Enhanced regulatory t cells and methods of use thereof

Abstract

Methods and compositions are provided for treating fibrosis (e.g., cardiac fibrosis) or other conditions associated with inflammation and / or fibrosis. The methods may include administering a nucleic acid encoding a sialic acid-binding immunoglobulin-like lectin 9 (Siglec-9) protein to a subject in need of treatment for fibrosis, wherein the Siglec-9 protein is expressed by the nucleic acid in the subject's regulatory T(T) receptor. reg (Expression in cells.) Methods and compositions may include T cells overexpressing Siglec-9. reg Cells to enhance T reg The ability of cells to target and express copper-amine oxidase 3 (AOC3)-containing cells (including fibroblasts or myofibroblasts). In some embodiments, the enhanced T reg Cells are targeted to myofibroblasts (e.g., cardiac myofibroblasts) with increased expression of fibrosis genes.

Description

[0001] Citation of relevant applications This application claims priority to U.S. Provisional Patent Application No. 63 / 551364, filed on February 8, 2024, the disclosure of which is incorporated herein by reference in its entirety.

[0002] Statement on federally funded research and development This invention was made with government support under grant number R01 HL124074 granted to Dr. Eduardo Marbán by the National Institutes of Health in the United States. The government holds certain rights to this invention.

[0003] References to sequence lists This application is submitted together with an electronic sequence list. The sequence list is provided as a file named SeqListing_CSMC024WO.xml, created on February 5, 2025, and measuring 4,933 bytes. The information in this electronic sequence list is incorporated herein by reference in its entirety. Background Technology Technical Field

[0005] Fibrosis is a condition affecting multiple tissues and poses a major medical challenge. It occurs when there is an excessive buildup of fibrous connective tissue, leading to organ dysfunction or failure. Common treatment strategies often focus on reducing inflammation—a key factor in the development of fibrosis—through immunosuppression. However, this approach can be counterproductive, as nonspecific immunosuppression may hinder the body's natural ability to repair and regenerate tissues, potentially exacerbating fibrosis. Summary of the Invention

[0006] Current immunotherapies aimed at treating fibrosis may lack the specificity required to avoid the known negative effects of existing technologies. Therefore, there is an urgent need to develop targeted therapies that can effectively modulate immune responses to prevent or alleviate fibrosis while avoiding the negative side effects of widespread immunosuppression. This article provides a method for treating fibrosis comprising administering a nucleic acid encoding a sialic acid-binding immunoglobulin-like lectin 9 (Siglec-9) protein to a subject requiring treatment for fibrosis, wherein the Siglec-9 protein is generated by the nucleic acid in the subject's regulatory T(T) receptor. reg It is expressed in cells. In some embodiments, the subject has a need for treatment of cardiac fibrosis.

[0007] This article also provides a method for treating conditions associated with inflammation and / or fibrosis, comprising administering a nucleic acid encoding a sialic acid-binding immunoglobulin-like lectin 9 (Siglec-9) protein to a subject in need of treatment for conditions associated with inflammation and / or fibrosis, wherein the Siglec-9 protein is generated by the nucleic acid in the subject's regulatory T(T) reg This article further provides a method for treating conditions associated with inflammation and / or fibrosis, comprising administering to a subject in need of treatment for conditions associated with inflammation and / or fibrosis a protein configured to induce sialic acid-binding immunoglobulin-like lectin 9 (Siglec-9) protein in the subject's regulatory T (T) cells. reg Nucleic acids expressed in cells. In some embodiments, the conditions associated with inflammation and / or fibrosis include cardiac inflammation and / or cardiac fibrosis. In some embodiments, the conditions associated with inflammation and / or fibrosis include myocardial infarction, hypertension, myocarditis, transplant rejection, cardiomyopathy, heart failure, and amyloidosis. In some embodiments, the conditions associated with inflammation and / or fibrosis are angiotensin II-induced conditions.

