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WO2024159138A1 - Lymphocytes t régulateurs de réparation modifiés - Google Patents

Lymphocytes t régulateurs de réparation modifiés Download PDF

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Publication number
WO2024159138A1
WO2024159138A1 PCT/US2024/013175 US2024013175W WO2024159138A1 WO 2024159138 A1 WO2024159138 A1 WO 2024159138A1 US 2024013175 W US2024013175 W US 2024013175W WO 2024159138 A1 WO2024159138 A1 WO 2024159138A1
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Prior art keywords
cell
nucleic acid
interleukin
nucleotide sequence
receptor
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Inventor
Payam ZARIN
Fabien Depis
Philippe KIEFFER-KWON
Tom WICKHAM
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Gentibio Inc
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Gentibio Inc
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Priority to EP24747885.2A priority Critical patent/EP4654981A1/fr
Publication of WO2024159138A1 publication Critical patent/WO2024159138A1/fr
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • C12N5/0637—Immunosuppressive T lymphocytes, e.g. regulatory T cells or Treg
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/20—Cellular immunotherapy characterised by the effect or the function of the cells
    • A61K40/22—Immunosuppressive or immunotolerising
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/545—IL-1
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7155—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • C—CHEMISTRY; METALLURGY
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    • C12N2510/00—Genetically modified cells
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • C—CHEMISTRY; METALLURGY
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
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    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • Cellular therapies using regulatory T cells may be useful to treat numerous types of diseases including acute and chronic inflammatory diseases or conditions.
  • Tregs Regulatory T cells 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.
  • compositions and methods for improved methods of producing repair Tregs and repair Treg populations for therapeutic use.
  • 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.
  • 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.
  • 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.
  • an IL-33 receptor e.g., ST2 and/or IL1RAP
  • an IL-18 receptor e.g., IL-18R1 (IL-18Ra) and/or IL18RAP (IL-18RP)
  • IL-18RP IL-18RP
  • 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.
  • Tregs 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).
  • 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.
  • 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).
  • ST2 interleukin 1 receptor-like 1
  • IL1RAP interleukin- 1 receptor accessory protein
  • 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).
  • ST2 supraphysiologic level of interleukin 1 receptor-like 1
  • IL1RAP interleukin- 1 receptor accessory protein
  • 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).
  • IL1RAP interleukin- 1 receptor accessory protein
  • 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).
  • ST2 interleukin 1 receptor-like 1
  • IL1RAP interleukin-1 receptor accessory protein
  • 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).
  • ST2 interleukin 1 receptor-like 1
  • IL1RAP interleukin-1 receptor accessory protein
  • 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).
  • ST2 interleukin 1 receptor-like 1
  • IL 1 RAP interleukin- 1 receptor accessory protein
  • 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).
  • IL1RAP interleukin- 1 receptor accessory protein
  • 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).
  • ST2 interleukin 1 receptor-like 1
  • IL1RAP interleukin-1 receptor accessory protein
  • 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).
  • IL1RAP interleukin-1 receptor accessory protein
  • 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).
  • ST2 interleukin 1 receptor-like 1
  • IL 1 RAP interleukin- 1 receptor accessory protein
  • 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).
  • IL-18R1 interleukin 18 receptor 1
  • IL-18RAP interleukin 18 receptor accessory protein
  • 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).
  • IL-18R1 interleukin 18 receptor 1
  • IL18RAP supraphysiologic level of interleukin 18 receptor accessory protein
  • 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).
  • IL-18R1 interleukin 18 receptor 1
  • IL18RAP interleukin- 18 receptor accessory protein
  • 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).
  • IL-18R1 interleukin 18 receptor 1
  • IL18RAP interleukin 18 receptor accessory protein
  • 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).
  • IL-18R1 interleukin 18 receptor 1
  • IL18RAP interleukin 18 receptor accessory protein
  • 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).
  • IL-18R1 interleukin 18 receptor 1
  • 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).
  • IL18RAP interleukin 18 receptor accessory protein
  • 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).
  • IL-18R1 interleukin 18 receptor 1
  • a membrane-bound interleukin-33 (mem-IL-33) protein comprising an interleukin-33 (IL-33) protein and a transmembrane domain.
  • the mem-IL-33 protein comprises a linker connecting the IL-33 protein to the transmembrane domain.
  • the linker is a glycine or glycine-serine linker.
  • the mem-IL-33 protein further comprises a hinge domain.
  • 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.
  • the transmembrane domain comprises a transmembrane anchor.
  • the transmembrane anchor comprises a CD94 anchor.
  • nucleic acid comprising an open reading frame (ORF) encoding the mem-IL-33.
  • nucleic acid further comprises a promoter operably linked to the ORF.
  • the promoter is a constitutive promoter.
  • the promoter is an MND promoter.
  • Some aspects relate to a vector comprising the nucleic acid encoding the mem-IL-33.
  • the vector is a lentiviral vector.
  • the vector is an adeno-associated viral (AAV) vector.
  • AAV adeno-associated viral
  • 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.
  • rlIL-33 receptor-linked interleukin-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).
  • IL-33 interleukin-33
  • ST2 interleukin 1 receptor-like 1
  • IL1RAP interleukin- 1 receptor accessory protein
  • polypeptide comprises IL-33 and ST2, wherein the IL-33 and ST2 are connected by the linker.
  • polypeptide comprises IL-33 and IL1RAP, wherein the IL-33 and IL1RAP are connected by the linker.
  • the linker is a glycine or glycine-serine linker.
  • the rIL-33 further comprises a hinge domain.
  • 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.
  • nucleic acid comprising an open reading frame (ORF) encoding the rIL-33.
  • nucleic acid further comprises a promoter operably linked to the ORF.
  • the promoter is a constitutive promoter.
  • the promoter is an MND promoter.
  • Some aspects relate to a vector comprising the nucleic acid encoding the rIL-33.
  • the vector is a lentiviral vector.
  • the vector is an adeno-associated viral (AAV) vector.
  • AAV adeno-associated viral
  • 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.
  • 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).
  • Treg regulatory T
  • IL-33 interleukin-33
  • 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.
  • a membrane-bound interleukin- 18 (mem-IL-18) protein comprising an interleukin- 18 (IL- 18) protein and a transmembrane domain.
  • the mem-IL-18 protein comprises a linker connecting the IL- 18 protein to the transmembrane domain.
  • the linker is a glycine or glycine-serine linker.
  • the mem-IL-18 protein further comprises a hinge domain.
  • 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.
  • the transmembrane domain comprises a transmembrane anchor.
  • the transmembrane anchor comprises a CD94 anchor.
  • nucleic acid comprising an open reading frame (ORF) encoding the mem-IL-18.
  • the nucleic acid further comprises a promoter operably linked to the ORF.
  • the promoter is a constitutive promoter.
  • the promoter is an MND promoter.
  • Some aspects relate to a vector comprising the nucleic acid encoding the mem-IL-18.
  • the vector is a lentiviral vector.
  • the vector is an adeno-associated viral (AAV) vector.
  • AAV adeno-associated viral
  • 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.
  • rlIL-18 receptor-linked interleukin- 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).
  • IL-18 interleukin- 18
  • IL-18R1 interleukin 18 receptor 1
  • IL18RAP interleukin- 18 receptor accessory protein
  • 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.
  • 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.
  • nucleic acid comprising an open reading frame (ORF) encoding the rIL-18.
  • nucleic acid further comprises a promoter operably linked to the ORF.
  • the promoter is a constitutive promoter.
  • the promoter is an MND promoter.
  • Some aspects relate to a vector comprising the nucleic acid encoding the rIL-18.
  • the vector is a lentiviral vector.
  • the vector is an adeno-associated viral (AAV) vector.
  • AAV adeno-associated viral
  • 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.
  • 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).
  • IL- 18 interleukin- 18
  • 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.
  • 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).
  • the Treg cells are cultured in the presence of 0.1 to 500 ng/mL IL-18.
  • wherein the Treg cells are cultured in the presence of 50 ng/mL IL-18.
  • 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.
  • CISC chemically induced signaling complex
  • 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.
  • the Treg cells are cultured in the presence of an anti-CD3 antibody and an anti-CD28 antibody.
  • 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.
  • compositions comprising a population of regulatory T (Treg) cells and IL-33.
  • the composition comprises 0.1 to 500 ng/mL IL-33. In some embodiments, the composition comprises 50 ng/mL IL-33.
  • compositions comprising a population of regulatory T (Treg) cells and IL-18.
  • the composition comprises 0.1 to 500 ng/mL IL-18. In some embodiments, the composition comprises 50 ng/mL IL-18.
  • 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.
  • CISC chemically induced signaling complex
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the heterologous promoter located downstream from the TSDR is an MND promoter.
  • 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.
  • CISC chemically induced signaling complex
  • 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
  • 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.
  • 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.
  • the cell expresses a chimeric antigen receptor (CAR).
  • the cell expresses a T cell receptor (TCR).
  • Some aspects relate to a method comprising administering the cell to a subject.
  • the cell is autologous to the subject.
  • 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.
  • the subject has or is at risk of developing an acute inflammatory disease or condition.
  • the acute inflammatory disease or condition is stroke, kidney injury, acute respiratory distress syndrome (ARDS), myocardial infarction, severe wounding, or a muscle injury.
  • ARDS acute respiratory distress syndrome
  • the subject has or is at risk of developing ARDS.
  • the subject has or is at risk of having a stroke.
  • the subject has or is at risk of developing kidney injury.
  • the kidney injury is an acute kidney injury.
  • the subject has or is at risk of developing fibrosis.
  • the fibrosis is pulmonary fibrosis.
  • 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.
  • 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.
  • at least 50% of engineered Treg cells comprise a genetic modification to stabilize FOXP3 expression.
  • 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.
  • 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.
  • 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.
  • hEngTregs human engineered T regulatory cells
  • 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. 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.
  • 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)).
  • 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.
  • 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.
  • mEngTregs murine engineered T regulatory cells
  • 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).
  • LPS lipopolysaccharides
  • Auto autologous
  • FIG. 7A shows bodyweight change over time (note that the top axis represents days).
  • FIG. 7B shows pulse oximetry data from Day 11
  • FIG. 7C shows lung weight measurements from Day 11.
  • 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.
  • 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.
  • 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.
  • FIG. 11 includes two graphs showing cell surface protein expression of human CCR4 on hEngTregs (left) and mouse CCR4 on mEngTregs (right).
  • 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.
  • FIG. 13 is a graph showing the ratio of lung to spleen engraftment of mEngTregs over time.
  • 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.
  • 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).
  • FIG. 16 is a schematic showing LPS-induced acute lung injury with allogeneic Fl adoptive cell transfer.
  • 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.
  • 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.
  • BALF bronchoalveolar lavage fluid
  • FIG. 19 shows lung histology and mean severity scores (by pathology) following mEngTreg administration. Samples were taken on Day 6 post- LPS administration.
  • 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+).
  • 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
  • mEngTregs are administered on day 1
  • 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.
  • 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.
  • 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.
  • tMCAO transient middle cerebral artery occlusion
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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).
  • 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.
  • 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.
  • 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.
  • FIG. 42A shows representative staining.
  • FIG. 42B shows CD39 expression.
  • FIG. 42C shows CD73 expression levels.
  • FIG. 42D shows CD39+CD73+ population frequency.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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. 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. 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.
  • 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.
  • 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.
  • 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.
  • repair Tregs are Tregs that express at least one repair receptor (e.g., the IL-33 receptor and/or the IL-18 receptor).
  • the engineered repair Tregs are engineered have stabilized FoxP3 expression.
  • 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.
  • CISC chemical-inducible signaling complex
  • 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.
  • alarmins e.g., IL-33 and IL- 18
  • 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).
  • inflammatory immune cells e.g., neutrophils, macrophages
  • AVG amphiregulin
  • KGF keratinocyte growth factor
  • TGF-P transforming growth factor beta
  • methods comprise inducing or increasing IL-33 and/or IL-18 signaling pathway activity in a Treg cell to generate engineered repair Tregs.
  • 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).
  • 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).
  • a target cell e.g, ex vivo, or in vivo.
  • 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.
  • IL-33 signaling is increased by inducing expression of one or more IL-33 receptor components (ST2 or IL1RAP).
  • 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 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).
  • 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.
  • 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).
  • IL-lR-associated kinases IRAKI, IRAK2, IRAK3, IRAK4
  • 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.
  • ox-LDL cardioprotective antioxidized low-density lipoprotein
  • IL-33 signaling in neuronal macrophages 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.
  • phagocytosis of extracellular matrix regulating synapse remodeling
  • Ap mitigating neuroinflammation
  • 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.
  • 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.
  • IL-18RP then binds to form a trimer.
  • MyD88 binds to the Toll-IL-1 receptor (TIR) domain of IL-18R1 and IL18RAP.
  • TIR Toll-IL-1 receptor
  • 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).
  • PI3K phosphatidylinositol-3 kinase
  • mTOR mammalian target of rapamycin
  • 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.
  • 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.
  • a promoter is operably linked to the sequence encoding ST2, IL1RAP, IL- 18R1, and/or IL18RAP, or functional derivative thereof.
  • the promoter is the natural promoter of the gene.
  • the promoter is heterologous to the gene.
  • the promoter is a constitutive promoter.
  • the promoter is an inducible promoter.
  • the cell is contacted with a reagent that promotes transcription through the inducible promoter.
  • 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.
  • 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.
  • a Kozak sequence is modified or replaced.
  • an exogenous Kozak sequence is inserted into the endogenous nucleic acid.
  • an endogenous promoter is modified or replaced.
  • an exogenous promoter is inserted into the endogenous nucleic acid.
  • an enhancer is modified or replaced.
  • an exogenous enhancer is inserted into the endogenous nucleic acid.
  • a ubiquitous chromatin opening element is inserted into the endogenous nucleic acid.
  • a stabilizing sequence e.g., woodchuck hepatitis virus posttranscriptional regulatory element (WPRE)
  • WPRE woodchuck hepatitis virus posttranscriptional regulatory element
  • 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.
  • 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
  • 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.
  • 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.
  • 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, 10 4 -fold, or 10 5 -fold relative to a sorted Treg that has not been genetically modified to increase ST2 expression.
  • the sorted Treg has not been genetically modified to increase FOXP3 expression.
  • the sorted Treg has an unmodified F0XP3 locus.
  • the sorted Treg has not been genetically modified.
  • 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, 10 4 -fold, or 10 5 -fold relative to a sorted Treg that has not been genetically modified to increase IL1RAP expression.
  • the sorted Treg has not been genetically modified to increase FOXP3 expression.
  • the sorted Treg has an unmodified F0XP3 locus.
  • the sorted Treg has not been genetically modified.
  • 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, 10 4 -fold, or 10 5 -fold relative to a sorted Treg that has not been genetically modified to increase IL-18R1 expression.
  • the sorted Treg has not been genetically modified to increase FOXP3 expression.
  • the sorted Treg has an unmodified F0XP3 locus.
  • the sorted Treg has not been genetically modified.
  • 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, 10 4 -fold, or 10 5 -fold relative to a sorted Treg that has not been genetically modified to increase IL18RAP expression.
  • the sorted Treg has not been genetically modified to increase FOXP3 expression.
  • the sorted Treg has an unmodified F0XP3 locus.
  • the sorted Treg has not been genetically modified.
  • any suitable method may be used to measure the extent of ST2, IL1RAP, IL-18R1, and/or IL18RAP expression in a cell.
  • 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.
  • qRT-PCR and/or RNAseq may be used to measure transcription of nucleotide sequences encoding ST2, IL1RAP, IL-18R1, and/or IL18RAP.
  • Some aspects relate to a plurality of engineered Tregs expressing ST2.
  • 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 + .
  • 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.
  • Some aspects relate to a plurality of engineered Tregs expressing IL1RAP.
  • 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 + .
  • 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 + .
  • 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,
  • Some aspects relate to a plurality of engineered Tregs expressing IL-18R1.
  • 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 + .
  • 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 + .
  • 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,
  • Some aspects relate to a plurality of engineered Tregs expressing IL18RAP.
  • 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 + .
  • 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 + .
  • 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.
  • any suitable method may be used to determine whether a cell is ST2+, IL1RAP+, IL- 18R1+, and/or IL18RAP+.
  • 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.
  • PBMCs e.g.. sorted Tregs
  • single-cell RNA sequencing methods may be used to measure the frequency of protein expression among the cells.
  • 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.
  • the cells are useful, for example, to mitigate and/or prevent certain signs and symptoms of inflammatory conditions.
  • 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.
  • CISC chemically inducible signaling complex
  • the second promoter is operably linked to a nucleotide sequence encoding ST2.
  • the first inserted promoter is operably linked to a nucleotide sequence encoding a soluble FRB domain.
  • 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.
  • the first inserted promoter is operably linked to a cDNA sequence encoding FOXP3.
  • 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
  • the second promoter is operably linked to a nucleotide sequence encoding ST2.
  • the first promoter is operably linked to a nucleotide sequence encoding a soluble FRB domain.
  • 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.
  • the first promoter is operably linked to a cDNA sequence encoding FOXP3.
  • 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.
  • CISC chemically inducible signaling complex
  • the second promoter is operably linked to a nucleotide sequence encoding ST2.
  • the first inserted promoter is operably linked to a nucleotide sequence encoding a soluble FRB domain.
  • 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.
  • the first inserted promoter is operably linked to a cDNA sequence encoding FOXP3.
  • 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.
  • CISC chemical
  • the second promoter is operably linked to a nucleotide sequence encoding ST2.
  • the first promoter is operably linked to a nucleotide sequence encoding a soluble FRB domain.
  • 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.
  • the first promoter is operably linked to a cDNA sequence encoding FOXP3.
  • an engineered cell comprising a modified F0XP3 locus and a modified second locus other than a F0XP3 locus
  • 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)
  • 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.
  • 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.
  • 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
  • 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.
  • the engineered Treg is a stabilized Treg.
  • 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.
  • Stabilized Tregs may be generated using any suitable method.
  • stabilized Tregs are generated by expressing the expression of one or more transcription factors.
  • CCAAT/enhancer binding protein CZEBP
  • 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.
  • certain nuclear factors may be overexpressed to increase FoxP3 expression.
  • 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.
  • 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.
  • 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.
  • the lineage commitment factor is selected from FOXP3, Helios, Ikaros.
  • a partial FOXP3 cDNA sequence is inserted in-frame with one or more exons of an endogenous F0XP3 coding sequence.
  • 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.
  • 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).
  • a 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.
  • the encoded gene products e.g., ST2, IL1RAP, IL-18R1, and/or IL18RAP
  • ST2, IL1RAP, IL-18R1, and/or IL18RAP may be expressed episomally.
  • the coding sequence encoding the repair mediator does not comprise an intron (e.g, the coding sequence is a cDNA sequence).
  • 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.
  • a gene product from a coding sequence without introns 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.
  • a method comprises contacting a cell with one or more nucleic acids to produce the genetically modified cell.
  • introducing a nucleic acid, protein, or vector into the cell comprises contacting the cell with the nucleic acid, protein, or vector, respectively.
  • the cell is in vitro.
  • the cell is ex vivo.
  • a cell is isolated from a subject, contacted with one or more nucleic acids, and administered to the same subject.
  • the cell is ex vivo.
  • a cell is isolated from a subject, contacted with one or more nucleic acids, and administered to a different subject.
  • the cell is in vivo.
  • the cell is modified extracorporeally.
  • the cell is a T cell.
  • the cell is a CD4+ T cell.
  • Nucleic acid molecules may be introduced into a cell using any suitable method.
  • viral or non-viral systems may be used to deliver transgenes into a cell.
  • 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.
  • a repair mediator e.g., ST2, IL1RAP, IL-18R1, and/or IL18RAP
  • 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).
  • 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.
  • 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.
  • 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.
  • the viral vector is a retrovirus.
  • the viral vector is a lentiviral vector.
  • the viral vector is a gamma-retroviral vector.
  • 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.
  • 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).
  • another protein e.g., ST2
  • the promoter is further operably linked to a nucleotide sequence encoding a selectable marker.
  • the selectable marker is LNGFR.
  • the vector comprises an MND promoter operably linked to a nucleotide sequence encoding ST2.
  • 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.
  • the vector comprises the nucleotide sequence of SEQ ID NO: 161.
  • the vector comprises an MND promoter operably linked to (i) a nucleotide sequence encoding ST2, and (ii) a selectable marker.
  • the selectable marker is LNGFR.
  • 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.
  • the vector comprises the nucleotide sequence of SEQ ID NO: 162.
  • the vector comprises an MND promoter operably linked to (i) a nucleotide sequence encoding ST2, and (ii) a nucleotide sequence encoding a CISC component.
  • the CISC component comprises an extracellular FKBP domain, an IL-2Ry transmembrane domain, and an IL-2Ry cytoplasmic domain.
  • the CISC component comprises an extracellular FRB domain, an IL-2RP transmembrane domain, and an IL-2RP cytoplasmic domain.
  • the nucleotide sequence encoding ST2 is 5' to the nucleotide sequence encoding the CISC component.
  • 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.
  • the vector comprises the nucleotide sequence of SEQ ID NO: 163.
  • the nucleotide sequence encoding ST2 is 3' to the nucleotide sequence encoding the CISC component.
  • 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.
  • the vector comprises the nucleotide sequence of SEQ ID NO: 164.
  • one or more vectors are adeno-associated viral (AAV) vectors.
  • AAV vectors is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11 vector.
  • one or more AAV vectors are AAV5 vectors.
  • one or more AAV vectors are AAV6 vectors.
  • 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.
  • the T cell is a T regulatory cell (Treg), such as a mammalian Treg.
  • the mammalian Treg expresses any one of the polypeptides.
  • 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 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.
  • the T cell is a CD4+ T cell.
  • the T cell is a CD8+ T cell.
  • 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).
  • 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.
  • 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;
  • LTR long terminal repeat
  • packaging genes viral Gag-Pol: Gag is a structural precursor protein, and Pol is a polymerase; and
  • Envelope gene may be pseudotyped to alter infectivity).
  • the three components described above 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.
  • 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.
  • 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 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.
  • a transfection reagent e.g., Polybrene (Merck®)
  • 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.
  • 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.
  • ST2 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.
  • 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:
  • the functional derivative has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
  • 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.
  • 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.
  • 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.
  • the encoded ST2 comprises the wild-type amino acid sequence of SEQ ID NO: 1.
  • the encoded ST2 consists of the wild-type amino acid sequence of SEQ ID NO: 1.
  • 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.
  • 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 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,
  • the functional derivative has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
  • 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.
  • 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.
  • 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.
  • the encoded ILlRAP comprises the wild-type amino acid sequence of SEQ ID NO: 2.
  • the encoded IL1RAP consists of the wild-type amino acid sequence of SEQ ID NO: 2.
  • a nucleotide sequence encoding ST2 is codon-optimized.
  • 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.
  • ST2 is encoded by nucleotide sequence of SEQ ID NO: 52.
  • ST2 may be expressed with any suitable signal peptide.
  • 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.
  • 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.
  • a nucleotide sequence encoding IL1RAP is codon-optimized.
  • 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.
  • IL1RAP is encoded by nucleotide sequence of SEQ ID NO: 54.
  • IL1RAP may be expressed with any suitable signal peptide.
  • the signal peptide is a wild-type IL1RAP signal peptide.
  • an IL1RAP signal peptide amino acid sequence is provided by SEQ ID NO: 44.
  • the signal peptide comprises the amino acid sequence of SEQ ID NO: 55.
  • 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.
  • the signal peptide is encoded by the nucleotide sequence of SEQ ID NO: 55.
  • IL-18R1 and/or IL18RAP expression is provided by SEQ ID NO: 44.
  • the signal peptide comprises the amino acid sequence of SEQ ID NO: 55.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the encoded IL-18R1 comprises the wild-type amino acid sequence of SEQ ID NO: 40.
  • the encoded IL-18R1 consists of the wild-type amino acid sequence of SEQ ID NO: 40.
  • 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.
  • 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.
  • 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.
  • 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.
  • the encoded IL18RAP comprises the wild-type amino acid sequence of SEQ ID NO: 41.
  • the encoded IL18RAP consists of the wild-type amino acid sequence of SEQ ID NO: 41.
  • a nucleotide sequence encoding IL-18R1 is codon-optimized.
  • 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.
  • IL-18R1 is encoded by nucleotide sequence of SEQ ID NO: 110.
  • IL-18R1 may be expressed with any suitable signal peptide.
  • the signal peptide is a wild-type IL-18R1 signal peptide.
  • an IL- 18R1 signal peptide amino acid sequence is provided by SEQ ID NO: 45.
  • the signal peptide comprises the amino acid sequence of SEQ ID NO: 45.
  • 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.
  • the signal peptide is encoded by the nucleotide sequence of SEQ ID NO: 111.
  • a nucleotide sequence encoding IL18RAP is codon-optimized.
  • 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.
  • IL18RAP is encoded by nucleotide sequence of SEQ ID NO: 112.
  • IL18RAP may be expressed with any suitable signal peptide.
  • the signal peptide is a wild-type IL18RAP signal peptide.
  • an IL18RAP signal peptide amino acid sequence is provided by SEQ ID NO: 46.
  • the signal peptide comprises the amino acid sequence of SEQ ID NO: 46.
  • 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.
  • the signal peptide is encoded by the nucleotide sequence of SEQ ID NO: 113.
  • 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
  • the functional derivative of the repair mediator 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).
  • a 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 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).
  • 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.
  • cytokines e.g., AREG and/or osteopontin
  • growth factors e.g., KGF, TGF-P
  • chemokine receptors e.g., CCR2, CCR5, CCR8
  • Nucleic acids comprising promoters operably linked to a coding sequence 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).
  • 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).
  • the sequence inserted into the targeted locus may replace all or part of the endogenous coding sequence encoding the polypeptide.
  • one or more mutations is introduced into the endogenous coding sequence to prevent translation of a full-length polypeptide from the endogenous coding sequence.
  • 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.
  • the targeted locus is a safe harbor locus.
  • the safe harbor locus is a HIPP 11 locus.
  • the safe harbor locus is a ROSA26 locus.
  • the safe harbor locus is an AAVS1 locus.
  • the targeted locus is a T cell receptor locus.
  • the T cell receptor locus is a TRAC locus.
  • the T cell receptor locus is a TRBC locus.
  • 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.
  • the nucleic acid, or vector comprising such a nucleic acid is not integrated into the genome of the cell.
  • a plasmid or artificial chromosome e.g., human artificial chromosome
  • the introduced vector or nucleic acid replicates independently of endogenous chromosomes.
  • more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of the vector are present in a genetically modified cell.
  • the number of copies of the vector in a cell exceeds the copy number of an individual chromosome in a cell.
  • 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.
  • a donor template comprising the promoter is introduced into the cell, and incorporated into the genome by homologous recombination.
  • 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.
  • a genomic coding sequence e.g., endogenous ST2, IL1RAP, IL-18R, or IL 18RAP coding sequence
  • 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).
  • the method further comprises modifying an endogenous coding sequence to which the promoter is operably linked.
  • 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).
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • a method comprises contacting a cell with one or more nucleic acids to produce the genetically modified cell.
  • introducing a nucleic acid, protein, or vector into the cell comprises contacting the cell with the nucleic acid, protein, or vector, respectively.
  • the cell is in vitro.
  • the cell is ex vivo.
  • a cell is isolated from a subject, contacted with one or more nucleic acids, and administered to the same subject.
  • the cell is ex vivo.
  • a cell is isolated from a subject, contacted with one or more nucleic acids, and administered to a different subject.
  • the cell is in vivo.
  • the cell is modified extracorporeally.
  • the cell is a T cell.
  • the cell is a CD4+ T cell.
  • 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.
  • 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.
  • 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.
  • 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 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,
  • the functional derivative has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
  • 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.
  • 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 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,
  • the functional derivative has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
  • 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.
  • 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.
  • 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.
