Engineered repair regulatory t cells

Abstract

Some aspects of the disclosure relate to compositions and methods for producing regulatory T cells (Tregs) with repair phenotypes by modifying cells to promote expression of at least one repair mediator.

Description

[0001] ENGINEERED REPAIR REGULATORY T CELLS

[0002] RELATED APPLICATIONS

[0003] This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63 / 441,717, filed January 27, 2023; U.S. Provisional Application No. 63 / 460,824, filed April 20, 2023; U.S. Provisional Application No. 63 / 469,824, filed May 30, 2023; and U.S. Provisional Application No. 63 / 592,137, filed October 20, 2023, the contents of each of which are incorporated by reference herein in their entirety.

[0004] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0005] The contents of the electronic Sequence Listing (G097170030WO00-SEQ-NTJ.xml; Size: 239,777 bytes; and Date of Creation: January 25, 2024) are herein incorporated by reference in their entirety.

[0006] BACKGROUND

[0007] Cellular therapies using regulatory T cells (Tregs) may be useful to treat numerous types of diseases including acute and chronic inflammatory diseases or conditions.

[0008] SUMMARY

[0009] Regulatory T cells (Tregs) having a repair phenotype (repair Tregs) play an important role in mediating inflammation control and tissue repair in acute and chronic inflammatory diseases and conditions. Endogenous human Tregs having this phenotype are present only at very low levels in the blood, and therefore cannot be effectively obtained in therapeutic numbers. In addition, endogenous Tregs may convert to an effector T cell (Teff) phenotype when exposed to inflammatory conditions.

[0010] Therefore, some aspects relate to compositions and methods for improved methods of producing repair Tregs and repair Treg populations for therapeutic use. In some embodiments, compositions and methods can stabilize repair Treg phenotypes, enhancing their therapeutic potential as cell therapies by increasing their potency and mitigating risks related to instability and potential transdifferentiation to effector T cell phenotypes. In some embodiments, compositions and methods can also be used to induce repair Treg populations in vivo, e.g., through transduction, transfection, or gene editing of endogenous T cells.

[0011] Compositions and methods are based, at least in part, on the recognition that enhancement of IL-33 and / or IL-18 signaling imparts a repair Treg phenotype. Some embodiments of methods involve enhancing IL-33 and / or IL- 18 signaling by engineering a cell (e.g., Treg) to express an IL-33 receptor (e.g., ST2 and / or IL1RAP) and / or an IL-18 receptor e.g., IL-18R1 (IL-18Ra) and / or IL18RAP (IL-18RP)), or a functional derivative of such a receptor. In some embodiments, a cell is engineered to constitutively express ST2, IL1RAP, IL-18R1, and / or IL18RAP or a functional derivative thereof. In some embodiments, a cell is engineered to express ST2, IL1RAP, IL-18R1, and / or IL18RAP or a functional derivative thereof transiently. In some embodiments, a cell is engineered to express ST2, IL1RAP, IL-18R1 and / or IL18RAP or a functional derivative thereof in a controllable manner (e.g., through use of an inducible promoter). In some embodiments, the cell is engineered to have a Treg phenotype. In contrast to sorting-based methods, which are limited by the number of Tregs available in a sample, such methods are scalable, enabling large-scale manufacturing of Tregs for therapeutic use. Ex vivo engineering of Tregs can be achieved using any source of Treg or immune cell made into a Treg cell, such as Tregs isolated from a donor, or Tregs induced from other autologous or allogeneic populations of cells (e.g., bulk peripheral T cells comprising CD3+, CD4+, and / or CD8+ cells). In some embodiments, the cell is a sorted Treg. Use of sorted Tregs has the advantage of limiting the number of modifications to a cell, as cells that already exhibit a Treg phenotype can be engineered to have a repair Treg phenotype.

[0012] Accordingly, some aspects relate to a cell that constitutively expresses interleukin 1 receptor-like 1 (ST2). Some aspects relate to a cell that constitutively expresses interleukin- 1 receptor accessory protein (IL1RAP). Some aspects relate to a cell that constitutively expresses interleukin 1 receptor-like 1 (ST2) and interleukin- 1 receptor accessory protein (IL1RAP).

[0013] Some aspects relate to a cell constitutively expressing a supraphysiologic level of interleukin 1 receptor-like 1 (ST2). Some aspects relate to a cell constitutively expressing a supraphysiologic level of interleukin- 1 receptor accessory protein (IL1RAP). Some aspects relate to a cell constitutively expressing a supraphysiologic level of interleukin 1 receptor-like 1 (ST2), and a supraphysiologic level of interleukin- 1 receptor accessory protein (IL1RAP).

[0014] Some aspects relate to a cell comprising a nucleic acid, wherein the nucleic acid comprises a unidirectional constitutive promoter operably linked to a nucleotide sequence encoding interleukin 1 receptor-like 1 (ST2). Some aspects relate to a cell comprising a nucleic acid, wherein the nucleic acid comprises a unidirectional constitutive promoter operably linked to a nucleotide sequence encoding interleukin- 1 receptor accessory protein (IL1RAP). Some aspects relate to a cell comprising: (i) a first nucleic acid comprising a first unidirectional constitutive promoter operably linked to a nucleotide sequence encoding interleukin 1 receptor-like 1 (ST2); and (ii) a second nucleic acid comprising a second unidirectional constitutive promoter operably linked to a nucleotide sequence encoding interleukin-1 receptor accessory protein (IL1RAP).

[0015] Some aspects relate to a cell comprising a heterologous promoter operably linked to an endogenous nucleotide sequence encoding interleukin 1 receptor-like 1 (ST2). Some aspects relate to a cell comprising a heterologous promoter operably linked to an endogenous nucleotide sequence encoding interleukin-1 receptor accessory protein (IL1RAP). Some aspects relate to a cell comprising: (i) a first nucleic acid comprising a first heterologous promoter operably linked to an endogenous nucleotide sequence encoding interleukin 1 receptor-like 1 (ST2); and (ii) a second nucleic acid comprising a second heterologous promoter operably linked to an endogenous nucleotide sequence encoding interleukin- 1 receptor accessory protein (IL 1 RAP).

[0016] Some aspects relate to a method comprising contacting a cell with a nucleic acid comprising a unidirectional constitutive promoter operably linked to a nucleotide sequence encoding interleukin 1 receptor-like 1 (ST2). Some aspects relate to a method comprising contacting a cell with a nucleic acid comprising a unidirectional constitutive promoter operably linked to a nucleotide sequence encoding interleukin- 1 receptor accessory protein (IL1RAP). Some aspects relate to a method comprising contacting a cell with: (i) a first nucleic acid comprising a first unidirectional constitutive promoter operably linked to a nucleotide sequence encoding interleukin 1 receptor-like 1 (ST2); and (ii) a second nucleic acid comprising a second unidirectional constitutive promoter operably linked to a nucleotide sequence encoding interleukin-1 receptor accessory protein (IL1RAP).

[0017] Some aspects relate to a method comprising contacting a cell with a nucleic acid comprising heterologous promoter, wherein the heterologous promoter is inserted into a nucleic acid of the cell genome, such that the heterologous promoter is operably linked to an endogenous nucleotide sequence encoding interleukin 1 receptor-like 1 (ST2). Some aspects relate to a method comprising contacting a cell with a nucleic acid comprising heterologous promoter, wherein the heterologous promoter is inserted into a nucleic acid of the cell genome, such that the heterologous promoter is operably linked to an endogenous nucleotide sequence encoding interleukin-1 receptor accessory protein (IL1RAP). Some aspects relate to a method comprising contacting a cell with a first nucleic acid comprising a first heterologous promoter and a second nucleic acid comprising a second heterologous promoter, wherein: (i) the first heterologous promoter is inserted into a first nucleic acid of the cell genome, such that the first heterologous promoter is operably linked to an endogenous nucleotide sequence encoding interleukin 1 receptor-like 1 (ST2); and (ii) the second heterologous promoter is inserted into a second nucleic acid of the cell genome, such that the second heterologous promoter is operably linked to an endogenous nucleotide sequence encoding interleukin- 1 receptor accessory protein (IL 1 RAP).

[0018] Some aspects relate to a cell that constitutively expresses interleukin 18 receptor 1 (IL-18R1). Some aspects relate to a cell that constitutively expresses interleukin 18 receptor accessory protein (IL18RAP). Some aspects relate to a cell that constitutively expresses interleukin 18 receptor 1 (IL-18R1) and interleukin 18 receptor accessory protein (IL18RAP).

[0019] Some aspects relate to a cell constitutively expressing a supraphysiologic level of interleukin 18 receptor 1 (IL-18R1). Some aspects relate to a cell constitutively expressing a supraphysiologic level of interleukin 18 receptor accessory protein (IL18RAP). Some aspects relate to a cell constitutively expressing a supraphysiologic level of interleukin 18 receptor 1 (IL-18R1), and a supraphysiologic level of interleukin 18 receptor accessory protein (IL18RAP).

[0020] Some aspects relate to a cell comprising a nucleic acid, wherein the nucleic acid comprises a unidirectional constitutive promoter operably linked to a nucleotide sequence encoding interleukin 18 receptor 1 (IL-18R1). Some aspects relate to a cell comprising a nucleic acid, wherein the nucleic acid comprises a unidirectional constitutive promoter operably linked to a nucleotide sequence encoding interleukin- 18 receptor accessory protein (IL18RAP). Some aspects relate to a cell comprising: (i) a first nucleic acid comprising a first unidirectional constitutive promoter operably linked to a nucleotide sequence encoding interleukin 18 receptor 1 (IL-18R1); and (ii) a second nucleic acid comprising a second unidirectional constitutive promoter operably linked to a nucleotide sequence encoding interleukin 18 receptor accessory protein (IL18RAP).

[0021] Some aspects relate to a cell comprising a heterologous promoter operably linked to an endogenous nucleotide sequence encoding interleukin 18 receptor 1 (IL-18R1). Some aspects relate to a cell comprising a heterologous promoter operably linked to an endogenous nucleotide sequence encoding interleukin 18 receptor accessory protein (IL18RAP). Some aspects relate to a cell comprising: (i) a first nucleic acid comprising a first heterologous promoter operably linked to an endogenous nucleotide sequence encoding interleukin 18 receptor 1 (IL-18R1); and (ii) a second nucleic acid comprising a second heterologous promoter operably linked to an endogenous nucleotide sequence encoding interleukin 18 receptor accessory protein (IL18RAP). Some aspects relate to a method comprising contacting a cell with a nucleic acid comprising a unidirectional constitutive promoter operably linked to a nucleotide sequence encoding interleukin 18 receptor 1 (IL-18R1). Some aspects relate to a method comprising contacting a cell with a nucleic acid comprising a unidirectional constitutive promoter operably linked to a nucleotide sequence encoding interleukin 18 receptor accessory protein (IL18RAP). Some aspects relate to a method comprising contacting a cell with: (i) a first nucleic acid comprising a first unidirectional constitutive promoter operably linked to a nucleotide sequence encoding interleukin 18 receptor 1 (IL-18R1); and (ii) a second nucleic acid comprising a second unidirectional constitutive promoter operably linked to a nucleotide sequence encoding interleukin 18 receptor accessory protein (IL18RAP). Some aspects relate to a method comprising contacting a cell with a nucleic acid comprising heterologous promoter, wherein the heterologous promoter is inserted into a nucleic acid of the cell genome, such that the heterologous promoter is operably linked to an endogenous nucleotide sequence encoding interleukin 18 receptor 1 (IL-18R1). Some aspects relate to a method comprising contacting a cell with a nucleic acid comprising heterologous promoter, wherein the heterologous promoter is inserted into a nucleic acid of the cell genome, such that the heterologous promoter is operably linked to an endogenous nucleotide sequence encoding interleukin 18 receptor accessory protein (IL18RAP). Some aspects relate to a method comprising contacting a cell with a first nucleic acid comprising a first heterologous promoter and a second nucleic acid comprising a second heterologous promoter, wherein: (i) the first heterologous promoter is inserted into a first nucleic acid of the cell genome, such that the first heterologous promoter is operably linked to an endogenous nucleotide sequence encoding interleukin 18 receptor 1 (IL-18R1); and (ii) the second heterologous promoter is inserted into a second nucleic acid of the cell genome, such that the second heterologous promoter is operably linked to an endogenous nucleotide sequence encoding interleukin 18 receptor accessory protein (IL18RAP).

[0022] Some aspects relate to a membrane-bound interleukin-33 (mem-IL-33) protein comprising an interleukin-33 (IL-33) protein and a transmembrane domain. In some embodiments, the mem-IL-33 protein comprises a linker connecting the IL-33 protein to the transmembrane domain. In some embodiments, the linker is a glycine or glycine-serine linker. In some embodiments, the mem-IL-33 protein further comprises a hinge domain. In some embodiments, the hinge domain comprises a polypeptide selected from the group consisting of: a hinge region of CD8a, a hinge region of a heavy chain of IgG, a hinge region of a heavy chain of IgD, and an extracellular region of an IL-10R 0 chain. In some embodiments, the transmembrane domain comprises a transmembrane anchor. In some embodiments, the transmembrane anchor comprises a CD94 anchor.

[0023] Some aspects relate to a nucleic acid comprising an open reading frame (ORF) encoding the mem-IL-33. In some embodiments, the nucleic acid further comprises a promoter operably linked to the ORF. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is an MND promoter. Some aspects relate to a vector comprising the nucleic acid encoding the mem-IL-33. In some embodiments, the vector is a lentiviral vector. In some embodiments, the vector is an adeno-associated viral (AAV) vector. Some aspects relate to a lipid nanoparticle comprising the nucleic acid.

[0024] Some aspects relate to a cell comprising the mem-IL-33. Some aspects relate to a cell comprising the nucleic acid encoding the mem-IL-33. Some aspects relate to a method comprising contacting a cell with the nucleic acid, vector, or lipid nanoparticle.

[0025] Some aspects relate to a receptor-linked interleukin-33 (rlIL-33) polypeptide comprising: (a) an interleukin-33 (IL-33) protein; (b) a linker; and (c) an interleukin 1 receptor-like 1 (ST2) or interleukin- 1 receptor accessory protein (IL1RAP).

[0026] In some embodiments, polypeptide comprises IL-33 and ST2, wherein the IL-33 and ST2 are connected by the linker. In some embodiments, polypeptide comprises IL-33 and IL1RAP, wherein the IL-33 and IL1RAP are connected by the linker. In some embodiments, the linker is a glycine or glycine-serine linker. In some embodiments, the rIL-33 further comprises a hinge domain. In some embodiments, the hinge domain comprises a polypeptide selected from the group consisting of: a hinge region of CD8a, a hinge region of a heavy chain of IgG, a hinge region of a heavy chain of IgD, and an extracellular region of an IL- 10R P chain.

[0027] Some aspects relate to a nucleic acid comprising an open reading frame (ORF) encoding the rIL-33. In some embodiments, the nucleic acid further comprises a promoter operably linked to the ORF. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is an MND promoter.

[0028] Some aspects relate to a vector comprising the nucleic acid encoding the rIL-33. In some embodiments, the vector is a lentiviral vector. In some embodiments, the vector is an adeno-associated viral (AAV) vector. Some aspects relate to a lipid nanoparticle comprising the nucleic acid encoding the rIL-33. Some aspects relate to a cell comprising the rIL-33. Some aspects relate to a cell comprising the nucleic acid encoding the rIL-33. Some aspects relate to a method comprising contacting a cell with the nucleic acid, vector, or lipid nanoparticle.

[0029] Some aspects relate to a method for producing a population of regulatory T (Treg) cells having a repair phenotype, the method comprising culturing a population of Treg cells in the presence of interleukin-33 (IL-33).

[0030] In some embodiments, the Treg cells are cultured in the presence of 0.1 to 500 ng / mL IL-33. In some embodiments, the Treg cells are cultured in the presence of 50 ng / mL IL-33.

[0031] Some aspects relate to a membrane-bound interleukin- 18 (mem-IL-18) protein comprising an interleukin- 18 (IL- 18) protein and a transmembrane domain. In some embodiments, the mem-IL-18 protein comprises a linker connecting the IL- 18 protein to the transmembrane domain. In some embodiments, the linker is a glycine or glycine-serine linker. In some embodiments, the mem-IL-18 protein further comprises a hinge domain. In some embodiments, the hinge domain comprises a polypeptide selected from the group consisting of: a hinge region of CD8a, a hinge region of a heavy chain of IgG, a hinge region of a heavy chain of IgD, and an extracellular region of an IL-10R 0 chain. In some embodiments, the transmembrane domain comprises a transmembrane anchor. In some embodiments, the transmembrane anchor comprises a CD94 anchor.

[0032] Some aspects relate to a nucleic acid comprising an open reading frame (ORF) encoding the mem-IL-18. In some embodiments, the nucleic acid further comprises a promoter operably linked to the ORF. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is an MND promoter. Some aspects relate to a vector comprising the nucleic acid encoding the mem-IL-18. In some embodiments, the vector is a lentiviral vector. In some embodiments, the vector is an adeno-associated viral (AAV) vector. Some aspects relate to a lipid nanoparticle comprising the nucleic acid.

[0033] Some aspects relate to a cell comprising the mem-IL-18. Some aspects relate to a cell comprising the nucleic acid encoding the mem-IL-18. Some aspects relate to a method comprising contacting a cell with the nucleic acid, vector, or lipid nanoparticle.

[0034] Some aspects relate to a receptor-linked interleukin- 18 (rlIL-18) polypeptide comprising: (a) an interleukin- 18 (IL-18) protein; (b) a linker; and (c) an interleukin 18 receptor 1 (IL-18R1) or interleukin- 18 receptor accessory protein (IL18RAP).

[0035] In some embodiments, the polypeptide comprises IL- 18 and IL-18R1, wherein the IL- 18 and IL-18R1 are connected by the linker. In some embodiments, polypeptide comprises IL- 18 and IL18RAP, wherein the IL- 18 and IL18RAP are connected by the linker. In some embodiments, the linker is a glycine or glycine-serine linker. In some embodiments, the rlL- 18 further comprises a hinge domain. In some embodiments, the hinge domain comprises a polypeptide selected from the group consisting of: a hinge region of CD8a, a hinge region of a heavy chain of IgG, a hinge region of a heavy chain of IgD, and an extracellular region of an IL-10R 0 chain.

[0036] Some aspects relate to a nucleic acid comprising an open reading frame (ORF) encoding the rIL-18. In some embodiments, the nucleic acid further comprises a promoter operably linked to the ORF. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is an MND promoter.

[0037] Some aspects relate to a vector comprising the nucleic acid encoding the rIL-18. In some embodiments, the vector is a lentiviral vector. In some embodiments, the vector is an adeno-associated viral (AAV) vector. Some aspects relate to a lipid nanoparticle comprising the nucleic acid encoding the rIL-18. Some aspects relate to a cell comprising the rIL-18. Some aspects relate to a cell comprising the nucleic acid encoding the rIL-18. Some aspects relate to a method comprising contacting a cell with the nucleic acid, vector, or lipid nanoparticle.

[0038] Some aspects relate to a method for producing a population of regulatory T (Treg) cells having a repair phenotype, the method comprising culturing a population of Treg cells in the presence of interleukin- 18 (IL- 18).

[0039] In some embodiments, the Treg cells are cultured in the presence of 0.1 to 500 ng / mL IL-18. In some embodiments, the Treg cells are cultured in the presence of 50 ng / mL IL-18.

[0040] Some aspects relate to a method for producing a population of regulatory T (Treg) cells having a repair phenotype, the method comprising culturing a population of Treg cells in the presence of interleukin- 18 (IL- 18). In some embodiments, the Treg cells are cultured in the presence of 0.1 to 500 ng / mL IL-18. In some embodiments, wherein the Treg cells are cultured in the presence of 50 ng / mL IL-18.

[0041] In some embodiments, the Treg cells are cultured in the presence of rapamycin, wherein the Treg cells comprise a nucleic acid encoding a first chemically induced signaling complex (CISC) component and a nucleic acid encoding a second CISC component, wherein the first CISC component comprises, in N-to-C-terminal order, an FKBP domain, an interleukin-2 receptor gamma (IL-2R.y) transmembrane domain, and an IL-2Ry cytoplasmic domain, and wherein the second CISC component comprises, in N-to-C-terminal order, an FRB domain, an interleukin-2 receptor beta (IL-2RP) transmembrane domain, and an IL-2RP cytoplasmic domain.

[0042] In some embodiments, the Treg cells are cultured in the presence of 0.01 - 100 nM rapamycin. In some embodiments, the Treg cells are cultured in the presence of 10 nM rapamycin.

[0043] In some embodiments, the Treg cells are cultured in the presence of an anti-CD3 antibody and an anti-CD28 antibody.

[0044] In some embodiments, the method comprises culturing the population of Treg cells in the presence of IL-2, IL-12, IL-21, IL-23, and / or TGF-p.

[0045] Some aspects relate to a composition comprising a population of regulatory T (Treg) cells and IL-33. In some embodiments, the composition comprises 0.1 to 500 ng / mL IL-33. In some embodiments, the composition comprises 50 ng / mL IL-33.

[0046] Some aspects relate to a composition comprising a population of regulatory T (Treg) cells and IL-18. In some embodiments, the composition comprises 0.1 to 500 ng / mL IL-18. In some embodiments, the composition comprises 50 ng / mL IL-18.

[0047] In some embodiments, composition comprises rapamycin, wherein one or more of the Treg cells comprise a nucleic acid encoding a first chemically induced signaling complex (CISC) component and a nucleic acid encoding a second CISC component, wherein the first CISC component comprises, in N-to-C-terminal order, an FKBP domain, an interleukin-2 receptor gamma (IL-2Ry) transmembrane domain, and an IL-2Ry cytoplasmic domain, and wherein the second CISC component comprises, in N-to-C-terminal order, an FRB domain, an interleukin-2 receptor beta (IL-2RP) transmembrane domain, and an IL-2RP cytoplasmic domain. In some embodiments, the composition comprises 0.01 - 100 nM rapamycin. In some embodiments, the composition comprises 10 nM rapamycin. In some embodiments, the composition further comprises an anti-CD3 antibody and an anti-CD28 antibody. In some embodiments, the composition further comprises IL-2, IL-12, IL-21, IL-23, and / or TGF-p.

[0048] In some embodiments, the cell is a stem cell or a T cell. In some embodiments, the cell is a CD4+ or CD8+ T cell. In some embodiments, the cell is a FoxP3+regulatory T (Treg) cell. In some embodiments, the cell expresses one or more markers selected from BATF, CTLA-4, HLA-DR, ICOS, Ki-67, TIGIT, TNFRSF18, CD25, CD39, CD49d, CD69, CD71, CD103, CD197. In some embodiments, the cell expresses one or more cytokines selected from IL-5, IL-10, IL-13, and TGF-p. In some embodiments, the cell produces soluble ST2 and / or osteopontin. In some embodiments, the cell expresses one or more chemokine receptors selected from CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR10, and CX3CR1. In some embodiments, the cell expresses CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR10, and CX3CR1. In some embodiments, the cell is a KLRG1+cell. In some embodiments, the cell is a NFIL3+cell. In some embodiments, the cell is a GATA3+cell. In some embodiments, the cell is a RLN3+cell. In some embodiments, the cell is a RAB4A+cell. In some embodiments, the cell is a LYN+cell. In some embodiments, the cell is a PTPN13+cell. In some embodiments, the cell is a TBC1 D4 cell. In some embodiments, the cell is a PPARy+cell.

[0049] In some embodiments, the cell comprises a heterologous promoter located in a nucleic acid of the cell genome: (i) downstream from a Treg-specific demethylated region (TSDR); and (ii) upstream from a first coding exon of an endogenous F0XP3 gene. In some embodiments, the heterologous promoter located downstream from the TSDR is an MND promoter.

[0050] In some embodiments, the cell comprises: (i) a nucleic acid encoding a first component of a chemically induced signaling complex (CISC), the first CISC component comprising: (a) a first extracellular domain comprising an FK506-binding protein domain that binds rapamycin; (b) a first transmembrane domain; and (c)a first cytoplasmic domain comprising an intracellular signaling domain of a first cytokine receptor; and (ii) a nucleic acid encoding a second CISC component, the second CISC component comprising: (a) a second extracellular domain comprising an FKBP-rapamycin-binding (FRB) domain; (b) a second transmembrane domain; and (c) a second cytoplasmic domain comprising an intracellular signaling domain of a second cytokine receptor, wherein the first and second CISC components dimerize in the presence of rapamycin.

[0051] In some embodiments, the first CISC component comprises, in N-to-C-terminal order, an FKBP domain, an interleukin-2 receptor gamma (IL-2RY) transmembrane domain, and an IL-2Ry cytoplasmic domain, and wherein the second CISC component comprises, in N-to-C- terminal order, an FRB domain, an interleukin-2 receptor beta (IL-2RP) transmembrane domain, and an IL-2RP cytoplasmic domain.

[0052] In some embodiments, the cell comprises a nucleic acid encoding a soluble FRB domain, wherein SEQ ID NO: 5 has at least 90% identity to the amino acid sequence of the soluble FRB domain. In some embodiments, the cell expresses a chimeric antigen receptor (CAR). In some embodiments, the cell expresses a T cell receptor (TCR).

[0053] Some aspects relate to a method comprising administering the cell to a subject. In some embodiments, the cell is autologous to the subject. In some embodiments, the cell is allogeneic to the subject. Some aspects relate to a method comprising administering the vector to a subject. Some aspects relate to a method comprising administering the lipid nanoparticle to a subject.

[0054] In some embodiments, the subject has or is at risk of developing an acute inflammatory disease or condition. In some embodiments, the acute inflammatory disease or condition is stroke, kidney injury, acute respiratory distress syndrome (ARDS), myocardial infarction, severe wounding, or a muscle injury. In some embodiments, the subject has or is at risk of developing ARDS. In some embodiments, the subject has or is at risk of having a stroke. In some embodiments, the subject has or is at risk of developing kidney injury. In some embodiments, the kidney injury is an acute kidney injury. In some embodiments, the subject has or is at risk of developing fibrosis. In some embodiments, the fibrosis is pulmonary fibrosis.

[0055] Some aspects relate to a composition comprising a plurality of engineered regulatory T (Treg) cells, at least 50% of the engineered Treg cells of the plurality expressing ST2 protein. In some embodiments, the engineered Treg cells expressing ST2 protein constitutively express ST2 protein. Some aspects relate to a composition comprising a plurality of engineered regulatory T (Treg) cells, at least 50% of the engineered Treg cells of the plurality expressing IL1RAP In some embodiments, the engineered Treg cells expressing IL1RAP constitutively express IL1RAP. Some aspects relate to a composition comprising a plurality of engineered regulatory T (Treg) cells, at least 50% of the engineered Treg cells of the plurality expressing IL-18R1. In some embodiments, the cells expressing IL-18R1 constitutively express IL-18R1. Some aspects relate to a composition comprising a plurality of engineered regulatory T (Treg) cells, at least 50% of the engineered Treg cells of the plurality expressing IL18RAP. In some embodiments, the engineered Treg cells expressing IL1RAP constitutively express IL18RAP.In some embodiments, at least 50% of engineered Treg cells comprise a genetic modification to stabilize FOXP3 expression. In some embodiments, the genetic modification to stabilize FOXP3 expression comprises a heterologous promoter inserted in a FOXP3 gene of the cell genome, wherein the heterologous promoter is downstream from a TSDR of the FOXP3 gene and upstream for a first coding exon of a FOXP3 gene.

[0056] BRIEF DESCRIPTION OF THE DRAWINGS

[0057] The following drawings form part of the present specification and are included to further demonstrate certain aspects, which can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. It is to be understood that the data illustrated in the drawings in no way limit the scope of the disclosure.

[0058] FIG. 1A is a schematic illustrating a cell that has been edited to express F0XP3, a chemically inducible signaling complex (CISC) for tunable IL-2 signaling, and ST2. FIG. IB shows exemplary mechanisms of action and applicability of human engineered T regulatory cells.

[0059] FIG. 2 is a series of plots demonstrating that hCCR4 expression is on the cell surface of human engineered T regulatory cells (hEngTregs) that have been edited to express F0XP3.

[0060] FIGs. 3A-3C are a series of flow cytometry plots showing that ST2 hEngTregs (human CD4+ cells transduced with a lentiviral vector encoding ST2 and then edited via an AAV vector system to express F0XP3 and CISC (IL-2)) express F0XP3 and ST2 at levels higher than mock-transfected human CD4+ cells and human CD4+ cells engineered to express F0XP3 and CISC (IL-2) (“Basic EngTregs”). FIG. 3 A shows the levels of F0XP3 (left) and ST2 (right) at Day 7 after transfection and on Day 18 (after selection by rapamycin). FIG. 3B shows populations of basic EngTregs (left peak) and ST2 EngTregs (right peak) on Day 18 (after rapamycin selection). FIG. 3C shows edited cells that were frozen and thawed staining as described in FIG. 3 A.

[0061] FIG. 4 is a graph showing levels of phosphorylated p65 following stimulation with IL-33 or PMA (control), demonstrating that ST2 EngTregs are responsive to IL-33 stimulation as compared to mock EngTregs (CD4+ cells engineered to express FOXP3 and CISC (IL-2)).

[0062] FIGs. 5A-5C provide graphs illustrating ST2 hEngTregs’ response to IL-33 stimulation. FIG. 5 A shows levels of Ki67 (a marker of proliferation) and CD71 (a marker of activation) in basic EngTregs and ST2 EngTregs with or without IL-33 stimulation. FIG. 5B shows, from left to right, the percentages of IL-13+ cells, TGF-P+ cells, and KGF+ cells from populations of basic EngTregs and ST2 EngTregs with or without IL-33 stimulation. FIG. 5C shows the percent of CCR8+ cells from populations of basic EngTregs and ST2 EngTregs with or without IL-33 stimulation.

[0063] FIG. 6 is a graph showing engraftment of IL-33 -treated murine engineered T regulatory cells (mEngTregs) (measured as the percent of CD45.2+ cells in murine bronchioalveolar lavage fluid, BALF) in mice following allogenic (“LPS + Allo mEngTreg”) and autologous (“LPS + Auto mEngTreg”) cell transfer using ST2 mEngTregs.

[0064] FIGs. 7A-7C show measurements of disease severity in mice challenged with lipopolysaccharides (LPS) (to induce acute lung injury) and then administered allogenic (“Allo”) or autologous (“Auto”) mEngTregs, or PBS (as a control). FIG. 7A shows bodyweight change over time (note that the top axis represents days). FIG. 7B shows pulse oximetry data from Day 11, and FIG. 7C shows lung weight measurements from Day 11.

[0065] FIGs. 8A-8B show neutrophil percentages and counts (concentrations) in mice challenged with LPS and then administered allogenic (“Allo”) or autologous (“Auto”) mEngTregs, or PBS (as a control). The percentages (left) and counts (rights) were determined in lung samples (FIG. 8 A) and B ALF samples (FIG. 8B) on Day 11.

[0066] FIGs. 9A-9B show measurements of alveolar macrophages (FIG. 9A) and CD11C+CD103+ dendritic cells (FIG. 9B) in BALF samples from mice challenged with LPS and then administered allogenic (“Allo”) or autologous (“Auto”) mEngTregs, or PBS (as a control). Samples were taken on Day 11.

[0067] FIG. 10 is a series of graphs showing cytokine responses in the BALF of mice challenged with LPS and then administered allogenic (“Allo”) or autologous (“Auto”) mEngTregs, or PBS (as a control). Samples were taken on Day 11.

[0068] FIG. 11 includes two graphs showing cell surface protein expression of human CCR4 on hEngTregs (left) and mouse CCR4 on mEngTregs (right).

[0069] FIG. 12 is a graph showing the percentage of CD45.2+ cells in a population of live CD4+ T cells in untreated mouse CD4+ T cells, thymic Tregs (endogenous Tregs), mEngTregs stimulated with IL-2, and mEngTregs stimulated with IL-2 and IL-33 in lung and spleen samples.

[0070] FIG. 13 is a graph showing the ratio of lung to spleen engraftment of mEngTregs over time.

[0071] FIG. 14 is a graph showing the percentage of CD45.2+ cells relative to live CD4+ cells in the spleen, lung, and BALF of mice on Day 6 and Day 13 after transduction with mEngTregs.

[0072] FIGs. 15A-15B demonstrate autologous mEngTreg efficacy in a bleomycin-induced acute lung injury mouse model. FIG. 15A shows the change in subject bodyweight following bleomycin treatment. FIG. 15B shows flow cytometry data, demonstrating that mEngTregs accumulate in the BALF (right plot), as compared to endogenous BALF -infiltrating nTregs (left plot).

[0073] FIG. 16 is a schematic showing LPS-induced acute lung injury with allogeneic Fl adoptive cell transfer.

[0074] FIGs. 17A-17B show murine engineered Treg (mEngTreg) expression of Foxp3, LNGFR, and other markers prior to adoptive cell transfer. FIG. 17A is a flow cytometry plot showing Day 7 transduced EngTreg expression of Foxp3 and transgene tag LNGFR. FIG. 17B shows expression of markers including CD25 and CCR4 by spleen-derived murine engineered Tregs.

[0075] FIG. 18 shows cytokine concentrations in the bronchoalveolar lavage fluid (BALF) of mice challenged with LPS and then administered allogenic (“Allo”) or autologous (“Auto”) mEngTregs, or PBS (control). Samples were taken on Day 6 post-LPS administration.

[0076] FIG. 19 shows lung histology and mean severity scores (by pathology) following mEngTreg administration. Samples were taken on Day 6 post- LPS administration.

[0077] FIGs. 20A-20D show comparisons of allogenic and autologous mEngTregs frequencies in LPS-induced acute lung injury. FIG. 20A shows the percentage of CD45.2+ cells relative to CD4+ cells in the BAL on Day 6 post- LPS administration. FIG. 20B shows frequencies of mEngTregs in the BAL, spleen, and lung on Day 11 post- LPS administration. FIG. 20C shows relative counts of CD45.2+ cells in BAL, lungs, and spleens of mice administered autologous or allogeneic murine EngTregs, at Day 6 post-LPS administration (or sham control administration). FIG. 20D shows representative gating for quantifying mEngTregs (CD45.2+) and host CD4+ cells (CD45.1+).

[0078] FIGs. 21A-21B show study design for measuring proliferation and engraftment of mEngTregs in a mouse model of LPS-induced ALI. FIG. 21A shows an overview of cell donor genotypes, recipient mouse genotypes, and administration of mEngTregs and / or LPS. LPS (or sham control) is administered on day 0, mEngTregs are administered on day 1, and mice are euthanized on day 5 to collect lung, bronchoalveolar lavage (BAL), spleen, mediastinal lymph nodes (mLNs), and colon samples to evaluate cell phenotypes and abundance. FIG. 2 IB shows representative gating for detecting mEngTreg by CD45.2 staining, and assaying mEngTreg proliferation by Ki67 staining.

[0079] FIGs. 22A-22C show mEngTreg engraftment and proliferation in the study described in FIG. 21 A. FIG. 22A shows presence of mEngTregs (CD45.2+) in BAL. FIG. 22B shows the proportion of CD4+ cells that were mEngTregs (CD45.2+) in BAL of each mouse. FIG. 22C shows intensity of Ki67 staining in autologous and allogeneic cells in spleens, mLNs, lungs, and BAL of mice administered mEngTregs.

[0080] FIG. 23 shows an overview of a mouse model of ischemic stroke by transient middle cerebral artery occlusion (tMCAO), as described in Rousselet et al., J Vis Exp. 2012. 69:4038.

