Enhanced regulatory t cells and methods of use thereof

Abstract

Methods and compositions for treating fibrosis (e.g., cardiac fibrosis), or other conditions associated with inflammation and / or fibrosis are provided. Methods can include administering a nucleic acid encoding a sialic acid-binding immunoglobulin-type lectin 9 (Siglec-9) protein to a subject in need of fibrosis treatment, where the Siglec-9 protein is expressed by the nucleic acid in regulatory T (T reg ) cells in the subject. Methods and compositions can include T reg cells that overexpress Siglec-9 to enhance the ability of T reg cells to target cells expressing amine oxidase, copper containing 3 (AOC3), including fibroblasts or myofibroblasts. In some embodiments, the enhanced T reg cells are targeted to myofibroblasts (e.g., cardiac myofibroblasts) with elevated expression of fibrotic genes.

Description

CSMC.024WO PATENT ENHANCED REGULATORY T CELLS AND METHODS OF USE THEREOF REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 551364, filed February 8, 2024, the disclosure of which is hereby incorporated by reference in its entirety. STATEMENT REGARDING FEDERALLY SPONSORED R&D

[0002] This invention was made with government support under Grant No. R01 HL124074, awarded to Dr. Eduardo Marbán by the National Institutes of Health. The Government has certain rights in the invention. REFERENCE TO SEQUENCE LISTING

[0003] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled SeqListing_CSMC024WO.xml created on February 5, 2025, which is 4,933 bytes in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety. BACKGROUND Field

[0004] Fibrosis, a condition affecting various tissues, presents a major healthcare challenge. This condition, along with associated pathologies, occurs when there is an excessive accumulation of fibrous connective tissue, potentially leading to organ dysfunction or failure. Common treatment strategies often focus on immunosuppression to reduce inflammation, a key factor in fibrosis development. However, this approach can be counterproductive, as non-specific immunosuppression may impede the body's natural ability to repair and regenerate tissue, potentially worsening fibrosis. SUMMARY

[0005] Current immunotherapies designed to treat fibrosis may lack the specificity needed to avoid the negative effects known in the art. As a result, there is a critical need for the development of targeted therapies that can effectively modulate immune response so as to prevent or mitigate fibrosis while avoiding the negative side effects of broad immunosuppression. Provided herein is a method of treating fibrosis, includingadministering 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 (Treg) cells in the subject. In some embodiments, the subject is need of cardiac fibrosis treatment.

[0006] 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 (Treg) cells in the subject. Further provided is 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. In some embodiments, the condition associated with inflammation and / or fibrosis includes cardiac inflammation and / or cardiac fibrosis. In some embodiments, the condition associated with inflammation and / or fibrosis includes myocardial infarction, hypertension, myocarditis, transplant rejection, cardiomyopathy, heart failure, and amyloidosis. In some embodiments, the condition associated with inflammation and / or fibrosis is an angiotensin II-induced condition.

[0007] In some embodiments, administering the nucleic acid encoding the Siglec- 9 protein includes administering to the subject a therapeutically effective amount of a composition comprising a population of Tregcells including the nucleic acid (or the nucleic acid encoding the Siglec-9 protein), optionally wherein the therapeutically effective amount of the composition comprises the Treg cells at 107cells / kg or more of body weight. In some embodiments, Tregcells of the population include a plasmid or a viral vector including the nucleic acid (or the nucleic acid encoding the Siglec-9 protein). In some embodiments, 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). In some embodiments, the viral vector includes an adenoviral vector, retroviral vector, or a lentiviral vector. In some embodiments, the population of Treg cells are derived from cells autologousto the subject. In some embodiments, the population of Treg cells are derived from cells allogeneic to the subject.

[0008] In some embodiments, 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 Tregcells in the subject. In some embodiments, the vehicle includes: a lipid nanoparticle (LNP); and a Treg cell-targeting moiety associated with the LNP. In some embodiments, the Treg cell-targeting moiety includes an antigen binding protein that binds a Tregcell marker. In some embodiments, the Tregcell marker is selected from: CD25 and / or Foxp3. In some embodiments, the method wherein the antigen binding protein is an antibody. In some embodiments, the antigen binding protein is selected from: an anti-CD25 monoclonal antibody and / or anti-Foxp3 monoclonal antibody.

[0009] In some embodiments, the nucleic acid includes mRNA encoding the Siglec-9 protein.

[0010] 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 angiotensin II-induced cardiac fibrosis. In some embodiments, the subject is in need of treating cardiac fibrosis subsequent to a myocardial infarction.

[0011] In some embodiments, the Siglec-9 protein includes an amino acid sequence at least 80% identical to SEQ ID NO: 2.

[0012] 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 (Treg) cells in the subject. Further provided is 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. In some embodiments, the nucleic acid encodes the membrane-bound fibroblast- specific binding protein. In some embodiments, the membrane-bound fibroblast-specificbinding protein includes sialic acid-binding immunoglobulin-type lectin 9 (Siglec-9). In some embodiments, the membrane-bound fibroblast-specific binding protein includes an amino acid sequence at least 80% identical to SEQ ID NO:2. In some embodiments, the condition associated with inflammation and / or fibrosis includes an inflammatory tissue including fibroblasts having elevated expression of amine oxidase, copper containing 3 (AOC3). In some embodiments, the fibroblasts having elevated expression of AOC3 include myofibroblasts. In some embodiments, the myofibroblasts include cardiac myofibroblasts.

[0013] In several embodiments, the method further includes: obtaining a population of immune cells including regulatory T (Treg) 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.

[0014] Also provided is 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 Tregcells. Also provided is 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. In some embodiments, the at least one vehicle includes a liposome, or a lipid nanoparticle (LNP). In some embodiments, the at least one vehicle includes a viral vector. In some embodiments, the viral vector includes an adenoviral vector, retroviral vector, or a lentiviral vector. In some embodiments, the nucleic acid includes a promoter operatively linked to anucleotide sequence encoding the Siglec9, wherein the promoter upregulates expression of Siglec9 in Treg cells.

[0015] In some embodiments, the subject includes at least one myofibroblast cell having elevated expression of AOC3 compared to other cell types. In some embodiments, the at least one myofibroblast cell is at least one cardiac myofibroblasts. In some embodiments, the subject is in need of treatment for fibrosis. In some embodiments, the fibrosis is a cardiac fibrosis. In some embodiments, the cardiac fibrosis is subsequent to a myocardial infarction.

[0016] Also provided is 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 Tregcells to a subject in need of inhibiting fibrotic gene expression. In some embodiments, the subject is in need of inhibiting fibrotic gene expression in cardiac myofibroblasts. In some embodiments, the fibrotic gene is at least one of Collagen I, α- smooth muscle actin (α-SMA), and / or amine oxidase, copper containing 3 (AOC3). In some embodiments, engineering includes delivering at least one vehicle including a nucleic acid encoding Siglec-9 to the population of Treg cells. In some embodiments, 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). In some embodiments, the nucleic acid includes mRNA. In some embodiments, overexpressing Siglec-9 includes upregulating expression of the mRNA.

[0017] In some embodiments, the subject is in need of treatment for fibrosis. In some embodiments, the fibrosis is a cardiac fibrosis. In some embodiments, the cardiac fibrosis is subsequent to a myocardial infarction.

[0018] In some embodiments, 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.

[0019] In several embodiments, administering includes intravenous or oral administration.

[0020] Also provided is an isolated nucleic acid, further including a nucleotide sequence encoding a sialic acid-binding immunoglobulin-like lectin 9 (Siglec-9) proteinoperatively linked to a promoter configured to express the Siglec-9 protein in a regulatory T (Treg) cell. In some embodiments, 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.

[0021] Also provided is a vector including the isolated nucleic acid of the present disclosure.

[0022] Also provided is a regulatory T (Treg) cell including the isolated nucleic acid, or the vector of the present disclosure.

[0023] Also provided is a composition 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. In some embodiments, vehicles of the population include a lipid nanoparticle (LNP). In some embodiments, the nucleic acid includes mRNA. In several embodiments, the Treg cell-targeting moiety includes an antigen binding protein that binds a Treg cell marker. In some embodiments, the Treg cell marker is selected from: CD25 and / or Foxp3. In some embodiments, the antigen binding protein is an antibody. In some embodiments, the antigen binding protein is selected from: an anti-CD25 monoclonal antibody and / or anti-Foxp3 monoclonal antibody.

[0024] Also provided is a composition including a population of enhanced regulatory T (Treg) cells overexpressing sialic acid-binding immunoglobulin-like lectin 9 (Siglec-9). In some embodiments, 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). In some embodiments, 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). In some embodiments, the plurality of nucleic acids includes a plurality of mRNA transcripts encoding Siglec-9. In some embodiments, the enhanced Treg cells exhibit Siglec-9-dependent interaction with cells expressing amine oxidase, copper containing 3 (AOC3). In some embodiments, the enhanced Tregcells exhibit Siglec-9-dependent interaction with cardiac myofibroblasts expressing AOC3.

[0025] Also provided is a composition of the present disclosure for the treatment of a condition associated with inflammation and / or fibrosis in a subject in need thereof.BRIEF DESCRIPTION OF THE DRAWINGS

[0026] FIG 1 illustrates an example non-limiting embodiment depicting a method of administering a nucleic acid to enhanced Treg cells that overexpress Siglec-9.

[0027] FIG. 2 illustrates graphical representations of data obtained from in vitro experiments of the effect of Angiotensin II concentrations on fibrotic gene expression.

[0028] FIG. 3 illustrates graphical representations of data obtained from in vitro experiments of the effect of Angiotensin II on fibrotic gene expression over time.

[0029] FIG. 4 illustrates graphical representations of data obtained from in vitro experiments of the effect of Angiotensin II concentrations and time on AOC3 expression; and the results of a western blot assay showing the in vitro upregulation of AOC3 in the presence of Angiotensin II.

[0030] FIG. 5 illustrates graphical representations of data obtained from in vitro experiments of Treg cells overexpressing Siglec-9.

[0031] FIG.6 illustrates a schematic diagram of in vitro experiments of Treg cells overexpressing Siglec-9 and data obtained from same.

[0032] FIG.7 illustrates a schematic diagram of in vitro experiments of enhanced Treg cells targeting to AOC3-expressing cardiac fibroblasts and data obtained from same.

[0033] FIG. 8 illustrates graphical representations of data from in vitro experiments of enhanced Treg cells targeting to AOC3-expressing cardiac fibroblasts.

[0034] FIG. 9 illustrates a schematic diagram of in vitro experiments testing the role of Siglec-9 in enhanced Tregcells targeting to AOC3-expressing cardiac fibroblasts and data obtained from same.

[0035] FIG. 10 illustrates graphical representations of data from in vitro experiments testing the role of Siglec-9 in enhanced Tregcells targeting to AOC3-expressing cardiac fibroblasts.

[0036] FIG.11 illustrates a schematic diagram of in vitro experiments testing the role of AOC3 in enhanced Tregcells targeting to AOC3-expressing cardiac fibroblasts and data obtained from same.

[0037] 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.

[0038] FIG. 13 illustrates data of upregulation of AOC3 and fibrotic genes observed from in vivo experiments using a myocardial infarction model.

[0039] 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.

[0040] FIG. 15 illustrates an in vivo experimental protocol for studying the anti- fibrotic effect of enhanced Treg cells.

[0041] FIG. 16 illustrates an exemplary non-limiting embodiment of the cellular processes by which Siglec-9 could promote Tregcell function by affecting immune cell signaling.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] FIG. 22 illustrates a graphical illustration of mRNA levels AOC3, collagen I, and α-SMA in heart tissues, demonstrating reduced fibrosis markers in siglec-9 Treg-treated groups.

[0048] FIG. 23 illustrates picrosirius red stained images and analysis of fibrosis areas, showing significant reduction in fibrosis with siglec-9 Treg treatment (n=5 per group).

[0049] FIG. 24 illustrates graphical representations of echocardiographic results illustrating improved cardiac function in siglec-9-Treg treated hearts (n=5 per group).

[0050] 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.

[0051] FIG. 26 shows quantitation of expression levels of the Western Blot shown in FIG.5. DETAILED DESCRIPTION

[0052] Therapies based on Regulatory T cells (Treg cells) offer targeted approach that can avoid the broader immunosuppressive effects typically associated with conventional immunotherapy treatment of cardiac fibrosis and related conditions.

[0053] Ischemic cardiomyopathy (ICM) 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. In later stages, fibrosis can also lead to remodeling of the heart, which is a process of maladaptive structural and functional changes that can further exacerbate HF. ICM is often associated with renal fibrosis, leading to chronic kidney disease. When ICM with HF with reduced ejection fraction (HFrEF) is complicated by chronickidney disease, mortality rises 3-fold. 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. Additionally, chronic HF leads to liver congestion, which can eventually progress to fibrosis and cirrhosis, disrupting metabolic functions and drug metabolism, further complicating patient management.

[0054] Methods of enhancing regulatory T (Treg) cells, and uses of the enhanced cells, are provided. Broadly, 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. As used herein, “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. In some embodiments, 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 Tregcells 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.

[0055] In various embodiments, the enhanced Tregcells 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. In some embodiments, 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. By leveraging the unique properties of Siglec-9, 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.

[0056] In some embodiments, 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). In some embodiments, the use of Tregcells in thesemethods 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. The methods described herein, including the use of LNPs for the targeted delivery of nucleic acids to Treg cells, provide a novel and effective way of treating inflammation- and / or fibrosis-related conditions, particularly those related to cardiac health, by harnessing the specificity and efficacy of enhanced Treg cells.

[0057] 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. In some embodiments, the vehicles can be designed to target Tregcells, facilitating the delivery and subsequent expression of Siglec-9 by Treg cells. In some further embodiments, the delivery vehicles can include liposomes, nanoparticles, lipid nanoparticles (LNPs), viral vectors, cell- penetrating peptides (CPPs), and extracellular vesicles (EVs). For example, each vehicle encapsulates a plurality of mRNA transcripts that encode Siglec-9, ensuring efficient transport and expression within Treg cells. Accordingly, in some embodiments, a population of enhanced Tregcells is produced, wherein the Tregcells are enhanced by their ability to overexpress Siglec-9. In some embodiments, such modification of Tregcells can significantly increase the therapeutic potential of Treg cells, including, but not limited to, in targeting and inhibiting fibrotic genes.

[0058] In some further embodiments, the enhanced Tregcells within the composition exhibit high-affinity binding to amine oxidase, copper containing 3 (AOC3), which is prominently expressed by cardiac myofibroblasts. In some embodiments, such specificity can mitigate and / or treats cardiac fibrosis, a condition often triggered by high blood pressure or myocardial infarction. For example, by focusing on AOC3, the enhanced Treg cells target fibrosis-causing cells and / or inhibit the expression of fibrotic genes, including, but not limited to, Collagen I, α-smooth muscle actin (α-SMA), and AOC3. 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.

