WO2025171122A1 - Lymphocytes t régulateurs améliorés et procédés d'utilisation associés - Google Patents

Lymphocytes t régulateurs améliorés et procédés d'utilisation associés

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Publication number
WO2025171122A1
WO2025171122A1 PCT/US2025/014776 US2025014776W WO2025171122A1 WO 2025171122 A1 WO2025171122 A1 WO 2025171122A1 US 2025014776 W US2025014776 W US 2025014776W WO 2025171122 A1 WO2025171122 A1 WO 2025171122A1
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WIPO (PCT)
Prior art keywords
cells
siglec
nucleic acid
fibrosis
treg
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PCT/US2025/014776
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English (en)
Inventor
Eduardo MARBÁN
Ke LIAO
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Cedars Sinai Medical Center
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Cedars Sinai Medical Center
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Publication of WO2025171122A1 publication Critical patent/WO2025171122A1/fr
Pending legal-status Critical Current
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • A61K35/761—Adenovirus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A61K38/178—Lectin superfamily, e.g. selectins
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/32—T-cell receptors [TCR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/416—Antigens related to auto-immune diseases; Preparations to induce self-tolerance
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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

Definitions

  • a method of treating fibrosis including administering a nucleic acid encoding a sialic acid-binding immunoglobulin-type lectin 9 (Siglec-9) protein to a subject in need of fibrosis treatment, wherein the Siglec-9 protein is expressed by the nucleic acid in regulatory T (T reg ) cells in the subject.
  • T reg regulatory T
  • the subject is need of cardiac fibrosis treatment.
  • Also provided is a method of treating a condition associated with inflammation and/or fibrosis including administering a nucleic acid encoding a sialic acid- binding immunoglobulin-type lectin 9 (Siglec-9) protein to a subject in need of treating a condition associated with inflammation and/or fibrosis, wherein the Siglec-9 protein is expressed by the nucleic acid in regulatory T (T reg ) cells in the subject.
  • T reg regulatory T
  • a method of treating a condition associated with inflammation and/or fibrosis comprising administering to a subject in need of treating a condition associated with inflammation and/or fibrosis a nucleic acid configured to induce expression of a sialic acid-binding immunoglobulin-type lectin 9 (Siglec-9) protein in regulatory T (Treg) cells of the subject.
  • the condition associated with inflammation and/or fibrosis includes cardiac inflammation and/or cardiac fibrosis.
  • the condition associated with inflammation and/or fibrosis includes myocardial infarction, hypertension, myocarditis, transplant rejection, cardiomyopathy, heart failure, and amyloidosis.
  • the method includes genetically modifying Treg cells with the plasmid or the viral vector including the nucleic acid (or the nucleic acid encoding the Siglec-9 protein).
  • the viral vector includes an adenoviral vector, retroviral vector, or a lentiviral vector.
  • the population of Treg cells are derived from cells autologous to the subject. In some embodiments, the population of Treg cells are derived from cells allogeneic to the subject.
  • the method further includes administering to the subject a vehicle including the nucleic acid (or the nucleic acid encoding the Siglec-9 protein), wherein the vehicle is configured to deliver the nucleic acid (or the nucleic acid encoding the Siglec-9 protein) to T reg cells in the subject.
  • the vehicle includes: a lipid nanoparticle (LNP); and a Treg cell-targeting moiety associated with the LNP.
  • the Treg cell-targeting moiety includes an antigen binding protein that binds a T reg cell marker.
  • the T reg cell marker is selected from: CD25 and/or Foxp3.
  • the method wherein the antigen binding protein is an antibody.
  • the antigen binding protein is selected from: an anti-CD25 monoclonal antibody and/or anti-Foxp3 monoclonal antibody.
  • the nucleic acid includes mRNA encoding the Siglec-9 protein.
  • the subject has suffered or is at risk of suffering from myocardial infarction.
  • the subject is in need of treating angiotensin II-induced cardiac fibrosis.
  • the subject is in need of treating cardiac fibrosis subsequent to a myocardial infarction.
  • the Siglec-9 protein includes an amino acid sequence at least 80% identical to SEQ ID NO: 2.
  • Also provided is a method of treating a condition associated with inflammation and/or fibrosis including administering a nucleic acid encoding a membrane- bound fibroblast-specific binding protein to a subject in need of treating a condition associated with inflammation and/or fibrosis, wherein the membrane-bound fibroblast- specific binding protein is expressed by the nucleic acid in regulatory T (T reg ) cells in the subject.
  • a method of treating a condition associated with inflammation and/or fibrosis comprising administering to a subject in need of treating a condition associated with inflammation and/or fibrosis a nucleic acid configured to induce expression of a membrane-bound fibroblast-specific binding protein in regulatory T (Treg) cells in the subject.
  • the nucleic acid encodes the membrane-bound fibroblast- specific binding protein.
  • the membrane-bound fibroblast-specific binding protein includes sialic acid-binding immunoglobulin-type lectin 9 (Siglec-9).
  • the membrane-bound fibroblast-specific binding protein includes an amino acid sequence at least 80% identical to SEQ ID NO:2.
  • the condition associated with inflammation and/or fibrosis includes an inflammatory tissue including fibroblasts having elevated expression of amine oxidase, copper containing 3 (AOC3).
  • the fibroblasts having elevated expression of AOC3 include myofibroblasts.
  • the myofibroblasts include cardiac myofibroblasts.
  • the method further includes: obtaining a population of immune cells including regulatory T (T reg ) cells; delivering the nucleic acid encoding the sialic acid-binding immunoglobulin-type lectin 9 (Siglec-9) protein to the Treg cells, whereby the Siglec-9 protein is overexpressed in the Treg cell; contacting the Treg cells overexpressing the Siglec-9 protein with at least one fibroblast cell and/or myofibroblast cell in a subject, wherein the at least one fibroblast cell and/or myofibroblast cell express amine oxidase, copper containing 3 (AOC3), to thereby treat the condition associated with inflammation and/or fibrosis.
  • T reg regulatory T
  • Siglec-9 sialic acid-binding immunoglobulin-type lectin 9
  • a method of targeted immune modulation of a subject including: obtaining a population of regulatory T cells (Treg); delivering at least one vehicle to a population of Treg cells, wherein the at least one vehicle includes a nucleic acid encoding sialic acid-binding immunoglobulin-type lectin-9 (Siglec-9) and that expresses Siglec-9 in the Treg cells, thereby producing a population of enhanced Treg cells overexpressing Siglec-9; and administering to a subject the population of enhanced T reg cells.
  • Treg regulatory T cells
  • Siglec-9 sialic acid-binding immunoglobulin-type lectin-9
  • a method of targeted immune modulation of a subject comprising: obtaining a population of regulatory T cells (Treg); delivering at least one vehicle to the population of Treg cells, wherein the at least one vehicle comprises a nucleic acid configured to induce expression of a sialic acid-binding immunoglobulin-type lectin-9 (Siglec-9) protein in the Treg cells of the population, thereby producing a population of enhanced Treg cells overexpressing Siglec-9; and administering to a subject the population of enhanced Treg cells.
  • the at least one vehicle includes a liposome, or a lipid nanoparticle (LNP).
  • the at least one vehicle includes a viral vector.
  • the viral vector includes an adenoviral vector, retroviral vector, or a lentiviral vector.
  • the nucleic acid includes a promoter operatively linked to a nucleotide sequence encoding the Siglec9, wherein the promoter upregulates expression of Siglec9 in Treg cells.
  • the subject includes at least one myofibroblast cell having elevated expression of AOC3 compared to other cell types.
  • the at least one myofibroblast cell is at least one cardiac myofibroblasts.
  • the subject is in need of treatment for fibrosis.
  • the fibrosis is a cardiac fibrosis.
  • the cardiac fibrosis is subsequent to a myocardial infarction.
  • a method of inhibiting fibrotic gene expression including: obtaining a population of Treg cells; engineering the Treg cells to overexpress Siglec-9, thereby producing enhanced Treg cells; and administering an effective amount of the enhanced T reg cells to a subject in need of inhibiting fibrotic gene expression.
  • the subject is in need of inhibiting fibrotic gene expression in cardiac myofibroblasts.
  • the fibrotic gene is at least one of Collagen I, ⁇ - smooth muscle actin ( ⁇ -SMA), and/or amine oxidase, copper containing 3 (AOC3).
  • engineering includes delivering at least one vehicle including a nucleic acid encoding Siglec-9 to the population of Treg cells.
  • the at least one vehicle includes at least one of a liposome, a nanoparticle, a lipid nanoparticle (LNP), a viral vector, a cell-penetrating peptide (CPP), and/or an extracellular vesicle (EV).
  • the nucleic acid includes mRNA.
  • overexpressing Siglec-9 includes upregulating expression of the mRNA.
  • the subject is in need of treatment for fibrosis.
  • the fibrosis is a cardiac fibrosis.
  • the cardiac fibrosis is subsequent to a myocardial infarction.
  • the techniques described herein relate to the method of any one of preceding claims, wherein the subject has upregulated expression of amine oxidase, copper containing 3 (AOC3) in cardiac myofibroblasts.
  • administering includes intravenous or oral administration.
  • an isolated nucleic acid further including a nucleotide sequence encoding a sialic acid-binding immunoglobulin-like lectin 9 (Siglec-9) protein operatively linked to a promoter configured to express the Siglec-9 protein in a regulatory T (Treg) cell.
  • Siglec-9 sialic acid-binding immunoglobulin-like lectin 9
  • the promoter is a regulatory T (Treg) cell-specific promoter selected from the group consisting of a Foxp3 promoter and an Interleukin-2 (IL-2) promoter.
  • Treg regulatory T
  • IL-2 Interleukin-2
  • a vector including the isolated nucleic acid of the present disclosure.
  • a regulatory T (T reg ) cell including the isolated nucleic acid, or the vector of the present disclosure.
  • compositions including a population of vehicles, each vehicle including: a nucleic acid encoding sialic acid-binding immunoglobulin-like lectin 9 (Siglec-9); and a regulatory T (Treg) cell-targeting moiety configured to deliver the nucleic acid encoding Siglec-9 to Treg cells.
  • vehicles of the population include a lipid nanoparticle (LNP).
  • the nucleic acid includes mRNA.
  • the Treg cell-targeting moiety includes an antigen binding protein that binds a Treg cell marker.
  • the Treg cell marker is selected from: CD25 and/or Foxp3.
  • the antigen binding protein is an antibody.
  • the antigen binding protein is selected from: an anti-CD25 monoclonal antibody and/or anti-Foxp3 monoclonal antibody.
  • a composition including a population of enhanced regulatory T (T reg ) cells overexpressing sialic acid-binding immunoglobulin-like lectin 9 (Siglec-9).
  • the enhanced Treg cells include at least one endocytosed vehicle, the at least one endocytosed vehicle including a plurality of nucleic acids encoding sialic acid-binding immunoglobulin-like lectin 9 (Siglec-9).
  • the at least one endocytosed vehicle includes at least one of a liposome, a nanoparticle, a lipid nanoparticle (LNP), a viral vector, a cell-penetrating peptide (CPP), and/or an extracellular vesicle (EV).
  • the plurality of nucleic acids includes a plurality of mRNA transcripts encoding Siglec-9.
  • the enhanced Treg cells exhibit Siglec-9-dependent interaction with cells expressing amine oxidase, copper containing 3 (AOC3).
  • the enhanced T reg cells exhibit Siglec-9-dependent interaction with cardiac myofibroblasts expressing AOC3.
  • FIG 1 illustrates an example non-limiting embodiment depicting a method of administering a nucleic acid to enhanced Treg cells that overexpress Siglec-9.
  • FIG. 2 illustrates graphical representations of data obtained from in vitro experiments of the effect of Angiotensin II concentrations on fibrotic gene expression.
  • FIG. 3 illustrates graphical representations of data obtained from in vitro experiments of the effect of Angiotensin II on fibrotic gene expression over time.
  • FIG. 3 illustrates graphical representations of data obtained from in vitro experiments of the effect of Angiotensin II on fibrotic gene expression over time.
  • FIG. 12 illustrates graphical representations of data from in vitro experiments testing the role of AOC3 in enhanced Treg cells targeting to AOC3-expressing cardiac fibroblasts.
  • FIG. 13 illustrates data of upregulation of AOC3 and fibrotic genes observed from in vivo experiments using a myocardial infarction model.
  • FIG. 14 illustrates a schematic diagram of and data from an experiment showing the post-myocardial infarction effects of enhanced Treg cells on fibrotic gene expression in cardiac fibroblasts.