  • 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.
  • a nucleotide sequence encoding ST2 is codon-optimized.
  • 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.
  • ST2 is encoded by nucleotide sequence of SEQ ID NO: 52.
  • ST2 may be expressed with any suitable signal peptide.
  • 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.
  • 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.
  • a nucleotide sequence encoding IL1RAP is codon-optimized.
  • 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.
  • IL1RAP is encoded by nucleotide sequence of SEQ ID NO: 54.
  • IL1RAP may be expressed with any suitable signal peptide.
  • the signal peptide is a wild-type IL1RAP signal peptide.
  • an IL1RAP signal peptide amino acid sequence is provided by SEQ ID NO: 44.
  • the signal peptide comprises the amino acid sequence of SEQ ID NO: 55.
  • 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.
  • the signal peptide is encoded by the nucleotide sequence of SEQ ID NO: 55.
  • IL-18R1 and/or IL18RAP expression is provided by SEQ ID NO: 44.
  • the signal peptide comprises the amino acid sequence of SEQ ID NO: 55.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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,
  • the functional derivative comprises a C-terminal fusion with another polypeptide.
  • a functional derivative lacks one or more domains or portions of wild-type IL-18R1 that are unnecessary for signaling.
  • the functional derivative has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
  • 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.
  • 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.
  • 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.
  • the encoded IL-18R1 comprises the wild-type amino acid sequence of SEQ ID NO: 40.
  • the encoded IL-18R1 consists of the wild-type amino acid sequence of SEQ ID NO: 40.
  • the encoded IL18RAP comprises the wild-type amino acid sequence of SEQ ID NO: 41.
  • the encoded IL18RAP consists of the wild-type amino acid sequence of SEQ ID NO: 41.
  • a nucleotide sequence encoding IL-18R1 is codon-optimized.
  • 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.
  • IL-18R1 is encoded by nucleotide sequence of SEQ ID NO: 110.
  • IL-18R1 may be expressed with any suitable signal peptide.
  • the signal peptide is a wild-type IL-18R1 signal peptide.
  • an IL- 18R1 signal peptide amino acid sequence is provided by SEQ ID NO: 45.
  • the signal peptide comprises the amino acid sequence of SEQ ID NO: 45.
  • 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.
  • the signal peptide is encoded by the nucleotide sequence of SEQ ID NO: 111.
  • a nucleotide sequence encoding IL18RAP is codon-optimized.
  • 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.
  • IL18RAP is encoded by nucleotide sequence of SEQ ID NO: 112.
  • IL18RAP may be expressed with any suitable signal peptide.
  • the signal peptide is a wild-type IL18RAP signal peptide.
  • an IL18RAP signal peptide amino acid sequence is provided by SEQ ID NO: 46.
  • the signal peptide comprises the amino acid sequence of SEQ ID NO: 46.
  • 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.
  • the signal peptide is encoded by the nucleotide sequence of SEQ ID NO: 113.
  • 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
  • the functional derivative of the repair mediator 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).
  • a 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 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • the flexible linker has the amino acid sequence GSTSGSGKPGSGEGSTKG (SEQ ID NO: 34).
  • the hinge is a flexible hinge.
  • the flexible hinge comprises a polypeptide selected from the following polypeptides or variants thereof:
  • the hinge region of the heavy chain of IgD (for example as set forth in SEQ ID NO: 33).
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the HLA-A2 domain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid sequence substitutions.
  • 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.
  • 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.
  • 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.
  • 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.
  • a linker e.g., a flexible linker
  • 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.
  • the amino acid sequence of the complete membrane-bound IL- 33 comprises a transmembrane domain, a linker, and an IL-33 protein.
  • the transmembrane anchor comprises a CD94 anchor.
  • the amino acid sequence of the complete membrane-bound IL-33 may be encoded by a nucleic acid molecule.
  • nucleic acid molecule comprising a nucleotide sequence encoding a membrane-bound IL-33.
  • the nucleic acid molecule is the sole nucleic acid molecule in the composition, z.e., the composition does not comprise additional nucleic acid molecules.
  • the nucleic acid comprises an open reading frame (ORF) encoding the mem-IL-33.
  • the nucleic acid further comprises a promoter operably linked to the ORF.
  • the promoter is a constitutive promoter.
  • the promoter is an MND promoter.
  • 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.
  • 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).
  • 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.
  • a viral vector comprising anyone of the nucleic acid molecules comprising a nucleotide sequence encoding a membrane-bound IL-33.
  • 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.
  • 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.
  • the viral vector is a retrovirus.
  • the viral vector is a lentiviral vector.
  • the viral vector is a gamma-retroviral vector.
  • 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.
  • 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.
  • Treg regulatory T cell
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the flexible linker has the amino acid sequence GSTSGSGKPGSGEGSTKG (SEQ ID NO: 34).
  • the hinge is a flexible hinge.
  • the flexible hinge comprises a polypeptide selected from the following polypeptides or variants thereof:
  • the hinge region of the heavy chain of IgD (for example as set forth in SEQ ID NO: 33).
  • 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).
  • 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.
  • 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.
  • connecting peptide referred to herein as “connecting peptide”; SEQ ID NO: 38
  • 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.
  • 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.
  • 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.
  • the transmembrane-intracellular region of mem-IL-18 is derived from IL18RAP.
  • 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.
  • the HLA-A2 domain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid sequence substitutions.
  • 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.
  • the CD28 domain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid sequence substitutions.
  • 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.
  • 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.
  • a linker e.g., a flexible linker
  • 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.
  • the amino acid of the complete membrane-bound IL- 18 comprises a transmembrane domain, a linker, and an IL-18 protein.
  • the transmembrane anchor comprises a CD94 anchor.
  • amino acid sequences of the complete membrane-bound IL-18 may be encoded by a nucleic acid molecule.
  • nucleic acid molecule comprising a nucleotide sequence encoding a membrane-bound IL- 18.
  • nucleic acid molecule is the sole nucleic acid molecule in the composition, z.e., the composition does not comprise additional nucleic acid molecules.
  • nucleic acid comprises an open reading frame (ORF) encoding the mem-IL-18.
  • nucleic acid further comprises a promoter operably linked to the ORF.
  • the promoter is a constitutive promoter.
  • the promoter is an MND promoter.
  • 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.
  • 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).
  • 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.
  • a viral vector comprising anyone of the nucleic acid molecules comprising a nucleotide sequence encoding a membrane-bound IL-18.
  • 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.
  • 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.
  • the viral vector is a retrovirus.
  • the viral vector is a lentiviral vector.
  • the viral vector is a gamma-retroviral vector.
  • 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.
  • 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.
  • Treg regulatory T cell
  • 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.
  • 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.
  • 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.
  • 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.
  • IL-33 is linked to ST2 and IL1RAP, or functional derivatives thereof.
  • 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).
  • the IL-33 portion comprises the wild-type amino acid sequence of SEQ ID NO: 39.
  • the IL-33 portion consists of the wild-type amino acid sequence of SEQ ID NO: 39.
  • 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.
  • 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.
  • the ST2 portion comprises the wild-type amino acid sequence of SEQ ID NO: 1.
  • the ST2 portion consists of the wild-type amino acid sequence of SEQ ID NO: 1.
  • 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.
  • the ST2 portion comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid sequence substitutions.
  • 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.
  • the IL1RAP portion comprises the wild-type amino acid sequence of SEQ ID NO: 2.
  • the IL1RAP portion consists of the wildtype amino acid sequence of SEQ ID NO: 2.
  • 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.
  • the IL1RAP portion comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid sequence substitutions.
  • 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.
  • the linker comprises a peptide (e.g., a human or artificial peptide).
  • the linker comprises a synthetic linker (e.g., a flexible linker) or an extracellular hinge domain.
  • the linker is a flexible linker or a rigid linker.
  • the linker is a flexible linker.
  • 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.
  • the Gly spacer comprises at least 3 Gly.
  • the Gly spacer comprises a sequence set forth as GGGS (SEQ ID NO: 8), GGGSGGG (SEQ ID NO: 9) or GGG.
  • 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.
  • rIL-33 further comprises a hinge.
  • IL-33 is linked to ST2 by a hinge.
  • IL-33 is linked to IL1RAP by a hinge.
  • IL-33 is linked to ST2 and IL1RAP by one or more hinges.
  • the hinge is a flexible hinge.
  • 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.
  • the hinge comprises a hinge region of CD8a.
  • the nucleic acid comprises an open reading frame (ORF) encoding the rIL-33 polypeptide.
  • ORF open reading frame
  • a promoter is operably linked to the ORF.
  • the promoter is a constitutive promoter.
  • the promoter is an MND promoter.
  • 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.
  • 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.
  • 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.
  • IL-18 is linked to IL-18R1 and IL18RAP, or functional derivatives thereof.
  • 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).
  • 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.
  • the IL-18 portion comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid sequence substitutions.
  • 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.
  • 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.
  • the IL-18R1 portion comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid sequence substitutions.
  • 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.
  • 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.
  • the IL18RAP portion comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid sequence substitutions.
  • 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.
  • the linker comprises a peptide (e.g., a human or artificial peptide).
  • the linker comprises a synthetic linker (e.g., a flexible linker) or an extracellular hinge domain.
  • the linker is a flexible linker or a rigid linker.
  • the linker is a flexible linker.
  • 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.
  • the Gly spacer comprises at least 3 Gly.
  • the Gly spacer comprises a sequence set forth as GGGS (SEQ ID NO: 8), GGGSGGG (SEQ ID NO: 9) or GGG.
  • 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.
  • rIL-18 further comprises a hinge.
  • IL-18 is linked to IL-18R1 by a hinge.
  • IL-18 is linked to IL18RAP by a hinge.
  • IL- 18 is linked to IL-18R1 and IL18RAP by one or more hinges.
  • the hinge is a flexible hinge.
  • 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.
  • the hinge comprises a hinge region of CD8a.
  • the nucleic acid comprises an open reading frame (ORF) encoding the rIL-18 polypeptide.
  • ORF open reading frame
  • a promoter is operably linked to the ORF.
  • the promoter is a constitutive promoter.
  • the promoter is an MND promoter.
  • 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).
  • 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
  • positive regulators of the IL-33/ST2 and/or IL-18 pathway e.g., ST2, IL1RAP, IL-18R1, and/or IL18RAP.
  • a gene encoding one or more positive regulators of the IL-33/ST2 and/or IL- 18 pathway is constitutively expressed in a cell, e.g., by delivery of nucleic acids comprising the gene into a subject.
  • ST2 is constitutively expressed in a cell.
  • IL1RAP is constitutively expressed in a cell.
  • ST2 and/or IL1RAP is constitutively expressed in a cell.
  • a method of producing repair Tregs in vivo comprises gene editing of a gene involved in IL-33 signaling and/or IL-18 signaling.
  • 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.
  • 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).
  • ST2 is constitutively expressed in a cell.
  • 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.
  • a gene encoding one or more positive regulators of the IL-33/ST2 and/or IL-18 pathways is constitutively expressed in a cell, e.g., by delivery of nucleic acids comprising the gene into the cell.
  • a method of producing repair Tregs ex vivo comprises gene editing of a gene involved in IL-33/ST2 and/or IL-18 signaling.
  • 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.
  • the cell is modified extracorporeally. In some embodiments, the cell is a T cell. In some embodiments, the cell is a CD4+ T cell.
  • 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).
  • the coding sequence is a cDNA encoding one repair mediator (e.g., ST2, IL1RAP, IL-18R1, and/or IL18RAP).
  • 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.
  • 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.
  • 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.
  • 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 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,
  • the functional derivative has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
  • 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.
  • 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 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,
  • the functional derivative has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
  • 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.
  • 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.
  • 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.
  • 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.
  • a nucleotide sequence encoding ST2 is codon-optimized.
  • 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.
  • ST2 is encoded by nucleotide sequence of SEQ ID NO: 52.
  • ST2 may be expressed with any suitable signal peptide.
  • 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.
  • 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.
  • a nucleotide sequence encoding IL1RAP is codon-optimized.
  • 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.
  • IL1RAP is encoded by nucleotide sequence of SEQ ID NO: 54.
  • IL1RAP may be expressed with any suitable signal peptide.
  • the signal peptide is a wild-type IL1RAP signal peptide.
  • an IL1RAP signal peptide amino acid sequence is provided by SEQ ID NO: 44.
  • the signal peptide comprises the amino acid sequence of SEQ ID NO: 55.
  • 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.
  • the signal peptide is encoded by the nucleotide sequence of SEQ ID NO: 55.
  • 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.
  • 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.
  • 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.
  • 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,
  • the functional derivative comprises a C-terminal fusion with another polypeptide.
  • a functional derivative lacks one or more domains or portions of wild-type IL-18R1 that are unnecessary for signaling.
  • the functional derivative has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
  • 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.
  • 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.
  • 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.
  • the encoded IL-18R1 comprises the wild-type amino acid sequence of SEQ ID NO: 40.
  • the encoded IL-18R1 consists of the wild-type amino acid sequence of SEQ ID NO: 40.
  • the encoded IL18RAP comprises the wild-type amino acid sequence of SEQ ID NO: 41.
  • the encoded IL18RAP consists of the wild-type amino acid sequence of SEQ ID NO: 41.
  • a nucleotide sequence encoding IL-18R1 is codon-optimized.
  • 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.
  • IL-18R1 is encoded by nucleotide sequence of SEQ ID NO: 110.
  • IL-18R1 may be expressed with any suitable signal peptide.
  • the signal peptide is a wild-type IL-18R1 signal peptide.
  • an IL- 18R1 signal peptide amino acid sequence is provided by SEQ ID NO: 45.
  • the signal peptide comprises the amino acid sequence of SEQ ID NO: 45.
  • 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.
  • the signal peptide is encoded by the nucleotide sequence of SEQ ID NO: 111.
  • a nucleotide sequence encoding IL18RAP is codon-optimized.
  • 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.
  • IL18RAP is encoded by nucleotide sequence of SEQ ID NO: 112.
  • IL18RAP may be expressed with any suitable signal peptide.
  • the signal peptide is a wild-type IL18RAP signal peptide.
  • an IL18RAP signal peptide amino acid sequence is provided by SEQ ID NO: 46.
  • the signal peptide comprises the amino acid sequence of SEQ ID NO: 46.
  • 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.
  • the signal peptide is encoded by the nucleotide sequence of SEQ ID NO: 113.
  • 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 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).
  • a repair mediator e.g., IL-18R
  • 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).
  • IL-18R 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 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).
  • 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.
  • cytokines e.g., AREG and/or osteopontin
  • growth factors e.g., KGF, TGF-P
  • chemokine receptors e.g., CCR2, CCR5, CCR8
  • the promoter and coding sequence encoding at least one repair mediator 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).
  • the cell comprises a promoter operably linked to a cDNA sequence encoding ST2 or a derivative thereof, inserted at a ST2 locus.
  • 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.
  • the promoter and coding sequence encoding at least one repair mediator are located in a safe harbor locus.
  • the safe harbor locus is a HIPP 11 locus.
  • the safe harbor locus is an AAVS1 locus.
  • the safe harbor locus is a ROSA26 locus.
  • 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.
  • the T cell receptor locus is a TRAC locus.
  • the T cell receptor locus is a TRBC locus.
  • 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.
  • a promoter inserted into a nucleic acid e.g., chromosome
  • 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.
  • 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.
  • 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.
  • 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 .
  • 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.
  • the inserted promoter is active promoting transcription of RNA encoding ST2, IL1RAP, IL-18R1, or IL18RAP, even under pro-inflammatory conditions.
  • the inserted promoter is a constitutive promoter.
  • the constitutive promoter is an EF-la, PGK, or MND promoter.
  • the constitutive promoter is an MND promoter.
  • the inserted promoter is an inducible promoter.
  • the promoter is inserted into an exon of the ST2, IL1RAP, IL- 18R1, or IL18RAP coding sequence, thereby creating a synthetic exon.
  • the coding sequence may be modified relative to the endogenous sequence, but still capable of encoding a ST2, IL1RAP, IL-18R1, or IL18RAP polypeptide.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 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,
  • the functional derivative has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
  • 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.
  • 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:
  • 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.
  • the functional derivative comprises an N-terminal fusion with another polypeptide.
  • the functional derivative comprises a C-terminal fusion with another polypeptide.
  • a functional derivative lacks one or more domains or portions of wild-type IL1RAP that are unnecessary for signaling.
  • 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.
  • 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.
  • 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.
  • a nucleotide sequence encoding ST2 is codon-optimized.
  • 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.
  • ST2 is encoded by nucleotide sequence of SEQ ID NO: 52.
  • ST2 may be expressed with any suitable signal peptide.
  • 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.
  • 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.
  • a nucleotide sequence encoding IL1RAP is codon-optimized.
  • 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.
  • IL1RAP is encoded by nucleotide sequence of SEQ ID NO: 54.
  • IL1RAP may be expressed with any suitable signal peptide.
  • the signal peptide is a wild-type IL1RAP signal peptide.
  • an IL1RAP signal peptide amino acid sequence is provided by SEQ ID NO: 44.
  • the signal peptide comprises the amino acid sequence of SEQ ID NO: 55.
  • 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.
  • the signal peptide is encoded by the nucleotide sequence of SEQ ID NO: 55.
  • 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.
  • 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.
  • 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.
  • 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.
  • a functional derivative lacks one or more domains or portions of wild-type IL-18R1 that are unnecessary for signaling.
  • 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.
  • 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.
  • 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.
  • the functional derivative comprises a C-terminal fusion with another polypeptide.
  • a functional derivative lacks one or more domains or portions of wild-type IL18RAP that are unnecessary for signaling.
  • 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.
  • 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.
  • the encoded IL-18R1 comprises the wild-type amino acid sequence of SEQ ID NO: 40.
  • the encoded IL-18R1 consists of the wild-type amino acid sequence of SEQ ID NO: 40.
  • the encoded IL18RAP comprises the wild-type amino acid sequence of SEQ ID NO: 41.
  • the encoded IL18RAP consists of the wild-type amino sequence of SEQ ID NO: 41.
  • a nucleotide sequence encoding IL-18R1 is codon-optimized.
  • 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.
  • IL-18R1 is encoded by nucleotide sequence of SEQ ID NO: 110.
  • IL-18R1 may be expressed with any suitable signal peptide.
  • the signal peptide is a wild-type IL-18R1 signal peptide.
  • an IL- 18R1 signal peptide amino acid sequence is provided by SEQ ID NO: 45.
  • the signal peptide comprises the amino acid sequence of SEQ ID NO: 45.
  • 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.
  • the signal peptide is encoded by the nucleotide sequence of SEQ ID NO: 111.
  • a nucleotide sequence encoding IL18RAP is codon-optimized.
  • 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.
  • IL18RAP is encoded by nucleotide sequence of SEQ ID NO: 112.
  • IL18RAP may be expressed with any suitable signal peptide.
  • 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.
  • the signal peptide comprises the amino acid sequence of SEQ ID NO: 46.
  • 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.
  • 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 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).
  • a repair mediator e.g., IL-18R
  • 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).
  • IL-18R 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 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).
  • 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.
  • cytokines e.g., AREG and/or osteopontin
  • growth factors e.g., KGF, TGF-P
  • chemokine receptors e.g., CCR2, CCR5, CCR8
  • 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.
  • a gene editing method comprises knocking out or inactivating an endogenous gene, such as by producing a chromosomal gene knockout in the genome.
  • 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.
  • a chromosomal gene knock-out or gene knock-in is made by chromosomal editing of a host cell.
  • Chromosomal editing can be performed using, for example, endonucleases.
  • endonucleases 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.
  • an endonuclease is capable of cleaving a nucleic acid sequence in a targeted gene, thereby inactivating or "knocking out" the targeted gene.
  • 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.
  • endonucleases for use in gene editing include zinc finger nucleases (ZFN), TALE-nucl eases (TALEN), RNA-guided nucleases, CRISPR-Cas nucleases, meganucleases, or megaTALs.
  • ZFN zinc finger nucleases
  • TALEN TALE-nucl eases
  • RNA-guided nucleases CRISPR-Cas nucleases
  • meganucleases or megaTALs.
  • 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).
  • HDR homology directed repair
  • NHEJ non-homologous end joining
  • 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.
  • a "zinc finger nuclease” 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.
  • ZFN zinc finger nuclease
  • 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).
  • 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).
  • HDR homology directed repair
  • 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.
  • NHEJ non-homologous end joining
  • a gene knockout or inactivation comprises an insertion, a deletion, a mutation or a combination thereof, made using a ZFN molecule.
  • TALEN transcription activator-like effector nuclease
  • 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.
  • RVD Repeat Variable Diresidue
  • 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.
  • 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.
  • 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.
  • a gene knockout comprises an insertion, a deletion, a mutation or a combination thereof, and made using a TALEN molecule.
  • 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.
  • 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.
  • CRISPR/Cas clustered regularly interspaced short palindromic repeats/Cas
  • Cas CRISPR/Cas, or Cas
  • 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 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.
  • 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).
  • 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.
  • a gene knockout or inactivation comprises an insertion, a deletion, a mutation or a combination thereof, and made using an RNA-guided nuclease.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • an exogenous nucleic acid sequence e.g., promoter, transgene, and/or combinations thereof
  • 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.
  • the double-stranded break is introduced by an RNA- guided nuclease in the presence of a gRNA at a PAM sequence of NGG.
  • the double-stranded break is introduced by an RNA-guided nuclease in the presence of a gRNA at a PAM sequence of NNNNCC.
  • 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.
  • 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.
  • 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.
  • the donor template is inserted following cleavage by an RNA-guided nuclease in the presence of a gRNA at a PAM sequence of NGG.
  • 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.
  • the gRNA comprises a spacer sequence of SEQ ID NO: 150.
  • 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.
  • 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.
  • 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.
  • the donor template is inserted following cleavage by an RNA-guided nuclease in the presence of a gRNA at a PAM sequence of NGG.
  • 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.
  • the gRNA comprises a spacer sequence of SEQ ID NO: 150.
  • 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.
  • 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.
  • the 5' homology arm comprises the nucleic acid sequence of SEQ ID NO: 143 or SEQ ID NO: 146
  • the 3' homology arm comprises the nucleic acid sequence of SEQ ID NO: 145.
  • the donor template is inserted following cleavage by an RNA-guided nuclease in the presence of a gRNA at a PAM sequence of NNNNCC.
  • 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.
  • the gRNA comprises a spacer sequence of SEQ ID NO: 151.
  • 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.
  • 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.
  • the 5' homology arm comprises the nucleic acid sequence of SEQ ID NO: 146
  • the 3' homology arm comprises the nucleic acid sequence of SEQ ID NO: 147.
  • the donor template is inserted following cleavage by an RNA-guided nuclease in the presence of a gRNA at a PAM sequence of NNNNCC.
  • 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.
  • the gRNA comprises a spacer sequence of SEQ ID NO: 151.
  • 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.
  • a donor template inserted into the FOXP3 locus comprises at least 90% sequence identity to SEQ ID NO: 165.
  • a donor template inserted into the FOXP3 locus comprises at least 95% sequence identity to SEQ ID NO: 165.
  • a donor template inserted into the FO P3 locus comprises the nucleotide sequence of SEQ ID NO: 165.
  • a donor template inserted into the FOXP3 locus consists of the nucleotide sequence of SEQ ID NO: 165.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the double-stranded break is introduced by an RNA-guided nuclease in the presence of a gRNA at a PAM sequence of NGG.
  • the double-stranded break is introduced by an RNA-guided nuclease in the presence of a gRNA at a PAM sequence of NNNNCC.
  • 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.
  • 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.
  • 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.
  • the donor template is inserted following cleavage by an RNA-guided nuclease in the presence of a gRNA at a PAM sequence of NGG.
  • 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.
  • the gRNA comprises a spacer sequence of SEQ ID NO: 159.
  • 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.
  • 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.
  • 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.
  • the donor template is inserted following cleavage by an RNA-guided nuclease in the presence of a gRNA at a PAM sequence of NNNNCC.
  • 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.
  • the gRNA comprises a spacer sequence of SEQ ID NO: 160.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • cleavage at a glyceraldehyde 3 -phosphate dehydrogenase (GAPDH) locus may prevent production of functional GAPDH, leading to death of cells that fail to express this essential protein.
  • GAPDH glyceraldehyde 3 -phosphate dehydrogenase
  • NHEJ DNA repair
  • 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.
  • 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.
  • 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.
  • 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.
  • the nucleic acid comprises a portion of an exon of the essential gene.
  • the nucleic acid comprises an exon of the essential gene.
  • the nucleic acid comprises two or more exons of the essential gene.
  • 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.
  • 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.
  • 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.
  • one or more C-terminal amino acids may be dispensable to the function of the essential gene product.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • the 2A motif is 3’ to the nucleotide sequence encoding the essential gene product, and 5’ to the heterologous protein-encoding nucleotide sequence.
  • the 2A motif is 3’ to the heterologous protein-encoding nucleotide sequence, and 5’ to the nucleotide sequence encoding the essential gene product.
  • the 2A motif is a P2A motif.
  • the 2A motif is a T2A motif.
  • the 2A motif is an E2A motif.
  • the 2A motif is an F2A motif.
  • multiple heterologous coding sequences are present in an inserted nucleic acid, or nucleic acid for insertion into a cell genome.
  • 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 heterologous coding sequences are present in the nucleic acid.
  • two or more heterologous coding sequences are separated by nucleotide sequences encoding 2A motifs.
  • separate pairs of heterologous coding sequences are separated by different nucleotide sequences encoding 2A motifs.
  • each heterologous coding sequence is separated by a different nucleotide sequence encoding a 2A motif.
  • the 2A motifs separating different proteins encoded by different heterologous coding sequences may be the same or different 2A motifs.
  • 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.
  • 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.
  • the nucleic acid comprises a nucleotide sequence encoding a chimeric antigen receptor (CAR).
  • the nucleic acid comprises a nucleotide sequence encoding a TCRa chain.
  • the nucleic acid comprises a nucleotide sequence encoding a TCRP chain.
  • the nucleic acid comprises a nucleotide sequence encoding a TCRa chain, and a nucleotide sequence encoding a TCRP chain.
  • 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.
  • the nucleic acid comprises a nucleotide sequence encoding a 5’ UTR.
  • the nucleic acid comprises a nucleotide sequence encoding a 3’ UTR.
  • the nucleic acid comprises a nucleotide sequence encoding a 3’ UTR and a polyAtail.
  • a transcribed polyAtail comprises at least 100 consecutive adenosine nucleotides.
  • a nucleic acid may be inserted at any suitable essential gene locus.
  • the essential gene locus is a GAPDH locus.
  • the essential gene locus is KIFll locus.
  • the essential gene locus is a TBP locus.
  • the gene locus is essential for Treg cell survival.
  • the gene locus is essential for maintenance of a Treg cell phenotype.
  • 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).
  • ZFN zinc finger nuclease
  • 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.
  • 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.
  • both homology arms are 100-2,000 nucleotides in length.
  • both homology arms are 300-1,000 nucleotides in length.
  • 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.
  • 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.
  • the nucleic acid is present in a lipid nanoparticle.
  • the lipid nanoparticle comprises the nuclease or a nucleic acid encoding the nuclease.
  • the lipid nanoparticle comprises a guide RNA or a nucleic acid encoding the guide RNA.
  • the nucleic acid encoding the nuclease is an mRNA.
  • a promoter e.g., heterologous promoter
  • 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.
  • a promoter is operably linked to an endogenous nucleotide sequence encoding ST2.
  • a promoter is operably linked to an endogenous nucleotide sequence encoding IL1RAP.
  • a promoter is operably linked to an endogenous nucleotide sequence encoding ST2 and/or IL1RAP.
  • 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).
  • 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.
  • an operably linked sequence e.g., a repair mediator polypeptide
  • 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.
  • the constitutive promoter is a strong promoter.
  • the constitutive promoter is a weak promoter.