[0081] FIGs. 24A-24C show the effect of antigen-specific and polyclonal mEngTregs on restoring sensorimotor function in a murine ischemic stroke model. Engineered Tregs were injected (1 million / mice, retro-orbital i.v.) into 12-week-old male recipient mice 5h after tMCAO. (FIG. 24A) The sensorimotor function was evaluated using the rotarod test up to 35 days after tMCAO. Data were expressed as the latency to fall off the rotating rod. (FIGs. 24B-24C) Adhesive removal test up to 35 days after tMCAO. Data were expressed as the latency to contact (FIG. 24B) and to remove (FIG. 24C) the tape from the impaired forepaw. Sham group (n=4); Vehicle (PBS) treatment group (n=8); MOG-specific Treg treatment group (n=7); polyclonal Treg treatment group (n=8). Data are expressed as mean ± standard deviation (SD). *p < 0.05, **p<0.01, ***p<0.001 between polyclonal Treg and PBS treatment groups; *p < 0.05, **p<0.01, between MOG-specific Treg and PBS treatment groups; ## p < 0.01 between polyclonal Treg and MOG-specific Treg groups; two-way repeated-measures ANOVA followed by Bonferroni post hoc. Differences between sham and stroke groups were not marked.

[0082] FIGs. 25A-25C show the effect of antigen-specific and polyclonal mEngTregs on restoring cognitive function in a murine ischemic stroke model. Engineered Tregs were injected (1 million / mice, retro-orbital z.v.) into 12-week-old male recipient mice 5h after tMCAO. (FIG. 25 A) Non-spatial memory was assessed 35d after tMCAO using the passive avoidance test. Latency until entry into the dark room from the light room was recorded. (FIG. 25B-25C) Novel object recognition (NOR) test 1 day before and 14 and 28 days after tMCAO. (FIG. 25B) The time spent around the novel object was calculated as the percentage of total exploration time. (FIG. 25C) The discrimination index (DI) is the time spent around the novel object minus the time spent on a familiar object divided by the time spent around both objects. *p < 0.05, **p<0.01, ***p<0.001 vs. sham; #p < 0.05, ###p<0.001, vs. PBS; two-way repeated-measures ANOVA followed by Bonferroni post hoc tests

[0083] FIGs. 26A-26D show phenotypes and expansion of cells edited for stable expression of FoxP3 and ST2. FIG. 26A shows CD25 and FoxP3 expression in each group at day 14 and day 20. FIG. 26B shows ST2 and IL1RAP at day 14 and day 20. FIG. 26C shows ST2 and IL1RAP expression at days 14 and 20 in cells cultured with IL-33. FIG. 26D shows kinetics of cell numbers in cells cultured with IL-33.

[0084] FIG. 27A shows collagen deposition (by trichrome staining) in mice administered LPS, followed by autologous or allogeneic mEngTregs. FIG. 27B shows mean lung interstitial fibrosis scores in the same mouse groups of FIG. 27A. Samples were collected at day 11 post-LPS administration. FIG. 28 shows proliferation of antigen-specific T effector cells incubated with CD4+ T cells or EngTregs, in the presence or absence of peptide antigen.

[0085] FIGs. 29A-29C show the Repair EngTreg process with in-process IL-33. FIG. 29A shows a schematic of the process timeline. FIGs. 29B-29C show the effects of expression of transgene and addition of in-process IL-33 on cell expansion (FIG. 29A) and cell viability (FIG. 29B).

[0086] FIG. 30 shows high ST2 and IL1RAP expression achieved in hEngTregs via lentiviral (LV) transduction. High expression of ST2 transgene was observed following Day 1 post-LV transduction, and high expression IL1RAP was observed following dual ST2 / IL1RAP LV transduction. No in-process IL-33 was added.

[0087] FIGs. 31A-31B show that phosphorylation of key signaling components revealed sufficiency of the ST2 transgene. Basic Tregs: CD4+FOXP3+; ST2 Tregs: CD4+FOXP3+ST2+; IL33R Tregs: CD4+FOXP3+ST2+IL1RAP+. FIG. 31A shows p65 NFkB and FIG. 3 IB shows p38 MAPK. Statistical analysis utilized were one-way ANOVA, Tukey’s multiple comparison test.

[0088] FIGs. 32A-32B show that expression of ST2 receptor on EngTreg plus incubation / production in IL-33 promotes enrichment of tissue Treg markers. FIG. 32A shows upregulation of tissue Treg markers in ST2 EngTregs with in-process IL-33, gated on Foxp3+ EngTregs. FIG. 32B shows hierarchical clustering (Pearson’s correlation) reveals segregation of ST2 EngTregs with in-process IL-33.

[0089] FIGs. 33A-33D show that CCR8 / GATA3 expression is highest in IL-33-primed ST2 Tregs at end-of-process. FIG. 33A shows that CCR8 and GATA3 expression appears to be highest in the ST2 / IL33R Tregs cultured in IL-33 during expansion. Data from 2 independent experiments gated on total FOXP3+ is shown. FIG. 33B shows CCR8 and GATA3 expression for fresh CD4s. FIGs. 33C-33D show CCR8 (FIG. 33C) and GAT A3 (FIG. 33D) expression in basic Tregs and ST2 Tregs cultured with and without IL-33.

[0090] FIGs. 34A-34D show that CCR5 expression is highest in IL-33-primed ST2 Tregs at end-of-process. FIG. 34A shows that CCR4 is high for all EngTregs samples, and CCR5 expression appears to be highest in the ST2 / IL33R Tregs cultured in IL-33 during expansion. FIG. 34B shows CCR4 and CCR5 expression for fresh CD4s. FIGs. 34C-34D show CCR4 (FIG. 34C) and CCR5 (FIG. 34D) expression in basic Tregs and ST2 Tregs cultured with and without IL-33.

[0091] FIGs. 35A-35D show that HLA-DR / CD39 expression was high in IL-33 -primed ST2 Tregs at end-of-process. FIG. 35A shows that HLA-DR and CD39 expression was highest in the ST2 / IL33R Tregs cultured in IL-33 during expansion. Data from 2 independent experiments gated on total FOXP3+ is shown. FIG. 35B shows CD39 and HLA-DR expression for fresh CD4s. FIGs. 35C-35D show CD39 (FIG. 35C) and HLA-DR (FIG. 35D) expression in basic Tregs and ST2 Tregs cultured with and without IL-33.

[0092] FIGs. 36A-36D show that CD71 / KL67 expression was high in IL-33-primed ST2 Tregs at end-of-process. FIG. 36A shows that CD71 expression appears to be highest in the ST2 / IL33R Tregs cultured in IL-33 during expansion. Data from 2 independent experiments gated on total FOXP3+ is shown. FIG. 36B shows CD71 and KI-67 expression for fresh CD4s. FIGs. 36C-36D show KI-67 (FIG. 36C) and CD71 (FIG. 36D) expression in basic Tregs and ST2 Tregs cultured with and without IL-33.

[0093] FIGs. 37A-37F show summary data demonstrating that higher expression of key chemokine receptors and tissue Treg transcription factors observed in ST2+ EngTregs with in-process IL-33. Data shown are gated on ST2+ FOXP3+. In the legend, “ip33” represents in-process IL-33. Harvest was conducted on Day 23. Expression is shown for targeting receptors CCR4, CCR5, and CCR8 (FIGs. 37A-37C, respectively) and tissue Treg transcription factors NFIL3, BATF, and GATA3 (FIGs. 37D-37F, respectively).

[0094] FIGs. 38A-38F show higher expression of Tissue Treg associated functional, activation and proliferation markers observed in ST2+ EngTregs with in-process IL-33. Data shown are gated on ST2+ FOXP3+ cells. In the legend, “ip33” represents in-process IL-33. Harvest was conducted on Day 23. Expression is shown for activation / proliferation markers HLA-DR, CD71, and KI67 (FIGs. 38A-38C, respectively) and functional mediators TIGIT, CD25, and CD39 (FIGs. 38D-38F, respectively).

[0095] FIGs. 39A-39C show that ST2+ Tregs express higher levels of IL-13 and can sequester IL-33. FIG. 39A shows that ST2+ and IL33R+ EngTregs sequester more of the pro- inflammatory alarmin IL-33 compared to basic EngTregs (not transduced to express ST2) following 24-hour co-culture with 50 ng / mL recombinant IL-33. FIGs. 39B-39C show that ST2 EngTregs exposed to in-process IL-33 express higher levels of repair mediator IL-13 following 72-hour culture with (FIG. 39C) or without (FIG. 39B) CD3 / 28 stimulation.

[0096] FIGs. 40A-40F show human ST2-expressing EngTregs are differentiated by Tissue Treg markers. FIG. 40A shows ST2-Tregs express higher levels of homing, activation and Tissue Treg functional markers. FIG. 40B shows the induction of NF-kB and MAPK signaling downstream of ST2 receptor observed only in ST2-Tregs cultured with IL-33. FIG. 40C shows the sequestration of IL-33 by ST2-expressing but not basic (ST2-) Tregs (1-day co-culture with IL-33). FIG. 40D shows a higher baseline expression of TNFR2 by ST2- expressing Tregs (assessed by flow cytometry, 3-day rest). FIG. 40E shows an upregulation of CD25 in response to TNF-a by ST2-Tregs. FIG. 40F shows an increased uptake of IL-2 by ST2 Tregs in response to TNF-a.

[0097] FIGs. 41A-41D show increased proportion of CCR8+ GATA3+ population, and marker intensity, in ST2-Tregs stimulated with anti-CD3 / anti-CD28 and IL-33. *2-way ANOVA, Tukey’s multiple comparisons test; p-value=0.0332(*), 0.0021(**), 0.0002(***), <0.0001(****). FIG. 41A shows representative staining. FIG. 41B shows CCR8 expression levels. FIG. 41C shows GAT A3 expression levels. FIG. 4 ID shows CCR8+GATA3+ population frequency.

[0098] FIGs. 42A-42D show increased proportion of CD73+ CD39+ population, and marker intensity, in ST2-Tregs stimulated with anti-CD3 / anti-CD28 and IL-33. *2-way ANOVA, Tukey’s multiple comparisons test; p-value=0.0332(*), 0.0021(**), 0.0002(***), <0.0001(****). FIG. 42A shows representative staining. FIG. 42B shows CD39 expression. FIG. 42C shows CD73 expression levels. FIG. 42D shows CD39+CD73+ population frequency.

[0099] FIGs. 43A-43D shows increased proportion of 0X40+ TIGIT+ population, and marker intensity, in ST2-Tregs stimulated with anti-CD3 / anti-CD28 and IL-33. FIG. 43 A shows representative staining. FIG. 43B shows TIGIT expression levels. FIG. 43C shows 0X40 expression levels. FIG. 43D shows TIGIT+OX40+ population frequency.

[0100] FIGs. 44A-44D shows reduced proportion of CD71+ CD25+ population, and marker intensity, in ST2-Tregs stimulated with anti-CD3 / anti-CD28 and IL-33. FIG. 44A shows representative staining. FIG. 44B shows CD71 expression levels. FIG. 44C shows CD25 expression levels. FIG. 44D shows CD71+ CD25+ population frequency.

[0101] FIGs. 45A-45B shows CCR8 and GATA3 marker expression and positivity in ST2- Tregs in response to IL-4 and IL-33 stimulation, with or without anti-CD3 / anti-CD28 antibody complexes (IC). FIG. 45A shows groups that are unstimulated, with IL-4, or with IL-4 and IL-33. FIG. 45B show groups that are anti-CD3 / anti-CD28 antibody complexes (IC) alone, with IL-4, or with IL-4 and IL-33.

[0102] FIGs. 46A-46B shows TNFR2 and ICOS marker expression and positivity in ST2- Tregs in response to IL-4 and IL-33 stimulation, with (FIG. 46A) or without (FIG. 46B) anti- CD3 / anti-CD28 antibody complexes (IC).

[0103] FIGs. 47A-47F show responses of Tregs and ST2-Tregs in response to IL-4 stimulation, IL-33 stimulation, IL-2 stimulation, TNF-a stimulation and / or anti-CD3 / anti- CD28 antibody complexes (IC). FIG. 47A shows TNFR2 and ICOS marker expression and positivity . FIG. 47B shows soluble ST2 (sST2) concentrations in supernatants. FIG. 47C shows osteopontin concentrations in supernatants. FIG. 47D shows IL-4 concentrations in supernatants, indicating the degree of IL-4 sequestration from the extracellular environment. FIG. 47E shows IL-2 concentrations in supernatants, indicating the degree of IL-2 sequestration. FIG. 47F shows TNF-a concentration in supernatants, indicating the degree of TNF-a sequestration.

[0104] FIGs. 48A-48G shows a cisplatin-induced acute kidney injury (AKI) model. FIG. 48A shows body weight change in mice administered PBS control or cisplatin. FIG. 48B shows serum blood urea nitrogen (BUN) concentration over time in the same mice. FIG. 48C shows neutrophil and monocyte frequencies among CD45+ cells in kidneys of the same mice. FIG. 48D shows kinetics of cytokine concentrations in kidneys following administration of 20 mg / kg cisplatin. FIG. 48E shows blood urea nitrogen (BUN) kinetics. FIG. 48F shows serum NGAL kinetics. FIG. 48G shows serum Kim-1 kinetics.

[0105] FIGs. 49A-49U provide an overview of AKI induction cell dosing, monitoring, assaying and the subsequent readouts of different conditions. FIG. 49A shows a timeline of treatment, sample collection, and monitoring of mice. Mice were administered PBS control, cisplatin, or cisplatin followed by ST2 EngTregs. FIG. 49B shows body weight change over time. FIG. 49C shows % body weight change at day 4 post-injury, relative to baseline. FIG. 49D shows spleen and individual kidney weights at day 4 post-injury. FIG. 49E shows counts of ST2 EngTregs in kidneys at day 4. FIG. 49F shows the percentage of CD4+ cells that are EngTregs (CD45.2+ donor cells). FIG. 49G shows representative gating for discriminating recipient mouse (CD45.1+) cells from donor EngTregs (CD45.2+) cells in the spleen and kidneys. FIG. 49H shows serum creatinine at day 4. Fig. 491 shows serum BUN at day 4. FIG. 49 J shows urine Kim-1 at day 4. FIG. 49K shows serum Kim-1 at day 4. FIG. 49L shows neutrophil frequency among CD45+ cells in kidneys at day 4. FIG. 49M shows monocyte frequency among CD45+ cells in kidneys at day 4. FIG. 49N shows representative gating for analysis of neutrophil and monocyte populations. FIG. 490 shows the correlation between kidney neutrophil frequency and urine BUN concentration. FIG. 49P shows macrophage frequencies in kidneys at day 4. FIG. 49Q shows kidney macrophage counts at day 4. FIG. 49R shows kidney macrophage antigen presentation (MHC-II+ frequency) at day 4. FIG. 49S shows kidney macrophage antigen presentation (MHC-II+ intensity) at day 4. FIG. 49T shows representative gating for analyzing macrophage numbers and MHC-II expression. FIG. 49U shows serum cytokine levels of IL-33, IL-la, IL-12p40, IL-23, TNF-a, and IL-6 at day 4. FIGs. 50A-50M provide an overview of a UIRI model of acute kidney injury, evaluating the preventive and therapeutic administration of EngTregs. FIG. 50A shows a timeline of study design. FIG. 50B shows survival over time. FIG. 50C shows glomerular filtration rates (GFRs) at day 21. FIG. 50D shows serum creatinine and BUN at day 10, and comparison to levels at day 28. FIG. 50E shows serum Kim-1 (kidney injury marker) at day 10. FIG. 50F shows injury assessed by histology. FIG. 50G shows representative histology images. FIG. 50H shows tubular degeneration / regeneration at day 28. FIG. 501 shows casts at day 28. FIG. 50J shows dilation at day 28. FIG. 50K shows inflammation at day 28. FIG. 50L shows fibrosis indicators at day 28, including scoring of H&E stained slides (left) and collagen content measured by hydroxyproline concentration (right). FIG. 50M shows medullary tubular necrosis scores at day 28.

[0106] FIGs. 51A-51C show improved gross sensorimotor performance in Fl treated tMCAO animals (increased latency to fall in rotarod test, FIGs. 50A and 50B) and improvement of memory deficit (step-through latency, FIG. 50C) by allogeneic mEngTregs in tMCAO stroke models.

[0107] FIGs. 52A-52B show effectiveness of allogeneic mEngTregs in a tMCAO stroke model. FIG. 51 A shows comparable efficacy of allogeneic and autologous EngTregs in adhesive removal and passive avoidance tests. FIG. 5 IB shows effectiveness of autologous EngTregs in rotarod and novel object recognition tests, with or without TCR modification.

[0108] FIGs. 53A-53D show results of an EngTreg treatment in a mouse model of bacterial acute respiratory distress syndrome (ARDS). Mice were intranasally inoculated with S. pneumoniae ATCC BAA-334 (TIGR4) and treated with EngTregs or Vehicle control. FIG. 53 A shows total protein levels in bronchoalveolar lavage fluid. FIG. 53B shows colony forming units in lungs. FIG. 53C shows clinical scores over time. FIG. 53D shows temperatures over time. Data shown for individual animals and as mean ± SEM (n=8 for each group except Naive group, where n=5 and Vehicle group, where n=7, as one animal was excluded due to low sample volume obtained). Data analyzed by one-way ANOVA followed by Dunnett' s multiple comparisons test, comparing each group against the Vehicle. Statistically significant differences shown where * indicates p<0.05.

[0109] DETAILED DESCRIPTION

[0110] Some aspects of the disclosure relate to methods and compositions for producing engineered repair regulatory T cells (repair Tregs). Repair Tregs are Tregs that express at least one repair receptor (e.g., the IL-33 receptor and / or the IL-18 receptor). In some embodiments, the engineered repair Tregs are engineered have stabilized FoxP3 expression. In some embodiments, the engineered repair Tregs further comprise a chemical-inducible signaling complex (CISC) that results in continued IL-2 signal transduction, promoting survival and proliferation of the engineered repair Tregs.

[0111] Without wishing to be bound by theory, it is thought that the engineered repair Tregs will exhibit a tailored response to specific inflammatory environmental cues, such as alarmins (e.g., IL-33 and IL- 18). In this way, it is thought that the engineered repair Tregs induce tolerance in the instance of overt inflammation, as the cells have been found to be important in mediating inflammation control and tissue repair in acute and chronic inflammatory diseases and conditions. In addition, the engineered repair Tregs may assist with the repair or regeneration of stromal tissue through the regulation of potentially damaging inflammatory immune cells (e.g., neutrophils, macrophages), and may promote induction of repair through growth factors and mediators such as amphiregulin (AREG), keratinocyte growth factor (KGF), and / or transforming growth factor beta (TGF-P).

[0112] In some embodiments, methods comprise inducing or increasing IL-33 and / or IL-18 signaling pathway activity in a Treg cell to generate engineered repair Tregs. In some embodiments, IL-33 signaling is induced or increased by expressing one or more components of the IL-33 signaling pathway (e.g., by expressing recombinant IL-33 receptor components, such as ST2 or IL1RAP) in a target cell (e.g., ex vivo, or in vivo). In some embodiments, IL- 18 signaling is increased by expressing one or more components of the IL- 18 signaling pathway (e.g., by expressing recombinant IL- 18 receptor components, such as IL-18R1 or IL18RAP) in a target cell (e.g, ex vivo, or in vivo). In some embodiments, the engineered repair Treg is engineered to express a transcription factor or activator of a repair pathway, such as GAT A3, PPARG, and / or BATF; cytokines (e.g, AREG, osteopontin); growth factors (e.g., KGF, TGF-P); and / or chemokine receptors (e.g., CCR2, CCR5, CCR8) that promote repair and regeneration. In some embodiments, IL-33 signaling is increased by inducing expression of one or more IL-33 receptor components (ST2 or IL1RAP). In some embodiments, IL-18 signaling is increased by inducing expression of one or more IL-33 receptor components (IL-18R1 or IL18RAP). Inducing expression of a protein (e.g., ST2) refers to causing an endogenous coding sequence encoding that protein to be transcribed and translated. Increasing expression of a protein may be accomplished through any suitable method, such as introducing a promoter (or enhancer or other regulatory element) to cause increased transcription of an endogenous coding sequence encoding the protein, providing an exogenous nucleic acid encoding the protein (e.g., mRNA comprising an open reading frame encoding the protein, or DNA comprising a promoter operably linked to a nucleotide sequence encoding the protein).

[0113] The IL-33 signaling pathway, or IL-33 / ST2 axis, on Tregs enhances transforming growth factor (TGF)-pi -mediated differentiation of Treg cells and provides a signal for Treg- cell accumulation and maintenance in inflamed tissues. In particular, IL-33 binds to the ST2 / IL-1 receptor accessory protein (IL-1RAP) heterodimer, recruiting MyD88 to its intracellular domain. MyD88 binding promotes Foxp3 and GATA3 expression, while also promoting Treg function and expansion through enhancing TGF-pi -mediated differentiation though a p38-dependent mechanism (Griesenauer et al., Front. Immunol., 2017). MyD88 binding also recruits IL-lR-associated kinases (IRAKI, IRAK2, IRAK3, IRAK4) and TRAF6, leading to either the NF-KB or AP-1 (via MAPK) pathway being activated (Chang et al., Transl. Perioper. & Pain Med., 2016). These signaling events promote a tissue repair phenotype in Tregs, which may promote tissue repair through multiple mechanisms including mediating tolerance to inflammation, negatively regulate potentially damaging inflammatory cells (e.g., neutrophils and macrophages), and produce growth factors and mediators including amphiregulin (AREG), keratinocyte growth factor (KGF), and / or TGF-p. Such Tregs stimulated by IL-33 exert cardioprotective effects, thereby mitigating the effects of atherosclerosis, through multiple mechanisms, such as reducing cell death of cardiomyocytes, expansion of M2 macrophages, a reparative macrophage subset, and inducing production of IL-5, which promotes generation of cardioprotective antioxidized low-density lipoprotein (ox-LDL) antibodies. See, e.g., Chen et al., Cell Physiol Biochem. 2018. 49(l):349-358; Miller et al., J Exp Med. 2008. 205:339-346. IL-33 signaling in neuronal macrophages (microglia) induces a similar protective response, including phagocytosis of extracellular matrix (regulating synapse remodeling), clearance of Ap (mitigating neuroinflammation), and preserving oligodendrocytes and oligodendrocyte precursor cells, thereby mitigating the effects of ischemic injury in stroke. Xie et al., Stroke. 2021. 52(6):2150-2161. Neuroprotective benefits of IL-33 in stroke are also associated with increased abundance of IL-10-producing Tregs. See, e.g., Zhang et al., JCI Insight. 2018. 3(18):e 121560.

[0114] Similarly, the IL-18 signaling pathway also results in activation of NF-KB and / or AP- 1. IL- 18 signals through its receptor, which comprises an interleukin 18 receptor 1 (IL-18R1 or IL-18Ra) chain, and an interleukin 18 receptor accessory protein (IL1RAP or IL-18RP) chain. Following the binding of IL- 18 to IL-18Ra, IL-18RP then binds to form a trimer. MyD88 binds to the Toll-IL-1 receptor (TIR) domain of IL-18R1 and IL18RAP. IRAKI and IRAK4 are the bound via the death domain of MyD88. TRAF6 then binds IRAKI, resulting in the degradation of inhibitor of KB (IKB) and translocation of phosphorylated p65 / p50 NF- KB into the nucleus. The MAPK cascade comprising Extracellular Signal-regulated Kinase (ERK), c-jun N-terminal kinase (JNK), and p38 is also activated, which induces IFN-y production promotes cell proliferation. IL-18 stimulation also induces the phosphorylation and activation of phosphatidylinositol-3 kinase (PI3K) / Akt / S6 and mammalian target of rapamycin (mTOR).

[0115] Accordingly, some aspects relate to cells having upregulated expression of ST2, IL1RAP, IL-18R1, and / or IL18RAP or a functional derivative thereof. ST2 is not expressed on many circulating T cell subsets (see, e.g., Griesenauer and Paczesny, Front Immunol. 2017. 8:475), and surface expression of each of IL-18R and IL1RAP is also low or absent on circulating T cells. Upregulation of ST2, IL1RAP, IL-18R1, and / or IL18RAP, expression in engineered Treg allows these engineered Tregs to also respond to IL-33, promoting tissue repair by the engineered Tregs. Expression of one or more of ST2, IL1RAP, IL-18R1, and / or IL18RAP may be upregulated by any suitable method. In some embodiments, an exogenous nucleic acid comprising a nucleotide sequence encoding ST2, IL1RAP, IL-18R1, and / or IL18RAP, or a functional derivative thereof, is introduced into the cell. In some embodiments, a promoter is operably linked to the sequence encoding ST2, IL1RAP, IL- 18R1, and / or IL18RAP, or functional derivative thereof. In some embodiments, the promoter is the natural promoter of the gene. In some embodiments, the promoter is heterologous to the gene. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is an inducible promoter. In some embodiments, the cell is contacted with a reagent that promotes transcription through the inducible promoter.

[0116] In some embodiments, the nucleic acid is inserted into a chromosome of the cell. In some embodiments, the nucleic acid is inserted by homology-directed repair following cleavage by a nuclease. In some embodiments, the nucleic acid is inserted by homologous recombination. In some embodiments, the nucleic acid is present on a viral vector that integrates into a chromosome. In some embodiments, the nucleic acid is expressed episomally. In some embodiments, the nucleic acid is present on a plasmid that is expressed episomally. In some embodiments, the nucleic acid is present on a viral vector that expresses an encoded sequence episomally.

[0117] In some embodiments, an endogenous nucleic acid sequence encoding ST2, IL1RAP, IL-18R1, and / or IL18RAP in a cell is modified to increase expression of ST2, IL1RAP, IL- 18R1, and / or IL18RAP relative to an unmodified cell. In some embodiments, a Kozak sequence is modified or replaced. In some embodiments, an exogenous Kozak sequence is inserted into the endogenous nucleic acid. In some embodiments, an endogenous promoter is modified or replaced. In some embodiments, an exogenous promoter is inserted into the endogenous nucleic acid. In some embodiments, an enhancer is modified or replaced. In some embodiments, an exogenous enhancer is inserted into the endogenous nucleic acid. In some embodiments, a ubiquitous chromatin opening element is inserted into the endogenous nucleic acid. In some embodiments, a stabilizing sequence (e.g., woodchuck hepatitis virus posttranscriptional regulatory element (WPRE)) may be inserted at the 3' region of the endogenous gene. These and other suitable methods for controlling expression of a desired gene allow for expression (e.g., constitutive expression) of ST2, IL1RAP, IL-18R1, and / or IL18RAP, and consequent production of Tregs that respond to the presence of IL-33. Tregs that stably express FoxP3 and also express ST2, IL1RAP, IL-18R1, and / or IL18RAP, having a resulting tissue reparative phenotype, are useful in multiple indications characterized by tissue damage, including stroke, myocardial infarction, acute swelling, severe wounding, muscle injuries, burn injuries, traumatic brain injury, acute respiratory distress syndrome (ARDS), pancreatic islet cell transplantation, asthma, hepatitis, primary sclerosing cholangitis, primary biliary cholangitis, polymyositis, Still’s disease, uveitis, ulcerative colitis, graft-versus-host disease (GvHD), tolerance induction for transplantation, transplant rejection, and sepsis.

[0118] In some embodiments, expression of ST2, IL1RAP, IL-18R1, and / or IL18RAP is increased relative to a Treg that has not been genetically modified. In some embodiments, expression of ST2, IL1RAP, IL-18R1 or IL18RAP is increased relative to a Treg that has not been genetically modified to increase expression of ST2, IL1RAP, IL-18R1 or IL18RAP, respectively. In some embodiments, expression of ST2, IL1RAP, IL-18R1, and / or IL18RAP is increased relative to a sorted Treg. In some embodiments, expression of ST2, IL1RAP, IL- 18R1, and / or IL18RAP is increased relative to a sorted Treg that has not been modified to stabilize FOXP3 expression. In some embodiments, expression of ST2, IL1RAP, IL-18R1, and / or IL18RAP is increased relative to a sorted Treg in which the F0XP3 locus has not been genetically modified.

[0119] In some embodiments, an engineered cell exhibits ST2 surface expression that is increased at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 1000-fold, 104-fold, or 105-fold relative to a sorted Treg that has not been genetically modified to increase ST2 expression. In some embodiments, the sorted Treg has not been genetically modified to increase FOXP3 expression. In some embodiments, the sorted Treg has an unmodified F0XP3 locus. In some embodiments, the sorted Treg has not been genetically modified.

[0120] In some embodiments, an engineered cell exhibits IL1RAP surface expression that is increased at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 1000-fold, 104-fold, or 105-fold relative to a sorted Treg that has not been genetically modified to increase IL1RAP expression. In some embodiments, the sorted Treg has not been genetically modified to increase FOXP3 expression. In some embodiments, the sorted Treg has an unmodified F0XP3 locus. In some embodiments, the sorted Treg has not been genetically modified.

[0121] In some embodiments, an engineered cell exhibits IL-18R1 surface expression that is increased at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 1000-fold, 104-fold, or 105-fold relative to a sorted Treg that has not been genetically modified to increase IL-18R1 expression. In some embodiments, the sorted Treg has not been genetically modified to increase FOXP3 expression. In some embodiments, the sorted Treg has an unmodified F0XP3 locus. In some embodiments, the sorted Treg has not been genetically modified.

[0122] In some embodiments, an engineered cell exhibits IL18RAP surface expression that is increased at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 1000-fold, 104-fold, or 105-fold relative to a sorted Treg that has not been genetically modified to increase IL18RAP expression. In some embodiments, the sorted Treg has not been genetically modified to increase FOXP3 expression. In some embodiments, the sorted Treg has an unmodified F0XP3 locus. In some embodiments, the sorted Treg has not been genetically modified.

[0123] Any suitable method may be used to measure the extent of ST2, IL1RAP, IL-18R1, and / or IL18RAP expression in a cell. As one example, western blot analysis of cell lysates may be used to quantify total amounts of ST2, IL1RAP, IL-18R1, and / or IL18RAP in a cell or sample of cells. As another example, qRT-PCR and / or RNAseq may be used to measure transcription of nucleotide sequences encoding ST2, IL1RAP, IL-18R1, and / or IL18RAP.

[0124] Some aspects relate to a plurality of engineered Tregs expressing ST2. In some embodiments, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of Tregs are ST2+. In some embodiments, the frequency of ST2+cells in the plurality of engineered Tregs is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 percentage points higher than the frequency of ST2+cells among sorted Tregs that have not been engineered.

[0125] Some aspects relate to a plurality of engineered Tregs expressing IL1RAP. In some embodiments, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of Tregs are IL1RAP+. In some embodiments, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of Tregs are FOXP3+IL1RAP+. In some embodiments, the frequency of IL1RAP+cells in the plurality of engineered Tregs is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,

[0126] 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 percentage points higher than the frequency of IL1RAP+cells among sorted Tregs that have not been engineered.

[0127] Some aspects relate to a plurality of engineered Tregs expressing IL-18R1. In some embodiments, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of Tregs are IL-18R1+. In some embodiments, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of Tregs are FOXP3+IL-18R1+. In some embodiments, the frequency of IL- 18R1+cells in the plurality of engineered Tregs is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,

[0128] 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 percentage points higher than the frequency of IL-18R1+cells among sorted Tregs that have not been engineered.

[0129] Some aspects relate to a plurality of engineered Tregs expressing IL18RAP. In some embodiments, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of Tregs are IL18RAP+. In some embodiments, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of Tregs are FOXP3+IL18RAP+. In some embodiments, the frequency of IL18RAP+cells in the plurality of engineered Tregs is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 percentage points higher than the frequency of IL18RAP+cells among sorted Tregs that have not been engineered.

[0130] Any suitable method may be used to determine whether a cell is ST2+, IL1RAP+, IL- 18R1+, and / or IL18RAP+. As an example, cells may be stained for surface expression of ST2, IL1RAP, IL-18R1, and / or IL18RAP by flow cytometry, and compared to PBMCs (e.g.. sorted Tregs) in which expression of such proteins is rare or absent. As another example, single-cell RNA sequencing methods may be used to measure the frequency of protein expression among the cells.

[0131] Some aspects relate to compositions, cells, nucleic acids, and vectors, and treatment modalities related to genetically modified cells, e.g., engineered repair Tregs. Engineered repair Tregs comprise any repair Treg that has one or more genetic modifications. Repair Tregs comprise natural repair Tregs and any cell that has been engineered to exhibit a repair Treg phenotype.

[0132] The cells are useful, for example, to mitigate and / or prevent certain signs and symptoms of inflammatory conditions.

[0133] Some aspects relate to an engineered cell comprising (1) a modified F0XP3 locus comprising a first inserted promoter operably linked to (i) a nucleotide sequence encoding a first component of a chemically inducible signaling complex (CISC), (ii) a nucleotide sequence encoding a second CISC component where the first and second CISC components dimerize in the presence of rapamycin or a rapalog to create a signaling-competent CISC and induce proliferation of the cell, and (iii) a nucleotide sequence encoding FOXP3 or a functional derivative thereof; and (2) an inserted nucleic acid comprising a second promoter operably linked to a nucleotide sequence encoding ST2, IL1RAP, IL-18R1, and / or IL18RAP, or a functional derivative thereof. In some embodiments, the second promoter is operably linked to a nucleotide sequence encoding ST2. In some embodiments, the first inserted promoter is operably linked to a nucleotide sequence encoding a soluble FRB domain. In some embodiments, the first inserted promoter is inserted downstream from a TSDR of the F0XP3 locus, and is operably linked to a first coding exon downstream of the TSDR. In some embodiments, the first inserted promoter is operably linked to a cDNA sequence encoding FOXP3.

[0134] Some aspects relate to methods of modifying cells by (1) inserting a first nucleic acid into a F0XP3 locus of the cell genome, where the first nucleic acid comprises a first promoter operably linked to (i) a nucleotide sequence encoding a first component of a chemically inducible signaling complex (CISC), and (ii) a nucleotide sequence encoding a second CISC component where the first and second CISC components dimerize in the presence of rapamycin or a rapalog to create a signaling-competent CISC and induce proliferation of the cell, and following insertion, the first promoter is operably linked to a nucleotide sequence encoding FOXP3 or a functional derivative thereof; and (2) contacting the cell with a second nucleic acid comprising a second promoter operably linked to a nucleotide sequence encoding ST2, IL1RAP, IL-18R1, and / or IL18RAP, or a functional derivative thereof. In some embodiments, the second promoter is operably linked to a nucleotide sequence encoding ST2. In some embodiments, the first promoter is operably linked to a nucleotide sequence encoding a soluble FRB domain. In some embodiments, the first promoter is inserted downstream from a TSDR of the F0XP3 locus, and is operably linked to a first coding exon downstream of the TSDR. In some embodiments, the first promoter is operably linked to a cDNA sequence encoding FOXP3.

[0135] Some aspects relate to an engineered cell comprising (1) a modified F0XP3 locus comprising a first inserted promoter operably linked to (i) a nucleotide sequence encoding a first component of a chemically inducible signaling complex (CISC), and (ii) a nucleotide sequence encoding FOXP3 or a functional derivative thereof; and (2) an inserted nucleic acid comprising a second promoter operably linked to (i) a nucleotide sequence encoding ST2, IL1RAP, IL-18R1, and / or IL18RAP, or a functional derivative thereof, and (ii) a nucleotide sequence encoding a second CISC component where the first and second CISC components dimerize in the presence of rapamycin or a rapalog to create a signaling-competent CISC and induce proliferation of the cell. In some embodiments, the second promoter is operably linked to a nucleotide sequence encoding ST2. In some embodiments, the first inserted promoter is operably linked to a nucleotide sequence encoding a soluble FRB domain. In some embodiments, the first inserted promoter is inserted downstream from a TSDR of the F0XP3 locus, and is operably linked to a first coding exon downstream of the TSDR. In some embodiments, the first inserted promoter is operably linked to a cDNA sequence encoding FOXP3.

[0136] Some aspects relate to methods of modifying cells by (1) inserting a first nucleic acid into a F0XP3 locus of the cell genome, where the first nucleic acid comprises a first promoter operably linked to (i) a nucleotide sequence encoding a first component of a chemically inducible signaling complex (CISC), and (ii) a nucleotide sequence encoding FOXP3 or a functional derivative thereof; and (2) contacting the cell with a second nucleic acid comprising a second promoter operably linked to (i) a nucleotide sequence encoding ST2, IL1RAP, IL-18R1, and / or IL18RAP, or a functional derivative thereof, and (ii) a nucleotide sequence encoding a second CISC component where the first and second CISC components dimerize in the presence of rapamycin or a rapalog to create a signaling- competent CISC and induce proliferation of the cell. In some embodiments, the second promoter is operably linked to a nucleotide sequence encoding ST2. In some embodiments, the first promoter is operably linked to a nucleotide sequence encoding a soluble FRB domain. In some embodiments, the first promoter is inserted downstream from a TSDR of the F0XP3 locus, and is operably linked to a first coding exon downstream of the TSDR. In some embodiments, the first promoter is operably linked to a cDNA sequence encoding FOXP3.