  • FIG. 15 illustrates an in vivo experimental protocol for studying the anti- fibrotic effect of enhanced Treg cells. [0041] FIG.
  • FIG. 16 illustrates an exemplary non-limiting embodiment of the cellular processes by which Siglec-9 could promote T reg cell function by affecting immune cell signaling.
  • FIG. 17 illustrates a schematic diagram of and data from a collagen contraction assay depicting reduced contraction in gels with siglec-9 engineered Tregs, indicating effective anti-fibrotic activity.
  • FIG. 18 illustrates schematic diagrams of and data from a collagen contraction assay depicting decreased effectiveness in reducing fibroblast contraction when siglec-9 is blocked, emphasizing the importance of siglec-9 in mediating Treg anti-fibrotic activity.
  • FIG. 17 illustrates a schematic diagram of and data from a collagen contraction assay depicting reduced effectiveness in reducing fibroblast contraction when siglec-9 is blocked, emphasizing the importance of siglec-9 in mediating Treg anti-fibrotic activity.
  • FIG. 19 illustrates schematic diagrams of and data from a collagen contraction assay after AOC3 neutralization showing decreased reduction in fibroblast contraction, further highlighting the critical role of the siglec-9-AOC3 interaction in targeting fibrotic processes.
  • FIG.20 is a schematic diagram showing a in vivo experimental design for assessing the therapeutic effects of enhanced Treg cells in a model of myocardial infarction.
  • FIG. 21 illustrates immunostaining analysis of heart sections showing infiltration of RFP-tagged siglec-9 engineered Tregs, with notable localization to AOC3- positive areas in MI hearts.
  • FIG. 22 illustrates a graphical illustration of mRNA levels AOC3, collagen I, and ⁇ -SMA in heart tissues, demonstrating reduced fibrosis markers in siglec-9 Treg-treated groups.
  • FIG.25 illustrates the results of a Western blot assay showing the in vitro upregulation of the SHP-1 pathway in siglec-9 engineered Tregs exposed to AOC3 showing phosphorylation levels of SHP-1, Pyk2 and Src.
  • FIG. 26 shows quantitation of expression levels of the Western Blot shown in FIG.5.
  • Reg cells Regulatory T cells
  • Ischemic cardiomyopathy refers to the progressive deterioration of heart function due to chronic ischemia, or reduced blood flow to the heart.
  • Fibrosis is a pathological process characterized by the excessive accumulation of extracellular matrix proteins and fibroblast differentiation into myofibroblasts, leading to the formation of scar tissue. Fibrosis plays a significant role in the development and progression of ICM. The underlying reduction in blood flow results in the accumulation of scar. Cardiac fibrosis plays various key roles as ICM progresses. Early on, the reduced blood flow increases the production of collagen and other extracellular matrix proteins. As ischemia continues, fibrosis becomes more extensive and can lead to the disruption of the heart's normal structure and function. The accumulation of fibrosis contributes to the development of heart failure (HF), a condition in which the heart is unable to pump blood efficiently, leading to shortness of breath, fatigue, and premature death.
  • HF heart failure
  • ICM is often associated with renal fibrosis, leading to chronic kidney disease.
  • HFrEF reduced ejection fraction
  • ICM is also frequently linked with multi-organ fibrosis, extending beyond the heart and kidneys to include the lungs and liver. This systemic fibrotic response can exacerbate overall disease burden, e.g., pulmonary fibrosis can impair gas exchange and increase the workload on an already compromised heart.
  • Treg regulatory T
  • these methods encompass administering a nucleic acid configured to induce expression in Treg cells (e.g., administering a nucleic acid encoding sialic acid-binding immunoglobulin-type lectin 9 (Siglec-9)) to Treg cells, thereby enhancing their functional properties, including, but not limited to, targeting and mitigating cardiac fibrosis.
  • a nucleic acid configured to induce expression in Treg cells (e.g., administering a nucleic acid encoding sialic acid-binding immunoglobulin-type lectin 9 (Siglec-9)) to Treg cells, thereby enhancing their functional properties, including, but not limited to, targeting and mitigating cardiac fibrosis.
  • induce expression denotes expression of a gene product at a level above (e.g., 1.2, 1.3, 1.4, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 9, 10, 20, 30, 40, 50 100 fold or more higher than) the level of expression without inducing expression of the gene product.
  • methods include delivering (or administering) a population of Treg cells, modified to express (or overexpress) Siglec-9, to a subject requiring treatment for cardiac fibrosis.
  • the enhancement of T reg cells may also involve, in some embodiments, using lipid nanoparticle (LNP) vehicles for effective delivery of the nucleic acid encoding Siglec-9 to Treg cells in the subject.
  • LNP lipid nanoparticle
  • the enhanced T reg cells demonstrate an increased ability to target and interact with fibroblast and/or myofibroblast cells, particularly those expressing amine oxidase, copper containing 3 (AOC3), in subjects suffering from cardiac fibrosis.
  • the administration of Treg cells modified to overexpress Siglec-9, regulate immune responses in subjects, especially those at risk of or having suffered myocardial infarction.
  • these enhanced Treg cells can be used to treat conditions associated with inflammation and/or fibrosis, including, but not limited, to cardiac fibrosis caused by myocardial infarction.
  • the use of Treg cells in these methods involves delivering nucleic acids configured to induce expression of Siglec-9 to Treg cells in a subject (e.g., without administering the cells).
  • the use of T reg cells in these methods involves delivery of nucleic acids encoding Siglec-9 to Treg cells in a subject without the direct administration of cells. This approach offers a potential advantage by minimizing inflammatory responses often associated with cell-based therapies.
  • a novel composition comprising a population of delivery vehicles, each containing nucleic acids that are configured to induce expression of sialic acid-binding immunoglobulin-like lectin 9 (Siglec-9) in Treg cells (e.g., nucleic acids that encode sialic acid-binding immunoglobulin-like lectin 9 (Siglec-9)), is also provided.
  • Siglec-9 sialic acid-binding immunoglobulin-like lectin 9
  • the vehicles can be designed to target T reg cells, facilitating the delivery and subsequent expression of Siglec-9 by Treg cells.
  • the delivery vehicles can include liposomes, nanoparticles, lipid nanoparticles (LNPs), viral vectors, cell- penetrating peptides (CPPs), and extracellular vesicles (EVs).
  • LNPs lipid nanoparticles
  • CPPs cell- penetrating peptides
  • EVs extracellular vesicles
  • each vehicle encapsulates a plurality of mRNA transcripts that encode Siglec-9, ensuring efficient transport and expression within Treg cells.
  • a population of enhanced T reg cells is produced, wherein the T reg cells are enhanced by their ability to overexpress Siglec-9.
  • such modification of T reg cells can significantly increase the therapeutic potential of Treg cells, including, but not limited to, in targeting and inhibiting fibrotic genes.
  • the enhanced T reg cells within the composition exhibit high-affinity binding to amine oxidase, copper containing 3 (AOC3), which is prominently expressed by cardiac myofibroblasts.
  • AOC3 amine oxidase, copper containing 3
  • such specificity can mitigate and/or treats cardiac fibrosis, a condition often triggered by high blood pressure or myocardial infarction.
  • the enhanced Treg cells target fibrosis-causing cells and/or inhibit the expression of fibrotic genes, including, but not limited to, Collagen I, ⁇ -smooth muscle actin ( ⁇ -SMA), and AOC3.
  • fibrotic genes including, but not limited to, Collagen I, ⁇ -smooth muscle actin ( ⁇ -SMA), and AOC3.
  • ⁇ -SMA ⁇ -smooth muscle actin
  • AOC3 fibrotic genes
  • This targeted approach not only addresses the underlying causes of cardiac fibrosis but also offers a novel therapeutic strategy for managing conditions caused by other fibrosis-associated genes and diseases.
  • the foregoing methods and compositions therefore, represent a significant advancement in the treatment of cardiac fibrosis and related conditions, offering a more targeted and effective immunotherapeutic treatment.
  • Vehicle or “delivery vehicle” as used herein refers to a molecule or collection of molecules suitable for delivering a nucleic acid into a cell.
  • a vehicle can be a plasmid, virus, or other nucleic acid construct, a liposome, nanoparticle, cell-penetrating peptide (CPP), or extracellular vesicle (EV), engineered to carry material into host cells.
  • CPP cell-penetrating peptide
  • EV extracellular vesicle
  • a vehicle can be used to introduce genetic information into an organism or cell line, for example, to study gene function, protein expression, or to develop gene therapy applications.
  • “Liposome” as used herein has its ordinary and customary meaning as understood by one of ordinary skill in the art, in view of the present disclosure.
  • Liposomes include spherical vesicles comprising one or more phospholipid bilayers, used to encapsulate and deliver materials to cells, and include conventional liposomes, multilamellar liposomes (MLVs), small unilamellar vesicles (SUVs), large unilamellar vesicles (LUVs), cationic liposomes, anionic liposomes, stealth liposomes, pH-sensitive liposomes, temperature-sensitive liposomes, and immunoliposomes.
  • MUVs multilamellar liposomes
  • SUVs small unilamellar vesicles
  • LUVs large unilamellar vesicles
  • cationic liposomes anionic liposomes
  • stealth liposomes pH-sensitive liposomes
  • temperature-sensitive liposomes temperature-sensitive liposomes
  • immunoliposomes immunoliposomes.
  • Nanoparticles include, without limitation, metallic nanoparticles (e.g., gold, silver), quantum dots, dendrimers, carbon-based nanoparticles (e.g., fullerenes, carbon nanotubes), lipid-based nanoparticles (e.g., “lipid nanoparticles” (LNP)), ceramic nanoparticles, polymeric nanoparticles, magnetic nanoparticles, solid lipid nanoparticles, nanostructured lipid carriers.
  • “Viral vector” as used herein is defined according to its ordinary and customary meaning as understood by one of ordinary skill in the art, in view of the present disclosure.
  • Viral vectors include adenoviral vectors, retroviral vectors, lentiviral vectors, adeno-associated viral vectors, herpes simplex viral vectors, poxviral vectors, baculoviral vectors, alphaviral vectors, rabies viral vectors, vaccinia viral vectors, foamy viral vectors, measles virus vectors, Sindbis viral vectors, and mumps virus vectors.
  • Cell-penetrating peptide (CPP) has its ordinary and customary meaning as understood by one of ordinary skill in the art, in view of the present disclosure.
  • CPPs include TAT peptides, penetratins, transportans, polyarginines, VP22s, SynB peptides, MPG peptides, pVECs, R9s, FHV peptides, MAPs, CADYs, Hph-1s, KLA peptides, Pep-1s, PTD-4s, R8s, GALAs, HIV-1 Tat peptides, TP10s, ANTP peptides, buforin IIs, K-FGFs, SV40 NLss, nona-arginines, azurins, hCT(9-32)-k7s, R7s, LMWPs, KALAs, PepFect14s, TP508s, fusogenic peptides, M918s, INF7s, Pep-7s, SynB3s, drosocins, SAP(1-8)s, LAH4s, BP100s, and
  • EVs include lipid bilayer structures generated by cells, and include exosomes, microvesicles, epididimosomes, argosomes, exosome-like vesicles, microparticles, promininosomes, prostasomes, dexosomes, texosomes, dex, tex, archeosomes and oncosomes.
  • Immune modulation refers to altering the activity of the adaptive or innate immune system of a vertebrate organism.
  • the transfection reagent comprises one or more of a liposome, lipid nanoparticle (LNP), an extracellular vesicle (EV), and a polyethylene glycol (PEG)-cationic lipid complex (PCLC).
  • the transfection reagent comprises EV derived from cardiosphere-derived cells (CDC).
  • the nucleic acid comprises a promoter operatively linked to a nucleotide sequence encoding the Siglec9, wherein the promoter upregulates expression of Siglec-9 in Treg cells.
  • the nucleic acid comprises a promoter operatively linked to a nucleotide sequence encoding an epigenetic modulator (e.g., a CRISPR activation system) configured to induce expression of siglec-9 from an endogenous locus in the T reg cells.
  • the promoter can be any suitable promoter for expressing the Siglec-9 protein in a Treg cell when the nucleic acid is incorporated into the cell (e.g., as described herein).
  • the promoter is a regulatory T (T reg ) cell-specific promoter.
  • the T reg cell-specific promoter is selected from the group consisting of a Foxp3 promoter and an Interleukin-2 (IL-2) promoter.