  • the constitutive promoter is an EF-la, PGK, or MND promoter.
  • another suitable promoter such as an SV40, CMV, UBC, SFFV, EFS, or CAGG promoter
  • the constitutive promoter is an MND promoter.
  • 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.
  • a promoter is a ubiquitous promoter. In some embodiments, the ubiquitous promoter is Reverse Orientation Splice Acceptor 26 (ROSA26) promoter.
  • ROSA26 Reverse Orientation Splice Acceptor 26
  • the promoter is a unidirectional promoter.
  • 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.
  • the unidirectional promoter drives transcription downstream of the promoter.
  • the unidirectional promoter drives transcription upstream of the promoter.
  • the unidirectional promoter is a unidirectional constitutive promoter.
  • the unidirectional constitutive promoter is operably linked to a nucleotide sequence encoding ST2.
  • 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.
  • Embodiments of methods for producing genetically modified cells 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).
  • the methods comprise editing an immune cell.
  • immune cells include B cells, T cells, ILCs, and NK cells.
  • 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.
  • 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.
  • 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 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 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.
  • 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.
  • 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.
  • the cell is a CD3+, CD4+, and/or CD8+ T cell.
  • the cell is a CD3+ T cell.
  • the cell is a CD4 CD8 T cell.
  • the Treg cell is a FoxP3+ Treg cell.
  • 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.
  • 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.
  • an engineered Treg is one that expresses a repair phenotype through modification, expression/over-expression, or manipulation of the IL33-ST2 axis.
  • the engineered Treg is a sorted engineered Treg.
  • a sorted engineered Treg refers to a sorted Treg that has been engineered to express a chimeric antigen receptor (CAR) or a transgenic T cell receptor (TCR).
  • 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 + CD25 hi CD127 10 or CD4 + CD25 hi CD127 lo CD45RA + .
  • a sorted Treg is engineered to upregulate expression of ST2, IL1RAP, IL- 18R1, and/or IL18RAP.
  • 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.
  • 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.
  • CISC chemically induced signaling complex
  • 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.
  • the engineered repair Treg comprises a Treg engineered to express FOXP3 and CISC (IL-2).
  • the cell is a human cell.
  • a cell as is isolated from 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).
  • the cell is obtained from peripheral blood.
  • the cell is obtained from umbilical cord blood.
  • 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.
  • a desired cell population e.g., CD3+ cells
  • the cell is in a subject.
  • the cell is in vivo.
  • Embodiments of genetically modified cells may be any suitable cell type.
  • the cell is a T cell, a precursor T cell, or a hematopoietic stem cell.
  • the cell is an NK-T cell (e.g., a FoxP3- NK-T cell or a FoxP3+ NK-T cell).
  • the cell is a regulatory B (Breg) cell (e.g., a FoxP3- B cell or a FoxP3+ B cell).
  • 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).
  • the cell is a CD25- T cell.
  • the cell is a regulatory T (Treg) cell.
  • Treg cells are Tri, Th3, CD8+CD28-, and Qa-1 restricted T cells.
  • the Treg cell is a FoxP3+ Treg cell.
  • 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.
  • the Treg cell is ST2+.
  • the Treg cell is IL-18R+.
  • a cell is genetically modified. In some embodiments, the cells have one or more phenotypes associated with ST2 signaling.
  • 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.
  • 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.
  • 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.
  • the cell expresses one or more chemokine receptors, selected from the group consisting of: CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR10, and CX3CR1.
  • the cell expresses CCR2.
  • the cell expresses CCR3.
  • the cell expresses CCR4.
  • the cell expresses CCR5.
  • the cell expresses CCR6.
  • the cell expresses CCR7.
  • the cell expresses CCR8.
  • the cell expresses CCR10.
  • the cell expresses CX3CR1.
  • the cell produces soluble ST2 and/or osteopontin. In some embodiments, the cell produces soluble ST2. In some embodiments, the cell produces osteopontin.
  • 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.
  • 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.
  • CISC chemically inducible signaling complex
  • CISC chemically inducible signaling complex
  • 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.
  • CISC chemically inducible signaling complex
  • CISC chemically inducible signaling complex
  • 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.
  • CISC chemically inducible signaling complex
  • CISC chemically inducible signaling complex
  • 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.
  • 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.
  • intracellular signaling domains include IL-2RP and IL-2Ry cytoplasmic domains and functional derivatives thereof.
  • an intracellular signaling domain of one CISC component comprises an IL-2RP cytoplasmic domain or a functional derivative thereof
  • an intracellular signaling domain of the other CISC component comprises an IL-2Ry cytoplasmic signaling domain or a functional derivative thereof.
  • dimerization of the CISC components induces phosphorylation of JAK1, JAK3, and/or STAT5 in the cell.
  • dimerization of the CISC components induces proliferation of the cell.
  • 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).
  • 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.
  • the truncated IL-2Rb domain comprises the amino acid sequence of PAALGKDTIPWLGHLLVGLSGAFGFIILVYLLINCRNTGPWLKKVLKCNTPDPSKFFS QLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQQDKVPEPASL SLNTDAYLSLQELQ (SEQ ID NO: 28).
  • the IL-2Ry cytoplasmic domain comprises the amino acid sequence of ERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEIPPKGGA LGEGPGASPCNQHSPYWAPPCYTLKPET (SEQ ID NO: 12).
  • transmembrane domains include IL-2RP, IL-2RY, erythropoietin (Epo), and thrombopoietin (Tpo) transmembrane domains.
  • 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).
  • one CISC component comprises an IL-2RP transmembrane domain
  • the other CISC component comprises an IL-2RY transmembrane domain.
  • an IL-2RP transmembrane domain comprises the amino acid sequence of IPWLGHLLVGLSGAFGFIILVYLLI (SEQ ID NO: 13).
  • an IL-2RY transmembrane domain comprises the amino acid sequence of VVISVGSMGLIISLLCVYFWL (SEQ ID NO: 14).
  • 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.
  • an extracellular binding domain of one CISC component comprises an FKBP domain
  • an extracellular binding domain of the other CISC component comprises an FRB domain.
  • the CISC components form a heterodimer in the presence of the CISC inducer molecule.
  • 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).
  • an FKBP domain comprises an F36V substitution, at a position corresponding to F36 of SEQ ID NO: 15 or SEQ ID NO: 16.
  • F36V 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.
  • an FRB domain comprises an amino acid sequence of EMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLM EAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISK (SEQ ID NO: 17).
  • 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.
  • an FRB domain comprises a K71P substitution, at a position corresponding to K71 of SEQ ID NO: 17.
  • 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.
  • a soluble FRB domain comprises an amino acid sequence of EMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLM EAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISK (SEQ ID NO: 18).
  • the soluble FRB domain consists of the amino acid sequence of EMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLM EAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISK (SEQ ID NO: 18).
  • 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.
  • 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.
  • the glycine spacer comprises at least 3 glycines.
  • the glycine spacer comprises a sequence set forth as GGGS (SEQ ID NO: 8), GGGSGGG (SEQ ID NO: 9) or GGG.
  • 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.
  • the hinge domain positions the extracellular domain close to the plasma membrane to minimize the potential for recognition by antibodies or binding fragments thereof.
  • the extracellular binding domain is located N-terminal to the hinge domain.
  • the hinge domain may be natural or synthetic.
  • a CISC component comprises a portion of an IL-2Ry extracellular domain between the extracellular binding domain and transmembrane domain.
  • the portion of the IL-2Ry extracellular domain comprises the amino acid sequence of GSNTSKENPFLFALEA (SEQ ID NO: 19).
  • a CISC component comprises a portion of an IL-2R[3 extracellular domain between the extracellular binding domain and transmembrane domain.
  • the portion of the IL-2R[3 extracellular domain comprises the amino acid sequence of GKDT (SEQ ID NO: 20).
  • the portion of the IL-2Ry extracellular domain comprises the amino acid sequence of QNLVIPWAPENLTLHKLSESQLELNWNNRFLNHCLEHLVQYRTDWDHSWTEQSVD YRHKFSLPSVDGQKRYTFRVRSRFNPLCGSAQHWSEWSHPIHWGSNTSKENPFLFAL EA (SEQ ID NO: 21).
  • a CISC component comprises a portion of an IL-2R[3 extracellular domain between the extracellular binding domain and transmembrane domain.
  • the portion of the IL-2R[3 extracellular domain comprises the amino acid sequence of KPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAP LLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKD T (SEQ ID NO: 22).
  • the CISC inducer molecule is rapamycin or a rapalog. In some embodiments, the CISC inducer molecule is rapamycin.
  • 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.
  • 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).
  • the third CISC component is a soluble protein that does not comprise a transmembrane domain.
  • the third CISC component comprises an intracellular FRB domain.
  • a third CISC component is a soluble protein comprising an FRB domain and lacking a transmembrane domain.
  • Nucleic acids encoding a first, second, and/or third CISC component may be comprised in one or more vectors.
  • a nucleic acid encoding a first CISC component is present on a separate vector from a nucleic acid encoding the second CISC component.
  • 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.
  • 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.
  • one or more vectors are viral vectors.
  • 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.
  • AAV adeno- associated viral
  • 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.
  • 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)).
  • 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.
  • 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.
  • one or more CISC components comprise an LCN2 signal peptide.
  • one or more CISC components comprise a CD8a signal peptide.
  • one or more CISC components comprise a GM-CSFRa signal peptide.
  • both CISC components comprise an LCN2 signal peptide.
  • both CISC components comprise a CD8a signal peptide.
  • both CISC components comprise a GM-CSFRa signal peptide.
  • 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.
  • 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.
  • the third CISC component does not comprise a signal peptide.
  • the third CISC component does not comprise a transmembrane domain.
  • 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.
  • an endogenous FOXP3 locus is modified in a cell, resulting in stabilized expression.
  • 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.
  • 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.
  • Such stabilized FoxP3 expression by heterologous promoter insertion allows stable FoxP3 expression even in inflammatory conditions, preventing transdifferentiation into a T effector cell.
  • the heterologous promoter may be inserted at any position between the endogenous promoter and the first coding exon of the F0XP3 coding sequence.
  • 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.
  • 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.
  • 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.
  • 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.
  • the nucleic acid comprising a heterologous promoter is comprised on a vector.
  • the vector is a viral vector.
  • the viral vector is an adeno-associated virus (AAV) vector.
  • the AAV vector is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11 vector.
  • the AAV vector is an AAV5 vector.
  • the AAV vector is an AAV6 vector.
  • 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).
  • additional coding sequences 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).
  • the sequence encoding FoxP3 is a cDNA sequence that does not comprise an intron.
  • 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.
  • 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.
  • 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.
  • 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.
  • TCRa TCRa
  • TRBC TCRP
  • a nuclease capable of cleaving the genome at a targeted locus, or a nucleic acid encoding the nuclease is introduced into the cell.
  • the nuclease 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.
  • 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
  • the nuclease may be any suitable nuclease, including a meganuclease, zinc finger nuclease, TALEN, or RNA-guided nuclease.
  • 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.”
  • a nucleic acid comprising a heterologous promoter operably linked to a sequence encoding FoxP3 or a functional derivative thereof is present on a vector.
  • the vector is a viral vector.
  • the vector is a lentiviral vector.
  • the vector is an adeno-associated virus (AAV) vector.
  • the AAV vector is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11 vector.
  • the AAV vector is an AAV5 vector.
  • the AAV vector is an AAV6 vector.
  • 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.
  • the heterologous promoter inserted into the F0XP3 locus or operably linked to the F0XP3 coding sequence may be any suitable promoter.
  • the promoter is a constitutive promoter.
  • the promoter is an MND, PGK, or EF-la promoter.
  • the promoter is an MND promoter.
  • the promoter is an inducible promoter.
  • the promoter is inducible by a drug or steroid.
  • 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”).
  • 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).
  • a ligand e.g., a small molecule such as rapamycin or an analog thereof.
  • 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.
  • 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.
  • 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.
  • a chimeric signaling receptor comprises more than one linker.
  • a polypeptide of a chimeric signaling receptor further comprises a signal peptide.
  • the first extracellular domain and second extracellular domains are dimerization domains that dimerize in the presence of a ligand.
  • 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.
  • 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).
  • a ligand that activates a chimeric signaling receptor is rapamycin or a rapalog.
  • Rapamycin a macrocyclic triene compound
  • the ligand that activates a chimeric signaling receptor is wild-type rapamycin (e.g., commercially available rapamycin, such as RAPAMUNE®).
  • the ligand that activates a chimeric signaling receptor comprises rapamycin having 1, 2, 3, 4, 5, or more modifications relative to wild-type rapamycin.
  • the methyl side group chain of the rapamycin may be modified (e.g., extended).
  • 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.
  • 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).
  • FKBP FK-binding protein 12
  • FKBP12-Rapamycin Binding domain of mTOR FKBP12-Rapamycin Binding domain of mTOR
  • the sequence of the FKBP domain is: GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVI RGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE (SEQ ID NO: 6).
  • 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.
  • the FKBP domain comprises up to 20 amino acid substitutions, up to 15 amino acid sequence substitutions, up to 10 amino acid substitutions.
  • the FKBP domain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid sequence substitutions.
  • the sequence of the FRB domain is EMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLM EAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISK (SEQ ID NO: 7).
  • 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.
  • 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.
  • the FRB domain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid sequence substitutions.
  • the FRB domain comprises an T74L mutation.
  • a first extracellular domain of a chimeric signaling receptor comprises a functional fragment of FKBP, the rapamycin binding domain of FK-binding protein 12.
  • 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).
  • a chimeric signaling receptor controls signaling that is instigated, propagated, or maintained by IL-33 or IL-18.
  • 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
  • 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.
  • a first extracellular domain comprises FKBP or a functional fragment thereof and a second extracellular domain comprises FRB or a functional fragment thereof.
  • a first membrane proximate domain of a chimeric signaling receptor for controlling ST2 signaling comprises an intracellular signaling domain or functional fragment of ST2
  • a second membrane proximate domain of a chimeric signaling receptor for controlling ST2 signaling comprises an intracellular signaling domain or functional fragment of IL1RAP.
  • a first membrane proximate domain of a chimeric signaling receptor for controlling ST2 signaling comprises an intracellular signaling domain or functional fragment of IL1RAP
  • a second membrane proximate domain of a chimeric signaling receptor for controlling ST2 signaling comprises an intracellular signaling domain or functional fragment of ST2.
  • 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.
  • a first polypeptide further comprises a first membrane distal signaling domain
  • 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.
  • a first extracellular domain comprises FKBP or a functional fragment thereof and a second extracellular domain comprises FRB or a functional fragment thereof.
  • a first membrane proximate domain of a chimeric signaling receptor for controlling IL18R signaling comprises an intracellular signaling domain or functional fragment of IL18R
  • a second membrane proximate domain of a chimeric signaling receptor for controlling IL18R signaling comprises an intracellular signaling domain or functional fragment of IL18RAP.
  • 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
  • a second membrane proximate domain of a chimeric signaling receptor for controlling IL18R signaling comprises an intracellular signaling domain or functional fragment of IL18R.
  • chimeric receptors for continuous IL-33 signaling.
  • chimeric receptors spontaneously dimerize, causing IL-33 signal transduction.
  • the chimeric receptors in some embodiments, comprises a first polypeptide and a second polypeptide.
  • the first chimeric receptor comprises a first polypeptide.
  • the second chimeric receptor comprises a second polypeptide.
  • 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.
  • 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.
  • 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.
  • the first polypeptide comprises a first extracellular domain, a first transmembrane domain, and a first intracellular domain.
  • 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.
  • the first intracellular domain comprises at least one intracellular domain of an ST2 and/or IL1RAP, or functional fragment thereof.
  • the first intracellular domain comprises an ST2 domain.
  • the first intracellular domain comprises an IL1RAP domain.
  • the first intracellular domain comprises an ST2 and an IL1RAP domain.
  • the second polypeptide comprises a second extracellular domain, a second transmembrane domain, and a second intracellular domain.
  • 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.
  • the second intracellular domain comprises at least one intracellular domain of an ST2 and/or IL1RAP or functional fragment thereof.
  • the second intracellular domain comprises an ST2 domain.
  • the second intracellular domain comprises an IL1RAP domain.
  • the second intracellular domain comprises an ST2 and an IL1RAP domain.
  • 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.
  • 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.
  • a chimeric signaling receptor comprises more than one linker.
  • a polypeptide of a chimeric signaling receptor further comprises a signal peptide.
  • the first and second chimeric receptors dimerize.
  • 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).
  • 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.
  • 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.
  • the first extracellular domain is a tintin-Zl domain.
  • the second extracellular domain is a tintin-Z2 domain.
  • 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.
  • 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.
  • the first extracellular domain is a Barnase domain.
  • the second extracellular domain is a Barstar domain.
  • the first extracellular domain is a Barnase domain and the second extracellular domain is a Barstar domain.
  • the first extracellular domain is a human pancreatic RNAse domain.
  • the second extracellular domain is a S-peptide domain.
  • the first extracellular domain is a human pancreatic RNAse domain and the second extracellular domain is a S-peptide domain.
  • 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).
  • the first and second chimeric receptors dimerize in the presence of a ligand.
  • the first and second chimeric receptor dimerization is induced by a ligand.
  • the first and second polypeptides dimerize in the presence of a ligand. In some embodiments, the first and second polypeptides dimerization is induced by a ligand. In some embodiments, the first extracellular domain and second extracellular domain are dimerization domains that dimerize in the presence of a ligand. In some embodiments, the first extracellular domain and second extracellular domain dimerization is induced by 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 in the presence of a ligand. In some embodiments, a first extracellular domain is different from a second extracellular domain.
  • the first and second extracellular domains heterodimerize in the presence of a ligand.
  • the ligand may be a small molecule, peptide, protein, or other biologic (e.g., comprising base pairs). See, e.g., Dang et al., Front Chem. 2022 Feb 8;10:829312.
  • the first extracellular domain is a FKBP domain.
  • the second extracellular domain is a CyPFas domain.
  • FKCsA induces the dimerization of the FKBP and CyPFas domains. See, e.g., Belsaw et al, Proc Natl Acad Sci U S A. 1996 May 14;93(10):4604-7.
  • the first extracellular domain is a GAI domain.
  • the second extracellular domain is a GID1 domain.
  • Gibberellin induces the dimerization of the GAI and GID1 domains. See, e.g., Miyamoto et al, Nat Chem Biol. ; 8(5): 465-470.
  • the first extracellular domain is an ABI domain.
  • the second extracellular domain is a PYL domain.
  • abscisic acid induces the dimerization of the ABI and PYL domains. See, e.g., Liang et al, Sci Signal. 2011 Mar 15;4(164):rs2.
  • the first extracellular domain is an SNAPTag domain.
  • the second extracellular domain is a HaloTag domain.
  • HaXS induces the dimerization of the SNAPTag and HaloTag domains. See, e.g., Erhart et al. Chem Biol. 2013 Apr 18;20(4):549-57.
  • the first extracellular domain is an Bclxl domain.
  • the second extracellular domain is a fab (e.g., AZ1) domain.
  • ATB-737 induces the dimerization of the Bclxl and fab (e.g., AZ1) domains. See, e.g., Hill et al., Nat Chem Biol. 2018 Feb;14(2): 112-117.
  • the first extracellular domain is a FKBP domain.
  • the second extracellular domain is a FKBP domain.
  • FK1012 induces the dimerization of the first FKBP and second FKBP domains. See, e.g., Spencer et al. 1993 Nov 12;262(5136): 1019-24.
  • the first extracellular domain is a first Cyclophilin domain.
  • the second extracellular domain is a second Cyclophilin domain.
  • (Cyclosporin A)2 induces the dimerization of the first Cyclophilin and second Cyclophilin domains. See, e.g., Belshaw et al., Chem Biol. 1996 Sep;3(9):731-8.
  • the first extracellular domain is a first GyrB domain.
  • the second extracellular domain is a second GyrB domain.
  • Coumermycin induces the dimerization of the first GyrB and second GyrB domains. See, e.g., Karlsson et al., Sci Rep. 2013;3:2716.
  • the first extracellular domain is linked to the first transmembrane domain via an extracellular linker (e.g., a flexible linker) or an extracellular hinge domain.
  • an extracellular linker that links an extracellular domain and a transmembrane domain comprises a peptide (e.g., a human or artificial peptide).
  • an extracellular linker that links an extracellular domain and a transmembrane domain comprises a synthetic linker (e.g., a flexible linker) or an extracellular hinge domain.
  • the linker is a flexible linker or a rigid linker. In some embodiments, the linker is a flexible linker.
  • 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.
  • the Gly spacer comprises at least 3 Gly.
  • the Gly spacer comprises a sequence set forth as GGGS (SEQ ID NO: 8), GGGSGGG (SEQ ID NO: 9) or GGG.
  • 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.
  • the first extracellular domain is linked to a first transmembrane domain through (G4S)xN or the extracellular hinge of ST2 or IL1RAP. In some embodiments, the first extracellular domain is linked to a first transmembrane domain through (G4S)XN. In some embodiments, the first extracellular domain is linked to a first transmembrane domain through the extracellular hinge of ST2. In some embodiments, the first extracellular domain is linked to a first transmembrane domain through the extracellular hinge of IL1RAP. In some embodiments, the first transmembrane domain is the transmembrane domain of ST2 or functional fragment thereof. In some embodiments, the first transmembrane domain is the transmembrane domain of IL1RAP or functional fragment thereof.
  • the second extracellular domain is linked to a second transmembrane domain through (G4S)xN or the extracellular hinge of ST2 or IL1RAP. In some embodiments, the second extracellular domain is linked to a second transmembrane domain through (G4S)xN. In some embodiments, the second extracellular domain is linked to a second transmembrane domain through the extracellular hinge of ST2. In some embodiments, the second extracellular domain is linked to a second transmembrane domain through the extracellular hinge of ILlRAP.In some embodiments, the second transmembrane domain is the transmembrane domain of ST2 or functional fragment thereof. In some embodiments, the second transmembrane domain is the transmembrane domain of IL1RAP or functional fragment thereof.
  • the amino acid sequence of a chimeric receptor or portion thereof comprises 1-30, 1-25, 1-20, 1-15, 1-10, or 1-5 additions, deletions, or substitutions relative to an amino acid sequence.
  • the first and second polypeptides allow the cell to respond to dimerization of the first and second extracellular domains in a manner similar to IL-33 stimulation.
  • nucleic acids that comprise polynucleotides that encode any one of the polypeptides (e.g., the first polypeptide and/or the second polypeptide).
  • a nucleic acid comprises a polynucleotide that encodes a first polypeptide of any one of the chimeric receptors for inducible IL-33 signaling.
  • a nucleic acid comprises a polynucleotide that encodes a second polypeptide of any one of the chimeric receptors for inducible IL-33 signaling.
  • a nucleic acid comprises a first polynucleotide that encodes a first polypeptide of any one of the chimeric receptors for inducible IL-33 signaling and a second polynucleotide that encodes a second polypeptide of any one of the engineered chimeric receptors for inducible IL-33 signaling.
  • nucleic acids comprising polynucleotides encoding one or more polypeptides of a chimeric receptor for inducible IL-33 signaling, are comprised in a vector.
  • a vector may be a plasmid or a viral vector.
  • a vector is a DNA vector (e.g., a linear or circular dsDNA vector).
  • a vector is an RNA vector (e.g., linear or circular RNA vector).
  • a method of making such a cell comprises transfecting a cell with any one or more of the nucleic acids comprising polynucleotides encoding one or more polypeptides of the chimeric receptors for inducible IL-33 signaling. Transfection may comprise the use of chemical reagents (e.g., a cationic polymer), lipids, or physical methods such as microinjection or electroporation.
  • chemical reagents e.g., a cationic polymer
  • lipids lipids
  • physical methods such as microinjection or electroporation.
  • transfection comprises contacting cells with a viral vector comprising a polynucleotide encoding one or more the polypeptides of the chimeric receptors for inducible IL-33 signaling.
  • methods of making a cell further comprises selection of cells comprising both a first polypeptide and a second polypeptide of the chimeric receptors for inducible IL-33 signaling by use of a ligand. Such a selection method can be used to select cells having a level of expression of both a first polypeptide and a second polypeptide of the chimeric receptors for inducible IL-33 signaling that is above a threshold value.
  • the chimeric receptors for inducible IL-33 signaling are expressed on an immune cell having a regulatory phenotype.
  • the immune cell is a Treg (e.g., having a repair phenotype).
  • a pharmaceutical composition comprising a plurality of cells expressing any one of the polypeptides of the chimeric receptors for inducible IL-33 signaling. Some aspects relate to a pharmaceutical composition comprising one or more nucleic acids comprising polynucleotides encoding one or more polypeptides of the chimeric receptors for inducible IL-33 signaling. In some embodiments, a pharmaceutical composition comprising cells further comprises a pharmaceutically acceptable carrier, diluent, or excipient. In some embodiments, a pharmaceutical composition is formulated for administration to a subject via any one of numerous routes, e.g., intravenous infusion, or by direct injection into a particular tissue or organ.
  • Some embodiments of methods for producing engineered cells include introducing a nucleic acid encoding an antigen-specific receptor (e.g., T cell receptor (TCR) or chimeric antigen receptor (CAR)) polypeptide, or portion thereof (e.g., a TCRa chain or TCRP chain), into a cell.
  • an antigen-specific receptor e.g., T cell receptor (TCR) or chimeric antigen receptor (CAR)
  • cells comprise a nucleic acid encoding an antigen-specific receptor.
  • nucleic acids and vectors e.g, for in vivo administration
  • a nucleic acid is inserted into, or comprises homology arms for directing insertion into, a TRAC locus or TRBC locus.
  • a nucleic acid is inserted into or designed for insertion into the TRAC or TRBC locus to capture the endogenous promoter.
  • Promoter capture includes the introduction of an exogenous sequence into a locus such that its expression is driven by the endogenous promoter.
  • a cell may be edited (ex vivo or in vivo) by inserting a nucleic acid molecule comprising a nucleic acid encoding an exogenous TCR or CAR or portion thereof into the TRAC or TRBC locus, where the nucleic acid encoding the TCR or CAR is inserted downstream of (e.g., 11 to 10,000 bp downstream from) the endogenous TRAC or TRBC promoter, such that the endogenous TRAC or TRBC promoter becomes operably linked to the inserted nucleic acid and drives expression of the exogenous TCR or CAR.
  • a nucleic acid molecule comprising a nucleic acid encoding an exogenous TCR or CAR or portion thereof into the TRAC or TRBC locus
  • the nucleic acid encoding the TCR or CAR is inserted downstream of (e.g., 11 to 10,000 bp downstream from) the endogenous TRAC or TRBC promoter, such that the endogenous TRAC or TRBC promoter becomes operably linked
  • a nucleic acid is inserted into or designed for insertion into a TRAC or TRBC locus, such that insertion disrupts expression of the endogenous TCRa or TCRP chain.
  • the coding sequence of the endogenous TCRa or TCRP chain, or a portion of the coding sequence is removed from the locus such that the endogenous TCRa or TCRP is not expressed in the cell.
  • the inserted nucleic acid comprises a heterologous promoter that drives expression of the inserted TCR or CAR.
  • a nucleic acid is inserted into or designed for insertion into a TRAC or TRBC locus to hijack the endogenous TRAC or TRBC gene with a heterologous promoter.
  • a cell may be edited by inserting a polynucleotide molecule comprising a promoter operably linked to (a) a nucleic acid encoding a full-length TCRP protein, and to a nucleic acid encoding TCRa variable (TRAV) and TCR joining (TRAJ) regions, where the coding sequences of the TRAV and TRAJ regions are inserted in-frame with the coding sequences encoding the TCRa constant regions, such that the inserted heterologous promoter controls transcription of a heterologous TCRP protein and transcription of a TCRa protein comprising heterologous TRAV/TRAJ amino acid sequences and an endogenous TCRa constant region amino acid sequence.