[0059] 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. Definitions

[0060] “Vehicle” or “delivery vehicle” as used herein refers to a molecule or collection of molecules suitable for delivering a nucleic acid into a cell. For example, 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. In some embodiments, 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. “Nanoparticle” 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. 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, without limitation, 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 hLF(1-11)s. “Extracellular vesicle” or “EV” as used herein have their ordinary and customary meaning as understood by one of ordinary skill in the art, in view of the present disclosure. 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.

[0061] “Immune modulation” as used herein refers to altering the activity of the adaptive or innate immune system of a vertebrate organism. Immune modulation can include enhanced or suppressed activity of one or more aspects of an immune response, compared to the immune response without the immune modulation. Immune modulation, e.g., targeted immune modulation, can include enhanced or suppressed activity of one aspect of an immune response without altering another aspect of the immune response. Targeted immune modulation can be specific to a tissue (e.g., heart-specific) and / or target specific processes (e.g., fibrosis).

[0062] “Immune cells” as used herein has its customary and ordinary meaning as understood by one of ordinary skill in the art in view of the present disclosure, and refer to specialized cells that mediate an immune response in a vertebrate organism. For example, immune cells can identify and neutralize pathogenic threats and dysfunctional or anomalous “self” cells. There are several types of immune cells, including, but not limited to, innate immune cells (e.g., neutrophils, macrophages, dendritic cells, natural killer cells) that provide immediate, non-specific defense mechanisms, and adaptive immune cells (e.g., lymphocytes such as B cells and T cells, including regulatory T cells) that mediate targeted, specific immune responses and establish immunological memory. These cell types often work synergistically and are essential for the detection, response to, and elimination of foreign material and aberrant self-physiology. This definition is inclusive of any modifications or variations to the standard biological functions or characteristics of immune cells, as may beemployed, referenced or understood by one of ordinary skill in the art, in view of the present disclosure.

[0063] “Regulatory T cells” or “Tregcells” are used interchangeably herein and have their customary and ordinary meaning as understood by one of ordinary skill in the art, in view of the present disclosure. Treg cells are anti-inflammatory T cells, and can be characterized by expression of cell surface markers that distinguish Tregcells from other immune cells (e.g., effector T cells, B cells, macrophages, NK cells, etc.). In some embodiments, Treg cells are CD4+and at least one of CD25+and FOXP3+. In some embodiments, Tregcells are CD4+ / CD25+ / CD127low. In some embodiments Tregcells are induced Treg cells (iTreg cells). In some embodiments, Treg cells, e.g., iTreg cells, are differentiated in vitro from naïve CD4+T cells. In some embodiments, Treg cells are Treg cells in vivo. “Enhanced Tregcells” as used herein refers to Tregcells that have been modified to express (e.g. overexpress) Siglec-9 protein, as described herein. In some embodiments, enhanced Treg cells express (e.g., overexpress) a plurality of membrane-bound Siglec-9 proteins, and exhibit targeted anti-inflammatory activity compared to Tregcells that have not been modified, in view of the present disclosure.

[0064] The term “effective amount” as used herein refers to the amount of a composition or an agent needed to provide the desired effect, e.g., cellular response, therapeutic effect, etc. The term “therapeutically effective amount” refers to an amount of a composition or therapeutic agent that is sufficient to provide, e.g., a particular anti- inflammatory effect when administered to a typical subject. A therapeutically effective amount as used herein, in various contexts, can include an amount sufficient to delay the development of a symptom of the disease, alter the course of a symptom disease (for example but not limited to, slowing the progression of a symptom of the disease), or reverse a symptom of the disease. The therapeutically effective amount may be administered in one or more doses of the therapeutic agent. The therapeutically effective amount may be administered in a single administration, or over a period of time in a plurality of doses.

[0065] “Subject,” as used herein refers to any vertebrate animal, including mammals and non-mammals. A subject can include primates, including humans, and non- primate mammals, such as rodents, domestic animals or game animals. Non-primate mammals can include mouse, rat, hamster, rabbit, dog, fox, wolf, cat, horse, cow, pig, sheep,goat, camel, deer, buffalo, bison, etc. Non-mammals can include bird (e.g., chicken, ostrich, emu, pigeon), reptile (e.g., snake, lizard, turtle), amphibian (e.g., frog, salamander), fish (e.g., salmon, cod, pufferfish, tuna), etc. The terms, “individual,” “patient,” and “subject” are used interchangeably herein.

[0066] “Treating” or “treatment” as used herein refers to altering one or more of the signs or symptoms of a condition, e.g., disease, described herein in a beneficial manner; improving or ameliorating other clinically accepted symptoms, e.g., by at least 2%, 3%, 4%, 5%, 10%, or more; or inducing a desired response, by performing methods described herein. Efficacy of treatment can be assessed, for example, by measuring a marker, indicator, symptom, and / or the incidence of a condition treated according to the methods described herein or any other measurable parameter appropriate, e.g., ventricular ejection fraction. Efficacy of treatment can also be measured by a failure of an individual to worsen as assessed by hospitalization, or need for medical interventions (e.g., progression of the disease is halted). Treatment includes any treatment of a disease in an individual or an animal (some non-limiting examples include a human or an animal) and includes: (1) inhibiting the disease, e.g., preventing a worsening of symptoms (e.g. pain or inflammation); or (2) relieving the severity of the disease, e.g., causing regression of symptoms. An therapeutically effective amount for the treatment of a disease means that amount which, when administered to a subject in need thereof, is sufficient to result in effective treatment as that term is defined herein, for that disease. Efficacy of an agent can be determined by assessing physical indicators of a condition or desired response, (e.g. heart activity). One skilled in the art can monitor efficacy of administration and / or treatment by measuring any one of such parameters, or any combination of parameters.

[0067] “Administering” as used herein can include any suitable routes of administering a therapeutic agent or composition as disclosed herein. Suitable routes of administration include, without limitation, oral, parenteral, intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), pulmonary, cutaneous, injection or topical administration. Administration can be local or systemic.

[0068] “Cardiac” or “-cardial” as used herein pertains to or is characteristic of the heart, and used herein to encompass structures, functions, pathologies, and treatments related to the heart, including but not limited to cardiac tissues, cells (such as cardiomyocytes,cardiac stromal cells), physiological processes (e.g., cardiac rhythm, myocardial contraction), and associated therapeutic methods. The scope of cardiac is inclusive of various modifications, innovations, or technological advancements aimed at diagnosing, treating, or researching heart-related conditions and functions.

[0069] “Fibrosis” as used herein has its customary and ordinary meaning as understood by one of ordinary skill in the art in view of the present disclosure, and refers to a pathology characterized by the excessive accumulation of extracellular matrix components in tissues or organs. Fibrosis can include any remodeling (e.g., pathological remodeling) of tissue (e.g., connective tissue, skeletal muscle, myocardium, skin), such as, but not limited to, deposition of fibrotic and / or fatty tissue, replacement of muscle tissue with fibrotic and / or fatty tissue, etc. Fibrosis can include, but is not limited to, inflammatory responses or inflammatory tissue trauma. For example, fibrosis often leads to scar tissue formation, which can impair tissue or organ function. Different organs can be affected by fibrosis, including but not limited to the lungs (pulmonary fibrosis), liver (cirrhosis), heart (myocardial fibrosis), and kidneys. “Fibrosis-associated diseases” as used herein has its customary and ordinary meaning as understood by one of ordinary skill in the art in view of the present disclosure, and refers to medical conditions resulting from and / or caused by fibrosis, or conditions that lead to fibrosis. For example, some fibrosis-associated diseases include idiopathic pulmonary fibrosis, cystic fibrosis, liver cirrhosis, systemic sclerosis, kidney fibrosis, Dupuytren’s contracture, nephrogenic systemic fibrosis, peritoneal fibrosis, retroperitoneal fibrosis, post- surgical adhesions, scleroderma, endomyocardial fibrosis, radiation-induced fibrosis, Asherman’s syndrome, pancreatic fibrosis, Peyronie’s disease. There are several fibrosis- associated diseases specific to the heart, including but not limited to myocardial fibrosis, cardiac fibrosis, endomyocardial fibrosis, hypertensive heart disease, ischemic heart disease, dilated cardiomyopathy, heart valve disease, congenital heart disease, arrhythmogenic right ventricular cardiomyopathy. “Myocardial infarction” as used herein has its customary and ordinary meaning as understood by one of ordinary skill in the art in view of the present disclosure, and refers to a medical disorder, commonly known as a “heart attack,” whereby blood flow to part of the heart is reduced or blocked, limiting oxygen supply to said part, and leading to tissue damage or death. Following a myocardial infarction, fibrosis of the heart can occur. Damaged heart myocytes are replaced with scar tissue, which is primarily composedof collagen. Scar tissue can reduce and / or impair cardiac function due to the lesser elasticity of scar tissue as compared to normal heart myocytes.

[0070] “Fibroblast” as used herein has its customary and ordinary meaning as understood by one of ordinary skill in the art in view of the present disclosure, and refers to a cell type associated with connective tissues. Fibroblasts most commonly synthesize components of the extracellular matrix, including collagen. Fibroblasts also play an important role in tissue regeneration. Fibroblasts differentiate within an organism in response to physiological or pathological conditions, and include, but are not limited to dermal fibroblasts, cardiac fibroblasts, pulmonary fibroblasts, bone marrow fibroblasts, periodontal ligament fibroblasts, adipose-derived fibroblasts, pancreatic fibroblasts, hepatic stellate cells (liver fibroblasts), intestinal fibroblasts, corneal fibroblasts. “Myofibroblast” as used herein has its customary and ordinary meaning as understood by one of ordinary skill in the art in view of the present disclosure, and refers to a specialized, contractile fibroblast, characterized by their expression of α-smooth muscle actin (α-SMA). Myofibroblasts develop particularly during wound healing and other fibrotic responses. Myofibroblasts secrete extracellular components, including, but not limited to, collagen, and fibronectin, as part of the process of an organism’s innate ability to regenerate tissue. Persistent activation is implicated in pathological fibrosis across various organs, including the heart.

[0071] “Gene expression” as used herein has its customary and ordinary meaning as understood by one of ordinary skill in the art in view of the present disclosure, and refers to the process by which genetic information encoded by a gene is transcribed into RNA (including mRNA) and subsequently translated into a peptide. Gene expression can refer to production of a gene product from a nucleic acid encoding the gene product, including, without limitation, production of mRNA from DNA encoding the mRNA, production of protein from DNA (e.g., via production of an mRNA), and production of protein from mRNA encoding the protein). This process includes but is not limited to, one or more of chromatin remodeling, DNA unpacking, histone modification, regulatory protein binding, transcription factor recruitment, 5’ capping, splicing, 3’ polyadenylation, nuclear export, mRNA editing, alternative splicing, mRNA localization, mRNA transport, RNA folding, RNA modifications, ribosome binding, ribonucleoprotein complex assembly, protein folding, protein sorting, post-translational modifications, phosphorylation, glycosylation, acetylation,ubiquitination, signal peptide cleavage, membrane targeting, formation of protein complexes, protein localization, protein degradation, targeting to lysosomes, autophagy, secretion, and subcellular compartmentalization. In some embodiments, gene expression can encompass both the natural expression of genes within an organism and the artificial manipulation of gene expression through recombinant DNA technology, gene editing tools or the use of inducible promoters. The definition is inclusive of quantitative and qualitative aspects of gene expression, as well as the use of gene expression for various applications, including but not limited to, therapeutic protein production, gene therapy, functional genomics studies, and synthetic biology.

[0072] “Overexpression” or “gene overexpression” as used herein refers to expression of a gene that exceeds a reference level of expression of the gene. The reference level can be, but is not limited to, the average or homeostatic levels of expression of a gene, e.g., in a given organism or cell. Overexpression can be achieved via any suitable option, such as (but not limited to) increased levels of transcription and / or translation. Overexpression can be induced or engineered through various means, e.g., the introduction of additional gene copies, promoter modification, and / or gene regulatory intervention. Overexpression encompasses both transient and stable overexpression, and may include endogenous gene upregulation or the introduction of exogenous genetic elements, with applications ranging from gene function analysis to disease therapeutics. In some embodiments, overexpression can be achieved by genetic modification of the cell. In some embodiments, overexpression can be achieved by epigenetic modification of the cell.

[0073] “Siglecs” as used herein refers to sialic acid-binding immunoglobulin-like lectins. Siglecs are a family of cell surface proteins primarily found on the surface of immune cells and are involved in cell-cell interactions and signaling. “Siglec-9” as used herein refers to sialic acid-binding immunoglobulin-like lectin 9. In some embodiments, Siglec-9 includes a mammalian Siglec-9. In some embodiments, Siglec-9 includes human Siglec-9 (Gene ID: 27180). In some embodiments, Siglec-9 protein has the amino acid sequence shown below (signal peptide underlined), with or without the signal peptide. 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 ltcqvtfpga svttnktvhl nvsyppqnlt241 mtvfqgdgtv stvlgngssl slpegqslrl vcavdavdsn pparlslswr gltlcpsqps 301 npgvlelpwv hlrdaaeftc raqnplgsqq vylnvslqsk atsgvtqgvv ggagatalvf 361 lsfcvifvvv rscrkksarp aagvgdtgie danavrgsas qgpltepwae dsppdqpppa 421 sarssvgege lqyaslsfqm vkpwdsrgqe atdteyseik ihr (SEQ ID NO:2)

[0074] “AOC3” as used herein refers to amine oxidase, copper containing 3. AOC3 may be used interchangeably with Vascular Adhesion Protein-1 (VAP-1). AOC3 is an enzyme that is integral to the immune system, e.g., by trafficking some immune cell types to areas of inflammation or infection. In some embodiments, AOC3 is a mammalian AOC3. In some embodiments, AOC3 includes human AOC3 (Gene ID: 8639).

[0075] As used herein the term “nucleic acid” or “oligonucleotide” refers to multiple nucleotides (e.g., molecules comprising a sugar (e.g. ribose or deoxyribose) linked to a phosphate group and to an exchangeable organic base, which is either a substituted pyrimidine (e.g. cytosine (C), thymidine (T) or uracil (U)) or a substituted purine (e.g. adenine (A) or guanine (G)). The term includes polynucleosides (i.e. a polynucleotide minus the phosphate) and any other organic base containing polymer. Purines and pyrimidines include but are not limited to adenine, cytosine, guanine, thymidine, inosine, 5- methylcytosine, 2-aminopurine, 2-amino-6-chloropurine, 2,6-diaminopurine, hypoxanthine, and other naturally and non-naturally occurring nucleobases, substituted and unsubstituted aromatic moieties. A nucleic acid can include any other suitable modifications. Thus, the term nucleic acid also encompasses nucleic acids with substitutions or modifications, such as in the bases and / or sugars.