  • the promoter is a constitutive promoter. In some embodiments, the promoter is a conditional promoter (e.g., a Tet-responsive promoter). [0096] In any of the methods of the present disclosure, in some embodiments, the method includes engineering T reg cells to express (e.g., overexpress) siglec-9 protein, thereby producing engineered or enhanced T reg cells. The T reg cells can be engineered using any suitable option. In some embodiments, the method includes delivering a nucleic acid of the present disclosure to the Treg cell. Delivering the nucleic acid to the Treg cell can be done using any suitable option for the delivery vehicle.
  • engineering the Treg cells includes delivering a nucleic acid of the present disclosure to the Treg cell under suitable conditions to induce expression of siglec-9 protein in the Treg cell.
  • delivering the nucleic acid to the T reg cell includes contacting an effective amount of the vehicle with a population of Treg cells under suitable conditions. Contacting at least one vehicle with at least one Treg cell can be done by any suitable option. In some embodiments, contacting is done in vitro or ex vivo. In some embodiments, contacting includes providing a plurality of vehicles in a culture medium in which Treg cells are being or will be maintained.
  • contacting can involve adding an amount, which can be expressed as a concentration, molarity, or quantity, of vehicles to the culture medium in which Treg cells are being or will be maintained.
  • vehicles can be provided in the culture medium at the start of culturing of the Treg cells.
  • vehicles can be added at regular intervals during culturing.
  • the method includes genetically modifying the Treg cell by, without limitation, transfection, transduction, electroporation, gene editing (e.g., CRISPR Cas9), etc., to thereby overexpress Siglec-9 in the T reg cells.
  • co-culturing can include providing a plurality of vehicles in a culture medium in which Treg cells are being or will be maintained. In some embodiments, co-culturing can involve adding an amount of vehicles to the culture medium in which Treg cells are being or will be maintained. In some embodiments, vehicles are provided in the culture medium at the start of culturing the Treg cells.
  • vehicles can be added at regular intervals during culturing.
  • Co-culturing vehicles with Treg cells can be done for a suitable period of time.
  • the period of time for co-culturing is the amount of time Treg cells are cultured in the presence of vehicles in the culture medium.
  • the period of time for co-culturing can be the amount of time Treg cells are cultured in a medium to which vehicles were added.
  • the period of time can be at least about 24 hours, about 36 hours, about 48 hours, about 60 hours, about 72 hours, about 4 days, about 5 days, about 1 week, about 2 weeks, about 1 month, about 2 months, about 3 months, or longer, or any amount of time in range defined by any two of the preceding values.
  • a suitable amount of the vehicle can be provided so as to induce overexpression of Siglec-9, and/or increased proliferation when the Treg cells are contacted with or co-cultured with a plurality of vehicles.
  • the ratio of T reg cells to vehicles provided in the culture medium can be any suitable ratio.
  • the number of the vehicle can be at least about 30 fold, about 40 fold, about 50 fold, about 75 fold, about 100 fold, about 125 fold, about 150 fold, about 200 fold, about 300 fold, about 400 fold, about 500 fold, about 600 fold, about 700 fold, about 800 fold, about 900 fold, about 1000 fold, about 1200 fold, about 1500 fold, about 2000 fold, about 3000 fold, about 4000 fold, about 5000 fold, about 7500 fold, about 10000 fold or more, or a fold amount in a range defined by any two of the preceding values, the number of T reg cells.
  • the vehicle provided at a ratio of Treg cells to vehicle particles of about 1:1000.
  • the MOI is, is about, or is at least 0.1, 0.2, 0.5, 1, 1.2, 1.5, 2, 2.2, 2.5, 3, 3.2, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 20, 50, 100 or more, or optionally the MOI is in a range defined by any two of the preceding values (e.g., 0.1-100, 0.1-10, 0.2-20, 1-5, 1-3, etc.).
  • contacting is done in vivo. In some embodiments, contacting is achieved in the subject, for example, by administering the plurality of vehicles to the subject, where the vehicle includes a Treg cell-targeting moiety associated therewith.
  • the method includes administering to the subject a vehicle comprising the nucleic acid encoding the Siglec-9 protein, wherein the vehicle is configured to deliver the nucleic acid encoding the Siglec-9 protein to Treg cells in the subject.
  • the vehicle includes the nucleic acid encoding the Siglec-9 protein that specifically expresses Siglec-9 protein in T reg cells in the subject. Any suitable option can be used to specifically express the Siglec-9 protein encoded by the nucleic acid in Treg cells in the subject.
  • the nucleic acid encoding the Siglec-9 protein is operatively linked to a T reg cell-specific promoter (e.g., a Foxp3 or IL-2 promoter).
  • the method includes administering to the subject a vehicle comprising the nucleic acid configured to induce expression (e.g., endogenous or exogenous expression) of the Siglec-9 protein in the T reg cell, wherein the vehicle is configured to deliver the nucleic acid to Treg cells in the subject.
  • the vehicle includes the nucleic acid configured to specifically induce expression (e.g., endogenous or exogenous expression) of the Siglec-9 protein. Any suitable option can be used to specifically induce expression (e.g., endogenous or exogenous expression) of the Siglec-9 protein by the nucleic acid in Treg cells in the subject.
  • any suitable Treg cell marker can be used to target the vehicle to Treg cells in the subject, such as, without limitation anti-CD25 monoclonal antibodies and/or anti-Foxp3 monoclonal antibodies.
  • the antigen binding protein is an antibody or antigen- binding fragment thereof that binds to the Treg cell marker.
  • the vehicle e.g., LNP
  • the antibody or antigen-binding fragment thereof that binds to the T reg cell marker, to thereby target the vehicle to T reg cells in the subject.
  • the method includes obtaining a population of immune cells comprising Treg cells; delivering the nucleic acid encoding the sialic acid- binding immunoglobulin-type lectin 9 (Siglec-9) protein to the T reg cells, whereby the Siglec- 9 protein is overexpressed in the Treg cell; contacting the Treg cells overexpressing the Siglec- 9 protein with at least one fibroblast cell and/or myofibroblast cell in a subject, wherein the at least one fibroblast cell and/or myofibroblast cell express amine oxidase, copper containing 3 (AOC3), to thereby treat the condition associated with inflammation and/or fibrosis.
  • Siglec-9 sialic acid- binding immunoglobulin-type lectin 9
  • the nucleic acid encoding the Siglec-9 protein is configured to overexpress the Siglec-9 protein in the T reg cells.
  • the population of immune cells is obtained from the subject (e.g., for later administration of autologous Treg cells). In some embodiments, the population of immune cells is allogeneic to the subject. [0104]
  • the engineered T reg cells and/or the vehicle containing the nucleic acid can be administered to the subject using any suitable option. Administration can be local or systemic. In some embodiments, administration is parenteral. Suitable option for administration include, without limitation, intravenous, intramuscular, subcutaneous, intra- arterial, intraperitoneal, or oral administration.
  • the engineered Treg cells and/or the vehicle containing the nucleic acid can be administered intravenously. In some embodiments, the engineered T reg cells and/or the vehicle containing the nucleic acid can be administered by infusion. In some embodiments, administering comprises intravenous or oral administration. [0105] Any suitable amount of the engineered T reg cells can be administered to the subject. In some embodiments, the method includes administering to the subject a therapeutically effective amount of the engineered Treg cells.
  • the method includes administering to the subject about 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , or more of the engineered T reg cells, or a number of cells in a range defined by any two of the preceding values (e.g., about 10 5 -10 12 cells, 10 6 -10 11 cells, 10 7 -10 10 cells, etc.).
  • the method includes administering to the subject about 10 3 cells/kg, 10 4 cells/kg, 10 5 cells/kg, 10 6 cells/kg, 10 7 cells/kg, 10 8 cells/kg, 10 9 cells/kg, or 10 10 cells/kg of body weight, or more, of the engineered Treg cells, or a body weight-based amount in a range defined by any two of the preceding values (e.g., about 10 3 -10 10 cells/kg, 10 4 -10 9 cells/kg, 10 5 -10 8 cells/kg, etc.). In some embodiments, the method includes administering to the subject the engineered Treg cells at 10 6 -10 9 cells/kg of body weight.
  • the method includes administering to the subject the engineered Treg cells at 10 6 -10 8 cells/kg of body weight. In some embodiments, the method includes administering to the subject the engineered Treg cells at about 10 7 cells/kg of body weight. [0106] In some embodiments, the method includes administering to the subject a therapeutically effective amount of the nucleic acid (e.g., as a cell-free composition).
  • the therapeutically effective amount of the nucleic acid includes about 0.01 ⁇ g, 0.02 ⁇ g, 0.05 ⁇ g, 0.1 ⁇ g, 0.2 ⁇ g, 0.5 ⁇ g, 1 ⁇ g, 2 ⁇ g, 3 ⁇ g, 4 ⁇ g, 5 ⁇ g, 6 ⁇ g, 7 ⁇ g, 8 ⁇ g, 9 ⁇ g, 10 ⁇ g, 15 ⁇ g, 20 ⁇ g, 25 ⁇ g, 30 ⁇ g, 40 ⁇ g, 50 ⁇ g, 75 ⁇ g, 100 ⁇ g, 125 ⁇ g, 150 ⁇ g, 175 ⁇ g, 200 ⁇ g, 250 ⁇ g, 300 ⁇ g, 400 ⁇ g, 500 ⁇ g, 600 ⁇ g, 700 ⁇ g, 800 ⁇ g, 900 ⁇ g, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 10 mg, 15 mg, 20 mg, 30 mg, 40 mg, 50 mg, 75 mg, 100 mg or more, or an amount in a range
  • the therapeutically effective amount of the nucleic acid includes about 0.001 mg/kg, 0.002 mg/kg, 0.005 mg/kg, 0.01 mg/kg, 0.02 mg/kg, 0.05 mg/kg, 0.1 mg/kg, 0.15 mg/kg, 0.2 mg/kg, 0.5 mg/kg, 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 60 mg/kg, 70 mg/kg, 80 mg/kg, 90 mg/kg, 100 mg/kg of body weight, or more, or an amount in a range defined by any two of the preceding values (e.g., 0.001 mg/kg-0.01 mg/kg, 0.01 mg/kg-0.1 mg/kg, 0.1 mg/kg-1 mg
  • the Siglec-9 protein can be expressed in the engineered T reg cell at any suitable level for treating the subject according to some embodiments of the present disclosure, and is generally expressed at a level higher than the level of expression of Siglec- 9 protein in a T reg cell that has not been modified (e.g., has not been modified to express or overexpress Siglec-9 protein as described herein).
  • expression of the Siglec-9 protein is increased by expression of the nucleic acid in the engineered T reg cell by, by about, or by at least 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 3, 3.2, 3.5, 4, 4.2, 4.5, 5, 5.2, 5.5, 6, 6.5, 7, 8, 9, 10 fold, or more, or by a fold increase that is in a range defined by any two of the preceding values (e.g., 1.5-10 fold, 2-8 fold, 3-6 fold, 4-6 fold, 1.5-2.5 fold, 1.6-2.4 fold, 1.7-2.3 fold, 1.8-2.2 fold, etc.), compared to a suitable reference (e.g., a T reg cell that has not been modified with the nucleic acid).
  • a suitable reference e.g., a T reg cell that has not been modified with the nucleic acid.
  • expression of the Siglec-9 protein is increased by expression of the nucleic acid in the engineered Treg cell by about 1.5-2.5 fold compared to a suitable reference (e.g., a T reg cell that has not been modified with the nucleic acid). In some embodiments, expression of the Siglec-9 protein is increased by expression of the nucleic acid in the Treg cell by 2-8 fold compared to a suitable reference (e.g., a T reg cell that has not been modified with the nucleic acid).
  • expression of the Siglec-9 protein is increased by expression of the nucleic acid in the engineered Treg cell by about 2 fold (e.g., by about 1.9 fold) compared to a suitable reference (e.g., a T reg cell that has not been modified with the nucleic acid). In some embodiments, expression of the Siglec-9 protein is increased by expression of the nucleic acid in the Treg cell by about 5 fold compared to a suitable reference (e.g., a Treg cell that has not been modified with the nucleic acid). In some embodiments, methods of the present disclosure can promote expression of Siglec-9 protein by the Treg cells.
  • the level of expression by the Treg cells contacted with or co-cultured with the delivery vehicles that includes the nucleic acids of the present disclosure, as described herein, can be compared to the level of Siglec-9 protein expression by a suitable control population of Treg cells.
  • the control population of Treg cells are Treg cells that have not been contacted with or co-cultured with vehicles.