  • TRAV TCRa variable
  • TRAJ TCR joining
  • This embodiment utilizes the endogenous 3’ regulatory region from the endogenous TRAC gene.
  • a similar approach may be used to hijack the endogenous TRBC locus, where the encoded full-length protein is a TCRa chain, and the nucleic acid further encodes TCRP variable and TCRP joining regions in-frame with TCRP constant regions.
  • a nucleic acid is inserted into a TRAC or TRBC locus without modifying the encoded amino acid sequence of the TCRa or TCRP protein, respectively. Insertion into a targeted locus may facilitate detection and/or sequencing of the modified locus (e.g., using targeted amplification), while maintaining specificity of the endogenous TCR.
  • a cell may be modified, while retaining expression of an endogenous TCR, which can allow for stimulation through exposure to a cognate peptide of the TCR and/or other suitable means of inducing TCR signal transduction, such as contact with anti-CD3/anti- CD28 reagents (e.g., beads or antibodies).
  • the amino acid sequence of the endogenous TCR is unmodified, and an inserted nucleic acid comprises a nucleotide sequence encoding a CAR.
  • an antigen-specific receptor is expressed episomally in a cell.
  • Episomal expression may be achieved by any suitable method, such as delivery of an RNA (e.g., mRNA or self-amplifying RNA) or DNA (e.g., plasmid or artificial chromosome) encoding the antigen-specific receptor.
  • RNA e.g., mRNA or self-amplifying RNA
  • DNA e.g., plasmid or artificial chromosome
  • the promoter may be any suitable promoter.
  • the promoter is a constitutive promoter.
  • the promoter is an MND promoter.
  • compositions and methods for engineering repair Tregs by inducing IL-33 signaling in cells relate to compositions and methods for engineering repair Tregs by inducing IL-33 signaling in cells.
  • the method for inducing IL-33 signaling comprises culturing T cells in the presence of IL-33.
  • the T cells are Tregs.
  • the Tregs are genetically modified Tregs.
  • genetic modification is the introduction of a nucleic acid into a cell. In some embodiments, the genetic modification is introduction of a nucleic acid by infection with a viral vector into a cell. In some embodiments, the genetic modification is introduction of an exogenous nucleic acid by electroporation into a cell. In some embodiments, the genetic modification is introduction of a nucleic acid by transfection into a cell. In some embodiments, the genetic modification is the introduction of a nucleic acid by transfection with lipid nanoparticles into a cell. In some embodiments, the genetic modification is the introduction of a nucleic acid with polymer nanoparticles into a cell.
  • the genetic modification is the introduction of a nucleic acid with calcium phosphate nano particles into a cell. In some embodiments, the genetic modification is the introduction of a nucleic acid by electroporation into a cell. In some embodiments, the genetic modification is the introduction of a nucleic acid by nucleofection into a cell. In some embodiments, the genetic modification is the introduction of a nucleic acid by biolistic delivery of DNA-coated microparticles into a cell.
  • genetic modification is the introduction of a vector into a cell. In some embodiments, the genetic modification is introduction of a vector by infection with a viral vector into a cell. In some embodiments, the genetic modification is introduction of a vector by electroporation into a cell. In some embodiments, the genetic modification is introduction of a nucleic acid by transfection into a cell. In some embodiments, the genetic modification is the introduction of a vector by transfection with lipid nanoparticles into a cell. In some embodiments, the genetic modification is the introduction of a vector with polymer nanoparticles into a cell. In some embodiments, the genetic modification is the introduction of a vector with calcium phosphate nano particles into a cell.
  • the genetic modification is the introduction of a vector by electroporation into a cell. In some embodiments, the genetic modification is the introduction of a vector by nucleofection into a cell. In some embodiments, the genetic modification is the introduction of a vector by biolistic delivery of DNA-coated microparticles into a cell.
  • genetic modification is the introduction of a nucleic acid into a cell.
  • Nucleic acids may be introduced into a cell using any suitable method.
  • genetic modification is the introduction of a vector into a cell.
  • Vectors may be introduced into a cell using any suitable method.
  • 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. Any suitable method may be used for gene editing.
  • RNA-guided nuclease e.g., Cas (e.g., Cas9) nuclease
  • ZFN zinc finger nuclease
  • TALEN transcription activator-like effector nuclease
  • meganuclease e.g., TALEN
  • a cell is transduced with a lentivirus (e.g., lentivirus-mediated gene editing).
  • the cell is transduced with a lentivirus comprising a promoter operably linked to a sequence encoding ST2 and/or IL 1 RAP.
  • the cell is transduced with a lentivirus comprising a promoter operably linked to a sequence encoding ST2.
  • the cell is transduced with a lentivirus encoding promotor operably linked to a sequence encoding IL1RAP.
  • the cell is transduced with a lentivirus comprising a promoter operably linked to a sequence encoding IL-18R1 and/or IL18RAP. In some embodiments, the cell is transduced with a lentivirus comprising a promoter operably linked to a sequence encoding IL-18R1. In some embodiments, the cell is transduced with a lentivirus comprising a promoter operably linked to a sequence encoding IL18RAP.
  • cells are transduced with a lentivirus no more than 24, no more than 18, no more than 12, no more than 8, no more than 6, no more than 4, or no more than 2 hours after CD3 and/or CD28 stimulation. Any suitable method may be used for CD3 and/or CD28 stimulation.
  • cells are exposed to anti-CD3/anti-CD28 beads.
  • cells are exposed to anti-CD3 beads.
  • cells are exposed to anti-CD28 beads.
  • cells are exposed to anti-CD3/anti-CD28 antibodies.
  • cells are exposed to anti-CD3 antibodies.
  • cells are exposed to anti-CD28 antibodies.
  • cells are transduced with a lentivirus 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 , 14, 15, 16 or 17 days after stimulation. In some embodiments, cells are transduced with a lentivirus 0-2, 2-4, 4-6, 6-8, 8-10, 10-12, 12-14, or 14-16 days after stimulation. In some embodiments, cells are transduced with a lentivirus 1-17, 2-17, 3-17, 4- 17, 5-17, 6-17, 7-17, 8-17, 9-17, 10-17, 11-17, 12-17, 13-17, 14-17, 15-17, or 16-17 days after stimulation.
  • cells are transduced with a lentivirus 0-17, 0-16, 0- 15, 0-14, 0-13, 0-12, 0-11, 0-10, 0-9, 0-8, 0-7, 0-6, 0-5, 0-4, 0-3, 0-2, or 0-1 days after stimulation.
  • cells are transduced with a lentivirus 0, 1, 2, or 3 weeks after stimulation.
  • cells are transduced with a lentivirus 0-3, 0-2, 0-1 weeks after stimulation.
  • cells are transduced with a lentivirus 1-3 or 2-3 weeks after stimulation.
  • cells are transduced with a lentivirus 0-1, 1-2, or 2-3 weeks after stimulation.
  • 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.
  • the T cells are cultured with a concentration of IL-33 between 0.1 to 500 ng/mL of IL-33.
  • T cells are cultured in the presence of 0.1 ng/ml, 0.2 ng/ml, 0.3 ng/ml, 0.4 ng/ml, 0.5 ng/mL, 1.0 ng/mL, 1.5 ng/mL, 2.0 ng/ml, 2.5 ng/mL, 3.0 ng/mL,3.5 ng/mL, 4.0 ng/mL, 4.5 ng/mL, 5.0 ng/mL, 5.5 ng/mL, 6.0 ng/mL, 6.5 ng/mL, 7.0 ng/mL, 7.5 ng/mL, 8.0 ng/mL, 8.5 ng/mL, 9.0 ng/mL, 9.5 ng/mL, 10.0 ng/mL, 15.0 ng/mL, 20 ng/mL, 25 ng/mL, 25
  • T cells are cultured in the presence of IL-33 with a concentration of 0.1-500 ng/ml, 0.5-500 ng/mL, 1.0-500 ng/mL, 5.0-500 ng/mL, 10-500 ng/mL, 20-500 ng/mL, 30-500 ng/mL, 40-500 ng/mL, 50-500 ng/mL, 60-500 ng/mL, 70-500 ng/mL, 80-500 ng/mL, 90-500 ng/mL, 100-500 ng/mL, 110-500 ng/mL, 120-500 ng/mL, 130-500 ng/mL, 140-500 ng/mL, 150-500 ng/mL, 160-500 ng/mL, 170-500 ng/mL, 180-500 ng/mL, 190-500 ng/mL, 200-500 ng/mL, 210-500 ng/mL, 220-500
  • T cells are cultured in the presence of IL-33 with a concentration of 0.1-0.5 ng/ml, 0.1-1.0 ng/mL, 0.1-1.5 ng/mL, 0.1- 2.0 ng/mL, 0.1-2.5 ng/mL, 0.1-3.0 ng/mL, 0.1-3.5 ng/mL, 0.1-4.0 ng/mL, 0.1-4.5 ng/mL, 0.1- 5.0 ng/mL, 0.1-6.0 ng/mL, 0.1-7.0 ng/mL, 0.1-8.0 ng/mL, 0.1-9.0 ng/mL, 0.1-10 ng/mL, 0.1- 20 ng/mL, 0.1-30 ng/mL, 0.1-40 ng/mL, 0.1-50 ng/mL, 0.1-60 ng/mL, 0.1-70 ng/mL, 0.1-80 ng/mL, 0.1-90 ng/mL, 0.1-100
  • T cells are cultured in the presence of IL-33 with a concentration of 0.5-450 ng/ml, 1.0-450 ng/mL, 10-450 ng/mL, 20-450 ng/mL, 30-450 ng/mL, 40-450 ng/mL, 50-450 ng/mL, 100-450 ng/mL, 150-450 ng/mL, 200-450 ng/mL, 250-450 ng/mL, 300-450 ng/mL, 350-450 ng/mL, or 400-450 ng/mL.
  • T cells are cultured in the presence of IL-33 with a concentration of 0.5-1.0 ng/ml, 1.0-10 ng/ml, 10-100 ng/mL, 50-100 ng/mL, 100-200 ng/mL, 150-250 ng/mL, 200-300 ng/mL, 250-350 ng/mL, or 350-400 ng/mL. In some embodiments, T cells are cultured in the presence of IL-33 with a concentration of 50 ng/mL.
  • T cells are cultured in the presence of IL-33 for between 1-20 days. In some embodiments, T cells are cultured in the presence of IL-33 for less than 1 day. In some embodiments, T cells are cultured in the presence of IL-33 for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days. In some embodiments, T cells are cultured in the presence of IL-33 for 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-13, 1-14,
  • T cells are cultured in the presence of IL-33 for 2-20, 3-30, 4-20, 5-20, 6-20, 7-20, 8-20, 9-20, 10-20, 11-20, 12-20, 13- 20, 14-20, 15-20, 16-20, 17-20, 18-20, or 19-20 days.
  • T cells are cultured in the presence of IL-33 for 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 2-11, 2-12, 2-13,
  • T cells are cultured in the presence of IL-33 for 3-4, 4-5, 5-6, 6-7, 7-8, 8-9, 9-10, 10-11, 11-12, 12-13, 13-14, 14-15, 15-16, 16-17, 17-18, or 18-19 days.
  • T cells are cultured in the presence of IL-33 for 1-5, 5-10, 10-15, or 15-20 days.
  • T cells are cultured in the presence of IL-33 for 1-4, 4-7, 7-10, 10-13, 13-16, or 16-19 days.
  • T cells are cultured in the presence of IL-33 for 14-16 days.
  • T cells are cultured in the presence of IL-33 before genetic modification. In some embodiments, T cells are cultured in the presence of IL-33 1-7 days before genetic modification. In some embodiments, T cells are cultured in the presence of IL- 33 1, 2, 3, 4, 5, 6, or 7 days before genetic modification. In some embodiments, T cells are cultured in the presence of IL-33 1-2, 1-3, 1-4, 1-5, or 1-6 days before genetic modification. In some embodiments, T cells are cultured in the presence of IL-33 2-7, 3-7, 4-7, 5-7, or 6-7 days before genetic modification. In some embodiments, T cells are cultured in the presence of IL-33 1-3, 2-4, 3-5, 4-6, or 5-7 days before genetic modification. In some embodiments, T cells are cultured in the presence of IL-33 at least 7 days before genetic modification.
  • T cells are cultured in the presence of IL-33 after genetic modification. In some embodiments, T cells are cultured in the presence of IL-33 1-7 days after genetic modification. In some embodiments, T cells are cultured in the presence of IL- 33 1, 2, 3, 4, 5, 6, or 7 days after genetic modification. In some embodiments, T cells are cultured in the presence of IL-33 1-2, 1-3, 1-4, 1-5, or 1-6 days before after modification. In some embodiments, T cells are cultured in the presence of IL-33 2-7, 3-7, 4-7, 5-7, or 6-7 days after genetic modification. In some embodiments, T cells are cultured in the presence of IL-33 1-3, 2-4, 3-5, 4-6, or 5-7 days after genetic modification. In some embodiments, T cells are cultured in the presence of IL-33 at least 7 days after genetic modification.
  • the cells are cultured in the presence of IL-33 and one or more additional cytokines. In some embodiments, the cells are cultured with IL-33 and TNF-a. In some embodiments, the cells are cultured with IL-33 and IL-2. In some embodiments, the cells are cultured with IL-33 and IL-4.
  • the cells are cultured in the presence of IL-18 and one or more additional cytokines. In some embodiments, the cells are cultured with IL-18 and TNF-a. In some embodiments, the cells are cultured with IL- 18 and IL-2. In some embodiments, the cells are cultured with IL-18 and IL-4.
  • T cells are cultured in the presence of rapamycin.
  • the T cells are Tregs.
  • the Tregs are genetically modified Tregs (e.g., engineered Tregs).
  • the engineered Tregs comprise a nucleic acid encoding a first CISC component and a nucleic acid encoding a second CISC component.
  • the first CISC component comprises in N-to-C-terminal order, and FKBP domain, an interleukin-2 receptor gamma (IL-2Ry) transmembrane domain, and an IL-2Ry cytoplasmic domain.
  • 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.
  • IL-2RP interleukin-2 receptor beta
  • T cells are cultured in the presence of rapamycin for between 1-20 days. In some embodiments, T cells are cultured in the presence of rapamycin for less than 1 day. In some embodiments, T cells are cultured in the presence of rapamycin for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days. In some embodiments, T cells are cultured in the presence of rapamycin for 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-13, 1-14, 1-15, 1-16, 1-17, 1-18, or 1-19 days.
  • T cells are cultured in the presence of rapamycin for 2-20, 3-30, 4-20, 5-20, 6-20, 7-20, 8-20, 9-20, 10-20, 11-20, 12-20, 13-20, 14-20, 15-20, 16-20, 17-20, 18-20, or 19-20 days. In some embodiments, T cells are cultured in the presence of rapamycin for 2-3, 2-4, 2-5, 2-6, 2-7, 2-
  • T cells are cultured in the presence of rapamycin for 3-4, 4-5, 5-6, 6-7, 7-8, 8-
  • T cells are cultured in the presence of rapamycin for 1-5, 5-10, 10-15, or 15-20 days. In some embodiments, T cells are cultured in the presence of rapamycin for 1-4, 4-7, 7-
  • T cells are cultured in the presence of rapamycin for 14-16 days.
  • T cells are cultured in the presence of rapamycin before genetic modification. In some embodiments, T cells are cultured in the presence of rapamycin 1-7 days before genetic modification. In some embodiments, T cells are cultured in the presence of rapamycin 1, 2, 3, 4, 5, 6, or 7 days before genetic modification. In some embodiments, T cells are cultured in the presence of rapamycin 1-2, 1-3, 1-4, 1-5, or 1-6 days before genetic modification. In some embodiments, T cells are cultured in the presence of rapamycin 2-7, 3-7, 4-7, 5-7, or 6-7 days before genetic modification. In some embodiments, T cells are cultured in the presence of rapamycin 1-3, 2-4, 3-5, 4-6, or 5-7 days before genetic modification. In some embodiments, T cells are cultured in the presence of rapamycin at least 7 days before genetic modification.
  • T cells are cultured in the presence of rapamycin after genetic modification. In some embodiments, T cells are cultured in the presence of rapamycin 1-7 days after genetic modification. In some embodiments, T cells are cultured in the presence of rapamycin 1, 2, 3, 4, 5, 6, or 7 days after genetic modification. In some embodiments, T cells are cultured in the presence of rapamycin 1-2, 1-3, 1-4, 1-5, or 1-6 days after genetic modification. In some embodiments, T cells are cultured in the presence of rapamycin 2-7, 3- 7, 4-7, 5-7, or 6-7 days after genetic modification. In some embodiments, T cells are cultured in the presence of rapamycin 1-3, 2-4, 3-5, 4-6, or 5-7 days after genetic modification. In some embodiments, T cells are cultured in the presence of rapamycin at least 7 days after genetic modification.
  • the T cells are cultured in the presence of rapamycin with a concentration between O.Ol-lOOnM rapamycin. In some embodiments, the T cells are cultured in the presence of rapamycin with a concentration of 0.01 nM, 0.02 nM, 0.03 nM, 0.04 nM, 0.05 nM, 0.06 nM, 0.07 nM, 0.08 nM, 0.09 nM, 0.1 nM, 0.2 nM, 0.3 nM, 0.4 nM, 0.5 nM, 0.6 nM, 0.7 nM, 0.8 nM, 0.9 nM, 1.0 nM, 1.5 nM, 2.0 nM, 2.5 nM, 3.0 nM, 3.5 nM, 4.0 nM, 4.5 nM, 5.0 nM, 5.5 nM, 6.0 nM, 6.5 nM, 7.0 nM, 7.5 nM, 8.0 n
  • the T cells are cultured in the presence of rapamycin with a concentration of 0.01-0.1 nM, 0.1-1 nM, 1-10 nM, or 10-100 nM. In some embodiments, the T cells are cultured in the presence of rapamycin with a concentration of 0.01-0.02 nM, 0.02- 0.03 nM, 0.03-0.04 nM, 0.04-0.05 nM, 0.05-0.06 nM, 0.06-0.07 nM, 0.07-0.08 nM, 0.08-0.09 nM, 0.09-0.1 nM, 0.1-0.2 nM, 0.2-0.3 nM, 0.3-0.4 nM, 0.4-0.5 nM, 0.5-0.6 nM, 0.6-0.7 nM, 0.7-0.8 nM, 0.8-0.9 nM, 0.9-1 nM, 1-2 nM, 2-3 nM, 3-4 nM, 4-5 nM, 5-6 nM
  • the T cells are cultured in the presence of rapamycin with a concentration of between 0.01-0.05 nM, 0.05-0.1 nM, 1-5 nM, or 5-10 nM. In some embodiments, the T cells are cultured in the presence of rapamycin with a concentration of 10 nM.
  • T cells are cultured in the presence of anti-CD3 antibodies and anti-CD28 antibodies. In some embodiments, T cells are cultured the presence of anti-CD3 antibodies. In some embodiments, T cells are cultured the presence of anti-CD28 antibodies. See e.g., Riddell and Greenberg J Immunol Methods. 1990 Apr 17; 128(2): 189-201.
  • T cells are cultured the presence of anti-CD3 beads and anti- CD28 beads. In some embodiments, T cells are cultured the presence of anti-CD3 beads. In some embodiments, T cells are cultured the presence of anti-CD28 beads. See e.g., Trickett et al., J Immunol Methods. 2003 Apr l;275(l-2):251-5 and Kalamasz et al. J Immunother. 2004 Sep-Oct;27(5):405-18.
  • T cells are cultured the presence of anti-CD3 antibodies before genetic modification. In some embodiments, T cells are cultured the presence of anti-CD3 antibodies 1-7 days before genetic modification. In some embodiments, T cells are cultured in the presence of anti-CD3 antibodies 1, 2, 3, 4, 5, 6, or 7 days before genetic modification. In some embodiments, T cells are cultured in the presence of anti-CD3 antibodies 1-2, 1-3, 1- 4, 1-5, or 1-6 days before genetic modification. In some embodiments, T cells are cultured in the presence of anti-CD3 antibodies 2-7, 3-7, 4-7, 5-7, or 6-7 days before genetic modification.
  • T cells are cultured in the presence of anti-CD3 antibodies 1-3, 2-4, 3-5, 4-6, or 5-7 days before genetic modification. In some embodiments, T cells are cultured in the presence of anti-CD3 antibodies at least 7 days before genetic modification.
  • T cells are cultured in the presence of anti-CD3 antibodies after genetic modification. In some embodiments, T cells are cultured in the presence of anti- CD3 antibodies 1-7 days after genetic modification. In some embodiments, T cells are cultured in the presence of anti-CD3 antibodies 1, 2, 3, 4, 5, 6, or 7 days after genetic modification. In some embodiments, T cells are cultured in the presence of anti-CD3 antibodies 1-2, 1-3, 1-4, 1-5, or 1-6 days after genetic modification. In some embodiments, T cells are cultured in the presence of anti-CD3 antibodies 2-7, 3-7, 4-7, 5-7, or 6-7 days after genetic modification.
  • T cells are cultured in the presence of anti-CD3 antibodies 1-3, 2-4, 3-5, 4-6, or 5-7 days after genetic modification. In some embodiments, T cells are cultured in the presence of anti-CD3 antibodies at least 7 days after genetic modification.
  • T cells are cultured in the presence of anti-CD28 antibody before genetic modification. In some embodiments, T cells are cultured in the presence of anti-CD28 antibodies 1-7 days before genetic modification. In some embodiments, T cells are cultured in the presence of anti-CD28 antibodies 1, 2, 3, 4, 5, 6, or 7 days before genetic modification. In some embodiments, T cells are cultured in the presence of anti-CD28 antibodies 1-2, 1-3, 1-4, 1-5, or 1-6 days before genetic modification. In some embodiments, T cells are cultured in the presence of anti-CD28 antibodies 2-7, 3-7, 4-7, 5-7, or 6-7 days before genetic modification.
  • T cells are cultured in the presence of anti-CD28 antibodies 1-3, 2-4, 3-5, 4-6, or 5-7 days before genetic modification. In some embodiments, T cells are cultured in the presence of anti-CD28 antibodies at least 7 days before genetic modification.
  • T cells are cultured in the presence of anti-CD28 antibody after genetic modification. In some embodiments, T cells are cultured in the presence of anti- CD28 antibodies 1-7 days after genetic modification. In some embodiments, T cells are cultured in the presence of anti-CD28 antibodies 1, 2, 3, 4, 5, 6, or 7 days after genetic modification. In some embodiments, T cells are cultured in the presence of anti-CD28 antibodies 1-2, 1-3, 1-4, 1-5, or 1-6 days after genetic modification. In some embodiments, T cells are cultured in the presence of anti-CD28 antibodies 2-7, 3-7, 4-7, 5-7, or 6-7 days after genetic modification.
  • T cells are cultured in the presence of anti-CD28 antibodies 1-3, 2-4, 3-5, 4-6, or 5-7 days after genetic modification. In some embodiments, T cells are cultured in the presence of anti-CD28 antibodies at least 7 after before genetic modification.
  • T cells are cultured in the presence of anti-CD3 beads before genetic modification. In some embodiments, T cells are cultured in the presence of anti-CD3 beads 1-7 days before genetic modification. In some embodiments, T cells are cultured in the presence of anti-CD3 beads 1, 2, 3, 4, 5, 6, or 7 days before genetic modification. In some embodiments, T cells are cultured in the presence of anti-CD3 beads 1-2, 1-3, 1-4, 1-5, or 1-6 days before genetic modification. In some embodiments, T cells are cultured in the presence of anti-CD3 beads 2-7, 3-7, 4-7, 5-7, or 6-7 days before genetic modification.
  • T cells are cultured in the presence of anti-CD3 beads 1-3, 2-4, 3-5, 4-6, or 5-7 days before genetic modification. In some embodiments, T cells are cultured in the presence of anti-CD3 beads at least 7 days before genetic modification.
  • T cells are cultured in the presence of anti-CD3 beads after genetic modification. In some embodiments, T cells are cultured in the presence of anti-CD3 beads 1-7 days after genetic modification. In some embodiments, T cells are cultured with anti-CD3 beads 1, 2, 3, 4, 5, 6, or 7 days after genetic modification. In some embodiments, T cells are cultured in the presence of anti-CD3 beads 1-2, 1-3, 1-4, 1-5, or 1-6 days after genetic modification. In some embodiments, T cells are cultured in the presence of anti-CD3 beads 2-7, 3-7, 4-7, 5-7, or 6-7 days after genetic modification.
  • T cells are cultured in the presence of anti-CD3 beads 1-3, 2-4, 3-5, 4-6, or 5-7 days after genetic modification. In some embodiments, T cells are cultured in the presence of anti-CD3 beads at least 7 days after genetic modification. In some embodiments, T cells are cultured in the presence of anti-CD28 beads before genetic modification. In some embodiments, T cells are cultured in the presence of anti-CD28 beads 1-7 days before genetic modification. In some embodiments, T cells are cultured in the presence of anti-CD28 beads 1, 2, 3, 4, 5, 6, or 7 days before genetic modification. In some embodiments, T cells are cultured in the presence of anti-CD28 beads 1-2, 1-3, 1-4, 1-5, or 1- 6 days before genetic modification.
  • T cells are cultured in the presence of anti-CD28 beads 2-7, 3-7, 4-7, 5-7, or 6-7 days before genetic modification. In some embodiments, T cells are cultured in the presence of anti-CD28 beads 1-3, 2-4, 3-5, 4-6, or 5-7 days before genetic modification. In some embodiments, T cells are cultured in the presence of anti-CD28 beads at least 7 days before genetic modification.
  • T cells are cultured in the presence of anti-CD28 beads after genetic modification. In some embodiments, T cells are cultured in the presence of anti-CD28 beads 1-7 days after genetic modification. In some embodiments, T cells are cultured in the presence of anti-CD28 beads 1, 2, 3, 4, 5, 6, or 7 days after genetic modification. In some embodiments, T cells are cultured in the presence of anti-CD28 beads 1-2, 1-3, 1-4, 1-5, or 1-
  • T cells are cultured in the presence of anti-CD28 beads 2-7, 3-7, 4-7, 5-7, or 6-7 days after genetic modification. In some embodiments, T cells are cultured in the presence of anti-CD28 beads 1-3, 2-4, 3-5, 4-6, or 5-
  • T cells are cultured in the presence of anti-CD28 beads at least 7 days after genetic modification.
  • the T cells are cultured with a concentration of IL- 18 between 0.1 to 500 ng/mL of IL-18. In some embodiments, T cells are cultured in the presence of 0.1 ng/ml, 0.2 ng/ml, 0.3 ng/ml, 0.4 ng/ml, 0.5 ng/mL, 1.0 ng/mL, 1.5 ng/mL, 2.0 ng/ml, 2.5 ng/mL, 3.0 ng/mL,3.5 ng/mL, 4.0 ng/mL, 4.5 ng/mL, 5.0 ng/mL, 5.5 ng/mL, 6.0 ng/mL, 6.5 ng/mL, 7.0 ng/mL, 7.5 ng/mL, 8.0 ng/mL, 8.5 ng/mL, 9.0 ng/mL, 9.5 ng/mL, 10.0 ng/mL, 15.0 ng/mL, 20 ng/mL, 25 ng
  • T cells are cultured in the presence of IL-18 with a concentration of 0.1-500ng/ml, 0.5-500 ng/mL, 1.0-500 ng/mL, 5.0-500 ng/mL, 10-500 ng/mL, 20-500 ng/mL, 30-500 ng/mL, 40-500 ng/mL, 50-500 ng/mL, 60-500 ng/mL, 70-500 ng/mL, 80-500 ng/mL, 90-500 ng/mL, 100-500 ng/mL, 110-500 ng/mL, 120-500 ng/mL, 130-500 ng/mL, 140-500 ng/mL, 150-500 ng/mL, 160-500 ng/mL, 170-500 ng/mL, 180-500 ng/mL, 190-500 ng/mL, 200-500 ng/mL, 210-500 ng/mL, 220-500 ng/mL,
  • T cells are cultured in the presence of IL-18 with a concentration of 0.1-0.5 ng/ml, 0.1-1.0 ng/mL, 0.1-1.5 ng/mL, 0.1- 2.0 ng/mL, 0.1-2.5 ng/mL, 0.1-3.0 ng/mL, 0.1-3.5 ng/mL, 0.1-4.0 ng/mL, 0.1-4.5 ng/mL, 0.1- 5.0 ng/mL, 0.1-6.0 ng/mL, 0.1-7.0 ng/mL, 0.1-8.0 ng/mL, 0.1-9.0 ng/mL, 0.1-10 ng/mL, 0.1- 20 ng/mL, 0.1-30 ng/mL, 0.1-40 ng/mL, 0.1-50 ng/mL, 0.1-60 ng/mL, 0.1-70 ng/mL, 0.1-80 ng/mL, 0.1-90 ng/mL, 0.1-100 ng
  • T cells are cultured in the presence of IL-18 with a concentration of 0.5-450 ng/ml, 1.0-450 ng/mL, 10-450 ng/mL, 20-450 ng/mL, 30-450 ng/mL, 40-450 ng/mL, 50-450 ng/mL, 100-450 ng/mL, 150-450 ng/mL, 200-450 ng/mL, 250-450 ng/mL, 300-450 ng/mL, 350-450 ng/mL, or 400-450 ng/mL.
