprises the nucleotide sequence of SEQ ID NO: 174, and the 3' homology arm comprises the nucleotide sequence of SEQ ID NO: 183.

[0466] In some embodiments, a nucleic acid for insertion into the FOXP3 locus comprises a 5' homology arm having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 186 and a 3' homology arm having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 194. In some embodiments, the 5' homology arm has at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 186, and the 3' homology arm has at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 194. In some embodiments, the 5' homology arm comprises the nucleotide sequence of SEQ ID NO: 186, and the 3' homology arm comprises the nucleotide sequence of SEQ ID NO: 194.

[0467] In some embodiments, a nucleic acid for insertion into the FOXP3 locus comprises a 5' homology arm having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 197 and a 3' homology arm having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 205. In some embodiments, the 5' homology arm has at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 197 and the 3' homology arm has at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 205. In some embodiments, the 5' homology arm comprises the nucleotide sequence of SEQ ID NO: 197 and the 3' homology arm comprises the nucleotide sequence of SEQ ID NO: 205.

[0468] In some embodiments, a nucleic acid for insertion into the FOXP3 locus comprises a 5' homology arm having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 208 and a 3' homology arm having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 217. In some embodiments, the 5' homology arm has at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 208, and the 3' homology arm has at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 217. In some embodiments, the 5' homology arm comprises the nucleotide sequence of SEQ ID NO: 208, and the 3' homology arm comprises the nucleotide sequence of SEQ ID NO: 217.

[0469] Nucleases and guide RNAs Some embodiments of the present disclosure relate to the use of nucleases to introduce double-strand breaks into the nucleic acid of a cell genome to edit the genome at a desired locus (for example, to facilitate the insertion of a donor template into the desired locus by homologous recombination repair).To achieve the editing of one or more genomic loci (for example, TRAC and / or FOXP3), any of a number of gene editing methods, gene editing systems, genome editing methods, and genome editing systems can be used.Examples of gene editing methods include, but are not limited to, the use of DNA endonucleases such as RNA-guided nucleases (for example, Cas nucleases (for example, Cas9 nucleases)), zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and meganucleases; transposon-mediated gene editing; serine integrase-mediated gene editing; and lentivirus-mediated gene editing.

[0470] In certain embodiments, chromosomal gene knockout or chromosomal gene knock-in (e.g., insertion) 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 capable of catalyzing the cleavage of phosphodiester bonds within a polynucleotide chain. "DNA endonuclease" refers to an endonuclease capable of catalyzing the cleavage of phosphodiester bonds within a DNA polynucleotide. In some embodiments, the endonuclease can cleave a nucleic acid sequence at a target locus to facilitate the insertion of an exogenous nucleic acid sequence into the target locus by homologous recombination. The endonuclease can be a natural endonuclease, a recombinant endonuclease, a genetic recombination endonuclease, or a fusion endonuclease. Examples of endonucleases used in gene editing include zinc finger nucleases (ZFNs), TALE nucleases (TALENs), RNA-guided nucleases, CRISPR-Cas nucleases, meganucleases, and megaTALs.

[0471] Nucleic acid strand breaks caused by endonucleases are typically double-strand breaks (DSBs), which are 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 that often results in changes at the break site in the DNA sequence, such as a substitution, deletion, or addition of at least one nucleotide. Non-homologous end joining (NHEJ) can also be used to "knock out" a target gene. When a double-strand break occurs, the presence of a donor template facilitates homology-directed repair (HDR).

[0472] 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 inserted 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.

[0473] 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.

[0474] The gene editing systems and methods described herein may utilize viral or non-viral vectors or cassettes and nucleases capable of site- or locus-specific gene editing. Such nucleases include RNA-guided nucleases, Cas nucleases (e.g., Cpf1 nuclease and Cas9 nuclease), meganucleases, TALENs, or ZFNs. Specific RNA-guided nucleases useful in some embodiments provided herein are disclosed in U.S. Patent No. 11,162,114, which is expressly incorporated herein by reference in its entirety. Examples of Cas nucleases include, but are not limited to, SpCas9, SaCas9, CjCas9, xCas9, C2c1, Cas13a / C2c2, C2c3, Cas13b, Cpf1, and variants thereof. Certain features useful for some embodiments provided herein are disclosed in WO2019 / 210057, which is expressly incorporated herein by reference in its entirety.