[0137] Some aspects relate to an engineered cell comprising a modified F0XP3 locus and a modified second locus other than a F0XP3 locus, where the modified F0XP3 locus comprises a first inserted promoter that is operably linked to (i) a nucleotide sequence encoding F0XP3 or a functional derivative thereof, and (ii) a nucleotide sequence encoding a first component of a chemically inducible signaling complex (CISC), and the second locus comprises a second inserted promoter operably linked to (i) a nucleotide sequence encoding ST2, IL1RAP, IL-18R1, and / or IL18RAP, or a functional derivative thereof, and (ii) a second component of the CISC, where the first and second CISC components dimerize in the presence of rapamycin or a rapalog to create a signaling-competent CISC and induce proliferation of the cell. In some embodiments, the second locus is a P2-microglobulin (B2M) locus. In some embodiments, the second locus is a TRAC locus. In some embodiments, the second promoter is operably linked to a nucleotide sequence encoding ST2. In some embodiments, the first inserted promoter is operably linked to a nucleotide sequence encoding a soluble FRB domain. In some embodiments, the first inserted promoter is inserted downstream from a TSDR of the F0XP3 locus, and is operably linked to a first coding exon downstream of the TSDR. In some embodiments, the first inserted promoter is operably linked to a cDNA sequence encoding FOXP3.

[0138] Some aspects relate to methods of modifying cells by (1) inserting a first nucleic acid into a F0XP3 locus of the cell genome, and (2) inserting a second nucleic acid into a second locus of the cell genome other than a F0XP3 locus, where the first nucleic acid comprises a first promoter operably linked to a nucleotide sequence encoding a first component of a chemically inducible signaling complex (CISC), and following insertion, the first promoter is operably linked a nucleotide sequence encoding F0XP3 or a functional derivative thereof, where the second nucleic acid comprises a second locus operably linked to a nucleotide sequence encoding ST2, IL1RAP, IL-18R1, and / or IL18RAP, or a functional derivative thereof, and (ii) a second component of the CISC, where the first and second CISC components dimerize in the presence of rapamycin or a rapalog to create a signaling- competent CISC and induce proliferation of the cell. In some embodiments, the second locus is a P2-microglobulin (B2M) locus. In some embodiments, the second locus is a TRAC locus. In some embodiments, the second promoter is operably linked to a nucleotide sequence encoding ST2. In some embodiments, the first promoter is operably linked to a nucleotide sequence encoding a soluble FRB domain. In some embodiments, the first promoter is inserted downstream from a TSDR of the F0XP3 locus, and is operably linked to a first coding exon downstream of the TSDR. In some embodiments, the first promoter is operably linked to a cDNA sequence encoding FOXP3.

[0139] In some embodiments, the engineered Treg is a stabilized Treg. In some embodiments, the stabilized Treg is a cell (e.g., stem cell, progenitor cell, T cell) that has been modified to exhibit and maintain a Treg phenotype.

[0140] Stabilized Tregs may be generated using any suitable method. For example, in some embodiments, stabilized Tregs are generated by expressing the expression of one or more transcription factors. As an example, the overexpression of CCAAT / enhancer binding protein (CZEBP), a transcription factor, may be used to withdraw suppression of FoxP3 expression to stabilize the Treg phenotype. Similarly, nuclear factors may be inhibited to increase FoxP3 expression. Examples of such nuclear factors include, but are not limited to, Spl, Rnf20, Rfx7, Srf, Elp2, Nsdl, Smarcbl, Klf2, Ctcf, and Satbl. Alternatively or additionally, certain nuclear factors may be overexpressed to increase FoxP3 expression. Examples of such nuclear factors include, but are not limited to, Usp22, Cbfb, Runxl, Myc, SS18, Med30, Atxn713, Med 12, Hnmpk, Zfp281, Taf51, Ddit3, Zmynd8, Med 14, Rad21, Dmapl, Medl l, Zkscan3, Foxpl, and Stat5b. In some embodiments, the expression of one or both of CDK8 and CDK19 reduced, deleted, or pharmacologically inhibited to stabilize a Treg. IL-2, IL-10, IL-35, and / or TNFR2 supplementation may additionally or alternatively be used to support the Treg phenotype.

[0141] In some embodiments, the engineered Treg comprises an exogenous nucleotide sequence encoding a lineage commitment factor that (i) promotes differentiation of a cell to a CD4+ Treg, and / or (ii) promoters the maintenance of the cell as a CD4+Treg. In some embodiments, the lineage commitment factor is selected from FOXP3, Helios, Ikaros. In some embodiments, a partial FOXP3 cDNA sequence is inserted in-frame with one or more exons of an endogenous F0XP3 coding sequence.

[0142] In some embodiments, the engineered Treg comprises an exogenous nucleotide sequence encoding FOXP3. In some embodiments, the engineered Treg comprises an exogenous nucleotide sequence comprising a promoter operably linked to a nucleotide sequence encoding FOXP3. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is an MND, EF-la, or PGK promoter. In some embodiments, the nucleotide sequence encoding FOXP3 is a FOXP3 cDNA sequence. Methods for expressing an exogenous coding sequence in a cell

[0143] Some aspects relate to methods of producing a genetically modified cell by introducing into the cell a nucleic acid comprising a promoter that is operably linked to a coding sequence encoding a repair mediator (e.g., ST2, IL1RAP, IL-18R1, and / or IL18RAP), and / or one or more functional derivatives of the repair mediator (e.g., ST2, IL1RAP, IL- 18R1, and / or IL18RAP). Such a nucleic acid may be integrated into the genome of a cell (e.g., by homologous recombination), such that the encoded gene products (e.g., ST2, IL1RAP, IL-18R1, and / or IL18RAP) are expressed from the genome. Alternatively, the encoded gene products (e.g., ST2, IL1RAP, IL-18R1, and / or IL18RAP) may be expressed episomally.

[0144] In some embodiments, the coding sequence encoding the repair mediator (e.g, ST2, IL 1 RAP, IL-18R1, and / or IL18RAP) does not comprise an intron (e.g, the coding sequence is a cDNA sequence). Transcription of mammalian genes generally yields RNA containing multiple exons of a coding sequence that are separated by intervening regions (introns), and processing of this RNA includes RNA splicing to remove introns, yielding an RNA comprising an open reading frame that is capable of being translated by ribosomes and tRNAs to produce the encoded polypeptide. Expression of a gene product from a coding sequence without introns (e.g., a cDNA sequence) abrogates the need for splicing, thereby allowing more rapid and efficient gene expression from an intron-deficient coding sequence relative to a coding sequence that must be spliced between transcription and translation.

[0145] In some embodiments, a method comprises contacting a cell with one or more nucleic acids to produce the genetically modified cell. In some embodiments, introducing a nucleic acid, protein, or vector into the cell comprises contacting the cell with the nucleic acid, protein, or vector, respectively. In some embodiments, the cell is in vitro. In some embodiments, the cell is ex vivo. In some embodiments, a cell is isolated from a subject, contacted with one or more nucleic acids, and administered to the same subject. In some embodiments, the cell is ex vivo. In some embodiments, a cell is isolated from a subject, contacted with one or more nucleic acids, and administered to a different subject. In some embodiments, the cell is in vivo. In some embodiments, the cell is modified extracorporeally. In some embodiments, the cell is a T cell. In some embodiments, the cell is a CD4+ T cell.

[0146] Nucleic acid molecules may be introduced into a cell using any suitable method. For example, viral or non-viral systems may be used to deliver transgenes into a cell. In some embodiments, the nucleic acid comprising a promoter that is operably linked to a coding sequence encoding a repair mediator (e.g., ST2, IL1RAP, IL-18R1, and / or IL18RAP), and / or one or more functional derivatives of the repair mediator (e.g., ST2, IL1RAP, IL-18R1, and / or IL18RAP) is delivered to a cell. The non-viral approaches include, but are not limited to, polymer nanoparticles, lipids, calcium phosphate, electroporation / nucleofection or biolistic delivery of DNA-coated microparticles (Matuskova and Durinikova, 2016. Advances in Molecular Retrovirology InTech).

[0147] Multiple vectors can be used in accordance with the nucleic acids and methods, depending on whether the DNA is integrated into chromatin of the host cell or not. Retroviral vectors such as those derived from gammaretroviruses or lentiviruses persist in the nucleus as integrated provirus and reproduce with cell division. Other types of vectors (e.g., those derived from herpesviruses or adenoviruses) remain in the cell in the episomal form.

[0148] In some embodiments, the viral vector is selected from a modified virus derived from a virus selected from the group consisting of a retrovirus, lentivirus, gammavirus, adenovirus, adeno-associated virus, pox virus, alphavirus, and herpes virus.

[0149] In some embodiments, the vector is a retrovirus, such as a modified gammavirus, lentivirus, murine stem cell virus, moloney murine leukemia virus, bovine leukaemia virus, Rous sarcoma virus, or spumavirus. In some embodiments, the viral vector is a retrovirus. In some embodiments, the viral vector is a lentiviral vector. In some embodiments, the viral vector is a gamma-retroviral vector.

[0150] In some embodiments, the vector comprises a promoter operably linked to a nucleotide sequence encoding ST2 or a functional derivative thereof. In some embodiments, the vector comprises a promoter operably linked to a nucleotide sequence encoding IL1RAP or a functional derivative thereof. In some embodiments, the vector comprises a promoter operably linked to a nucleotide sequence encoding IL-18R1 or a functional derivative thereof. In some embodiments, the vector comprises a promoter operably linked to a nucleotide sequence encoding IL18RAP or a functional derivative thereof. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is selected from an MND promoter, a PGK promoter, and an EF- la promoter. In some embodiments, the promoter is further operably linked to a nucleotide sequence encoding a CISC component. In some embodiments, the CISC component comprises an extracellular FKBP domain, an IL- 2Ry transmembrane domain, and an IL-2RY cytoplasmic domain. In some embodiments, a nucleotide sequence encoding a 2A motif is present between the nucleotide sequence encoding the CISC component and a nucleotide sequence encoding another protein (e.g., ST2). In some embodiments, the promoter is further operably linked to a nucleotide sequence encoding a selectable marker. In some embodiments, the selectable marker is LNGFR. In some embodiments, the vector comprises an MND promoter operably linked to a nucleotide sequence encoding ST2. In some embodiments, the vector comprises a nucleotide sequence with at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleotide sequence of SEQ ID NO: 161. In some embodiments, the vector comprises the nucleotide sequence of SEQ ID NO: 161.

[0151] In some embodiments, the vector comprises an MND promoter operably linked to (i) a nucleotide sequence encoding ST2, and (ii) a selectable marker. In some embodiments, the selectable marker is LNGFR. In some embodiments, the vector comprises a nucleotide sequence with at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleotide sequence of SEQ ID NO: 162. In some embodiments, the vector comprises the nucleotide sequence of SEQ ID NO: 162.

[0152] In some embodiments, the vector comprises an MND promoter operably linked to (i) a nucleotide sequence encoding ST2, and (ii) a nucleotide sequence encoding a CISC component. In some embodiments, the CISC component comprises an extracellular FKBP domain, an IL-2Ry transmembrane domain, and an IL-2Ry cytoplasmic domain. In some embodiments, the CISC component comprises an extracellular FRB domain, an IL-2RP transmembrane domain, and an IL-2RP cytoplasmic domain.

[0153] In some embodiments, the nucleotide sequence encoding ST2 is 5' to the nucleotide sequence encoding the CISC component. In some embodiments, the vector comprises a nucleotide sequence with at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleotide sequence of SEQ ID NO: 163. In some embodiments, the vector comprises the nucleotide sequence of SEQ ID NO: 163.

[0154] In some embodiments, the nucleotide sequence encoding ST2 is 3' to the nucleotide sequence encoding the CISC component. In some embodiments, the vector comprises a nucleotide sequence with at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleotide sequence of SEQ ID NO: 164. In some embodiments, the vector comprises the nucleotide sequence of SEQ ID NO: 164.

[0155] In some embodiments, one or more vectors are adeno-associated viral (AAV) vectors. In some embodiments, one or more AAV vectors is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11 vector. In some embodiments, one or more AAV vectors are AAV5 vectors. In some embodiments, one or more AAV vectors are AAV6 vectors.

[0156] In some embodiments, the polypeptide is the sole polypeptide encoded by the nucleotide sequence, z.e., the nucleic acid molecule of the viral vector does not encode additional different proteins but may comprise additional control elements such as promoters and terminators. Some aspects relate to a composition comprising the viral vector. Some aspects relate to a regulatory T cell comprising any one of the nucleic acid molecules, or the viral vector. In some embodiments, the T cell is a T regulatory cell (Treg), such as a mammalian Treg.

[0157] In some embodiments, the mammalian Treg expresses any one of the polypeptides.

[0158] In some embodiments, the mammalian Treg is a human Treg.

[0159] Some aspects relate to a method of preparing allogeneic or autologous Tregs with a repair Treg phenotype, the method comprising contacting T cells with the nucleic acid molecule comprising a nucleotide sequence encoding any one of the polypeptides, or a viral vector comprising it, thereby endowing said T cells with a repair Treg phenotype, and thus preparing Tregs with a repair phenotype. In some embodiments, the T cell is a CD4+ T cell. In some embodiments, the T cell is a CD8+ T cell.

[0160] Preparation of CD4+ T cells may be performed using any suitable method (e.g., separation of human CD4+ T cells followed by preparation of recombinant retroviral stock and retroviral transduction of human CD4+ T cells).

[0161] Recombinant retroviral and lentiviral vectors may be prepared and used to transduce cells using any suitable method, such as use of a commercial kit including packaging cells, plasmids and transfection reagents, which are offered by many companies, including Invitrogen®, Sigma®, Clontech®, Cell Biolabs®, SBI®, Genecopoeia® and many others. The methods are thus performed along with the guidelines supplied with the commercial kits.

[0162] In short, according to a non-limiting example taught by the y-Retrovirus Guide of Addgene, the following components are used: (a) y-Retroviral transfer plasmid encoding a transgene of interest: The transgene sequence is flanked by long terminal repeat (LTR) sequences, which facilitate integration of the transfer plasmid sequences into the host genome. Typically, it is the sequences between and including the LTRs that is integrated into the host genome upon viral transduction; (b) Packaging genes (viral Gag-Pol): Gag is a structural precursor protein, and Pol is a polymerase; and (c) Envelope gene (may be pseudotyped to alter infectivity). As a non-limiting example, the three components described above (envelope, packaging, and transfer) are supplied by three types of plasmids, which are co-transfected into a 293T packaging cell line. This system allows for flexibility to pseudotype y-retrovirus using different envelopes to modify tropism. Different envelope plasmids can direct the production of virus with various tropisms.

[0163] As an example, recombinant retroviral stock may be prepared by cloning, taking the resulting plasmid as well as a plasmid carrying gag / pol and a plasmid carrying env, and transfecting HEK293T cells. In some embodiments, the plasmid may comprise any combination of a nucleotide sequence encoding a protein (mem-IL-33, ST2, IL1RAP, IL- 18R1, and / or IL18RAP), Gag / Pol, and Env. A further non-limiting example of methods for transducing human cells (e.g., CD4+ T cells), includes combining the viral supernatant with a transfection reagent (e.g., Polybrene (Merck®)), adding the composition to RetroNectin®- (Takara®) coated wells and spinning down. The resulting supernatant is then removed and CD4+ T cells are added and placed in an incubator for transfection.

[0164] In some embodiments, the cells are T cells. In some embodiments, the cells are CD4+ T cells. In some embodiments, the cells are ex vivo. In some embodiments, the cells are in vivo. In some embodiments, the cells are extracorporeal.

[0165] ST2 and / or IL 1 RAP expression

[0166] Some embodiments of the methods comprise introducing a nucleic acid comprising a promoter operably linked to a nucleic acid sequence encoding ST2 and / or IL1RAP, or a functional derivative thereof, into a cell. Some embodiments of the methods comprise introducing a nucleic acid comprising a promoter operably linked to a nucleic acid sequence encoding ST2, or a functional derivative thereof, into a cell. The functional derivative of ST2 may include a protein that has a substantial activity of a wild-type ST2, or increased activity relative to wild-type ST2. One of ordinary skill in the art may use any suitable method (e.g., phosphorylation assays and / or assaying changes in gene expression following stimulation with ST2) to test the functionality or activity of a ST2 or derivative thereof. The functional derivative of ST2 may also include any ST2 or fragment thereof that has conservative substitutions of one or more amino acid residues relative to full-length, wild-type ST2 as set forth in SEQ ID NO: 1.

[0167] In some embodiments, the functional derivative has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 substitutions relative to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the functional derivative has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 insertions relative to the amino acid sequence of SEQ ID NO:

[0168] 1. In some embodiments, the functional derivative has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,

[0169] 14, 15, 16, 17, 18, 19, or 20 deletions relative to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the functional derivative comprises an N-terminal fusion with another polypeptide. In some embodiments, the functional derivative comprises a C-terminal fusion with another polypeptide. In some embodiments, a functional derivative lacks one or more domains or portions of wild-type ST2 that are unnecessary for signaling.

[0170] In some embodiments, the encoded ST2 or functional derivative thereof has about or at least 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or more of the activity exhibited by a wild-type ST2. In some embodiments, the encoded ST2 comprises an amino acid sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or up to 100% sequence identity to the amino acid sequence of wildtype human ST2 set forth as SEQ ID NO: 1. In some embodiments, the encoded ST2 comprises the wild-type amino acid sequence of SEQ ID NO: 1. In some embodiments, the encoded ST2 consists of the wild-type amino acid sequence of SEQ ID NO: 1.

[0171] Some embodiments of the methods comprise introducing a nucleic acid comprising a promoter operably linked to a nucleic acid sequence encoding IL1RAP, or a functional derivative thereof, into a cell. The functional derivative of IL1RAP may include a protein that has a substantial activity of a wild-type IL1RAP, or increased activity relative to wild-type IL1RAP. One of ordinary skill in the art may use any suitable method (e.g., phosphorylation assays and / or assaying changes in gene expression following stimulation with IL1RAP) to test the functionality or activity of a IL 1 RAP or derivative thereof. The functional derivative of IL1RAP may also include any IL1RAP or fragment thereof that has conservative substitutions of one or more amino acid residues relative to full-length, wild-type IL1RAP as set forth in SEQ ID NO: 2.

[0172] In some embodiments, the functional derivative has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 substitutions relative to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the functional derivative has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,

[0173] 13, 14, 15, 16, 17, 18, 19, or 20 insertions relative to the amino acid sequence of SEQ ID NO:

[0174] 2. In some embodiments, the functional derivative has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,

[0175] 14, 15, 16, 17, 18, 19, or 20 deletions relative to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the functional derivative comprises an N-terminal fusion with another polypeptide. In some embodiments, the functional derivative comprises a C-terminal fusion with another polypeptide. In some embodiments, a functional derivative lacks one or more domains or portions of wild-type IL1RAP that are unnecessary for signaling.

[0176] In some embodiments, the encoded IL1RAP or functional derivative thereof has about or at least 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or more of the activity exhibited by a wild-type IL1RAP. In some embodiments, the encoded IL1RAP comprises an amino acid sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or up to 100% sequence identity to the amino acid sequence of wild-type human IL1RAP set forth as SEQ ID NO: 2. In some embodiments, the encoded ILlRAPcomprises the wild-type amino acid sequence of SEQ ID NO: 2. In some embodiments, the encoded IL1RAP consists of the wild-type amino acid sequence of SEQ ID NO: 2.

[0177] In some embodiments, a nucleotide sequence encoding ST2 is codon-optimized. In some embodiments, ST2 is encoded by a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence of SEQ ID NO: 52. In some embodiments, ST2 is encoded by nucleotide sequence of SEQ ID NO: 52. ST2 may be expressed with any suitable signal peptide. In some embodiments, the signal peptide is a wild-type ST2 signal peptide. An example of an ST2 signal peptide amino acid sequence is provided by SEQ ID NO: 43. In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 43. In some embodiments, the signal peptide is encoded by a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence of SEQ ID NO: 53. In some embodiments, the signal peptide is encoded by the nucleotide sequence of SEQ ID NO: 53.

[0178] In some embodiments, a nucleotide sequence encoding IL1RAP is codon-optimized. In some embodiments, IL1RAP is encoded by a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence of SEQ ID NO: 54. In some embodiments, IL1RAP is encoded by nucleotide sequence of SEQ ID NO: 54. IL1RAP may be expressed with any suitable signal peptide. In some embodiments, the signal peptide is a wild-type IL1RAP signal peptide. An example of an IL1RAP signal peptide amino acid sequence is provided by SEQ ID NO: 44. In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 55. In some embodiments, the signal peptide is encoded by a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence of SEQ ID NO: 55. In some embodiments, the signal peptide is encoded by the nucleotide sequence of SEQ ID NO: 55. IL-18R1 and / or IL18RAP expression

[0179] Some embodiments of the methods comprise introducing a nucleic acid comprising a promoter operably linked to a nucleic acid sequence encoding IL-18R1 and / or IL18RAP, or a functional derivative thereof, into a cell. Some embodiments of the methods comprise introducing a nucleic acid comprising a promoter operably linked to a nucleic acid sequence encoding IL-18R1, or a functional derivative thereof, into a cell. The functional derivative of IL-18R1 may include a protein that has a substantial activity of a wild-type IL-18R1, or increased activity relative to wild-type IL-18R1. One of ordinary skill in the art may use any suitable method (e.g., phosphorylation assays and / or assaying changes in gene expression following stimulation with IL-18R1) to test the functionality or activity of an IL-18R1 or derivative thereof. The functional derivative of IL-18R1 may also include any IL-18R1 or fragment thereof that has conservative substitutions of one or more amino acid residues relative to full-length, wild-type IL-18R1 of SEQ ID NO: 40.

[0180] In some embodiments, the functional derivative has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 substitutions relative to the amino acid sequence of SEQ ID NO: 40. In some embodiments, the functional derivative has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 insertions relative to the amino acid sequence of SEQ ID NO: 40. In some embodiments, the functional derivative has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 deletions relative to the amino acid sequence of SEQ ID NO: 40. In some embodiments, the functional derivative comprises a C-terminal fusion with another polypeptide. In some embodiments, a functional derivative lacks one or more domains or portions of wild-type IL-18R1 that are unnecessary for signaling.

[0181] In some embodiments, the encoded IL-18R1 or functional derivative thereof has about or at least 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or more of the activity exhibited by a wild-type IL-18R1 of SEQ ID NO: 40. In some embodiments, the encoded IL-18R1 comprises an amino acid sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or up to 100% sequence identity to the amino acid sequence of wild-type human IL-18R1 of SEQ ID NO: 40. In some embodiments, the encoded IL-18R1 comprises the wild-type amino acid sequence of SEQ ID NO: 40. In some embodiments, the encoded IL-18R1 consists of the wild-type amino acid sequence of SEQ ID NO: 40.

[0182] Some embodiments of the methods comprise introducing a nucleic acid comprising a promoter operably linked to a nucleic acid sequence encoding IL18RAP, or a functional derivative thereof, into a cell. The functional derivative of IL18RAP may include a protein that has a substantial activity of a wild-type IL18RAP, or increased activity relative to wildtype IL18RAP. One of ordinary skill in the art may use any suitable method (e.g., phosphorylation assays and / or assaying changes in gene expression following stimulation with IL18RAP) to test the functionality or activity of an IL18RAP or derivative thereof. The functional derivative of IL18RAP may also include any IL18RAP or fragment thereof that has conservative substitutions of one or more amino acid residues relative to full-length, wild-type IL18RAP of SEQ ID NO: 41.

[0183] In some embodiments, the functional derivative has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 substitutions relative to the amino acid sequence of SEQ ID NO: 41. In some embodiments, the functional derivative has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 insertions relative to the amino acid sequence of SEQ ID NO: 41. In some embodiments, the functional derivative has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 deletions relative to the amino acid sequence of SEQ ID NO: 41. In some embodiments, the functional derivative comprises a C-terminal fusion with another polypeptide. In some embodiments, a functional derivative lacks one or more domains or portions of wild-type IL18RAP that are unnecessary for signaling.

[0184] In some embodiments, the encoded IL18RAP or functional derivative thereof has about or at least 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or more of the activity exhibited by a wild-type IL18RAP of SEQ ID NO: 41. In some embodiments, the encoded IL18RAP comprises an amino acid sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or up to 100% sequence identity to the amino acid sequence of wild-type human IL18RAP of SEQ ID NO: 41. In some embodiments, the encoded IL18RAP comprises the wild-type amino acid sequence of SEQ ID NO: 41. In some embodiments, the encoded IL18RAP consists of the wild-type amino acid sequence of SEQ ID NO: 41.

[0185] In some embodiments, a nucleotide sequence encoding IL-18R1 is codon-optimized. In some embodiments, IL-18R1 is encoded by a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence of SEQ ID NO: 110. In some embodiments, IL-18R1 is encoded by nucleotide sequence of SEQ ID NO: 110. IL-18R1 may be expressed with any suitable signal peptide. In some embodiments, the signal peptide is a wild-type IL-18R1 signal peptide. An example of an IL- 18R1 signal peptide amino acid sequence is provided by SEQ ID NO: 45. In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 45. In some embodiments, the signal peptide is encoded by a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence of SEQ ID NO: 111. In some embodiments, the signal peptide is encoded by the nucleotide sequence of SEQ ID NO: 111.

[0186] In some embodiments, a nucleotide sequence encoding IL18RAP is codon-optimized. In some embodiments, IL18RAP is encoded by a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence of SEQ ID NO: 112. In some embodiments, IL18RAP is encoded by nucleotide sequence of SEQ ID NO: 112. IL18RAP may be expressed with any suitable signal peptide. In some embodiments, the signal peptide is a wild-type IL18RAP signal peptide. An example of an IL18RAP signal peptide amino acid sequence is provided by SEQ ID NO: 46. In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 46. In some embodiments, the signal peptide is encoded by a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence of SEQ ID NO: 113. In some embodiments, the signal peptide is encoded by the nucleotide sequence of SEQ ID NO: 113.

[0187] Other repair mediators ’ expression

[0188] Some embodiments of the methods comprise introducing a nucleic acid comprising a promoter operably linked to a nucleic acid sequence encoding a repair mediator (e.g, IL- 18R), or a functional derivative thereof, into a cell. The functional derivative of the repair mediator (e.g, IL-18R) may include a protein that has a substantial activity of a wild-type repair mediator (e.g., IL-18R), or increased activity relative to the wild-type repair mediator (e.g., IL-18R). One of ordinary skill in the art may use any suitable method to test the functionality or activity of a repair mediator (e.g., IL-18R) or derivative thereof. The functional derivative of a repair mediator (e.g., IL-18R) may also include any repair mediator (e.g., IL-18R) or fragment thereof that has conservative substitutions of one or more amino acid residues relative to full-length, wild-type repair mediator (e.g., IL-18R).

[0189] Other repair mediators include a transcription factor or activator of a repair pathway, such as GATA3, PPARG, and / or BATF; cytokines (e.g., AREG and / or osteopontin); growth factors (e.g., KGF, TGF-P); and / or chemokine receptors (e.g., CCR2, CCR5, CCR8) that promote repair and regeneration. See, e.g, Wohlfert et al., J Clin Invest. 2011. 121(11):4503— 4515 (discussing GATA3); Miao et al., Cell Commun Signal. 2022. 20(l):48 (discussing PPARgamma); Trujillo-Ochoa et al, Nat Rev Immunol . 2023. 23(12):842-856 (discussing BATF); Zhang et al., Cell Physiol Biochem. 2017. 43 (6) :2155-2169 (discussing AREG, KGF, and chemokine receptors); Shi et al. , Immunity. 2021. 54(7): 1527-1542 (discussing osteopontin); Konkel et aL, Immunity. 2017. 46:660-674 (discussing TGF-P).

[0190] Targeted loci

[0191] Nucleic acids comprising promoters operably linked to a coding sequence (e.g., a nucleic acid sequence encoding ST2, IL1RAP IL-18R1, or IL18RAP) may be inserted into a targeted locus, such that a population of genetically modified cells contain the inserted sequences at a consistent location of the genome. Such consistency is useful, for example, in screening cells and cell populations by analyzing the targeted locus (e.g., by PCR amplification of genomic DNA using primers flanking the insertion site).

[0192] In some embodiments, the nucleic acid comprising a promoter that is introduced into the cell is inserted at a targeted locus. The targeted locus may correspond to one or more polypeptides encoded by the nucleic acid (e.g., a nucleic acid encoding ST2 is inserted at a ST2 locus or a nucleic acid encoding IL-18R1 is inserted at an IL18R1 locus). In such embodiments, the sequence inserted into the targeted locus may replace all or part of the endogenous coding sequence encoding the polypeptide. In some embodiments, one or more mutations (e.g., nonsense mutation) is introduced into the endogenous coding sequence to prevent translation of a full-length polypeptide from the endogenous coding sequence. In some embodiments, all or part of the endogenous coding sequence is removed from the genome by insertion of the promoter and coding sequence on the inserted nucleic acid.

[0193] In some embodiments, the targeted locus is a safe harbor locus. In some embodiments, the safe harbor locus is a HIPP 11 locus. In some embodiments, the safe harbor locus is a ROSA26 locus. In some embodiments, the safe harbor locus is an AAVS1 locus. In some embodiments, the targeted locus is a T cell receptor locus. In some embodiments, the T cell receptor locus is a TRAC locus. In some embodiments, the T cell receptor locus is a TRBC locus.

[0194] In some embodiments, nucleic acids may be integrated in a non-targeted manner (e.g., by use of a lentiviral vector), such that a population of genetically modified cells contains diverse integration sites.

[0195] In some embodiments, the nucleic acid, or vector comprising such a nucleic acid, is not integrated into the genome of the cell. For example, a plasmid or artificial chromosome (e.g., human artificial chromosome) may be introduced into the cell, with the promoter driving transcription of the operably linked coding sequence from the plasmid or artificial chromosome, without integration of the vector into the chromosome. In some embodiments, the introduced vector or nucleic acid replicates independently of endogenous chromosomes. In some embodiments, more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of the vector are present in a genetically modified cell. In some embodiments, the number of copies of the vector in a cell exceeds the copy number of an individual chromosome in a cell.

[0196] Methods for stabilizing expression of an endogenous gene in a cell

[0197] Some aspects relate to methods of producing a genetically modified cell by inserting a promoter into a nucleic acid of a cell genome (e.g., by homologous recombination) upstream from a coding sequence of an endogenous ST2, IL1RAP, IL-18R1, or IL18RAP gene on the nucleic acid, such that the inserted promoter becomes operably linked to a coding sequence of the endogenous ST2, IL1RAP, IL-18R, or IL18RAP gene. In some embodiments, a donor template comprising the promoter is introduced into the cell, and incorporated into the genome by homologous recombination.

[0198] Insertion of a promoter into the genome allows regulation of a genomic coding sequence (e.g., endogenous ST2, IL1RAP, IL-18R, or IL 18RAP coding sequence) in a desired manner, depending on the type and placement of the promoter. For example, placement of a promoter downstream from an endogenous regulatory element may bypass endogenous regulatory mechanisms (e.g., silencing of the gene in certain conditions, and expression in other conditions), allowing constitutive expression (e.g., by insertion of a constitutive promoter) or tunable expression (e.g., by insertion of an inducible or regulatable promoter and administration of an inducing agent for that promoter).

[0199] In some embodiments, the method further comprises modifying an endogenous coding sequence to which the promoter is operably linked. Such modifications may correct one or more endogenous mutations (e.g., to restore protein function to that of a wild-type protein), remove one more endogenous sequence elements (e.g., introns), or introduce one or more mutations to improve protein function (e.g., inhibit or bypass endogenous regulation).

[0200] Insertion of a promoter may modify the coding sequence expressed from the genome. For example, promoter insertion downstream of one or more exons may shorten the sequence of the protein expressed from the endogenous gene. The insertion of a promoter with an additional nucleic acid sequence (e.g., including an in-frame START codon and optionally additional in-frame codons) may incorporate additional amino acids into the N-terminus of the encoded polypeptide. The inclusion of a nucleic acid sequence downstream from the promoter on the donor template may replace a nucleic acid sequence in the endogenous ST2 or IL18R1 gene, thereby altering the coding sequence and amino acid sequence of the encoded polypeptide. In other embodiments, the promoter is inserted upstream from the first coding exon of the endogenous coding sequence, and the amino acid sequence of the encoded ST2 or IL-18R1 polypeptide is not altered by insertion of the promoter.

[0201] Modification of an endogenous coding sequence to substitute desired amino acids may be accomplished by any suitable method. In some embodiments, the donor template comprising the promoter further comprises a homology arm comprising a modified coding sequence or portion thereof, such that integration of the donor template into the cell genome replaces the codons encoding the substituted amino acids with codons encoding the desired amino acids. In some embodiments, the homology arm comprising the modified coding sequence or portion thereof comprises a homologous nucleic acid sequence downstream from the modified coding sequence or portion thereof, where the homologous nucleic acid sequence is identical to an endogenous sequence downstream from the endogenous coding sequence to be modified, to promote homologous recombination.

[0202] In some embodiments, the endogenous coding sequence is modified before insertion of the promoter. In some embodiments, the endogenous coding sequence is modified after insertion of the promoter (e.g., by incorporation of a second donor template by a second homologous recombination event).

[0203] In some embodiments, a method comprises contacting a cell with one or more nucleic acids to produce the genetically modified cell. In some embodiments, introducing a nucleic acid, protein, or vector into the cell comprises contacting the cell with the nucleic acid, protein, or vector, respectively. In some embodiments, the cell is in vitro. In some embodiments, the cell is ex vivo. In some embodiments, a cell is isolated from a subject, contacted with one or more nucleic acids, and administered to the same subject. In some embodiments, the cell is ex vivo. In some embodiments, a cell is isolated from a subject, contacted with one or more nucleic acids, and administered to a different subject. In some embodiments, the cell is in vivo. In some embodiments, the cell is modified extracorporeally. In some embodiments, the cell is a T cell. In some embodiments, the cell is a CD4+ T cell.

[0204] ST2 and / or IL 1 RAP expression

[0205] Some embodiments of the methods comprise inserting a promoter into the genome of a cell upstream from a coding sequence of an endogenous ST2 gene and / or IL1RAP gene, such that the inserted promoter is operably linked to the coding sequence of the endogenous ST2 gene or the IL1RAP gene. In some embodiments, the method further comprises modifying a coding sequence of the endogenous ST2 gene and / or the endogenous IL1RAP gene. Such modifications may remove one or more introns and / or mutate one or more exons of the endogenous ST2 gene and / or IL1RAP gene.

[0206] In some embodiments, the modified coding sequence encodes a functional derivative of ST2 or IL1RAP. The functional derivative of ST2 or IL1RAP may include a protein that has a substantial activity of a wild-type ST2 or IL1RAP, or increased activity relative to wildtype ST2 or IL1RAP. One of ordinary skill in the art may use any suitable method to test the functionality or activity of a ST2 or IL1RAP or derivative thereof. The functional derivative of ST2 or IL1RAP may also include any ST2 or IL1RAP or fragment thereof that has conservative substitutions of one or more amino acid residues relative to full-length, wildtype ST2 or IL1RAP as set forth in SEQ ID NOs: 1 and 2, respectively.

[0207] In some embodiments, the functional derivative has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 substitutions relative to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the functional derivative has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,

[0208] 13, 14, 15, 16, 17, 18, 19, or 20 insertions relative to the amino acid sequence of SEQ ID NO:

[0209] 1. In some embodiments, the functional derivative has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,

[0210] 14, 15, 16, 17, 18, 19, or 20 deletions relative to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the functional derivative comprises an N-terminal fusion with another polypeptide. In some embodiments, the functional derivative comprises a C-terminal fusion with another polypeptide. In some embodiments, a functional derivative lacks one or more domains or portions of wild-type ST2 that are unnecessary for signaling.