  • T cells are cultured in the presence of IL- 18 with a concentration of 0.5-1.0 ng/ml, 1.0-10 ng/ml, 10-100 ng/mL, 50-100 ng/mL, 100-200 ng/mL, 150-250 ng/mL, 200-300 ng/mL, 250-350 ng/mL, or 350-400 ng/mL. In some embodiments, T cells are cultured in the presence of IL- 18 with a concentration of 50 ng/mL.
  • T cells are cultured in the presence of IL- 18 for between 1-20 days. In some embodiments, T cells are cultured in the presence of IL- 18 for less than 1 day. In some embodiments, T cells are cultured in the presence of IL-18 for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days. In some embodiments, T cells are cultured in the presence of IL- 18 for 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-13, 1-14,
  • T cells are cultured in the presence of IL-18 for 2-20, 3-30, 4-20, 5-20, 6-20, 7-20, 8-20, 9-20, 10-20, 11-20, 12-20, 13- 20, 14-20, 15-20, 16-20, 17-20, 18-20, or 19-20 days.
  • T cells are cultured in the presence of IL-18 for 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 2-11, 2-12, 2-13,
  • T cells are cultured in the presence of IL- 18 for 3-4, 4-5, 5-6, 6-7, 7-8, 8-9, 9-10, 10-11, 11-12, 12-13, 13-14, 14-15, 15-16, 16-17, 17-18, or 18-19 days.
  • T cells are cultured in the presence of IL-18 for 1-5, 5-10, 10-15, or 15-20 days.
  • T cells are cultured in the presence of IL-18 for 1-4, 4-7, 7-10, 10-13, 13-16, or 16-19 days.
  • T cells are cultured in the presence of IL-18 for 14-16 days.
  • T cells are cultured in the presence of IL-18 before genetic modification. In some embodiments, T cells are cultured in the presence of IL- 18 1-7 days before genetic modification. In some embodiments, T cells are cultured in the presence of IL- 18 1, 2, 3, 4, 5, 6, or 7 days before genetic modification. In some embodiments, T cells are cultured in the presence of IL-18 1-2, 1-3, 1-4, 1-5, or 1-6 days before genetic modification. In some embodiments, T cells are cultured in the presence of IL-18 2-7, 3-7, 4-7, 5-7, or 6-7 days before genetic modification. In some embodiments, T cells are cultured in the presence of IL-18 1-3, 2-4, 3-5, 4-6, or 5-7 days before genetic modification. In some embodiments, T cells are cultured in the presence of IL- 18 at least 7 days before genetic modification.
  • T cells are cultured in the presence of IL- 18 after genetic modification. In some embodiments, T cells are cultured in the presence of IL- 18 1-7 days after genetic modification. In some embodiments, T cells are cultured in the presence of IL- 18 1, 2, 3, 4, 5, 6, or 7 days after genetic modification. In some embodiments, T cells are cultured in the presence of IL-18 1-2, 1-3, 1-4, 1-5, or 1-6 days before after modification. In some embodiments, T cells are cultured in the presence of IL-18 2-7, 3-7, 4-7, 5-7, or 6-7 days after genetic modification. In some embodiments, T cells are cultured in the presence of IL-18 1-3, 2-4, 3-5, 4-6, or 5-7 days after genetic modification. In some embodiments, T cells are cultured in the presence of IL-18 at least 7 days after genetic modification.
  • T cells are cultured in the presence of one or more cytokines.
  • the cytokines are IL-2, IL-12, IL-21, IL-23, and/or TGF-p.
  • T cells are cultured in the presence of IL-2.
  • T cells are cultured in the presence of IL-12.
  • T cells are cultured in the presence of IL-21.
  • T cells are cultured in the presence of IL-23.
  • T cells are cultured in the presence of TGF-p.
  • the method for inducing IL-33 signaling comprises a composition of T cells and IL-33.
  • the T cells are Tregs.
  • the Tregs are engineered Tregs.
  • the method for inducing IL- 18 signaling comprises a composition of T cells and IL-18.
  • the T cells are Tregs.
  • the Tregs are engineered Tregs.
  • engineered Tregs are genetically modified.
  • engineered Tregs are genetically modified Tregs.
  • genetic modification is the introduction of a nucleic acid (e.g., one or more nucleic acids) into a cell. Nucleic acids may be introduced into a cell by any suitable method. In some embodiments, the genetic modification is introduction of a nucleic acid by infection with a viral vector into a cell.
  • the genetic modification is introduction of an exogenous nucleic acid by electroporation into a cell. In some embodiments, the genetic modification is introduction of a nucleic acid by transfection into a cell. In some embodiments, the genetic modification is the introduction of a nucleic acid by transfection with lipid nanoparticles into a cell. In some embodiments, the genetic modification is the introduction of a nucleic acid with polymeric nanoparticles into a cell. In some embodiments, the genetic modification is the introduction of a nucleic acid with calcium phosphate nanoparticles into a cell. In some embodiments, the genetic modification is the introduction of a nucleic acid by electroporation into a cell.
  • the genetic modification is the introduction of a nucleic acid by nucleofection into a cell. In some embodiments, the genetic modification is the introduction of a nucleic acid by biolistic delivery of DNA-coated microparticles into a cell.
  • genetic modification is the introduction of a vector into a cell.
  • Vectors may be introduced using any suitable method.
  • the genetic modification is introduction of a vector by infection with a viral vector into a cell.
  • the genetic modification is introduction of a vector by electroporation into a cell.
  • the genetic modification is introduction of a vector by transfection into a cell.
  • the genetic modification is the introduction of a vector by transfection with lipid nanoparticles into a cell.
  • the genetic modification is the introduction of a vector with polymeric nanoparticles into a cell.
  • the genetic modification is the introduction of a vector with calcium phosphate nanoparticles into a cell.
  • the genetic modification is the introduction of a vector by electroporation into a cell. In some embodiments, the genetic modification is the introduction of a vector by nucleofection into a cell. In some embodiments, the genetic modification is the introduction of a vector by biolistic delivery of DNA-coated microparticles into a cell.
  • 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. Any suitable method may be used for gene editing.
  • RNA-guided nuclease e.g., Cas (e.g., Cas9) nuclease
  • ZFN zinc finger nuclease
  • TALEN transcription activator-like effector nuclease
  • meganuclease e.g., TALEN
  • compositions comprising T cells and IL-33.
  • a composition comprises T cells and IL-33.
  • the composition comprises T cells and IL-33, with an IL-33 concentration of 0.1 to 500 ng/mL.
  • the composition comprises T cells and IL-33, with an IL-33 concentration of 0.1 ng/ml, 0.2 ng/ml, 0.3 ng/ml, 0.4 ng/ml, 0.5 ng/mL, 1.0 ng/mL, 1.5 ng/mL, 2.0 ng/ml, 2.5 ng/mL, 3.0 ng/mL,3.5 ng/mL, 4.0 ng/mL, 4.5 ng/mL, 5.0 ng/mL, 5.5 ng/mL, 6.0 ng/mL, 6.5 ng/mL, 7.0 ng/mL, 7.5 ng/mL, 8.0 ng/mL, 8.5 ng/mL, 9.0 ng/mL, 9.5 ng/mL, 10.0 ng/mL, 15.0 ng/mL, 20 ng/mL, 25 ng/mL, 30 ng/mL, 35 ng/mL, 40 ng/mL,
  • the composition comprises T cells and IL-33, with an IL-33 concentration of 0.1-500 ng/ml, 0.5-500 ng/mL, 1.0-500 ng/mL, 5.0-500 ng/mL, 10-500 ng/mL, 20-500 ng/mL, 30-500 ng/mL, 40-500 ng/mL, 50-500 ng/mL, 60-500 ng/mL, 70-500 ng/mL, 80-500 ng/mL, 90-500 ng/mL, 100-500 ng/mL, 110-500 ng/mL, 120-500 ng/mL, 130-500 ng/mL, 140-500 ng/mL, 150-500 ng/mL, 160-500 ng/mL, 170-500 ng/mL, 180-500 ng/mL, 190-500 ng/mL, 200-500 ng/mL, 210-500 ng/mL, 220-
  • the composition comprises T cells and IL-33, with an IL-33 concentration of 0.1-0.5 ng/ml 0.1-1.0 ng/mL, 0.1-1.5 ng/mL,0.1-2.0 ng/mL, 0.1-2.5 ng/mL, 0.1-3.0 ng/mL, 0.1-3.5 ng/mL, 0.1-4.0 ng/mL, 0.1-4.5 ng/mL, 0.1-5.0 ng/mL, 0.1-6.0 ng/mL,0.1-7.0 ng/mL, 0.1-8.0 ng/mL, 0.1-9.0 ng/mL, 0.1-10 ng/mL, 0.1-20 ng/mL, 0.1-30 ng/mL, 0.1-40 ng/mL, 0.1-50 ng/mL, 0.1-60 ng/mL, 0.1-70 ng/mL, 0.1-80 ng/mL, 0.1-90 ng/mL, 0.1-100 ng/mL
  • the composition comprises T cells and IL-33, with an IL-33 concentration of 0.5-450 ng/ml, 1.0-450 ng/mL, 10-450 ng/mL, 20-450 ng/mL, 30-450 ng/mL, 40-450 ng/mL, 50-450 ng/mL, 100-450 ng/mL, 150-450 ng/mL, 200-450 ng/mL, 250-450 ng/mL, 300-450 ng/mL, 350-450 ng/mL, or 400-450 ng/mL.
  • an IL-33 concentration of 0.5-450 ng/ml, 1.0-450 ng/mL, 10-450 ng/mL, 20-450 ng/mL, 30-450 ng/mL, 40-450 ng/mL, 50-450 ng/mL, 100-450 ng/mL, 150-450 ng/mL, 200-450 ng/mL, 250-450 ng/mL,
  • the composition comprises T cells and IL-33, with an IL-33 concentration of 0.5-1.0 ng/ml, 1.0-10 ng/ml, 10-100 ng/mL, 50-100 ng/mL, 100-200 ng/mL, 150-250 ng/mL, 200-300 ng/mL, 250-350 ng/mL, or 350-400 ng/mL.
  • the composition comprises T cells and IL-33, with an IL-33 concentration of 50 ng/mL.
  • the composition comprises T cells and IL-33 cultured for 1-20 days. In some embodiments, the composition comprises T cells and IL-33 cultured for less than 1 day. In some embodiments, the composition comprises T cells and IL-33 cultured for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days. In some embodiments, the composition comprises T cells and IL-33 cultured for 1-2, 1-3, 1-4, 1-5, 1- 6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-13, 1-14, 1-15, 1-16, 1-17, 1-18, or 1-19 days.
  • the composition comprises T cells and IL-33 cultured for 2-20, 3-30, 4-20, 5- 20, 6-20, 7-20, 8-20, 9-20, 10-20, 11-20, 12-20, 13-20, 14-20, 15-20, 16-20, 17-20, 18-20, or 19-20 days.
  • the composition comprises T cells and IL-33 cultured for 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 2-11, 2-12, 2-13, 2-14, 2-15, 2-16, 2-17, 2-18, 2-19, or
  • the composition comprises T cells and IL-33 cultured for
  • the composition comprises T cells and IL-33 cultured for 1-5, 5-10, 10-15, or 15-20 days. In some embodiments, the composition comprises T cells and IL-33 cultured for 1-4, 4-7, 7-10, 10-13, 13-16, or 16-19 days. In some embodiments, the composition comprises T cells and IL-33 cultured for 14-16 days.
  • the composition comprises T cells and IL-33 cultured before genetic modification. In some embodiments, the composition comprises T cells and IL-33 cultured for 1-7 days before genetic modification. In some embodiments, the composition comprises T cells and IL-33 cultured for 1, 2, 3, 4, 5, 6, or 7 days before genetic modification. In some embodiments, the composition comprises T cells and IL-33 cultured for IL-33 1-2, 1-3, 1-4, 1-5, or 1-6 days before genetic modification. In some embodiments, the composition comprises T cells and IL-33 cultured for 2-7, 3-7, 4-7, 5-7, or 6-7 days before genetic modification.
  • the composition comprises T cells and IL-33 cultured for 1-3, 2-4, 3-5, 4-6, or 5-7 days before genetic modification. In some embodiments, the composition comprises T cells and IL-33 cultured for at least 7 days before genetic modification.
  • the composition comprises T cells and IL-33 cultured after genetic modification. In some embodiments, the composition comprises T cells and IL-33 cultured for 1-7 days after genetic modification. In some embodiments, the composition comprises T cells and IL-33 cultured for 1, 2, 3, 4, 5, 6, or 7 days after genetic modification. In some embodiments, the composition comprises T cells and IL-33 cultured for 1-2, 1-3, 1- 4, 1-5, or 1-6 days before after modification. In some embodiments, the composition comprises T cells and IL-33 cultured for 2-7, 3-7, 4-7, 5-7, or 6-7 days after genetic modification. In some embodiments, the composition comprises T cells and IL-33 cultured for 1-3, 2-4, 3-5, 4-6, or 5-7 days after genetic modification. In some embodiments, the composition comprises T cells and IL-33 cultured for at least 7 days after genetic modification.
  • the composition comprises T cells and rapamycin.
  • the T cells are Tregs.
  • the Tregs are engineered Tregs.
  • the engineered Tregs comprise a nucleic acid encoding a first CISC component and a nucleic acid encoding a second CISC component.
  • the first CISC component comprises in N-to-C-terminal order, and FKBP domain, an interleukin-2 receptor gamma (TL-2Ry ) transmembrane domain, and an IL-2Ry cytoplasmic domain.
  • 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.
  • IL-2RP interleukin-2 receptor beta
  • the composition comprises T cells and rapamycin cultured for between 1-20 days. In some embodiments, the composition comprises T cells and rapamycin cultured for less than 1 day. In some embodiments, the composition comprises T cells and rapamycin cultured for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days. In some embodiments, the composition comprises T cells and rapamycin cultured for 1- 2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-13, 1-14, 1-15, 1-16, 1-17, 1-18, or 1- 19 days.
  • the composition comprises T cells and rapamycin cultured for 2-20, 3-30, 4-20, 5-20, 6-20, 7-20, 8-20, 9-20, 10-20, 11-20, 12-20, 13-20, 14-20, 15-20, 16-20, 17-20, 18-20, or 19-20 days.
  • the composition comprises T cells and rapamycin cultured for 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 2-11, 2-12, 2-13, 2-14, 2- 15, 2-16, 2-17, 2-18, 2-19, or 2-20 days.
  • the composition comprises T cells and rapamycin cultured for 3-4, 4-5, 5-6, 6-7, 7-8, 8-9, 9-10, 10-11, 11-12, 12-13, 13-14, 14-15, 15-16, 16-17, 17-18, or 18-19 days.
  • the composition comprises T cells and rapamycin cultured for 1-5, 5-10, 10-15, or 15-20 days.
  • the composition comprises T cells and rapamycin cultured for 1-4, 4-7, 7-10, 10-13, 13-16, or 16-19 days.
  • the composition comprises T cells and rapamycin cultured for 14-16 days.
  • the composition comprises T cells and rapamycin cultured before genetic modification. In some embodiments, the composition comprises T cells and rapamycin cultured 1-7 days before genetic modification. In some embodiments, the composition comprises T cells and rapamycin cultured 1, 2, 3, 4, 5, 6, or 7 days before genetic modification. In some embodiments, the composition comprises T cells and rapamycin cultured 1-2, 1-3, 1-4, 1-5, or 1-6 days before genetic modification. In some embodiments, the composition comprises T cells and rapamycin cultured 2-7, 3-7, 4-7, 5-7, or 6-7 days before genetic modification. In some embodiments, the composition comprises T cells and rapamycin cultured 1-3, 2-4, 3-5, 4-6, or 5-7 days before genetic modification. In some embodiments, the composition comprises T cells and rapamycin cultured at least 7 days before genetic modification.
  • the composition comprises T cells and rapamycin cultured after genetic modification. In some embodiments, the composition comprises T cells and rapamycin cultured 1-7 days after genetic modification. In some embodiments, the composition comprises T cells and rapamycin cultured 1, 2, 3, 4, 5, 6, or 7 days after genetic modification. In some embodiments, the composition comprises T cells and rapamycin cultured 1-2, 1-3, 1-4, 1-5, or 1-6 days after genetic modification. In some embodiments, the composition comprises T cells and rapamycin cultured 2-7, 3-7, 4-7, 5-7, or 6-7 days after genetic modification. In some embodiments, the composition comprises T cells and rapamycin cultured 1-3, 2-4, 3-5, 4-6, or 5-7 days after genetic modification. In some embodiments, the composition comprises T cells and rapamycin cultured at least 7 days after genetic modification.
  • the composition comprises T cells and rapamycin, with a rapamycin concentration of O.Ol-lOOnM. In some embodiments, the composition comprises T cells and rapamycin, with a rapamycin concentration of 0.01 nM, 0.02 nM, 0.03 nM, 0.04 nM, 0.05 nM, 0.06 nM, 0.07 nM, 0.08 nM, 0.09 nM, 0.1 nM, 0.2 nM, 0.3 nM, 0.4 nM, 0.5 nM, 0.6 nM, 0.7 nM, 0.8 nM, 0.9 nM, 1.0 nM, 1.5 nM, 2.0 nM, 2.5 nM, 3.0 nM, 3.5 nM, 4.0 nM, 4.5 nM, 5.0 nM, 5.5 nM, 6.0 nM, 6.5 nM, 7.0 nM, 7.5 nM, 8.0 nM,
  • the composition comprises T cells and rapamycin, with a rapamycin concentration of 0.01-0.1 nM, 0.1-1 nM, 1-10 nM, or 10-100 nM.
  • the composition comprises T cells and rapamycin, with a rapamycin concentration of 0.01-0.02 nM, 0.02-0.03 nM, 0.03-0.04 nM, 0.04-0.05 nM, 0.05-0.06 nM, 0.06-0.07 nM, 0.07-0.08 nM, 0.08-0.09 nM, 0.09-0.1 nM, 0.1-0.2 nM, 0.2-0.3 nM, 0.3-0.4 nM, 0.4-0.5 nM, 0.5-0.6 nM, 0.6-0.7 nM, 0.7-0.8 nM, 0.8-0.9 nM, 0.9-1 nM, 1-2 nM, 2-3 nM, 3-4 nM, 4-5 nM, 5-6
  • the composition comprises T cells and rapamycin, with a rapamycin concentration of 0.01-0.05 nM, 0.05-0.1 nM, 1-5 nM, or 5-10 nM. In some embodiments, the composition comprises T cells and rapamycin, with a rapamycin concentration of 10 nM.
  • the composition comprises T cells, anti-CD3 antibodies, and anti-CD28 antibodies. In some embodiments, the composition comprises T cells and anti- CD3 antibodies. In some embodiments, the composition comprises T cells and anti-CD28 antibodies. See e.g., Riddell and Greenberg J Immunol Methods. 1990 Apr 17; 128(2): 189- 201.
  • the composition comprises T cells, anti-CD3 beads, and anti- CD28 beads. In some embodiments, the composition comprises T cells and anti-CD3 beads. In some embodiments, the composition comprises T cells and anti-CD28 beads. See e.g., Trickett et al. , J Immunol Methods. 2003 Apr 1 ;275(1 -2):251 -5 and Kalamasz et al. J Immunother. 2004 Sep-Oct;27(5):405-18.
  • the composition comprises T cells and anti-CD3 antibodies cultured before genetic modification. In some embodiments, the composition comprises T cells and anti-CD3 antibodies cultured 1-7 days before genetic modification. In some embodiments, the composition comprises T cells and anti-CD3 antibodies cultured 1, 2, 3, 4, 5, 6, or 7 days before genetic modification. In some embodiments, the composition comprises T cells and anti-CD3 antibodies cultured 1-2, 1-3, 1-4, 1-5, or 1-6 days before genetic modification. In some embodiments, the composition comprises T cells and anti-CD3 antibodies cultured 2-7, 3-7, 4-7, 5-7, or 6-7 days before genetic modification. In some embodiments, the composition comprises T cells and anti-CD3 antibodies cultured 1-3, 2-4, 3-5, 4-6, or 5-7 days before genetic modification.
  • the composition comprises T cells and anti-CD3 antibodies cultured at least 7 days before genetic modification. In some embodiments, the composition comprises T cells and anti-CD3 antibodies cultured after genetic modification. In some embodiments, the composition comprises T cells and anti-CD3 antibodies cultured 1-7 days after genetic modification. In some embodiments, the composition comprises T cells and anti-CD3 antibodies cultured 1, 2, 3, 4, 5, 6, or 7 days after genetic modification. In some embodiments, the composition comprises T cells and anti- CD3 antibodies cultured 1-2, 1-3, 1-4, 1-5, or 1-6 days after genetic modification. In some embodiments, the composition comprises T cells and anti-CD3 antibodies cultured 2-7, 3-7, 4-7, 5-7, or 6-7 days after genetic modification.
  • the composition comprises T cells and anti-CD3 antibodies cultured 1-3, 2-4, 3-5, 4-6, or 5-7 days after genetic modification. In some embodiments, the composition comprises T cells and anti-CD3 antibodies cultured at least 7 days after genetic modification.
  • the composition comprises T cells and anti-CD28 antibodies cultured before genetic modification. In some embodiments, the composition comprises T cells and anti-CD28 antibodies cultured 1-7 days before genetic modification. In some embodiments, the composition comprises T cells and anti-CD28 antibodies cultured 1, 2, 3, 4, 5, 6, or 7 days before genetic modification. In some embodiments, the composition comprises T cells and anti-CD28 antibodies cultured 1-2, 1-3, 1-4, 1-5, or 1-6 days before genetic modification. In some embodiments, the composition comprises T cells and anti-CD28 antibodies cultured 2-7, 3-7, 4-7, 5-7, or 6-7 days before genetic modification. In some embodiments, the composition comprises T cells and anti-CD28 antibodies cultured 1-3, 2-4, 3-5, 4-6, or 5-7 days before genetic modification. In some embodiments, the composition comprises T cells and anti-CD28 antibodies cultured at least 7 days before genetic modification.
  • the composition comprises T cells and anti-CD28 antibodies cultured after genetic modification. In some embodiments, the composition comprises T cells and anti-CD28 antibodies cultured 1-7 days after genetic modification. In some embodiments, the composition comprises T cells and anti-CD28 antibodies cultured 1, 2, 3, 4, 5, 6, or 7 days after genetic modification. In some embodiments, the composition comprises T cells and anti-CD28 antibodies cultured 1-2, 1-3, 1-4, 1-5, or 1-6 days after genetic modification. In some embodiments, the composition comprises T cells and anti-CD28 antibodies cultured 2-7, 3-7, 4-7, 5-7, or 6-7 days after genetic modification. In some embodiments, the composition comprises T cells and anti-CD28 antibodies cultured 1-3, 2-4, 3-5, 4-6, or 5-7 days after genetic modification. In some embodiments, the composition comprises T cells and anti-CD28 antibodies cultured at least 7 after before genetic modification.
  • the composition comprises T cells and anti-CD3 beads cultured before genetic modification. In some embodiments, the composition comprises T cells and anti-CD3 beads cultured 1-7 days before genetic modification. In some embodiments, the composition comprises T cells and anti-CD3 beads cultured 1, 2, 3, 4, 5, 6, or 7 days before genetic modification. In some embodiments, the composition comprises T cells and anti-CD3 beads cultured 1-2, 1-3, 1-4, 1-5, or 1-6 days before genetic modification. In some embodiments, the composition comprises T cells and anti-CD3 beads cultured 2-7, 3-7, 4-7, 5-7, or 6-7 days before genetic modification. In some embodiments, the composition comprises T cells and anti-CD3 beads cultured 1-3, 2-4, 3-5, 4-6, or 5-7 days before genetic modification. In some embodiments, the composition comprises T cells and anti-CD3 beads cultured at least 7 days before genetic modification.
  • the composition comprises T cells and anti-CD3 beads cultured after genetic modification. In some embodiments, the composition comprises T cells and anti-CD3 beads cultured 1-7 days after genetic modification. In some embodiments, the composition comprises T cells and anti-CD3 beads cultured 1, 2, 3, 4, 5, 6, or 7 days after genetic modification. In some embodiments, the composition comprises T cells and anti-CD3 beads cultured 1-2, 1-3, 1-4, 1-5, or 1-6 days after genetic modification. In some embodiments, the composition comprises T cells and anti-CD3 beads cultured 2-7, 3-7, 4-7, 5-7, or 6-7 days after genetic modification. In some embodiments, the composition comprises T cells and anti-CD3 beads cultured 1-3, 2-4, 3-5, 4-6, or 5-7 days after genetic modification. In some embodiments, the composition comprises T cells and anti-CD3 beads cultured at least 7 days after genetic modification.
  • the composition comprises T cells and anti-CD28 beads cultured before genetic modification. In some embodiments, the composition comprises T cells and anti-CD28 beads cultured 1-7 days before genetic modification. In some embodiments, the composition comprises T cells and anti-CD28 beads cultured 1, 2, 3, 4, 5, 6, or 7 days before genetic modification. In some embodiments, the composition comprises T cells and anti-CD28 beads cultured 1-2, 1-3, 1-4, 1-5, or 1-6 days before genetic modification. In some embodiments, the composition comprises T cells and anti-CD28 beads cultured 2-7, 3-7, 4-7, 5-7, or 6-7 days before genetic modification. In some embodiments, the composition comprises T cells and anti-CD28 beads cultured 1-3, 2-4, 3-5, 4-6, or 5-7 days before genetic modification. In some embodiments, the composition comprises T cells and anti-CD28 beads cultured at least 7 days before genetic modification.
  • the composition comprises T cells and anti-CD28 beads cultured after genetic modification. In some embodiments, the composition comprises T cells and anti-CD28 beads cultured 1-7 days after genetic modification. In some embodiments, the composition comprises T cells and anti-CD28 beads cultured 1, 2, 3, 4, 5, 6, or 7 days after genetic modification. In some embodiments, the composition comprises T cells and anti- CD28 beads cultured 1-2, 1-3, 1-4, 1-5, or 1-6 days after genetic modification. In some embodiments, the composition comprises T cells and anti-CD28 beads cultured 2-7, 3-7, 4-7, 5-7, or 6-7 days after genetic modification. In some embodiments, the composition comprises T cells and anti-CD28 beads cultured 1-3, 2-4, 3-5, 4-6, or 5-7 days after genetic modification. In some embodiments, the composition comprises T cells and anti-CD28 beads cultured at least 7 days after genetic modification.