[0475] As used herein, the term "clustered regularly interspaced short palindromic repeats / Cas (CRISPR / Cas or 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 several types (e.g., type I, type II, type III, and type V) 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 deliver a transgene flanked by homologous sequences in a donor template to 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).Furthermore, the region of the guide RNA that is complementary to the target site can be modified or programmed to target the desired sequence (Xie et al., PLOS One 9:e100448, 2014; US Patent Application Publication No. 2014 / 0068797; US Patent Application Publication No. 2014 / 0186843; US Patent No. 8,697,359; and PCT Publication WO 2015 / 071474; all of which are incorporated herein by reference). Examples of CRISPR / Cas nucleases include, but are not limited to, Cas9, SaCas9, CjCas9, xCas9, C2C1, Cas13a / C2c2, C2c3, Cas13b, Cpf1, and variants thereof. Furthermore, the gene editing methods and gene editing systems described herein may use other RNA-guided nucleases that can introduce double-strand breaks in DNA by cleaving at the PAM sequence adjacent to the target sequence in the DNA in the presence of a guide RNA containing a spacer sequence complementary to the target sequence. In some embodiments, the RNA-guided nuclease is a nuclease having a protospacer adjacent motif (PAM) sequence represented by 5'-NNNNCC-3' (i.e., a nuclease that cleaves dsDNA at the PAM sequence). Exemplary RNA-guided nucleases having a PAM sequence represented by NNNNCC are described, for example, in International Application PCT / US2019 / 035373, published as PCT Publication WO2019 / 236566 (these documents are incorporated herein by reference in their entirety). In some embodiments, an RNA-guided nuclease localizes to the DNA of a target sequence in the presence of a gRNA having the nucleotide sequence of SEQ ID NO: 237 and cleaves the DNA at the PAM sequence designated NGG, where the poly-N portion of SEQ ID NO: 237 is a proto-spacer sequence complementary to the target DNA sequence. In some embodiments, an RNA-guided nuclease localizes to the DNA of a target sequence in the presence of a gRNA having the nucleotide sequence of SEQ ID NO: 238 and cleaves the DNA at the PAM sequence designated NNNNCC, where the poly-N portion of SEQ ID NO: 238 is a proto-spacer sequence complementary to the target DNA sequence.In some embodiments, the RNA-guided nuclease, in the presence of a gRNA having the nucleotide sequence of SEQ ID NO: 239, localizes to the DNA of the target sequence and cleaves the DNA at the PAM sequence designated NNNNCC, where the polyN portion of SEQ ID NO: 239 is a protospacer sequence complementary to the target DNA sequence.

[0476] In some embodiments, the gene knockout or inactivation comprises an insertion, deletion, mutation, or a combination thereof, introduced using an RNA-guided nuclease. Exemplary gRNA sequences and methods using the sequences for knocking out endogenous genes encoding proteins in immune cells include those described in Ren et al., Clin Cancer Res. 2017. 23(9):2255-2266, all of which are expressly incorporated herein by reference in their entirety.

[0477] In some embodiments, genetic modification involves inserting an exogenous nucleic acid sequence (e.g., a heterologous promoter, a transgene, and / or a combination thereof) into the cellular genome, wherein an RNA-guided nuclease introduces a double-stranded break in the cellular genome, and the exogenous nucleic acid sequence is introduced into the cellular genome by homology-directed repair.

[0478] In some embodiments, the genetic modification comprises inserting an exogenous nucleic acid (e.g., a donor template) into the TRAC locus of the cell genome, the donor template comprising a 5' homologous arm and a 3' homologous arm, each homologous arm having homology to a nucleotide sequence at the TRAC locus, thereby introducing a double-stranded break at the TRAC locus prior to insertion of the exogenous nucleic acid into the TRAC locus. In some embodiments, in the presence of the gRNA, an RNA-guided nuclease introduces a double-stranded break at the PAM sequence designated NGG. In some embodiments, in the presence of the gRNA, an RNA-guided nuclease introduces a double-stranded break at the PAM sequence designated NNNNCC.