[0211] In some embodiments, the functional derivative has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 substitutions relative to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the functional derivative has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,

[0212] 13, 14, 15, 16, 17, 18, 19, or 20 insertions relative to the amino acid sequence of SEQ ID NO:

[0213] 2. In some embodiments, the functional derivative has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,

[0214] 14, 15, 16, 17, 18, 19, or 20 deletions relative to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the functional derivative comprises an N-terminal fusion with another polypeptide. In some embodiments, the functional derivative comprises a C-terminal fusion with another polypeptide. In some embodiments, a functional derivative lacks one or more domains or portions of wild-type IL1RAP that are unnecessary for signaling.

[0215] In some embodiments, the encoded ST2 or IL1RAP or functional derivative thereof has about or at least 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or more of the activity exhibited by a wild-type ST2 or IL1RAP. In some embodiments, the encoded ST2 or IL1RAP comprises an amino acid sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or up to 100% sequence identity to the amino acid sequence of wild-type human ST2 or IL1RAP set forth as SEQ ID NOs: 1 and 2, respectively. In some embodiments, the encoded ST2 comprises the wild-type amino acid sequence of SEQ ID NO: 1. In some embodiments, the encoded ST2 consists of the wild-type amino acid sequence of SEQ ID NO: 1. In some embodiments, the encoded IL1RAP comprises the wild-type amino acid sequence of SEQ ID NO: 2. In some embodiments, the encoded IL1RAP consists of the wild-type amino acid sequence of SEQ ID NO: 2.

[0216] In some embodiments, a nucleotide sequence encoding ST2 is codon-optimized. In some embodiments, ST2 is encoded by a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence of SEQ ID NO: 52. In some embodiments, ST2 is encoded by nucleotide sequence of SEQ ID NO: 52. ST2 may be expressed with any suitable signal peptide. In some embodiments, the signal peptide is a wild-type ST2 signal peptide. An example of an ST2 signal peptide amino acid sequence is provided by SEQ ID NO: 43. In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 43. In some embodiments, the signal peptide is encoded by a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence of SEQ ID NO: 53. In some embodiments, the signal peptide is encoded by the nucleotide sequence of SEQ ID NO: 53.

[0217] In some embodiments, a nucleotide sequence encoding IL1RAP is codon-optimized. In some embodiments, IL1RAP is encoded by a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence of SEQ ID NO: 54. In some embodiments, IL1RAP is encoded by nucleotide sequence of SEQ ID NO: 54. IL1RAP may be expressed with any suitable signal peptide. In some embodiments, the signal peptide is a wild-type IL1RAP signal peptide. An example of an IL1RAP signal peptide amino acid sequence is provided by SEQ ID NO: 44. In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 55. In some embodiments, the signal peptide is encoded by a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence of SEQ ID NO: 55. In some embodiments, the signal peptide is encoded by the nucleotide sequence of SEQ ID NO: 55. IL-18R1 and / or IL18RAP expression

[0218] Some embodiments of the methods comprise inserting a promoter into the genome of a cell upstream from a coding sequence of an endogenous IL18R1 gene and / or IL18RAP gene, such that the inserted promoter is operably linked to the coding sequence of the endogenous IL18R1 gene or the IL18RAP gene. In some embodiments, the method further comprises modifying a coding sequence of the endogenous IL18R1 gene and / or the endogenous IL18RAP gene. Such modifications may remove one or more introns and / or mutate one or more exons of the endogenous IL18R1 gene and / or IL18RAP gene.

[0219] In some embodiments, the modified coding sequence encodes a functional derivative of IL-18R1 or IL18RAP. The functional derivative of IL-18R1 or IL18RAP may include a protein that has a substantial activity of a wild-type IL-18R1 or IL18RAP, or increased activity relative to wild-type IL-18R1 or IL18RAP. One of ordinary skill in the art may use any suitable method to test the functionality or activity of an IL-18R1 or IL18RAP or derivative thereof. The functional derivative of IL-18R1 or IL18RAP may also include any IL-18R1 or IL18RAP or fragment thereof that has conservative substitutions of one or more amino acid residues relative to full-length, wild-type IL-18R1 or IL18RAP as set forth in SEQ ID NOs: 40 and 41, respectively.

[0220] In some embodiments, the functional derivative has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 substitutions relative to the amino acid sequence of SEQ ID NO: 40. In some embodiments, the functional derivative has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 insertions relative to the amino acid sequence of SEQ ID NO: 40. In some embodiments, the functional derivative has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,

[0221] 12, 13, 14, 15, 16, 17, 18, 19, or 20 deletions relative to the amino acid sequence of SEQ ID NO: 40. In some embodiments, the functional derivative comprises a C-terminal fusion with another polypeptide. In some embodiments, a functional derivative lacks one or more domains or portions of wild-type IL-18R1 that are unnecessary for signaling.

[0222] In some embodiments, the functional derivative has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,

[0223] 13, 14, 15, 16, 17, 18, 19, or 20 substitutions relative to the amino acid sequence of SEQ ID NO: 41. In some embodiments, the functional derivative has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 insertions relative to the amino acid sequence of SEQ ID NO: 41. In some embodiments, the functional derivative has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 deletions relative to the amino acid sequence of SEQ ID NO: 41. In some embodiments, the functional derivative comprises a C-terminal fusion with another polypeptide. In some embodiments, a functional derivative lacks one or more domains or portions of wild-type IL18RAP that are unnecessary for signaling.

[0224] In some embodiments, the encoded IL-18R1 or IL18RAP or functional derivative thereof has about or at least 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or more of the activity exhibited by a wild-type IL-18R1 or IL18RAP as set forth in SEQ ID NOs: 40 and 41, respectively. In some embodiments, the encoded IL- 18R1 or IL18RAP comprises an amino acid sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or up to 100% sequence identity to the amino acid sequence of wild-type human IL-18R1 or IL18RAP as set forth in SEQ ID NOs: 40 and 41, respectively. In some embodiments, the encoded IL-18R1 comprises the wild-type amino acid sequence of SEQ ID NO: 40. In some embodiments, the encoded IL-18R1 consists of the wild-type amino acid sequence of SEQ ID NO: 40. In some embodiments, the encoded IL18RAP comprises the wild-type amino acid sequence of SEQ ID NO: 41. In some embodiments, the encoded IL18RAP consists of the wild-type amino acid sequence of SEQ ID NO: 41.

[0225] In some embodiments, a nucleotide sequence encoding IL-18R1 is codon-optimized. In some embodiments, IL-18R1 is encoded by a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence of SEQ ID NO: 110. In some embodiments, IL-18R1 is encoded by nucleotide sequence of SEQ ID NO: 110. IL-18R1 may be expressed with any suitable signal peptide. In some embodiments, the signal peptide is a wild-type IL-18R1 signal peptide. An example of an IL- 18R1 signal peptide amino acid sequence is provided by SEQ ID NO: 45. In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 45. In some embodiments, the signal peptide is encoded by a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence of SEQ ID NO: 111. In some embodiments, the signal peptide is encoded by the nucleotide sequence of SEQ ID NO: 111.

[0226] In some embodiments, a nucleotide sequence encoding IL18RAP is codon-optimized. In some embodiments, IL18RAP is encoded by a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence of SEQ ID NO: 112. In some embodiments, IL18RAP is encoded by nucleotide sequence of SEQ ID NO: 112. IL18RAP may be expressed with any suitable signal peptide. In some embodiments, the signal peptide is a wild-type IL18RAP signal peptide. An example of an IL18RAP signal peptide amino acid sequence is provided by SEQ ID NO: 46. In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 46. In some embodiments, the signal peptide is encoded by a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence of SEQ ID NO: 113. In some embodiments, the signal peptide is encoded by the nucleotide sequence of SEQ ID NO: 113.

[0227] Other repair mediators ’ expression

[0228] Some embodiments of the methods comprise introducing a nucleic acid comprising a promoter operably linked to a nucleic acid sequence encoding a repair mediator (e.g, IL- 18R), or a functional derivative thereof, into a cell. The functional derivative of the repair mediator (e.g, IL-18R) may include a protein that has a substantial activity of a wild-type repair mediator (e.g., IL-18R), or increased activity relative to the wild-type repair mediator (e.g., IL-18R). One of ordinary skill in the art may use any suitable method to test the functionality or activity of a repair mediator (e.g., IL-18R) or derivative thereof. The functional derivative of a repair mediator (e.g., IL-18R) may also include any repair mediator (e.g., IL-18R) or fragment thereof that has conservative substitutions of one or more amino acid residues relative to full-length, wild-type repair mediator (e.g., IL-18R). Other repair mediators include a transcription factor or activator of a repair pathway, such as GAT A3, PPARG, and / or BATF; cytokines (e.g., AREG and / or osteopontin); growth factors (e.g., KGF, TGF-P); and / or chemokine receptors (e.g., CCR2, CCR5, CCR8) that promote repair and regeneration. See, e.g., Wohlfert et al., J Clin Invest. 2011. 121(11):4503— 4515 (discussing GATA3); Miao et al., Cell Commun Signal. 2022. 20(l):48 (discussing PPARgamma); Trujillo-Ochoa et al, Nat Rev Immunol. 2023. 23(12):842-856 (discussing BATF); Zhang et al., Cell Physiol Biochem. 2017. 43 (6) :2155-2169 (discussing AREG, KGF, and chemokine receptors); Shi et al., Immunity. 2021. 54(7): 1527-1542 (discussing osteopontin); Konkel et al., Immunity. 2017. 46:660-674 (discussing TGF-P).

[0229] Membrane-bound IL-33

[0230] Some aspects relate to a membrane-anchored derivative of IL-33 (mem-IL-33), and / or a nucleic acid encoding a mem-IL-33. Some aspects relate to a cell comprising a mem-IL-33. Some aspects relate to a cell comprising a nucleic acid encoding a mem-IL-33. Native IL-33 is a ligand and, without wishing to be bound by theory, it is thought that imparting a functional ligand on its membrane-anchored form offers abundant IL-33 signaling, thereby maintaining a repair Treg phenotype. Some aspects relate to an isolated nucleic acid molecule comprising a nucleotide sequence encoding an IL-33 linked to a transmembrane-intracellular region, optionally through a hinge and / or a linker, referred to herein as mem-IL-33.

[0231] In some embodiments, the isolated nucleic acid molecule does not comprise a nucleotide sequence encoding for additional different proteins except for mem -IL-33 but may comprise additional control elements such as promoters and terminators.

[0232] In principle, to provide flexibility, the flexible linkers generally comprise small, nonpolar (e.g., Gly) or polar (e.g., Ser or Thr) amino acids, such an underlying sequence of alternating Gly and Ser residues. Solubility of the linker and associated IL-33 may be enhanced by including charged residues, e.g., two positively charged residues (Lys) and one negatively charged residue (Glu). The linker may vary from 2 to 31 amino acids. In some embodiments, the linker may vary for each condition so that the linker does not impose any constraints on the conformation or interactions of the linked partners in lengths, such as between 12 and 18 residues.

[0233] In some embodiments, the flexible linker has the amino acid sequence GSTSGSGKPGSGEGSTKG (SEQ ID NO: 34).

[0234] In some embodiments, the hinge is a flexible hinge. In some embodiments, the flexible hinge comprises a polypeptide selected from the following polypeptides or variants thereof:

[0235] • The hinge region of CD8a, (for example as set forth in SEQ ID NO: 31)

[0236] • The hinge region of the heavy chain of IgG (for example as set forth in SEQ ID NO: 32); and

[0237] • The hinge region of the heavy chain of IgD (for example as set forth in SEQ ID NO: 33).

[0238] In some embodiments, the second flexible linker comprises a 21 amino acid sequence comprising the amino acid sequence Gly4Ser(Gly3Ser)2 (referred to herein as “short linker”; SEQ ID NO: 36).

[0239] In some embodiments, the second flexible linker consists of a 28 amino acid spacer comprising the amino acid sequence Gly4Ser(Gly3Ser)2Ser2(Gly3Ser)3 (referred to herein as “long linker”; SEQ ID NO:37) and the connecting peptide of SEQ ID NO: 38.

[0240] In some embodiments, the flexible linker further comprises an 8 amino acid bridge of the sequence SSQPTIPI (referred to herein as “connecting peptide”; SEQ ID NO: 38) derived from the membrane-proximal part of the connecting peptide of HLA-A2. In some embodiments, the transmembrane-intracellular region (transmembrane anchor domain) of the mem-IL-33 is derived from the heavy chain of a human MHC class I molecule selected from an HLA-A, HLA-B or HLA-C molecule. In some embodiments, the MHC class I molecule is HLA-A2 (as set forth in SEQ ID NO: 29); human CD28 (as set forth in SEQ ID NO: 30); human ST2 chain (as set forth in UniProt Accession No. Q01638; e.g., encoded by a nucleotide sequence as set forth in GenBank Accession No. D12763.1) or fragment thereof; or human IL1RAP chain (as set forth in UniProt Accession No. Q9NPH3; e.g., encoded by a nucleotide sequence as set forth in GenBank Accession No. AB006537.1) or fragment thereof. In some embodiments, the transmembrane-intracellular region of mem- IL-33 is derived from HLA-A2. In some embodiments, the transmembrane-intracellular region of mem-IL-33 is derived from CD28. In some embodiments, the transmembrane- intracellular region of mem-IL-33 is derived from ST2. In some embodiments, the transmembrane-intracellular region of mem-IL-33 is derived from IL1RAP. In some embodiments, the HLA-A2 domain comprises up to 20 amino acid substitutions, up to 15 amino acid sequence substitutions, up to 10 amino acid substitutions, or up to 5 amino acid substitutions. In some embodiments, the HLA-A2 domain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid sequence substitutions. In some embodiments, the CD28 domain comprises up to 20 amino acid substitutions, up to 15 amino acid sequence substitutions, up to 10 amino acid substitutions, or up to 5 amino acid substitutions. In some embodiments, the CD28 domain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid sequence substitutions. In some embodiments, the ST2 domain comprises up to 20 amino acid substitutions, up to 15 amino acid sequence substitutions, up to 10 amino acid substitutions, or up to 5 amino acid substitutions. In some embodiments, the ST2 domain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid sequence substitutions. In some embodiments, the IL1RAP domain comprises up to 20 amino acid substitutions, up to 15 amino acid sequence substitutions, up to 10 amino acid substitutions, or up to 5 amino acid substitutions. In some embodiments, the IL1RAP domain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid sequence substitutions.

[0241] In some embodiments, the mem-IL-33 is fused to an ST2 extracellular domain. In some embodiments, the mem-IL-33 is fused to a IL1RAP extracellular domain. In some embodiments, the mem-IL-33 is fused to an ST2 extracellular domain and / or a IL1RAP extracellular domain via a linker (e.g., a flexible linker). In some embodiments, the mem-IL- 33 is fused to the N-terminus of an ST2 extracellular domain and / or the N-terminus of a IL1RAP extracellular domain. Mem-IL-33 proteins are not limited to specific amino acid sequences, but may also be variants of these polypeptides or have amino acid sequences that are substantially identical to those described in this section. A “variant” of a polypeptide having a reference amino acid sequence is modified at one or more amino acid residues relative to the reference amino acid sequence, yet still retains the biological activity of the polypeptide having the reference amino acid sequence. A “substantially identical” amino acid sequence as used herein refers to a sequence that differs from a reference sequence by one or more conservative or nonconservative amino acid substitutions, deletions, or insertions, particularly when such a substitution occurs at a site that is not the active site of the molecule, and provided that the polypeptide essentially retains its functional properties. A conservative amino acid substitution, for example, substitutes one amino acid with another of the same class, e.g., substitution of one hydrophobic amino acid with another hydrophobic amino acid, a polar amino acid with another polar amino acid, a basic amino acid with another basic amino acid, or an acidic amino acid with another acidic amino acid. One or more amino acids can be deleted from the peptide, thus obtaining a fragment thereof without significantly altering its biological activity.

[0242] In some embodiments, the amino acid sequence of the complete membrane-bound IL- 33, comprises a transmembrane domain, a linker, and an IL-33 protein. In some embodiments, the transmembrane anchor comprises a CD94 anchor.

[0243] The amino acid sequence of the complete membrane-bound IL-33 may be encoded by a nucleic acid molecule.

[0244] Some aspects relate to a composition comprising a nucleic acid molecule comprising a nucleotide sequence encoding a membrane-bound IL-33. In certain embodiments the nucleic acid molecule is the sole nucleic acid molecule in the composition, z.e., the composition does not comprise additional nucleic acid molecules. In some embodiments, the nucleic acid comprises an open reading frame (ORF) encoding the mem-IL-33. In some embodiments, the nucleic acid further comprises a promoter operably linked to the ORF. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is an MND promoter.

[0245] The nucleic acid molecules may be delivered to cells for the purpose of enforcing a repair Treg phenotype. The nucleic acid molecules may be introduced into a cell using any suitable method. For example, viral or non-viral systems may be used to deliver transgenes into a cell. The non-viral approaches include, but are not limited to, polymer nanoparticles, lipids, calcium phosphate, electroporation / nucleofection or biolistic delivery of DNA-coated microparticles (Matuskova and Durinikova, 2016. Advances in Molecular Retrovirology InTech).

[0246] Multiple vectors can be used in accordance with the nucleic acids and methods for introduction, depending on whether the DNA is integrated into chromatin of the host cell or not. Retroviral vectors such as those derived from gammaretroviruses or lentiviruses persist in the nucleus as integrated provirus and reproduce with cell division. Other types of vectors (e.g., those derived from herpesviruses or adenoviruses) may remain in the cell in the episomal form.

[0247] Some aspects relate to a viral vector comprising anyone of the nucleic acid molecules comprising a nucleotide sequence encoding a membrane-bound IL-33. In some embodiments, the viral vector is selected from a modified virus derived from a virus selected from the group consisting of a retrovirus, lentivirus, gammavirus, adenovirus, adeno-associated virus, pox virus, alphavirus, and herpes virus.

[0248] In some embodiments, the vector is a retrovirus, such as a modified gammavirus, lentivirus, murine stem cell virus, moloney murine leukemia virus, bovine leukaemia virus, Rous sarcoma virus, or spumavirus. In some embodiments, the viral vector is a retrovirus. In some embodiments, the viral vector is a lentiviral vector. In some embodiments, the viral vector is a gamma-retroviral vector.

[0249] In some embodiments, the nucleic acid molecule is the sole polypeptide encoded by the nucleotide sequence, z.e., the nucleic acid molecule of the viral vector does not encode additional different proteins, but may comprise additional control elements such as promoters and terminators.

[0250] Some aspects relate to a composition comprising the viral vector. Some aspects relate to a mammalian regulatory T cell (Treg) comprising any one of the nucleic acid molecules, or the viral vector. In some embodiments, the mammalian Treg expresses a membrane bound IL-33 on its surface. In some embodiments, the mammalian Treg is a human Treg.

[0251] Some aspects relate to a method of preparing allogeneic or autologous Tregs with a repair Treg phenotype, the method comprising contacting CD4+ T cells with the nucleic acid molecule comprising a nucleotide sequence encoding a membrane-bound IL-33, or a viral vector comprising it, thereby endowing said CD4+ T cells with a repair Treg phenotype, and thus preparing Tregs with a repair phenotype. Membrane-bound IL-18

[0252] Some aspects relate to a membrane-anchored derivative of IL-18 (mem-IL-18), and / or a nucleic acid encoding a mem-IL-18. Some aspects relate to a cell comprising a mem-IL-18. Some aspects relate to a cell comprising a nucleic acid encoding a mem-IL-18. Native IL-18 is a ligand and, without wishing to be bound by theory, it is thought that imparting a functional ligand on its membrane-anchored form offers abundant IL- 18 signaling, thereby maintaining a repair Treg phenotype.

[0253] Some aspects relate to an isolated nucleic acid molecule comprising a nucleotide sequence encoding an IL-18 linked to a transmembrane-intracellular region, optionally through a hinge and / or a linker, referred to herein as mem-IL-18.

[0254] In some embodiments, the isolated nucleic acid molecule does not comprise a nucleotide sequence encoding for additional different proteins except for mem-IL-18 but may comprise additional control elements such as promoters and terminators.

[0255] In principle, to provide flexibility, the flexible linkers generally comprise small, nonpolar (e.g., Gly) or polar (e.g., Ser or Thr) amino acids, such an underlying sequence of alternating Gly and Ser residues. Solubility of the linker and associated IL-18 may be enhanced by including charged residues, e.g., two positively charged residues (Lys) and one negatively charged residue (Glu). The linker may vary from 2 to 31 amino acids. In some embodiments, the linker may vary for each condition so that the linker does not impose any constraints on the conformation or interactions of the linked partners in lengths, such as between 12 and 18 residues.

[0256] In some embodiments, the flexible linker has the amino acid sequence GSTSGSGKPGSGEGSTKG (SEQ ID NO: 34).

[0257] In some embodiments, the hinge is a flexible hinge. In some embodiments, the flexible hinge comprises a polypeptide selected from the following polypeptides or variants thereof:

[0258] • The hinge region of CD8a, (for example as set forth in SEQ ID NO: 31)

[0259] • The hinge region of the heavy chain of IgG (for example as set forth in SEQ ID NO: 32); and

[0260] • The hinge region of the heavy chain of IgD (for example as set forth in SEQ ID NO: 33).

[0261] In some embodiments, the second flexible linker comprises a 21 amino acid sequence comprising the amino acid sequence Gly4Ser(Gly3Ser)2 (referred to herein as “short linker”; SEQ ID NO: 36). In some embodiments, the second flexible linker consists of a 28 amino acid spacer comprising the amino acid sequence Gly4Ser(Gly3Ser)2Ser2(Gly3Ser)3 (referred to herein as “long linker”; SEQ ID NO:37) and the connecting peptide of SEQ ID NO: 38.

[0262] In some embodiments, the flexible linker further comprises an 8 amino acid bridge of the sequence SSQPTIPI (referred to herein as “connecting peptide”; SEQ ID NO: 38) derived from the membrane-proximal part of the connecting peptide of HLA-A2.

[0263] In some embodiments, the transmembrane-intracellular region (transmembrane anchor domain) of the mem-IL-18 is derived from the heavy chain of a human MHC class I molecule selected from an HLA-A, HLA-B or HLA-C molecule. In some embodiments, the MHC class I molecule is HLA-A2 (as set forth in SEQ ID NO: 29); human CD28 (as set forth in SEQ ID NO: 30); human IL-18R1 chain (as set forth in UniProt Accession No. Q13478; e.g., encoded by a nucleotide sequence as set forth in GenBank Accession No. AY192162.1) or fragment thereof; or human IL18RAP chain (as set forth in UniProt Accession No. 095256; e.g., encoded by a nucleotide sequence as set forth in GenBank Accession No. AF077346.1) or fragment thereof. In some embodiments, the transmembrane-intracellular region of mem-IL-18 is derived from HLA-A2. In some embodiments, the transmembrane- intracellular region of mem-IL-18 is derived from CD28. In some embodiments, the transmembrane-intracellular region of mem-IL-18 is derived from IL-18R1. In some embodiments, the transmembrane-intracellular region of mem-IL-18 is derived from IL18RAP. In some embodiments, the HLA-A2 domain comprises up to 20 amino acid substitutions, up to 15 amino acid sequence substitutions, up to 10 amino acid substitutions, or up to 5 amino acid substitutions. In some embodiments, the HLA-A2 domain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid sequence substitutions. In some embodiments, the CD28 domain comprises up to 20 amino acid substitutions, up to 15 amino acid sequence substitutions, up to 10 amino acid substitutions, or up to 5 amino acid substitutions. In some embodiments, the CD28 domain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid sequence substitutions. In some embodiments, the IL-18R1 domain comprises up to 20 amino acid substitutions, up to 15 amino acid sequence substitutions, up to 10 amino acid substitutions, or up to 5 amino acid substitutions. In some embodiments, the IL-18R1 domain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid sequence substitutions. In some embodiments, the IL18RAP domain comprises up to 20 amino acid substitutions, up to 15 amino acid sequence substitutions, up to 10 amino acid substitutions, or up to 5 amino acid substitutions. In some embodiments, the IL18RAP domain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid sequence substitutions. In some embodiments, the mem-IL-18 is fused to an IL-18R1 extracellular domain. In some embodiments, the mem-IL-18 is fused to a IL-18RAP extracellular domain. In some embodiments, the mem-IL-18 is fused to an IL18R1 extracellular domain and / or a IL18RAP extracellular domain via a linker (e.g., a flexible linker). In some embodiments, the mem-IL- 18 is fused to the N-terminus of an IL-18R1 extracellular domain and / or the N-terminus of a IL18RAP extracellular domain.

[0264] Mem-IL-18 proteins are not limited to specific amino acid sequences but may also be variants of these polypeptides or have amino acid sequences that are substantially identical to those described in this section. A “variant” of a polypeptide is modified at one or more amino acid residues, relative to a reference amino acid sequence, yet still retains the biological activity of a polypeptide having the reference amino acid sequence. A “substantially identical” amino acid sequence as used herein refers to a sequence that differs from a reference sequence by one or more conservative or non-conservative amino acid substitutions, deletions, or insertions, particularly when such a substitution, deletion, or insertion occurs at a site that is not the active site of the molecule, and provided that the polypeptide essentially retains its functional properties. A conservative amino acid substitution, for example, substitutes one amino acid with another of the same class, e.g., substitution of one hydrophobic amino acid with another hydrophobic amino acid, a polar amino acid with another polar amino acid, a basic amino acid with another basic amino acid or an acidic amino acid with another acidic amino acid. One or more amino acids can be deleted from the peptide, thus obtaining a fragment thereof without significantly altering its biological activity.

[0265] In some embodiments, the amino acid of the complete membrane-bound IL- 18 comprises a transmembrane domain, a linker, and an IL-18 protein. In some embodiments, the transmembrane anchor comprises a CD94 anchor.

[0266] The amino acid sequences of the complete membrane-bound IL-18 may be encoded by a nucleic acid molecule.

[0267] Some aspects relate to a composition comprising a nucleic acid molecule comprising a nucleotide sequence encoding a membrane-bound IL- 18. In certain embodiments the nucleic acid molecule is the sole nucleic acid molecule in the composition, z.e., the composition does not comprise additional nucleic acid molecules. In some embodiments, the nucleic acid comprises an open reading frame (ORF) encoding the mem-IL-18. In some embodiments, the nucleic acid further comprises a promoter operably linked to the ORF. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is an MND promoter.

[0268] The nucleic acid molecules may be delivered to cells for the purpose of enforcing a repair Treg phenotype. The nucleic acid molecules may be introduced into a cell using any suitable method. For example, viral or non-viral systems may be used to deliver transgenes into a cell. The non-viral approaches include, but are not limited to, polymer nanoparticles, lipids, calcium phosphate, electroporation / nucleofection or biolistic delivery of DNA-coated microparticles (Matuskova and Durinikova, 2016. Advances in Molecular Retrovirology InTech).

[0269] Multiple vectors can be used in accordance with the nucleic acids and methods for introduction, depending on whether the DNA is integrated into chromatin of the host cell or not. Retroviral vectors such as those derived from gammaretroviruses or lentiviruses persist in the nucleus as integrated provirus and reproduce with cell division. Other types of vectors (e.g., those derived from herpesviruses or adenoviruses) may remain in the cell in the episomal form.

[0270] Some aspects relate to a viral vector comprising anyone of the nucleic acid molecules comprising a nucleotide sequence encoding a membrane-bound IL-18. In some embodiments, the viral vector is selected from a modified virus derived from a virus selected from the group consisting of a retrovirus, lentivirus, gammavirus, adenovirus, adeno-associated virus, pox virus, alphavirus, and herpes virus.

[0271] In some embodiments, the vector is a retrovirus, such as a modified gammavirus, lentivirus, murine stem cell virus, moloney murine leukemia virus, bovine leukaemia virus, Rous sarcoma virus, or spumavirus. In some embodiments, the viral vector is a retrovirus. In some embodiments, the viral vector is a lentiviral vector. In some embodiments, the viral vector is a gamma-retroviral vector.

[0272] In some embodiments, the nucleic acid molecule is the sole polypeptide encoded by the nucleotide sequence, z.e., the nucleic acid molecule of the viral vector does not encode additional different proteins but may comprise additional control elements such as promoters and terminators.

[0273] Some aspects relate to a composition comprising the viral vector. Some aspects relate to a mammalian regulatory T cell (Treg) comprising any one of the nucleic acid molecules, or the viral vector. In some embodiments, the mammalian Treg expresses a membrane-bound IL- 18 on its surface. In some embodiments, the mammalian Treg is a human Treg. Some aspects relate to a method of preparing allogeneic or autologous Tregs with a repair Treg phenotype, the method comprising contacting CD4+ T cells with the nucleic acid molecule comprising a nucleotide sequence encoding a membrane-bound IL- 18, or a viral vector comprising it, thereby endowing said CD4+ T cells with a repair Treg phenotype, and thus preparing Tregs with a repair phenotype.

[0274] IL-33 linked to ST2 and / or IL1RAP

[0275] Some aspects relate to an IL-33 (as set forth in UniProt Accession No. A0A1I9RI51; e.g., encoded by a nucleotide sequence as set forth in GenBank Accession No. AY905581.1) linked to either ST2 and / or IL1RAP, or a functional derivative thereof. An IL-33 linked to ST2 and / or IL1RAP is referred to as a receptor-linked IL-33 (rIL-33). Some aspects relate to a nucleic acid encoding an rIL-33. Some aspects relate to a cell comprising an rIL-33. Some aspects relate to a cell comprising a nucleic acid encoding an rIL-33.

[0276] In some embodiments IL-33 is linked to ST2 (as set forth in UniProt Accession No. Q01638; e.g., encoded by a nucleotide sequence as set forth in GenBank Accession No. D12763.1, or SEQ ID NO: 1), or a functional derivative thereof. In some embodiments, IL-33 is linked to IL1RAP (as set forth in UniProt Accession No. Q9NPH3; e.g., encoded by a nucleotide sequence as set forth in GenBank Accession No. AB006537.1, or SEQ ID NO: 2), or a functional derivative thereof, into a cell. In some embodiments, IL-33 is linked to ST2 and IL1RAP, or functional derivatives thereof.

[0277] In some embodiments, the IL-33 portion comprises an amino acid sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or up to 100% sequence identity to the amino acid sequence of wild-type human IL-33 set forth in SEQ ID NO: 39 (UniProt Accession No. A0A1I9RI51). In some embodiments, the IL-33 portion comprises the wild-type amino acid sequence of SEQ ID NO: 39. In some embodiments, the IL-33 portion consists of the wild-type amino acid sequence of SEQ ID NO: 39. In some embodiments, the IL-33 portion comprises up to 20 amino acid substitutions, up to 15 amino acid sequence substitutions, up to 10 amino acid substitutions, or up to 5 amino acid substitutions. In some embodiments, the IL-33 portion comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid sequence substitutions.

[0278] In some embodiments, the ST2 portion comprises an amino acid sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or up to 100% sequence identity to the amino acid sequence of wild-type human ST2 set forth as SEQ ID NO: 1. In some embodiments, the ST2 portion comprises the wild-type amino acid sequence of SEQ ID NO: 1. In some embodiments, the ST2 portion consists of the wild-type amino acid sequence of SEQ ID NO: 1. In some embodiments, the ST2 portion comprises up to 20 amino acid substitutions, up to 15 amino acid sequence substitutions, up to 10 amino acid substitutions, or up to 5 amino acids. In some embodiments, the ST2 portion comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid sequence substitutions.

[0279] In some embodiments, the IL1RAP portion comprises an amino acid sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or up to 100% sequence identity to the amino acid sequence of wild-type human IL1RAP set forth as SEQ ID NO: 2. In some embodiments, the IL1RAP portion comprises the wild-type amino acid sequence of SEQ ID NO: 2. In some embodiments, the IL1RAP portion consists of the wildtype amino acid sequence of SEQ ID NO: 2. In some embodiments, the IL1RAP portion comprises up to 20 amino acid substitutions, up to 15 amino acid sequence substitutions, up to 10 amino acid substitutions, or up to 5 amino acid substitutions. In some embodiments, the IL1RAP portion comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid sequence substitutions.

[0280] In some embodiments, IL-33 is connected to ST2 by a linker. In some embodiments, IL-33 is connected to IL1RAP by a linker. In some embodiments, IL-33 is connected to ST2 and IL1RAP by a linker.

[0281] In some embodiments, the linker comprises a peptide (e.g., a human or artificial peptide). In some embodiments, the linker comprises a synthetic linker (e.g., a flexible linker) or an extracellular hinge domain. In some embodiments, the linker is a flexible linker or a rigid linker. In some embodiments, the linker is a flexible linker. In some embodiments, the linker comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids, such as Gly, or a number of amino acids, such as Gly, within a range defined by any two of the aforementioned numbers. In some embodiments, the Gly spacer comprises at least 3 Gly. In some embodiments, the Gly spacer comprises a sequence set forth as GGGS (SEQ ID NO: 8), GGGSGGG (SEQ ID NO: 9) or GGG. In principle, to provide flexibility, the flexible linkers generally comprise small, non-polar (e.g., Gly) or polar (e.g., Ser or Thr) amino acids, such an underlying sequence of alternating Gly and Ser residues. Solubility of the linker and associated chimeric signaling receptor may be enhanced by including charged residues; e.g., two positively charged residues (Lys) and one negatively charged residue (Glu). The linker may vary from 2 to 31 amino acids, suitable for each condition so that the linker does not impose any constraints on the conformation or interactions of the linked partners in lengths, such as between 12 and 18 residues.

[0282] In some embodiments, rIL-33 further comprises a hinge. In some embodiments, IL-33 is linked to ST2 by a hinge. In some embodiments, IL-33 is linked to IL1RAP by a hinge. In some embodiments, IL-33 is linked to ST2 and IL1RAP by one or more hinges. In some embodiments, the hinge is a flexible hinge. In some embodiments, the hinge domain comprises a polypeptide selected from the group consisting of: a hinge region of CD8a, a hinge region of a heavy chain of IgG, a hinge region of a heavy chain of IgD, and an extracellular region of an IL-10R P chain. In some embodiments, the hinge comprises a hinge region of CD8a.

[0283] In some embodiments, the nucleic acid comprises an open reading frame (ORF) encoding the rIL-33 polypeptide. In some embodiments, a promoter is operably linked to the ORF. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is an MND promoter.

[0284] IL-18 linked to IL-18R1 and / or IL18RAP

[0285] Some aspects relate to an IL- 18 (as set forth in UniProt Accession No. Q 14116; e.g., encoded by a nucleotide sequence as set forth in GenBank Accession No. AY044641.1) linked to either IL-18R1 and / or IL18RAP, or a functional derivative thereof. An IL-18 linked to IL-18R1 and / or IL18RAP is referred to as a receptor-linked IL-18 (rIL-18). Some aspects relate to a nucleic acid encoding an rIL-18. Some aspects relate to a cell comprising an rlL- 18. Some aspects relate to a cell comprising a nucleic acid encoding an rIL-18.

[0286] In some embodiments, IL- 18 is linked to IL-18R1 (as set forth in UniProt Accession No. QI 3478; e.g., encoded by a nucleotide sequence as set forth in GenBank Accession No. AY192162.1) or a functional derivative thereof. In some embodiments, IL-18 is linked to IL18RAP (as set forth in UniProt Accession No. 095256; e.g., encoded by a nucleotide sequence as set forth in GenBank Accession No. AF077346.1), or a functional derivative thereof, into a cell. In some embodiments, IL-18 is linked to IL-18R1 and IL18RAP, or functional derivatives thereof.