[0008] In some embodiments, administering a nucleic acid encoding the Siglec-9 protein comprises administering to the subject a therapeutically effective amount of a composition, the composition comprising a T-cell containing the nucleic acid (or a nucleic acid encoding the Siglec-9 protein). reg Cell population, optionally, wherein the therapeutically effective amount of composition comprises 10 7 The T cells / kg body weight or more reg Cells. In some embodiments, the T reg The cell population contains a plasmid or viral vector containing the said nucleic acid (or nucleic acid encoding the Siglec-9 protein). In some embodiments, the method includes using a plasmid or viral vector containing the said nucleic acid (or nucleic acid encoding the Siglec-9 protein) on T cells. reg Cells undergo gene modification. In some embodiments, the viral vector includes an adenovirus vector, a retrovirus vector, or a lentivirus vector. In some embodiments, the T... reg The cell population is derived from the subject's own cells. In some embodiments, the T... reg The cell population was derived from allogeneic cells of the subject.

[0009] In some embodiments, the method further includes administering to the subject a vector containing the nucleic acid (or nucleic acid encoding the Siglec-9 protein), wherein the vector is configured to deliver the nucleic acid (or nucleic acid encoding the Siglec-9 protein) to the subject's T cells. regCells. In some embodiments, the carrier comprises: lipid nanoparticles (LNPs); and T cells associated with the LNPs. reg Cell-targeting component. In some embodiments, the T reg Cell-targeting components include binding to T reg Antigen-binding proteins of cell markers. In some embodiments, the T... reg The 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 anti-CD25 monoclonal antibody and / or anti-Foxp3 monoclonal antibody.

[0010] In some embodiments, the nucleic acid includes mRNA encoding the Siglec-9 protein.

[0011] In some embodiments, the subject has a history of or is at risk of having a myocardial infarction. In some embodiments, the subject needs treatment for angiotensin II-induced cardiac fibrosis. In some embodiments, the subject needs treatment for post-myocardial infarction cardiac fibrosis.

[0012] In some embodiments, the Siglec-9 protein comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 2.

[0013] This article also provides a method for 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 treatment for a condition associated with inflammation and / or fibrosis, wherein the membrane-bound fibroblast-specific binding protein is generated by the nucleic acid in the subject's regulatory T(T) reg This article further provides a method for treating conditions associated with inflammation and / or fibrosis, comprising administering to a subject in need of treatment for conditions associated with inflammation and / or fibrosis a protein configured to induce membrane-bound fibroblast-specific binding in the subject's regulatory T(T) cells. regThe nucleic acid expressed in the cell. In some embodiments, the nucleic acid encodes the membrane-bound fibroblast-specific binding protein. In some embodiments, the membrane-bound fibroblast-specific binding protein includes sialic acid-binding immunoglobulin-like lectin 9 (Siglec-9). In some embodiments, the membrane-bound fibroblast-specific binding protein comprises an amino acid sequence with at least 80% identity to SEQ ID NO: 2. In some embodiments, the condition associated with inflammation and / or fibrosis includes inflammatory tissue comprising fibroblasts with elevated copper-containing amine oxidase 3 (AOC3) expression. In some embodiments, the fibroblasts with elevated AOC3 expression include myofibroblasts. In some embodiments, the myofibroblasts include cardiac myofibroblasts.

[0014] In some embodiments, the method further includes: obtaining a modulating T (T reg ) immune cell population; delivery of nucleic acid encoding sialic acid-binding immunoglobulin-like lectin 9 (Siglec-9) protein to the T cells. reg Cells, thereby allowing the Siglec-9 protein to be present in the T cells. reg Overexpression in cells; causing T cells to overexpress Siglec-9 protein reg The cells are in contact with at least one fibroblast and / or myofibroblast of the subject, wherein the at least one fibroblast and / or myofibroblast expresses copper-containing amino oxidase 3 (AOC3), thereby treating the condition associated with the inflammation and / or fibrosis.

[0015] This article also provides a method for targeted immune modulation of subjects, including: obtaining regulatory T cells (T cells). reg ) group; towards T reg Cell population delivery of at least one vector, wherein the at least one vector contains nucleic acid encoding sialic acid-binding immunoglobulin-type lectin-9 (Siglec-9) and is located in the T cell population. reg Cells express Siglec-9, thereby producing enhanced T cells that overexpress Siglec-9. reg Cell populations; and administration of the enhanced T to the subject reg Cell population. This article also provides a method for targeted immune modulation of subjects, including: obtaining regulatory T cells (T cells). reg ) group; to the T reg Cell population delivery of at least one vector, wherein the at least one vector comprises a protein configured to induce sialic acid binding of immunoglobulin-type lectin-9 (Siglec-9) protein in the T cells. reg Nucleic acid expressed in the cell population leads to the production of enhanced T cells overexpressing Siglec-9.reg Cell populations; and administration of the enhanced T to the subject reg Cell population. In some embodiments, the at least one vector comprises liposomes or lipid nanoparticles (LNPs). In some embodiments, the at least one vector comprises a viral vector. In some embodiments, the viral vector comprises an adenovirus vector, a retroviral vector, or a lentiviral vector. In some embodiments, the nucleic acid comprises a promoter operatively linked to a nucleotide sequence encoding Siglec9, wherein the promoter upregulates T... reg Expression of Siglec9 in cells.