[0076] Polypeptide or nucleic acid molecules of the present disclosure may share a certain degree of sequence similarity or identity with the reference molecules (e.g., reference polypeptides or reference polynucleotides), for example, with art-described molecules (e.g., engineered or designed molecules or wild-type molecules). The term “identity” as known in the art, refers to a relationship between the sequences of two or more polypeptides or polynucleotides, as determined by comparing the sequences. In the art, identity also means the degree of sequence relatedness between them as determined by the number of matches between strings of two or more amino acid residues or nucleic acid residues. Identity measures the percent of identical matches between the smaller of two or more sequences with gap alignments (if any) addressed by a particular mathematical model or computer program (e.g., “algorithms”). Identity of related peptides can be readilycalculated by known methods. “% identity” as it applies to polypeptide or polynucleotide sequences is defined as the percentage of residues (amino acid residues or nucleic acid residues) in the candidate amino acid or nucleic acid sequence that are identical with the residues in the amino acid sequence or nucleic acid sequence of a second sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity. Any suitable methods and computer programs for the alignment can be used. It is understood that identity depends on a calculation of percent identity but may differ in value due to gaps and penalties introduced in the calculation. Generally, variants of a particular polynucleotide or polypeptide have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% but less than 100% sequence identity to that particular reference polynucleotide or polypeptide as determined by sequence alignment programs and parameters described herein and known to those skilled in the art. Such tools for alignment include those of the BLAST suite (Stephen F. Altschul, et al (1997), “Gapped BLAST and PSI-BLAST: a new generation of protein database search programs”. Nucleic Acids Res.25:3389-3402). Another popular local alignment technique is based on the Smith-Waterman algorithm (Smith, T. F. & Waterman, M. S. (1981) “Identification of common molecular subsequences.” J. Mol. Biol. 147:195-197.) A general global alignment technique based on dynamic programming is the Needleman-Wunsch algorithm (Needleman, S. B. & Wunsch. C. D. (1970) “A general method applicable to the search for similarities in the amino acid sequences of two proteins.” J. Mol. Biol. 48:443- 453.). More recently a Fast Optimal Global Sequence Alignment Algorithm (FOGSAA) has been developed that purportedly produces global alignment of nucleotide and protein sequences faster than other optimal global alignment methods, including the Needleman- Wunsch algorithm. Other tools are described herein, specifically in the definition of “identity” below.

[0077] The term “identity” refers to the overall relatedness between polymeric molecules, for example, between polynucleotide molecules (e.g. DNA molecules and / or RNA molecules) and / or between polypeptide molecules. Calculation of the percent identity of two polynucleic acid sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second nucleic acid sequences for optimal alignment and non-identical sequencescan be disregarded for comparison purposes). In certain embodiments, the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the length of the reference sequence. The nucleotides at corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a suitable mathematical algorithm. For example, the percent identity between two nucleic acid sequences can be determined using methods such as those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects. Smith. D. W., ed., Academic Press. New York, 1993; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; each of which is incorporated herein by reference. For example, the percent identity between two nucleic acid sequences can be determined using the algorithm of Meyers and Miller (CABIOS, 1989, 4:11-17), which has been incorporated into the ALIGN program (version 2.0) using a PAM 120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. The percent identity between two nucleic acid sequences can, alternatively, be determined using the GAP program in the GCG software package using an NWSgapdna.CMP matrix. Methods commonly employed to determine percent identity between sequences include, but are not limited to those disclosed in Carillo, H., and Lipman, D., SIAM J Applied Math., 48:1073 (1988); incorporated herein by reference. Techniques for determining identity are codified in publicly available computer programs. Exemplary computer software to determine homology between two sequences include, but are not limited to, GCG program package, Devereux, J., et al., Nucleic Acids Research, 12(1), 387 (1984)), BLASTP, BLASTN, and FASTA Altschul, S. F. et al., J. Molec. Biol., 215, 403 (1990)).

[0078] “Isolated” as used herein with reference to an isolated biomolecule, e.g., a nucleic acid, has the ordinary and customary meaning to one of ordinary skill in the art in view of the present disclosure. An isolated biomolecule, e.g., an isolated nucleic acid, is generally in a non-natural environment, or in an environment that the biomolecule would otherwise not have been without human intervention of the biomolecule or its environment. In some embodiments, an isolated biomolecule is not inside a cell or an organism.

[0079] As used herein, the phrase “physiologically compatible” and “pharmaceutically acceptable” are employed interchangeably herein to refer to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0080] Definitions of common terms in cell biology and molecular biology can be found in “The Merck Manual of Diagnosis and Therapy”, 19th Edition, published by Merck Research Laboratories, 2006 (ISBN 0-91 1910-19-0); Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0- 632-02182-9); Benjamin Lewin, Genes X, published by Jones & Bartlett Publishing, 2009 (ISBN-10: 0763766321); Kendrew et al. (eds.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081- 569-8) and Current Protocols in Protein Sciences 2009, Wiley Intersciences, Coligan et al., eds.

[0081] As used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[0082] The singular terms “a,” “an,” and “the” include plural referents unless context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise. The abbreviation, “e.g.” is used herein to indicate a non-limiting example. Thus, the abbreviation “e.g.” is synonymous with the term “for example.” The term “about” as used herein to, for example, define the values and ranges of molecular weights means that the indicated values and / or range limits can vary within ±20%, e.g., within ±15%, within ±10%, including within ±5%. The use of “about”before a number includes the number itself. For example, “about 5” provides express support for “5.” Numbers provided in ranges include overlapping ranges and integers in between; for example a range of 1-4 and 5-7 includes for example, 1-7, 1-6, 1-5, 2-5, 2-7, 4-7, 1, 2, 3, 4, 5, 6 and 7. METHODS

[0083] Provided herein is a method of treating a condition associated with inflammation and / or fibrosis (e.g., cardiac fibrosis), comprising administering a nucleic acid (e.g., a therapeutically effective amount of a nucleic acid) encoding a sialic acid-binding immunoglobulin-type lectin 9 (Siglec-9) protein to a subject in need of treating the condition associated with inflammation and / or fibrosis (e.g., cardiac fibrosis), wherein the Siglec-9 protein is expressed by the nucleic acid in regulatory T (Treg) cells in the subject. In some embodiments, administering the nucleic acid encoding the Siglec-9 protein includes administering a population of Treg cells (e.g., a therapeutically effective amount of a composition that includes a population of Tregcells) that are engineered (e.g., genetically modified) to express (e.g., overexpress) the Siglec-9 protein, as described herein. In some embodiments, administering the nucleic acid encoding the Siglec-9 protein includes administering a vehicle (e.g., lipid nanoparticle (LNP)) comprising the nucleic acid encoding the Siglec-9 protein to the subject, where the vehicle is configured to deliver the nucleic acid encoding the Siglec-9 protein to Treg cells in the subject, as described herein. In some embodiments, the method of treating fibrosis includes administering a nucleic acid (e.g., a therapeutically effective amount of 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 (Treg) cells in the subject. As used herein, “encode” denotes a nucleic acid (e.g., DNA or RNA) having sequence information that specifies a gene product through transcription (e.g., an RNA gene product) and / or translation (e.g., a protein gene product), unless indicated otherwise. For example, a DNA molecule “encoding” a protein gene product denotes that the DNA molecule has nucleotide sequence information that specifies a protein gene product (via sequence information of a RNA gene product (e.g., mRNA) encoded by the DNA molecule).

[0084] Also provided is a method of treating a condition associated with inflammation and / or fibrosis (e.g., cardiac fibrosis), comprising administering to a subject in need of treating the condition associated with inflammation and / or fibrosis (e.g., cardiac fibrosis) a nucleic acid (e.g., a therapeutically effective amount of a nucleic acid) a configured to induce expression of a sialic acid-binding immunoglobulin-type lectin 9 (Siglec-9) protein in regulatory T (Treg) cells of the subject. In some embodiments, administering the nucleic acid includes administering a therapeutically effective amount of a composition that includes a population of Treg cells that are engineered (e.g., genetically modified) to express (e.g., overexpress) the Siglec-9 protein, as described herein. In some embodiments, administering the nucleic acid includes administering a vehicle (e.g., lipid nanoparticle (LNP)) comprising the nucleic acid to the subject, where the vehicle is configured to deliver the nucleic acid to Tregcells in the subject, as described herein. In some embodiments, the method of treating fibrosis includes administering a nucleic acid (e.g., a therapeutically effective amount of 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 in need of fibrosis treatment. In some embodiments, the method includes administering a nucleic acid (e.g., a therapeutically effective amount of a nucleic acid) encoding a sialic acid-binding immunoglobulin-type lectin 9 (Siglec-9) protein to the subject in need of treatment for fibrosis.

[0085] Fibrosis that can be treated by the present methods includes, without limitation, cardiac, pulmonary, hepatic, and colorectal fibrosis. In some embodiments, the fibrosis is cardiac fibrosis. In some embodiments, cardiac fibrosis is related to progression of ischemic cardiomyopathy (ICM).

[0086] In some embodiments, the Siglec-9 protein that is expressed (e.g., overexpressed) in the Tregcell is encoded by a nucleotide sequences that is, is about, or is at least 75, 80, 85, 90, 92, 95, 97, 99, or about 100%, or a percentage in a range defined by any two of the preceding values (e.g., 75-100%, 80-99%, 85-95%, 87-97%, etc.) identical to SEQ ID NO: 1, as provided below: cctgaggaacagacgttccctcgcggccctggcacctctaaccccagacatgctgctgctgct gctgcccctgctctgggggagggagagggcggaaggacagacaagtaaactgctgacgatgca gagttccgtgacggtgcaggaaggcctgtgtgtccatgtgccctgctccttctcctacccctc gcatggctggatttaccctggcccagtagttcatggctactggttccgggaaggggccaatacagaccaggatgctccagtggccacaaacaacccagctcgggcagtgtgggaggagactcggga ccgattccacctccttggggacccacataccgagaattgcaccctgagcatcagagatgccag aagaagtgatgcggggagatacttctttcgtatggagaaaggaagtataaaatggaattataa acatcaccggctctctgtgaatgtgacagccttgacccacaggcccaacatcctcatcccagg caccctggagtccggctgcccccagaatctgacctgctctgtgccctgggcctgtgagcaggg gacaccccctatgatctcctggatagggacctccgtgtcccccctggacccctccaccacccg ctcctcggtgctcaccctcatcccacagccccaggaccatggcaccagcctcacctgtcaggt gaccttccctggggccagcgtgaccacgaacaagaccgtccatctcaacgtgtcctacccgcc tcagaacttgaccatgactgtcttccaaggagacggcacagtatccacagtcttgggaaatgg ctcatctctgtcactcccagagggccagtctctgcgcctggtctgtgcagttgatgcagttga cagcaatccccctgccaggctgagcctgagctggagaggcctgaccctgtgcccctcacagcc ctcaaacccgggggtgctggagctgccttgggtgcacctgagggatgaagctgaattcacctg cagagctcagaaccctctcggctctcagcaggtctacctgaacgtctccctgcagagcaaagc cacatcaggagtgactcagggggtggtcgggggagctggagccacagccctggtcttcctgtc cttctgcgtcatcttcgttgtagtgaggtcctgcaggaagaaatcggcaaggccagcagcggg cgtgggagatacgggcatagaggatgcaaacgctgtcaggggttcagcctctcaggggcccct gactgaaccttgggcagaagacagtcccccagaccagcctcccccagcttctgcccgctcctc agtgggggaaggagagctccagtatgcatccctcagcttccagatggtgaagccttgggactc gcggggacaggaggccactgacaccgagtactcggagatcaagatccacagatgagaaactgc agagactcaccctgattgagggatcacagcccctccaggcaagggagaagtcagaggctgatt cttgtagaattaacagccctcaacgtgatgagctatgataacactatgaattatgtgcagagt gaaaagcacacaggctttagagtcaaagtatctcaaacctgaatccacactgtgccctccctt ttatttttttaactaaaagacagacaaattcctaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa aaaaaa (SEQ ID NO:1)

[0087] In some embodiments, the Siglec-9 protein is encoded by a nucleotide sequence of SEQ ID NO: 1 (with or without the 5’ or 3’ UTR). The 5’ UTR is single underlined, and the 3’ UTR is double underlined above.

[0088] In some embodiments, the Siglec-9 protein that is expressed (e.g., overexpressed) in the Tregcell has an amino acid sequences that is, is about, or is at least 75, 80, 85, 90, 92, 95, 97, 98, 99, or about 100%, or a percentage in a range defined by any two of the preceding values (e.g., 75-100%, 80-99%, 85-95%, 87-97%, etc.) identical to SEQ ID NO: 2 (with or without the signal peptide). 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 ltcqvtfpga svttnktvhl nvsyppqnlt 241 mtvfqgdgtv stvlgngssl slpegqslrl vcavdavdsn pparlslswr gltlcpsqps301 npgvlelpwv hlrdaaeftc raqnplgsqq vylnvslqsk atsgvtqgvv ggagatalvf 361 lsfcvifvvv rscrkksarp aagvgdtgie danavrgsas qgpltepwae dsppdqpppa 421 sarssvgege lqyaslsfqm vkpwdsrgqe atdteyseik ihr (SEQ ID NO:2) In some embodiments, the Siglec-9 protein as an amino acid sequence at least 90% identical to SEQ ID NO:2 (without the signal peptide). In some embodiments, the Siglec-9 protein as an amino acid sequence at least 95% identical to SEQ ID NO:2 (without the signal peptide). In some embodiments, the Siglec-9 protein as an amino acid sequence at least 98% identical to SEQ ID NO:2 (without the signal peptide). In some embodiments, the Siglec-9 protein as an amino acid sequence at least 99% identical to SEQ ID NO:2 (without the signal peptide). In some embodiments, the Siglec-9 protein has the amino acid sequence of SEQ ID NO: 2 (without the signal peptide). In some embodiments, the Siglec-9 protein has the amino acid sequence of residues 18-463 of SEQ ID NO: 2.

[0089] In some embodiments, the method includes expressing or overexpressing siglec-9 from a nucleic acid exogenous to the Treg cell. In some embodiments, the nucleic acid encodes the Siglec-9 protein, or includes a nucleotide sequence that encodes the Siglec-9 protein. The nucleic acid encoding the Siglec-9 protein can be any suitable nucleic acid. In some embodiments, the nucleic acid includes RNA (e.g., mRNA) encoding the Siglec-9 protein. In some embodiments, the nucleic acid includes DNA encoding the Siglec-9 protein.

[0090] In some embodiments, the method includes expressing or overexpressing siglec-9 from an endogenous (e.g., genomic) locus of the Treg cell. In some embodiments, the nucleic acid encodes an epigenetic modulator that is configured to induce expression of (e.g., overexpress) Siglec-9 protein from an endogenous siglec-9 locus (e.g., a genomic locus of the siglec-9 gene) in the Treg cell. Any suitable epigenetic modulator can be used to induce expression of (e.g., overexpress) Siglec-9 protein from the endogenous siglec-9 locus. Suitable options include, without limitation, a CRISPR activation system configured to target the siglec-9 locus.

[0091] In some embodiments, the nucleic acid is comprised in a vector (e.g., a plasmid, viral vector, etc.). In some embodiments, the vector is a plasmid (e.g., a lentiviral vector). For example, the plasmid can comprise a lentiviral vector (e.g., pLenti-GIII-CMV). In some alternative embodiments, the vector can be adenovirus vectors, adeno-associated virus (AAV) vectors, retroviral vectors, a transposon vector, and like vectors known to those skilled in the relevant art will recognize.