[0287] In some embodiments, the IL-18 portion of the IL-18 linked to IL-18R1 or IL-18 linked to IL18RAP comprises an amino acid sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or up to 100% sequence identity to the amino acid sequence of wild-type human IL-18 (UniProt Accession No. Q14116). In some embodiments, the IL- 18 portion comprises up to 20 amino acid substitutions, up to 15 amino acid sequence substitutions, up to 10 amino acid substitutions, or up to 5 amino acid substitutions. In some embodiments, the IL-18 portion comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid sequence substitutions.

[0288] In some embodiments, the IL-18R1 portion of the IL- 18 linked to IL-18R1 comprises an amino acid sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or up to 100% sequence identity to the amino acid sequence of wild-type human IL-18R1. In some embodiments, the IL-18R1 portion comprises up to 20 amino acid substitutions, up to 15 amino acid sequence substitutions, up to 10 amino acid substitutions, or up to 5 amino acids. In some embodiments, the IL-18R1 portion comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid sequence substitutions.

[0289] In some embodiments, the IL18RAP portion of the IL- 18 linked to IL18RAP comprises an amino acid sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or up to 100% sequence identity to the amino acid sequence of wildtype human IL18RAP. In some embodiments, the IL18RAP portion comprises up to 20 amino acid substitutions, up to 15 amino acid sequence substitutions, up to 10 amino acid substitutions, or up to 5 amino acid substitutions. In some embodiments, the IL18RAP portion comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid sequence substitutions.

[0290] In some embodiments, IL-18 is connected to IL-18R1 by a linker. In some embodiments, IL-18 is connected to IL18RAP by a linker. In some embodiments, IL-18 is connected to IL-18R1 and IL18RAP by one or more linkers.

[0291] In some embodiments, the linker comprises a peptide (e.g., a human or artificial peptide). In some embodiments, the linker comprises a synthetic linker (e.g., a flexible linker) or an extracellular hinge domain. In some embodiments, the linker is a flexible linker or a rigid linker. In some embodiments, the linker is a flexible linker. In some embodiments, the linker comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids, such as Gly, or a number of amino acids, such as Gly, within a range defined by any two of the aforementioned numbers. In some embodiments, the Gly spacer comprises at least 3 Gly. In some embodiments, the Gly spacer comprises a sequence set forth as GGGS (SEQ ID NO: 8), GGGSGGG (SEQ ID NO: 9) or GGG. In principle, to provide flexibility, the flexible linkers generally comprise small, non-polar (e.g., Gly) or polar (e.g., Ser or Thr) amino acids, such an underlying sequence of alternating Gly and Ser residues. Solubility of the linker and associated chimeric signaling receptor may be enhanced by including charged residues; e.g., two positively charged residues (Lys) and one negatively charged residue (Glu). The linker may vary from 2 to 31 amino acids, suitable for each condition so that the linker does not impose any constraints on the conformation or interactions of the linked partners in lengths, such as between 12 and 18 residues.

[0292] In some embodiments, rIL-18 further comprises a hinge. In some embodiments, IL-18 is linked to IL-18R1 by a hinge. In some embodiments, IL-18 is linked to IL18RAP by a hinge. In some embodiments, IL- 18 is linked to IL-18R1 and IL18RAP by one or more hinges. In some embodiments, the hinge is a flexible hinge. In some embodiments, the hinge domain comprises a polypeptide selected from the group consisting of: a hinge region of CD8a, a hinge region of a heavy chain of IgG, a hinge region of a heavy chain of IgD, and an extracellular region of an IL-10R P chain. In some embodiments, the hinge comprises a hinge region of CD8a.

[0293] In some embodiments, the nucleic acid comprises an open reading frame (ORF) encoding the rIL-18 polypeptide. In some embodiments, a promoter is operably linked to the ORF. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is an MND promoter.

[0294] In vivo methods

[0295] Some embodiments of producing Tregs with repair function are performed in vivo by administering to a subject reagents and / or compositions that induce or upregulate the IL- 33 / ST2 and / or IL-18 pathway in cells (e.g., immune cells of the subject such as CD3+, CD4+ or CD8+ cells) by expressing or inducing expression and / or activity of positive regulators of the IL-33 / ST2 and / or IL-18 pathway (e.g., ST2, IL1RAP, IL-18R1, and / or IL18RAP). In some embodiments, a gene encoding one or more positive regulators of the IL-33 / ST2 and / or IL- 18 pathway (e.g., ST2, IL1RAP, IL-18R1, and / or IL18RAP) is constitutively expressed in a cell, e.g., by delivery of nucleic acids comprising the gene into a subject. In some embodiments, ST2 is constitutively expressed in a cell. In some embodiments, IL1RAP is constitutively expressed in a cell. In some embodiments, ST2 and / or IL1RAP is constitutively expressed in a cell. In some embodiments, a method of producing repair Tregs in vivo comprises gene editing of a gene involved in IL-33 signaling and / or IL-18 signaling. In some embodiments, a gene involved in IL-33 signaling and / or IL-18 signaling is a positive regulator and gene editing is performed to induce constitutive production of the positive regulator. Compositions to administer may include nucleic acids, e.g., comprised in vectors (e.g., viral or non-viral vectors) or formulated using nanoparticles, that encode constitutively active positive regulators of the IL-33 signaling and / or IL-18 signaling pathways.

[0296] Ex vivo methods

[0297] Some embodiments of methods of producing Tregs with repair function are performed ex vivo, e.g., in isolated immune cells that are transfected with, contacted with, or treated with reagents and / or compositions that induce or upregulate the IL-33 signaling and / or IL- 18 signaling pathways in cells (e.g., immune cells of the subject such as CD3+, CD4+ or CD8+ cells) by expressing or inducing expression and / or activity of positive regulators of the IL-33 / ST2 and / or IL-18 pathways (e.g., ST2, IL1RAP, IL-18R1, and / or IL18RAP). In some embodiments, ST2 is constitutively expressed in a cell. In some embodiments, IL1RAP is constitutively expressed in a cell. In some embodiments, ST2 and IL1RAP are constitutively expressed in a cell. In some embodiments, IL-18R1 is constitutively expressed in a cell. In some embodiments, IL18RAP is constitutively expressed in a cell. In some embodiments, IL-18R1 and IL18RAP are constitutively expressed in a cell.

[0298] Cells thus engineered are then administered to a subject. In some embodiments, a gene encoding one or more positive regulators of the IL-33 / ST2 and / or IL-18 pathways (e.g., ST2, IL1RAP, IL-18R1 and / or IL18RAP) is constitutively expressed in a cell, e.g., by delivery of nucleic acids comprising the gene into the cell. In some embodiments, a method of producing repair Tregs ex vivo comprises gene editing of a gene involved in IL-33 / ST2 and / or IL-18 signaling. In some embodiments, a gene involved in the IL-33 / ST2 and / or IL-18 signaling pathways is a positive regulator and gene editing is performed to induce constitutive production of the positive regulator.

[0299] In some embodiments, the cell is modified extracorporeally. In some embodiments, the cell is a T cell. In some embodiments, the cell is a CD4+ T cell.

[0300] Cells expressing an exogenous coding sequence

[0301] Some aspects relate to cells comprising a promoter operably linked to a coding sequence encoding at least one repair mediator (e.g., ST2, IL1RAP, IL-18R1, and / or IL18RAP). In some embodiments, the coding sequence is a cDNA encoding one repair mediator (e.g., ST2, IL1RAP, IL-18R1, and / or IL18RAP). ST2 and / or IL 1 RAP expression

[0302] Some embodiments of cells comprise a promoter inserted into the genome of a cell upstream from a coding sequence of an endogenous ST2 gene and / or IL1RAP gene, such that the inserted promoter is operably linked to the coding sequence of the endogenous ST2 gene or the IL1RAP gene. In some embodiments, the coding sequence of the endogenous ST2 gene and / or the endogenous IL1RAP gene is modified. Such modifications may remove one or more introns and / or mutate one or more exons of the endogenous ST2 gene and / or IL1RAP gene.

[0303] In some embodiments, the modified coding sequence encodes a functional derivative of ST2 or IL1RAP. The functional derivative of ST2 or IL1RAP may include a protein that has a substantial activity of a wild-type ST2 or IL1RAP, or increased activity relative to wildtype ST2 or IL1RAP. One of ordinary skill in the art may use any suitable method to test the functionality or activity of a ST2 or IL1RAP or derivative thereof. The functional derivative of ST2 or IL1RAP may also include any ST2 or IL1RAP or fragment thereof that has conservative substitutions of one or more amino acid residues relative to full-length, wildtype ST2 or IL1RAP as set forth in SEQ ID NOs: 1 and 2, respectively.

[0304] In some embodiments, the functional derivative has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 substitutions relative to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the functional derivative has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,

[0305] 13, 14, 15, 16, 17, 18, 19, or 20 insertions relative to the amino acid sequence of SEQ ID NO:

[0306] 1. In some embodiments, the functional derivative has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,

[0307] 14, 15, 16, 17, 18, 19, or 20 deletions relative to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the functional derivative comprises an N-terminal fusion with another polypeptide. In some embodiments, the functional derivative comprises a C-terminal fusion with another polypeptide. In some embodiments, a functional derivative lacks one or more domains or portions of wild-type ST2 that are unnecessary for signaling.

[0308] In some embodiments, the functional derivative has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 substitutions relative to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the functional derivative has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,

[0309] 13, 14, 15, 16, 17, 18, 19, or 20 insertions relative to the amino acid sequence of SEQ ID NO:

[0310] 2. In some embodiments, the functional derivative has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,

[0311] 14, 15, 16, 17, 18, 19, or 20 deletions relative to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the functional derivative comprises an N-terminal fusion with another polypeptide. In some embodiments, the functional derivative comprises a C-terminal fusion with another polypeptide. In some embodiments, a functional derivative lacks one or more domains or portions of wild-type IL1RAP that are unnecessary for signaling.

[0312] In some embodiments, the encoded ST2 or IL1RAP or functional derivative thereof has about or at least 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or more of the activity exhibited by a wild-type ST2 or IL1RAP. In some embodiments, the encoded ST2 or IL1RAP comprises an amino acid sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or up to 100% sequence identity to the amino acid sequence of wild-type human ST2 or IL1RAP set forth as SEQ ID NOs: 1 and 2, respectively. In some embodiments, the encoded ST2 comprises the wild-type amino acid sequence of SEQ ID NO: 1. In some embodiments, the encoded ST2 consists of the wild-type amino acid sequence of SEQ ID NO: 1. In some embodiments, the encoded IL1RAP comprises the wild-type amino acid sequence of SEQ ID NO: 2. In some embodiments, the encoded IL1RAP consists of the wild-type amino acid sequence of SEQ ID NO: 2.

[0313] In some embodiments, a nucleotide sequence encoding ST2 is codon-optimized. In some embodiments, ST2 is encoded by a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence of SEQ ID NO: 52. In some embodiments, ST2 is encoded by nucleotide sequence of SEQ ID NO: 52. ST2 may be expressed with any suitable signal peptide. In some embodiments, the signal peptide is a wild-type ST2 signal peptide. An example of an ST2 signal peptide amino acid sequence is provided by SEQ ID NO: 43. In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 43. In some embodiments, the signal peptide is encoded by a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence of SEQ ID NO: 53. In some embodiments, the signal peptide is encoded by the nucleotide sequence of SEQ ID NO: 53.

[0314] In some embodiments, a nucleotide sequence encoding IL1RAP is codon-optimized. In some embodiments, IL1RAP is encoded by a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence of SEQ ID NO: 54. In some embodiments, IL1RAP is encoded by nucleotide sequence of SEQ ID NO: 54. IL1RAP may be expressed with any suitable signal peptide. In some embodiments, the signal peptide is a wild-type IL1RAP signal peptide. An example of an IL1RAP signal peptide amino acid sequence is provided by SEQ ID NO: 44. In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 55. In some embodiments, the signal peptide is encoded by a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence of SEQ ID NO: 55. In some embodiments, the signal peptide is encoded by the nucleotide sequence of SEQ ID NO: 55.

[0315] IL-18R1 and / or IL18RAP expression

[0316] Some embodiments of cells comprise a promoter inserted into the genome of a cell upstream from a coding sequence of an endogenous IL18R1 gene and / or IL18RAP gene, such that the inserted promoter is operably linked to the coding sequence of the endogenous IL18R1 gene or the IL18RAP gene. In some embodiments, the coding sequence of the endogenous IL18R1 gene and / or the endogenous IL18RAP gene is modified. Such modifications may remove one or more introns and / or mutate one or more exons of the endogenous IL18R1 gene and / or IL18RAP gene.

[0317] In some embodiments, the modified coding sequence encodes a functional derivative of IL-18R1 or IL18RAP. The functional derivative of IL-18R1 or IL18RAP may include a protein that has a substantial activity of a wild-type IL-18R1 or IL18RAP, or increased activity relative to wild-type IL-18R1 or IL18RAP. One of ordinary skill in the art may use any suitable method to test the functionality or activity of an IL-18R1 or IL18RAP or derivative thereof. The functional derivative of IL-18R1 or IL18RAP may also include any IL-18R1 or IL18RAP or fragment thereof that has conservative substitutions of one or more amino acid residues relative to full-length, wild-type IL-18R1 or IL18RAP as set forth in SEQ ID NOs: 40 and 41, respectively.

[0318] In some embodiments, the functional derivative has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 substitutions relative to the amino acid sequence of SEQ ID NO: 40. In some embodiments, the functional derivative has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 insertions relative to the amino acid sequence of SEQ ID NO: 40. In some embodiments, the functional derivative has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,

[0319] 12, 13, 14, 15, 16, 17, 18, 19, or 20 deletions relative to the amino acid sequence of SEQ ID NO: 40. In some embodiments, the functional derivative comprises a C-terminal fusion with another polypeptide. In some embodiments, a functional derivative lacks one or more domains or portions of wild-type IL-18R1 that are unnecessary for signaling.

[0320] In some embodiments, the functional derivative has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,

[0321] 13, 14, 15, 16, 17, 18, 19, or 20 substitutions relative to the amino acid sequence of SEQ ID NO: 41. In some embodiments, the functional derivative has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 insertions relative to the amino acid sequence of SEQ ID NO: 41. In some embodiments, the functional derivative has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 deletions relative to the amino acid sequence of SEQ ID NO: 41. In some embodiments, the functional derivative comprises a C-terminal fusion with another polypeptide. In some embodiments, a functional derivative lacks one or more domains or portions of wild-type IL18RAP that are unnecessary for signaling.

[0322] In some embodiments, the encoded IL-18R1 or IL18RAP or functional derivative thereof has about or at least 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or more of the activity exhibited by a wild-type IL-18R1 or IL18RAP as set forth in SEQ ID NOs: 40 and 41, respectively. In some embodiments, the encoded IL- 18R1 or IL18RAP comprises an amino acid sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or up to 100% sequence identity to the amino acid sequence of wild-type human IL-18R1 or IL18RAP as set forth in SEQ ID NOs: 40 and 41, respectively. In some embodiments, the encoded IL-18R1 comprises the wild-type amino acid sequence of SEQ ID NO: 40. In some embodiments, the encoded IL-18R1 consists of the wild-type amino acid sequence of SEQ ID NO: 40. In some embodiments, the encoded IL18RAP comprises the wild-type amino acid sequence of SEQ ID NO: 41. In some embodiments, the encoded IL18RAP consists of the wild-type amino acid sequence of SEQ ID NO: 41.

[0323] In some embodiments, a nucleotide sequence encoding IL-18R1 is codon-optimized. In some embodiments, IL-18R1 is encoded by a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence of SEQ ID NO: 110. In some embodiments, IL-18R1 is encoded by nucleotide sequence of SEQ ID NO: 110. IL-18R1 may be expressed with any suitable signal peptide. In some embodiments, the signal peptide is a wild-type IL-18R1 signal peptide. An example of an IL- 18R1 signal peptide amino acid sequence is provided by SEQ ID NO: 45. In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 45. In some embodiments, the signal peptide is encoded by a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence of SEQ ID NO: 111. In some embodiments, the signal peptide is encoded by the nucleotide sequence of SEQ ID NO: 111.

[0324] In some embodiments, a nucleotide sequence encoding IL18RAP is codon-optimized. In some embodiments, IL18RAP is encoded by a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence of SEQ ID NO: 112. In some embodiments, IL18RAP is encoded by nucleotide sequence of SEQ ID NO: 112. IL18RAP may be expressed with any suitable signal peptide. In some embodiments, the signal peptide is a wild-type IL18RAP signal peptide. An example of an IL18RAP signal peptide amino acid sequence is provided by SEQ ID NO: 46. In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 46. In some embodiments, the signal peptide is encoded by a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence of SEQ ID NO: 113. In some embodiments, the signal peptide is encoded by the nucleotide sequence of SEQ ID NO: 113.

[0325] Other repair mediators ’ expression

[0326] Some embodiments of cells comprise an inserted nucleic acid comprising a promoter operably linked to a nucleic acid sequence encoding a repair mediator (e.g, IL-18R), or a functional derivative thereof. The functional derivative of the repair mediator (e.g, IL-18R) may include a protein that has a substantial activity of a wild-type repair mediator (e.g., IL- 18R), or increased activity relative to the wild-type repair mediator (e.g., IL-18R). One of ordinary skill in the art may use any suitable method to test the functionality or activity of a repair mediator (e.g., IL-18R) or derivative thereof. The functional derivative of a repair mediator (e.g., IL-18R) may also include any repair mediator (e.g., IL-18R) or fragment thereof that has conservative substitutions of one or more amino acid residues relative to full- length, wild-type repair mediator (e.g., IL-18R).

[0327] Other repair mediators include a transcription factor or activator of a repair pathway, such as GATA3, PPARG, and / or BATF; cytokines (e.g., AREG and / or osteopontin); growth factors (e.g., KGF, TGF-P); and / or chemokine receptors (e.g., CCR2, CCR5, CCR8) that promote repair and regeneration. See, e.g, Wohlfert et al., J Clin Invest. 2011. 121(11):4503— 4515 (discussing GATA3); Miao et al., Cell Commun Signal. 2022. 20(l):48 (discussing PPARgamma); Trujillo-Ochoa et al, Nat Rev Immunol . 2023. 23(12):842-856 (discussing BATF); Zhang et al., Cell Physiol Biochem. 2017. 43 (6) :2155-2169 (discussing AREG, KGF, and chemokine receptors); Shi et al., Immunity. 2021. 54(7): 1527-1542 (discussing osteopontin); Konkel et al., Immunity. 2017. 46:660-674 (discussing TGF-P). Inserted locus

[0328] In some embodiments, the promoter and coding sequence encoding at least one repair mediator (e.g., ST2, IL1RAP, IL-18R1, and / or IL18RAP), or functional derivative thereof, are located in a locus corresponding to the polypeptide encoded by the coding sequence (e.g., a promoter and coding sequence encoding ST2 are inserted in a ST2 locus of the cell genome or a promoter and coding sequence encoding IL-18R1 are inserted in a IL18Rl locus of the cell genome). In some embodiments, the cell comprises a promoter operably linked to a cDNA sequence encoding ST2 or a derivative thereof, inserted at a ST2 locus. In some embodiments, the cell comprises a promoter operably linked to a cDNA sequence encoding IL-18R1 or a derivative thereof, inserted at an IL18R1 locus. In some embodiments, the cell comprises a promoter operably linked to a cDNA sequence encoding IL1RAP or a derivative thereof, inserted a a III RAP locus. In some embodiments, the cell comprises a promoter operably linked to a cDNA sequence encoding IL18RAP or a derivative thereof, inserted at an IL18RAPlocus. In some embodiments, the cell comprises a promoter operably linked to a cDNA sequence encoding IL-18R or a derivative thereof, inserted at an IL-18R locus.

[0329] In some embodiments, the promoter and coding sequence encoding at least one repair mediator (e.g., ST2, IL1RAP, IL-18R1, and / or IL18RAP), or functional derivative thereof, are located in a safe harbor locus. In some embodiments, the safe harbor locus is a HIPP 11 locus. In some embodiments, the safe harbor locus is an AAVS1 locus. In some embodiments, the safe harbor locus is a ROSA26 locus.

[0330] In some embodiments, the promoter and coding sequence encoding ST2, IL1RAP, IL- 18R1 and / or IL18RAP, or functional derivative thereof, are located in a T cell receptor locus. In some embodiments, the T cell receptor locus is a TRAC locus. In some embodiments, the T cell receptor locus is a TRBC locus.

[0331] Cells for stabilized expression of an endogenous coding sequence

[0332] Some aspects relate to cells comprising a promoter inserted into a nucleic acid e.g., chromosome) of the cell genome, upstream from a coding sequence of an endogenous ST2, IL1RAP, IL18R1, or IL18RAP gene, such that the inserted promoter is operably linked to a coding sequence encoding ST2, IL1RAP, IL-18R1, and / or IL18RAP.

[0333] In some embodiments, the promoter is inserted into the genome at the endogenous promoter. Insertion of a promoter into the endogenous promoter may entirely remove endogenous promoter, or inactivate the endogenous promoter, depending on the sequence of the donor polynucleotide used in homology-directed repair. For example, a donor template may comprise, in 5 '-to-3 ' order, a sequence corresponding to a portion of the endogenous promoter, the promoter, and a sequence corresponding to a sequence downstream from the endogenous promoter, which, when incorporated by homology-directed repair, creates a chromosome containing the portion of the endogenous promoter followed by the inserted promoter.

[0334] In some embodiments, the promoter is inserted downstream from the endogenous promoter, and upstream from the first coding exon of the ST2, IL1RAP, IL-18R1. or IL18RAP coding sequence. The promoter may be inserted at any position between the endogenous promoter and the first coding exon of the ST2, IL1RAP, IL-18R1, or IL18RAP coding sequence. In some embodiments, the promoter is inserted 1-10,000, 10-1,000, 10-100, 10- 5,000, 20-4,000, 30-3,000, 40-2,000, 50-1,000, 60-750, 70-500, 80-400, 90-300, 100-200, 1-1,000, 1,000-2,000, 2,000-3,000, 3,000-4,000, 4,000-5,000, 5,000-6,000, 6,000-7,000, 7,000-8,000, 8,000-9,000, or 9,000-10,000 nucleotides downstream from the endogenous promoter of ST2, IL1RAP, II.-18R1. or IL18RAP . In some embodiments, the promoter is inserted 1-10,000, 10-1,000, 10-100, 10-5,000, 20-4,000, 30-3,000, 40-2,000, 50-1,000, 60-750, 70-500, 80-400, 90-300, 100-200, 1-1,000, 1,000-2,000, 2,000-3,000, 3,000- 4,000, 4,000-5,000, 5,000-6,000, 6,000-7,000, 7,000-8,000, 8,000-9,000, or 9,000-10,000 nucleotides upstream from the first coding exon of the ST2, 11.1 RAP. II.-18R1. or IL18RAP coding sequence.

[0335] In some embodiments, the inserted promoter is active promoting transcription of RNA encoding ST2, IL1RAP, IL-18R1, or IL18RAP, even under pro-inflammatory conditions. In some embodiments, the inserted promoter is a constitutive promoter. In some embodiments, the constitutive promoter is an EF-la, PGK, or MND promoter. In some embodiments, the constitutive promoter is an MND promoter. In some embodiments, the inserted promoter is an inducible promoter.

[0336] In some embodiments, the promoter is inserted into an exon of the ST2, IL1RAP, IL- 18R1, or IL18RAP coding sequence, thereby creating a synthetic exon. In such embodiments, the coding sequence may be modified relative to the endogenous sequence, but still capable of encoding a ST2, IL1RAP, IL-18R1, or IL18RAP polypeptide. For example, one or more codons of the endogenous coding sequence may be replaced by nucleotides of the inserted promoter, and a downstream codon may be replaced by a start (AUG) codon, such that the inserted promoter mediates transcription of an mRNA that encodes a modified ST2, IL1RAP, IL-18R7, or IL 18RAP that is shorter than the endogenous form.

[0337] ST2 and / or IL 1 RAP expression

[0338] Some embodiments of cells comprise a promoter inserted into the genome of a cell upstream from a coding sequence of an endogenous ST2 gene and / or IL1RAP gene, such that the inserted promoter is operably linked to the coding sequence of the endogenous ST2 gene or the IL1RAP gene. In some embodiments, the coding sequence of the endogenous ST2 gene and / or the endogenous IL1RAP gene is modified. Such modifications may remove one or more introns and / or mutate one or more exons of the endogenous ST2 gene and / or IL1RAP gene.

[0339] In some embodiments, the modified coding sequence encodes a functional derivative of ST2 or IL1RAP. The functional derivative of ST2 or IL1RAP may include a protein that has a substantial activity of a wild-type ST2 or IL1RAP, or increased activity relative to wildtype ST2 or IL1RAP. One of ordinary skill in the art may use any suitable method to test the functionality or activity of a ST2 or IL1RAP or derivative thereof. The functional derivative of ST2 or IL1RAP may also include any ST2 or IL1RAP or fragment thereof that has conservative substitutions of one or more amino acid residues relative to full-length, wildtype ST2 or IL1RAP as set forth in SEQ ID NOs: 1 and 2, respectively.

[0340] In some embodiments, the functional derivative has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 substitutions relative to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the functional derivative has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,

[0341] 13, 14, 15, 16, 17, 18, 19, or 20 insertions relative to the amino acid sequence of SEQ ID NO:

[0342] 1. In some embodiments, the functional derivative has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,

[0343] 14, 15, 16, 17, 18, 19, or 20 deletions relative to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the functional derivative comprises an N-terminal fusion with another polypeptide. In some embodiments, the functional derivative comprises a C-terminal fusion with another polypeptide. In some embodiments, a functional derivative lacks one or more domains or portions of wild-type ST2 that are unnecessary for signaling.

[0344] In some embodiments, the functional derivative has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 substitutions relative to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the functional derivative has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 insertions relative to the amino acid sequence of SEQ ID NO:

[0345] 2. In some embodiments, the functional derivative has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 deletions relative to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the functional derivative comprises an N-terminal fusion with another polypeptide. In some embodiments, the functional derivative comprises a C-terminal fusion with another polypeptide. In some embodiments, a functional derivative lacks one or more domains or portions of wild-type IL1RAP that are unnecessary for signaling.

[0346] In some embodiments, the encoded ST2 or IL1RAP or functional derivative thereof has about or at least 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or more of the activity exhibited by a wild-type ST2 or IL1RAP. In some embodiments, the encoded ST2 or IL1RAP comprises an amino acid sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or up to 100% sequence identity to the amino acid sequence of wild-type human ST2 or IL1RAP set forth as SEQ ID NOs: 1 and 2, respectively. In some embodiments, the encoded ST2 comprises the wild-type amino acid sequence of SEQ ID NO: 1. In some embodiments, the encoded ST2 consists of the wild-type amino acid sequence of SEQ ID NO: 1. In some embodiments, the encoded IL1RAP comprises the wild-type amino acid sequence of SEQ ID NO: 2. In some embodiments, the encoded IL1RAP consists of the wild-type amino acid sequence of SEQ ID NO: 2.

[0347] In some embodiments, a nucleotide sequence encoding ST2 is codon-optimized. In some embodiments, ST2 is encoded by a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence of SEQ ID NO: 52. In some embodiments, ST2 is encoded by nucleotide sequence of SEQ ID NO: 52. ST2 may be expressed with any suitable signal peptide. In some embodiments, the signal peptide is a wild-type ST2 signal peptide. An example of an ST2 signal peptide amino acid sequence is provided by SEQ ID NO: 43. In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 43. In some embodiments, the signal peptide is encoded by a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence of SEQ ID NO: 53. In some embodiments, the signal peptide is encoded by the nucleotide sequence of SEQ ID NO: 53.

[0348] In some embodiments, a nucleotide sequence encoding IL1RAP is codon-optimized. In some embodiments, IL1RAP is encoded by a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence of SEQ ID NO: 54. In some embodiments, IL1RAP is encoded by nucleotide sequence of SEQ ID NO: 54. IL1RAP may be expressed with any suitable signal peptide. In some embodiments, the signal peptide is a wild-type IL1RAP signal peptide. An example of an IL1RAP signal peptide amino acid sequence is provided by SEQ ID NO: 44. In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 55. In some embodiments, the signal peptide is encoded by a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence of SEQ ID NO: 55. In some embodiments, the signal peptide is encoded by the nucleotide sequence of SEQ ID NO: 55.

[0349] IL-18R1 and / or IL18RAP expression

[0350] Some embodiments of cells comprise a promoter inserted into the genome of a cell upstream from a coding sequence of an endogenous IL-18R1 gene and / or IL18RAP gene, such that the inserted promoter is operably linked to the coding sequence of the endogenous IL-18R1 gene or the IL18RAP gene. In some embodiments, the coding sequence of the endogenous IL-18R1 gene and / or the endogenous IL18RAP gene is modified. Such modifications may remove one or more introns and / or mutate one or more exons of the endogenous IL-18R1 gene and / or IL18RAP gene.

[0351] In some embodiments, the modified coding sequence encodes a functional derivative of IL-18R1 or IL18RAP. The functional derivative of IL-18R1 or IL18RAP may include a protein that has a substantial activity of a wild-type IL-18R1 or IL18RAP, or increased activity relative to wild-type IL-18R1 or IL18RAP. One of ordinary skill in the art may use any suitable method to test the functionality or activity of an IL-18R1 or IL18RAP or derivative thereof. The functional derivative of IL-18R1 or IL18RAP may also include any IL-18R1 or IL18RAP or fragment thereof that has conservative substitutions of one or more amino acid residues relative to full-length, wild-type IL-18R1 or IL18RAP as set forth in SEQ ID NOs: 40 and 41, respectively.

[0352] In some embodiments, the functional derivative has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 substitutions relative to the amino acid sequence of SEQ ID NO: 40. In some embodiments, the functional derivative has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 insertions relative to the amino acid sequence of SEQ ID NO: 40. In some embodiments, the functional derivative has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 deletions relative to the amino acid sequence of SEQ ID NO: 40. In some embodiments, the functional derivative comprises a C-terminal fusion with another polypeptide. In some embodiments, a functional derivative lacks one or more domains or portions of wild-type IL-18R1 that are unnecessary for signaling. In some embodiments, the functional derivative has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 substitutions relative to the amino acid sequence of SEQ ID NO: 41. In some embodiments, the functional derivative has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 insertions relative to the amino acid sequence of SEQ ID NO: 41. In some embodiments, the functional derivative has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 deletions relative to the amino acid sequence of SEQ ID NO: 41. In some embodiments, the functional derivative comprises a C-terminal fusion with another polypeptide. In some embodiments, a functional derivative lacks one or more domains or portions of wild-type IL18RAP that are unnecessary for signaling.

[0353] In some embodiments, the encoded IL-18R1 or IL18RAP or functional derivative thereof has about or at least 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or more of the activity exhibited by a wild-type IL-18R1 or IL18RAP as set forth in SEQ ID NOs: 40 and 41, respectively. In some embodiments, the encoded IL- 18R1 or IL18RAP comprises an amino acid sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or up to 100% sequence identity to the amino acid sequence of wild-type human IL-18R1 or IL18RAP as set forth in SEQ ID NOs: 40 and 41, respectively. In some embodiments, the encoded IL-18R1 comprises the wild-type amino acid sequence of SEQ ID NO: 40. In some embodiments, the encoded IL-18R1 consists of the wild-type amino acid sequence of SEQ ID NO: 40. In some embodiments, the encoded IL18RAP comprises the wild-type amino acid sequence of SEQ ID NO: 41. In some embodiments, the encoded IL18RAP consists of the wild-type amino sequence of SEQ ID NO: 41.

[0354] In some embodiments, a nucleotide sequence encoding IL-18R1 is codon-optimized. In some embodiments, IL-18R1 is encoded by a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence of SEQ ID NO: 110. In some embodiments, IL-18R1 is encoded by nucleotide sequence of SEQ ID NO: 110. IL-18R1 may be expressed with any suitable signal peptide. In some embodiments, the signal peptide is a wild-type IL-18R1 signal peptide. An example of an IL- 18R1 signal peptide amino acid sequence is provided by SEQ ID NO: 45. In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 45. In some embodiments, the signal peptide is encoded by a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence of SEQ ID NO: 111. In some embodiments, the signal peptide is encoded by the nucleotide sequence of SEQ ID NO: 111. In some embodiments, a nucleotide sequence encoding IL18RAP is codon-optimized. In some embodiments, IL18RAP is encoded by a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence of SEQ ID NO: 112. In some embodiments, IL18RAP is encoded by nucleotide sequence of SEQ ID NO: 112. IL18RAP may be expressed with any suitable signal peptide. In some embodiments, the signal peptide is a wild-type IL18RAP signal peptide. An example of an IL18RAP signal peptide amino acid sequence is provided by SEQ ID NO: 46. In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 46. In some embodiments, the signal peptide is encoded by a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence of SEQ ID NO: 113. In some embodiments, the signal peptide is encoded by the nucleotide sequence of SEQ ID NO: 113.

[0355] Other repair mediators ’ expression

[0356] Some embodiments of cells comprise an nucleic acid comprising a promoter operably linked to a nucleic acid sequence encoding a repair mediator (e.g, IL-18R), or a functional derivative thereof. The functional derivative of the repair mediator (e.g, IL-18R) may include a protein that has a substantial activity of a wild-type repair mediator (e.g., IL-18R), or increased activity relative to the wild-type repair mediator (e.g., IL-18R). One of ordinary skill in the art may use any suitable method to test the functionality or activity of a repair mediator (e.g., IL-18R) or derivative thereof. The functional derivative of a repair mediator (e.g., IL-18R) may also include any repair mediator (e.g., IL-18R) or fragment thereof that has conservative substitutions of one or more amino acid residues relative to full-length, wild-type repair mediator (e.g., IL-18R).

[0357] Other repair mediators include a transcription factor or activator of a repair pathway, such as GATA3, PPARG, and / or BATF; cytokines (e.g., AREG and / or osteopontin); growth factors (e.g., KGF, TGF-P); and / or chemokine receptors (e.g., CCR2, CCR5, CCR8) that promote repair and regeneration. See, e.g, Wohlfert et al., J Clin Invest. 2011. 121(11):4503— 4515 (discussing GATA3); Miao et al., Cell Commun Signal. 2022. 20(l):48 (discussing PPARgamma); Trujillo-Ochoa et al, Nat Rev Immunol . 2023. 23(12):842-856 (discussing BATF); Zhang et al., Cell Physiol Biochem. 2017. 43 (6) :2155-2169 (discussing AREG, KGF, and chemokine receptors); Shi et al., Immunity. 2021. 54(7): 1527-1542 (discussing osteopontin); Konkel et al., Immunity. 2017. 46:660-674 (discussing TGF-P). Nucleases, guide RNAs, and homology arms

[0358] Some aspects relate to the use of nucleases to introduce a double-stranded break into nucleic acid of a cell genome and edit the genome at a desired locus (e.g., to promote integration of a donor template at the locus by homology-directed repair and / or inactivate a targeted gene). Any one of multiple gene- or genome- editing methods can used to accomplish editing of one or more loci e.g., ST2, IL1RAP, IL18R1, IL18RAP, TRAC, TRBC, AAVS1, and / or HIPP 11 . Non-limiting examples of gene editing methods include use of a DNA endonuclease such as an RNA-guided nuclease (e.g., Cas (e.g., Cas9) nuclease), zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), or meganuclease; transposon-mediated gene editing; serine integrase-mediated gene editing; and lentivirus-mediated gene editing. In some embodiments, a gene editing method comprises knocking out or inactivating an endogenous gene, such as by producing a chromosomal gene knockout in the genome. As used herein, the term "chromosomal gene knockout" refers to a genetic alteration, inactivation, or introduced inhibitory agent in a host cell that prevents (e.g., reduces, delays, suppresses, or abrogates) production, by the host cell, of a functionally active endogenous polypeptide product. Alterations resulting in a chromosomal gene knockout or inactivation can include, for example, introduced nonsense mutations (including the formation of premature stop codons), missense mutations, gene deletion, or strand breaks.