[0016] In some embodiments, the subject includes at least one myofibroblast with elevated AOC3 expression compared to other cell types. In some embodiments, the at least one myofibroblast is at least one cardiac myofibroblast. In some embodiments, the subject has a need for treatment of fibrosis. In some embodiments, the fibrosis is cardiac fibrosis. In some embodiments, the cardiac fibrosis is secondary to myocardial infarction.

[0017] This article also provides a method for inhibiting fibrosis gene expression, including: obtaining T reg Cell population; for the T reg Cells were engineered to overexpress Siglec-9, thereby producing enhanced T cells. reg Cells; and administration of an effective amount of the enhanced T to a subject who requires inhibition of fibrosis gene expression. reg Cells. In some embodiments, the subject has a need to suppress the expression of fibrosis genes in cardiac myofibroblasts. In some embodiments, the fibrosis gene is at least one of type I collagen, α-smooth muscle actin (α-SMA), and / or copper-containing amino oxidase 3 (AOC3). In some embodiments, the engineering includes introducing the T cells... reg A cell population delivers at least one vector containing a nucleic acid encoding Siglec-9. In some embodiments, the at least one vector comprises at least one of liposomes, nanoparticles, lipid nanoparticles (LNPs), viral vectors, cell-penetrating peptides (CPPs), and / or extracellular vesicles (EVs). In some embodiments, the nucleic acid comprises mRNA. In some embodiments, overexpression of Siglec-9 includes upregulating the expression of the mRNA.

[0018] In some embodiments, the subject requires treatment for fibrosis. In some embodiments, the fibrosis is cardiac fibrosis. In some embodiments, the cardiac fibrosis is secondary to myocardial infarction.

[0019] In some embodiments, the techniques described herein relate to the method described in any of the preceding claims, wherein the subject has upregulated expression of copper-containing amine oxidase 3 (AOC3) in cardiac myofibroblasts.

[0020] In some embodiments, administration includes intravenous or oral administration.

[0021] This article also provides an isolated nucleic acid that further comprises a nucleotide sequence encoding a sialic acid-binding immunoglobulin-like lectin 9 (Siglec-9) protein, which is operatively linked to a protein configured to be used in regulatory T (T reg The promoter for expressing the Siglec-9 protein in cells. In some embodiments, the promoter is a regulatory T (T) promoter selected from the Foxp3 promoter and the interleukin-2 (IL-2) promoter. reg Cell-specific promoters.

[0022] This article also provides a vector comprising the isolated nucleic acid of this disclosure.

[0023] This document also provides a regulatory T(T) component, including the isolated nucleic acid or vector of this disclosure. reg )cell.

[0024] This article also provides a composition comprising a group of vectors, each vector comprising: a nucleic acid encoding sialic acid-binding immunoglobulin-like lectin 9 (Siglec-9); and a nucleic acid configured to deliver the Siglec-9-encoding nucleic acid to a T-cell matrix. reg Regulatory T cells (T) reg Cell-targeting component. In some embodiments, the carrier of the cell population comprises lipid nanoparticles (LNPs). In some embodiments, the nucleic acid comprises mRNA. In several embodiments, the T... reg Cell-targeting components include binding to T reg Antigen-binding proteins of cell markers. In some embodiments, the T... reg The 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 anti-CD25 monoclonal antibody and / or anti-Foxp3 monoclonal antibody.