[0092] With reference to FIG. 1, a non-limiting embodiment of a method of the present disclosure is provided. A method 100 of treating fibrosis (e.g., cardiac fibrosis), comprising delivering a nucleic acid 120 encoding a sialic acid-binding immunoglobulin- type lectin 9 (Siglec-9) protein to a population of immune cells (e.g., Treg cells), the method including obtaining at least one vehicle 110 that includes the nucleic acid encoding Siglec 9; and obtaining at least one immune cell comprising a regulatory T (Treg) cells 101. The nucleic acid encoding Siglec 9 can express Siglec 9 protein in the Treg cell. In some embodiments, the method includes delivering the vehicle to a population of Treg cell. In some embodiments, the population of Tregcell is in a subject, and delivering the vehicle includes administering the vehicle to the subject. In some embodiments, the population of Treg cells is provided in vitro. In some embodiments, the method includes contacting (e.g., in vivo or in vitro) at least a portion of the at least one vehicle 110 with the at least one Tregcell 101 for a period of time, where the contacting induces the incorporation of the vehicle 110 into the cytoplasm 102 of a Treg cell 101. In some embodiments, after the vehicle 110 is incorporated into the cytoplasm of a Tregcell 101, the vehicle can be encapsulated by a membrane 130 derived from a Tregcell membrane. Degradation of the membrane 130 encapsulating the vehicle 110 may follow, and thereafter degradation of the vehicle 115, releasing nucleic acids 121 enclosed therein into the cytoplasm 102. The at least one nucleic acid in the cytoplasm 121 may interact with at least one ribosome 140. A nucleic acid 122 and ribosome 140 complex is subsequently formed, followed by translation of the nucleic acid 122, thereby producing at least one polypeptide protein precursor 150, including, but not limited to, a polypeptide precursor of Siglec-9. Post-translation modification of the polypeptide precursor 150 may follow, whereby further modification and Treg cell reconfiguration results in, e.g., at least one Siglec-9 protein 160 to be expressed on the membrane of the Tregcell. In some embodiments, the nucleic acid induces expression (e.g., functional expression) of Siglec-9 protein at a level that is greater than the level of expression of native Siglec-9 protein by the Treg cell. In some embodiments, Siglec-9 is overexpressed in the modified Tregcell. In some embodiments, the method includes contacting at least one membrane-bound Siglec-9 protein 160 with at least one target cell 170, including but not limited to a fibroblast cell, a cardiac fibroblast cell, or a myocardial fibroblast cell, wherein enhanced targeting of the target cell 170 is due to the specificity of Siglec-9160 for amineoxidase, copper containing 3 (AOC3) proteins 180 that decorate the membrane of the target cell 170. In some embodiments, the target cell expresses AOC3 at an elevated level compared to a non-target cell and / or a cell that has not been induced to upregulated fibrotic genes.

[0093] Also provided is a method of targeted immune modulation of a subject, comprising: obtaining a population of regulatory T cells (Treg); delivering at least one vehicle (e.g., delivery vehicle) to the population of Treg cells, wherein the at least one vehicle comprises a nucleic acid encoding sialic acid-binding immunoglobulin-type lectin-9 (Siglec- 9) protein and Siglec-9 is expressed by the nucleic acid in the Tregcells 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. Also provided is 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, thereby producing a population of enhanced Tregcells overexpressing Siglec-9; and administering to a subject the population of enhanced Treg cells. In some embodiments, the nucleic acid encodes siglec-9 protein that is expressed in the Tregcells. In some embodiments, the nucleic acid encodes an epigenetic modulator (e.g., a CRISPR activation system) that induces expression from an endogenous siglec-9 locus in the Treg cells.

[0094] In some embodiments, targeted immune modulation includes selective immunosuppression of any suitable target tissue that has upregulated expression of fibrotic genes (e.g., due to inflammation, tissue damage, etc.). In some embodiments, the tissue targeted by the enhanced Treg cells includes, without limitation, cardiac, pulmonary, hepatic, and / or colorectal tissue. In some embodiments, targeted immune modulation includes selective immunosuppression of cardiac tissue via the targeting of Treg cells to cardiomyocytes expressing AOC3 membrane-bound proteins.

[0095] In any method of the present disclosure, in some embodiments, the vehicle (or delivery vehicle) is any suitable vehicle for delivering the nucleic acid to the Treg cell. In some embodiments, the vehicle includes, without limitation, a liposome, a nanoparticle, a lipid nanoparticle (LNP), a viral vector, a cell-penetrating peptide (CPP), and / or anextracellular vesicle (EV). In some embodiments, the vehicle includes, a polyethylene glycol (PEG)-cationic lipid complex (PCLC). In some embodiments, the viral vector includes an adenoviral vector, retroviral vector, or a lentiviral vector. In some embodiments, the vehicle is a transfection reagent. In several embodiments, 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). In several embodiments, the transfection reagent comprises EV derived from cardiosphere-derived cells (CDC). In some embodiments, 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. In some embodiments, 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 Tregcells. 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). In some embodiments, the promoter is a regulatory T (Treg) cell-specific promoter. In some embodiments the Tregcell-specific promoter is selected from the group consisting of a Foxp3 promoter and an Interleukin-2 (IL-2) promoter. In some embodiments, 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 Tregcells to express (e.g., overexpress) siglec-9 protein, thereby producing engineered or enhanced Tregcells. The Tregcells 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. In some embodiments, 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. In some embodiments, delivering the nucleic acid to the Tregcell 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, contactingincludes providing a plurality of vehicles in a culture medium in which Treg cells are being or will be maintained. In some embodiments, 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. In some embodiments, vehicles can be provided in the culture medium at the start of culturing of the Treg cells. In some embodiments, vehicles can be added at regular intervals during culturing. In some embodiments, 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 Tregcells. In some embodiments, the method includes transducing the Treg cells with a viral vector that includes the nucleic acid encoding Siglec9. In some embodiments, the method includes transducing the Treg cells with a viral vector that includes the nucleic acid configured to induce expression of Siglec-9 protein in the Tregcells, as provided herein.

[0097] Contacting Treg cells with vehicles can be done for any suitable period of time. In some embodiments, the period of time for contacting can be the amount of time the Tregcells are cultured in the presence of vehicles in the culture medium. In some embodiments, the period of time for contacting is the amount of time the Treg cells are cultured in a medium to which vehicles are added. In some embodiments, 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.

[0098] In some embodiments, 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. In some embodiments, vehicles can be added at regular intervals during culturing.

[0099] Co-culturing vehicles with Treg cells can be done for a suitable period of time. In some embodiments, 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. In some embodiments,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. In some embodiments, 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.

[0100] In some embodiments, 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 Tregcells to vehicles provided in the culture medium can be any suitable ratio. In some embodiments, 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 Tregcells. In some embodiments, the vehicle provided at a ratio of Treg cells to vehicle particles of about 1:1000. In several embodiments, the ratio is about 1:100, about 1:200, about 1:300, about 1:400, about 1:500, about 1:600, about 1:700, about 1:800, about 1:900, about 1:1000, about 1000:1, about 900:1, about 800:1, about 700:1, about 600:1, about 500:1, about 400:1, about 300:1, about 200:1, about 100:1, or any ratio between those listed. In some embodiments, contacting involves contacting an effective amount of the delivery vehicle (e.g., viral vector) with the Tregcells at a suitable multiplicity of infection (MOI). In some embodiments, 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.).

[0101] In some embodiments, 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. In some embodiments, the method includes administering to the subject a vehicle comprising the nucleic acid encoding the Siglec-9 protein, wherein the vehicle is configured to deliverthe nucleic acid encoding the Siglec-9 protein to Treg cells in the subject. In some embodiments, the vehicle includes the nucleic acid encoding the Siglec-9 protein that specifically expresses Siglec-9 protein in Tregcells 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. In some embodiments, the nucleic acid encoding the Siglec-9 protein is operatively linked to a Tregcell-specific promoter (e.g., a Foxp3 or IL-2 promoter). In some embodiments, 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 Tregcell, wherein the vehicle is configured to deliver the nucleic acid to Treg cells in the subject. In some embodiments, 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. In some embodiments, the nucleic acid configured to specifically induce expression (e.g., endogenous or exogenous expression) of the Siglec-9 protein is operatively linked to a Tregcell-specific promoter (e.g., a Foxp3 or IL-2 promoter).

[0102] In some embodiments, the vehicle comprises: a lipid nanoparticle (LNP); and a Tregcell-targeting moiety associated with the LNP. In some embodiments, the Tregcell-targeting moiety includes an antigen binding protein that binds a Tregcell marker. 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. In some embodiments, the antigen binding protein is an antibody or antigen- binding fragment thereof that binds to the Treg cell marker. In some embodiments, the vehicle (e.g., LNP) is conjugated with the antibody or antigen-binding fragment thereof that binds to the Tregcell marker, to thereby target the vehicle to Tregcells in the subject.

[0103] In some embodiments, 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 Tregcells, 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. In some embodiments, the nucleic acid encoding the Siglec-9 protein is configured to overexpress the Siglec-9 protein in the Tregcells. In some embodiments, 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 Tregcells 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. In some embodiments, the engineered Treg cells and / or the vehicle containing the nucleic acid can be administered intravenously. In some embodiments, the engineered Tregcells 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 Tregcells 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. In some embodiments, the method includes administering to the subject about 105, 106, 107, 108, 109, 1010, 1011, 1012, or more of the engineered Tregcells, or a number of cells in a range defined by any two of the preceding values (e.g., about 105-1012cells, 106-1011cells, 107-1010cells, etc.). In some embodiments, the method includes administering to the subject about 103cells / kg, 104cells / kg, 105cells / kg, 106cells / kg, 107cells / kg, 108cells / kg, 109cells / kg, or 1010cells / 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 103-1010cells / kg, 104-109cells / kg, 105-108cells / kg, etc.). In some embodiments, the method includes administering to the subject the engineered Treg cells at 106-109cells / kg of body weight. In some embodiments, the method includes administering to the subject the engineered Treg cells at 106-108cells / kg of body weight. In some embodiments, the method includes administering to the subject the engineered Treg cells at about 107cells / 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). Insome embodiments, the therapeutically effective amount of the nucleic acid includes about 0.01 µg, 0.02 µg, 0.05 µg, 0.1 µg, 0.2 µg, 0.5 µg, 1 µg, 2 µg, 3 µg, 4 µg, 5 µg, 6 µg, 7 µg, 8 µg, 9 µg, 10 µg, 15 µg, 20 µg, 25 µg, 30 µg, 40 µg, 50 µg, 75 µg, 100 µg, 125 µg, 150 µg, 175 µg, 200 µg, 250 µg, 300 µg, 400 µg, 500 µg, 600 µg, 700 µg, 800 µg, 900 µg, 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 any two of the preceding values (e.g., 0.01 µg-0.1 µg, 0.1 µg-1 µg, 1 µg-10 µg, 10 µg-100 µg, 100 µg-1 mg, 1 mg-10mg, 10mg-100mg). In some embodiments, 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 / kg, 1 mg / kg-10 mg / kg, 10 mg / kg-100 mg / kg).

[0107] The Siglec-9 protein can be expressed in the engineered Tregcell 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 Tregcell that has not been modified (e.g., has not been modified to express or overexpress Siglec-9 protein as described herein). In some embodiments, expression of the Siglec-9 protein is increased by expression of the nucleic acid in the engineered Tregcell 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 Tregcell 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 engineered Treg cell by about 1.5-2.5 fold compared to a suitable reference (e.g., a Tregcell 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 Tregcell that has not been modified with the nucleic acid). In someembodiments, 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 Tregcell 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. In some embodiments, the control population of Treg cells are Treg cells that have not been contacted with or co-cultured with vehicles. In some embodiments, 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 Tregcells, e.g., Tregcells that have not been contacted or co-cultured with vehicles. In some embodiments, 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., Tregcells, 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., Tregcells, not contacted with or co-cultured with vehicles. In some embodiments, 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. In some embodiments, Siglec-9 protein expression by the engineered Tregcells 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. In some embodiments, theexpression level is determined using, without limitation, Western blot, ELISA, flow cytometry, fluorescence microscopy, etc.

[0108] Also provided is 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. In some embodiments, the nucleic acid encodes the membrane-bound fibroblast-specific binding protein. In some embodiments, 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. In some embodiments, 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 (Treg) cells in the subject. As used herein, “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 Tregcell with a fibroblast. In some embodiments, the protein construct is known to bind a fibroblast-specific cell-surface marker or target protein. In some embodiments, expression or overexpression of the membrane-bound fibroblast- specific binding protein by the regulatory T (Treg) cells in the subject promotes anti- inflammatory activity of the Tregcells 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. In some embodiments, the fibroblast-specific target protein is a gene or protein that is upregulated in the target tissue under inflammatory and / or fibrotic conditions. In some embodiments, the fibroblast-specific target protein is AOC3. In some embodiments, the membrane-bound fibroblast-specific binding protein comprises an amino acid sequence that is, is about, or is at least 75, 80, 85, 90, 92, 95, 97, 98, 99, or about 100%, identical to SEQ ID NO: 2 (with or without the signal peptide), or optionally the amino acid sequence is identical to SEQ ID NO:2 at a percentagein a range defined by any two of the preceding values (e.g., 75-100%, 80-99%, 85-95%, 87- 97%, etc.). In some embodiments, the membrane-bound fibroblast-specific binding protein is Siglec-9.

[0109] The target tissue can be any suitable tissue of the subject. In some embodiments, the target tissue is, without limitation, tissue of the heart, lung, brain, peripheral nervous system, liver, spleen, kidney, pancreas, bladder, muscle, stomach, intestine, colon, testes, prostate, ovary, uterus, skin. In some embodiments, the target tissue includes cardiac, pulmonary, hepatic, or colorectal tissue. In some embodiments, the tissue is cardiac tissue. In some embodiments, fibrotic genes is expressed (or is upregulated) in the target tissue. In some embodiments, the fibrotic gene expression in the target tissue is caused by (or is related to) an autoimmune response or disease. In some embodiments, the target tissue is cardiac tissue affected by the proliferation of cardiac fibroblast cells and / or myocardial fibroblast cells. In some embodiments, the target tissue is affected by a myocardial infarction. In some embodiments, the target tissue includes cells expressing one or more fibroblast-specific markers. In some embodiments, the target tissue has pathological expression (e.g., overexpression) of one or more fibroblast-specific markers. In some embodiments, the cells of the target tissue have elevated level of expression of one or more fibroblast-specific markers, compared to non-pathological or healthy tissue. In some embodiments, a larger fraction of cells of the target tissue express the one or more fibroblast- specific markers, compared to non-pathological or healthy tissue. In some embodiments, the target tissue has pathological expression (e.g., overexpression at the cellular level and / or expression by larger fraction of cells of the target tissue) of AOC3 membrane-bound proteins. In some embodiments, the target tissue is comprised of cells expressing (e.g., overexpressing) AOC3 membrane-bound proteins. In some embodiments, the target tissue includes cardiac myofibroblasts having pathological expression (e.g., overexpression at the cellular level and / or expression by larger fraction of cells of the target tissue) of AOC3. In some embodiments, the target tissue includes cardiac myofibroblasts expressing (e.g., overexpressing) AOC3. In some embodiments, the target tissue includes pulmonary myofibroblasts having pathological expression (e.g., overexpression at the cellular level and / or expression by larger fraction of cells of the target tissue) of AOC3. In some embodiments, the target tissue includes pulmonary myofibroblasts expressing (e.g.,overexpressing) AOC3. In some embodiments, the target tissue includes cells expressing (e.g., overexpressing) AOC3 and one or more other markers. In some embodiments, the target tissue includes colorectal myofibroblasts having pathological expression (e.g., overexpression at the cellular level and / or expression by larger fraction of cells of the target tissue) of AOC3 and NKX2-3. In some embodiments, the target tissue includes colorectal myofibroblasts expressing (e.g., overexpressing) AOC3 and NKX2-3.