[0359] In certain embodiments, a chromosomal gene knock-out or gene knock-in (e.g., insertion) is made by chromosomal editing of a host cell. Chromosomal editing can be performed using, for example, endonucleases. As used herein "endonuclease" refers to an enzyme capable of catalyzing cleavage of a phosphodiester bond within a polynucleotide chain. A DNA endonuclease refers to an endonuclease that is capable of catalyzing cleavage of a phosphodiester bond within a DNA polynucleotide. In certain embodiments, an endonuclease is capable of cleaving a nucleic acid sequence in a targeted gene, thereby inactivating or "knocking out" the targeted gene. In some embodiments, an endonuclease is capable of cleaving a nucleic acid sequence in a targeted locus, promoting insertion of an exogenous nucleic acid sequence into the targeted locus by homologous recombination. An endonuclease may be a naturally occurring, recombinant, genetically modified, or fusion endonuclease. Examples of endonucleases for use in gene editing include zinc finger nucleases (ZFN), TALE-nucl eases (TALEN), RNA-guided nucleases, CRISPR-Cas nucleases, meganucleases, or megaTALs.

[0360] The nucleic acid strand breaks caused by DNA endonucleases are typically doublestrand breaks (DSB), which may be commonly repaired through the distinct mechanisms of homology directed repair (HDR) by homologous recombination, or by 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(1 l):a012740, PMID 24097900). During HDR / homologous recombination, a donor nucleic acid molecule may be used for a donor gene "knock-in", for target gene "knock-out", and optionally to inactivate a target gene through a donor gene knock in or target gene knock out event. NHEJ is an error-prone repair process that often results in changes to the DNA sequence at the site of the cleavage, e.g., a substitution, deletion, or addition of at least one nucleotide. NHEJ may be used to "knockout" a target gene. HDR is favored by the presence of a donor template at the time of DSB formation.

[0361] As used herein, a "zinc finger nuclease" (ZFN) refers to a fusion protein comprising a zinc finger DNA-binding domain fused to a non-specific DNA cleavage domain, such as a Fokl endonuclease. Each zinc finger motif of about 30 amino acids binds to about 3 base pairs of DNA, and amino acids at certain residues can be changed to alter 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). Multiple zinc finger motifs can be linked in tandem to create binding specificity to desired DNA sequences, such as regions having a length ranging from about 9 to about 18 base pairs. By way of background, ZFNs mediate genome editing by catalyzing the formation of a site-specific DNA double strand break (DSB) in the genome, and targeted integration of a transgene comprising flanking sequences homologous to the genome at the site of DSB is facilitated by homology directed repair (HDR). Alternatively, a DSB generated by a ZFN can result in knock out of target gene via repair by non-homologous end joining (NHEJ), which is an error-prone cellular repair pathway that results in the insertion or deletion of nucleotides at the cleavage site. In certain embodiments, a gene knockout or inactivation comprises an insertion, a deletion, a mutation or a combination thereof, made using a ZFN molecule.

[0362] As used herein, a "transcription activator-like effector nuclease" (TALEN) refers to a fusion protein comprising a TALE DNA-binding domain and a DNA cleavage domain, such as a Fokl endonuclease. A "TALE DNA binding domain" or "TALE" is composed of one or more TALE repeat domains / units, each generally having a highly conserved 33-35 amino acid sequence with divergent 12th and 13th amino acids. The TALE repeat domains are involved in binding of the TALE to a target DNA sequence. The divergent amino acid residues, referred to as the Repeat Variable Diresidue (RVD), correlate with specific nucleotide recognition. The natural (canonical) code for DNA recognition of these TALEs has been determined such that an HD (histidine-aspartic acid) sequence at positions 12 and 13 of the TALE leads to the TALE binding to cytosine (C), NG (asparagine-glycine) binds to a T nucleotide, NI (asparagine-isoleucine) to A, NN (asparagine-asparagine) binds to a G or A nucleotide, and NG (asparagine-glycine) binds to a T nucleotide. Non-canonical (atypical) RVDs are also known (see, e.g., U.S. Patent Publication No. US 2011 / 0301073, which atypical RVDs are incorporated by reference herein in their entirety). TALENs can be used to direct site-specific double-strand breaks (DSB) in the genome of T cells. Non- homologous end joining (NHEJ) ligates DNA from both sides of a double-strand break in which there is little or no sequence overlap for annealing, thereby introducing errors that knock out gene expression. Alternatively, homology directed repair (HDR) can introduce a transgene at the site of DSB providing homologous flanking sequences are present in the donor template containing the transgene. In certain embodiments, a gene knockout comprises an insertion, a deletion, a mutation or a combination thereof, and made using a TALEN molecule.

[0363] Gene-editing systems and methods may make use of viral or non-viral vectors or cassettes, as well as nucleases that allow site-specific or locus-specific gene-editing, such as RNA-guided nucleases, Cas nucleases (e.g., Cpfl or Cas9 nucleases), meganucleases, TALENs, or ZFNs. Certain RNA-guided nucleases useful with some embodiments are disclosed in U.S. Patent No. 11,162,114, which is expressly incorporated by reference herein in its entirety. Non-limiting examples of Cas nucleases include SpCas9, SaCas9, CjCas9, xCas9, C2cl, Casl3a / C2c2, C2c3, Casl3b, Cpfl, and variants thereof. Certain features useful with some embodiments are disclosed in WO 2019 / 210057, which is expressly incorporated by reference in its entirety.

[0364] As used herein, a "clustered regularly interspaced short palindromic repeats / Cas" (CRISPR / Cas, or Cas) nuclease system refers to a system that employs a CRISPR RNA (crRNA)-guided Cas nuclease to recognize target sites within a genome (known as protospacers) via base-pairing complementarity and then to cleave the DNA if a short, conserved protospacer associated motif (PAM) immediately follows 3’ of the complementary target sequence. CRISPR / Cas systems are classified into types (e.g., type I, type II, type III, and type V) based on the sequence and structure of the Cas nucleases. The crRNA-guided surveillance complexes in types I and III need multiple Cas subunits. The Type II system, the most studied, comprises at least three components: an RNA-guided Cas9 nuclease, a crRNA, and a trans-acting crRNA (tracrRNA). The tracrRNA comprises a duplex forming region. A crRNA and a tracrRNA form a duplex that is capable of interacting with a Cas9 nuclease and guiding the Cas9 / crRNA:tracrRNA complex to a specific site on the target DNA via Watson- Crick base-pairing between the spacer on the crRNA and the protospacer on the target DNA upstream from a PAM. Cas9 nuclease cleaves a double-stranded break within a region defined by the crRNA spacer. Repair by NHEJ results in insertions and / or deletions which disrupt expression of the targeted locus. Alternatively, a donor template transgene with homologous flanking sequences can be introduced at the site of DSB via homology directed repair (HDR). The crRNA and tracrRNA can be engineered into a single guide RNA (sgRNA or gRNA) (see, e.g., Jinek et al., Science 337:816-21, 2012). Further, the region of the guide RNA complementary to the target site can be altered or programed to target a desired sequence (Xie etal., PLOS One 9:el00448, 2014; U.S. Pat. Appl. Pub. No. US 2014 / 0068797, U.S. Pat. Appl. Pub. No. US 2014 / 0186843; U.S. Pat. No. 8,697,359, and PCT Publication No. WO 2015 / 071474; each of which is incorporated by reference). Nonlimiting examples of CRISPR / Cas nucleases include Cas9, SaCas9, CjCas9, xCas9, C2C1, Casl3a / C2c2, C2c3, Casl3b, Cpfl, and variants thereof.

[0365] In some embodiments, a gene knockout or inactivation comprises an insertion, a deletion, a mutation or a combination thereof, and made using an RNA-guided nuclease. Examples of gRNA sequences and methods of using the same to knock out endogenous genes that encode immune cell proteins include those described in Ren et aL, Clin Cancer Res. 2017. 23(9):2255-2266, the gRNAs, Cas9 DNAs, vectors, and gene knockout techniques of which are hereby expressly incorporated by reference in their entirety. Other RNA-guided nucleases capable of introducing a double-stranded break in DNA in the presence of a guide RNA comprising a spacer sequence complementary to a target sequence of the DNA, by cleaving at a PAM sequence adjacent to the target sequence on the DNA, may also be used in gene editing methods and systems described herein. In some embodiments, the RNA-guided nuclease is a nuclease having (z.e., cleaving dsDNA at) a protospacer-adjacent motif (PAM) sequence of 5'-NNNNCC-3'. Examples RNA-guided nucleases having a PAM sequence of NNNNCC are described, e.g., in International Application No. PCT / US2019 / 035373, published as PCT Publication No. WO 2019 / 236566, which is incorporated by reference herein in its entirety. In some embodiments, the RNA-guided nuclease cleaves DNA at a PAM sequence of NGG, and localizes to DNA at a target sequence in the presence of a gRNA having the nucleotide sequence of 174, where the polyN stretch of SEQ ID NO: 174 is the protospacer sequence complementary to the target DNA sequence. In some embodiments, the RNA-guided nuclease cleaves DNA at a PAM sequence of NNNNCC, and localizes to DNA at a target sequence in the presence of a gRNA having the nucleotide sequence of SEQ ID NO: 175, where the polyN stretch of SEQ ID NO: 175 is the protospacer sequence complementary to the target DNA sequence. In some embodiments, the RNA-guided nuclease cleaves DNA at a PAM sequence of NNNNCC, and localizes to DNA at a target sequence in the presence of a gRNA having the nucleotide sequence of SEQ ID NO: 176, where the polyN stretch of SEQ ID NO: 176 is the protospacer sequence complementary to the target DNA sequence.

[0366] In some embodiments, a gene modification comprises an insertion of an exogenous nucleic acid sequence (e.g., promoter, transgene, and / or combinations thereof) into the genome of a cell, where an RNA-guided nuclease introduces a double-stranded break in the genome and the exogenous nucleic acid sequence is introduced into the genome by homology-directed repair.

[0367] In some embodiments, a genetic modification comprises insertion of an exogenous nucleic acid e.g., donor template) into the F0XP3 locus of a cell genome, where the donor template comprises a 5' homology arm and a 3' homology arm, each having homology to nucleotide sequences within the F0XP3 locus, such that the exogenous nucleic acid is inserted into the F0XP3 locus following introduction of a double-stranded break within the F0XP3 locus. In some embodiments, the double-stranded break is introduced by an RNA- guided nuclease in the presence of a gRNA at a PAM sequence of NGG. In some embodiments, the double-stranded break is introduced by an RNA-guided nuclease in the presence of a gRNA at a PAM sequence of NNNNCC.

[0368] 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: 138, the 3' homology arm comprises a nucleotide sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO: 139. In some embodiments, the 5' homology arm comprises a nucleotide sequence having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 138, the 3' homology arm comprises a nucleotide sequence having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 139. In some embodiments, the 5' homology arm comprises the nucleic acid sequence of SEQ ID NO: 138 and the 3' homology arm comprises the nucleic acid sequence of SEQ ID NO: 139. In some embodiments, the donor template is inserted following cleavage by an RNA-guided nuclease in the presence of a gRNA at a PAM sequence of NGG. In some embodiments, the gRNA comprises a spacer sequence with no more than 3, no more than 2, or no more than 1 mismatches relative to SEQ ID NO: 150. In some embodiments, the gRNA comprises a spacer sequence of SEQ ID NO: 150. 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: 140, the 3' homology arm comprises a nucleotide sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO: 141. In some embodiments, the 5' homology arm comprises a nucleotide sequence having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 140, the 3' homology arm comprises a nucleotide sequence having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 141. In some embodiments, the 5' homology arm comprises the nucleic acid sequence of SEQ ID NO: 140 and the 3' homology arm comprises the nucleic acid sequence of SEQ ID NO: 141. In some embodiments, the donor template is inserted following cleavage by an RNA-guided nuclease in the presence of a gRNA at a PAM sequence of NGG. In some embodiments, the gRNA comprises a spacer sequence with no more than 3, no more than 2, or no more than 1 mismatches relative to SEQ ID NO: 150. In some embodiments, the gRNA comprises a spacer sequence of SEQ ID NO: 150.

[0369] 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: 143 or SEQ ID NO: 146, the 3' homology arm comprises a nucleotide sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO: 145. In some embodiments, the 5' homology arm comprises a nucleotide sequence having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 143 or SEQ ID NO: 146, the 3' homology arm comprises a nucleotide sequence having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 145. In some embodiments, the 5' homology arm comprises the nucleic acid sequence of SEQ ID NO: 143 or SEQ ID NO: 146, and the 3' homology arm comprises the nucleic acid sequence of SEQ ID NO: 145. In some embodiments, the donor template is inserted following cleavage by an RNA-guided nuclease in the presence of a gRNA at a PAM sequence of NNNNCC. In some embodiments, the gRNA comprises a spacer sequence with no more than 3, no more than 2, or no more than 1 mismatches relative to SEQ ID NO: 151. In some embodiments, the gRNA comprises a spacer sequence of SEQ ID NO: 151.

[0370] 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: 146, the 3' homology arm comprises a nucleotide sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO: 147. In some embodiments, the 5' homology arm comprises a nucleotide sequence having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 146, the 3' homology arm comprises a nucleotide sequence having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 147. In some embodiments, the 5' homology arm comprises the nucleic acid sequence of SEQ ID NO: 146, and the 3' homology arm comprises the nucleic acid sequence of SEQ ID NO: 147. In some embodiments, the donor template is inserted following cleavage by an RNA-guided nuclease in the presence of a gRNA at a PAM sequence of NNNNCC. In some embodiments, the gRNA comprises a spacer sequence with no more than 3, no more than 2, or no more than 1 mismatches relative to SEQ ID NO: 151. In some embodiments, the gRNA comprises a spacer sequence of SEQ ID NO: 151.

[0371] In some embodiments, a donor template inserted into the F0XP3 locus comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 165. In some embodiments, a donor template inserted into the FOXP3 locus comprises at least 90% sequence identity to SEQ ID NO: 165. In some embodiments, a donor template inserted into the FOXP3 locus comprises at least 95% sequence identity to SEQ ID NO: 165. In some embodiments, a donor template inserted into the FO P3 locus comprises the nucleotide sequence of SEQ ID NO: 165. In some embodiments, a donor template inserted into the FOXP3 locus consists of the nucleotide sequence of SEQ ID NO: 165.

[0372] In some embodiments, a donor template inserted into the FOXP3 locus comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 166. In some embodiments, a donor template inserted into the FOXP3 locus comprises at least 90% sequence identity to SEQ ID NO: 166. In some embodiments, a donor template inserted into the FOXP3 locus comprises at least 95% sequence identity to SEQ ID NO: 166. In some embodiments, a donor template inserted into the FO P3 locus comprises the nucleotide sequence of SEQ ID NO: 166. In some embodiments, a donor template inserted into the FOXP3 locus consists of the nucleotide sequence of SEQ ID NO: 166.

[0373] In some embodiments, a donor template inserted into the FOXP3 locus comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 167. In some embodiments, a donor template inserted into the FOXP3 locus comprises at least 90% sequence identity to SEQ ID NO: 167. In some embodiments, a donor template inserted into the FOXP3 locus comprises at least 95% sequence identity to SEQ ID NO: 167. In some embodiments, a donor template inserted into the FO P3 locus comprises the nucleotide sequence of SEQ ID NO: 167. In some embodiments, a donor template inserted into the FOXP3 locus consists of the nucleotide sequence of SEQ ID NO: 167. In some embodiments, a donor template inserted into the FOXP 3 locus comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 168. In some embodiments, a donor template inserted into the FOXP 3 locus comprises at least 90% sequence identity to SEQ ID NO: 168. In some embodiments, a donor template inserted into the FOXP3 locus comprises at least 95% sequence identity to SEQ ID NO: 168. In some embodiments, a donor template inserted into the FOXP3 locus comprises the nucleotide sequence of SEQ ID NO: 168. In some embodiments, a donor template inserted into the FOXP3 locus consists of the nucleotide sequence of SEQ ID NO: 168.

[0374] In some embodiments, a donor template inserted into the FOXP3 locus comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 169. In some embodiments, a donor template inserted into the FOXP3 locus comprises at least 90% sequence identity to SEQ ID NO: 169. In some embodiments, a donor template inserted into the FOXP3 locus comprises at least 95% sequence identity to SEQ ID NO: 169. In some embodiments, a donor template inserted into the FOXP 3 locus comprises the nucleotide sequence of SEQ ID NO: 169. In some embodiments, a donor template inserted into the FOXP3 locus consists of the nucleotide sequence of SEQ ID NO: 169.

[0375] In some embodiments, a donor template inserted into the FOXP 3 locus comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 170. In some embodiments, a donor template inserted into the FOXP3 locus comprises at least 90% sequence identity to SEQ ID NO: 170. In some embodiments, a donor template inserted into the FOXP 3 locus comprises at least 95% sequence identity to SEQ ID NO: 170. In some embodiments, a donor template inserted into the FOXP 3 locus comprises the nucleotide sequence of SEQ ID NO: 170. In some embodiments, a donor template inserted into the FOXP3 locus consists of the nucleotide sequence of SEQ ID NO: 170.

[0376] In some embodiments, a donor template inserted into the FOXP 3 locus comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 171. In some embodiments, a donor template inserted into the FOXP3 locus comprises at least 90% sequence identity to SEQ ID NO: 171. In some embodiments, a donor template inserted into the FOXP 3 locus comprises at least 95% sequence identity to SEQ ID NO: 171. In some embodiments, a donor template inserted into the FOXP 3 locus comprises the nucleotide sequence of SEQ ID NO: 171. In some embodiments, a donor template inserted into the FOXP3 locus consists of the nucleotide sequence of SEQ ID NO: 171.

[0377] In some embodiments, a genetic modification comprises insertion of an exogenous nucleic acid (e.g., donor template) into the TRAC locus of a cell genome, where the donor template comprises a 5' homology arm and a 3' homology arm, each having homology to nucleotide sequences within the TRAC locus, such that the exogenous nucleic acid is inserted into the TRAC locus following introduction of a double-stranded break within the TRAC locus. In some embodiments, the double-stranded break is introduced by an RNA-guided nuclease in the presence of a gRNA at a PAM sequence of NGG. In some embodiments, the double-stranded break is introduced by an RNA-guided nuclease in the presence of a gRNA at a PAM sequence of NNNNCC.

[0378] 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: 153, the 3' homology arm comprises a nucleotide sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO: 154. In some embodiments, the 5' homology arm comprises a nucleotide sequence having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 153, the 3' homology arm comprises a nucleotide sequence having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 154. In some embodiments, the 5' homology arm comprises the nucleic acid sequence of SEQ ID NO: 153 and the 3' homology arm comprises the nucleic acid sequence of SEQ ID NO: 154. In some embodiments, the donor template is inserted following cleavage by an RNA-guided nuclease in the presence of a gRNA at a PAM sequence of NGG. In some embodiments, the gRNA comprises a spacer sequence with no more than 3, no more than 2, or no more than 1 mismatches relative to SEQ ID NO: 159. In some embodiments, the gRNA comprises a spacer sequence of SEQ ID NO: 159.

[0379] 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: 156, the 3' homology arm comprises a nucleotide sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO: 157. In some embodiments, the 5' homology arm comprises a nucleotide sequence having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 156, the 3' homology arm comprises a nucleotide sequence having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 157. In some embodiments, the 5' homology arm comprises the nucleic acid sequence of SEQ ID NO: 156 and the 3' homology arm comprises the nucleic acid sequence of SEQ ID NO: 157. In some embodiments, the donor template is inserted following cleavage by an RNA-guided nuclease in the presence of a gRNA at a PAM sequence of NNNNCC. In some embodiments, the gRNA comprises a spacer sequence with no more than 3, no more than 2, or no more than 1 mismatches relative to SEQ ID NO: 160. In some embodiments, the gRNA comprises a spacer sequence of SEQ ID NO: 160.

[0380] In some embodiments, a donor template inserted into the TRAC locus comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 172. In some embodiments, a donor template inserted into the TRAC locus comprises at least 90% sequence identity to SEQ ID NO: 172. In some embodiments, a donor template inserted into the TRAC locus comprises at least 95% sequence identity to SEQ ID NO: 172. In some embodiments, a donor template inserted into the TRAC locus comprises the nucleotide sequence of SEQ ID NO: 172. In some embodiments, a donor template inserted into the TRAC locus consists of the nucleotide sequence of SEQ ID NO: 172.

[0381] In some embodiments, a donor template inserted into the TRAC locus comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 173. In some embodiments, a donor template inserted into the TRAC locus comprises at least 90% sequence identity to SEQ ID NO: 173. In some embodiments, a donor template inserted into the TRAC locus comprises at least 95% sequence identity to SEQ ID NO: 173. In some embodiments, a donor template inserted into the TRAC locus comprises the nucleotide sequence of SEQ ID NO: 173. In some embodiments, a donor template inserted into the TRAC locus consists of the nucleotide sequence of SEQ ID NO: 173.

[0382] Selection by Essential Gene Exon Knock-In (SLEEK)

[0383] Some embodiments of engineered cells comprise an inserted nucleic acid encoding at least a portion of an essential gene inserted into a locus comprising the essential gene. Some embodiments of methods of editing an engineered cell comprise inserting a nucleic acid encoding a portion of an essential gene into a locus comprising the essential gene. Some embodiments of nucleic acids comprise a nucleotide sequence encoding at least a portion of an essential gene and homology arms that direct insertion of the nucleic acid into a locus comprising the essential gene. Selection by Essential-gene Exon Knock-in (SLEEK) is described in Allen etal., Nat Biotechnol. 2023. doi: 10.1038 / s41587-023-01779-8. Insertion of at least a portion of an essential gene (e.g., an exon of an essential gene) allows selection to address the preference of some cells for non-homologous end joining (NHEJ) over homology-directed repair (HDR). As used herein, an “essential gene” refers to a gene with no alternatives in the cell, that could be expressed to compensate for loss of expression by the essential gene, where failure to express the product of the essential gene (i) inhibits cellular replication, (ii) results in cell death, and / or (iii) in a Treg cell, reduces in loss of the Treg cell phenotype e.g., loss of F0XP3 expression). Essential genes may be those involved in key cellular processes such as central metabolism or transcription. In some embodiments, an essential gene is associated with central metabolism. In some embodiments, an essential gene is associated with transcription. In some embodiments, an essential gene is associated with protein folding. In some embodiments, an essential gene is associated with translocation. In some embodiments, an essential gene is associated with quality control. In some embodiments, an essential gene encodes a chaperone protein. In some embodiments, an essential gene encodes a cytoskeletal protein. In some embodiments, an essential gene encodes a cytoskeletal factor. In some embodiments, an essential gene encodes a protein involved in DNA repair. In some embodiments, an essential gene is GAPDH. In some embodiments, an essential gene is KIF11. In some embodiments, an essential gene is TBP.

[0384] The cells, methods, and / or nucleic acids described in this section involve targeted genome editing, such as editing using a targeted nuclease (e.g., RNA-guided nuclease (e.g., Cas9), TALEN, ZFN, and / or meganuclease). Targeted chromosomal cleavage by a nuclease at an essential gene locus allows disruption of the essential gene, if a corresponding correction (e.g., insertion of a donor nucleic acid compensating for the disruption) does not occur. For example, cleavage at a glyceraldehyde 3 -phosphate dehydrogenase (GAPDH) locus, and consequent indel introduction by DNA repair (e.g., by NHEJ) may prevent production of functional GAPDH, leading to death of cells that fail to express this essential protein. But, where a donor nucleic acid targeting an essential gene locus, and comprising a nucleotide sequence encoding at least a portion of the essential gene, is inserted into the chromosome at the cleaved locus by homology-directed repair, restored expression of the essential gene avoids such cell death. This editing approach thus allows for highly efficient introduction of one or more transgenes into a cell, by insertion of a nucleic acid at an essential gene locus, where the nucleic acid comprises (i) the transgene(s), and (ii) at least a portion of the essential gene, such that expression of the essential gene occurs in cells comprising the inserted nucleic acid, and does not occur in cells lacking the inserted nucleic acid.

[0385] Accordingly, some embodiments of cells comprise an inserted nucleic acid at an essential gene locus, where the inserted nucleic acid comprises (i) a heterologous proteinencoding nucleotide sequence, and (ii) at least a portion of the essential gene. Some embodiments of methods of editing a cell comprise contacting a cell with a nucleic acid comprising (i) a first homology arm, (ii) a second homology arm, each homology arm having homology to an essential gene locus, (iii) a heterologous protein-encoding nucleotide sequence, and (iv) at least a portion of the essential gene. Some embodiments of nucleic acids comprise (i) a first homology arm, (ii) a second homology arm, each homology arm having homology to an essential gene locus, (iii) a heterologous protein-encoding nucleotide sequence, and (iv) at least a portion of the essential gene.

[0386] An inserted nucleic acid in a cell genome, or nucleic acid for insertion into a cell genome, may comprise any suitable portion of an essential gene. In some embodiments, the nucleic acid comprises a portion of an exon of the essential gene. In some embodiments, the nucleic acid comprises an exon of the essential gene. In some embodiments, the nucleic acid comprises two or more exons of the essential gene. In some embodiments, the nucleic acid comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 exons of the essential gene. A nucleic acid comprising multiple exons of the essential gene may comprise the exons in series without intervening nucleotides (e.g., without introns). The nucleic acid may comprise the last exon of the essential gene, with a stop codon downstream of the last codon encoding an amino acid of the protein encoded by the essential gene. The nucleic acid may comprise the first coding exon of the essential gene. The first coding exon of the essential gene may be the first exon present on mRNA encoded by the essential gene, or a different exon, depending on the location of the start codon of the open reading frame encoding the protein encoded by the essential gene.

[0387] In some embodiments, the nucleic acid comprises a mutated exon, or mutated portion of an exon, of the essential gene. The mutated exon or exon portion may be codon-optimized for expression in a cell. The mutated exon or exon portion may comprise fewer codons than the naturally occurring form of the exon (e.g., encoding a shorter amino acid sequence). The mutated exon or exon portion may lack a stop codon that is present in the natural exon. The mutated exon or exon portion may lack a start codon that is present in the natural exon. Whereas a start codon is necessary in the wild-type gene to initiate translation, the encoded methionine may be dispensable to the essential gene product. In such instances, an inserted nucleic acid may comprise the heterologous protein-encoding nucleotide sequence in-frame with a mutant form of the first coding exon of the essential gene, where the start codon and one or more subsequent codons present in the naturally occurring first coding exon are not present in the mutant form of the first coding exon. Similarly, one or more C-terminal amino acids may be dispensable to the function of the essential gene product. In such instances, an inserted nucleic acid may comprise the heterologous protein-encoding nucleotide sequence in-frame with a mutant form of the last exon of the essential gene, and optionally mutant forms of one or more upstream exons of the essential gene, where one or more codons encoding C-terminal amino acids are absent from the mutant forms of the exons. In some embodiments, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 C-terminal amino acids of the essential gene product are not encoded by a cell genome comprising the inserted nucleic acid. In some embodiments, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 N- terminal amino acids of the essential gene product are not encoded by a cell genome comprising the inserted nucleic acid.

[0388] An inserted nucleic acid in a cell genome, or nucleic acid for insertion into a cell genome, may comprise the heterologous protein-encoding nucleotide sequence upstream from the first coding exon of the essential gene. An inserted nucleic acid in a cell genome, or nucleic acid for insertion into a cell genome, may comprise the heterologous proteinencoding nucleotide sequence downstream from the last coding exon of the essential gene. In some embodiments, the heterologous protein-encoding nucleotide sequence, when inserted into a cell genome, is in-frame with the essential gene, such that a single transcribed RNA encodes the essential gene product and heterologous protein(s). In some embodiments, a nucleotide sequence encoding a 2A motif is present in-frame between the nucleotide sequence encoding the essential gene product and the heterologous protein-encoding nucleotide sequence. In some embodiments, the 2A motif is 3’ to the nucleotide sequence encoding the essential gene product, and 5’ to the heterologous protein-encoding nucleotide sequence. In some embodiments, the 2A motif is 3’ to the heterologous protein-encoding nucleotide sequence, and 5’ to the nucleotide sequence encoding the essential gene product. In some embodiments, the 2A motif is a P2A motif. In some embodiments, the 2A motif is a T2A motif. In some embodiments, the 2A motif is an E2A motif. In some embodiments, the 2A motif is an F2A motif.

[0389] In some embodiments, multiple heterologous coding sequences are present in an inserted nucleic acid, or nucleic acid for insertion into a cell genome. In some embodiments, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 heterologous coding sequences are present in the nucleic acid. In some embodiments, two or more heterologous coding sequences are separated by nucleotide sequences encoding 2A motifs. In some embodiments, separate pairs of heterologous coding sequences are separated by different nucleotide sequences encoding 2A motifs. In some embodiments, each heterologous coding sequence is separated by a different nucleotide sequence encoding a 2A motif. Separation of multiple heterologous coding sequences by different nucleotide sequences encoding 2A motifs reduces the likelihood of recombination that could occur between identical nucleotide sequences encoding 2A motifs, leading to excision of a heterologous coding sequence from the cell genome. The 2A motifs separating different proteins encoded by different heterologous coding sequences may be the same or different 2A motifs.

[0390] In some embodiments, the nucleic acid comprises a nucleotide sequence encoding FOXP3. In some embodiments, the nucleic acid comprises a nucleotide sequence encoding a first CISC component as described in the “Chemically Induced Signaling Complex” section. In some embodiments, the nucleic acid comprises a nucleotide sequence encoding a second CISC component as described in the “Chemically Induced Signaling Complex” section. In some embodiments the nucleic acid comprises a nucleotide sequence encoding a first CISC component, and a nucleotide sequence encoding a second CISC component. In some embodiments, the nucleic acid comprises a nucleotide sequence encoding a third CISC component (z.e., soluble FRB domain) as described in the “Chemically Induced Signaling Complex” section. In some embodiments, the nucleic acid comprises a nucleotide sequence encoding a chimeric antigen receptor (CAR). In some embodiments, the nucleic acid comprises a nucleotide sequence encoding a TCRa chain. In some embodiments, the nucleic acid comprises a nucleotide sequence encoding a TCRP chain. In some embodiments, the nucleic acid comprises a nucleotide sequence encoding a TCRa chain, and a nucleotide sequence encoding a TCRP chain.

[0391] An inserted nucleic acid in a cell genome, or nucleic acid for insertion into a cell genome, may comprise an exogenous stop codon, a nucleotide sequence encoding a 5’ untranslated region (UTR), a nucleotide sequence encoding a 3’ untranslated region (UTR), and / or a nucleotide sequence encoding a polyAtail. In some embodiments, the nucleic acid comprises a nucleotide sequence encoding a 5’ UTR. In some embodiments, the nucleic acid comprises a nucleotide sequence encoding a 3’ UTR. In some embodiments, the nucleic acid comprises a nucleotide sequence encoding a 3’ UTR and a polyAtail. In some embodiments, a transcribed polyAtail comprises at least 100 consecutive adenosine nucleotides.

[0392] A nucleic acid may be inserted at any suitable essential gene locus. In some embodiments, the essential gene locus is a GAPDH locus. In some embodiments, the essential gene locus is KIFll locus. In some embodiments, the essential gene locus is a TBP locus. In some embodiments, the gene locus is essential for Treg cell survival. In some embodiments, the gene locus is essential for maintenance of a Treg cell phenotype.

[0393] Any suitable nuclease may be used for targeted cleavage. In some embodiments, a cell is edited by an RNA-guided nuclease. In some embodiments, the RNA-guided nuclease is a Cas endonuclease. In some embodiments, the RNA-guided nuclease is a Cas9 endonuclease. In some embodiments, the RNA-guided nuclease is a Cpfl endonuclease. In some embodiments, the nuclease is a meganuclease. In some embodiments, the nuclease is a TALEN. In some embodiments, the nuclease is a MegaTAL. In some embodiments, the nuclease is a zinc finger nuclease (ZFN).

[0394] The homology arms may be the same length, have similar lengths (within 100 bp of each other), or different lengths. In some embodiments, one or both homology arms have a length of 100-2,000 bp, 200-2,000 bp, 400-1,500 bp, or 500-1,000 bp. In some embodiments, one or both homology arms are 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, both homology arms are 100-2,000 nucleotides in length. In some embodiments, both homology arms are 300-1,000 nucleotides in length. In some embodiments, both homology arms are 300-700 nucleotides in length. In some embodiments, both homology arms are 300-500 nucleotides in length. In some embodiments, both homology arms are 500-700 nucleotides in length. In some embodiments, both homology arms are 700-1,000 nucleotides in length.

[0395] In some embodiments, the nucleic acid is a single-stranded DNA (ssDNA). In some embodiments, the nucleic acid is a closed-ended DNA (ceDNA). In some embodiments, the nucleic acid is present in a vector. Any suitable vector may comprise the nucleic acid. In some embodiments, the vector is a viral vector. In some embodiments, the vector is an integrase-deficient lentiviral vector (IDLV). In some embodiments, the vector is an adeno- associated viral (AAV) vector. In some embodiments, the AAV vector is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11 vector. In some embodiments, the AAV vector is an AAV5 vector. In some embodiments, the AAV vector is an AAV6 vector.

[0396] In some embodiments, the nucleic acid is present in a lipid nanoparticle. In some embodiments, the lipid nanoparticle comprises the nuclease or a nucleic acid encoding the nuclease. In some embodiments, the lipid nanoparticle comprises a guide RNA or a nucleic acid encoding the guide RNA. In some embodiments, the nucleic acid encoding the nuclease is an mRNA.

[0397] Promoters and uses thereof

[0398] Embodiments of the compositions, cells, nucleic acids, vectors, and methods that contemplate use of a promoter (e.g., heterologous promoter) may use any suitable promoter. In some embodiments, the promoter on the introduced nucleic acid is active, promoting transcription of RNA encoding ST2, IL1RAP, IL-18R1, or IL18RAP, even under pro- inflammatory conditions. In some embodiments, a promoter is operably linked to an endogenous nucleotide sequence encoding ST2. In some embodiments, a promoter is operably linked to an endogenous nucleotide sequence encoding IL1RAP. In some embodiments, a promoter is operably linked to an endogenous nucleotide sequence encoding ST2 and / or IL1RAP. In some embodiments, the promoter is a heterologous promoter. A heterologous promoter, when operably linked to a coding sequence, refers to a promoter that is not operably linked to that coding sequence in nature. A heterologous promoter may also refer to a promoter that controls episomal expression of an introduced nucleic acid sequence. A heterologous promoter may be derived from a different location in the cell genome. A heterologous promoter may be a synthetic promoter that is not found in nature in any organism. A heterologous promoter may be obtained from another species (e.g., virus, bacterium). In some embodiments, a heterologous promoter is operably linked to an endogenous nucleotide sequence encoding ST2. In some embodiments, a heterologous promoter is operably linked to an endogenous nucleotide sequence encoding IL1RAP. In some embodiments, a heterologous promoter is operably linked to an endogenous nucleotide sequence encoding ST2 and / or IL1RAP. In some embodiments, the promoter is a constitutive promoter, which promotes transcription of an operably linked sequence (e.g., a repair mediator polypeptide) at a consistent rate. In some embodiments, a constitutively active promoter promotes transcription of an operably linked sequence (e.g., a repair mediator polypeptide) at a supraphy si ologi cal rate. Constitutive promoters may be strong promoters, which promote transcription at a higher rate than an endogenous promoter, or weak promoters, which promote transcription at a lower rate than a strong or endogenous promoter. In some embodiments, the constitutive promoter is a strong promoter. In some embodiments, the constitutive promoter is a weak promoter. In some embodiments, the constitutive promoter is an EF-la, PGK, or MND promoter. In some embodiments, another suitable promoter, such as an SV40, CMV, UBC, SFFV, EFS, or CAGG promoter, is used. In some embodiments, the constitutive promoter is an MND promoter. In some embodiments, the promoter is an inducible promoter. Inducible promoters promote transcription of an operably linked sequence in response to the presence of an activating signal, or the absence of a repressor signal. In some embodiments, the inducible promoter is inducible by a drug or steroid. In some embodiments, a promoter is a ubiquitous promoter. In some embodiments, the ubiquitous promoter is Reverse Orientation Splice Acceptor 26 (ROSA26) promoter. In some embodiments, the promoter is a unidirectional promoter. In some embodiments, the unidirectional promoter drives expression in either a forward (e.g., directing transcription downstream from the promoter) or reverse direction (e.g., directing transcription upstream from the promoter), but not both. In some embodiments, the unidirectional promoter drives transcription downstream of the promoter. In some embodiments, the unidirectional promoter drives transcription upstream of the promoter. In some embodiments, the unidirectional promoter is a unidirectional constitutive promoter. In some embodiments, the unidirectional constitutive promoter is operably linked to a nucleotide sequence encoding ST2. In some embodiments, the unidirectional constitutive promoter is operably linked to a nucleotide sequence encoding IL1RAP. In some embodiments, a unidirectional constitutive promoter is operably linked to a nucleotide sequence encoding ST2 and IL1RAP. In some embodiments, the unidirectional constitutive promoter is operably linked to a nucleotide sequence encoding IL-18R1. In some embodiments, the unidirectional constitutive promoter is operably linked to a nucleotide sequence encoding IL18RAP. In some embodiments, a unidirectional constitutive promoter is operably linked to a nucleotide sequence encoding IL-18R1 and IL18RAP.