  • compositions comprising T cells and IL-18.
  • a composition comprises T cells and IL-18.
  • the composition comprises T cells and IL-18, with an IL-18 concentration of 0.1 to 500 ng/mL.
  • the composition comprises T cells and IL- 18, with an IL- 18 concentration of 0.1 ng/ml, 0.2 ng/ml, 0.3 ng/ml, 0.4 ng/ml, 0.5 ng/mL, 1.0 ng/mL, 1.5 ng/mL, 2.0 ng/ml, 2.5 ng/mL, 3.0 ng/mL,3.5 ng/mL, 4.0 ng/mL, 4.5 ng/mL, 5.0 ng/mL, 5.5 ng/mL, 6.0 ng/mL, 6.5 ng/mL, 7.0 ng/mL, 7.5 ng/mL, 8.0 ng/mL, 8.5 ng/mL, 9.0 ng/mL, 9.5 ng/mL, 10.0 ng/mL, 15.0 ng/mL, 20 ng/mL, 25 ng/mL, 30 ng/mL, 35 ng/mL, 40 ng/mL, 45
  • the composition comprises T cells and IL- 18, with an IL- 18 concentration of 0.1-500 ng/ml, 0.5-500 ng/mL, 1.0-500 ng/mL, 5.0-500 ng/mL, 10-500 ng/mL, 20-500 ng/mL, 30-500 ng/mL, 40-500 ng/mL, 50-500 ng/mL, 60-500 ng/mL, 70-500 ng/mL, 80-500 ng/mL, 90-500 ng/mL, 100-500 ng/mL, 110-500 ng/mL, 120-500 ng/mL, 130-500 ng/mL, 140-500 ng/mL, 150-500 ng/mL, 160-500 ng/mL, 170-500 ng/mL, 180-500 ng/mL, 190-500 ng/mL, 200-500 ng/mL, 210-500 ng/mL, 220-500
  • the composition comprises T cells and IL-18, with an IL-18 concentration of 0.1-0.5 ng/ml 0.1-1.0 ng/mL, 0.1-1.5 ng/mL,0.1-2.0 ng/mL, 0.1-2.5 ng/mL, 0.1-3.0 ng/mL, 0.1-3.5 ng/mL, 0.1-4.0 ng/mL, 0.1-4.5 ng/mL, 0.1-5.0 ng/mL, 0.1-6.0 ng/mL,0.1-7.0 ng/mL, 0.1-8.0 ng/mL, 0.1-9.0 ng/mL, 0.1-10 ng/mL, 0.1-20 ng/mL, 0.1-30 ng/mL, 0.1-40 ng/mL, 0.1-50 ng/mL, 0.1-60 ng/mL, 0.1-70 ng/mL, 0.1-80 ng/mL, 0.1-90 ng/mL, 0.1-100 ng/mL,
  • the composition comprises T cells and IL- 18, with an IL- 18 concentration of 0.5-450 ng/ml, 1.0-450 ng/mL, 10-450 ng/mL, 20-450 ng/mL, 30-450 ng/mL, 40-450 ng/mL, 50-450 ng/mL, 100-450 ng/mL, 150-450 ng/mL, 200-450 ng/mL, 250-450 ng/mL, 300-450 ng/mL, 350-450 ng/mL, or 400-450 ng/mL.
  • an IL- 18 concentration of 0.5-450 ng/ml, 1.0-450 ng/mL, 10-450 ng/mL, 20-450 ng/mL, 30-450 ng/mL, 40-450 ng/mL, 50-450 ng/mL, 100-450 ng/mL, 150-450 ng/mL, 200-450 ng/mL, 250-450 ng/mL, 300
  • the composition comprises T cells and IL-18, with an IL-18 concentration of 0.5-1.0 ng/ml, 1.0-10 ng/ml, 10-100 ng/mL, 50-100 ng/mL, 100-200 ng/mL, 150-250 ng/mL, 200-300 ng/mL, 250-350 ng/mL, or 350-400 ng/mL.
  • the composition comprises T cells and IL- 18, with an IL- 18 concentration of 50 ng/mL.
  • the composition comprises T cells and IL-18 cultured for 1-20 days. In some embodiments, the composition comprises T cells and IL- 18 cultured for less than 1 day. In some embodiments, the composition comprises T cells and IL-18 cultured for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days. In some embodiments, the composition comprises T cells and IL-18 cultured for 1-2, 1-3, 1-4, 1-5, 1- 6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-13, 1-14, 1-15, 1-16, 1-17, 1-18, or 1-19 days.
  • the composition comprises T cells and IL-18 cultured for 2-20, 3-30, 4-20, 5- 20, 6-20, 7-20, 8-20, 9-20, 10-20, 11-20, 12-20, 13-20, 14-20, 15-20, 16-20, 17-20, 18-20, or 19-20 days.
  • the composition comprises T cells and IL- 18 cultured for 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 2-11, 2-12, 2-13, 2-14, 2-15, 2-16, 2-17, 2-18, 2-19, or
  • the composition comprises T cells and IL- 18 cultured for
  • the composition comprises T cells and IL- 18 cultured for 1-5, 5-10, 10-15, or 15-20 days. In some embodiments, the composition comprises T cells and IL-18 cultured for 1-4, 4-7, 7-10, 10-13, 13-16, or 16-19 days. In some embodiments, the composition comprises T cells and IL-18 cultured for 14-16 days.
  • the composition comprises T cells and IL- 18 cultured before genetic modification. In some embodiments, the composition comprises T cells and IL- 18 cultured for 1-7 days before genetic modification. In some embodiments, the composition comprises T cells and IL-18 cultured for 1, 2, 3, 4, 5, 6, or 7 days before genetic modification. In some embodiments, the composition comprises T cells and IL- 18 cultured for IL-18 1-2, 1-3, 1-4, 1-5, or 1-6 days before genetic modification. In some embodiments, the composition comprises T cells and IL-18 cultured for 2-7, 3-7, 4-7, 5-7, or 6-7 days before genetic modification. In some embodiments, the composition comprises T cells and IL-18 cultured for 1-3, 2-4, 3-5, 4-6, or 5-7 days before genetic modification. In some embodiments, the composition comprises T cells and IL- 18 cultured for at least 7 days before genetic modification.
  • the composition comprises T cells and IL- 18 cultured after genetic modification. In some embodiments, the composition comprises T cells and IL- 18 cultured for 1-7 days after genetic modification. In some embodiments, the composition comprises T cells and IL- 18 cultured for 1, 2, 3, 4, 5, 6, or 7 days after genetic modification. In some embodiments, the composition comprises T cells and IL-18 cultured for 1-2, 1-3, 1- 4, 1-5, or 1-6 days before after modification. In some embodiments, the composition comprises T cells and IL-18 cultured for 2-7, 3-7, 4-7, 5-7, or 6-7 days after genetic modification. In some embodiments, the composition comprises T cells and IL- 18 cultured for 1-3, 2-4, 3-5, 4-6, or 5-7 days after genetic modification. In some embodiments, the composition comprises T cells and IL- 18 cultured for at least 7 days after genetic modification.
  • the composition comprises T cells and one or more cytokines.
  • the cytokines are IL-2, IL-12, IL-21, IL-23, and/or TGF-p.
  • the composition comprises T cells and IL-2.
  • the composition comprises T cells and IL-12.
  • the composition comprises T cells and IL-21.
  • the composition comprises T cells and IL-23.
  • the composition comprises T cells and TGF-p. Measurement of IL-33R/ST2 and/or IL-18R expression and signaling
  • IL-33R/ST2 signaling may be evaluated using any suitable method.
  • suitable methods include measuring phosphorylation, signal transduction, and activity.
  • phosphorylation, signal transduction, and/or activity may be measured using Phos-Flow, Western Blot, activity assays (e.g., kinase activity assays, enzyme assays, or enzyme kinetic assays), ELISA, immunohistochemistry, immunofluorescence, mass spectrometry, or flow cytometry.
  • phosphorylation of p65 NFKB
  • phosphorylation of p38 is measured.
  • IL- 18 signaling may be evaluated using any suitable method.
  • suitable methods include measuring phosphorylation, signal transduction, and activity.
  • phosphorylation, signal transduction, and/or activity may be measured using Phos- Flow, Western Blot, activity assays e.g., kinase activity assays, enzyme assays, or enzyme kinetic assays), ELISA, immunohistochemistry, immunofluorescence, mass spectrometry, or flow cytometry.
  • phosphorylation of p65 NFKB
  • phosphorylation of p38 is measured.
  • expression of a gene or protein is measured. In some embodiments, expression of a gene is measured. In some embodiments, expression of a gene is measured by any suitable method. In some embodiments, expression of a protein is measured. In some embodiments, expression of a protein is measured by any suitable method. In some embodiments, protein production is measured. In some embodiments, protein production is measured by any suitable method. For example, expression of a gene or protein, or protein production, may be measured using Western Blot, ELISA, immunohistochemistry, immunofluorescence, mass spectrometry, or flow cytometry. In some embodiments, RNA is measured. In some embodiments, RNA is measured by QPCR or rtPCR. In some embodiments, RNA is measured by any suitable method.
  • the methods are used to measure expression levels of IL-13. In some embodiments, the methods are used to measure expression of ST2. In some embodiments, the methods are used to measure expression of IL1RAP. In some embodiments, the methods are used to measure expression of IL-18R. In some embodiments, the methods are used to measure expression of IL-18Ra. In some embodiments, the methods are used to measure expression of IL-18Rp. In some embodiments, the methods are used to measure expression of GAT A3. In some embodiments, the methods are used to measure expression of CCR8. In some embodiments, the methods are used to measure expression of CCR5. In some embodiments, the methods are used to measure expression of CCR4. In some embodiments, the methods are used to measure expression of CD39.
  • the methods are used to measure expression of HLA-DR. In some embodiments, the methods are used to measure expression of CD71. In some embodiments, the methods are used to measure expression of KI67. In some embodiments, the methods are used to measure expression of TIGIT. In some embodiments, expression is protein expression. In some embodiments, expression is RNA expression.
  • Some embodiments of the engineered cells exhibit increased expression of one or more cytokines or other proteins, relative to a cell that has not been engineered (e.g., a cell that is not genetically modified, and/or a cell that is not a Treg).
  • expression of IL-13 is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, relative to a cell that has not been engineered.
  • IL- 13 expression is increased by at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 300, 400, or 500 times, relative to a cell that has not been engineered.
  • expression of ST2 is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, relative to a cell that has not been engineered.
  • ST2 expression is increased by at least 10, 20, 30, 40, 50, 100, 200, 300, 400, or 500 times, relative to a cell that has not been engineered. In some embodiments, ST2 expression is increased by at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 300, 400, or 500 times, relative to a cell that has not been engineered. In some embodiments, expression of IL1RAP is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, relative to a cell that has not been engineered.
  • IL1RAP expression is increased by at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 300, 400, or 500 times, relative to a cell that has not been engineered.
  • IL-18R1 expression is increased by at least 10, 20, 30, 40, 50, 100, 200, 300, 400, or 500 times, relative to a cell that has not been engineered.
  • IL- 18R1 expression is increased by at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 300, 400, or 500 times, relative to a cell that has not been engineered.
  • expression of IL-18R1 is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, relative to a cell that has not been engineered. In some embodiments, IL-18R1 expression is increased by at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 300, 400, or 500 times, relative to a cell that has not been engineered. In some embodiments, IL18RAP expression is increased by at least 10, 20, 30, 40, 50, 100, 200, 300, 400, or 500 times, relative to a cell that has not been engineered.
  • IL18RAP expression is increased by at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 300, 400, or 500 times, relative to a cell that has not been engineered.
  • expression of CCR5 is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, relative to a cell that has not been engineered.
  • CCR5 expression is increased by at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 300, 400, or 500 times, relative to a cell that has not been engineered.
  • expression of CCR8 is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, relative to a cell that has not been engineered. In some embodiments, CCR8 expression is increased by at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 300, 400, or 500 times, relative to a cell that has not been engineered. In some embodiments, expression of BATF is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, relative to a cell that has not been engineered. In some embodiments, BATF expression is increased by at least 2, 3, 4, 5,
  • HLA-DR is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, relative to a cell that has not been engineered. In some embodiments, HLA-DR expression is increased by at least 2, 3, 4, 5, 6,
  • expression of CD71 is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, relative to a cell that has not been engineered. In some embodiments, CD71 expression is increased by at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 300, 400, or 500 times, relative to a cell that has not been engineered. In some embodiments, expression of KI67 is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, relative to a cell that has not been engineered.
  • KI67 expression is increased by at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 300, 400, or 500 times, relative to a cell that has not been engineered.
  • expression of TIGIT is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, relative to a cell that has not been engineered.
  • TIGIT expression is increased by at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 300, 400, or 500 times, relative to a cell that has not been engineered.
  • expression of CD25 is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, relative to a cell that has not been engineered. In some embodiments, CD25 expression is increased by at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 300, 400, or 500 times, relative to a cell that has not been engineered. In some embodiments, expression of CD39 is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, relative to a cell that has not been engineered. In some embodiments, CD39 expression is increased by at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 300, 400, or 500 times, relative to a cell that has not been engineered.
  • compositions for producing engineered cells are provided.
  • compositions for administering to a subject so that the compositions target particular cells (e.g., immune cells or pluripotent cells) and upregulate IL-33 and/or IL-18 signaling so as to be engineered into Treg cells in vivo.
  • Such compositions 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 and/or IL-18 pathways.
  • such compositions include nucleic acids encoding constitutively active members of the IL-33 and/or IL-18 pathways that positively regulate the pathway.
  • compositions for making Tregs comprise a nucleic acid that induces or increases expression of ST2 or IL- 18R1.
  • CD4+ T cells or CD8+ T cells are enriched from a biological sample such as peripheral blood mononuclear cells (PBMC) prior to gene editing (e.g., dual editing).
  • PBMC peripheral blood mononuclear cells
  • enriched CD4+ T cells or CD8+ T cells are non-specifically activated (e.g., with solid-phase immobilized anti-CD3 and anti-CD28 antibodies) prior to gene editing.
  • a composition for making Tregs comprises one or more nucleic acids encoding T cell receptors (TCRs), chimeric antigen receptors (CARs) to target particular cells, enhance suppressive function, or both.
  • a composition comprises a nucleic acid encoding IL-2.
  • a composition for making Tregs comprises a nucleic acid encoding a constitutively active IL- 10 (see, e.g., WO 2019/180724, which is incorporated herein by reference in its entirety), CISC components, and/or a soluble FRB protein untethered from mTOR (see, e.g., WO 2018/111834, WO 2019/210057, and WO 2020/264039, each of which is incorporated herein by reference in its entirety).
  • a nucleic acid (e.g., for introduction into a cell or administration to a subject) is comprised in a vector.
  • vector is used to refer to any molecule (e.g., nucleic acid, plasmid) or arrangement of molecules (e.g., virus) used to transfer coding information to a host cell.
  • expression vector refers to a vector that is suitable for introduction of a host cell and contains nucleic acid sequences that direct and/or control expression of introduced heterologous nucleic acid sequences. Expression includes, but is not limited to, processes such as transcription, translation, and RNA splicing, if introns are present.
  • Non-limiting examples of vectors include artificial chromosomes, minigenes, cosmids, plasmids, phagemids, and viral vectors.
  • Non-limiting examples of viral vectors include lentiviral vectors, retroviral vectors, herpesvirus vectors, adenovirus vectors, and adeno-associated viral vectors.
  • one or more vectors comprising nucleic acids are lentiviral vectors.
  • cells are transduced with more than one vector (e.g., lentiviral vector).
  • a cell is transduced with a first lentiviral vector encoding ST2 and a second lentiviral vector encoding IL1RAP, and, optionally, a third lentiviral vector encoding IL-18R.
  • one or more vectors are adenoviral vectors.
  • one or more vectors are adeno-associated viral (AAV) vectors.
  • one or more AAV vectors is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11 vector.
  • one or more AAV vectors are AAV5 vectors.
  • one or more AAV vectors are AAV6 vectors.
  • nucleic acids may include genomic sequences, extra-genomic and plasmid-encoded sequences and smaller engineered gene segments that express, or may be adapted to express, proteins, polypeptides, peptides and the like. Such segments may be naturally isolated or modified synthetically by the skilled person.
  • polynucleotides may be singlestranded (coding or antisense) or double-stranded, and may be DNA (genomic, cDNA or synthetic) or RNA molecules.
  • RNA molecules may include HnRNA molecules, which contain introns and correspond to a DNA molecule in a one-to-one manner, and mRNA molecules, which do not contain introns. Additional coding or non-coding sequences may, but need not, be present within a polynucleotide, and a polynucleotide may, but need not, be linked to other molecules and/or support materials.
  • Polynucleotides may comprise a native sequence or may comprise a sequence encoding a variant or derivative of such a sequence.
  • polynucleotide variants may have substantial identity to a reference polynucleotide sequence encoding an immunomodulatory polypeptide.
  • a polynucleotide may be a polynucleotide comprising at least 70% sequence identity, preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher, sequence identity or a sequence identity that is within a range defined by any two of the aforementioned percentages as compared to a reference polynucleotide sequence such as a sequence encoding a polypeptide, using any suitable method (e.g., BLAST analysis using standard parameters, as described below).
  • BLAST analysis using standard parameters, as described below.
  • polynucleotide variants will contain one or more substitutions, additions, deletions and/or insertions, preferably such that the binding affinity of a polypeptide variant of a given polypeptide which is capable of a specific binding interaction with another molecule and is encoded by the variant polynucleotide is not substantially diminished relative to a polypeptide encoded by a polynucleotide sequence specifically set forth herein.
  • nucleic acid sequences are codon-optimized for expression in a cell.
  • Codon usage tables are readily available, for example, at the “Codon Usage Database” available at www.kazusa.or.jp.
  • Codon-optimized coding regions can be designed by various methods known to those skilled in the art.
  • polynucleotides, or fragments thereof regardless of the length of the coding sequence itself, may be combined with other DNA sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably. It is therefore contemplated that a nucleic acid fragment of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol.
  • polynucleotide segments with total lengths of or about of 10,000, 5000, 3000, 2,000, 1,000, 500, 200, 100, or 50 base pairs in length, and the like, (including all intermediate lengths) are contemplated to be useful.
  • two sequences are said to be “identical” if the sequence of nucleotides in the two sequences is the same when aligned for maximum correspondence, as described below. Comparisons between two sequences are typically performed by comparing the sequences over a comparison window to identify and compare local regions of sequence similarity.
  • a “comparison window” as used herein, refers to a segment of at least or at least about 20 contiguous positions, usually 30 to 75, or 40 to 50, in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
  • Optimal alignment of sequences for comparison may be conducted using the Megalign program in the Lasergene suite of bioinformatics software (DNASTAR, Inc., Madison, WI), using default parameters.
  • This program embodies several alignment schemes described in the following references: Dayhoff, M.O. (1978) A model of evolutionary change in proteins - Matrices for detecting distant relationships. In Dayhoff, M.O. (ed.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington DC Vol. 5, Suppl. 3, pp. 345-358; Hein J., Unified Approach to Alignment and Phylogenes, pp. 626-645 (1990); Methods in Enzymology vol.
  • optimal alignment of sequences for comparison may be conducted by the local identity algorithm of Smith and Waterman, Add. APL. Math 2:482 (1981), by the identity alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity methods of Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85: 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, WI), or by inspection.
  • BLAST and BLAST 2.0 are described in Altschul et al.. Nucl Acids Res . 1977. 25:3389-3402, and Altschul et al.. J Mol Biol. 1990. 215:403-410, respectively.
  • BLAST and BLAST 2.0 can be used, for example with the parameters described below, to determine percent sequence identity among two or more the polynucleotides.
  • Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.
  • cumulative scores can be calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always ⁇ 0). Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment.
  • the “percentage of sequence identity” is determined by comparing two optimally aligned sequences over a window of comparison of at least 20 positions, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (e.g., gaps) of 20 percent or less, usually 5 to 15 percent, or 10 to 12 percent, as compared to the reference sequences (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • additions or deletions e.g., gaps
  • the percentage is calculated by determining the number of positions at which the identical nucleic acid bases occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the reference sequence (e.g., the window size) and multiplying the results by 100 to yield the percentage of sequence identity.
  • Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art. These and related techniques and procedures may be generally performed according to conventional methods as described in various general and more specific references that are cited and discussed throughout the present specification. Unless specific definitions are provided, the nomenclature utilized in connection with, and the laboratory procedures and techniques of, molecular biology, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry are those well- known and commonly used in the art. Standard techniques may be used for recombinant technology, molecular biological, microbiological, chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.
  • compositions comprising a cell, vector, or nucleic acid, and a pharmaceutically acceptable excipient or carrier.
  • Such pharmaceutical compositions are formulated, for example, for systemic administration, or administration to target tissues.
  • “Acceptable” means that the excipient (carrier) must be compatible with the active ingredient of the composition (and preferably, capable of stabilizing the active ingredient) and not deleterious to the subject to be treated.
  • the precise nature of the carrier or other material may depend on the route of administration, e.g., parenteral, intramuscular, intradermal, sublingual, buccal, ocular, intranasal, subcutaneous, intrathecal, intratumoral, oral, vaginal, or rectal. See, e.g., Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. K. E. Hoover.
  • the pharmaceutical compositions to be used for in vivo administration must be sterile, with the exception of any cells, viruses, and/or viral vectors being used to achieve a biological effect (e.g., immunosuppression). This is readily accomplished by, for example, filtration through sterile filtration membranes.
  • the pharmaceutical compositions may be placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
  • the pharmaceutical compositions can be formulated for intramuscular injection, intravenous injection, intradermal injection, or subcutaneous injection.
  • compositions can comprise pharmaceutically acceptable carriers, buffer agents, excipients, salts, or stabilizers in the form of lyophilized formulations or aqueous solutions. See, e.g., Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. K. E. Hoover).
  • Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations used, and may comprise buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine,
  • the pharmaceutical composition comprises lipid nanoparticles which can be prepared by suitable methods, such as described in Epstein et al., Proc Natl Acad Sci USA .1985. 82:3688; Hwang et al. Proc Natl Acad Sci USA. 1980. 77:4030; and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Pat. No. 5,013,556. Particularly useful liposomes can be generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE).
  • PEG-PE PEG-derivatized phosphatidylethanolamine
  • Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter.
  • Lipids used in the formulation of lipid nanoparticles for delivering nucleic acids include ionizable amino lipids, non-cationic lipids, sterols, and polyethylene glycol-modified lipids. See, e.g., Buschmann et al., Vaccines. 2021. 9(1):65.
  • the nucleic acid is surrounded by the lipids of the lipid nanoparticle and present in the interior of the lipid nanoparticle.
  • the nucleic acid is dispersed throughout the lipids of the lipid nanoparticle.
  • the lipid nanoparticle comprises an ionizable amino lipid, a non-cationic lipid, a sterol, and/or a polyethylene glycol (PEG)-modified lipid.
  • compositions can be in unit dosage forms such as tablets, pills, capsules, powders, granules, solutions or suspensions, or suppositories, for oral, parenteral or rectal administration, or administration by inhalation or insufflation.
  • the principal active ingredient can be mixed with a pharmaceutical carrier, e.g., conventional tableting ingredients such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, and other pharmaceutical diluents, e.g., water, to form a solid preformulation composition containing a homogeneous mixture, or a non-toxic pharmaceutically acceptable salt thereof.
  • a pharmaceutical carrier e.g., conventional tableting ingredients such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, and other pharmaceutical diluents, e.g., water
  • a pharmaceutical carrier e.g., conventional tableting ingredients such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphat
  • the tablets or pills of the novel composition can be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action.
  • the tablet or pill can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former.
  • the two components can be separated by an enteric layer that serves to resist disintegration in the stomach and permits the inner component to pass intact into the duodenum or to be delayed in release.
  • enteric layers or coatings such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol and cellulose acetate.
  • compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable, aqueous or organic solvents, or mixtures thereof, and powders.
  • the liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as set out above.
  • the compositions are administered by the oral or nasal respiratory route for local or systemic effect.
  • compositions in preferably sterile pharmaceutically acceptable solvents may be nebulized by use of gases. Nebulized solutions may be breathed directly from the nebulizing device or the nebulizing device may be attached to a face mask, tent or intermittent positive pressure breathing machine. Solution, suspension, or powder compositions may be administered, preferably orally or nasally, from devices which deliver the formulation in an appropriate manner.
  • compositions may be useful for treating a subject that has or is at risk of developing an inflammatory, autoimmune, or allergic condition or disease.
  • a subject having or at risk of developing an inflammatory, autoimmune, or allergic condition or disease may be identified by ascertaining the presence and/or absence of one or more risk factors, diagnostic indicators, or prognostic indications. The determination may be made based on clinical, cellular, or serologic findings, including flow cytometry, serology, and/or DNA analyses.
  • the pharmaceutical compositions can include a therapeutically effective amount of any cell, vector, and/or nucleic acid.
  • the pharmaceutical composition includes a cell, vector, or nucleic acid at any of the doses.
  • a “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result.
  • the therapeutically effective amount may vary according to factors such as the age, sex, and weight of the individual, and the ability of the cell, nucleic acid, or vector to effect a desired response in the subject.
  • a therapeutically effective amount reduces the severity of a disease.
  • a therapeutically effective amount reduces the duration of a disease.
  • a therapeutically effective amount delays the progression of a disease.
  • a therapeutically effective amount alleviates one or more symptoms of a disease.
  • a therapeutically effective amount prevents recurrence of a disease.
  • a therapeutically effective amount delays recurrence of a disease.
  • compositions can be prepared in accordance with known techniques. See, e.g., Remington, The Science And Practice of Pharmacy (21st ed., Philadelphia, Lippincott, Williams & Wilkins, 2005).
  • cells, vectors, or nucleic acids may be admixed with a pharmaceutically acceptable excipient, and the resulting composition is administered to a subject.
  • the carrier must be acceptable in the sense of being compatible with any other ingredients in the formulation and must not be deleterious to the subject.
  • the carrier can be a solid or a liquid, or both, and can be formulated with the compound as a unitdose formulation.
  • a pharmaceutical composition comprises cells at a dose of about 10 4 to about IO 10 cells/kg. In some embodiments, the pharmaceutical composition comprises cells at a dose of about: 10 4 to 10 5 , 10 5 to 10 6 , 10 6 to 10 7 , 10 7 to 10 8 , 10 8 to 10 9 , or 10 9 to IO 10 cells/kg.
  • a pharmaceutical composition comprises cells at a dose of about 0.1 x 10 6 , 0.2 x 10 6 , 0.3 x 10 6 , 0.4 x 10 6 , 0.5 x 10 6 , 0.6 x 10 6 , 0.7 x 10 6 , 0.8 x 10 6 , 0.9 x 10 6 , 1.0 x 10 6 , 1.1 x 10 6 , 1.2 x 10 6 , 1.3 x 10 6 , 1.4 x 10 6 , 1.5 x 10 6 , 1.6 x 10 6 , 1.7 x
  • a pharmaceutical composition comprises an effective amount of a vector or nucleic acid. In some examples, the pharmaceutical composition comprises about 0.1 mg/kg to about 3 mg/kg of the vector or nucleic acid. In some embodiments, the pharmaceutical composition comprises about 0.1 mg/kg, about 0.25 mg/kg, about 0.5 mg/kg, about 0.75 mg/kg, about 1.0 mg/kg, about 1.5 mg/kg, about 2.0 mg/kg, about 2.5 mg/kg, or about 3.0 mg/kg of the vector or nucleic acid.