[0110] In some embodiments, the fibroblast-specific target protein includes, without limitation, Collagen I, α-smooth muscle actin (α-SMA), and amine oxidase, copper containing 3 (AOC3). In some embodiments, the membrane-bound fibroblast-specific binding protein includes sialic acid-binding immunoglobulin-type lectin 9 (Siglec-9). In some embodiments, the condition associated with inflammation and / or fibrosis includes an inflammatory tissue (e.g., inflammatory cardiac tissue) containing fibroblasts (e.g., cardiac myofibroblasts) having elevated expression of amine oxidase, copper containing 3 (AOC3). AOC3 may be expressed by cells or a tissue targeted by the enhanced or modified Treg cells at any suitable level. In some embodiments, AOC3 is expressed in the target cell or tissue at or at about 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12 fold higher level, or more, or at a fold higher level of expression in a range defined by any two of the preceding values (e.g., 4-12 fold, 5-11 fold, 6-10 fold, 7-9 fold, etc.), compared to a suitable reference (e.g., a cardiac myofibroblast or cardiac tissue that is not fibrotic). In some embodiments, AOC3 is expressed in the target cell or tissue at about 9.0-9.5-fold higher (e.g., 9.3-fold higher) level compared to a suitable reference (e.g., a cardiac myofibroblast or cardiac tissue that is not fibrotic).

[0111] Also provided is a method of inhibiting fibrotic gene expression, comprising: obtaining a population of Treg cells; engineering the Treg cells to overexpress Siglec-9, thereby producing enhanced Tregcells; and administering the enhanced Tregcells (or an effective amount of the enhanced Treg cells) to a subject in need of inhibiting fibrotic gene expression in cardiac myofibroblasts. In some embodiments, the fibrotic gene is at least one of Collagen I, α-smooth muscle actin (α-SMA), and / or amine oxidase, copper containing 3 (AOC3). In some embodiments, the subject in need of inhibiting fibrotic gene expression in cardiac myofibroblasts is a subject in need of treatment for cardiac inflammation and / or cardiac fibrosis. In some embodiments, the cardiac inflammation and / or cardiac fibrosis issubsequent to a myocardial infarction. In some embodiments, the cardiac inflammation and / or cardiac fibrosis is associated with ICM. In some embodiments, the subject in need of inhibiting fibrotic gene expression in cardiac myofibroblasts is a subject in need of treatment myocardial infarction, hypertension, myocarditis, transplant rejection, cardiomyopathy, heart failure, and amyloidosis.

[0112] In some embodiments, engineering comprises delivering at least one vehicle comprising a nucleic acid configured to induce expression of Siglec-9 protein in the population of Treg cells. In some embodiments, engineering comprises delivering at least one vehicle comprising a nucleic acid encoding Siglec-9 protein to the population of Tregcells. . In some embodiments, engineering comprises delivering at least one vehicle comprising a nucleic acid encoding an epigenetic modulator (e.g., a CRISPR activation system) configured to induce expression of Siglec-9 protein from an endogenous locus of the Tregcells. In some embodiments, engineering comprises contacting an effective amount of the at least one vehicle with Treg cells under conditions sufficient to deliver the nucleic acid to the Treg cells. In some embodiments, 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). In some embodiments, overexpressing Siglec-9 comprises upregulating expression of mRNA encoding Siglec-9. In some embodiments, the subject is in need of treatment for fibrosis (e.g., cardiac fibrosis). In some embodiments, the subject is in need of treatment for cardiac fibrosis due to or subsequent to a myocardial infarction. In some embodiments, the subject is in need of treatment for cardiac fibrosis associated with ICM.

[0113] The present methods, as described below, can effectively address the production of fibroblast cells and myofibroblast cells caused by trauma to tissue, e.g., cardiac tissue. In some embodiments, cardiac tissue trauma can be caused by myocardial infarctions. In some embodiments, the proliferation of fibroblast cells and myofibroblast cells, e.g., cardiac myofibroblasts, can occur subsequent to or concurrent with a myocardial infarction. As used herein, 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. In some embodiments, the increased fibroblast cells and myofibroblast cells activitycan be a response to the damage caused by the myocardial infarction, potentially leading to reduced cardiac function or fibrosis.

[0114] A variety of Tregcells can be used in the methods and compositions of the present disclosure. In some embodiments, the Treg cells are human Treg cells. In some embodiments, Treg cells can be obtained from an individual, e.g., a donor (e.g., for allogeneic use), a patient (e.g., for autologous use). Any suitable option can be used to obtain Tregcells from an individual. Immune cells, e.g., Treg 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. In some embodiments, immune cells can be isolated as peripheral blood mononuclear cells (PBMC) from a blood sample obtained from the peripheral blood of a subject. Immune cells, e.g., Treg cells, may also be obtained from a unit of blood obtained from an apheresis or leukapheresis procedure.

[0115] In some embodiments, a population of Tregcells can be obtained by enriching a population of immune cells (e.g., immune cells from a peripheral blood sample from a subject) for Tregcells. Any suitable option for obtaining Tregcells, e.g., an enriched population of Tregcells can be used, e.g., sorting for Tregcells using one or more markers, as described herein. Any suitable option can be used to enrich the population of immune cells for Tregcells. In some embodiments, Tregcells can be isolated from the population of immune cells. In some embodiments, immune cells other than Tregcells (e.g., CD8+T cells) can be depleted from the population of immune cells. In some embodiments, 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. In some embodiments, Tregcells can be sorted based on expression of at least CD4 and CD25. The sorting can involve, without limitation, flow cytometry or magnetic sorting. In some embodiments, the Treg cells are CD4+CD25+. In some embodiments, Tregcells are CD4+CD25+CD127low. In some embodiments, the population can be enriched for CD4+FOXP3+cells. In some embodiments, 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.

[0116] As used herein the “+” indicates positivity for the given marker, which according to some embodiments is expression above a detectable threshold level for that marker. In several embodiments, the expression is upregulated as compared to a T cell not subjected to the methods disclosed herein.

[0117] 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. In some embodiments, 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. In some embodiments, 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).

[0118] In some embodiments, 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. When Tregcells overexpressing Siglec-9, i.e., enhanced Tregcells, are administered to a subject, such Treg cells can influence the behavior of fibroblast cells and myofibroblast cells in target tissues. In some embodiments, 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.

[0119] In some embodiments, the administration of enhanced Treg cells to a subject can reduce the proliferation of fibroblast cells and / or myofibroblast cells in the targettissue, which can be a response to tissue damage or stress. In some further embodiments, 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.

[0120] In some further embodiments, 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.

[0121] In some embodiments, 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. In some embodiments, 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.

[0122] In some embodiments, fibroblast proliferation can be related to an autoimmune response or disease. In some embodiments, 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 granulomatosis, Behçet’s disease, autoimmune uveitis, polymyositis, autoimmune lymphoproliferative syndrome, Churg-Strauss syndrome, mixed connective tissue disease, autoimmune pancreatitis, primary sclerosing cholangitis,autoimmune encephalitis. In some embodiments, the autoimmune disease can be a cardiac autoimmune disease, e.g., autoimmune myocarditis.

[0123] In some embodiments, administering to a patient the Tregcells, e.g., the enhanced Treg cells, according to methods of the present disclosure 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. In some embodiments, 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. In some embodiments, administering to a subject enhanced Treg cells, according to methods of the present disclosure, can restore or reduce damage to a target tissue. In some embodiments, administering to a patient enhanced Tregcells 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.

[0124] In some embodiments, the enhanced Treg cells, when administered to a subject with cardiac fibrosis can restore or improve cardiac function in the patient. In some embodiments, the cardiac function can be measured by ejection fraction, e.g., left ventricular ejection fraction. In some embodiments, cardiac function can be measured by the average cardiac function of a patient having cardiac fibrosis. In some embodiments, the ejection fraction 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 ejection fraction before administering the Tregcells.

[0125] In some embodiments, the present methods include treating conditions associated with inflammation and / or fibrosis. In some embodiments, the condition associated with inflammation and / or fibrosis includes, without limitation, inflammationand / or fibrosis of the heart, skeletal muscle, or skin. In some embodiments, the conditions associated with inflammation and / or fibrosis includes aging. In some embodiments, the conditions treated by the present treatment methods are a symptom and / or sequelae of an infection. In some embodiments, the inflammatory condition is a chronic condition.

[0126] In some embodiments, a treatment method of the present disclosure treats any one or more of a variety of inflammatory conditions. In some embodiments, the inflammatory condition is one that is responsive to the anti-inflammatory effect of IL-10. In some embodiments, 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)).

[0127] In some embodiments, the inflammation can be subsequent to or concurrent with an infectious disease. The inflammation can be related to a variety of infectious diseases. In some embodiments, the infectious disease is associated with myocardial injury. In some embodiments, the heart condition includes acute myocarditis associated with the infectious disease. In some embodiment, the inflammatory condition includes a cytokine storm, or hyperinflammation, associate with the infectious disease. In some embodiments, the inflammatory condition includes acute lung injury or acute respiratory distress syndrome (ARDS).

[0128] In some embodiments, 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). In some embodiments, 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.

[0129] In some embodiments, the inflammation is subsequent to or concurrent with an infection by a virus, e.g., a DNA or RNA virus. In some embodiments, the virus is an RNA virus, e.g., a single or double-stranded virus. In some embodiments, the RNA virus is a positive sense, single-stranded RNA virus. In some embodiments, the virus belongs to the Nidovirales order. In some embodiments, the virus belongs to the Coronaviridae family. In some embodiments, the virus belongs to the alphacoronavirus, betacoronavirus, gammacoronavirus or deltacoronavirus genus. In some embodiments, the alphacoronavirus is, without limitation, human coronavirus 229E, human coronavirus NL63 or transmissible gastroenteritis virus (TGEV). In some embodiments, 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. In some embodiments, the gammacoronavirus is infectious bronchitis virus (IBV).

[0130] In some embodiments, the inflammation and / or fibrosis is related to an autoimmune disease, graft-versus-host disease (GVHD) or an organ transplant. In some embodiments, 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, rheumatoid arthritis, sarcoidosis, scleroma, spondyloarthropathy, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia and ulcerative colitis. In some embodiments, the autoimmune disease is scleroderma or systemic sclerosis.

[0131] In some embodiments, the inflammation is related to a bone marrow transplantation. In some embodiments, the inflammation is related to allograft rejection following tissue transplantation. In some embodiments, the inflammation is related to cardiomyopathy, heart failure and / or amyloidosis. In some embodiments, the autoimmune disease is a cardiac autoimmune disease, e.g., autoimmune myocarditis. Non-limitingexamples 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)). COMPOSITIONS

[0132] Provided herein are compositions suitable for use in a method of treating a subject, as described herein. Provided is a nucleic acid (e.g., an isolated nucleic acid) comprising 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 Tregcell when the nucleic acid is incorporated into the cell (e.g., as described herein). In some embodiments, the promoter is a regulatory T (Treg) cell-specific promoter. In some embodiments the Treg cell-specific promoter is selected from the group consisting of a Foxp3 promoter and an Interleukin-2 (IL-2) promoter. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is a conditional promoter. Any suitable conditional promoter can be used. In some embodiments, the conditional promoter is a chemically inducible promoter. In some embodiments, the conditional promoter includes a Tet-responsive promoter.

[0133] The composition includes any suitable amount of the nucleic acid. In some embodiments, 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). In some embodiments, the composition includes, includes about, or includes at least 0.01 µg, 0.02 µg, 0.05 µg, 0.1 µg, 0.2 µg, 0.5 µg, 1 µg, 2 µg, 3 µg, 4 µg, 5 µg, 6 µg, 7 µg, 8 µg, 9 µg, 10 µg, 15 µg, 20 µg, 25 µg, 30 µg, 40 µg, 50 µg, 75 µg, 100 µg, 125 µg, 150 µg, 175 µg, 200 µg, 250 µg, 300 µg, 400 µg, 500 µg, 600 µg, 700 µg, 800 µg, 900 µg, 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 any two of the preceding values (e.g., 0.01 µg-0.1 µg, 0.1 µg-1 µg, 1 µg-10 µg, 10 µg-100 µg, 100 µg-1 mg, 1 mg-10mg, 10mg-100mg) of the nucleic acid. In some embodiments, the composition includes, includes about, or includes at least 0.01 µg / mL, 0.02 µg / mL, 0.05 µg / mL, 0.1µg / mL, 0.2 µg / mL, 0.5 µg / mL, 1 µg / mL, 2 µg / mL, 3 µg / mL, 4 µg / mL, 5 µg / mL, 6 µg / mL, 7 µg / mL, 8 µg / mL, 9 µg / mL, 10 µg / mL, 15 µg / mL, 20 µg / mL, 25 µg / mL, 30 µg / mL, 40 µg / mL, 50 µg / mL, 75 µg / mL, 100 µg / mL, 125 µg / mL, 150 µg / mL, 175 µg / mL, 200 µg / mL, 250 µg / mL, 300 µg / mL, 400 µg / mL, 500 µg / mL, 600 µg / mL, 700 µg / mL, 800 µg / mL, 900 µg / mL, 1 mg / mL, or an amount in a range defined by any two of the preceding values (e.g., 0.01 µg / mL-1 mg / mL, 0.05-100 µg / mL, 0.1-10 µg / mL, 0.02-200 µg / mL, etc.) of the nucleic acid.

[0134] Also provided is a vector (e.g., a plasmid, a viral vector) that includes the nucleic acid (with or without the promoter) that encodes the Siglec-9 protein (e.g., having the sequence of SEQ ID NO:2).

[0135] Also provided is a regulatory T (Treg) cell (e.g., an isolated Treg cell) that includes the nucleic acid and / or the vector. In some embodiments, the Tregcell 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.