[0399] Cell types

[0400] Embodiments of methods for producing genetically modified cells (e.g., by in vitro or ex vivo gene editing, and / or administration of compositions, vectors, or nucleic acids to a subject for in vivo editing) may use any suitable cell type as a material for, e.g., introduction of nucleic acids, vectors, and / or compositions. It is to be understood that methods that comprise manipulation of CD4+ cells can be applied to other types of cells (e.g., CD8+ cells). In some embodiments, the methods comprise editing an immune cell. Non-limiting examples of immune cells include B cells, T cells, ILCs, and NK cells. In some embodiments, the methods comprise editing CD3+ cells, thereby producing edited CD3+ cells, including CD4+ and CD8+ Treg cells. In some embodiments, the methods comprise editing CD4+ T cells, thereby producing CD4+ Treg cells. In some embodiments, the methods comprise editing CD8+ T cells, thereby producing CD8+ Treg cells. In some embodiments, the methods comprise editing NK 1.1+ T cells, thereby producing NK 1.1+ Treg cells.

[0401] In some embodiments, the methods comprise editing a stem cell. In some embodiments, the methods comprise editing a pluripotent stem cell. In some embodiments, the methods comprise editing CD34+ hematopoietic stem cells (HSCs). In some embodiments, the methods comprise editing induced pluripotent stem cells (iPSCs). Edited stem cells may be matured in vitro to produce Treg cells, or administered to a subject to allow in vivo development into Treg cells. Edited stem cells may be matured into CD3+ Treg cells, CD4+ Treg cells, CD8+ Treg cells, NK1.1+ Treg cells, or a combination thereof.

[0402] In some embodiments, a method comprises editing a T cell. A T cell or T lymphocyte is an immune system cell that matures in the thymus and produces a T cell receptor (TCR), e.g., an antigen-specific heterodimeric cell surface receptor typically comprised of an a-P heterodimer or a y-5 heterodimer. T cells of a given clonality typically express only a single TCR clonotype that recognizes a specific antigenic epitope presented by a syngeneic antigen- presenting cell in the context of a major histocompatibility complex-encoded determinant. T cells can be naive ("TN"; not exposed to antigen; increased expression of CD62L, CCR7, CD28, CD3, CD 127, and CD45RA, and decreased or no expression of CD45RO as compared to TCM (described herein)), memory T cells (TM) (antigen experienced and long-lived), including stem cell memory T cells, and effector cells (antigen-experienced, cytotoxic). TM can be further divided into subsets of central memory T cells (TCM, expresses CD62L, CCR7, CD28, CD95, CD45RO, and CD127) and effector memory T cells (TEM, express CD45RO, decreased expression of CD62L, CCR7, CD28, and CD45RA). Effector T cells (TE) refers to antigen-experienced CD8+ cytotoxic T lymphocytes that express CD45RA, have decreased expression of CD62L, CCR7, and CD28 as compared to TCM, and are positive for granzyme and perforin. Helper T cells (TH) are CD4+ cells that influence the activity of other immune cells by releasing cytokines. CD4+ T cells can activate and suppress an adaptive immune response, and which of those two functions is induced will depend on the presence of other cells and signals. T cells can be collected using known techniques, and the various subpopulations or combinations thereof can be enriched or depleted by known techniques, for example, using antibodies that specifically recognize one or more T cell surface phenotypic markers, by affinity binding to antibodies, flow cytometry, fluorescence activated cell sorting (FACS), or immunomagnetic bead selection.

[0403] Other examples of T cells include regulatory T cells (Tregs, also known as suppressor T cells), such as CD4+ CD25+ (FoxP3+) regulatory T cells and Tregl7 cells, as well as Tri, Th3, CD8+CD28-, or Qa-1 restricted T cells. In some embodiments, the cell is a CD3+, CD4+, and / or CD8+ T cell. In some embodiments, the cell is a CD3+ T cell. In some embodiments, the cell is a CD4 CD8 T cell. In some embodiments, the Treg cell is a FoxP3+ Treg cell. In some embodiments, the Treg cell expresses CTLA-4, LAG-3, CD25, CD39, CD27, CD70, CD357 (GITR), neuropilin-1, galectin-1, and / or IL-2Ra on its surface. In some embodiments, the Treg is an engineered Treg; that is, a Treg that has been manipulated so that its genetic, epigenetic, and / or phenotypic identity is altered relative to an appropriate reference cell such as otherwise identical cell that has not been so manipulated. In one example, an engineered Treg is one that expresses a repair phenotype through modification, expression / over-expression, or manipulation of the IL33-ST2 axis. In some embodiments, the engineered Treg is a sorted engineered Treg. A sorted engineered Treg, as used herein, refers to a sorted Treg that has been engineered to express a chimeric antigen receptor (CAR) or a transgenic T cell receptor (TCR). In some embodiments, the Treg is not a sorted Treg, wherein a sorted Treg is isolated from a human sample based on the following cell surface markers: CD4+CD25hiCD12710or CD4+CD25hiCD127loCD45RA+. In some embodiments, a sorted Treg is engineered to upregulate expression of ST2, IL1RAP, IL- 18R1, and / or IL18RAP. In some embodiments, a sorted T cell is engineered to upregulate expression of ST2, IL1RAP, IL-18R1, and / or IL18RAP. In some embodiments, a sorted T cell is engineered to stabilize FoxP3 expression and to upregulate expression of ST2, IL1RAP, IL-18R1, and / or IL18RAP. In some embodiments, a sorted T cell is engineered to express a CAR and to upregulate expression of ST2, IL1RAP, IL-18R1, and / or IL18RAP. In some embodiments, the sorted Treg does not have a genetically modified F0XP3 locus. In some embodiments, the sorted Treg has not been genetically modified to alter FOXP3 expression. In some embodiments, the sorted Treg does not express an exogenous F0XP3 coding sequence. In some embodiments, the sorted Treg has not been genetically modified to express a chemically induced signaling complex (CISC). In some embodiments, the sorted Treg does not express proteins that dimerize in the presence of rapamycin or a rapalog resulting in IL-2 signaling.

[0404] In some embodiments, the Treg is an induced Treg (iTreg) or a peripheral Treg (pTreg); as used herein, an iTreg and a pTreg refer to a stem cell that has been stimulated (e.g., in the presence of TGF-P and IL-2), in vitro and in vivo, respectively. In some embodiments, the engineered repair Treg comprises a Treg engineered to express FOXP3 and CISC (IL-2).

[0405] In some embodiments, the cell is a human cell. In some embodiments, a cell as is isolated from a biological sample. A biological sample may be a sample from a subject (e.g., a human subject) or a composition produced in a lab (e.g., a culture of cells). A biological sample obtained from a subject make be a liquid sample (e.g., blood or a fraction thereof, a bronchial lavage, cerebrospinal fluid, or urine), or a solid sample (e.g., a piece of tissue). In some embodiments, the cell is obtained from peripheral blood. In some embodiments, the cell is obtained from umbilical cord blood. In some embodiments, the cell is obtained by sorting cells of peripheral blood to obtain a desired cell population (e.g., CD3+ cells), and one or more cells of the sorted population are modified. In some embodiments, the cell is in a subject. In some embodiments, the cell is in vivo.

[0406] Embodiments of genetically modified cells may be any suitable cell type. In some embodiments, the cell is a T cell, a precursor T cell, or a hematopoietic stem cell. In some embodiments, the cell is an NK-T cell (e.g., a FoxP3- NK-T cell or a FoxP3+ NK-T cell). In some embodiments, the cell is a regulatory B (Breg) cell (e.g., a FoxP3- B cell or a FoxP3+ B cell). In some embodiments, the cell is a CD4+ T cell (e.g., a FoxP3-CD4+ T cell or a FoxP3+CD4+ T cell) or a CD8+ T cell (e.g., a FoxP3-CD8+ T cell or a FoxP3+CD8+ T cell). In some embodiments, the cell is a CD25- T cell. In some embodiments, the cell is a regulatory T (Treg) cell. Non-limiting examples of Treg cells are Tri, Th3, CD8+CD28-, and Qa-1 restricted T cells. In some embodiments, the Treg cell is a FoxP3+ Treg cell. In some embodiments, the Treg cell expresses CTLA-4, LAG-3, CD25, CD39, CD27, CD70, CD357 (GITR), neuropilin-1, galectin-1, and / or IL-2Ra on its surface. In some embodiments, the Treg cell is ST2+. In some embodiments, the Treg cell is IL-18R+.

[0407] In some embodiments, a cell is genetically modified. In some embodiments, the cells have one or more phenotypes associated with ST2 signaling.

[0408] In some embodiments, the cell is a stem cell. In some embodiments, the cell is a T cell. In some embodiments, the T cell is a FoxP3+ Treg. In some embodiments, the Treg is a genetically modified Treg. In some embodiments, the cell is a CD4+ cell. In some embodiments, the cell is a CD8+ cell. In some embodiments, the cell expresses one or more markers selected from BATF, CTLA-4, HLA-DR, ICOS, Ki-67, TIGIT, TNFRSF18, CD25, CD39, CD49d, CD69, CD71, CD103, CD197. In some embodiments, the cell expresses BATF. In some embodiments, the cell expresses CTLA-4. In some embodiments, the cell expresses HLA-DR. In some embodiments, the cell expresses ICOS. In some embodiments, the cell expresses Ki-67. In some embodiments, the cell expresses TIGIT. In some embodiments, the cell expresses TNFRSF18. In some embodiments, the cell expresses CD25. In some embodiments, the cell expresses CD39. In some embodiments, the cell expresses CD49d. In some embodiments, the cell expresses CD69. In some embodiments, the cell expresses CD71. In some embodiments, the cell expresses CD 103. In some embodiments, the cell expresses CD 197.

[0409] In some embodiments, the cell expresses one or more cytokines, selected from the group consisting of: IL-5, IL-10, IL-13, and TGF-p. In some embodiments, the cell expresses IL-5. In some embodiments, the cell expresses IL- 10. In some embodiments, the cell expresses IL-13. In some embodiments, the cell expresses TGF-p.

[0410] In some embodiments, the cell expresses one or more chemokine receptors, selected from the group consisting of: CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR10, and CX3CR1. In some embodiments, the cell expresses CCR2. In some embodiments, the cell expresses CCR3. In some embodiments, the cell expresses CCR4. In some embodiments, the cell expresses CCR5. In some embodiments, the cell expresses CCR6. In some embodiments, the cell expresses CCR7. In some embodiments, the cell expresses CCR8. In some embodiments, the cell expresses CCR10. In some embodiments, the cell expresses CX3CR1.

[0411] In some embodiments, the cell produces soluble ST2 and / or osteopontin. In some embodiments, the cell produces soluble ST2. In some embodiments, the cell produces osteopontin.

[0412] In some embodiments, the cell is a KLRG1+ cell. In some embodiments, the cell is a NFIL3+ cell. In some embodiments, the cell is a GATA3+ cell. In some embodiments, the cell is a RLN3+ cell. In some embodiments, the cell is a RAB4A+ cell. In some embodiments, the cell is a LYN+ cell. In some embodiments, the cell is a PTPN13+ cell. In some embodiments, the cell is a TCB1D4- cell. In some embodiments, the cell is a PPARy+ cell.

[0413] Chemically induced signaling complexes (CISC)

[0414] Some embodiments of methods of modifying cells comprise introducing into the cell one or more nucleic acids that collectively comprise (1) a first nucleic acid sequence encoding a first chemically inducible signaling complex (CISC) component, and (2) a second nucleic acid sequence encoding a second chemically inducible signaling complex (CISC) component, each CISC component comprising (a) an extracellular binding domain that is capable of binding to a CISC inducer molecule, (b) a transmembrane domain, and (c) an intracellular signaling domain, such that binding of the first and second CISC components to the CISC inducer molecule results in dimerization of the CISC components and a signal transduction event in the cell. Similarly, some embodiments of cells comprise (1) a first nucleic acid sequence encoding a first chemically inducible signaling complex (CISC) component, and (2) a second nucleic acid sequence encoding a second chemically inducible signaling complex (CISC) component, each CISC component comprising (a) an extracellular binding domain that is capable of binding to a CISC inducer molecule, (b) a transmembrane domain, and (c) an intracellular signaling domain, such that binding of the first and second CISC components to the CISC inducer molecule results in dimerization of the CISC components and a signal transduction event in the cell. Additionally, some nucleic acids and vectors comprise (1) a first nucleic acid sequence encoding a first chemically inducible signaling complex (CISC) component, and / or (2) a second nucleic acid sequence encoding a second chemically inducible signaling complex (CISC) component, each CISC component comprising (a) an extracellular binding domain that is capable of binding to a CISC inducer molecule, (b) a transmembrane domain, and (c) an intracellular signaling domain, such that binding of the first and second CISC components to the CISC inducer molecule results in dimerization of the CISC components and a signal transduction event in a cell.

[0415] Expression of CISC components in a cell allows selective induction of signaling in a cell by manipulation of the presence and / or concentration of the CISC inducer molecule. Such controllable induction of signaling allows, for example, selective expansion of cells expressing both CISC components, where the signal transduction event results in proliferation of the cell. In some embodiments, where two nucleic acids, each encoding a different CISC component, are introduced into the cell, such selective expansion allows for selection of cells that contain both nucleic acids, as contacting a cell comprising only one CISC component would not induce dimerization with the absent second CISC component.

[0416] Non-limiting examples of intracellular signaling domains include IL-2RP and IL-2Ry cytoplasmic domains and functional derivatives thereof. In some embodiments, an intracellular signaling domain of one CISC component comprises an IL-2RP cytoplasmic domain or a functional derivative thereof, and an intracellular signaling domain of the other CISC component comprises an IL-2Ry cytoplasmic signaling domain or a functional derivative thereof. In some embodiments, dimerization of the CISC components induces phosphorylation of JAK1, JAK3, and / or STAT5 in the cell. In some embodiments, dimerization of the CISC components induces proliferation of the cell. In some embodiments, the IL-2RP cytoplasmic domain comprises the amino acid sequence of NCRNTGPWLKKVLKCNTPDPSKFF SQLS SEHGGD VQKWLS SPFPS S SF SPGGLAPEIS PLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEACQVY FTYDPYSEEDPDEGVAGAPTGSSPQPLQPLSGEDDAYCTFPSRDDLLLFSPSLLGGPSP PSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAG EEVPDAGPREGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLV (SEQ ID NO: 11). In some embodiments, the IL-2RP cytoplasmic domain is truncated, relative to a wild-type IL-2RP cytoplasmic domain. An example of an truncated IL-2RP cytoplasmic domain that retains signal transduction activity is described, for example, in Cook etal., Mol Ther. 2023. S1525-0016(23)00255-l. In some embodiments, the truncated IL-2Rb domain comprises the amino acid sequence of PAALGKDTIPWLGHLLVGLSGAFGFIILVYLLINCRNTGPWLKKVLKCNTPDPSKFFS QLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQQDKVPEPASL SLNTDAYLSLQELQ (SEQ ID NO: 28). In some embodiments, the IL-2Ry cytoplasmic domain comprises the amino acid sequence of ERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEIPPKGGA LGEGPGASPCNQHSPYWAPPCYTLKPET (SEQ ID NO: 12).

[0417] Non-limiting examples of transmembrane domains include IL-2RP, IL-2RY, erythropoietin (Epo), and thrombopoietin (Tpo) transmembrane domains. In some embodiments, the transmembrane domain of a CISC component is derived from the same protein as the intracellular signaling domain of the CISC component (e.g., a CISC component comprising an IL-2RP intracellular domain comprises an IL-2RP transmembrane domain). In some embodiments, one CISC component comprises an IL-2RP transmembrane domain, and the other CISC component comprises an IL-2RY transmembrane domain. In some embodiments, an IL-2RP transmembrane domain comprises the amino acid sequence of IPWLGHLLVGLSGAFGFIILVYLLI (SEQ ID NO: 13). In some embodiments, an IL-2RY transmembrane domain comprises the amino acid sequence of VVISVGSMGLIISLLCVYFWL (SEQ ID NO: 14).

[0418] Non-limiting examples of extracellular binding domains capable of binding a CISC inducer molecule include an FK506-binding protein (FKBP) domain and an FKBP- rapamycin-binding (FRB) domain. FKBP and FRB domains are capable of binding to rapamycin or rapalogs, such as those described below. In some embodiments, an extracellular binding domain of one CISC component comprises an FKBP domain, and an extracellular binding domain of the other CISC component comprises an FRB domain. In some embodiments, the CISC components form a heterodimer in the presence of the CISC inducer molecule. In some embodiments, an FKBP domain comprises an amino acid sequence of GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVI RGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLGE (SEQ ID NO: 15). In some embodiments, an FKBP domain comprises an amino acid sequence of GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVI RGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE (SEQ ID NO: 16) (Glyl07). In some embodiments, an FKBP domain comprises an F36V substitution, at a position corresponding to F36 of SEQ ID NO: 15 or SEQ ID NO: 16. Such a substitution allows the FKBP domain to interact with derivatives of rapamycin, such as API 903. Clackson et al., Proc Natl Acad Sci USA. 1998. 95(18): 10437-10442.

[0419] In some embodiments, an FRB domain comprises an amino acid sequence of EMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLM EAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISK (SEQ ID NO: 17). In some embodiments, an FRB domain comprises a T74L substitution, at a position corresponding to T74 of SEQ ID NO: 17. This T74L substitution corresponds to a T2098L substitution in the FRB domain as present on full-length mTOR. In some embodiments, an FRB domain comprises a K71P substitution, at a position corresponding to K71 of SEQ ID NO: 17. In some embodiments, an FRB domain comprises a K71T or K71 A substitution, at a position corresponding to K71 of SEQ ID NO: 17. In some embodiments, an FRB domain comprises a W77F substitution, at a position corresponding to W77 of SEQ ID NO: 17. In some embodiments, an FRB domain comprises a K71P substitution, a T74L substitution, and a W77F substitution. Such substitutions allow the FRB domain to interact with derivatives of rapamycin, such as AP21967. Stankunas etal., Mol Cell. 2003. 12(6): 1615-1624; Bayle et al., Chem Biol. 2006. 13(l):99-107.

[0420] In some embodiments, a soluble FRB domain comprises an amino acid sequence of EMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLM EAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISK (SEQ ID NO: 18). In some embodiments, the soluble FRB domain consists of the amino acid sequence of EMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLM EAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISK (SEQ ID NO: 18).

[0421] Each of the extracellular binding domains, transmembrane domains, and intracellular signaling domains of the CISC components may be connected to another domain of the same CISC component by a linker. In some embodiments, the linker comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids, such as glycines, or a number of amino acids, such as glycine, within a range defined by any two of the aforementioned numbers. In some embodiments, the glycine spacer comprises at least 3 glycines. In some embodiments, the glycine spacer comprises a sequence set forth as GGGS (SEQ ID NO: 8), GGGSGGG (SEQ ID NO: 9) or GGG.

[0422] An extracellular binding domain may be connected to a transmembrane domain by a hinge domain. A hinge refers to a domain that links the extracellular binding domain to the transmembrane domain, and may confer flexibility to the extracellular binding domain. In some embodiments, the hinge domain positions the extracellular domain close to the plasma membrane to minimize the potential for recognition by antibodies or binding fragments 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. In some embodiments, a CISC component comprises a portion of an IL-2Ry extracellular domain between the extracellular binding domain and transmembrane domain. In some embodiments, the portion of the IL-2Ry extracellular domain comprises the amino acid sequence of GSNTSKENPFLFALEA (SEQ ID NO: 19). In some embodiments, a CISC component comprises a portion of an IL-2R[3 extracellular domain between the extracellular binding domain and transmembrane domain. In some embodiments, the portion of the IL-2R[3 extracellular domain comprises the amino acid sequence of GKDT (SEQ ID NO: 20).

[0423] In some embodiments, the portion of the IL-2Ry extracellular domain comprises the amino acid sequence of QNLVIPWAPENLTLHKLSESQLELNWNNRFLNHCLEHLVQYRTDWDHSWTEQSVD YRHKFSLPSVDGQKRYTFRVRSRFNPLCGSAQHWSEWSHPIHWGSNTSKENPFLFAL EA (SEQ ID NO: 21). In some embodiments, a CISC component comprises a portion of an IL-2R[3 extracellular domain between the extracellular binding domain and transmembrane domain. In some embodiments, the portion of the IL-2R[3 extracellular domain comprises the amino acid sequence of KPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAP LLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKD T (SEQ ID NO: 22).

[0424] In some embodiments, the CISC inducer molecule is rapamycin or a rapalog. In some embodiments, the CISC inducer molecule is rapamycin. Non-limiting examples of rapalogs include everolimus, CCI-779, C20-methallylrapamycin, C16-(S)-3-methylindolerapamycin, C16-iRap, C16-(S)-7-methylindolerapamycin, AP21967, Cl 6- (S)Butylsulfonamidorapamycin, AP23050, sodium mycophenolic acid, benidipine hydrochloride, AP1903, and AP23573, and metabolites or derivatives thereof.

[0425] In some embodiments, a method comprises introducing into a cell a nucleic acid encoding a third CISC component that is capable of binding to the CISC inducer molecule. Such CISC components are useful, for example, for binding to the intracellular CISC inducer molecules (e.g., intracellular rapamycin), thereby preventing the bound CISC inducer molecule 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 comprise a transmembrane domain. In some embodiments, the third CISC component comprises an intracellular FRB domain. In some embodiments, a third CISC component is a soluble protein comprising an FRB domain and lacking a transmembrane domain.

[0426] Nucleic acids encoding a first, second, and / or third CISC component may be comprised in one or more vectors. In some embodiments, a nucleic acid encoding a first CISC component is present on a separate vector from a nucleic acid encoding the second CISC component. In some embodiments, a nucleic acid encoding the third CISC component is present on the same vector as a nucleic acid encoding the first or second CISC component. In other embodiments, a nucleic acid encoding the third CISC component is present on a distinct vector from nucleic acids encoding the first and / or second CISC components. In some embodiments, one or more vectors are viral vectors. In some embodiments, one or more vectors are lentiviral vectors. In some embodiments, one or more vectors are adeno- associated viral (AAV) vectors. In some embodiments, one or more AAV vectors is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11 vector. In some embodiments, one or more AAV vectors are AAV5 vectors. In some embodiments, one or more AAV vectors are AAV6 vectors.

[0427] In some embodiments, a CISC component comprises an amino acid sequence with 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 as SEQ ID NO: 3, 4, or 26. In some embodiments, one or more CISC components further comprise a signal peptide. The signal peptide may be any suitable signal peptide directs the translated CISC component to the cell membrane. Non-limiting examples of signal peptides include an LCN2 signal peptide (MPLGLLWLGLALLGALHAQA (SEQ ID NO: 23)), a CD8a signal peptide (MALPVTALLLPLALLLHAARPILWH (SEQ ID NO: 24)), and GM-CSFRa signal peptide (MLLLVTSLLLCELPHPAFLLI (SEQ ID NO: 25)).

[0428] In some embodiments, one CISC component comprises an amino acid sequence with 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 as SEQ ID NO: 3 or 27, and the other CISC component comprises an amino acid sequence with 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 as SEQ ID NO: 4 or 26. In some embodiments, each CISC component further comprises a signal peptide, which may have the same or different amino acid sequences. The signal peptides may be any suitable signal peptide that directs the translated CISC component to the cell membrane. In some embodiments, one or more CISC components comprise an LCN2 signal peptide. In some embodiments, one or more CISC components comprise a CD8a signal peptide. In some embodiments, one or more CISC components comprise a GM-CSFRa signal peptide. In some embodiments, both CISC components comprise an LCN2 signal peptide. In some embodiments, both CISC components comprise a CD8a signal peptide. In some embodiments, both CISC components comprise a GM-CSFRa signal peptide.

[0429] In some embodiments, a third CISC component comprises an amino acid sequence with 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 as SEQ ID NO: 5. In some embodiments, a third CISC component consists of an amino acid sequence with 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 as SEQ ID NO: 5. In some embodiments, the third CISC component does not comprise a signal peptide. In some embodiments, the third CISC component does not comprise a transmembrane domain.

[0430] Stabilized FoxP3 expression

[0431] Some embodiments of methods of modifying cells comprise introducing a genetic modification in a cell that stabilizes expression of FoxP3. Similarly, some embodiments of cells comprise a genetic modification that stabilizes or increases FoxP3expression, relative to an unmodified cell. Additionally, some embodiments of nucleic acids and vectors stabilize FoxP3 expression in a cell.

[0432] In some embodiments, an endogenous FOXP3 locus is modified in a cell, resulting in stabilized expression. For example, in some embodiments, a heterologous promoter is inserted within or downstream from a Treg-specific demethylated region (TSDR) in the genome, and upstream from a first coding exon of an endogenous FOXP3 coding sequence. In some embodiments, a promoter is inserted downstream from the TSDR, and within or upstream from the first coding exon of FOXP3. Insertion of a heterologous promoter in this manner bypasses endogenous regulation of FOXP3 by the TSDR, which can become methylated in inflammatory conditions, inhibiting transcription of the endogenous FOXP3 coding sequence from the endogenous FOXP3 promoter located upstream from the TSDR. Thus, such stabilized FoxP3 expression by heterologous promoter insertion allows stable FoxP3 expression even in inflammatory conditions, preventing transdifferentiation into a T effector cell.

[0433] The heterologous promoter may be inserted at any position between the endogenous promoter and the first coding exon of the F0XP3 coding sequence. In some embodiments, the heterologous promoter is inserted 1-10,000, 10-1,000, 10-100, 10-5,000, 20-4,000, 30- 3,000, 40-2,000, 50-1,000, 60-750, 70-500, 80-400, 90-300, 100-200, 1-1,000, 1,000- 2,000, 2,000-3,000, 3,000-4,000, 4,000-5,000, 5,000-6,000, 6,000-7,000, 7,000-8,000, 8,000-9,000, or 9,000-10,000 nucleotides downstream from the TSDR of FOXP3. In some embodiments, the heterologous promoter is inserted 1-10,000, 10-1,000, 10-100, 10-5,000, 20-4,000, 30-3,000, 40-2,000, 50-1,000, 60-750, 70-500, 80-400, 90-300, 100-200, 1- 1,000, 1,000-2,000, 2,000-3,000, 3,000-4,000, 4,000-5,000, 5,000-6,000, 6,000-7,000, 7,000-8,000, 8,000-9,000, or 9,000-10,000 nucleotides upstream from the first coding exon of the FOXP3 coding sequence. In some embodiments, the heterologous promoter is inserted into the first coding exon, such that a synthetic first coding exon is created, where the synthetic first coding exon differs from the endogenous first coding exon but still comprises a start codon that is in-frame with the FOXP3 coding sequence of downstream FOXP3 exons. In some embodiments, the heterologous promoter is inserted into the TSDR, such that the TSDR is modified and does not inhibit transcription of the endogenous FOXP3 coding sequence in inflammatory conditions.

[0434] In some embodiments, the nucleic acid comprising a heterologous promoter is comprised on a vector. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is an adeno-associated virus (AAV) vector. In some embodiments, the AAV vector is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11 vector. In some embodiments, the AAV vector is an AAV5 vector. In some embodiments, the AAV vector is an AAV6 vector.

[0435] In some embodiments, a nucleic acid comprising a promoter operably linked to a nucleic acid sequence encoding FoxP3 or a functional derivative thereof is introduced into the cell. Expression of a heterologous promoter and sequence encoding FoxP3 is useful, for example, for expressing functional FoxP3 in cells containing genomic mutations in the FOXP3 coding sequence (e.g., cells from subjects having IPEX syndrome). Additionally, additional coding sequences (e.g., encoding a ST2, IL1RAP, IL-18R1, or IL18RAP protein) may be included in a nucleic acid, such that the heterologous promoter controls transcription of RNA encoding FoxP3 sequence and one or more other proteins (e.g., ST2, IL1RAP, IL- 18R1, and / or IL18RAP). In some embodiments, the sequence encoding FoxP3 is a cDNA sequence that does not comprise an intron.

[0436] The introduced nucleic acid may be integrated into the genome at a targeted locus (e.g., by homologous recombination), integrated in a non-targeted manner (e.g., by delivery on a lentiviral vector), or not integrated. In some embodiments, the nucleic acid comprises a 5' homology arm that is upstream from the promoter, and a 3' homology arm that is downstream from the nucleic acid sequence encoding FoxP3, and both homology arms have homology to a targeted locus in a genome. Such homology arms promote insertion of the nucleic acid into the genome at the targeted locus by homologous recombination. The homology arms may be the same length, have similar lengths (within 100 bp of each other), or different lengths. In some embodiments, one or both homology arms have a length of 200- 2,000 bp, 400-1,500 bp, 500-1,000 bp. In some embodiments, one or both homology arms are 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.

[0437] In some embodiments, the nucleic acid is inserted at a FOXP3 locus in the genome. In some embodiments, the nucleic acid is inserted at a non-FOXP3 locus. In some embodiments, the targeted locus is a safe harbor locus. In some embodiments, the safe harbor locus is an AAVS1 locus, a HIPP 11 locus, or a ROSA26 locus. In some embodiments, the nucleic acid is inserted at a TCRa (TRAC) locus. In some embodiments, the nucleic acid is inserted at a TCRP (TRBC) locus.

[0438] In some embodiments, a nuclease capable of cleaving the genome at a targeted locus, or a nucleic acid encoding the nuclease (e.g., an mRNA) is introduced into the cell. Following delivery of the nuclease or transcription of the nuclease inside the cell, the nuclease introduces a double-stranded break at the targeted locus, thereby promoting integration of a donor template (e.g., nucleic acid comprising a promoter and sequence encoding FoxP3, or nucleic acid comprising a heterologous promoter for promoting transcription of an endogenous FOXP3 coding sequence) into the genome at the targeted locus by homology-directed repair. The nuclease may be any suitable nuclease, including a meganuclease, zinc finger nuclease, TALEN, or RNA-guided nuclease. In embodiments where an RNA-guided nuclease (or nucleic acid encoding an RNA-guided nuclease) is delivered, a guide RNA (or nucleic acid encoding a guide RNA) comprising a spacer sequence complementary to a genomic sequence at the targeted locus is introduced into the cell. A gRNA or nucleic acid encoding a gRNA may be introduced into the cell with the nuclease or nucleic acid encoding the nuclease, or introduced separately (e.g., in a separate vector or delivery vehicle). The RNA-guided nuclease may be any suitable RNA-guided nuclease, such as those described in the section entitled “Nucleases.”

[0439] In some embodiments, a nucleic acid comprising a heterologous promoter operably linked to a sequence encoding FoxP3 or a functional derivative thereof is present on a vector. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a lentiviral vector. In some embodiments, the vector is an adeno-associated virus (AAV) vector. In some embodiments, the AAV vector is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11 vector. In some embodiments, the AAV vector is an AAV5 vector. In some embodiments, the AAV vector is an AAV6 vector. In some embodiments, the vector is a plasmid. In some embodiments, the vector is bacterial artificial chromosome. In some embodiments, the vector is human artificial chromosome. In some embodiments, the vector integrates into a chromosome of the genome, and RNA encoding FoxP3 is transcribed from the genome of the cell. In other embodiments, the vector does not integrate into a chromosome, and the sequence encoding FoxP3 is expressed episomally.

[0440] The heterologous promoter inserted into the F0XP3 locus or operably linked to the F0XP3 coding sequence may be any suitable promoter. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is an MND, PGK, or EF-la promoter. In some embodiments, the promoter is an MND promoter. In some embodiments, the promoter is an inducible promoter. In some embodiments, the promoter is inducible by a drug or steroid.

[0441] Rapamycin-Induced Signal Enhancement (RISE) Complex

[0442] In some embodiments, an engineered repair Treg may comprise a rapamycin-induced signal enhancement (RISE) complex, either in addition to, or in place of, the engineered ST2 and / or IL-18R. RISE is a chimeric signaling receptor that is activatable by rapamycin or a rapalog. Activation of a RISE complex leads to signaling through the IL33 / ST2 axis (“RISE33”) or the IL-18 pathway (“RISE18”).

[0443] Therefore, some aspects relate to a chimeric signaling receptor comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises a first extracellular domain, a first transmembrane domain, and a first membrane proximal signaling domain, and the second polypeptide comprises a second extracellular domain, a second transmembrane domain, and a second membrane proximal signaling domain, such that the first and second extracellular binding domains are capable of binding a ligand (e.g., a small molecule such as rapamycin or an analog thereof).

[0444] In some embodiments, a polypeptide of a chimeric signaling receptor (e.g., first polypeptide or a second polypeptide) further comprises an extracellular linker between an extracellular domain and a transmembrane domain. In some embodiments, a polypeptide of a chimeric signaling receptor (e.g., first polypeptide or a second polypeptide) further comprises a membrane proximal linker between a transmembrane domain and a membrane proximal signaling domain. In some embodiments, a polypeptide of a chimeric signaling receptor (e.g., first polypeptide or a second polypeptide) further comprises a membrane distal linker between a membrane proximal signaling domain and a membrane distal signaling domain. In some embodiments, a chimeric signaling receptor comprises more than one linker. In some embodiments, a polypeptide of a chimeric signaling receptor further comprises a signal peptide.

[0445] In some embodiments, the first extracellular domain and second extracellular domains are dimerization domains that dimerize in the presence of a ligand. In some embodiments, a first extracellular domain is the same as a second extracellular domain and the first and second extracellular domains homodimerize upon binding to a ligand. In some embodiments, a first extracellular domain is different from a second extracellular domain and the first and second extracellular domains heterodimerize upon binding to a ligand. The ligand may be a small molecule, peptide, protein, or other biologic (e.g., comprising base pairs). In some embodiments, a ligand that activates a chimeric signaling receptor is rapamycin or a rapalog. Rapamycin, a macrocyclic triene compound, is also known as sirolimus and is commercially available (e.g., RAPAMUNE®). In some embodiments, the ligand that activates a chimeric signaling receptor is wild-type rapamycin (e.g., commercially available rapamycin, such as RAPAMUNE®). In some embodiments, the ligand that activates a chimeric signaling receptor comprises rapamycin having 1, 2, 3, 4, 5, or more modifications relative to wild-type rapamycin. For example, in some embodiments, the methyl side group chain of the rapamycin may be modified (e.g., extended). In some embodiments, the rapalog is everolimus, CCI-779, C20-methallylrapamycin, C16-(S)-3-methylindolerapamycin, C16- iRap, C16-(S)-7-methylindolerapamycin, AP21967, C16-(S)Butylsulfonamidorapamycin, AP23050, sodium mycophenolic acid, benidipine hydrochloride, AP1903, AP23573, or a metabolite or derivative thereof.

[0446] In some embodiments, the first extracellular domain comprises the rapamycin binding domain of FK-binding protein 12 (FKBP), and the second extracellular domain comprises the rapamycin binding domain of FKBP12-Rapamycin Binding domain of mTOR (FRB). In some embodiments, the sequence of the FKBP domain is: GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVI RGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE (SEQ ID NO: 6). In some embodiments, the FKBP domain may comprise a polypeptide having at least 50% identity (e.g., at least 50, at least 60, at least 70, at least 80, at least 90, at least 95, at least 98, at least 99, or at least 99.5% identity) to SEQ ID NO: 6. In some embodiments, the FKBP domain comprises up to 20 amino acid substitutions, up to 15 amino acid sequence substitutions, up to 10 amino acid substitutions. In some embodiments, the FKBP domain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid sequence substitutions.