  • pharmaceutical composition comprises about 0.1 mg/kg to about 0.25 mg/kg, about 0.25 mg/kg to about 0.5 mg/kg, about 0.5 mg/kg to about 0.75 mg/kg, about 0.75 mg/kg to about 1.0 mg/kg, about 1.0 mg/kg to about 1.5 mg/kg, about 1.5 mg/kg to about 2.0 mg/kg, about 2.0 mg/kg to about 2.5 mg/kg, or about 2.5 mg/kg to about 3.0 mg/kg of the vector or nucleic acid.
  • the pharmaceutical composition comprises a vector or nucleic acid encapsulated within a lipid nanoparticle.
  • lipid nanoparticles comprise at least one cationic lipid, at least one non-cationic lipid, and at least one conjugated lipid.
  • lipid nanoparticles can comprise from about 50 mol % to about 85 mol % of a cationic lipid, from about 13 mol % to about 49.5 mol % of a non-cationic lipid, and from about 0.5 mol % to about 10 mol % of a lipid conjugate, and are produced in such a manner as to have a non- lamellar e.g., non-bilayer) morphology.
  • lipid nanoparticles can comprise from about 40 mol % to about 85 mol % of a cationic lipid, from about 13 mol % to about 49.5 mol % of a non-cationic lipid, and from about 0.5 mol % to about 10 mol % of a lipid conjugate and are produced in such a manner as to have a non-lamellar (e.g., non-bilayer) morphology.
  • a non-lamellar e.g., non-bilayer
  • Cationic lipids can include, for example, one or more of the following: palmitoyi- oleoyl-nor-arginine (PONA), MPDACA, GUADACA, ((6Z,9Z,28Z,31Z)-heptatriaconta- 6,9,28,31-tetraen- 19-yl 4-(dimethylamino)butanoate) (MC3), LenMC3, CP-LenMC3, y- LenMC3, CP-y-LenMC3, MC3MC, MC2MC, MC3 Ether, MC4 Ether, MC3 Amide, Pan- MC3, Pan-MC4 and Pan MC5, l,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA),
  • DLin-EG-DMA N,N-dioleyl- N,N-dimethylammonium chloride
  • DODAC N,N-dioleyl- N,N-dimethylammonium chloride
  • DODMA l,2-dioleyloxy-N,N-dimethylaminopropane
  • DMDMA l,2-distearyloxy-N,N-dimethylaminopropane
  • DOTMA N,N-distearyl-N,N- dimethylammonium bromide (DDAB), N-(l-(2,3-dioleoyloxy)propyl)-N,N,N- trimethylammonium chloride (DOTAP), 3-(N-(N',N'-dimethylaminoethane)- carbamoyl)
  • the cationic lipid can also be DLinDMA, DLin-K-C2-DMA (“XTC2”), MC3, LenMC3, CP-LenMC3, y-LenMC3, CP-y- LenMC3, MC3MC, MC2MC, MC3 Ether, MC4 Ether, MC3 Amide, Pan-MC3, Pan-MC4, Pan MC5, or mixtures thereof.
  • XTC2 DLin-K-C2-DMA
  • the cationic lipid may comprise from about 50 mol % to about 90 mol %, from about 50 mol % to about 85 mol %, from about 50 mol % to about 80 mol %, from about 50 mol % to about 75 mol %, from about 50 mol % to about 70 mol %, from about 50 mol % to about 65 mol %, or from about 50 mol % to about 60 mol % of the total lipid present in the particle.
  • the cationic lipid may comprise from about 40 mol % to about 90 mol %, from about 40 mol % to about 85 mol %, from about 40 mol % to about 80 mol %, from about 40 mol % to about 75 mol %, from about 40 mol % to about 70 mol %, from about 40 mol % to about 65 mol %, or from about 40 mol % to about 60 mol % of the total lipid present in the particle.
  • the non-cationic lipid may comprise, e.g., one or more anionic lipids and/or neutral lipids.
  • the non-cationic lipid comprises one of the following neutral lipid components: (1) cholesterol or a derivative thereof; (2) a phospholipid; or (3) a mixture of a phospholipid and cholesterol or a derivative thereof.
  • Examples of cholesterol derivatives include, but are not limited to, cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, and mixtures thereof.
  • the phospholipid may be a neutral lipid including, but not limited to, dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), palmitoyloleyol-phosphatidylglycerol (POPG), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine, dielaidoylphosphatidylethanolamine (DEPE), stearoyloleoyl-phosphatidylethanolamine (SOPE), egg phosphatidyl
  • the non-cationic lipid may comprise from about 10 mol % to about 60 mol %, from about 15 mol % to about 60 mol %, from about 20 mol % to about 60 mol %, from about 25 mol % to about 60 mol %, from about 30 mol % to about 60 mol %, from about 10 mol % to about 55 mol %, from about 15 mol % to about 55 mol %, from about 20 mol % to about 55 mol %, from about 25 mol % to about 55 mol %, from about 30 mol % to about 55 mol %, from about 13 mol % to about 50 mol %, from about 15 mol % to about 50 mol % or from about 20 mol % to about 50 mol % of the total lipid present in the particle.
  • the non-cationic lipid is a mixture of a phospholipid and
  • the conjugated lipid that inhibits aggregation of particles may comprise, e.g., one or more of the following: a polyethyleneglycol (PEG)-lipid conjugate, a polyamide (ATTA)- lipid conjugate, a cationic-polymer-lipid conjugates (CPLs), or mixtures thereof.
  • the nucleic acid-lipid particles comprise either a PEG-lipid conjugate or an ATTA-lipid conjugate.
  • the PEG-lipid conjugate or ATTA-lipid conjugate is used together with a CPL.
  • the conjugated lipid that inhibits aggregation of particles may comprise a PEG-lipid including, e.g., a PEG-diacylglycerol (DAG), a PEG dialkyloxypropyl (DAA), a PEG-phospholipid, a PEG-ceramide (Cer), or mixtures thereof.
  • the PEG-DAA conjugate may be PEG-di lauryl oxy propyl (C 12), a PEG- dimyristyloxypropyl (C14), a PEG-dipalmityloxypropyl (C16), a PEG-distearyloxypropyl (Cl 8), or mixtures thereof.
  • Additional PEG-lipid conjugates suitable for use include, but are not limited to, mPEG2000-l,2-di-0-alkyl-sn3-carbomoylglyceride (PEG-C-DOMG).
  • PEG-C-DOMG mPEG2000-l,2-di-0-alkyl-sn3-carbomoylglyceride
  • the synthesis of PEG- C-DOMG is described in PCT Application No. PCT/US08/88676.
  • Yet additional PEG-lipid conjugates suitable for use include, without limitation, l-[8'-(l,2-dimyristoyl-3-propanoxy)- carboxamido-3 ',6'-dioxaoctanyl]carbamoyl-co-methyl-poly(ethylene glycol) (2KPEG-DMG).
  • 2KPEG-DMG The synthesis of 2KPEG-DMG is described in U.S. Pat. No. 7,404
  • the conjugated lipid that inhibits aggregation of particles may comprise from about 0.1 mol % to about 2 mol %, from about 0.5 mol % to about 2 mol %, from about 1 mol % to about 2 mol %, from about 0.6 mol % to about 1.9 mol %, from about 0.7 mol % to about 1.8 mol %, from about 0.8 mol % to about 1.7 mol %, from about 1 mol % to about 1.8 mol %, from about 1.2 mol % to about 1.8 mol %, from about 1.2 mol % to about 1.8 mol %, from about 1.2 mol % to about 1.7 mol %, from about 1.3 mol % to about 1.6 mol %, from about 1.4 mol % to about 1.5 mol %, or about 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 mol % (or any fraction thereof
  • the PEG moiety has an average molecular weight of about 2,000 Daltons.
  • the conjugated lipid that inhibits aggregation of particles may comprise from about 5.0 mol % to about 10 mol %, from about 5 mol % to about 9 mol %, from about 5 mol % to about 8 mol %, from about 6 mol % to about 9 mol %, from about 6 mol % to about 8 mol %, or about 5 mol %, 6 mol %, 7 mol %, 8 mol %, 9 mol %, or 10 mol % (or any fraction thereof or range therein) of the total lipid present in the particle.
  • the PEG moiety has an average molecular weight of about 750 Daltons.
  • the composition may comprise amphoteric liposomes, which contain at least one positive and at least one negative charge carrier, which differs from the positive one, the isoelectric point of the liposomes being between 4 and 8. This objective is accomplished owing to the fact that liposomes are prepared with a pH-dependent, changing charge.
  • Liposomal structures with the desired properties are formed, for example, when the amount of membrane-forming or membrane-based cationic charge carriers exceeds that of the anionic charge carriers at a low pH and the ratio is reversed at a higher pH. This is always the case when the ionizable components have a pKa value between 4 and 9. As the pH of the medium drops, all cationic charge carriers are more charged and all anionic charge carriers lose their charge.
  • Cationic compounds are useful in amphoteric liposomes.
  • strongly cationic compounds can include, for example: DC-Chol 3-P-[N-(N',N'-dimethylmethane) carbamoyl] cholesterol, TC-Chol 3- -[N-(N', N', N'-trimethylaminoethane) carbamoyl cholesterol, BGSC bisguanidinium-spermidine-cholesterol, BGTC bis-guadinium-tren- cholesterol, DOTAP (l,2-dioleoyloxypropyl)-N,N,N-trimethylammonium chloride, DOSPER ( 1,3 -di oleoyloxy -2-(6-carboxy-spermyl)-propylarnide, DOTMA (1,2-dioleoyloxypropyl)- N,N,N-trimethylamronium chloride) (Lipofectin®), DORIE l,2-diole
  • weakly cationic compounds include, without limitation: His-Chol (histaminyl-cholesterol hemisuccinate), Mo-Chol (morpholine-N-ethylamino-cholesterol hemi succinate), or histidinyl-PE.
  • neutral compounds include, without limitation: cholesterol, ceramides, phosphatidyl cholines, phosphatidyl ethanolamines, tetraether lipids, or diacyl glycerols.
  • Anionic compounds useful for amphoteric liposomes include those non-cationic compounds previously.
  • examples of weakly anionic compounds can include: CHEMS (cholesterol hemi succinate), alkyl carboxylic acids with 8 to 25 carbon atoms, or diacyl glycerol hemi succinate.
  • Additional weakly anionic compounds can include the amides of aspartic acid, or glutamic acid and PE as well as PS and its amides with glycine, alanine, glutamine, asparagine, serine, cysteine, threonine, tyrosine, glutamic acid, aspartic acid or other amino acids or aminodicarboxylic acids.
  • the esters of hydroxycarboxylic acids or hydroxy dicarboxylic acids and PS are also weakly anionic compounds.
  • amphoteric liposomes may contain a conjugated lipid.
  • conjugated lipids include, without limitation, PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates (e.g., PEG- CerC14 or PEG-CerC20), PEG-modified dialkylamines and PEG-modified 1,2- diacyloxypropan-3 -amines.
  • PEG-modified diacylglycerols and dialkylglycerols are particularly examples.
  • the neutral lipids may comprise from about 10 mol % to about 60 mol %, from about 15 mol % to about 60 mol %, from about 20 mol % to about 60 mol %, from about 25 mol % to about 60 mol %, from about 30 mol % to about 60 mol %, from about 10 mol % to about 55 mol %, from about 15 mol % to about 55 mol %, from about 20 mol % to about 55 mol %, from about 25 mol % to about 55 mol %, from about 30 mol % to about 55 mol %, from about 13 mol % to about 50 mol %, from about 15 mol % to about 50 mol % or from about 20 mol % to about 50 mol % of the total lipid present in the particle.
  • the conjugated lipid that inhibits aggregation of particles may comprise from about 0.1 mol % to about 2 mol %, from about 0.5 mol % to about 2 mol %, from about 1 mol % to about 2 mol %, from about 0.6 mol % to about 1.9 mol %, from about 0.7 mol % to about 1.8 mol %, from about 0.8 mol % to about 1.7 mol %, from about 1 mol % to about 1.8 mol %, from about 1.2 mol % to about 1.8 mol %, from about 1.2 mol % to about 1.8 mol %, from about 1.2 mol % to about 1.7 mol %, from about 1.3 mol % to about 1.6 mol %, from about 1.4 mol % to about 1.5 mol %, or about 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 mol % (or any fraction thereof
  • the PEG moiety has an average molecular weight of about 2,000 Daltons.
  • the conjugated lipid that inhibits aggregation of particles may comprise from about 5.0 mol % to about 10 mol %, from about 5 mol % to about 9 mol %, from about 5 mol % to about 8 mol %, from about 6 mol % to about 9 mol %, from about 6 mol % to about 8 mol %, or about 5 mol %, 6 mol %, 7 mol %, 8 mol %, 9 mol %, or 10 mol % (or any fraction thereof or range therein) of the total lipid present in the particle.
  • the PEG moiety has an average molecular weight of about 750 Daltons.
  • the remaining balance of the amphoteric liposome can comprise a mixture of cationic compounds and anionic compounds formulated at various ratios.
  • the ratio of cationic to anionic lipid may selected in order to achieve the desired properties of nucleic acid encapsulation, zeta potential, pKa, or other physicochemical property that is at least in part dependent on the presence of charged lipid components.
  • the lipid nanoparticles have a composition that specifically enhances delivery and uptake in stem cells, hematopoietic cells, or T cells.
  • the pharmaceutical composition comprises an effective amount of a lipid nanoparticle formulation, wherein the lipid nanoparticle formulation comprises a vector or nucleic acid.
  • the lipid nanoparticle formulation comprises about 0.1 mg/kg to about 3 mg/kg of the vector or nucleic acid.
  • the lipid nanoparticle formulation comprises about 0.1 mg/kg, about 0.25 mg/kg, about 0.5 mg/kg, about 0.75 mg/kg, about 1.0 mg/kg, about 1.5 mg/kg, about 2.0 mg/kg, about 2.5 mg/kg, or about 3.0 mg/kg of the vector or nucleic acid.
  • the lipid nanoparticle formulation comprises about 0.1 mg/kg to about 0.25 mg/kg, about 0.25 mg/kg to about 0.5 mg/kg, about 0.5 mg/kg to about 0.75 mg/kg, about 0.75 mg/kg to about 1.0 mg/kg, about 1.0 mg/kg to about 1.5 mg/kg, about 1.5 mg/kg to about 2.0 mg/kg, about 2.0 mg/kg to about 2.5 mg/kg, or about 2.5 mg/kg to about 3.0 mg/kg of the vector or nucleic acid.
  • the pharmaceutical composition comprises an effective amount of a lipid nanoparticle formulation comprising a donor template comprising a template nucleic acid, wherein lipid nanoparticle formulation comprises about 0.1 mg/kg to about 3 mg/kg of the donor polynucleotide. In some embodiments, the lipid nanoparticle formulation comprises about 0.1 mg/kg, about 0.25 mg/kg, about 0.5 mg/kg, about 0.75 mg/kg, about 1.0 mg/kg, about 1.5 mg/kg, about 2.0 mg/kg, about 2.5 mg/kg, or about 3.0 mg/kg of the donor polynucleotide.
  • the lipid nanoparticle formulation comprises about 0.1 mg/kg to about 0.25 mg/kg, about 0.25 mg/kg to about 0.5 mg/kg, about 0.5 mg/kg to about 0.75 mg/kg, about 0.75 mg/kg to about 1.0 mg/kg, about 1.0 mg/kg to about 1.5 mg/kg, about 1.5 mg/kg to about 2.0 mg/kg, about 2.0 mg/kg to about 2.5 mg/kg, or about 2.5 mg/kg to about 3.0 mg/kg of the donor polynucleotide.
  • pharmaceutical compositions can further comprise one or more additional agents useful in the treatment of an inflammatory condition or disease in a subject.
  • a method comprises administering to a subject any one of the cells.
  • a method comprises administering to the subject a cell that had previously been obtained from that subject before being administered (z.e., the cell is an autologous cell).
  • a method comprises (i) isolation of cells from a subject; (ii) processing the cells by any method (e.g., gene editing or introducing a vector); and (iii) administering the processed cells to the same subject.
  • a method comprises administering to the subject a cell that had previously been obtained from a different subject than the one to whom the cell is administered (i.e., the cell is an allogeneic cell).
  • a method comprises (i) isolation of cells from a first subject; (ii) processing the cells by any method (e.g., gene editing or introducing a vector); and (iii) administering the processed cells to a second subject.
  • Some embodiments of the methods, cells, systems, and compositions include any of the cells, vectors, nucleic acids, or lipid nanoparticles, for use as a medicament.
  • the cell, vector, nucleic acid, or lipid nanoparticle is for use in a method of preventing, treating, inhibiting, or ameliorating an inflammatory condition or disease in a subject.
  • a cell is for use in a method of preventing, treating, inhibiting, or ameliorating an inflammatory condition or disease in a subject.
  • the cell is autologous to the subject (i.e., derived from the subject).
  • the cell is allogeneic to the subject (i.e., derived from a different subject).
  • a cell, vector, nucleic acid, or lipid nanoparticle is used for treating, preventing, inhibiting, or ameliorating an autoinflammatory, ischemic, and/or inflammatory condition or disease in a subject.
  • the subject has or is at risk of developing an inflammatory condition or disease.
  • the inflammatory condition or disease is selected from stroke, myocardial infarction, acute swelling, severe wounding, acute kidney injury, fibrosis, muscle injuries, burn injuries, traumatic brain injury, acute lung injury (ALI), acute respiratory distress syndrome (ARDS), pancreatic islet cell transplantation, asthma, hepatitis, primary sclerosing cholangitis, primary biliary cholangitis, polymyositis, Still’s disease, uveitis, ulcerative colitis, Crohn’s disease, dermatitis, psoriasis, psoriatic arthritis, graft-versus-host disease (GvHD), tolerance induction for transplantation, transplant rejection, and sepsis.
  • stroke myocardial infarction
  • acute swelling severe wounding
  • acute kidney injury fibrosis
  • muscle injuries burn injuries
  • traumatic brain injury acute lung injury (ALI), acute respiratory distress syndrome (ARDS)
  • ARDS acute respiratory distress syndrome
  • pancreatic islet cell transplantation asthma, hepati
  • a cell, vector, nucleic acid, or lipid nanoparticle is used for treating, preventing, treating, inhibiting, or ameliorating fibrosis in a subject.
  • the subject has fibrosis.
  • the subject is at elevated risk for developing fibrosis.
  • Fibrosis is characterized by overgrowth, hardening, and/or scarring of various tissues, which follows deposition of excess extracellular matrix (ECM) components such as collagen. Fibrosis typically results from chronic inflammation, where inflammation occurs together with tissue remodeling and repair processes.
  • ECM extracellular matrix
  • causes of fibrotic conditions may vary, and include the presence of external stimuli, such as pathogens and allergens, that trigger inflammation.
  • Inflammation leading to fibrosis may also result from mechanical injury, such as blunt force or surgery.
  • Autoimmune reactions are also characterized by inflammation, and chronic inflammation in such conditions may result in fibrosis.
  • Any of infections, toxins, drugs, trauma, and recurrent inflammation cause epithelial or endothelial damage, which activate innate and adaptive immune responses, leading to inflammation and immune cell recruitment, as well as fibroblast (e.g., myofibroblast) activation, which results in tissue repair and remodeling.
  • fibroblast e.g., myofibroblast activation
  • Fibrotic conditions share several common features, including the activation of myofibroblasts, which produce ECM components (including collagen and fibronectin) and mediate fibrotic tissue remodeling. See, e.g., Wynn and Ramalingam, Nat Med. 2012. 18(7): 1028-1040.
  • an engineered cell suppresses activation of myofibroblasts.
  • an engineered cell suppresses production of one or more ECM components.
  • an engineered cell suppresses collagen production.
  • an engineered cell suppresses collagen deposition.
  • an engineered cell suppresses fibronectin production.
  • an engineered cell suppresses fibroblast proliferation.
  • an engineered cell reduces the number of circulating fibrocytes in a subject.
  • TGF-[31 transforming growth factor
  • PDGF platelet- derived growth factor
  • VEGF vascular endothelial growth factor
  • FGF fibroblast growth factor
  • a subject has an elevated level of one or more of TGF-[31, PDGF, VEGF, and FGF relative to a baseline level.
  • the concentration of TGF-[31, PDGF, VEGF, and FGF is elevated in the subject’s blood, compared to a baseline level in blood.
  • the concentration of TGF-[31, PDGF, VEGF, and FGF is elevated in an organ of the subject, compared to a baseline level in the organ. In some embodiments, the concentration of TGF-[31, PDGF, VEGF, and FGF is elevated in a tissue of the subject, compared to a baseline level in the tissue. Any suitable method may be used to measure TGF-[31, PDGF, VEGF, and FGF levels in blood, organs, or tissues, such as ELISA or Luminex. In some embodiments, the level of TGF-[31, PDGF, VEGF, and FGF is elevated in the lung, heart, stomach, small intestine, large intestine, kidney, liver, or pancreas of the subject.
  • an engineered cell reduces the level of one or more of TGF- [31, PDGF, VEGF, and FGF in the subject. In some embodiments, an engineered cell reduces the level of one or more of TGF-[31, PDGF, VEGF, and FGF in the subject’s blood. In some embodiments, an engineered cell reduces the level of one or more of TGF-[31, PDGF, VEGF, and FGF in an organ of the subject. In some embodiments, the organ is a lung. In some embodiments, the organ is a heart. In some embodiments, the organ is a kidney. In some embodiments, the organ is a liver. In some embodiments, the organ is a stomach. In some embodiments, the organ is the small intestine. In some embodiments, the organ is the large intestine.
  • the fibrosis is post-inflammatory fibrosis.
  • Post-inflammatory fibrosis is characterized by excess deposition of ECM components following inflammation.
  • the post-inflammatory fibrosis is associated with ARDS.
  • the fibrosis is pulmonary post-inflammatory fibrosis.
  • the fibrosis is idiopathic pulmonary fibrosis.
  • the fibrosis is renal fibrosis. Multiple causes can contribute to renal fibrosis, including acute kidney injury.
  • the inflammatory condition is due to an inflammatory disease, such as asthma, systemic lupus erythematosus, rheumatoid arthritis, reactive arthritis, spondy arthritis, systemic vasculitis, insulin dependent diabetes mellitus, multiple sclerosis, experimental allergic encephalomyelitis, Sjogren's syndrome, graft versus host disease and scleroderma. Autoimmune diseases may also result in inflammation.
  • an inflammatory disease such as asthma, systemic lupus erythematosus, rheumatoid arthritis, reactive arthritis, spondy arthritis, systemic vasculitis, insulin dependent diabetes mellitus, multiple sclerosis, experimental allergic encephalomyelitis, Sjogren's syndrome, graft versus host disease and scleroderma.
  • Autoimmune diseases may also result in inflammation.
  • autoimmune diseases include, but are not limited to, myasthenia gravis, Guillain-Barre disease, primary biliary cirrhosis, hepatitis, hemolytic anemia, uveitis, Grave's disease, pernicious anemia, thrombocytopenia, Hashimoto's thyroiditis, oophoritis, orchitis, adrenal gland diseases, anti-phospholipid syndrome, Wegener's granulomatosis, Behcet's disease, polymyositis, dermatomyositis, multiple sclerosis, vitiligo, ankylosing spondylitis, Pemphigus vulgaris, psoriasis, dermatitis herpetiformis, Addison's disease, Goodpasture's syndrome, Basedow's disease, thrombopenia purpura, allergy and cardiomyopathy.
  • the inflammatory condition is due to a cancer, such as lymphoma (Hodgkins and non-Hodgkins) B-cell lymphoma, T-cell lymphoma, leukemia such as myeloid leukemia and other types of leukemia, mycosis fungoides, carcinoma, adenocarcinoma, sarcoma, glioma, blastoma, neuroblastoma, plasmacytoma, histiocytoma, melanoma, adenoma, hypoxic tumor, myeloma, AIDS-related lymphoma or AIDS-related sarcoma, metastatic cancer, bladder cancer, brain cancer, nervous system cancer, squamous cell carcinoma of the head and neck, neuroblastoma, glioblastoma, ovarian cancer, skin cancer, liver cancer, squamous cell carcinomas of the mouth, throat, larynx, and lung, colon cancer, cervical cancer, breast cancer, cervical carcinoma, epitopedi
  • inflammation is caused by an infection, such as a viral infection (e.g., Herpes simplex virus type-1, Herpes simplex virus type-2, Cytomegalovirus, Epstein- Barr virus, Varicella-zoster virus, Human herpes virus 6, Human herpes virus 7, Human herpes virus 8, Variola virus, Vesicular stomatitis virus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis D virus, Hepatitis E virus, Rhinovirus, Coronavirus, Influenza virus A, Influenza virus B, Measles virus, Polyomavirus, Human Papillomavirus, Respiratory syncytial virus, Adenovirus, Coxsackie virus, Dengue virus, Mumps virus, Poliovirus, Rabies virus, Rous sarcoma virus, Yellow fever virus, Ebola virus, Marburg virus, Lassa fever virus, Eastern Equine Encephalitis virus, Japanese Encephalitis virus, St
  • inflammation is caused by an infection, such as a bacterial infection (e.g., caused by Listeria monocytogenes, Listeria ivanovii, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus equi, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus viridans group, Bacillus anthracis, B.
  • a bacterial infection e.g., caused by Listeria monocytogenes, Listeria ivanovii, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus equi, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus viridans group, Bacillus anthracis, B.
  • subtilis Peptostreptococcus sp., Peptostreptococcus sp., Actinomyces israelii, Clostridium tetani, Clostridium perfringens, Clostridium botulinum, Pseudomonas aeruginosa, Campylobacter sp., Vibrio cholerae, Ehrlichia sp., Actinobacillus pleuropneumoniae , Pasteurella haemolytica, Pasteur ella multocida, Legionella pneumophila, Salmonella typhi, Shigella sp., Brucella abortus, other Brucella species, Chlamydia trachomatis, Chlamydia psittaci, Chlamydia pneumoniae, Coxiella burnetti, Escherichia coli, Neiserria meningitidis, Neiserria gonorrheae, Ha
  • the subject has or has had a stroke. In some embodiments, the subject has or has had a hemorrhagic stroke. In some embodiments has or has had an ischemic stroke. Stroke is a condition characterized by blockage of blood flow to the brain (ischemic stroke) or leakage of blood into the intracranial cavity (hemorrhagic stroke). Many pathologies are associated with ischemic stroke, but leading causes include large vessel disease, small vessel disease, and cardioembolism. Hemorrhagic stroke is typically caused by rupture of small arteries, hypertensive vasculopathy, cerebral amyloid angiopathy, and coagulopathy. See, e.g., Tadi P, Lui F. Acute Stroke. [Updated 2023 Feb 28], In: StatPearls [Internet], Treasure Island (FL): StatPearls Publishing; 2023 Jan. NCBI Books NBK535369.
  • the inflammatory disease is acute respiratory distress syndrome (ARDS).
  • ARDS is characterized by damage to the alveolar-endothelial interface, resulting in loss of epithelial barrier integrity. Such damage may be caused by an external insult, such as a bacterial, viral, or eukaryotic parasite, the presence of an irritant.
  • ARDS may be caused by pulmonary inflammation, such as inflammation associated with an autoimmune condition. Tissue damage in ARDS can cause depletion of stem cells within the lung, such as type II alveolar epithelial cells, which impairs tissue repair processes following ARDS. Such aberrant repair processes following pulmonary inflammation and ARDS can facilitate pulmonary fibrosis.
  • engineered cells may be administered to subjects experiencing or at elevated risk for ARDS, to treat or prevent pulmonary fibrosis.
  • engineered cells may be administered to subjects experiencing, or that have recently experienced, inflammation, to treat or prevent post-inflammatory fibrosis.
  • Treatment or prevention of fibrosis may be characterized, for example, by reduced deposition of collagen, or a reduced rate of collagen deposition, relative to an untreated control subject. Any suitable method may be used to evaluate deposition of collagen or other extracellular matrix components, such as trichrome blue staining.
  • the subject has ARDS.
  • the subject is at elevated risk for ARDS.
  • the subject has a history of ARDS.