[0479] In some embodiments, the 5' homology arm comprises a nucleotide sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO: 85, and the 3' homology arm comprises a nucleotide sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO: 93. In some embodiments, the 5' homology arm comprises the nucleic acid sequence of SEQ ID NO: 85, and the 3' homology arm comprises the nucleic acid s...

Claims

1. 1. A method for producing genetically modified CD4+ regulatory T (Treg) cells, comprising: contacting a first nucleic acid and a second nucleic acid with a CD4+ T cell; (i) the first nucleic acid is (a) a first 5' homology arm having homology to a first nucleic acid sequence at the TRAC locus of the genome of the cell; (b) the first MND promoter; (c) a nucleotide sequence encoding a first chemical-inducible signaling complex (CISC) component comprising: (1) an extracellular binding domain comprising the rapamycin-binding domain of FK506-binding protein 12 (FKBP); (2) a transmembrane domain of IL-2Rγ; and (3) an intracellular domain comprising the cytoplasmic domain of IL-2Rγ or a functional fragment thereof; (d) a nucleotide sequence encoding the TCR β chain of a TCR specific for the type 1 diabetes (T1D)-associated peptide IGRP; (e) a nucleotide sequence encoding at least a portion of a TCR alpha chain, wherein the at least a portion of the TCR alpha chain comprises a TCR alpha variable (TRAV) region and a TCR alpha joining (TRAJ) region, and is in frame with the constant region of an endogenous TCR alpha, such that the TCR alpha chain and the TCR beta chain together form an IGRP-specific TCR, which is expressed from the TRAC locus; a nucleotide sequence in which the TCR alpha chain comprises an alpha CDR1 having the amino acid sequence of SEQ ID NO: 1, an alpha CDR2 having the amino acid sequence of SEQ ID NO: 2, and an alpha CDR3 having the amino acid sequence of SEQ ID NO: 3, and the TCR beta chain comprises a beta CDR1 having the amino acid sequence of SEQ ID NO: 4, a beta CDR2 having the amino acid sequence of SEQ ID NO: 5, and a beta CDR3 having the amino acid sequence of SEQ ID NO: 6; and (f) a first 3' homology arm having homology to a second nucleic acid sequence at the TRAC locus downstream of the first nucleic acid sequence at the TRAC locus; Including, the first MND promoter is operably linked to a nucleotide sequence set forth in (i)(c), (i)(d), and (i)(e); and (ii) the second nucleic acid is (a) a second 5' homology arm having homology to a first nucleic acid sequence at a FOXP3 locus in the genome of the cell; (b) a second MND promoter; (c) a nucleotide sequence encoding a second CISC component comprising: (1) an extracellular binding domain comprising the FKBP-rapamycin binding (FRB) domain of mTOR; (2) a transmembrane domain of IL-2Rβ; and (3) a cytoplasmic domain of IL-2Rβ or a functional fragment thereof; (d) a nucleotide sequence encoding a cytosolic FRB domain that binds rapamycin and does not contain a transmembrane domain; and (e) a second 3' homologous arm having homology to a second nucleic acid sequence at the FOXP3 locus downstream of the first nucleic acid sequence at the FOXP3 locus and downstream of a Treg-specific demethylation region (TSDR) at the FOXP3 locus; Including, The method, wherein the second MND promoter is operably linked to the nucleotide sequence described in (ii)(c), the nucleotide sequence described in (ii)(d), and the first coding exon of the FOXP3 locus.

2. The TCR alpha chain comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 9, and the TCR beta chain comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:

10. The method of claim 1.

3. The method of claim 1, comprising contacting the CD4+ T cells with an RNA-guided DNA endonuclease, a first guide RNA (gRNA) comprising a spacer sequence that targets the TRAC locus, and a second gRNA comprising a spacer sequence that targets the FOXP3 locus.

4. 4. The method of claim 3, wherein the RNA-guided DNA endonuclease is Cas9.

5. The method described in claim 1, characterized in that the genetically modified Treg suppresses the proliferation of IGRP-specific effector T cells.