  • Siglec-9 protein expression by the engineered Treg cells is increased by, by about, or by at least 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 8-fold or more, or by a fold amount in a range defined by any two of the preceding values, as compared to a suitable control population of T reg cells, e.g., T reg cells that have not been contacted or co-cultured with vehicles.
  • Siglec-9 protein expression by the engineered Treg cells is at least 1.5-fold higher as compared to Siglec-9 protein production by a population of immune cells, e.g., T reg cells, not contacted with or co-cultured with the vehicles. In some embodiments, Siglec-9 protein expression by the engineered Treg cells is at least 2-fold higher as compared to Siglec-9 protein expression by a population of immune cells, e.g., T reg cells, not contacted with or co-cultured with vehicles.
  • Siglec-9 protein expression by the engineered Treg cells is 4-6 fold higher as compared to Siglec-9 protein expression by a population of immune cells, e.g., Treg cells, not contacted with or co-cultured with vehicles.
  • Siglec-9 protein expression by the engineered T reg cells is about 5-fold higher as compared to Siglec-9 protein expression by a population of immune cells, e.g., Treg cells, not contacted with or co-cultured with vehicles.
  • the expression level can be determined, if desired, using any suitable option. In some embodiments, the expression level is based on the whole-cell expression level. In some embodiments, the expression level is based on a cell-surface expression level.
  • the expression level is determined using, without limitation, Western blot, ELISA, flow cytometry, fluorescence microscopy, etc.
  • a method of treating a condition associated with inflammation and/or fibrosis comprising administering to a subject in need of treating a condition associated with inflammation and/or fibrosis a nucleic acid configured to induce expression of a membrane-bound fibroblast-specific binding protein in regulatory T (T reg ) cells in the subject.
  • T reg regulatory T
  • the nucleic acid encodes the membrane-bound fibroblast-specific binding protein.
  • the nucleic acid encodes an epigenetic modulator (e.g., a CRISPR activation system) that induces expression of the membrane-bound fibroblast-specific binding protein from an endogenous locus of the Treg cells.
  • the method of treating a condition associated with inflammation and/or fibrosis includes administering a nucleic acid encoding a membrane- bound fibroblast-specific binding protein to a subject in need of treating a condition associated with inflammation and/or fibrosis, wherein the membrane-bound fibroblast- specific binding protein is expressed by the nucleic acid in regulatory T (T reg ) cells in the subject.
  • T reg regulatory T
  • membrane-bound fibroblast-specific binding protein denotes a protein construct that can be expressed by a Treg cell, generally on the plasma membrane, and that can promote interaction of the T reg cell with a fibroblast.
  • the protein construct is known to bind a fibroblast-specific cell-surface marker or target protein.
  • expression or overexpression of the membrane-bound fibroblast-specific binding protein by the regulatory T (T reg ) cells in the subject promotes anti- inflammatory activity of the T reg cells at a target tissue specified by the membrane-bound fibroblast-specific binding protein.
  • the membrane-bound fibroblast-specific binding protein can bind to any suitable fibroblast-specific target protein that is a marker for a condition associated with inflammation and/or fibrosis in a target tissue.
  • the fibroblast-specific target protein is a gene or protein that is upregulated in the target tissue under inflammatory and/or fibrotic conditions.
  • the fibroblast-specific target protein is AOC3.
  • the proliferation of fibroblast cells and myofibroblast cells can occur subsequent to or concurrent with a myocardial infarction.
  • the production of fibroblast cells and myofibroblast cells subsequent to a myocardial infarction refers to the process that occurs or continues after the initial cardiac event.
  • the increased fibroblast cells and myofibroblast cells activity can be a response to the damage caused by the myocardial infarction, potentially leading to reduced cardiac function or fibrosis.
  • T reg cells can be used in the methods and compositions of the present disclosure.
  • the Treg cells are human Treg cells.
  • Immune cells for use in methods of the present disclosure may be isolated from a biological sample taken from a mammalian subject, such as a human subject, originating from a number of sources, including for example, peripheral blood (e.g., peripheral blood mononuclear cells), bone marrow, thymus, tissue biopsy, tumor, lymph node tissue, gut associated lymphoid tissue, mucosa associated lymph node tissue, spleen tissue or any other lymphoid tissue and tumors.
  • peripheral blood e.g., peripheral blood mononuclear cells
  • PBMC peripheral blood mononuclear cells
  • Immune cells may also be obtained from a unit of blood obtained from an apheresis or leukapheresis procedure.
  • a population of T reg cells can be obtained by enriching a population of immune cells (e.g., immune cells from a peripheral blood sample from a subject) for T reg cells. Any suitable option for obtaining T reg cells, e.g., an enriched population of T reg cells can be used, e.g., sorting for T reg cells using one or more markers, as described herein. Any suitable option can be used to enrich the population of immune cells for T reg cells.
  • T reg cells can be isolated from the population of immune cells.
  • immune cells other than T reg cells can be depleted from the population of immune cells.
  • enriching includes sorting cells of the population of immune cells based on expression of markers, e.g., cell surface markers, that differentiate between types of immune cells.
  • T reg cells can be sorted based on expression of at least CD4 and CD25. The sorting can involve, without limitation, flow cytometry or magnetic sorting.
  • the Treg cells are CD4 + CD25 + .
  • T reg cells are CD4 + CD25 + CD127 low .
  • the population can be enriched for CD4 + FOXP3 + cells.
  • Treg cells can be obtained or isolated from a population of immune cells (e.g., immune cells from a peripheral blood sample from a subject) and are expanded in vitro.
  • the “+” indicates positivity for the given marker, which according to some embodiments is expression above a detectable threshold level for that marker.
  • the expression is upregulated as compared to a T cell not subjected to the methods disclosed herein.
  • the subject can be any suitable individual or patient who is in need of treating a condition associated with inflammation and/or fibrosis. In some embodiments, the subject is suffering from the condition, or is suffering from the consequences of the condition.
  • the subject is diagnosed as having the condition associated with inflammation and/or fibrosis. In some embodiments, the subject is at risk of having the condition. In some embodiments, the subject is at risk of having the condition based on one or more of the subject’s family history, genetic predisposition, lifestyle, and medical history. In some embodiments, the subject has suffered or is at risk of suffering from myocardial infarction. In some embodiments, the subject is in need of treating cardiac fibrosis subsequent to a myocardial infarction. In some embodiments, the subject is in need of treating cardiac fibrosis associated with ICM.
  • the subject has at least one myofibroblast cell having elevated expression of AOC3 compared to other cell types (and/or compared to a myofibroblast cell that does not have upregulated fibrotic gene expression).
  • the overexpression and/or enhanced Siglec-9 production by Treg cells contacted with or co-cultured with at least one vehicle and/or a plurality of vehicles, as disclosed herein can promote the regulation of fibroblast cell and myofibroblast cell proliferation.
  • T reg cells overexpressing Siglec-9 i.e., enhanced T reg cells
  • Treg cells can influence the behavior of fibroblast cells and myofibroblast cells in target tissues.
  • enhanced Treg cells can decrease the expression of fibrotic genes, thereby inhibiting myofibroblast transdifferentiation and reducing fibrosis. This modulation can be compared to a suitable control, such as the level of fibroblast and myofibroblast proliferation in the target tissue before administering the Treg cells, or the expected or average level of such cell proliferation in a patient who has not received the enhanced Treg cells.
  • the administration of enhanced Treg cells to a subject can reduce the proliferation of fibroblast cells and/or myofibroblast cells in the target tissue, which can be a response to tissue damage or stress.
  • the enhanced Treg cells when administered, may improve the function of a target tissue impaired by fibroblast and myofibroblast proliferation.
  • the improvement in tissue function could be assessed compared to a suitable control, such as the function of the target tissue before administering the enhanced Treg cells or the average function of a tissue impaired by excessive fibroblast and myofibroblast proliferation.
  • the administration of enhanced Treg cells may restore or improve cardiac function in patients with conditions involving abnormal fibroblast and myofibroblast activity, such as in the case of cardiac remodeling following myocardial infarction. Cardiac function could be evaluated in terms of parameters including, without limitation, the ejection fraction of the patient, for example, left ventricular ejection fraction.
  • the fibroblast production can be subsequent to or concurrent with wound healing, tissue repair, inflammatory responses, fibrotic diseases, autoimmune disorders, chronic infections, radiation exposure, genetic conditions, and/or aging.
  • fibroblast production can result in myofibroblast production, which is subsequent to or concurrent with myocardial infarction, heart failure, chronic hypertension, cardiomyopathies, heart valve diseases, chronic stress, neurohormonal activation, heart tissue aging, ischemic heart disease, cardiac arrhythmias, or amyloidosis.
  • fibroblast proliferation can be related to an autoimmune response or disease.
  • fibroblast proliferation can be related to one or more of the following diseases or conditions: systemic lupus erythematosus, rheumatoid arthritis, scleroderma, dermatomyositis, Sjögren’s syndrome, autoimmune thyroiditis, Graves’ disease, autoimmune hepatitis, multiple sclerosis, psoriasis, vitiligo, type 1 diabetes mellitus, autoimmune myocarditis, Crohn’s disease, ulcerative colitis, pemphigus vulgaris, ankylosing spondylitis, Addison’s disease, alopecia areata, autoimmune hemolytic anemia, celiac disease, Goodpasture’s syndrome, Hashimoto’s thyroiditis, idiopathic thrombocytopenic purpura, myasthenia gravis, pernicious anemia, primary biliary cirrhosis, Guillain-Barré syndrome, Wegener’s granul
  • the autoimmune disease can be a cardiac autoimmune disease, e.g., autoimmune myocarditis.
  • administering to a patient the T reg cells, e.g., the enhanced Treg cells can restore or improve function of a target tissue, e.g., heart, lung, kidney, liver, etc., of the patient that was impaired by the cardiac fibrosis.
  • the function of the target tissue may be improved compared to a suitable baseline value, e.g., function of the target tissue before administering the enhanced Treg cells, the average function of the target tissue impaired by the cardiac fibrosis, etc.
  • the function of the target tissue can be increased by at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or more, or by a percentage in a range defined by any two of the preceding values, compared to a suitable baseline value, e.g., the function before administering the enhanced Treg cells.
  • administering to a subject enhanced Treg cells can restore or reduce damage to a target tissue.
  • administering to a patient enhanced T reg cells can reduce the damage to the target tissue by at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or more, or by a percentage in a range defined by any two of the preceding values.
  • the enhanced Treg cells when administered to a subject with cardiac fibrosis can restore or improve cardiac function in the patient.
  • the cardiac function can be measured by ejection fraction, e.g., left ventricular ejection fraction.
  • cardiac function can be measured by the average cardiac function of a patient having cardiac fibrosis.
  • the conditions treated by the present treatment methods are a symptom and/or sequelae of an infection.
  • the inflammatory condition is a chronic condition.
  • a treatment method of the present disclosure treats any one or more of a variety of inflammatory conditions.
  • the inflammatory condition is one that is responsive to the anti-inflammatory effect of IL-10.
  • the inflammatory condition includes viral infection, sepsis, arthritis (rheumatoid arthritis, juvenile rheumatoid arthritis, psoriatic arthritis), multiple sclerosis, pemphigus, and type 1 diabetes (also referred to as insulin-dependent diabetes mellitus (IDDM)).
  • IDDM insulin-dependent diabetes mellitus
  • the inflammation can be subsequent to or concurrent with an infectious disease.
  • the inflammation can be related to a variety of infectious diseases.
  • the infectious disease is associated with myocardial injury.
  • the heart condition includes acute myocarditis associated with the infectious disease.
  • the inflammatory condition includes a cytokine storm, or hyperinflammation, associate with the infectious disease.
  • the inflammatory condition includes acute lung injury or acute respiratory distress syndrome (ARDS).
  • the infectious disease can be an infection by, without limitation, one or more of the following pathogens: viruses (including but not limited to coronavirus, human immunodeficiency virus, herpes simplex virus, papilloma virus, parainfluenza virus, influenza virus, hepatitis virus, Coxsackie Virus, herpes zoster virus, measles virus, mumps virus, rubella, rabies virus, hemorrhagic viral fevers, H1N1, and the like), prions, parasites, fungi, mold, yeast and bacteria (both gram-positive and gram- negative).
  • viruses including but not limited to coronavirus, human immunodeficiency virus, herpes simplex virus, papilloma virus, parainfluenza virus, influenza virus, hepatitis virus, Coxsackie Virus, herpes zoster virus, measles virus, mumps virus, rubella, rabies virus, hemorrhagic viral fevers
  • pathogens include, without limitation, Candida albicans, Aspergillus niger, Escherichia coli (E. coli), Pseudomonas aeruginosa (P. aeruginosa), and Staphylococcus aureus (S. aureus), Group A streptococci, S. pneumoniae, Mycobacterium tuberculosis, Campylobacter jejuni, Salmonella, Shigella, and a variety of drug resistant bacteria.