  • Symptoms of ARDS include, without limitation, shortness of breath, rapid breathing, low blood oxygen, labored breathing, fatigue, confusion, and cough.
  • Risk factors for ARDS include, without limitation advanced age, female gender, smoking, alcohol use, aortic vascular surgery, cardiovascular surgery, traumatic brain injury, pancreatitis, pulmonary contusion, infectious pneumonia, and use of certain drugs (radiation agents, chemotherapeutic agents, and/or amiodarone). See, e.g., Diamond M, Peniston HL, Sanghavi DK, et al. Acute Respiratory Distress Syndrome. [Updated 2023 Apr 6], In: StatPearls [Internet], Treasure Island (FL): StatPearls Publishing; 2023 Jan.
  • a cell or method is effective for preventing one or more symptoms of ARDS in a subject. In some embodiments, a cell or method is effective for reducing the severity of one or more symptoms of ARDS in a subject. In some embodiments, a cell or method is effective for reducing the frequency of one or more symptoms of ARDS in a subject. In some embodiments, a composition, pharmaceutical composition, or method is effective for terminating one or more symptoms of ARDS in a subject.
  • the inflammatory disease is acute lung injury (ALI).
  • ALI acute lung injury
  • Acute lung injury is characterized by sudden onset of inflammation and increased permeability in the lungs, leading to impaired oxygenation and respiratory failure.
  • the subject has ALI. In some embodiments, the subject is at elevated risk for ALI. In some embodiments, the subject has a history of ALI. Symptoms of ALI include, without limitation, difficulty breathing, rapid breathing, hypoxemia, cough, chest pain, restlessness, fatigue, fever, decreased breath, and confusion. Risk factors of ALI, include, without limitation, transplant, transfusion, near drowning, infection, sepsis, trauma, aspiration, inhalation of toxic substances, ischemia-reperfusion injury, chronic lung diseases (e.g., COPD), and pancreatitis.
  • the ALI is trauma-related ALI. In some embodiments, the ALI is sepsis-induced ALI. In some embodiments, the ALI is pneumonia-associated ALI. In some embodiments, the ALI is aspiration-induced ALI. In some embodiments, the ALI is inhalation-related ALI. In some embodiments, the ALI is drug-induced ALI. In some embodiments, the ALI is near-drowning-related ALI. In some embodiments, the ALI is reperfusion injury-related ALI. In some embodiments, the ALI is transfusion-related ALI.
  • a cell or method is effective for preventing one or more symptoms of ALI in a subject. In some embodiments, a cell or method is effective for reducing the severity of one or more symptoms of ALI in a subject. In some embodiments, a cell or method is effective for reducing the frequency of one or more symptoms of ALI in a subject. In some embodiments, a composition, pharmaceutical composition, or method is effective for terminating one or more symptoms of ALI in a subject.
  • the subject is infected with a bacterium.
  • the bacterium is Streptococcus pneumoniae, Staphylococcus aureus, Klebsiella pneumoniae, Legionella pneumophila, Pseudomonas aeruginosa, o Haemophilus influenzae.
  • the subject is infected with a virus.
  • the virus is an influenza virus, adenovirus, respiratory syncytial virus (RSV), herpesvirus, hantavirus, measles virus, or a coronavirus.
  • the subject is infected with a fungus.
  • the subject is infected with a mold.
  • the subject is infected with a parasite.
  • the subject is infected with a Plasmodium parasite.
  • the subject has malaria-associated ARDS (MA- ARDS).
  • a cell or method is effective for reducing the volume of fluid in the lungs of the subject. In some embodiments, a cell or method is effective in reducing vascular permeability in the lungs of the subject. In some embodiments, a cell or method is effective for reducing infiltration of one or more immune cells into alveoli of the subject. In some embodiments, a cell or method is effective for reducing the concentration of one or more inflammatory cytokines in bronchoalveolar fluid of the subject.
  • a cell, vector, nucleic acid, or lipid nanoparticle is used for treating, preventing, treating, inhibiting, or ameliorating acute kidney injury in a subject.
  • Acute kidney injury refers to a reduction in kidney function, as measured by glomerular filtration rate. Signs of acute kidney injury include increased creatinine, and/or decreased urine volume. Acute kidney injury may lead to accumulation of water, sodium, and other metabolites, as well as other electrolyte imbalances.
  • causes of acute injury include reduced blood flow to the kidney (prerenal acute kidney injury); tubular necrosis or interstitial nephritis, renal ischemia, sepsis, and nephrotoxins (renal acute kidney injury; and obstructions such as renal/uretal calculi, tumors, blood clots, or urethral obstruction (post- renal acute kidney injury).
  • prerenal acute kidney injury tubular necrosis or interstitial nephritis, renal ischemia, sepsis, and nephrotoxins
  • renal acute kidney injury renal acute kidney injury
  • obstructions such as renal/uretal calculi, tumors, blood clots, or urethral obstruction
  • an engineered cell reduces inflammation in the kidney of the subject. Inflammation contributes to the pathogenesis of acute kidney injury, and so engineered cells may reduce inflammation, thereby mitigating the pathology of acute kidney injury.
  • an engineered cell reduces the abundance of adenosine triphosphate (ATP) in the kidney of the subject. Damaged cells release ATP, which may act as a danger signal to other innate immune cells.
  • Treg cells expressing the pair of CD39 and CD73 convert ATP to the nucleoside adenosine, which is anti-inflammatory.
  • an engineered cell produces one or more anti-inflammatory cytokines that reduce inflammation in the kidney of the subject.
  • an engineered cell suppresses proliferation of one or more immune cells in the kidney of the subject.
  • the subject has acute kidney injury. In some embodiments, the subject is at elevated risk for acute kidney injury. In some embodiments, the subject has a history of acute kidney injury. Symptoms of acute kidney injury include, without limitation, increased serum creatinine, increased blood urea nitrogen (BUN), increased serum Kim-1, increased urinary Kim-1, and reduced glomerular filtration rate (GFR).
  • BUN blood urea nitrogen
  • GFR reduced glomerular filtration rate
  • Risk factors for acute kidney injury include, without limitation, reduced blood volume (e.g., due to dehydration, severe bleeding, fluid loss), low blood pressure (e.g., due to shock, infection, heart failure), reduced renal blood flow (e.g., renal artery stenosis), acute tubular necrosis (e.g., due to ischemia or exposure to renotoxic drugs, such as cisplatin), glomuleronephritis (inflammation of glomuleri), interstitial nephritis (inflammation between tubules), urinary tract obstruction (e.g, due to tumor, kidney stone, or enlarged prostate), bladder outlet obstruction, advanced age, diabetes, hypertension, heart failure, liver disease, and use of nephrotoxic drugs (e.g., NS AIDs, contrast agents).
  • reduced blood volume e.g., due to dehydration, severe bleeding, fluid loss
  • low blood pressure e.g., due to shock, infection, heart failure
  • reduced renal blood flow
  • the subject has received cardiac surgery. Cardiac surgery is implicated in the development of acute kidney injury, or cardiac surgery-associated acute kidney injury (CSA-AKI). See, e.g., O’Neal et al., Crit Care. 2016. 20: 187.
  • the subject has received cardiac surgery within 90, 80, 70, 60, 50, 40, 30, 25, 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 day of administration of an engineered cell.
  • an engineered cell is administered within 90, 80, 70, 60, 50, 40, 30, 25, 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 day prior to cardiac surgery.
  • the subject has received cardiac surgery but not developed acute kidney injury. In some embodiments, the subject has received cardiac surgery, and is identified as being at elevated risk for acute kidney injury on the basis of one or more biomarkers. See, e.g., Zou et al., Front Physiol. 2022. 13:960059. In some embodiments, the subject has elevated serum creatinine. In some embodiments, the subject has increased blood urea nitrogen. In some embodiments, the subject has increased serum Kim-1. In some embodiments, the subject has increased urinary Kim-1. In some embodiments, the subject has received cisplatin. Cisplatin is implicated in the development of acute kidney injury, or cisplatin-induced acute kidney injury.
  • the subject has received cisplatin within 90, 80, 70, 60, 50, 40, 30, 25, 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5,
  • an engineered cell is administered within 90, 80, 70, 60, 50, 40, 30, 25, 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6,
  • a cell or method is effective for preventing one or more symptoms of acute kidney injury in a subject. In some embodiments, a cell or method is effective for reducing the severity of one or more symptoms of acute kidney injury in a subject. In some embodiments, a cell or method is effective for reducing the frequency of one or more symptoms of acute kidney injury in a subject. In some embodiments, a composition, pharmaceutical composition, or method is effective for terminating one or more symptoms of acute kidney injury in a subject.
  • a cell or method is effective for reducing serum creatinine in a subject. In some embodiments, a cell or method is effective for reducing blood urea nitrogen in a subject. In some embodiments, a cell or method is effective for reducing serum Kim-1 in a subject. In some embodiments, a cell or method is effective for reducing urinary Kim-1 in a subject. In some embodiments, a cell or method is effective for increasing glomerular filtration rate in a subject.
  • the inflammatory disease is multiple sclerosis (MS).
  • MS multiple sclerosis
  • CNS central nervous system
  • Multiple sclerosis is a disease of the central nervous system (CNS) that is characterized by chronic inflammation, demyelination, gliosis, and neuronal loss. These and other symptoms arise in connection with degradation of myelin sheaths surrounding neurons, impairing CNS function.
  • CNS central nervous system
  • it is expected that exposure to osteopontin produced by engineered Treg cells is beneficial for inducing a reparative phenotype in microglia, thereby facilitating repair of damaged CNS tissue (e.g., brain white matter).
  • the subject has multiple sclerosis. In some embodiments, the subject is at elevated risk for multiple sclerosis. In some embodiments, the subject has a history of multiple sclerosis. In some embodiments, the subject has a history of multiple sclerosis.
  • the multiple sclerosis is relapsing-remitting multiple sclerosis. In some embodiments, the multiple sclerosis is primary progressive multiple sclerosis. In some embodiments, the multiple sclerosis is secondary progressive multiple sclerosis. In some embodiments, the multiple sclerosis is progressive-relapsing multiple sclerosis. In some embodiments, the subject has had a clinically isolated episode of neurological symptoms (clinically isolated syndrome).
  • Symptoms of multiple sclerosis include, without limitation, fatigue, vision problems, numbness or tingling, muscle weakness, muscle stiffness, muscle spasms, disrupted coordination, dizziness, vertigo, bladder dysfunction, bowel dysfunction, difficulty concentrating, impaired memory, pain, difficulty speaking, and difficulty swallowing.
  • Risk factors for multiple sclerosis include, without limitation, a family history of multiple sclerosis, female sex, Northern European ancestry, low vitamin D levels, Epstein-Barr virus (EBV) infection, ans smoking. See, e.g., Tafti D, Ehsan M, Xixis KL. Multiple Sclerosis. [Updated 2022 Sep 7], In: StatPearls [Internet], Treasure Island (FL): StatPearls Publishing; 2024 Jan-.
  • the subject has had an ischemic injury.
  • Ischemic injury may follow a reduction or complete interruption of blood supply to an organ or tissue. Reduction in blood flow can cause reduced nutrient and/or oxygen availability, thereby impairing cellular function.
  • Ischemia can occur in multiple organs and tissues, such as the heart, brain, and kidneys.
  • myocardial ischemia is associated with conditions such as angina or myocardial infarction.
  • Cerebral ischemia is associated with conditions like stroke or transient ischemic attack.
  • Peripheral ischemia may occur in limbs, leading to conditions such as peripheral artery disease or ischemic limb injuries.
  • Organ ischemia may occur in vital organs, such as the kidneys.
  • the inflammatory disease is myocardial infarction.
  • the inflammatory condition is acute swelling, for example, from an impact injury.
  • the inflammatory condition is associated with a burn (e.g., a first degree, second degree, and/or third degree burn).
  • the inflammatory condition is traumatic brain injury.
  • the inflammatory condition is due to severe wounding.
  • the inflammatory condition is due to a muscle injury.
  • the inflammatory condition is associated with pancreatic islet cell transplantation.
  • the inflammatory disease is asthma.
  • the inflammatory disease is hepatitis.
  • the inflammatory disease is primary sclerosing cholangitis. In some embodiments, the inflammatory disease is primary biliary cholangitis. In some embodiments, the inflammatory disease is Still’s disease. In some embodiments, the inflammatory disease is uveitis. In some embodiments, the inflammatory disease is graft-versus-host disease (GvHD). In some embodiments, the inflammatory condition is tolerance induction for transplantation. In some embodiments, the inflammatory condition is transplant rejection. In some embodiments, the inflammatory disease is sepsis.
  • the cell expresses an antigen-specific receptor (e.g., T cell receptor or chimeric antigen receptor) that is specific to an antigen associated with the inflammatory condition or disease.
  • an antigen-specific receptor e.g., T cell receptor or chimeric antigen receptor
  • a cell, vector, nucleic acid, or lipid nanoparticle may be administered between 1 and 14 days over a 30-day period. In some embodiments, doses may be provided 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days over a 60-day period. Alternate protocols may be appropriate for individual subjects.
  • a suitable dose is an amount of a compound that, when administered as described above, is capable of detectably altering or ameliorating symptoms, or decreases at least one indicator of autoimmune, allergic or other inflammatory immune activity in a statistically significant manner by at least 10-50% relative to the basal (e.g., untreated) level, which can be monitored by measuring specific levels of blood components, for example, detectable levels of circulating immunocytes and/or other inflammatory cells and/or soluble inflammatory mediators including proinflammatory cytokines.
  • rapamycin or a rapalog is administered to the subject before the administration of cells, in conjunction with cells, and/or following the administration of cells.
  • Administration of rapamycin or a rapalog that is capable of inducing dimerization of the CISC components on the surface of a cell results in continued IL-2 signal transduction in vivo, promoting survival and proliferation of the CISC-expressing cell without the undesired effects that would be caused by IL-2 administration, such as activation of other T cells.
  • the rapamycin or rapalog that is administered 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, and AP23573, or a metabolite or derivative thereof.
  • the rapamycin or rapalog is administered at a dose of 0.001 mg/kg to 10 mg/kg body mass of the subject, or a dose between 0.001 mg/kg and 10 mg/kg. In some embodiments, the rapamycin or rapalog is administered at a dose of 0.001 mg/kg to 0.01 mg/kg, 0.01 mg/kg to 0.1 mg/kg, 0.1 mg/kg to 1 mg/kg, or 1 mg/kg to 10 mg/kg. In some embodiments, the rapamycin or rapalog is administered in a separate composition from the cells. In some embodiments, the rapamycin or rapalog is administered in multiple doses.
  • the rapamycin or rapalog is administered for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 or more days. In some embodiments, the rapamycin or rapalog is administered for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more weeks. In some embodiments, the subject is a human. In some embodiments, the administration of the rapamycin or rapalog results in prolonged survival of the administered cells, relative to a subject that is not administered rapamycin or a rapalog. In some embodiments, the administration of the rapamycin or rapalog increases the frequency of cells circulating in the peripheral blood of a subject, relative to a subject that is not administered rapamycin or a rapalog.
  • an appropriate dosage and treatment regimen provides the cells, vectors, nucleic acids, or lipid nanoparticles in an amount sufficient to provide therapeutic and/or prophylactic benefit.
  • a response can be monitored by establishing an improved clinical outcome (e.g., more frequent remissions, complete or partial, or longer disease-free survival) in treated subjects as compared to non-treated subjects.
  • Decreases e.g., reductions having statistical significance when compared to a relevant control
  • preexisting immune responses to an antigen associated with an autoimmune, allergic, or other inflammatory condition generally correlate with an improved clinical outcome.
  • Such immune responses may generally be evaluated using standard leukocyte and/or lymphocyte cell surface marker or cytokine expression, proliferation, cytotoxicity or released cytokine assays, which are routine in the art and may be performed using samples obtained from a subject before and after therapy.
  • an engineered cell is administered intravenously. In some embodiments, an engineered cell is delivered to a lung of a subject. In some embodiments, an engineered cell homes to the lung after administration. In some embodiments, an engineered cell is delivered to a kidney of a subject. In some embodiments, an engineered cell homes to the kidney after administration. In some embodiments, an engineered cell is delivered to the central nervous system. In some embodiments, an engineered cell homes to the central nervous system after administration. In some embodiments, an engineered cell is delivered to the peripheral nervous system. In some embodiments, an engineered cell homes to the peripheral nervous system after administration.
  • an engineered cell traffics to a site of tissue damage in the subject after administration. In some embodiments, an engineered cell traffics to a site having an elevated concentration of IL-33 after administration. In some embodiments, an engineered cell traffics to a site having an elevated concentration of IL- 18 after administration.
  • the subject is a human. In some embodiments, the subject is an animal. In some embodiments, the animal is a research animal. In some embodiments, the animal is a domesticated animal. In some embodiments, the animal is a rodent. In some embodiments, the rodent is a mouse, rat, guinea pig, chinchilla, or hamster. In some embodiments, the animal is a dog, cat, rabbit, guinea pig, hamster, or ferret. In some embodiments, the animal is a bovine, swine, llama, alpaca, sheep, or goat.
  • Example 1 Sustained Induction of hCCR4 of Human Engineered T Regulatory Cells (hEngTregs)
  • This Example evaluated the in vitro expression patterns of human CCR4 (hCCR4) on human engineered T regulatory cells (hEngTregs).
  • CD4+ cells were transduced with vectors encoding FOXP3 (“single-edited (FOXP3)”) and levels of hCCR4 were measured via flow cytometry on Day 7 and Day 11 post-transduction.
  • FOXP3 single-edited
  • mock CD4+ Single-edited
  • unstained cells were used as controls.
  • the results are shown in FIG. 2, and demonstrate that the edited cells expressed hCCR4. As hCCR4 expression is induced by FOXP3, the results indicate that the edited cells express FOXP3 at levels greater than the control groups, and that FOXP3 expression is sustained.
  • Human ST2 engineered T regulatory cells (ST2 hEngTregs) were generated as follows. On Day 0, CD4+ T cells were thawed and stimulated with a 3: 1 ratio of beads (anti- CD3:CD28). Next, while the cells were being activated by the beads, the cells were transduced with a lentiviral vector encoding ST2 on Day 1 in the presence of LentiBoost B (diluted 1 : 100) in 2 mL. High cell density (0.75 x 10 6 /mL) was maintained during the transduction process. Following transduction, the culture was supplemented with IL-2 daily. The cells were de-beaded on Day 3.
  • LentiBoost B diluted 1 : 100
  • the cells were electroporated with ribonucleoprotein (RNP) and edited with an AAV vector system.
  • RNP ribonucleoprotein
  • cells were electroporated with 80 pmol Cas9, 400 pmol T9 gRNA per IxlO 6 cells and then transduced with 50000 VG per cell at 5xl0 6 cells/mL in serum-free media for two hours on a rotator.
  • Cells were then recovered in regular media with IL-2 at 0.5xl0 6 cells/mL. From Day 7 until the end of the protocol, rapamycin was added to maintain a concentration of 10 nM (media was changed every other day).
  • the cells were restimulated with the anti- CD3/CD28 bead mixture.
  • FIG. 3A the ST2 EngTregs and Tregs engineered to express FOXP3 and CISC (IL-2) (“basic EngTregs”) were found to express approximately the same level of FOXP3 (left side of FIG. 3 A), while the ST2 hEngTregs were found to express ST2, whereas the basic EngTregs did not (right side of FIG. 3 A). That is, on Day 18 (with rapamycin selection), greater than 85% of EngTregs remained ST2-positive (right side of FIG. 3 A). This is further illustrated in FIG.
  • the ST2 hEngTregs were rested for four days without exposure to beads or cytokines. On Day 5, the cells were stimulated with 50 ng/mL of IL-33 or PMA (as a control) for 10 minutes. Cells were then fixed with CytoFix, permeabilized in BD Perm III, and stained for phosphorylated p65. As NFkB p65 phosphorylation is a downstream IL-33 signaling event, an increase in phospho- P65 indicates responsiveness to IL-33. The results are shown in FIG.
  • FIG. 5A-5C demonstrate that ST2 hEngTregs, but not basic EngTregs, show high p65 phosphorylation after IL-33 stimulation.
  • the ST2 hEngTregs (stimulated with anti-CD3/CD28 +/- IL-33) were further compared to the basic EngTregs (stimulated with anti-CD3/CD28 +/- IL-33), as shown in FIGs. 5A-5C.
  • IL-33 was found to enhance activation and proliferation of ST2 hEngTregs relative to basic EngTregs (FIG. 5A), as well as to promote more IL-13, KGF (a tissue repair cytokine), and TGF-P expression (FIG. 5B).
  • ST2 hEngTregs stimulated with IL- 33 were also found to have upregulated CCR8, demonstrating that a non-CCR4 chemokine receptor is also upregulated (FIG. 5C).
  • CCR8 is a brain-homing chemokine, demonstrating that ST hEngTregs may home to specific tissues, for example, the brain, in inflammatory contexts.
  • hEngTregs Immunogenicity of hEngTregs was evaluated by incubating either hEngTregs, or conventional bulk CD4+ T cells, expressing HLA-A*02 with T effector cells expressing a TCR specific to an HLA-A*02-restricted peptide, in the presence or absence of the peptide (FIG. 28).
  • T effector cell proliferation occurred following incubation with hEngTregs than with conventional CD4+ T cells, indicating that hEngTregs are less immunogenic, and therefore less likely to be rejected following engraftment, than conventional CD4+ T cells. This reduced susceptibility to rejection supports use of allogeneic hEngTregs for therapeutic applications.
  • mice were used to characterize ST2 EngTreg performance in the context of acute lung injury (ALI).
  • murine engineered Tregs mEngTregs
  • CD4+ T cells were isolated from lymphoid organs of male C57BL/6 wildtype (autologous) or CB6F1 (allogeneic, C57BL/6 x BALB/c Fl cross) donor mice.
  • the CD4+ T cells were stimulated with anti-CD3:CD28 beads and supplemented with IL-2.
  • CRISPR-Cas9 technology with AAV delivery was used to insert a MND promoter upstream from the first coding exon of the endogenous F0XP3 gene and downstream from the Treg-specific demethylated region (TSDR).
  • TSDR Treg-specific demethylated region
  • mEngTregs were then cultured with IL-2 and IL-33 to help promote “tissue Treg” and “core Treg” effector phenotypes in absence of CISC (IL-2 signaling) and ST2 reagents in the murine system. Enrichment was column-based and performed via positive selection for LNGFR-expressing cells. At Day 7 post-transduction, mEngTregs expressed high levels of Foxp3 and ST2 transgene tag LNGFR (FIG. 17A). Splenocyte-derived mEngTregs expressed markers including CD25 and CCR4 prior to adoptive cell transfer (FIG. 17B).
  • the ALI model was established as follows and is illustrated in FIG. 16. Lung injury in recipient mice was induced via intratracheal LPS instillation (5mg/kg intratracheally) at Day 0. mEngTregs were adoptively transferred on day 1 via intravenous tail-vein injection (2 million cells per mouse in a volume of lOOpl formulated in PBS + 2% FBS). On days 4, 6, and 11, spleen, lung, and bronchoalveolar lavage fluid (BALF) were collected and mEngTreg engraftment was assessed by flow cytometry. Bodyweights were monitored and blood oxygen content was determined by pulse-oximetry on days 6 and 11. The right lung lobe was also measured on Day 11 at experimental end-point. Splenocytes from B6 x Balb/c mice (Fl) were used as a surrogate for allogeneic mEngTregs.
  • B6 x Balb/c mice Fl
  • the mEngTreg-treated animals also showed fewer inflammatory infiltrates e.g., neutrophils) in lung and BAL tissue (FIGs. 8 A and 8B, respectively). These subjects also demonstrated recovery of alveolar macrophages (FIG. 9A), which are important for tissue repair. The mEngTreg-treated animals also showed dendritic cell numbers that were comparable to healthy controls at Day 11 (FIG. 9B).
  • mEngTreg-treated LPS ALI mice had a lower concentration of several inflammatory cytokines in BALF samples as measured by cytokine bead assay (CB A) on Day 6 (FIG. 18) and on Day 11 (FIG. 10). Increased CCR4 ligands, CCL17 and CCL22, were detected at the site of injury. The data show reduction of both ligands as disease was resolved.
  • CB A cytokine bead assay
  • mice were administered LPS (day 0) to establish ALI, administered autologous or allogeneic mEngTregs (day 1), and euthanized (day 5) to collect lungs, BAL, spleen, mediastinal lymph nodes (mLN), and colons for analysis of mEngTregs (FIG. 21A).
  • Cells were stained for live/dead, Ki67, EdU, CD4, CD25, CD45.1, CD45.2, ST2, LNGFR, and FoxP3.
  • mEngTregs were produced from murine CD45.2+ donors, allowing quantification of mEngTregs by measuring CD45.2+ cells (FIG. 21B).
  • mEngTreg proliferation was measured by Ki67 staining (FIG. 21B). Greater levels of mEngTregs were observed in mice administered LPS, indicating that engraftment is higher in inflammatory environments (FIG. 22A). Both autologous (derived from B6 donor) and allogeneic (derived from a CB6F1 (C57BL/6 x BALB/c Fl cross) donor) engrafted to similar extents in LPS- treated mice (FIG. 22B).
  • mice were administered LPS (day 0) to establish ALI, administered autologous or allogeneic mEngTregs (day 1), and euthanized (day 11) to collect lungs, which were sectioned and Trichome stained to evaluate collagen deposition, a measure of lung fibrosis (FIG. 27A). While LPS-challenged mice exhibited substantial collagen deposition, lungs of mice administered either autologous or allogeneic mEngTregs resembled naive control mice not challenged with LPS (FIG. 27A), with markedly lower interstitial fibrosis scores compared to mice administered LPS alone (FIG. 27B). These results indicate that engineered Tregs mitigate collagen deposition and fibrosis associated with local inflammation.
  • Lung homing was determined by measuring levels of CCR4, a chemokine receptor known to promote lung homing.
  • CCR4 a chemokine receptor known to promote lung homing.
  • hEngTregs the population of CCR4+ cells was found to be increased relative to CD4+ cells following stimulation with IL-2 and rapamycin (left graph, FIG. 11).
  • ST2 mEngTregs stimulated with IL-2 and IL- 33 (right graph, FIG. 11).
  • mEngTregs were primed ex vivo with IL- 33 and, five days later, superior enrichment in the lung was observed, as evidenced by the presence of donor (CD45.2+) cells in the lung samples assessed by flow cytometry (FIG. 12).
  • a time course experiment was also performed, and it was observed that mEngTregs transition from preferential homing to the spleen early to preferential lung homing within a week (FIG. 13).
  • engraftment was compared in the spleen, lung, and BALF on Day 6 and Day 13 after transduction with mEngTregs. As can be seen in FIG. 14, there appears to be specific homing of adoptively transferred cells to desired areas in the context of ALI (lung and BALF).
  • Example 5 Autologous mEngTreg Efficacy in a Bleomycin-induced ALI Model mEngTregs were produced as described above using BALB/c CD45.1+ T cells (donor cells). Mice (BALB/c; CD45.2+) received bleomycin treatment (6U/kg intratracheally (i.t.)) on Day 0. On Day 1, mice were administered IxlO 6 mEngTregs cultured with IL-2 (Group 1) or IxlO 6 mEngTregs cultured with IL-2 and IL-33 (Group 2). The controls were administered bleomycin and PBS (Group 3) or PBS and PBS (Group 4). On Day 7, samples were obtained and analyzed.

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Abstract

Certains aspects de la divulgation concernent des compositions et des procédés de production de lymphocytes T régulateurs (Treg) avec des phénotypes de réparation par modification de cellules pour favoriser l'expression d'au moins un médiateur de réparation.
PCT/US2024/013175 2023-01-27 2024-01-26 Lymphocytes t régulateurs de réparation modifiés Ceased WO2024159138A1 (fr)

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