6. A genetically modified Treg cell produced by the method of claim 1.

7. The method described in claim 1, wherein the first CISC component and the second CISC component form a heterodimer in the presence of rapamycin.

8. The method described in claim 7, further comprising a step of contacting the genetically modified Treg with rapamycin.

9. A genetically modified Treg cell population produced by the method of claim 8.

10. A genetically engineered regulatory T (Treg) cell, comprising: a first nucleic acid inserted into a TRAC locus in the genome of a cell; and a second nucleic acid inserted into a FOXP3 locus in the genome of the cell; (i) the TRAC locus into which the first nucleic acid has been inserted is (a) the first MND promoter; (b) an exogenous nucleotide sequence encoding a first chemical-induced signaling complex (CISC) component comprising: (1) an extracellular binding domain comprising the rapamycin-binding domain of FK506-binding protein 12 (FKBP); (2) a transmembrane domain of IL-2Rγ; and (3) an intracellular domain comprising the cytoplasmic domain of IL-2Rγ or a functional fragment thereof; (c) an exogenous nucleotide sequence encoding an exogenous TCR β chain of a TCR specific for the type 1 diabetes (T1D)-associated peptide IGRP; and (d) an exogenous nucleotide sequence encoding at least a portion of a TCR alpha chain, wherein the at least a portion of the TCR alpha chain comprises a TCR alpha variable (TRAV) region and a TCR alpha joining (TRAJ) region, and is in frame with the endogenous TCR alpha constant region, such that the TCR alpha chain and the TCR beta chain together form an IGRP-specific TCR, which is expressed from the TRAC locus; an exogenous nucleotide sequence, wherein the TCR α chain comprises an αCDR1 having the amino acid sequence of SEQ ID NO: 1, an αCDR2 having the amino acid sequence of SEQ ID NO: 2, and an αCDR3 having the amino acid sequence of SEQ ID NO: 3, and the TCR β chain comprises a βCDR1 having the amino acid sequence of SEQ ID NO: 4, a βCDR2 having the amino acid sequence of SEQ ID NO: 5, and a βCDR3 having the amino acid sequence of SEQ ID NO: 6; Including, the first MND promoter is operably linked to a nucleotide sequence according to (i)(b), (i)(c), and (i)(d); and (ii) the FOXP3 locus into which the second nucleic acid has been inserted is (a) the second MND promoter; (b) a nucleotide sequence encoding a second CISC component comprising: (1) an extracellular binding domain comprising the FKBP-rapamycin binding (FRB) domain of mTOR; (2) a transmembrane domain of IL-2Rβ; and (3) a cytoplasmic domain of IL-2Rβ or a functional fragment thereof; and (c) a nucleotide sequence encoding a cytosolic FRB domain that binds rapamycin and does not contain a transmembrane domain; Including, A cell, wherein the second MND promoter is inserted downstream of the Treg-specific demethylation region (TSDR) of the FOXP3 locus and is operably linked to the nucleotide sequence described in (ii)(b), the nucleotide sequence described in (ii)(c), and the first coding exon of FOXP3 of the FOXP3 locus.

11. The TCR α chain comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:9, and the TCR β chain comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:

10. The genetically modified Treg cell of claim 10.

12. A genetically modified Treg cell as described in claim 10, wherein the first CISC component and the second CISC component form a heterodimer in the presence of rapamycin.

13. A pharmaceutical composition comprising the genetically modified Treg cells of claim 6, or the genetically modified Treg cells of any one of claims 10 to 12, or the genetically modified Treg cell population of claim 9, and a pharmaceutically acceptable excipient.

14. The pharmaceutical composition of claim 13, which is a pharmaceutical composition for treating type 1 diabetes in a subject in need thereof.

15. The pharmaceutical composition of claim 14, wherein the genetically modified Treg cells are autologous cells of the subject.

16. The pharmaceutical composition of claim 14, wherein the treatment of type 1 diabetes further comprises administering rapamycin to the subject.

17. 15. The pharmaceutical composition of claim 14, wherein the subject is a human.

18. The pharmaceutical composition described in claim 14, characterized in that the genetically modified Treg suppresses the proliferation of IGRP-specific effector T cells in the subject.

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