[0025] This article also provides a composition comprising an enhanced regulatory T (T) cell overexpressing sialic acid-binding immunoglobulin-like lectin 9 (Siglec-9). reg Cell population. In some embodiments, the enhanced T regThe cell contains at least one endocytic carrier, said at least one endocytic carrier comprising a plurality of nucleic acids encoding sialic acid-binding immunoglobulin-like lectin 9 (Siglec-9). In some embodiments, said at least one endocytic carrier comprises at least one of liposomes, nanoparticles, lipid nanoparticles (LNPs), viral vectors, cell-penetrating peptides (CPPs), and / or extracellular vesicles (EVs). In some embodiments, said plurality of nucleic acids comprises a plurality of mRNA transcripts encoding Siglec-9. In some embodiments, the enhanced T... reg The cells exhibited a Siglec-9-dependent interaction with cells expressing copper-containing amino oxidase 3 (AOC3). In some embodiments, the enhanced T... reg The cells exhibited Siglec-9-dependent interactions with AOC3-expressing cardiac myofibroblasts.

[0026] This document also provides a composition disclosed herein for treating conditions associated with inflammation and / or fibrosis in subjects of need. Attached Figure Description

[0027] Figure 1 A non-limiting example implementation is shown, depicting the process of adding an enhanced T to a molecule overexpressing Siglec-9. reg Methods for administering nucleic acids to cells.

[0028] Figure 2 A graphical representation of data obtained from in vitro experiments on the effect of angiotensin II concentration on fibrosis gene expression is shown.

[0029] Figure 3 A graphical representation of data obtained from in vitro experiments on the effects of angiotensin II on the expression of fibrosis genes over time is shown.

[0030] Figure 4 Graphical representations of data obtained from in vitro experiments on the effects of angiotensin II concentration and time on AOC3 gene expression are shown; along with the results of Western blot analysis demonstrating the in vitro upregulation of AOC3 in the presence of angiotensin II.

[0031] Figure 5 This shows T from overexpressing Siglec-9 reg Graphical representation of data obtained from in vitro cell experiments.

[0032] Figure 6 T cells overexpressing Siglec-9 were shown. reg A schematic diagram of the in vitro cell experiment and the data obtained from it.

[0033] Figure 7 Enhanced T is shownreg A schematic diagram of an in vitro experiment targeting AOC3-expressing fibroblasts and the data obtained therefrom.

[0034] Figure 8 Enhanced T is shown reg Graphical representation of data from in vitro experiments targeting AOC3-expressing fibroblasts.

[0035] Figure 9 The test of Siglec-9 in enhanced T2000 was shown. reg A schematic diagram of an in vitro experiment demonstrating the role of cell-targeted AOC3 expression in fibroblasts, and the data obtained therefrom.

[0036] Figure 10 The test of Siglec-9 in enhanced T2000 was shown. reg Graphical representation of in vitro experimental data on the role of cell-targeted AOC3 expression in fibroblasts.

[0037] Figure 11 The test of AOC3 in enhanced T is shown. reg A schematic diagram of an in vitro experiment demonstrating the role of cell-targeted AOC3 expression in fibroblasts, and the data obtained therefrom.

[0038] Figure 12 The test of AOC3 in enhanced T is shown. reg Graphical representation of in vitro experimental data on the role of cell-targeted AOC3 expression in fibroblasts.

[0039] Figure 13 Data on the upregulation of AOC3 and fibrosis genes observed in in vivo experiments using a myocardial infarction model are presented.

[0040] Figure 14 The enhanced T is shown reg A schematic diagram of the experiment on the effects of cell-mediated effects on the expression of fibrosis genes in fibroblasts after myocardial infarction, and the data obtained therefrom.

[0041] Figure 15 This demonstrates the research on enhanced T reg In vivo experimental protocol for the anti-fibrotic effect of cells.

[0042] Figure 16 This suggests that Siglec-9 may promote T cells by influencing immune cell signaling. reg An exemplary, non-limiting implementation of cellular processes of cell function.

[0043] Figure 17 The diagram depicts a T-shaped structure engineered with Siglec-9. regA schematic diagram of the collagen shrinkage experiment with reduced shrinkage in the gel and the data obtained therefrom, showing that it exhibits effective anti-fibrotic activity.

[0044] Figure 18 The diagram illustrates a reduced collagen contraction assay demonstrating the decreased effect of siglec-9 blockade on fibroblast contraction, along with the data obtained therefrom. It highlights the role of siglec-9 in mediating T... reg Importance of anti-fibrotic activity.

[0045] Figure 19 A schematic diagram of the collagen contraction experiment after AOC3 neutralization and the data obtained therefrom are shown, demonstrating a reduction in fibroblast contraction, further highlighting the key role of the Siglec-9-AOC3 interaction in targeting the fibrosis process.