  • the inflammation is subsequent to or concurrent with an infection by a virus, e.g., a DNA or RNA virus.
  • the virus is an RNA virus, e.g., a single or double-stranded virus.
  • the RNA virus is a positive sense, single-stranded RNA virus.
  • the virus belongs to the Nidovirales order.
  • the virus belongs to the Coronaviridae family.
  • the virus belongs to the alphacoronavirus, betacoronavirus, gammacoronavirus or deltacoronavirus genus.
  • the alphacoronavirus is, without limitation, human coronavirus 229E, human coronavirus NL63 or transmissible gastroenteritis virus (TGEV).
  • the betacoronavirus is, without limitation, Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), SARS-CoV-2 (COVID-19), Middle Eastern Respiratory Syndrome Coronavirus (MERS-CoV), human coronavirus HKU1, or human coronavirus OC43.
  • the gammacoronavirus is infectious bronchitis virus (IBV).
  • IBV infectious bronchitis virus
  • the inflammation and/or fibrosis is related to an autoimmune disease, graft-versus-host disease (GVHD) or an organ transplant.
  • the inflammation is related to one or more of the following diseases or conditions: Behçet’s disease, polymyositis/dermatomyositis, autoimmune cytopenias, autoimmune myocarditis, primary liver cirrhosis, Goodpasture’s syndrome, autoimmune meningitis, Sjögren’s syndrome, systemic lupus erythematosus, Addison’s disease, alopecia greata, ankylosing spondylitis, autoimmune hepatitis, autoimmune mumps, Crohn's disease, insulin-dependent diabetes mellitus, dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, hemolytic anemia, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoi
  • the autoimmune disease is scleroderma or systemic sclerosis.
  • the inflammation is related to a bone marrow transplantation.
  • the inflammation is related to allograft rejection following tissue transplantation.
  • the inflammation is related to cardiomyopathy, heart failure and/or amyloidosis.
  • the autoimmune disease is a cardiac autoimmune disease, e.g., autoimmune myocarditis.
  • Non-limiting examples of autoimmune diseases or disorders include autoimmune myocarditis, conditions involving infiltration of T cells and chronic inflammatory responses, arthritis (rheumatoid arthritis, juvenile rheumatoid arthritis, psoriatic arthritis), multiple sclerosis, pemphigus, and type 1 diabetes (also referred to as insulin-dependent diabetes mellitus (IDDM)).
  • IDDM insulin-dependent diabetes mellitus
  • nucleic acid e.g., an isolated nucleic acid
  • a nucleotide sequence encoding a sialic acid-binding immunoglobulin-like lectin 9 (Siglec-9) protein operatively linked to a promoter configured to express the Siglec-9 protein in a regulatory T (Treg) cell.
  • the promoter can be any suitable promoter for expressing the Siglec-9 protein in a T reg cell when the nucleic acid is incorporated into the cell (e.g., as described herein).
  • the promoter is a regulatory T (Treg) cell-specific promoter.
  • the Treg cell-specific promoter is selected from the group consisting of a Foxp3 promoter and an Interleukin-2 (IL-2) promoter.
  • the promoter is a constitutive promoter.
  • the promoter is a conditional promoter. Any suitable conditional promoter can be used.
  • the conditional promoter is a chemically inducible promoter.
  • the conditional promoter includes a Tet-responsive promoter.
  • the composition includes any suitable amount of the nucleic acid.
  • the composition includes a therapeutically effective amount of the nucleic acid (e.g., to treat a condition associated with inflammation and/or fibrosis when administered to a subject in need thereof).
  • the composition includes, includes about, or includes at least 0.01 ⁇ g, 0.02 ⁇ g, 0.05 ⁇ g, 0.1 ⁇ g, 0.2 ⁇ g, 0.5 ⁇ g, 1 ⁇ g, 2 ⁇ g, 3 ⁇ g, 4 ⁇ g, 5 ⁇ g, 6 ⁇ g, 7 ⁇ g, 8 ⁇ g, 9 ⁇ g, 10 ⁇ g, 15 ⁇ g, 20 ⁇ g, 25 ⁇ g, 30 ⁇ g, 40 ⁇ g, 50 ⁇ g, 75 ⁇ g, 100 ⁇ g, 125 ⁇ g, 150 ⁇ g, 175 ⁇ g, 200 ⁇ g, 250 ⁇ g, 300 ⁇ g, 400 ⁇ g, 500 ⁇ g, 600 ⁇ g, 700 ⁇ g, 800 ⁇ g, 900 ⁇ g, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 10 mg, 15 mg, 20 mg, 30 mg, 40 mg, 50 mg, 75 mg, 100 mg or more, or an amount in a range defined by
  • the composition includes, includes about, or includes at least 0.01 ⁇ g/mL, 0.02 ⁇ g/mL, 0.05 ⁇ g/mL, 0.1 ⁇ g/mL, 0.2 ⁇ g/mL, 0.5 ⁇ g/mL, 1 ⁇ g/mL, 2 ⁇ g/mL, 3 ⁇ g/mL, 4 ⁇ g/mL, 5 ⁇ g/mL, 6 ⁇ g/mL, 7 ⁇ g/mL, 8 ⁇ g/mL, 9 ⁇ g/mL, 10 ⁇ g/mL, 15 ⁇ g/mL, 20 ⁇ g/mL, 25 ⁇ g/mL, 30 ⁇ g/mL, 40 ⁇ g/mL, 50 ⁇ g/mL, 75 ⁇ g/mL, 100 ⁇ g/mL, 125 ⁇ g/mL, 150 ⁇ g/mL, 175 ⁇ g/mL, 200 ⁇ g/mL, 250 ⁇ g/mL
  • a vector e.g., a plasmid, a viral vector
  • a regulatory T (Treg) cell e.g., an isolated Treg cell
  • the T reg cell has been genetically modified (e.g., using the nucleic acid or the vector of the present disclosure) to express (e.g., overexpress) the Siglec-9 protein.
  • composition that includes a population of enhanced (or engineered) T reg cells overexpressing sialic acid-binding immunoglobulin-like lectin 9 (Siglec-9).
  • Siglec-9 protein expression by the enhanced or engineered T reg cells is increased by, by about, or by at least 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4- fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 8-fold, 9 fold, 10 fold or more, or by a fold amount in a range defined by any two of the preceding values (e.g., 1.5-10 fold, 2-8 fold, 3-7 fold, 4-6 fold, etc.), as compared to a suitable reference, e.g., T reg cells that have not been engineered to overexpress siglec-9 protein.
  • a suitable reference e.g., T reg cells that have not been engineered to overexpress siglec-9 protein.
  • Siglec-9 expression by the enhanced or engineered Treg cells is at least 1.5-fold higher as compared to a suitable reference, e.g., Treg cells that have not been engineered to overexpress siglec-9 protein.
  • Siglec-9 expression by the enhanced or engineered T reg cells is at least 2- fold higher as compared to a suitable reference, e.g., Treg cells that have not been engineered to overexpress siglec-9 protein.
  • Siglec-9 expression by the enhanced or engineered T reg cells is 4-6 fold higher as compared to a suitable reference, e.g., T reg cells that have not been engineered to overexpress siglec-9 protein.
  • Siglec- 9 expression by the enhanced or engineered Treg cells is about 5-fold higher as compared to a suitable reference, e.g., T reg cells that have not been engineered to overexpress siglec-9 protein.
  • the enhanced Treg cells have been genetically modified (e.g., using the nucleic acid or the vector) to express (e.g., overexpress) the Siglec-9 protein.
  • the enhanced T reg cells have endocytosed vehicles that include a plurality of nucleic acids encoding sialic acid-binding immunoglobulin-like lectin 9 (Siglec-9).
  • the enhanced Treg cells comprise at least one endocytosed vehicle, the at least one endocytosed vehicle comprising a plurality of nucleic acids encoding sialic acid- binding immunoglobulin-like lectin 9 (Siglec-9).
  • the endocytosed vehicle comprises at least one of a liposome, a nanoparticle, a lipid nanoparticle (LNP), a viral vector, a cell-penetrating peptide (CPP), and/or an extracellular vesicle (EV).
  • the plurality of nucleic acids comprises a plurality of mRNA transcripts encoding Siglec-9.
  • the enhanced T reg cells exhibit Siglec-9-dependent interaction with cells expressing amine oxidase, copper containing 3 (AOC3).
  • the interaction with AOC-3-expressing cells mediated by Siglec-9 expressed by the enhanced T reg cells is a functional interaction (e.g., one that allows the enhanced T reg cells to suppress the immune response in the tissue that contains the AOC-3-expressing cells).
  • the composition can include any suitable number of the enhanced T reg cells.
  • the composition includes a therapeutically effective amount of the enhanced Treg cells (e.g., to treat a condition associated with inflammation and/or fibrosis when administered to a subject in need thereof).
  • the composition includes, includes at least, or includes about 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , or a number in a range defined by two of the preceding values (e.g., about 10 3 -10 12 , 10 4 -10 11 , 10 5 -10 10 , 10 6 -10 9 , etc.) enhanced Treg cells.
  • the composition can include any suitable concentration of the enhanced T reg cells.
  • the composition includes, includes at least, or includes about 10 3 /mL, 10 4 /mL, 10 5 /mL, 10 6 /mL, 10 7 /mL, 10 8 /mL, 10 9 /mL, 10 10 /mL, 10 11 /mL, 10 12 /mL, or a concentration in a range defined by two of the preceding values (e.g., about 10 3 -10 12 /mL, 10 4 -10 11 /mL, 10 5 -10 10 /mL, 10 6 -10 9 /mL, etc.) enhanced Treg cells.
  • the composition includes a substantially pure population of the enhanced Treg cells, compared to other cells.
  • compositions comprising a population of vehicles, each vehicle comprising a nucleic acid encoding sialic acid-binding immunoglobulin-like lectin 9 (Siglec-9); and a regulatory T (T reg ) cell-targeting moiety configured to deliver the nucleic acid encoding Siglec-9 to Treg cells.
  • the vehicle includes a lipid nanoparticle (LNP).
  • the vehicle is a transfection reagent (e.g., liposome, etc.).
  • the T reg cell-targeting moiety comprises an antigen binding protein that binds a Treg cell marker.
  • the antigen binding protein is selected from: anti-CD25 monoclonal antibodies and/or anti-Foxp3 monoclonal antibodies.
  • the vehicle is a viral vector.
  • the composition is a pharmaceutical or therapeutic composition.
  • the composition includes pharmaceutically acceptable excipient.
  • the composition is a cell-free composition, e.g., the composition is substantially free of cells such as Treg cells.
  • materials which can serve as pharmaceutically acceptable excipients include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc; (8) cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12) esters, such as ethyl ole
  • engineered Treg cells overexpressing Siglec-9 can be used as a therapeutic approach for diseases where inflammation and tissue fibrosis are the primary drivers of pathology, including myocardial infarction (heart attack), hypertension, transplant rejection, myocarditis, cardiomyopathy, heart failure, and amyloidosis.
  • Treg cells overexpressing Siglec-9 specifically target AOC3-expressing fibroblasts, leading to the inhibition of cardiac fibrosis.
  • the approach is generalizable to target fibrosis in other organs.
  • Cardiac fibrosis and associated conditions like myocardial infarction (MI) present significant health challenges.
  • Cardiac fibrosis characterized by the excessive accumulation of fibrous connective tissue in the heart, can lead to reduced cardiac function and other heart-related diseases. While inflammation often precedes fibrosis, the therapeutic approach of using immunosuppression to reduce inflammation can be a double-edged sword.
  • Non-specific immunosuppression although effective in reducing inflammation, can inadvertently impair the body's ability to repair and regenerate tissue. This impairment might, in certain contexts, exacerbate fibrosis.
  • Treg cell-based therapies which are potential candidates for this purpose, often suffer from a lack of specificity, leading to the aforementioned non-specific immunosuppressive effects.
  • the present disclosure focuses on enhancing the specificity of Treg cells. By engineering Treg cells to overexpress Siglec-9, the present methods can enhance the specificity of these cells to target cardiac myofibroblasts, where AOC3 is highly expressed.