[0136] Also provided is a composition that includes a population of enhanced (or engineered) Tregcells overexpressing sialic acid-binding immunoglobulin-like lectin 9 (Siglec-9). In some embodiments, Siglec-9 protein expression by the enhanced or engineered Tregcells 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., Tregcells that have not been engineered to overexpress siglec-9 protein. In some embodiments, 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. In some embodiments, Siglec-9 expression by the enhanced or engineered Tregcells 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. In some embodiments, Siglec-9 expression by the enhanced or engineered Tregcells is 4-6 fold higher as compared to a suitable reference, e.g., Tregcells that have not been engineered to overexpress siglec-9 protein. In some embodiments, Siglec- 9 expression by the enhanced or engineered Treg cells is about 5-fold higher as compared to a suitable reference, e.g., Tregcells that have not been engineered to overexpress siglec-9protein. In some embodiments, 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. In some embodiments, the enhanced Tregcells have endocytosed vehicles that include a plurality of nucleic acids encoding sialic acid-binding immunoglobulin-like lectin 9 (Siglec-9). In some embodiments, 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). In some embodiments, 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). In some embodiments, the plurality of nucleic acids comprises a plurality of mRNA transcripts encoding Siglec-9.

[0137] In some embodiments, the enhanced Tregcells exhibit Siglec-9-dependent interaction with cells expressing amine oxidase, copper containing 3 (AOC3). In some embodiments, the interaction with AOC-3-expressing cells mediated by Siglec-9 expressed by the enhanced Tregcells is a functional interaction (e.g., one that allows the enhanced Tregcells to suppress the immune response in the tissue that contains the AOC-3-expressing cells).

[0138] The composition can include any suitable number of the enhanced Tregcells. In some embodiments, 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). In some embodiments, the composition includes, includes at least, or includes about 103, 104, 105, 106, 107, 108, 109, 1010, 1011, 1012, or a number in a range defined by two of the preceding values (e.g., about 103-1012, 104-1011, 105-1010, 106-109, etc.) enhanced Treg cells. The composition can include any suitable concentration of the enhanced Tregcells. In some embodiments, the composition includes, includes at least, or includes about 103 / mL, 104 / mL, 105 / mL, 106 / mL, 107 / mL, 108 / mL, 109 / mL, 1010 / mL, 1011 / mL, 1012 / mL, or a concentration in a range defined by two of the preceding values (e.g., about 103-1012 / mL, 104-1011 / mL, 105-1010 / mL, 106-109 / mL, etc.) enhanced Treg cells. In some embodiments, the composition includes a substantially pure population of the enhanced Treg cells, compared to other cells.

[0139] Also provided is 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 Treg cells. In some embodiments, the vehicle includes a lipid nanoparticle (LNP). In some embodiments, the vehicle is a transfection reagent (e.g., liposome, etc.). In some embodiments, the Tregcell-targeting moiety comprises an antigen binding protein that binds a Treg cell marker. In some embodiments, the antigen binding protein is selected from: anti-CD25 monoclonal antibodies and / or anti-Foxp3 monoclonal antibodies. In some embodiments, the vehicle is a viral vector.

[0140] In some embodiments, the composition is a pharmaceutical or therapeutic composition. In some embodiments the composition includes pharmaceutically acceptable excipient. In some embodiments, the composition is a cell-free composition, e.g., the composition is substantially free of cells such as Treg cells. Some non-limiting examples of 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 oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer’s solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and / or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids (23) serum component, such as serum albumin, HDL and LDL; (22) C2-C12 alcohols, such as ethanol; and (23) other non-toxic compatible substances employed in pharmaceutical formulations. Additional Embodiments

[0141] Further, non-limiting embodiments of the present disclosure are provided below.

[0142] In some embodiments, 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. In some embodiments, Treg cells overexpressing Siglec-9 specifically target AOC3-expressing fibroblasts, leading to the inhibition of cardiac fibrosis. In some embodiments, the approach is generalizable to target fibrosis in other organs.

[0143] 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. The crux of the problem lies in the lack of targeted therapies that can modulate the immune response in a manner that specifically prevents or reduces fibrosis without inducing broad, non-specific immunosuppression. 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.

[0144] In some embodiments, provided herein is a targeted and effective solution to the problem of cardiac fibrosis and its associated conditions. In some embodiments, 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. In some embodiments, 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 theimmune 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. Broad Therapeutic Potential: The present methods and compositions, while primarily focused on MI, can be applied to other fibrosis-associated diseases.

[0148] In some embodiments, 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 α-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 superior anti-fibrotic effects of engineered Tregs with OE-Siglec-9 on cardiac myofibroblasts via AOC3 interaction.

[0153] 5. Observations of upregulation of AOC3 and fibrotic genes post-MI in vivo.

[0154] 6. The heightened efficacy of Siglec-9 engineered Tregs in downregulating fibrotic gene expression post-MI compared to traditional Tregs.

[0155] In some embodiments, 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.

[0158] 3. Broad Therapeutic Potential: Beyond just MI, the present disclosure includes treating other fibrosis-associated diseases.

[0159] 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 (Treg) cells in the subject. 2. The method of embodiment 1, wherein the subject is in need of cardiac fibrosis treatment. 3. A method of treating a condition associated with inflammation and / or fibrosis, comprising 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 (Treg) cells in the subject. 3a. 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. 4. The method of embodiment 3 or 3a, wherein the condition associated with inflammation and / or fibrosis comprises cardiac inflammation and / or cardiac fibrosis. 5. 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. The method of any one of embodiments 3-5, wherein the condition associated with inflammation and / or fibrosis is an angiotensin II-induced condition. 7. The method of any one of the preceding embodiments, wherein 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.8. The method of embodiment 7, wherein 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. 9. 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. 10. The method of embodiment 8 or 9, wherein the viral vector comprises an adenoviral vector, retroviral vector, or a lentiviral vector. 11. The method of any one of embodiments 7-10, wherein the population of Treg cells are derived from cells autologous to the subject. 12. The method of any one of embodiments 7-10, wherein the population of Treg cells are derived from cells allogeneic to the subject. 13. The method of 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 Tregcells in the subject. 14. The method of embodiment 13, wherein the vehicle comprises: a lipid nanoparticle (LNP); and a Treg cell-targeting moiety associated with the LNP. 15. The method of embodiment 13 or 14, wherein the Treg cell-targeting moiety comprises an antigen binding protein that binds a Treg cell marker. 16. The method of embodiment 15, wherein the Treg cell marker is selected from: CD25 and / or Foxp3. 17. The method of embodiment 15 or 16, wherein the antigen binding protein is an antibody. 18. The method of any one of embodiments 15-17, wherein the antigen binding protein is selected from: an anti-CD25 monoclonal antibody and / or anti-Foxp3 monoclonal antibody. 19. The method of any one of embodiments 12-17, wherein the nucleic acid comprises mRNA encoding the Siglec-9 protein. 20. The method of any one of the preceding embodiments, wherein the subject has suffered or is at risk of suffering from myocardial infarction.21. The method of any one of the preceding embodiments, wherein the subject is in need of treating angiotensin II-induced cardiac fibrosis. 22. The method of any one of the preceding embodiments, wherein the subject is in need of treating cardiac fibrosis subsequent to a myocardial infarction. 23. The method of any one of the preceding embodiments, wherein the Siglec-9 protein comprises an amino acid sequence at least 80% identical to SEQ ID NO: 2. 24. 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. 24a. 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. 25. The method of embodiment 24 or 24a, wherein the membrane-bound fibroblast-specific binding protein comprises sialic acid-binding immunoglobulin-type lectin 9 (Siglec-9). 26. The method of embodiment 24 or 24a, wherein the membrane-bound fibroblast-specific binding protein comprises an amino acid sequence at least 80% identical to SEQ ID NO:2. 27. The method of any one of embodiments 24-26, wherein the condition associated with inflammation and / or fibrosis comprises an inflammatory tissue comprising fibroblasts having elevated expression of amine oxidase, copper containing 3 (AOC3). 28. The method of embodiment 27, wherein the fibroblasts having elevated expression of AOC3 comprises myofibroblasts. 29. The method of embodiment 28, wherein the myofibroblasts comprises cardiac myofibroblasts. 30. The method of any one of embodiments 24-29, further comprising: obtaining a population of immune cells comprising regulatory T (Treg) cells;delivering the nucleic acid or 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 Tregcell; 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. 31. 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 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 Treg cells. 31a. 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 Tregcells of the population, thereby producing a population of enhanced Treg cells overexpressing Siglec-9; and administering to a subject the population of enhanced Tregcells, optionally wherein the nucleic acid encodes the Siglec-9 protein. 32. The method of embodiment 31 or 31a, wherein the at least one vehicle comprises a liposome, or a lipid nanoparticle (LNP). 33. The method of embodiment 31 or 31a, wherein the at least one vehicle comprises a viral vector. 34. The method of embodiment 33, wherein the viral vector comprises an adenoviral vector, retroviral vector, or a lentiviral vector. 35. The method of any one of embodiments 31-34, wherein the nucleic acid comprises a promoter operatively linked to a nucleotide sequence encoding the Siglec9, wherein the promoter upregulates expression of Siglec9 in Tregcells.36. The method of any one of embodiments 31-35, wherein 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 Tregcells; engineering the Treg cells to overexpress Siglec-9, thereby producing enhanced Treg cells; and administering the enhanced Tregcells to a subject in need of inhibiting fibrotic gene expression. 42. The method of embodiment 41, wherein the fibrotic gene is at least one of Collagen I, α-smooth muscle actin (α-SMA), and / or amine oxidase, copper containing 3 (AOC3). 43. The method of embodiment 41 or 42, wherein engineering comprises: delivering at least one vehicle comprising a nucleic acid encoding Siglec-9 to the population of Tregcells, 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 method of embodiment 43, wherein 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). 45. The method of embodiment 43 or 44, wherein the nucleic acid comprises mRNA. 46. The method of embodiment 45, wherein overexpressing Siglec-9 comprises upregulating expression of the mRNA.47. The method of any one of embodiments 41-46, wherein the subject is in need of treatment for fibrosis. 48. The method of embodiment 47, wherein the fibrosis is a cardiac fibrosis. 49. The method of embodiment 48, wherein the cardiac fibrosis is subsequent to a myocardial infarction. 50. The method of any one of preceding embodiments, wherein the subject has upregulated expression of amine oxidase, copper containing 3 (AOC3) in cardiac myofibroblasts. 51. The method of any one of preceding embodiments, wherein the administering comprises intravenous or oral administration. 52. An isolated nucleic acid comprising 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. 53. The isolated nucleic acid of embodiment 52, wherein 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. 54. A vector comprising the nucleic acid of embodiment 52 or 53. 55. A regulatory T (Treg) cell comprising the isolated nucleic acid of embodiment 52 or 53, or the vector of embodiment 54, optionally wherein the Tregcell is a human Tregcell. 56. 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 Tregcells, optionally wherein the Tregcells are human Tregcells. 57. The composition of embodiment 56, wherein vehicles of the population comprise a lipid nanoparticle (LNP). 58. The composition of embodiment 56 or 57, wherein the nucleic acid comprises mRNA. 59. The composition of any one of embodiments 56-58, wherein the Treg cell- targeting moiety comprises an antigen binding protein that binds a Tregcell marker.60. The composition of embodiment 59, wherein the Treg cell marker is selected from: CD25 and / or Foxp3. 61. The composition of embodiment 59 or 60, wherein the antigen binding protein is an antibody. 62. The composition of any one of embodiments 59-61, wherein the antigen binding protein is selected from: an anti-CD25 monoclonal antibody and / or anti-Foxp3 monoclonal antibody. 63. 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. 64. The composition of embodiment 63, wherein 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). 65. The composition of embodiment 64, wherein 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). 66. The composition of embodiment 64 or 65, wherein the plurality of nucleic acids comprises a plurality of mRNA transcripts encoding Siglec-9. 67. The composition of any one of embodiments 63-66, wherein the enhanced Tregcells exhibit Siglec-9-dependent interaction with cells expressing amine oxidase, copper containing 3 (AOC3). 68. The composition of embodiment 67, wherein the enhanced Tregcells exhibit Siglec-9-dependent interaction with cardiac myofibroblasts expressing AOC3. 69. The composition of any one of embodiments 56-68 for the treatment of a condition associated with inflammation and / or fibrosis in a subject in need thereof. 70. Use of the Treg cells of embodiment 55 or the composition of any one of embodiments 56-68 for the treatment of a condition associated with inflammation and / or fibrosis in a subject in need thereof. 71. Use of the 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.

[0160] The technology described herein is further illustrated by the following examples, which in no way should be construed as being further limiting. EXAMPLES Example 1:

[0161] This non-limiting example shows that Angiotensin II induces fibrotic genes in primary mouse cardiac fibroblast. Angiotensin II dose-dependently induces upregulation of fibrotic genes in mouse cardiac fibroblastadministered to isolated primary mouse cardiac fibroblast (MCF) cells at different concentrations. The expression levels of fibrosis-associated genes, specifically Collagen I and α-SMA, were then measured over time in response to 10 μM administration of Angiotensin II to MCF cells.

[0163] 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 μM and 10 μM, were administered to MCF cells. A control treatment is also shown, where no Angiotensin II was administered to MCF cells. After culturing MCF cells for 24 hours, mRNA was isolated from MCF cells to quantify the fold increase in mRNA transcripts for Collagen I 210 and α- 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 α-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 cardiacupregulation of fibrotic genes in mouse cardiac cells, primary mouse cardiac fibroblast (MCF) cells were exposed to Angiotensin II over time. The expression levels of fibrosis-associated genes, specifically Collagen I and α-SMA, were then measured to assess their correlation with increasing concentrations of Angiotensin II.

[0165] FIG. 3 illustrates a graphical representation 300 of data obtained from in vitro experiments of the effect of 10 μM 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. At each time point, mRNA was isolated from MCF cells to quantify the fold increase in mRNA transcripts for Collagen I 310 and α-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 α-SMA 320 over time when 10 μM Angiotensin II was administered to the MCF cells. Angiotensin II does- and time-dependently induces expression of AOC3 in mouse cardiac fibroblast

[0166] To determine whether Angiotensin II induces a dose-dependent and time- dependent upregulation of AOC3 in mouse cardiac cells, primary mouse cardiac fibroblast (MCF) cells were isolated and exposed to Angiotensin II. The expression level of AOC3 was then measured per dosage or over time in response to administration of Angiotensin II to MCF cells.

[0167] 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 μM and 10 μM, 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 μM Angiotensin II to MCF cells. A control treatment (Ctrl) is also shown, where no Angiotensin II was administered to MCF cells. In both experiments, 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.

[0168] To confirm the positive correlation between AOC3 upregulation the presence of Angiotensin II, a western blot assay 430 was performed. The results of a westernblot 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 β actin (Act). Example 2:

[0169] This non-limiting example 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 Tregtargeting of AOC3-expressing cardiac fibroblasts.

[0170] To determine whether Siglec-9 binding to AOC3 facilitates Treg targeting of AOC3-expressing cardiac fibroblasts, enhanced Tregcells 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 ltcqvtfpga svttnktvhl nvsyppqnlt 241 mtvfqgdgtv stvlgngssl slpegqslrl vcavdavdsn pparlslswr gltlcpsqps 301 npgvlelpwv hlrdaaeftc raqnplgsqq vylnvslqsk atsgvtqgvv ggagatalvf 361 lsfcvifvvv rscrkksarp aagvgdtgie danavrgsas qgpltepwae dsppdqpppa 421 sarssvgege lqyaslsfqm vkpwdsrgqe atdteyseik ihr (SEQ ID NO:2)

[0171] Mouse Treg cells were obtained and transduced with the lentiviral vector to generate enhanced Tregcells. 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 β actin control. A 5-fold increase in expression levels was confirmed by Western blot (FIG. 26).