[0046] Figure 20 To evaluate the enhanced T reg A schematic diagram of the in vivo experimental design for the therapeutic effect of cells in a myocardial infarction model.

[0047] Figure 21 Immunostaining analysis of cardiac slices is shown, revealing RFP-labeled Siglec-9-engineered T cells. reg The infiltration was clearly localized in the AOC3-positive area of ​​the heart with myocardial infarction.

[0048] Figure 22 A graphical representation of the mRNA levels of AOC3, type I collagen, and α-SMA in cardiac tissue confirms the presence of Siglec-9 T. reg The treatment group showed a decrease in fibrosis markers.

[0049] Figure 23 The image shows Sirius red staining and analysis of fibrotic areas, demonstrating the use of Siglec-9 T reg The treatment significantly reduced fibrosis (n=5 per group).

[0050] Figure 24 The accompanying graph illustrates the echocardiographic results, showing the Siglec-9-T... reg Improved cardiac function in the treated heart (n=5 per group).

[0051] Figure 25 The results of Western blot analysis are shown, demonstrating the effects of exposure to AOC3 on Siglec-9-engineered T cells. reg The SHP-1 pathway was upregulated in vivo, showing the phosphorylation levels of SHP-1, Pyk2, and Src.

[0052] Figure 26 It shows Figure 5Quantitative analysis of protein expression levels shown in the Western blot. Detailed Implementation

[0053] Based on regulatory T cells (T reg Cellular therapy offers a targeted approach that avoids the broader immunosuppressive effects typically associated with conventional immunotherapies for cardiac fibrosis and related diseases.

[0054] Ischemic cardiomyopathy (ICM) is a progressive deterioration of cardiac function caused by chronic ischemia or reduced cardiac blood flow. Fibrosis is a pathological process characterized by the excessive accumulation of extracellular matrix proteins and the differentiation of fibroblasts into myofibroblasts, leading to scar tissue formation. Fibrosis plays a crucial role in the occurrence and development of ICM. Reduced blood flow leads to scar accumulation. As ICM progresses, cardiac fibrosis plays several key roles. In the early stages, reduced blood flow increases the production of collagen and other extracellular matrix proteins. As ischemia persists, fibrosis becomes more widespread and may lead to the destruction of normal cardiac structure and function. Fibrotic accumulation contributes to the development of heart failure (HF), a condition in which the heart is unable to pump blood effectively, leading to shortness of breath, fatigue, and premature death. In later stages, fibrosis can also lead to cardiac remodeling, a process of maladaptive structural and functional changes that can further exacerbate heart failure. ICM is often associated with renal fibrosis, leading to chronic kidney disease. When heart failure with reduced ejection fraction (HFrEF) type ICM is complicated by chronic kidney disease, the mortality rate increases threefold. ICM is also frequently associated with multi-organ fibrosis, including not only the heart and kidneys, but also the lungs and liver. This systemic fibrotic response can exacerbate the overall disease burden; for example, pulmonary fibrosis can impair gas exchange and increase the workload of an already damaged heart. Furthermore, chronic heart failure can lead to hepatic congestion, which may eventually progress to fibrosis and cirrhosis, disrupting metabolic function and drug metabolism, further complicating patient management.

[0055] This article provides enhanced T(T) reg Methods for regulating cells and their applications. In general, these methods include... reg Cell administration was configured to induce T regNucleic acids expressed in cells (e.g., administration of nucleic acids encoding sialic acid-binding immunoglobulin-like lectin 9 (Siglec-9)) enhance their functional properties, including but not limited to targeting and alleviating cardiac fibrosis. As used herein, “inducible expression” means an expression level of a gene product that is higher than the expression level without inducible gene product expression (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 times or more higher). In some embodiments, the method includes delivering (or administering) a T-cell modified to express (or overexpress) Siglec-9 to a subject in need of treatment for cardiac fibrosis. reg Cell population. In some implementations, T reg Cellular enhancement may also involve the use of lipid nanoparticle (LNP) carriers to efficiently deliver nucleic acids encoding Siglec-9 to the subject's T cells. reg In cells.