  • this targeted approach ensures: [0145] 1. Enhanced Specificity: The engineered Treg cells can specifically target cardiac myofibroblasts, reducing the risk of non-specific immunosuppression and its associated drawbacks. Traditional drugs and therapies either broadly suppress the immune system or target only a few inflammatory mediators. In contrast, in some embodiments, the engineered Treg cells of the present disclosure offer a more precise modulation of the immune response, focusing on the root causes of fibrosis without causing broad immunosuppression. [0146] 2. Effective Fibrosis Mitigation: By targeting the root causes of fibrosis, the present methods and compositions offer a therapeutic approach to mitigate myocardial infarction (MI) and other fibrosis-associated diseases. [0147] 3.
  • MI myocardial infarction
  • the present methods and compositions while primarily focused on MI, can be applied to other fibrosis-associated diseases.
  • the key findings supporting the present methods and compositions include: [0149] 1. Angiotensin II's role in dose-dependently and time-dependently inducing the upregulation of fibrotic genes (CollagenI and ⁇ -SMA) as well as AOC3 in MCF. [0150] 2. The binding of Siglec-9 to AOC3, which facilitates the precise targeting of Treg cells to AOC3-expressing cardiac fibroblasts. [0151] 3. Enhanced inhibition of fibrotic gene expression in cardiac fibroblasts by Tregs overexpressing Siglec-9. [0152] 4.
  • the present disclosure provides: [0156] 1. Targeted Immune Modulation: The present disclosure offers a targeted therapeutic approach, addressing the challenge of non-specific immunosuppression seen with natural Treg cells. [0157] 2.
  • Siglec-9 Overexpression The overexpression of Siglec-9 in Treg cells enhances their specificity, directly targeting the root cause (cardiac myofibroblasts) of cardiac fibrosis.
  • 3. Broad Therapeutic Potential Beyond just MI, the present disclosure includes treating other fibrosis-associated diseases.
  • Additional non-limiting embodiments of the present disclosure are provided by the following numbered embodiments. 1. A method of treating fibrosis, comprising administering a nucleic acid encoding a sialic acid-binding immunoglobulin-type lectin 9 (Siglec-9) protein to a subject in need of fibrosis treatment, wherein the Siglec-9 protein is expressed by the nucleic acid in regulatory T (T reg ) cells in the subject. 2.
  • T reg regulatory T
  • a method of treating a condition associated with inflammation and/or fibrosis comprising administering to a subject in need of treating a condition associated with inflammation and/or fibrosis a nucleic acid configured to induce expression of a sialic acid- binding immunoglobulin-type lectin 9 (Siglec-9) protein in regulatory T (Treg) cells of the subject.
  • Siglec-9 sialic acid- binding immunoglobulin-type lectin 9
  • the condition associated with inflammation and/or fibrosis comprises cardiac inflammation and/or cardiac fibrosis.
  • the method of embodiment 3, 3a, or 4, wherein the condition associated with inflammation and/or fibrosis comprises myocardial infarction, hypertension, myocarditis, transplant rejection, cardiomyopathy, heart failure, and amyloidosis. 6.
  • administering the nucleic acid or the nucleic acid encoding the Siglec-9 protein comprises administering to the subject a population of Treg cells comprising the nucleic acid encoding the Siglec-9 protein.
  • Treg cells of the population comprise a plasmid or a viral vector comprising the nucleic acid or the nucleic acid encoding the Siglec- 9 protein.
  • the method of embodiment 8, comprising genetically modifying Treg cells with the plasmid or the viral vector comprising the nucleic acid or the nucleic acid encoding the Siglec-9 protein.
  • the viral vector comprises an adenoviral vector, retroviral vector, or a lentiviral vector.
  • the population of Treg cells are derived from cells autologous to the subject.
  • the population of Treg cells are derived from cells allogeneic to the subject. 13.
  • any one of embodiments 1-6 comprising administering to the subject a vehicle comprising the nucleic acid or the nucleic acid encoding the Siglec-9 protein, wherein the vehicle is configured to deliver the nucleic acid or the nucleic acid encoding the Siglec-9 protein to T reg cells in the subject.
  • the vehicle comprises: a lipid nanoparticle (LNP); and a Treg cell-targeting moiety associated with the LNP.
  • the Treg cell-targeting moiety comprises an antigen binding protein that binds a Treg cell marker.
  • the Treg cell marker is selected from: CD25 and/or Foxp3. 17.
  • the antigen binding protein is an antibody.
  • the antigen binding protein is selected from: an anti-CD25 monoclonal antibody and/or anti-Foxp3 monoclonal antibody.
  • the nucleic acid comprises mRNA encoding the Siglec-9 protein.
  • a method of treating a condition associated with inflammation and/or fibrosis comprising administering a nucleic acid encoding a membrane-bound fibroblast-specific binding protein to a subject in need of treating a condition associated with inflammation and/or fibrosis, wherein the membrane-bound fibroblast-specific binding protein is expressed by the nucleic acid in regulatory T (Treg) cells in the subject.
  • Treg regulatory T
  • a method of targeted immune modulation of a subject comprising: obtaining a population of regulatory T cells (T reg ); delivering at least one vehicle to the population of Treg cells, wherein the at least one vehicle comprises a nucleic acid configured to induce expression of a sialic acid-binding immunoglobulin-type lectin-9 (Siglec-9) protein in the T reg cells of the population, thereby producing a population of enhanced Treg cells overexpressing Siglec-9; and administering to a subject the population of enhanced T reg cells, optionally wherein the nucleic acid encodes the Siglec-9 protein.
  • the at least one vehicle comprises a liposome, or a lipid nanoparticle (LNP).
  • the at least one vehicle comprises a viral vector.
  • the viral vector comprises an adenoviral vector, retroviral vector, or a lentiviral vector.
  • the nucleic acid comprises a promoter operatively linked to a nucleotide sequence encoding the Siglec9, wherein the promoter upregulates expression of Siglec9 in T reg cells.
  • the subject comprises at least one myofibroblast cell having elevated expression of AOC3 compared to other cell types. 37.
  • the method of embodiment 36, wherein the at least one myofibroblast cell is at least one cardiac myofibroblasts. 38. The method of any one of embodiments 31-37, wherein the subject is in need of treatment for fibrosis. 39. The method of embodiment 38, wherein the fibrosis is a cardiac fibrosis. 40. The method of embodiment 39, wherein the cardiac fibrosis is subsequent to a myocardial infarction. 41. A method of inhibiting fibrotic gene expression, comprising: obtaining a population of T reg cells; engineering the Treg cells to overexpress Siglec-9, thereby producing enhanced Treg cells; and administering the enhanced T reg cells to a subject in need of inhibiting fibrotic gene expression. 42.
  • fibrotic gene is at least one of Collagen I, ⁇ -smooth muscle actin ( ⁇ -SMA), and/or amine oxidase, copper containing 3 (AOC3).
  • engineering comprises: delivering at least one vehicle comprising a nucleic acid encoding Siglec-9 to the population of T reg cells, optionally wherein the nucleic acid comprises mRNA; or delivering at least one vehicle comprising a nucleic acid configured to induce expression of Siglec-9 protein in the population of Treg cells. 44.
  • the at least one vehicle comprises at least one of a liposome, a nanoparticle, a lipid nanoparticle (LNP), a viral vector, a cell- penetrating peptide (CPP), and/or an extracellular vesicle (EV).
  • the nucleic acid comprises mRNA.
  • overexpressing Siglec-9 comprises upregulating expression of the mRNA.
  • Siglec-9 sialic acid-binding immunoglobulin-like lectin 9
  • the isolated nucleic acid of embodiment 52 wherein the promoter is a regulatory T (T reg ) cell-specific promoter selected from the group consisting of a Foxp3 promoter and an Interleukin-2 (IL-2) promoter.
  • T reg regulatory T
  • a vector comprising the nucleic acid of embodiment 52 or 53.
  • a regulatory T (T reg ) cell comprising the isolated nucleic acid of embodiment 52 or 53, or the vector of embodiment 54, optionally wherein the T reg cell is a human T reg cell.
  • a composition comprising a population of vehicles, each vehicle comprising: a nucleic acid encoding sialic acid-binding immunoglobulin-like lectin 9 (Siglec-9); and a regulatory T (Treg) cell-targeting moiety configured to deliver the nucleic acid encoding Siglec-9 to T reg cells, optionally wherein the T reg cells are human T reg cells.
  • vehicles of the population comprise a lipid nanoparticle (LNP).
  • LNP lipid nanoparticle
  • the nucleic acid comprises mRNA.
  • the Treg cell- targeting moiety comprises an antigen binding protein that binds a T reg cell marker. 60.
  • a composition comprising a population of enhanced regulatory T (Treg) cells overexpressing sialic acid-binding immunoglobulin-like lectin 9 (Siglec-9), optionally wherein the Treg cells are human Treg cells.
  • Treg enhanced regulatory T
  • Siglec-9 sialic acid-binding immunoglobulin-like lectin 9
  • the enhanced Treg cells comprise at least one endocytosed vehicle, the at least one endocytosed vehicle comprising a plurality of nucleic acids encoding sialic acid-binding immunoglobulin-like lectin 9 (Siglec-9).
  • the at least one endocytosed vehicle comprises at least one of a liposome, a nanoparticle, a lipid nanoparticle (LNP), a viral vector, a cell-penetrating peptide (CPP), and/or an extracellular vesicle (EV).
  • the plurality of nucleic acids comprises a plurality of mRNA transcripts encoding Siglec-9.
  • AOC3 amine oxidase, copper containing 3
  • Treg cells of embodiment 55 or the composition of any one of embodiments 56-68 for preparation of a medicament for the treatment of a condition associated with inflammation and/or fibrosis in a subject in need thereof.
  • 72. The method, isolated nucleic acid, vector, Treg cell, or composition of any one of the preceding embodiments, wherein the Siglec-9 is human Siglec-9.
  • Siglec-9 is human Siglec-9.
  • FIG. 2 illustrates a graphical representation 200 of data obtained from in vitro experiments of the effect of Angiotensin II concentrations on fibrotic gene expression. Data illustrate three concentrations of Angiotensin II, specifically, 100nM, 1 ⁇ M and 10 ⁇ M, were administered to MCF cells. A control treatment is also shown, where no Angiotensin II was administered to MCF cells.
  • mRNA was isolated from MCF cells to quantify the fold increase in mRNA transcripts for Collagen I 210 and ⁇ - SMA 220 relative the control.
  • the data illustrate that there was a positive correlation between the mRNA expression fold increase for both Collagen I 210 and ⁇ -SMA 220 as the concentration of Angiotesin II administered to the MCF cells increased.
  • Angiotensin II time-dependently induces upregulation of fibrotic genes in mouse cardiac upregulation of fibrotic genes in mouse cardiac cells, primary mouse cardiac fibroblast (MCF) cells were exposed to Angiotensin II over time.
  • FIG. 3 illustrates a graphical representation 300 of data obtained from in vitro experiments of the effect of 10 ⁇ M Angiotensin II on fibrotic gene expression over time. Data illustrate expression levels were measured at three time points, specifically, 6 hours, 12 hours, and 24 hours after administration of Angiotensin II to MCF cells. A control treatment (Ctrl) is also shown, where no Angiotensin II was administered to MCF cells.
  • mRNA was isolated from MCF cells to quantify the fold increase in mRNA transcripts for Collagen I 310 and ⁇ -SMA 320 relative the control.
  • Data illustrate that there was a positive correlation between the mRNA expression fold increase for both Collagen I 310 and ⁇ -SMA 320 over time when 10 ⁇ M Angiotensin II was administered to the MCF cells.
  • Angiotensin II does- and time-dependently induces expression of AOC3 in mouse cardiac fibroblast [0166]
  • MCF primary mouse cardiac fibroblast
  • FIG. 4 illustrates graphical representations 400 of data obtained from in vitro experiments of the effect of Angiotensin II concentrations and time on AOC3 expression.
  • 410 illustrates three concentrations of Angiotensin II, specifically, 100nM, 1 ⁇ M and 10 ⁇ M, were administered to MCF cells.
  • a control treatment (Ctrl) is also shown, where no Angiotensin II was administered to MCF cells.
  • 420 illustrates expression levels were measured at three time points, specifically, 6 hours, 12 hours, and 24 hours after administration of 10 ⁇ M Angiotensin II to MCF cells.
  • a control treatment (Ctrl) is also shown, where no Angiotensin II was administered to MCF cells.
  • mRNA was isolated from MCF cells to quantify the fold increase in mRNA transcripts for AOC3 relative the control. Data illustrate how there is a positive correlation between the mRNA expression fold increase for AOC3 with respect to dosage and time.