[0172] Regulatory T cells (Treg) were obtained from a mouse spleen using the CD4+CD25+ Treg cell isolation kit (Cat. No.130-091-041, from MILTENYI BIOTEC™). A spleen was placed in a sterile dish with phosphate-buffered saline (PBS) + 1 mM EDTA. Any excess tissue was removed and the spleen was minced using a syringe plunger to release splenocytes. Next, a single-cell suspension was prepared by passing the minced tissue through a 70 µm 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 µm nylon mesh to remove any clumps. The total number of cells was determined, followed by resuspension of the cell pellet in 40 µL of buffer per 10⁷ cells. The cells were labelled by adding 10 µL of CD4+CD25+CD127 dim / – T Cell Biotin-Antibody Cocktail II per 10⁷ cells. The solution was thoroughly mixed and incubated for 10 minutes at 2°C to 8 °C. Next, 10 µL of buffer, 20 µL of Anti-Biotin MicroBeads, and 20 µL of CD20 MicroBeads per 10⁷ total cells, was added to the solution, followed by mixing and incubation for another 15 minutes at 2°C to 8 °C. The cells were then washed by adding 1 mL to 2 mL of buffer per 10⁷ cells and centrifuged at 300 × g for 10 minutes. The supernatant was then completely aspirated and resuspended such that there were approximately 10⁸ cells in 500 µL 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.

[0174] For positive selection of CD4+CD25+CD127 dim / – Treg cells, an MS Column was used. The column was rinse with 500 µL of buffer, the cell suspension applied, and the flow-through collected. The column was washed with 3×500 µL 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 skillin 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 Tregcells for further analysis or experimentation.

[0176] FIG.6 illustrates an immunoprecipitation assay 600 demonstrating Siglec- 9 expressed by Treg cells binding to AOC3 expressed by cardiac fibroblasts (CFs). Enhanced Tregcells were co-cultured with cardiac fibroblasts overexpressing AOC3. Following co- culturing, 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. For example, 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 β actin proteins 640 remained consistent.

[0177] To determine whether Siglec-9 binding to AOC3 facilitates Tregtargeting of AOC3-expressing cardiac fibroblasts, a cell adhesion assay was performed. FIG. 7 illustrates a cell adhesion assay 700 for studying the role of Siglec-9 and AOC3 interactions in enhanced Tregcells. 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 Tregcells only expressed GFP. The enhanced Tregcells expressing GFP (or control Tregcells) were spread onto the surface of the MCF monolayer (with or without exposure to Ang II) 740. Next, 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.

[0178] Exposure of MCF cells to Ang II increased the amount of control Treg cells bound to the MCF monolayer. A greater number of enhanced Treg cells bound to the Ang II- exposed MCF monolayer compared the number of control Tregcells bound to the Ang II- exposed MCF monolayer. These results support a model for Siglec-9-expressed on Treg cells facilitating Treg cell targeting of AOC3-expressing cardiac fibroblasts.Overexpression of Siglec-9 in Treg cells enhances inhibition of fibrotic gene expression in cardiac fibroblasts

[0179] To determine whether overexpression of Siglec-9 in Tregcells enhances inhibition of fibrotic gene expression in cardiac fibroblasts enhanced Treg cells were co- cultured with cardiac fibroblasts. Following co-culturing, cells were lysed and mRNA collected.

[0180] 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 α-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 α-SMA by Ang II. Overexpressing Siglec-9 in Treg cells enhanced the suppression of fibrotic gene expression by the Treg cells. Example 3:

[0181] This non-limiting example shows the role of Siglec-9 interactions with AOC3 in the engineered Tregcell-mediated anti-fibrotic effects on cardiac fibroblasts.

[0182] 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 Tregcells.

[0183] 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. As described above, a MCF monolayer was prepared and exposed to an Angiotensin II solution 920 to induce MCF fibrosis. Control MCF cells were not exposed to Ang II. Mouse 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. Next, Treg cells were allowed to adhere to the MCF monolayer 950. The MCF monolayer was washed to remove any unbound cells 960. Theamount of cells bound to the MCF monolayer was determined by fluorescent microscopy 970.

[0184] Anti-Siglec-9 antibody treatment of the Tregcells abolished the increased binding of enhanced Treg cells to Ang II-exposed MCF monolayer, whereas the IgG control had no effect. Thus, targeting of enhanced Treg cells to AOC3-expressing cardiac fibroblasts was dependent on Siglec-9 binding.

[0185] 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 α- SMA 1020), compared to IgG control. Thus, targeting of enhanced Treg cells to AOC3- expressing cardiac fibroblasts is dependent on Siglec-9 binding.

[0186] Cell adhesion assays 1100 and co-culturing experiments were performed using an anti-AOC3 antibody to treat the MCF monolayer (with or without exposure to Ang II) before combining with the Tregcells (FIGs. 11 and 12). A MCF monolayer was prepared and exposed to an Angiotensin II solution 1120 to induce MCF fibrosis. Control MCF cells were not exposed to Ang II. The Ang II-treated or control MCF cells were then treated with either IgG control or an anti-AOC3 antibody (“anti-AOC3”). Enhanced Tregcells overexpressed Siglec-9 and were engineered to express GFP 1130. The enhanced Tregcells expressing GFP were spread onto the surface of the MCF monolayer (with or without exposure to Ang II, treated with anti-AOC3 antibody or IgG) 1140. Next, Tregcells were allowed to adhere to the MCF monolayer 1150. The MCF monolayer was washed to remove any unbound cells 1160. The amount of cells bound to the MCF monolayer was determined by fluorescent microscopy 1170.

[0187] Similar to blocking Siglec-9, the anti-AOC3 antibody treatment of the MCF monolayer abolished the increased binding of enhanced Treg cells to the Ang II-exposed MCF monolayer, whereas the IgG control had no effect. Thus, targeting of enhanced Treg cells to AOC3-expressing cardiac fibroblasts was dependent on AOC3 binding.

[0188] FIG.12 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-AOC3 antibody reduced the inhibitory effect ofthe enhanced Treg cells on expression of fibrotic gene expression (Collagen I 1210 and α- SMA 1220), compared to IgG control. Thus, targeting of enhanced Treg cells to AOC3- expressing cardiac fibroblasts is dependent on AOC3 binding.

[0189] These results indicate that targeting of enhanced Treg cells to AOC3- expressing cardiac fibroblasts was dependent on Siglec-9 and AOC3 interactions. Example 4:

[0190] This non-limiting example 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 Tregcells

[0191] To determine whether enhanced Treg cells engineered to overexpress Siglec-9 show superior ability to downregulate fibrotic gene expression post-myocardial infarction as compared to normal Tregcells, gene expression levels were quantified using qPCR and western blot assays.

[0192] 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. For fibrosis-associated genes AOC31310, Collagen I 1320, and α- 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 (β-Actin). 1340 illustrates that post-myocardial infarction, fibrosis- gene expression increases over time.

[0193] FIG.14 illustrates the experimental design 1400 and data of the enhanced efficacy of enhanced Tregcells in downregulating in vivo fibrotic gene expression post- myocardial infarction compared to normal Treg cells. The effect of Treg cells when administered in vivo over a time course of 21 days was studied. 2 x 105per dose of control Tregcells (not over-expressing Siglec-9) or enhanced Tregcells (OE-sig9-Treg) wereadministered to animals post-myocardial infarction, every 7 days for a total of three doses. Another set of animals were administered vehicle control (no Treg cells). After 21 days, heart tissue was collected and western blot assays and qPCR were performed to analyze the expression level fibrosis-associated genes.

[0194] Gene expression was measured as fold change relative to a control. The expression level of four genes, AOC31430, Collagen I 1440, α-SMA 1450, and Foxp31460, were tested in the presence of a vehicle, normal Treg cells, and enhanced Treg cells (Sig-Treg) after 21 days. Fibrosis-associated genes, Collagen I 1440 and α-SMA 1450, demonstrate dramatically decreased expression levels post-myocardial infarction and in the presence of enhanced Treg cells. 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 Tregcells, 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 Tregcells in downregulating fibrosis-associated genes, e.g., Collagen I and α-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. 15 illustrates an in vivo protocol 1500 for studying the in vivo anti- fibrotic effect of enhanced Tregcells. A subject, such as a mouse, is infused with Angiotensin II at 1 mg kg-1day-1(Day 0). Enhanced Treg cells are administered to the subject by infusion on Days 3, 7, and 14. On Day 21, one or more of the following is assessed: Treg cell infiltration / accumulation in the injured myocardium; Siglec9-Tregtargeting 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 (ΔEF); area of fibrosis (PS red)).Example 6:

[0197] This non-limiting example shows in vivo therapeutic effects of enhanced Tregcells on cardiac fibrosis induced by myocardial infarction.

[0198] 2 x 105per dose of enhanced Treg cells (OE-sig9-Treg) (or control Treg cells (not over-expressing Siglec-9)) are administered to animals post-myocardial infarction, every 7 days for a total of three doses. Another set of animals are administered vehicle control (no Treg cells). After 21 days, one or more of the following is assessed: Treg cell infiltration / accumulation in the injured myocardium; Siglec9-Treg targeting to AOC3+ myofibroblast in the heart; expression level of IL-10 / Foxp3 in Tregcells; expression of fibrotic genes and proteins by myofibroblasts in the heart; cardiac function and pathology (e.g., ejection fraction (ΔEF); area of fibrosis (PS red)). Example 7:

[0199] This non-limiting example provides a model for Siglec-9-mediated enhancement of Tregfunction. Siglec-9 may promote Tregfunction by increasing release of interleukin 10 (IL-10)

[0200] FIG. 16 illustrates an exemplary embodiment of the cellular processes 1600 by which Siglec-9 promotes Tregcell function by affecting immune cell signaling. In some embodiments, 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. Activation of SHP-1 may lead to expression of cytokines, e.g., interleukin 10 (IL-10). The SHP-1-Siglec-9 protein complex may promote enhanced Treg function by increasing the release of IL-10. IL-10 may promote Tregfunction, 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.

[0202] 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. 18, blocking siglec-9 with a neutralizing antibody significantly reduces the myofibroblast contraction 1820 in the collagen contraction assay 1810 emphasizing the critical role of siglec-9 in mediating these effects. Additionally, FIG. 19 shows that neutralizing AOC3 similarly diminishes myofibroblast contraction 1920 in the collagen contraction assay 1910 corroborating the importance of the siglec-9-AOC3 interaction in targeting and attenuating fibrotic processes. 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.

[0204] In vivo studies substantiated the targeted anti-fibrotic potential of siglec-9 engineered Tregcells in a MI model, using left anterior descending (LAD) artery ligation (FIG. 20). I.V. injected siglec-9 engineered Treg cells, identifiable by their RFP tag, selectively localized with AOC3+ cells in the infarct zone (FIG. 21), indicating targeted engagement of engineered Tregcells with AOC3-expressing myofibroblasts in the injured heart. Quantitative PCR (FIG.22) further supports that AOC32210 is upregulated in MI, and that siglec-9 engineered Tregcells are the most effective in reducing fibrotic gene expression, 2220, 2230.

[0205] The functional impact of the siglec-9 engineered Treg cells on heart tissue remodeling and recovery was explored. Shown in FIG. 23, Picrosirius red staining of heart tissue 2310 showed a significant reduction in the fibrotic area in MI hearts treated with both types of Treg cells compared to the vehicle group. Notably, hearts treated with siglec-9 engineered Treg cells exhibited a greater reduction in fibrosis 2320, highlighting their superior efficacy in mitigating fibrotic tissue deposition.

[0206] Echocardiographic assessments provide quantitative evidence of improved cardiac function. Data in FIG.24 indicate that siglec-9 engineered Treg cells more effectively enhanced the ejection fraction 2410, with better preservation of heart function post-MIcompared to non-engineered Treg cells. These findings suggest that beyond their role in targeting and modulating fibrotic pathways, 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 Tregcells 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 α-SMA, both in vitro and in vivo. Adhesion assays have confirmed that siglec-9 engineered Treg cells have a significantly enhanced capacity to adhere to fibrotic regions, predominantly driven by their interaction with AOC3. Blocking this interaction diminishes Tregcells’ adhesion and fibrotic gene suppression capabilities, underscoring the essential role of the siglec-9-AOC3 interaction in mediating these effects. Additionally, in vivo experiments using a mouse MI model demonstrate that these engineered Tregcells not only reduce fibrosis but also contribute to improved cardiac function, as evidenced by enhanced left ventricular ejection fraction (LVEF). Example 10

[0209] This non-limiting example shows characterization of the functional impact and molecular mechanisms of siglec-9 overexpression in Tregcells under AOC3 (myofibroblast marker) stimulation.

[0210] The influence of overexpressing siglec-9 in Treg cells on the cells’ reaction to AOC3, a marker of myofibroblasts, is assessed.

[0211] Human Cells: Human Tregcells (hTregs) purchased from IQ Biosciences (Cat # IQB-Hu1-iTr-1), are obtained from normal volunteers participating in an IRB- approved donor program. The vendor provides certificates attesting to purity and identity by flow cytometry (90% CD4+CD25+(90%CD127, 85%% FOXP3+)), and negative assays for HIV, HCV, and Hepatitis B.

[0212] Vectors: 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-viralplasmid transfection of siglec-9 in target cells (e.g., FIGs. 5, 7). Alternatively, 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. (cat# LV590, 43776061-GFP & C129-RFP, respectively). Testing the effect of siglec-9 protein overexpression on the functional regulation of hTregs under stimulation by the myofibroblast marker AOC3, focusing on IL-10 production, Treg cell lineage stabilization, migration, and suppression assays

[0213] The effect of siglec-9 overexpression on critical Tregcell functions is investigated using the following assays.

[0214] a) IL-10 Production: 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; b) 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 Tregcells and control Treg cells post-AOC3 stimulation; c) Treg cell Migration Assay: Migration towards AOC3 will be assessed using a trans-well migration setup. This test will measure the ability of siglec-9 overexpressed Tregcells to move towards an AOC3 gradient, indicative of their targeted response capabilities. d) Suppressive Activity Assessment: The ability of siglec-9 overexpressed Treg cells to suppress effector T cell (Teff) cell proliferation 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.

[0215] Siglec-9 overexpressing Treg cells will demonstrate enhanced anti- inflammatory responses, primarily through increased IL-10 production, improved stability of Tregcell 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. Collectively, these effects underscore the potential of siglec-9 modified Tregcells to modulate fibrotic pathways effectively. Different vector designs or promoters may be explored to enhance siglec-9 expression. Additionally, comprehensive cytokine profiling could be conducted to understand the broad spectrum of inflammatory or regulatorycytokines affected by siglec-9 overexpression, providing a more complete view of the engineered Treg cells’ functional alterations. Example 11:

[0216] This non-limiting example dissects the role of the molecular mechanisms through which the SHP-1 signaling pathway mediates siglec-9 function of Tregcell under AOC3 stimulation. This Example is related to Example 10.