[0447] In some embodiments, the sequence of the FRB domain is EMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLM EAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISK (SEQ ID NO: 7). In some embodiments, the FRB domain may comprise a polypeptide having at least 50% identity (e.g., at least 50, at least 60, at least 70, at least 80, at least 90, at least 95, at least 98, at least 99, or at least 99.5% identity) to SEQ ID NO: 7. In some embodiments, the FRB domain comprises up to 20 amino acid substitutions, up to 15 amino acid sequence substitutions, up to 10 amino acid substitutions, or up to 5 amino acid substitutions. In some embodiments, the FRB domain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid sequence substitutions.

[0448] In one embodiment, the FRB domain comprises an T74L mutation. In some embodiments, a first extracellular domain of a chimeric signaling receptor comprises a functional fragment of FKBP, the rapamycin binding domain of FK-binding protein 12. In some embodiments, a second extracellular domain of a chimeric signaling receptor comprises a functional fragment of FRB, the rapamycin binding domain of FKBP12-Rapamycin Binding domain of mTOR. A functional fragment of FKBP or FRB is a fragment that dimerizes with a counterpart fragment upon binding to a ligand (e.g., rapamycin or a rapalog).

[0449] In some embodiments, a chimeric signaling receptor controls signaling that is instigated, propagated, or maintained by IL-33 or IL-18. In some embodiments, a signaling domain of a chimeric signaling receptor (e.g., a first membrane proximal signaling domain, second membrane proximal signaling domain, a first membrane distal signaling domain, or second membrane distal signaling domain) comprises an intracellular signaling domain or functional fragment of a receptor for IL-33 or IL-18.

[0450] For example, in some embodiments a chimeric signaling receptor for controlling ST2 signaling comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises a first extracellular domain, a first transmembrane domain, and a first membrane proximal signaling domain, and the second polypeptide comprises a second extracellular domain, a second transmembrane domain, and a second membrane proximal signaling domain. In some embodiments, a first extracellular domain comprises FKBP or a functional fragment thereof and a second extracellular domain comprises FRB or a functional fragment thereof. In some embodiments, a first membrane proximate domain of a chimeric signaling receptor for controlling ST2 signaling comprises an intracellular signaling domain or functional fragment of ST2, and a second membrane proximate domain of a chimeric signaling receptor for controlling ST2 signaling comprises an intracellular signaling domain or functional fragment of IL1RAP. In some embodiments, a first membrane proximate domain of a chimeric signaling receptor for controlling ST2 signaling comprises an intracellular signaling domain or functional fragment of IL1RAP, and a second membrane proximate domain of a chimeric signaling receptor for controlling ST2 signaling comprises an intracellular signaling domain or functional fragment of ST2.

[0451] In some embodiments, a chimeric signaling receptor for controlling IL18R signaling comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises a first extracellular domain, a first transmembrane domain, and a first membrane proximal signaling domain, and the second polypeptide comprises a second extracellular domain, a second transmembrane domain, and a second membrane proximal signaling domain. In some embodiments, a first polypeptide further comprises a first membrane distal signaling domain, and a second polypeptide further comprises a second membrane distal signaling domain. IL18R domains may be comprised in either the membrane proximate signaling domains of the receptor polypeptides, or in the membrane distal signaling domains of the receptor polypeptides.

[0452] In some embodiments, a first extracellular domain comprises FKBP or a functional fragment thereof and a second extracellular domain comprises FRB or a functional fragment thereof. In some embodiments, a first membrane proximate domain of a chimeric signaling receptor for controlling IL18R signaling comprises an intracellular signaling domain or functional fragment of IL18R, and a second membrane proximate domain of a chimeric signaling receptor for controlling IL18R signaling comprises an intracellular signaling domain or functional fragment of IL18RAP. In some embodiments, a first membrane proximate domain of a chimeric signaling receptor for controlling IL-18R signaling comprises an intracellular signaling domain or functional fragment of IL18RAP, and a second membrane proximate domain of a chimeric signaling receptor for controlling IL18R signaling comprises an intracellular signaling domain or functional fragment of IL18R.

[0453] Engineered ST2 and / or IL1RAP Receptor for Continuous or Inducible IL-33 Signaling

[0454] Some aspects relate to engineered chimeric receptors for continuous IL-33 signaling. In some embodiments, chimeric receptors spontaneously dimerize, causing IL-33 signal transduction. The chimeric receptors, in some embodiments, comprises a first polypeptide and a second polypeptide. In some embodiments, the first chimeric receptor comprises a first polypeptide. In some embodiments, the second chimeric receptor comprises a second polypeptide.

[0455] In some embodiments, the methods of modifying cells comprise introducing into the cell one or more nucleic acids that collectively comprise (1) a first nucleic acid sequence encoding the first polypeptide (2) and a second nucleic acid encoding the second polypeptide. Similarly, some embodiments of cells comprise (1) a first nucleic acid sequence encoding the first polypeptide (2) and a second nucleic acid encoding the second polypeptide. Additionally, some nucleic acids and vectors comprise (1) a first nucleic acid sequence encoding the first polypeptide (2) and a second nucleic acid encoding the second polypeptide.

[0456] In some embodiments, the first polypeptide comprises a first extracellular domain, a first transmembrane domain, and a first intracellular domain. In some embodiments, the first extracellular domain is linked to the first transmembrane domain via a linker (e.g., a flexible linker) or a first extracellular hinge domain. In some embodiments, the first intracellular domain comprises at least one intracellular domain of an ST2 and / or IL1RAP, or functional fragment thereof. In some embodiments, the first intracellular domain comprises an ST2 domain. In some embodiments, the first intracellular domain comprises an IL1RAP domain. In some embodiments, the first intracellular domain comprises an ST2 and an IL1RAP domain. In some embodiments, the second polypeptide comprises a second extracellular domain, a second transmembrane domain, and a second intracellular domain. In some embodiments, the second extracellular domain is linked to the second transmembrane domain via a linker (e.g., a flexible linker) or a second extracellular hinge domain. In some embodiments, the second intracellular domain comprises at least one intracellular domain of an ST2 and / or IL1RAP or functional fragment thereof. In some embodiments, the second intracellular domain comprises an ST2 domain. In some embodiments, the second intracellular domain comprises an IL1RAP domain. In some embodiments, the second intracellular domain comprises an ST2 and an IL1RAP domain.

[0457] In some embodiments, a polypeptide of a chimeric signaling receptor (e.g., first polypeptide or a second polypeptide) further comprises an extracellular linker between an extracellular domain and a transmembrane domain. In some embodiments, a polypeptide of a chimeric signaling receptor (e.g., first polypeptide or a second polypeptide) further comprises an intracellular linker between a transmembrane domain and an intracellular signaling domain. In some embodiments, a chimeric signaling receptor comprises more than one linker. In some embodiments, a polypeptide of a chimeric signaling receptor further comprises a signal peptide. In some embodiments, the first and second chimeric receptors dimerize. In some embodiments, the first and second polypeptides dimerize. In some embodiments, the first extracellular domain and second extracellular domains dimerize. In some embodiments, the first extracellular domain and second extracellular domains are dimerization domains. In some embodiments, a first extracellular domain is the same as a second extracellular domain. In some embodiments, the first and second extracellular domains homodimerize. In some embodiments, a first extracellular domain is different from a second extracellular domain. In some embodiments, and the first and second extracellular domains heterodimerize. In some embodiments, the first extracellular domain and second extracellular domain spontaneously dimerize (z.e., dimerization occurs in the absence of a ligand). In some embodiments, a first extracellular domain is the same as a second extracellular domain. In some embodiments, the first and second extracellular domains homodimerize without the presence of a ligand. In some embodiments, a first extracellular domain is different from a second extracellular domain. In some embodiments, the first and second extracellular domains heterodimerize without the presence of a ligand.

[0458] In some embodiments, spontaneous dimerization is caused by ZIP domains. In some embodiments, spontaneous dimerization is caused by bZIP domains. In some embodiments, the first and second extracellular domains are ZIP domains. In some embodiments, the first and second ZIP domains dimerize to form a leucine zipper. In some embodiments, the first extracellular domain is a bZIP domain. In some embodiments, the second extracellular domain is a bZIP domain. In some embodiments, the first and second extracellular bZIP domains are the same. In some embodiments, the first and second extracellular bZIP domains are different. In some embodiments, the first and second bZIP domains dimerize to form a leucine zipper.

[0459] In some embodiments, the first extracellular domain is a tintin-Zl domain. In some embodiments, the second extracellular domain is a tintin-Z2 domain. In some embodiments, both the first and second extracellular domains are tintin-Z2 domains. See, e.g., Garcia- Manyes et al. J Biol Chem. 2012 Jun 8;287(24):20240-7.

[0460] In some embodiments, the first extracellular domain is a DDD1 domain. In some embodiments, the second extracellular domain is an ADI domain. In some embodiments, the first extracellular domain is a DDD1 domain and the second extracellular domain is an ADI domain.

[0461] In some embodiments, the first extracellular domain is a Barnase domain. In some embodiments, the second extracellular domain is a Barstar domain. In some embodiments, the first extracellular domain is a Barnase domain and the second extracellular domain is a Barstar domain.

[0462] In some embodiments, the first extracellular domain is a human pancreatic RNAse domain. In some embodiments, the second extracellular domain is a S-peptide domain. In some embodiments, the first extracellular domain is a human pancreatic RNAse domain and the second extracellular domain is a S-peptide domain. In some embodiments, the dimerization domains are inducible dimerization domains. Inducible dimerization occurs in the presence of a ligand that is not part of either chimeric receptor (e.g., a small molecule or third protein or peptide). In some embodiments, the first and second chimeric receptors dimerize in the presence of a ligand. In some embodiments, the first and second chimeric receptor dimerization is ind...

Claims

CLAIMSWhat is claimed is:

1. A cell that constitutively expresses interleukin 1 receptor-like 1 protein (ST2).

2. A cell that constitutively expresses interleukin- 1 receptor accessory protein (IL1RAP).

3. A cell that constitutively expresses interleukin 1 receptor-like 1 protein (ST2) and interleukin-1 receptor accessory protein (IL1RAP).

4. A cell constitutively expressing a supraphysiologic level of interleukin 1 receptor-like 1 protein (ST2).

5. A cell constitutively expressing a supraphysiologic level of interleukin- 1 receptor accessory protein (IL 1 RAP).

6. A cell constitutively expressing a supraphysiologic level of interleukin 1 receptor-like 1 protein (ST2), and a supraphysiologic level of interleukin- 1 receptor accessory protein (IL1RAP).

7. A cell comprising a nucleic acid, wherein the nucleic acid comprises a unidirectional constitutive promoter operably linked to a nucleotide sequence encoding interleukin 1 receptor-like 1 protein (ST2).

8. A cell comprising a nucleic acid, wherein the nucleic acid comprises a unidirectional constitutive promoter operably linked to a nucleotide sequence encoding interleukin- 1 receptor accessory protein (IL 1 RAP).

9. A cell comprising:(i) a first nucleic acid comprising a first unidirectional constitutive promoter operably linked to a nucleotide sequence encoding interleukin 1 receptor-like 1 protein (ST2); and(ii) a second nucleic acid comprising a second unidirectional constitutive promoter operably linked to a nucleotide sequence encoding interleukin- 1 receptor accessory protein (IL 1 RAP) .

10. A cell comprising a heterologous promoter operably linked to an endogenous nucleotide sequence encoding interleukin 1 receptor-like 1 protein (ST2).

11. A cell comprising a heterologous promoter operably linked to an endogenous nucleotide sequence encoding interleukin-1 receptor accessory protein (IL1RAP).

12. A cell comprising:(i) a first nucleic acid comprising a first heterologous promoter operably linked to an endogenous nucleotide sequence encoding interleukin 1 receptor-like 1 protein (ST2); and(ii) a second nucleic acid comprising a second heterologous promoter operably linked to an endogenous nucleotide sequence encoding interleukin- 1 receptor accessory protein (IL 1 RAP) .

13. A method comprising contacting a cell with a nucleic acid comprising a unidirectional constitutive promoter operably linked to a nucleotide sequence encoding interleukin 1 receptor-like 1 protein (ST2).

14. A method comprising contacting a cell with a nucleic acid comprising a unidirectional constitutive promoter operably linked to a nucleotide sequence encoding interleukin- 1 receptor accessory protein (IL 1 RAP).

15. A method comprising contacting a cell with:(i) a first nucleic acid comprising a first unidirectional constitutive promoter operably linked to a nucleotide sequence encoding interleukin 1 receptor-like 1 protein (ST2); and(ii) a second nucleic acid comprising a second unidirectional constitutive promoter operably linked to a nucleotide sequence encoding interleukin- 1 receptor accessory protein (IL 1 RAP) .

16. A method comprising contacting a cell with a nucleic acid comprising heterologous promoter, wherein the heterologous promoter is inserted into a nucleic acid of the cell genome, such that the heterologous promoter is operably linked to an endogenous nucleotide sequence encoding interleukin 1 receptor-like 1 protein (ST2).

17. A method comprising contacting a cell with a nucleic acid comprising heterologous promoter, wherein the heterologous promoter is inserted into a nucleic acid of the cell genome, such that the heterologous promoter is operably linked to an endogenous nucleotide sequence encoding interleukin-1 receptor accessory protein (IL1RAP).

18. A method comprising contacting a cell with a first nucleic acid comprising a first heterologous promoter and a second nucleic acid comprising a second heterologous promoter, wherein:(i) the first heterologous promoter is inserted into a first nucleic acid of the cell genome, such that the first heterologous promoter is operably linked to an endogenous nucleotide sequence encoding interleukin 1 receptor-like 1 protein (ST2); and(ii) the second heterologous promoter is inserted into a second nucleic acid of the cell genome, such that the second heterologous promoter is operably linked to an endogenous nucleotide sequence encoding interleukin-1 receptor accessory protein (IL1RAP).

19. A cell that constitutively expresses interleukin 18 receptor 1 (IL-18R1).

20. A cell that constitutively expresses interleukin 18 receptor accessory protein (IL18RAP).

21. A cell that constitutively expresses interleukin 18 receptor 1 (IL-18R1) and interleukin 18 receptor accessory protein (IL18RAP).

22. A cell constitutively expressing a supraphysiologic level of interleukin 18 receptor 1 (IL-18R1).

23. A cell constitutively expressing a supraphysiologic level of interleukin 18 receptor accessory protein (IL18RAP).

24. A cell constitutively expressing a supraphysiologic level of interleukin 18 receptor 1 (IL-18R1), and a supraphysiologic level of interleukin 18 receptor accessory protein (IL18RAP).

25. A cell comprising a nucleic acid, wherein the nucleic acid comprises a unidirectional constitutive promoter operably linked to a nucleotide sequence encoding interleukin 18 receptor 1 (IL-18R1).

26. A cell comprising a nucleic acid, wherein the nucleic acid comprises a unidirectional constitutive promoter operably linked to a nucleotide sequence encoding interleukin- 18 receptor accessory protein (IL18RAP).

27. A cell comprising:(i) a first nucleic acid comprising a first unidirectional constitutive promoter operably linked to a nucleotide sequence encoding interleukin 18 receptor 1 (IL-18R1); and(ii) a second nucleic acid comprising a second unidirectional constitutive promoter operably linked to a nucleotide sequence encoding interleukin 18 receptor accessory protein (IL18RAP).

28. A cell comprising a heterologous promoter operably linked to an endogenous nucleotide sequence encoding interleukin 18 receptor 1 (IL-18R1).

29. A cell comprising a heterologous promoter operably linked to an endogenous nucleotide sequence encoding interleukin 18 receptor accessory protein (IL18RAP).

30. A cell comprising:(i) a first nucleic acid comprising a first heterologous promoter operably linked to an endogenous nucleotide sequence encoding interleukin 18 receptor 1 (IL-18R1); and(ii) a second nucleic acid comprising a second heterologous promoter operably linked to an endogenous nucleotide sequence encoding interleukin 18 receptor accessory protein (IL18RAP).

31. A method comprising contacting a cell with a nucleic acid comprising a unidirectional constitutive promoter operably linked to a nucleotide sequence encoding interleukin 18 receptor 1 (IL-18R1).

32. A method comprising contacting a cell with a nucleic acid comprising a unidirectional constitutive promoter operably linked to a nucleotide sequence encoding interleukin 18 receptor accessory protein (IL18RAP).

33. A method comprising contacting a cell with:(i) a first nucleic acid comprising a first unidirectional constitutive promoter operably linked to a nucleotide sequence encoding interleukin 18 receptor 1 (IL-18R1); and(ii) a second nucleic acid comprising a second unidirectional constitutive promoter operably linked to a nucleotide sequence encoding interleukin 18 receptor accessory protein (IL18RAP).

34. A method comprising contacting a cell with a nucleic acid comprising heterologous promoter, wherein the heterologous promoter is inserted into a nucleic acid of the cell genome, such that the heterologous promoter is operably linked to an endogenous nucleotide sequence encoding interleukin 18 receptor 1 (IL-18R1).

35. A method comprising contacting a cell with a nucleic acid comprising heterologous promoter, wherein the heterologous promoter is inserted into a nucleic acid of the cell genome, such that the heterologous promoter is operably linked to an endogenous nucleotide sequence encoding interleukin 18 receptor accessory protein (IL18RAP).

36. A method comprising contacting a cell with a first nucleic acid comprising a first heterologous promoter and a second nucleic acid comprising a second heterologous promoter, wherein:(i) the first heterologous promoter is inserted into a first nucleic acid of the cell genome, such that the first heterologous promoter is operably linked to an endogenous nucleotide sequence encoding interleukin 18 receptor 1 (IL-18R1); and(ii) the second heterologous promoter is inserted into a second nucleic acid of the cell genome, such that the second heterologous promoter is operably linked to an endogenous nucleotide sequence encoding interleukin 18 receptor accessory protein (IL18RAP).

37. A membrane-bound interleukin-33 (mem-IL-33) protein comprising an interleukin-33 (IL-33) protein and a transmembrane domain.

38. The mem-IL-33 of claim 37, wherein the mem-IL-33 protein comprises a linker connecting the IL-33 protein to the transmembrane domain.

39. The mem-IL-33 of claim 38, wherein the linker is a glycine or glycine-serine linker.

40. The mem-IL-33 of any one of claims 37-39, wherein the mem-IL-33 protein further comprises a hinge domain.

41. The mem-IL-33 of claim 40, wherein the hinge domain comprises a polypeptide selected from the group consisting of: a hinge region of CD8a, a hinge region of a heavy chain of IgG, a hinge region of a heavy chain of IgD, and an extracellular region of an IL- 10R P chain.

42. The mem-IL-33 of any one of claims 37-41, wherein the transmembrane domain comprises a transmembrane anchor.

43. The mem-IL-33 of claim 42, wherein the transmembrane anchor comprises a CD94 anchor.

44. A nucleic acid comprising an open reading frame (ORF) encoding the mem-IL-33 of any one of claims 37-43.

45. The nucleic acid of claim 44, further comprising a promoter operably linked to the ORF.

46. The nucleic acid of claim 45, wherein the promoter is a constitutive promoter.

47. The nucleic acid of claim 45 or 46, wherein the promoter is an MND promoter.

48. A vector comprising the nucleic acid of any one of claims 44-47.

49. The vector of claim 48, wherein the vector is a lentiviral vector.

50. The vector of claim 48, wherein the vector is an adeno-associated viral (AAV) vector.

51. A lipid nanoparticle comprising the nucleic acid of any one of claims 44-47.

52. A cell comprising the mem-IL-33 of any one of claims 37-43.

53. A cell comprising the nucleic acid of any one of claims 44- 47.

54. A method comprising contacting a cell with the nucleic acid of any one of claims 44- 47, vector of any one of claims 48-50, or lipid nanoparticle of claim 51.

55. A membrane-bound interleukin- 18 (mem-IL-18) protein comprising an interleukin- 18 (IL- 18) protein and a transmembrane domain.

56. The mem-IL-18 of claim 55, wherein the mem-IL-18 protein comprises a linker connecting the IL- 18 protein to the transmembrane domain.

57. The mem-IL-18 of claim 56, wherein the linker is a glycine or glycine-serine linker.

58. The mem-IL-18 of any one of claims 55-57, wherein the mem-IL-18 protein further comprises a hinge domain.

59. The mem-IL-18 of claim 58, wherein the hinge domain comprises a polypeptide selected from the group consisting of: a hinge region of CD8a, a hinge region of a heavy chain of IgG, a hinge region of a heavy chain of IgD, and an extracellular region of an IL- 10R P chain.

60. The mem-IL-18 of any one of claims 55-59, wherein the transmembrane domain comprises a transmembrane anchor.

61. The mem-IL-18 of claim 60, wherein the transmembrane anchor comprises a CD94 anchor.

62. A nucleic acid comprising an open reading frame (ORF) encoding the mem-IL-18 of any one of claims 55-61.

63. The nucleic acid of claim 62, further comprising a promoter operably linked to the ORF.

64. The nucleic acid of claim 63, wherein the promoter is a constitutive promoter.

65. The nucleic acid of claim 62 or 63, wherein the promoter is an MND promoter.

66. A vector comprising the nucleic acid of any one of claims 62-65.

67. The vector of claim 66, wherein the vector is a lentiviral vector.

68. The vector of claim 66, wherein the vector is an adeno-associated viral (AAV) vector.

69. A lipid nanoparticle comprising the nucleic acid of any one of claims 62-65.

70. A cell comprising the mem-IL-18 of any one of claims 55-61.

71. A cell comprising the nucleic acid of any one of claims 62-65.

72. A method comprising contacting a cell with the nucleic acid of any one of claims 62- 65, vector of any one of claims 66-68, or lipid nanoparticle of claim 69.

73. A receptor-linked interleukin-33 (rIL-33) polypeptide comprising:(a) an interleukin-33 (IL-33) protein;(b) a linker; and(c) an interleukin 1 receptor-like 1 protein (ST2) or interleukin- 1 receptor accessory protein (IL 1 RAP).

74. The rIL-33 polypeptide of claim 73, wherein polypeptide comprises IL-33 and ST2, wherein the IL-33 and ST2 are connected by the linker.

75. The rIL-33 polypeptide of claim 73, wherein polypeptide comprises IL-33 and IL 1 RAP, wherein the IL-33 and IL 1 RAP are connected by the linker.

76. The rIL-33 polypeptide of any one of claims 73-75, wherein the linker is a glycine or glycine-serine linker.

77. The rIL-33 polypeptide of any one of claims 73-76, wherein the rIL-33 further comprises a hinge domain.

78. The rIL-33 polypeptide of claim 77, wherein the hinge domain comprises a polypeptide selected from the group consisting of: a hinge region of CD8a, a hinge region of a heavy chain of IgG, a hinge region of a heavy chain of IgD, and an extracellular region of an IL-10R 0 chain.

79. A nucleic acid comprising an open reading frame (ORF) encoding the rIL-33 polypeptide of any one of claims 73-78.

80. The nucleic acid of claim 79, further comprising a promoter operably linked to the ORF.

81. The nucleic acid of claim 80, wherein the promoter is a constitutive promoter.

82. The nucleic acid of claim 80 or 81, wherein the promoter is an MND promoter.

83. A vector comprising the nucleic acid of any one of claims 79- 82.

84. The vector of claim 83, wherein the vector is a lentiviral vector.

85. The vector of claim 83, wherein the vector is an adeno-associated viral (AAV) vector.

86. A lipid nanoparticle comprising the nucleic acid of any one of claims 79- 82.

87. A cell comprising the rIL-33 polypeptide of any one of claims 73-78.

88. A cell comprising the nucleic acid of any one of claims 79-83.

89. A method comprising contacting a cell with the nucleic acid of any one of claims 79- 83, vector of any one of claims 83-85, or lipid nanoparticle of claim 86.

90. A receptor-linked interleukin- 18 (rIL-18) polypeptide comprising:(a) an interleukin- 18 (IL- 18) protein;(b) a linker; and(c) an interleukin 18 receptor 1 (IL18R1) or interleukin- 18 receptor accessory protein (IL18RAP).

91. The rIL-18 polypeptide of claim 90, wherein polypeptide comprises IL- 18 and IL18R1, wherein the IL-18 and IL18R1 are connected by the linker.

92. The rIL-18 polypeptide of claim 90, wherein polypeptide comprises IL- 18 and IL18RAP, wherein the IL- 18 and IL18RAP are connected by the linker.

93. The rIL-18 polypeptide of any one of claims 90-92, wherein the linker is a glycine or glycine-serine linker.

94. The rIL-18 polypeptide of any one of claims 90-93, wherein the rIL-18 polypeptide further comprises a hinge domain.

95. The rIL-18 polypeptide of claim 94, wherein the hinge domain comprises a polypeptide selected from the group consisting of: a hinge region of CD8a, a hinge region of a heavy chain of IgG, a hinge region of a heavy chain of IgD, and an extracellular region of an IL-10R 0 chain.

96. A nucleic acid comprising an open reading frame (ORF) encoding the rIL-18 polypeptide of any one of claims 90-95.

97. The nucleic acid of claim 96, further comprising a promoter operably linked to the ORF.

98. The nucleic acid of claim 97, wherein the promoter is a constitutive promoter.

99. The nucleic acid of claim 97 or 98, wherein the promoter is an MND promoter.

100. A vector comprising the nucleic acid of any one of claims 96- 99.

101. The vector of claim 100, wherein the vector is a lentiviral vector.

102. The vector of claim 100, wherein the vector is an adeno-associated viral (AAV) vector.

103. A lipid nanoparticle comprising the nucleic acid of any one of claims 96-99.

104. A cell comprising the rIL-18 polypeptide of any one of claims 90-95.

105. A cell comprising the nucleic acid of any one of claims 96-99.

106. A method comprising contacting a cell with the nucleic acid of any one of claims 96- 99, vector of any one of claims 100-102, or lipid nanoparticle of claim 103.

107. A method for producing a population of regulatory T (Treg) cells having a repair phenotype, the method comprising culturing a population of Treg cells in the presence of interleukin-33 (IL-33).

108. The method of claim 107, wherein the Treg cells are cultured in the presence of 0.1 to 500 ng / mL IL-33.

109. The method of claim 107 or 108, wherein the Treg cells are cultured in the presence of 50 ng / mL IL-33.

110. A method for producing a population of regulatory T (Treg) cells having a repair phenotype, the method comprising culturing a population of Treg cells in the presence of interleukin- 18 (IL-18).

111. The method of claim 110, wherein the Treg cells are cultured in the presence of 0.1 to 500 ng / mL IL-18.

112. The method of claim 110 or 111, wherein the Treg cells are cultured in the presence of 50 ng / mL IL-18.

113. The method of any one of claims 107-112, wherein the Treg cells are cultured in the presence of rapamycin, wherein the Treg cells comprise a nucleic acid encoding a first chemically induced signaling complex (CISC) component and a nucleic acid encoding a second CISC component, wherein the first CISC component comprises, in N-to-C-terminal order, an FKBP domain, an interleukin-2 receptor gamma (IL-2Ry) transmembrane domain, and an IL-2Ry cytoplasmic domain, and wherein the second CISC component comprises, in N-to-C-terminal order, an FRB domain, an interleukin-2 receptor beta (IL-2RP) transmembrane domain, and an IL-2RP cytoplasmic domain.

114. The method of claim 113, wherein the Treg cells are cultured in the presence of 0.01 - 100 nM rapamycin.

115. The method of claim 113 or 114, wherein the Treg cells are cultured in the presence of 10 nM rapamycin.

116. The method of any one of claims 107-115, wherein the Treg cells are cultured in the presence of an anti-CD3 antibody and an anti-CD28 antibody.

117. The method of any one of claims 107-116, wherein the method comprises culturing the population of Treg cells in the presence of IL-2, IL-12, IL-21, IL-23, and / or TGF-p.

118. A composition comprising a population of regulatory T (Treg) cells and IL-33.

119. The composition of claim 118, wherein the composition comprises 0.1 to 500 ng / mL IL-33.

120. The composition of claim 118 or 119, wherein the composition comprises 50 ng / mL IL-33.

121. A composition comprising a population of regulatory T (Treg) cells and IL-18.

122. The composition of claim 118, wherein the composition comprises 0.1 to 500 ng / mL IL-18.

123. The composition of claim 118 or 119, wherein the composition comprises 50 ng / mL IL-18.

124. The composition of any one of claims 118-123, wherein composition comprises rapamycin, wherein one or more of the Treg cells comprise a nucleic acid encoding a first chemically induced signaling complex (CISC) component and a nucleic acid encoding a second CISC component, wherein the first CISC component comprises, in N-to-C-terminal order, an FKBP domain, an interleukin-2 receptor gamma (IL-2Ry) transmembrane domain, and an IL-2Ry cytoplasmic domain, and wherein the second CISC component comprises, in N-to-C-terminal order, an FRB domain, an interleukin-2 receptor beta (IL-2RP) transmembrane domain, and an IL-2RP cytoplasmic domain.

125. The composition of claim 124, wherein the composition comprises 0.01 - 100 nM rapamycin.

126. The composition of claim 124 or 125, wherein the composition comprises 10 nM rapamycin.

127. The composition of any one of claims 123-125, further comprising an anti-CD3 antibody and an anti-CD28 antibody.

128. The composition of any one of claims 124-127, further comprising IL-2, IL-12, IL- 21, IL-23, and / or TGF-p.

129. The cell of any one of claims 1-36, 52, 53, 70, 71, 87, 88, 104, or 105, wherein the cell is a stem cell or a T cell.

130. The cell of claim 129, wherein the cell is a CD4+ or CD8+ T cell.

131. The cell of claim 129 or 130, wherein the cell is a FoxP3+regulatory T (Treg) cell.

132. The cell of any one of claims 129-131, wherein the cell expresses one or more markers selected from BATF, CTLA-4, HLA-DR, ICOS, Ki-67, TIGIT, TNFRSF18, CD25, CD39, CD49d, CD69, CD71, CD103, CD197.

133. The cell of any one of claims 129-132, wherein the cell expresses one or more cytokines selected from IL-5, IL-10, IL-13, and TGF-p.

134. The cell of any one of claims 129-133, wherein the cell produces soluble ST2 and / or osteopontin.

135. The cell of any one of claims 129-134, wherein the cell expresses one or more chemokine receptors selected from CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR10, and CX3CR1.

136. The cell of any one of claims 129-135, wherein the cell expresses CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR10, and CX3CR1.

137. The cell of any one of claims 129-136, wherein the cell is a KLRG1+cell.

138. The cell of any one of claims 129-137, wherein the cell is a NFIL3+cell.

139. The cell of any one of claims 129-138, wherein the cell is a GATA3+cell.

140. The cell of any one of claims 129-139, wherein the cell is a RLN3+cell.

141. The cell of any one of claims 129-140, wherein the cell is a RAB4A+cell.

142. The cell of any one of claims 129-141, wherein the cell is a LYN+cell.

143. The cell of any one of claims 129-142, wherein the cell is a PTPN13+cell.

144. The cell of any one of claims 129-143, wherein the cell is a TBC1 D4 cell.

145. The cell of any one of claims 129-144, wherein the cell is a PPARy+cell.

146. The cell of any one of claims 129-145, wherein the cell comprises a heterologous promoter located in a nucleic acid of the cell genome:(i) downstream from a Treg-specific demethylated region (TSDR); and(ii) upstream from a first coding exon of an endogenous FOXP3 gene.

147. The cell of claim 146, wherein the heterologous promoter located downstream from the TSDR is an MND promoter.

148. The cell of any one of claims 129-147, wherein the cell comprises:(i) a nucleic acid encoding a first component of a chemically induced signaling complex (CISC), the first CISC component comprising:(a) a first extracellular domain comprising an FK506-binding protein domain that binds rapamycin;(b) a first transmembrane domain; and(c) a first cytoplasmic domain comprising an intracellular signaling domain of a first cytokine receptor; and(ii) a nucleic acid encoding a second CISC component, the second CISC component comprising:(a) a second extracellular domain comprising an FKBP-rapamycin-binding (FRB) domain;(b) a second transmembrane domain; and(c) a second cytoplasmic domain comprising an intracellular signaling domain of a second cytokine receptor, wherein the first and second CISC components dimerize in the presence of rapamycin.

149. The cell of claim 148, wherein the first CISC component comprises, in N-to-C- terminal order, an FKBP domain, an interleukin-2 receptor gamma (IL-2Ry) transmembrane domain, and an IL-2Ry cytoplasmic domain, and wherein the second CISC component comprises, in N-to-C-terminal order, an FRB domain, an interleukin-2 receptor beta (IL-2RP) transmembrane domain, and an IL-2RP cytoplasmic domain.

150. The cell of claim 148 or 149, wherein the cell comprises a nucleic acid encoding a soluble FRB domain, wherein SEQ ID NO: 5 has at least 90% identity to an amino acid sequence of the soluble FRB domain.

151. The cell of any one of claims 1-36, 52, 53, 70, 71, 87, 88, 104, 105, or 129-150, wherein the cell expresses a chimeric antigen receptor (CAR).

152. The cell of any one of claims 1-36, 52, 53, 70, 71, 87, 88, 104, 105, or 129-150, wherein the cell expresses a T cell receptor (TCR).

153. A method comprising administering the cell of any one of claims 1-36, 52, 53, 70, 71, or 128-151 to a subject.

154. The method of claim 153, wherein the cell is autologous to the subject.

155. The method of claim 153, wherein the cell is allogeneic to the subject.

156. A method comprising administering the vector of any one of claims 48-50, 66-68, 83-85, or 100-102 to a subject.

157. A method comprising administering the lipid nanoparticle of any one of claims 51, 69,86, or 103 to a subject.

158. The method of any one of claims 153-157, wherein the subject has or is at risk of developing an acute inflammatory disease or condition.

159. The method of claim 158, wherein the acute inflammatory disease or condition is stroke, kidney injury, acute respiratory distress syndrome (ARDS), myocardial infarction, severe wounding, or a muscle injury.

160. The method of claim 158, wherein the subject has or is at risk of developing ARDS.

161. The method of claim 158, wherein the subject has or is at risk of having a stroke.

162. The method of claim 158, wherein the subject has or is at risk of developing kidney injury.

163. The method of claim 162, wherein the kidney injury is an acute kidney injury.

164. The method of any one of claims 153-157, wherein the subject has or is at risk of developing fibrosis.

165. The method of claim 164, wherein the fibrosis is pulmonary fibrosis.

166. A composition comprising a plurality of engineered regulatory T (Treg) cells, at least 50% of the engineered Treg cells of the plurality expressing ST2 protein.

167. The composition of claim 166, wherein the engineered Treg cells expressing ST2 protein constitutively express ST2 protein.

168. A composition comprising a plurality of engineered regulatory T (Treg) cells, at least 50% of the engineered Treg cells of the plurality expressing IL1RAP.

169. The composition of claim 168, wherein the engineered Treg cells expressing IL1RAP constitutively express IL1RAP.

170. A composition comprising a plurality of engineered regulatory T (Treg) cells, at least 50% of the engineered Treg cells of the plurality expressing IL-18R1.

171. The composition of claim 170, wherein the engineered Treg cells expressing IL-18R1 constitutively express IL-18R1.

172. A composition comprising a plurality of engineered regulatory T (Treg) cells, at least 50% of the engineered Treg cells of the plurality expressing IL18RAP.

173. The composition of claim 172, wherein the engineered Treg cells expressing IL18RAP constitutively express IL18RAP.

174. The composition of any one of claims 166-173, wherein at least 50% of the engineered Treg cells comprise a genetic modification to stabilize FOXP3 expression.

175. The composition of claim 174, wherein the genetic modification to stabilize FOXP3 expression comprises a heterologous promoter inserted in a FOXP3 gene of the cell genome, wherein the heterologous promoter is downstream from a TSDR of the FOXP3 gene and upstream for a first coding exon of a FOXP3 gene.

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