  • a western blot assay 430 was performed.
  • Example 2 The results of a western blot assay illustrate upregulation of AOC3 relative to a negative control for which no Angiotensin II was applied, and relative to a positive control for which the administration of Angiotensin II had no effect on expression levels of ⁇ actin (Act).
  • Example 2 shows that Siglec-9 binding to AOC3 and overexpression in Treg cells facilitates Treg cell targeting of AOC3-expressing cardiac fibroblasts. Siglec-9 binding to AOC3 facilitates T reg targeting of AOC3-expressing cardiac fibroblasts. [0170] To determine whether Siglec-9 binding to AOC3 facilitates Treg targeting of AOC3-expressing cardiac fibroblasts, enhanced T reg cells overexpressing Siglec-9 were prepared.
  • a lentiviral vector (pLenti-GIII-CMV) that contains the SIGLEC9 gene was obtained from Applied Biological Materials Inc. (catalog number 43776061).
  • the SIGLEC9 gene, or Homo sapiens sialic acid binding Ig-like lectin 9, was represented as a 1,707 base pair mRNA sequence (NCBI Accession Number BC035365; SEQ ID NO:1).
  • the SIGLEC9 protein encoded by the lentiviral vector had the predicted amino acid sequence of SEQ ID NO:2, shown below (predicted signal peptide underlined): 1 mlllllpllw greraegqts klltmqssvt vqeglcvhvp csfsypshgw iypgpvvhgy 61 wfregantdq dapvatnnpa ravweetrdr fhllgdphtk nctlsirdar rsdagryffr 121 mekgsikwny khhrlsvnvt althrpnili pgtlesgcpq nltcsvpwac eqgtppmisw 181 igtsvspldp sttrssvltl ipqpqdhgts ltcq
  • FIG. 5 illustrates a western blot assay 500 demonstrating the ability to generate Treg cells that overexpress Siglec-9.
  • 510 demonstrates the increased expression of Siglec-9 in Treg cells, thereby producing enhanced Treg cells.
  • 520 shows a ⁇ actin control.
  • a 5-fold increase in expression levels was confirmed by Western blot (FIG. 26).
  • Reg Regulatory T cells
  • spleen was minced using a syringe plunger to release splenocytes.
  • a single-cell suspension was prepared by passing the minced tissue through a 70 ⁇ m cell strainer into a tube. After straining, cells were washed with PBS, centrifuged at 300 ⁇ g for 10 minutes to form a cell pellet, and the supernatant was carefully removed. [0173] For effective magnetic labeling, the cells were passed through a 30 ⁇ m nylon mesh to remove any clumps. The total number of cells was determined, followed by resuspension of the cell pellet in 40 ⁇ L of buffer per 107 cells.
  • the supernatant was then completely aspirated and resuspended such that there were approximately 108 cells in 500 ⁇ L of buffer.
  • An LD Column in a MACS Separator was used for magnetic separation/depletion. The column was rinsed with 2 mL of buffer, the cell suspension applied, and the flow-through containing the pre-enriched CD4+ cell fraction was collected. The column was washed with 2 ⁇ 1 mL of buffer.
  • an MS Column was used for positive selection of CD4+CD25+CD127 dim/– Treg cells. The column was rinse with 500 ⁇ L of buffer, the cell suspension applied, and the flow-through collected. The column was washed with 3 ⁇ 500 ⁇ L of buffer.
  • the column was then removed from the separator and the magnetically labeled cells were flushed out with 1 mL of buffer. For higher purity, the eluted fraction was passed through a second new MS Column. [0175] Throughout this process, no pause was taken between each step and the cells and solutions were maintained in cold conditions (e.g., 2°C to 8 °C) to maintain cell viability and prevent non-specific labeling. As would be understood by one having the skill in the relevant art, the above volumes of reagents and buffers can be varied based on the number of cells undergoing isolation and preparation. The above protocol yields a purified sample of T reg cells for further analysis or experimentation.
  • FIG.6 illustrates an immunoprecipitation assay 600 demonstrating Siglec- 9 expressed by Treg cells binding to AOC3 expressed by cardiac fibroblasts (CFs).
  • CFs cardiac fibroblasts
  • Enhanced T reg cells were co-cultured with cardiac fibroblasts overexpressing AOC3.
  • cells were lysed and immunoprecipitation was performed against Siglec-9 protein.
  • a western blot assay was performed on the immunoprecipitates to determine targeted binding of Siglec-9 to AOC3.
  • Western blot analysis 610 of the immunoprecipitation assay 600 demonstrates that Siglec-9 expressed by Treg cells binds to AOC3 expressed by CFs.
  • the binding can be visualized by adding a sequence encoding GFP to the lentiviral vector such that GFP is co-expressed with Siglec-9 in cells transduced with the lentiviral vector.
  • Analysis of the input sample confirmed overexpression of AOC3 620 by the CFs, while expression of Siglec-9 proteins 630 or ⁇ actin proteins 640 remained consistent.
  • FIG. 7 illustrates a cell adhesion assay 700 for studying the role of Siglec-9 and AOC3 interactions in enhanced T reg cells.
  • a MCF monolayer was prepared and exposed to Angiotensin II (Ang II) solution 720 to induce MCF fibrosis.
  • Control MCF cells were not exposed to Ang II.
  • Enhanced Treg cells overexpressing Siglec-9 and engineered to express GFP were prepared 730.
  • Control T reg cells only expressed GFP.
  • the enhanced T reg cells expressing GFP (or control T reg cells) were spread onto the surface of the MCF monolayer (with or without exposure to Ang II) 740.
  • Treg cells were allowed to adhere to the MCF monolayer 750.
  • the MCF monolayer was washed to remove any unbound cells 760.
  • the amount of cells bound to the MCF monolayer was determined by fluorescent microscopy 770.
  • FIG. 8 illustrates graphical representations 800 of data obtained from in vitro experiments of Treg cells overexpressing Siglec-9 exhibiting enhanced inhibition of fibrotic gene expression in cardiac fibroblasts.
  • the fold change in mRNA transcription was measured relative to control for fibrotic genes Collagen I 810 and ⁇ -SMA 820.
  • Angiotensin II increased fibrotic gene expression in cardiac fibroblasts.
  • Co-culturing Treg cells with Ang II-exposed cardiac fibroblasts abrogated the increase in expression of fibrotic genes, Collagen I and ⁇ -SMA by Ang II.
  • Overexpressing Siglec-9 in Treg cells enhanced the suppression of fibrotic gene expression by the Treg cells.
  • Example 3 shows the role of Siglec-9 interactions with AOC3 in the engineered T reg cell-mediated anti-fibrotic effects on cardiac fibroblasts.
  • Cell adhesion assays and co-culturing experiments were performed to determine the role of Siglec-9 interaction with AOC3 on the cardiac fibroblast-targeting and anti-fibrotic effects of enhanced T reg cells.
  • FIG.9 illustrates a cell adhesion assay 900 for studying the role of Siglec- 9 and AOC3 interactions in the cardiac fibroblast-targeting and anti-fibrotic effects of enhanced Treg cells.
  • a MCF monolayer was prepared and exposed to an Angiotensin II solution 920 to induce MCF fibrosis.
  • mice Treg cells were prepared as described in Example 2.
  • Enhanced Treg cells overexpressed Siglec-9 and were engineered to express GFP 930.
  • the enhanced Treg cells were then treated with either IgG control or an anti-Siglec-9 antibody (“anti-Siglec-9”).
  • the enhanced Treg cells expressing GFP were spread onto the surface of the MCF monolayer (with or without exposure to Ang II) 940.
  • Treg cells were allowed to adhere to the MCF monolayer 950.
  • the MCF monolayer was washed to remove any unbound cells 960.
  • the amount of cells bound to the MCF monolayer was determined by fluorescent microscopy 970.
  • FIG.10 illustrates graphical representations of data obtained from in vitro cell adhesion assays for probing the role of Siglec-9 interacting with AOC3 on fibrotic gene expression. The data demonstrate that anti-Siglec-9 antibody reduced the inhibitory effect of the enhanced Treg cells on expression of fibrotic gene expression (Collagen I 1010 and ⁇ - SMA 1020), compared to IgG control.
  • Example 4 shows the role of upregulating AOC3 and fibrotic genes after a myocardial infarction in vivo.
  • Enhanced Treg cells engineered to overexpress Siglec-9 show superior ability to downregulate fibrotic gene expression post-myocardial infarction as compared to normal T reg cells
  • gene expression levels were quantified using qPCR and western blot assays.
  • FIG. 13 illustrates data of upregulation 1300 of AOC3 and fibrotic genes observed from in vivo experiments using a myocardial infarction mice model.
  • Gene expression was measured relative to a control sham, measured over time as the fold increase relative to the control.
  • fibrosis-associated genes AOC31310, Collagen I 1320, and ⁇ - SMA 1330, gene expression increased over time as a result of a myocardial infarction induced in the mouse models. Expression levels were measured at 72 hours (D3), 168 hours (D7) and 504 hours (D21).
  • a western blot assay was also performed to illustrate the increase in expression of the above fibrosis-associated genes relative to a negative (sham) control and a positive control gene ( ⁇ -Actin).
  • AOC3 1430 expression levels were not significantly different from expression levels of AOC3 in the presence of the vehicle or normal (non-enhanced) Treg cells.
  • Foxp31460 expression levels dramatically increase in the presence of enhanced T reg cells, suggesting it may play a role in immune regulation and fibrosis development.
  • Expression data were confirmed by a western blot assay 1410, which further demonstrates the efficacy of enhanced T reg cells in downregulating fibrosis-associated genes, e.g., Collagen I and ⁇ -SMA, post-myocardial infarction.
  • Example 5 [0195] This non-limiting example shows in vivo therapeutic effects of enhanced Treg cells on Angiotensin II-induced cardiac fibrosis. [0196] FIG.
  • FIG. 15 illustrates an in vivo protocol 1500 for studying the in vivo anti- fibrotic effect of enhanced T reg cells.
  • a subject such as a mouse, is infused with Angiotensin II at 1 mg kg -1 day -1 (Day 0).
  • Enhanced Treg cells are administered to the subject by infusion on Days 3, 7, and 14.
  • Treg cell infiltration/accumulation in the injured myocardium Siglec9-T reg targeting to AOC3+ myofibroblast in the heart; expression level of IL-10/Foxp3 in Treg cells; expression of fibrotic genes and proteins by myofibroblasts in the heart; cardiac function and pathology (eg., ejection fraction ( ⁇ EF); area of fibrosis (PS red)).
  • ⁇ EF ejection fraction
  • PS red area of fibrosis
  • cardioprotection 1610 is achieved by administering to a subject at least one enhanced Treg cell.
  • the enhanced Treg cell overexpresses Siglec-9 proteins, which can activate, e.g., a tyrosine phosphatase such as a Src Homology 2 domain-containing Phosphatase-1 (SHP-1) pathway, upon Siglec-9 binding AOC3 expressed by, e.g., myocardial fibroblast cells.
  • SHP-1 Src Homology 2 domain-containing Phosphatase-1
  • Activation of SHP-1 may lead to expression of cytokines, e.g., interleukin 10 (IL-10).
  • IL-10 interleukin 10
  • the SHP-1-Siglec-9 protein complex may promote enhanced Treg function by increasing the release of IL-10.
  • IL-10 may promote T reg function, thereby further mitigating cardiac fibrosis.
  • IL-10 may promote Treg cell lineage stability.
  • Example 8 [0201] This non-limiting example assesses the anti-fibrotic efficacy of siglec-9 engineered Tregs in targeting AOC3-expressing cardiac myofibroblasts.
  • FIG. 17 utilizes a collagen contraction assay 1710 to show that siglec-9 engineered Treg cells are more effective than non-engineered Treg cells in reversing AngII- induced fibroblast contraction 1720, further validating their functional superiority in anti- fibrotic interventions. As shown in FIG.
  • Example 9 [0203] This non-limiting example evaluates the in vivo anti-fibrotic effects of siglec-9 engineered Treg cells in a mouse myocardial infarction (MI) model. This Example relates to Example 4.
  • siglec-9 engineered Treg cells may also contribute to significant functional improvements in cardiac performance. [0207] These findings show that siglec-9 engineered Treg cells, through their targeted action on AOC3-expressing myofibroblasts, effectively mitigated cardiac fibrosis. [0208] As shown in the above Examples, siglec-9 engineered T reg cells can selectively target AOC3-expressing cardiac myofibroblasts, a critical component in the pathology of ICM. Through targeted modulation of the AOC3 pathway, these engineered Treg cells effectively reduce the expression of key fibrotic markers such as collagen I and ⁇ -SMA, both in vitro and in vivo.