[0217] Preliminary data demonstrate that AOC3 interacts with siglec-9, corroborating siglec-9’s role as a functional ligand for AOC3 (FIG. 6). This interaction prompts downstream activation of the SHP-1 signaling pathway, a response augmented in the presence of AOC3 as evidenced by increased phosphorylation levels of SHP-12510, Pyk2 2520, and Src 2530 in siglec-9 engineered Tregcells (FIG. 25). The SHP-1 pathway is downstream of siglec-9 and significantly influences IL-10 production and Foxp3 expression, pivotal elements in the immunoregulatory capabilities of Treg cells. Based on these insights, AOC3-siglec-9 interaction may directly modulate Tregcell function, potentially enhancing their antifibrotic effects through precise molecular pathways. Example 12

[0218] 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.

[0219] The effect of AOC3-siglec-9 interaction to directly modulates Tregcell functions is investigated using the following assays.

[0220] 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. This measure assesses the impact of AOC3-siglec-9 interaction disruption on downstream signaling pathways. b) 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 Tregcell function. Insome 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 Tregcell stability. d) 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.

[0221] 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 Tregcells. Similarly, the stability of Tregcell lineage markers such as Foxp3, CD25, and CTLA-4 may decrease, reflecting impaired Treg cell functionality. Additionally, the effectiveness of these Tregcells in suppressing Teff cell proliferation may be reduced, further affirming the critical role of the AOC3-siglec-9 interaction in maintaining Tregcell 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 Tregcell 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. Moreover, 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

[0222] This non-limiting example shows optimization of siglec-9 overexpression levels in Tregcells for enhanced anti-fibrotic efficacy.

[0223] 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. b) Optimization of expression levels: Determine the optimal siglec-9 expression by titrating the vector and Doxycycline concentration in Treg cell cultures and assessing the resulting expression levels quantitatively. 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 α-SMA) in cardiac fibroblasts, using PCR and WB for quantification. 3) 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 Tregcells’ capability to modulate fibrotic responses effectively. Example 14

[0225] This non-limiting example shows designing lentiviral vectors for controlled overexpression of siglec-9 in Tregcells.

[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. The vector demonstrates targeted siglec-9 expression levels reaching 3-10 times those of non-engineered Treg cells without affecting cell viability.

[0227] 1. 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. 2. Initial Titration of Doxycycline: a) Transduce a population of Tregcells with the lentiviral vector; b) Exposedifferent groups of transduced Treg cells to varying concentrations of doxycycline (e.g., 0.1 µg / mL, 0.5 µg / mL, 1 µg / mL, and 2 µg / 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. Optimization of Doxycycline Dosing: a) Select the doxycycline concentration that initiates the desired baseline expression level (e.g., 3X); b) Incrementally increase the doxycycline concentration to fine-tune the expression to reach 5X and 10X levels; c) Confirm the reproducibility of expression levels across multiple experiments and batches of Treg cells. 4. Validation of Expression Levels: Perform Western blot and qPCR to validate siglec-9 protein and mRNA levels, respectively, ensuring they align with the expected multiples of baseline expression.

[0228] A lentiviral vector with a Tet-responsive promoter allowing controllable siglec-9 expression linked to a GFP reporter for easy monitoring is constructed. The initial titration of doxycycline establishes a dose-response curve, demonstrating predictable siglec-9 overexpression levels at different doxycycline concentrations. Additionally, incremental adjustments of doxycycline concentration cane fine-tune siglec-9 expression to targeted levels (3X, 5X, 10X) without compromising Tregcell viability and functionality. Alternative inducible systems include inducible CRISPR activation systems. Example 15

[0229] This non-limiting example shows determining the optimal siglec-9 expression level to effectively inhibit fibrosis.

[0230] The impact of varying siglec-9 expression levels on Tregcells’ capacity to inhibit Angiotensin II-induced proliferation of cardiac fibroblasts and reduce fibrotic markers is assessed. 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. Concurrently, a significant decrease in the levels of fibrotic markers such ascollagen type I and α-SMA (e.g., of at least a 30% reduction) at the minimal effective level of siglec-9, as determined by PCR and WB analyses, is observed. In quantifying gel contraction, gels with the lowest effective siglec-9 expression may exhibit reduction (e.g., at least ~40% reduction) in contraction compared to gels with non-engineered Treg cells. Example 16

[0232] This non-limiting example shows therapeutic application of siglec-9 engineered Treg cells in preclinical ICM models.

[0233] The results above indicate that Siglec-9 engineered Tregcells effectively mitigate cardiac fibrosis and improve cardiac function. To effectively mimic the ICM phenotype, a more chronic, nonreperfused MI model is used. 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.

[0234] The minimal effective dose is determined and heart function recovery post-treatment is assessed in ICM mouse models using the following options.

[0235] a) Fibrosis quantification: Picrosirius red staining for fibrotic area visualization, WB for fibrotic protein markers (Collagen I and αSMA), and qPCR for gene expression levels of these markers. b) Heart function and structure Analysis: Measurement of LVEF, and end-diastolic and end-systolic volumes, via echocardiography. c) Inflammation assessment: H&E staining for general inflammation, Immunohistochemistry (IHC) for specific inflammatory cells (CD68, CD3), ELISA for inflammatory cytokines (TNF-α, IL-6, IL-1β), and qPCR for inflammatory gene expression.

[0236] 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. A dose that results in improvement (e.g., at least a 20% improvement) in LVEF and achieves reduction (e.g., a minimum of 25% reduction) in inflammatory markers, including cellularinfiltration and cytokine levels, is determined. Additional markers or more sensitive assays may be used to better capture the anti-inflammatory effects of the treatment.

[0237] The therapeutic impact of engineered Tregcells on cardiac function, structure and safety in a pig model of chronic ICM is assessed using the following options.

[0238] a) LVEF measurement: Employ cine MRI to calculate LVEF by determining end-diastolic and end-systolic volumes. b) Scar size measurement: Use Late Gadolinium Enhancement (LGE) MRI to identify and measure myocardial scars. c) 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-α, IL-6, IL-1β) in heart tissue or serum via ELISA. Gene expression analysis: Use qPCR to measure mRNA levels of inflammatory markers.

[0239] Optimally-dosed engineered 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.

[0240] All patents and other publications; including literature references, issued patents, published patent applications, and co-pending patent applications; cited throughout this application are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the technology described herein. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.

[0241] The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specificembodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize. For example, while method steps or functions are presented in a given order, alternative embodiments may perform functions in a different order, or functions may be performed substantially concurrently. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the disclosure can be modified, if necessary, to employ the compositions, functions and concepts of the above references and application to provide yet further embodiments of the disclosure. Moreover, due to biological functional equivalency considerations, some changes can be made in protein structure without affecting the biological or chemical action in kind or amount. These and other changes can be made to the disclosure in light of the detailed description. All such modifications are intended to be included within the scope of the appended claims.

[0242] Specific elements of any of the foregoing embodiments can be combined or substituted for elements in other embodiments. Furthermore, while advantages associated with certain embodiments of the disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages to fall within the scope of the disclosure.

Claims

WHAT IS CLAIMED IS:

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 (Treg) cells in the subject.

2. The method of claim 1, wherein the subject is in need of cardiac fibrosis treatment.

3. 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.

4. The method of claim 3, wherein the nucleic acid encodes the Siglec-9 protein expressed by the Treg cells in the subject, optionally wherein the nucleic acid comprises mRNA encoding the Siglec-9 protein.

5. The method of claim 3, wherein the condition associated with inflammation and / or fibrosis comprises cardiac inflammation and / or cardiac fibrosis.

6. The method of claim 3, wherein the condition associated with inflammation and / or fibrosis comprises myocardial infarction, hypertension, myocarditis, transplant rejection, cardiomyopathy, heart failure, and amyloidosis.

7. The method of claim 3, wherein the condition associated with inflammation and / or fibrosis is an angiotensin II-induced condition.

8. The method of claim 3, wherein administering the nucleic acid comprises administering to the subject a therapeutically effective amount of a composition comprising a population of Tregcells comprising the nucleic acid, optionally wherein the therapeutically effective amount of the composition comprises the Treg cells at 107cells / kg or more of body weight.

9. The method of claim 8, wherein Tregcells of the population comprise a plasmid or a viral vector comprising the nucleic acid, optionally wherein the viral vector comprises an adenoviral vector, retroviral vector, or a lentiviral vector.

10. The method of claim 9, comprising genetically modifying Treg cells with the plasmid or the viral vector comprising the nucleic acid.

11. The method of claim 3, wherein the population of Tregcells are derived from cells autologous to the subject.

12. The method of claim 3, wherein the population of Treg cells are derived from cells allogeneic to the subject.

13. The method of claim 3, comprising administering to the subject a vehicle comprising the nucleic acid, wherein the vehicle is configured to deliver the nucleic acid to Tregcells in the subject.

14. The method of claim 13, wherein the vehicle comprises: a lipid nanoparticle (LNP); and a Treg cell-targeting moiety associated with the LNP.

15. The method of claim 14, wherein the Tregcell-targeting moiety comprises an antigen binding protein that binds a Treg cell marker, optionally wherein the Treg cell marker is selected from: CD25 and / or Foxp3.

16. The method of claim 15, wherein the antigen binding protein is an antibody, optionally wherein the antigen binding protein is selected from: an anti-CD25 monoclonal antibody and / or anti-Foxp3 monoclonal antibody.

17. The method of claim 1, wherein the subject has suffered or is at risk of suffering from myocardial infarction.

18. The method of claim 1, wherein the subject is in need of treating angiotensin II-induced cardiac fibrosis.

19. The method of claim 1, wherein the subject is in need of treating cardiac fibrosis subsequent to a myocardial infarction.

20. The method of claim 3, wherein the Siglec-9 protein comprises an amino acid sequence at least 80% identical to SEQ ID NO:

2.

21. 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, optionally wherein the nucleic acid encodes the membrane-bound fibroblast-specific binding protein.

22. The method of claim 21, wherein the membrane-bound fibroblast-specific binding protein comprises an amino acid sequence at least 80% identical to SEQ ID NO:2, optionally wherein the membrane-bound fibroblast-specific binding protein comprises sialic acid-binding immunoglobulin-type lectin 9 (Siglec-9).

23. The method of claim 21, wherein the condition associated with inflammation and / or fibrosis comprises an inflammatory tissue comprising fibroblasts having elevated expression of amine oxidase, copper containing 3 (AOC3).

24. The method of claim 23, wherein the fibroblasts having elevated expression of AOC3 comprises myofibroblasts, optionally wherein the myofibroblasts comprises cardiac myofibroblasts.

25. The method of claim 21, further comprising: obtaining a population of immune cells comprising regulatory T (Treg) cells; delivering the nucleic acid to the Treg cells, whereby the Siglec-9 protein is overexpressed in the Treg cell; contacting the Tregcells 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 expresses amine oxidase, copper containing 3 (AOC3), to thereby treat the condition associated with inflammation and / or fibrosis.

26. 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 Tregcells, 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 Tregcells overexpressing Siglec-9; and administering to a subject the population of enhanced Treg cells, optionally wherein the nucleic acid encodes the Siglec-9 protein.

27. The method of claim 26, wherein the at least one vehicle comprises a liposome, a lipid nanoparticle (LNP), or a viral vector, optionally wherein the viral vector comprises an adenoviral vector, retroviral vector, or a lentiviral vector.

28. The method of claim 26, wherein the nucleic acid comprises a promoter operatively linked to a nucleotide sequence encoding the Siglec9 protein, wherein the promoter upregulates expression of the Siglec9 protein in the Tregcells.

29. The method of claim 26, wherein the subject comprises at least one myofibroblast cell having elevated expression of AOC3 compared to other cell types, optionally wherein the at least one myofibroblast cell is at least one cardiac myofibroblasts.

30. The method of claim 26, wherein the subject is in need of treatment for fibrosis.

31. The method of claim 30, wherein the fibrosis is a cardiac fibrosis, optionally wherein the cardiac fibrosis is subsequent to a myocardial infarction.

32. A method of inhibiting fibrotic gene expression, comprising: obtaining a population of Tregcells; engineering the Treg cells to overexpress Siglec-9 protein, thereby producing enhanced Treg cells; and administering an effective amount of the enhanced Tregcells to a subject in need of inhibiting fibrotic gene expression, optionally wherein the fibrotic gene is at least one of Collagen I, α-smooth muscle actin (α-SMA), and / or amine oxidase, copper containing 3 (AOC3).

33. The method of claim 32, wherein engineering comprises: delivering at least one vehicle comprising a nucleic acid encoding Siglec-9 protein to the population of Tregcells, 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.

34. The method of claim 33, wherein 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).

35. The method of claim 33, comprising upregulating expression of the mRNA to overexpress the Siglec-9 protein.

36. The method of claim 32, wherein the subject is in need of treatment for fibrosis, optionally wherein the fibrosis is a cardiac fibrosis.

37. The method of claim 36, wherein the cardiac fibrosis is subsequent to a myocardial infarction.

38. The method of claim 3, wherein the subject has upregulated expression of amine oxidase, copper containing 3 (AOC3) in cardiac myofibroblasts.

39. The method of claim 1, wherein the administering comprises intravenous or oral administration.

40. An isolated nucleic acid comprising 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, optionally wherein 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.

41. A vector comprising the isolated nucleic acid of claim 40.

42. A regulatory T (Treg) cell comprising the isolated nucleic acid of claim 40, optionally wherein the Treg cell is a human Treg cell.

43. 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 Tregcells, optionally wherein the Tregcells are human Tregcells.

44. The composition of claim 43, wherein vehicles of the population comprise a lipid nanoparticle (LNP).

45. The composition of claim 43, wherein the nucleic acid comprises mRNA.

46. The composition of claim 43, wherein the Treg cell-targeting moiety comprises an antigen binding protein that binds a Tregcell marker, optionally wherein the Tregcell marker is selected from: CD25 and / or Foxp3.

47. The composition of claim 46, wherein the antigen binding protein is an antibody, optionally wherein the antigen binding protein is selected from: an anti-CD25 monoclonal antibody and / or anti-Foxp3 monoclonal antibody.

48. A composition comprising a population of enhanced regulatory T (Treg) cells overexpressing sialic acid-binding immunoglobulin-like lectin 9 (Siglec-9), optionally wherein the Tregcells are human Tregcells.

49. The composition of claim 48, wherein 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).

50. The composition of claim 49, wherein 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).

51. The composition of claim 49, wherein the plurality of nucleic acids comprises a plurality of mRNA transcripts encoding Siglec-9.

52. The composition of claim 48, wherein the enhanced Tregcells exhibit Siglec- 9-dependent interaction with cells expressing amine oxidase, copper containing 3 (AOC3), optionally wherein the enhanced Treg cells exhibit Siglec-9-dependent interaction with cardiac myofibroblasts expressing AOC3.

53. The method, isolated nucleic acid, vector, Tregcell, or composition of any one of the preceding claims, wherein the Siglec-9 is human Siglec-9.