  • key fibrotic markers such as collagen I and ⁇ -SMA
  • Siglec-9 expression will be driven by a CMV promoter, co- expressed with a GFP or RFP reporter, in a lentiviral vector that can be used for non-viral plasmid transfection of siglec-9 in target cells (e.g., FIGs. 5, 7).
  • the vector is packaged into lentiviral particles for high efficiency transduction and stably integrated expression. Transient transfection is primarily used, as hTregs are primary cells not requiring long-term genetic modification.
  • the pLenti-CMV-GFP-2A-Puro-Blank Vector & siglec-9 Lentivirus vector are purchased from Applied Biological Materials, Inc.
  • IL-10 levels will be quantified using enzyme-linked immunosorbent assay (ELISA) and qRT-PCR for mRNA expression in siglec-9 overexpressing Treg cells and control Treg cells post-AOC3 stimulation;
  • Treg cell Lineage Stability The expression of Treg cell-specific lineage markers (FoxP3, CD25, CTLA-4) will be evaluated using WB and flow cytometry in siglec-9 overexpressing T reg cells and control Treg cells post-AOC3 stimulation;
  • Treg cell Migration Assay Migration towards AOC3 will be assessed using a trans-well migration setup.
  • siglec-9 overexpressed Treg cells will be evaluated using a co-culture proliferation assay, utilizing CFSE staining and flow cytometry to track Teff cell division in the presence of AOC3.
  • Siglec-9 overexpressing Treg cells will demonstrate enhanced anti- inflammatory responses, primarily through increased IL-10 production, improved stability of T reg cell lineage markers (FoxP3, CD25, CTLA-4), and more effective migration towards AOC3 gradients. This targeted action may also lead to significant suppression of Teff cell proliferation.
  • Example 11 This non-limiting example dissects the role of the molecular mechanisms through which the SHP-1 signaling pathway mediates siglec-9 function of T reg cell under AOC3 stimulation. This Example is related to Example 10.
  • AOC3-siglec-9 interaction may directly modulate T reg cell function, potentially enhancing their antifibrotic effects through precise molecular pathways.
  • Example 12 This non-limiting example shows characterization of the role of the SHP-1 signaling pathway in siglec-9 mediated function of Treg cell under AOC3 stimulation. This Example is related to Examples 10 and 11.
  • the effect of AOC3-siglec-9 interaction to directly modulates T reg cell functions is investigated using the following assays.
  • a) SHP-1 Pathway Activation Activation of SHP-1 pathway components will be evaluated using WB to detect phosphorylation status of SHP-1, Pyk2, and Src, following blocking of AOC3 or siglec-9 interactions with neutralizing antibodies.
  • IL-10 Cytokine Release Assays Following the blockade of AOC3- siglec-9 interactions or SHP-1 knockdown in siglec-9 overexpressing Treg cells, IL-10 levels will be quantified using ELISA and qRT-PCR. This assay may confirm the reduction in anti- inflammatory cytokine production, demonstrating the pathway’s role in T reg cell function. In some embodiments, a 50% reduction in IL-10 compared to controls indicates a significant pathway disruption.
  • c) Lineage Stability Assays The stability of Treg cell lineage markers (Foxp3, CD25, CTLA-4) will be examined using flow cytometry and WB after blocking the AOC3-siglec-9 interaction or SHP-1 knockdown. In some embodiments, a minimum 20% reduction in marker expression will be considered a successful outcome, indicating changes in T reg cell stability.
  • Teff Suppression Assays The effectiveness of siglec-9 overexpressing Treg cell in suppressing Teff cell proliferation will be assessed using a co- culture proliferation assay. This includes CFSE staining and flow cytometry to evaluate Teff cell division in the presence of blocked AOC3 or siglec-9.
  • a reduction in suppression efficiency (e.g., by at least 30%) compared to controls may demonstrate the critical role of AOC3-siglec-9 interaction in Treg cell suppressive function.
  • Blocking the AOC3-siglec-9 interaction with neutralizing antibodies leads to a reduction in SHP-1 pathway activation, evidenced by decreased phosphorylation levels of SHP-1, Pyk2, and Src. This disruption is expected to result in diminished IL-10 production, indicating a reduction in the anti-inflammatory capabilities of siglec-9 overexpressing T reg cells.
  • the stability of T reg cell lineage markers such as Foxp3, CD25, and CTLA-4 may decrease, reflecting impaired Treg cell functionality.
  • T reg cells effectiveness of these T reg cells in suppressing Teff cell proliferation may be reduced, further affirming the critical role of the AOC3-siglec-9 interaction in maintaining T reg cell regulatory function.
  • Knockdown of SHP-1 using siRNA may mirror these effects by similarly inhibiting the SHP-1 pathway, thereby validating its essential role in the mediation of T reg cell functions through the AOC3-siglec-9 interaction.
  • Alternative approaches include employing additional molecular tools such as CRISPR-Cas9 to genetically knock out AOC3 or siglec-9 in Treg cells to study the impact on the SHP-1 pathway and Treg cell functions.
  • expanding the range of assessed functions to include additional regulatory and activation markers could help in providing a broader understanding of the impact of pathway disruption on Treg cell capabilities.
  • Example 13 shows optimization of siglec-9 overexpression levels in T reg cells for enhanced anti-fibrotic efficacy.
  • Siglec-9 overexpression levels in Treg cells are optimized using the following options.
  • [0224] a) Vector construction and validation: construction of a lentiviral vector with a controllable promoter for siglec-9 overexpression. Efficiency and expression levels will be validated through WB and flow cytometry.
  • c) Functional assays Evaluate the effects of varying siglec-9 expression levels on Treg cell mediate antifibrosis effect: 1) Fibroblast proliferation inhibition: Assess the impact of varied siglec-9 expression levels (3X, 5X, and 10X) on the capacity of Treg cell to inhibit Angiotensin II-induced proliferation of cardiac fibroblasts. Use quantitative assays to establish a direct correlation between increased siglec-9 levels and decreased proliferation of stimulated fibroblasts.2) Fibrotic marker reduction: Evaluate the effect of different siglec- 9 expression levels on the reduction of fibrotic markers (collagen type I and ⁇ -SMA) in cardiac fibroblasts, using PCR and WB for quantification.
  • fibrotic markers collagen type I and ⁇ -SMA
  • Collagen gel contraction assay Measure the contraction of collagen gels containing Angiotensin II-exposed cardiac fibroblasts co-cultured with varying levels of siglec-9 overexpressed Treg cells. This assay will demonstrate the T reg cells’ capability to modulate fibrotic responses effectively.
  • Example 14 This non-limiting example shows designing lentiviral vectors for controlled overexpression of siglec-9 in T reg cells. [0226] A lentiviral vector with a controllable promoter (the Tet-On and Tet-Off systems use doxycycline to control gene expression) for siglec-9 is developed, titration experiments to optimize multiplicity of Infection (MOI) is performed, and overexpression levels is validated via PCR and WB.
  • MOI multiplicity of Infection
  • the vector demonstrates targeted siglec-9 expression levels reaching 3-10 times those of non-engineered Treg cells without affecting cell viability.
  • Vector Construction Construct a lentiviral vector containing the siglec- 9 gene under the control of a Tet-responsive promoter. Incorporate a reporter gene (e.g., GFP) linked to siglec-9 to facilitate quantification of expression levels.
  • GFP reporter gene
  • Initial Titration of Doxycycline a) Transduce a population of T reg cells with the lentiviral vector; b) Expose different groups of transduced Treg cells to varying concentrations of doxycycline (e.g., 0.1 ⁇ g/mL, 0.5 ⁇ g/mL, 1 ⁇ g/mL, and 2 ⁇ g/mL) to establish a dose-response curve. c) Measure expression levels at 24-, 48-, and 72-hours post-doxycycline exposure using qPCR and Western blot to determine the kinetics of siglec-9 expression.3.
  • concentrations of doxycycline e.g., 0.1 ⁇ g/mL, 0.5 ⁇ g/mL, 1 ⁇ g/mL, and 2 ⁇ g/mL
  • a correlation between escalated siglec-9 expression levels (3X, 5X, and 10X) and a reduction in the proliferation of Angiotensin II-stimulated cardiac fibroblasts may be observed. Furthermore, reduced collagen gel contraction in cultures with Treg cells overexpressing siglec-9 (3X, 5X, and 10X) may be observed, indicating effective inhibition of Angiotensin II-exposed cardiac fibroblast contractility. [0231] In some embodiments, a minimum of a 50% reduction in proliferation rates at the lowest effective siglec-9 expression level compared to control groups may be achieved.
  • the mouse ICM model is also used to conduct efficient and controlled dosing studies to determine the optimal therapeutic dosage.
  • the optimal dosage in a pig model which closely mimics human cardiac physiology and disease, is tested. This model is critical for translating preclinical findings into clinical applications.
  • the optimal dose identified using mouse models can be translated to the pig model on a weight-to-weight basis.
  • the minimal effective dose is determined and heart function recovery post-treatment is assessed in ICM mouse models using the following options.
  • Fibrosis quantification Picrosirius red staining for fibrotic area visualization, WB for fibrotic protein markers (Collagen I and ⁇ SMA), and qPCR for gene expression levels of these markers.
  • Heart function and structure Analysis Measurement of LVEF, and end-diastolic and end-systolic volumes, via echocardiography.
  • Inflammation assessment H&E staining for general inflammation, Immunohistochemistry (IHC) for specific inflammatory cells (CD68, CD3), ELISA for inflammatory cytokines (TNF- ⁇ , IL-6, IL-1 ⁇ ), and qPCR for inflammatory gene expression.
  • the minimal effective dose (optimal dose) of engineered Treg cells that leads to reduction (e.g., at least a 30% reduction) in fibrosis, with corresponding decreases (e.g., of 20-30%) in protein and gene expression levels associated with fibrosis is identified.
  • the therapeutic impact of engineered T reg cells on cardiac function, structure and safety in a pig model of chronic ICM is assessed using the following options.
  • LVEF measurement Employ cine MRI to calculate LVEF by determining end-diastolic and end-systolic volumes.
  • Scar size measurement Use Late Gadolinium Enhancement (LGE) MRI to identify and measure myocardial scars.
  • Inflammation assessment Histopathological Analysis: Utilize Hematoxylin and Eosin (H&E) staining to evaluate cellular infiltration.
  • IHC Detect specific inflammatory cells (macrophages and T cells) using CD68 and CD3 antibodies.
  • Cytokine quantification Measure inflammatory cytokines (TNF- ⁇ , IL-6, IL-1 ⁇ ) in heart tissue or serum via ELISA.
  • Treg cells may significantly improve LVEF and reduce scar size by LGE, indicating effective cardiac recovery. Inflammatory markers may decrease, e.g., by at least 25%, demonstrating reduced cardiac inflammation. 3-fold lower and 3-fold higher doses may be examined to fine-tune the dosing to ensure optimal efficacy and safety.
  • the safety profile confirms no adverse effects on survival, blood parameters, or heart rhythm.

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Abstract

L'invention concerne des procédés et des compositions pour le traitement de la fibrose (par exemple, la fibrose cardiaque) ou d'autres états associés à une inflammation et/ou à une fibrose. Les procédés peuvent comprendre l'administration d'un acide nucléique codant pour une protéine de lectine 9 de type immunoglobuline de liaison à l'acide sialique (Siglec-9) à un sujet ayant besoin d'un traitement contre la fibrose, la protéine Siglec-9 étant exprimée par l'acide nucléique dans des lymphocytes T régulateurs (Treg) chez le sujet. L'invention concerne des procédés et des compositions pouvant comprendre des lymphocytes Treg qui surexpriment Siglec-9 pour améliorer la capacité de lymphocytes Treg à des cellules cibles exprimant une amine oxydase à cuivre 3 (AOC3), comprenant des fibroblastes ou des myofibroblastes. Dans certains modes de réalisation, les lymphocytes Treg améliorés sont ciblés sur des myofibroblastes (par exemple, des myofibroblastes cardiaques) avec une expression élevée de gènes fibrotiques.
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US12584127B2 (en) 2012-08-13 2026-03-24 Cedars-Sinai Medical Center Exosomes and micro-ribonucleic acids for tissue regeneration
US12544409B2 (en) 2014-10-03 2026-02-10 Cedars-Sinai Medical Center Cardiosphere-derived cells and exosomes secreted by such cells in the treatment of muscular dystrophy

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