[0056] In various embodiments, the enhanced T reg The cells exhibited an enhanced ability to target and interact with fibroblasts and / or myofibroblasts in subjects with cardiac fibrosis, particularly those expressing copper-containing amino oxidase 3 (AOC3). In some embodiments, T cells modified to overexpress Siglec-9 were administered. reg Cells can modulate the immune response of subjects, especially those at risk of or who have already experienced a myocardial infarction. By utilizing the unique properties of Siglec-9, these enhanced T cells... reg Cells can be used to treat diseases associated with inflammation and / or fibrosis, including but not limited to cardiac fibrosis caused by myocardial infarction.

[0057] In some implementations, T is used in these methods. reg Cells are involved in delivering T cells into the subject's body. reg Cell delivery is configured to induce Siglec-9 expression of nucleic acids (e.g., without cell administration). In some embodiments, T is used in these methods. reg Cells are involved in delivering T cells into the subject's body. reg Cellular delivery of nucleic acids encoding Siglec-9 without direct cell administration offers a potential advantage by minimizing the inflammatory response typically associated with cell therapy. The method described herein includes targeted delivery of nucleic acids to T cells using LNPs. reg Cells, by utilizing enhanced T reg The specificity and effectiveness of the cells provide a novel and effective way to treat inflammation and / or fibrosis-related diseases, especially those related to heart health.

[0058] This article also provides a novel composition comprising a group of delivery vectors, each vector containing substances configured to induce T... reg Cells express nucleic acids that encode sialic acid-binding immunoglobulin-like lectin 9 (Siglec-9) (e.g., nucleic acids encoding sialic acid-binding immunoglobulin-like lectin 9 (Siglec-9)). In some embodiments, these vectors can be programmed to target T cells. reg Cells, promoting T reg Cellular delivery and subsequent expression of Siglec-9. In a further embodiment, the delivery vector may include liposomes, nanoparticles, lipid nanoparticles (LNPs), viral vectors, cell-penetrating peptides (CPPs), and extracellular vesicles (EVs). For example, each vector encapsulates multiple mRNA transcripts encoding Siglec-9, ensuring their expression in T cells. reg Efficient intracellular transport and expression. Therefore, in some embodiments, enhanced T cells can be prepared. reg Cell population, wherein the T reg Cells are enhanced through their ability to overexpress Siglec-9. In some embodiments, T... reg Such modifications to cells can significantly increase T reg The therapeutic potential of cells, including but not limited to targeting and inhibiting fibrosis genes.

[0059] In some further embodiments, the enhanced T in the composition reg Cells exhibit a high affinity binding to copper-containing amine oxidase 3 (AOC3), which is significantly expressed in cardiac myofibroblasts. In some embodiments, this specificity can alleviate and / or treat cardiac fibrosis, a condition commonly caused by hypertension or myocardial infarction. For example, by focusing on AOC3, enhanced T cells... reg Cells can target cells that cause fibrosis and / or inhibit the expression of fibrosis genes, including but not limited to type I collagen, α-smooth muscle actin (α-SMA), and AOC3. This targeted approach not only addresses the root cause of cardiac fibrosis but also provides a novel therapeutic strategy for managing symptoms caused by other fibrosis-related genes and diseases.

[0060] Therefore, the above-described methods and compositions represent a significant advancement in the treatment of cardiac fibrosis and related diseases, providing a more targeted and effective immunotherapy approach.

[0061] definition As used herein, “vector” or “delivery vector” refers to a molecule or collection of molecules suitable for delivering nucleic acids into cells. For example, a vector may be a plasmid, virus or other nucleic acid construct, liposome, nanoparticle, cell-penetrating peptide (CPP), or extracellular vesicle (EV) engineered to carry substances into host cells. In some embodiments, vectors may 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” has the common and customary meaning as understood by those skilled in the art in conjunction with this disclosure. Liposomes include spherical vesicles composed of one or more phospholipid bilayers for encapsulating and delivering substances into cells, including conventional liposomes, multilayer 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” is defined herein according to the common and customary meaning as understood by those skilled in the art in conjunction with this disclosure. Nanoparticles include, but are not limited to, metal nanoparticles (such as gold and silver), quantum dots, dendritic polymers, carbon-based nanoparticles (such as fullerenes and carbon nanotubes), lipid-based nanoparticles (such as "lipid nanoparticles" (LNPs)), ceramic nanoparticles, polymer nanoparticles, magnetic nanoparticles, solid lipid nanoparticles, and nanostructured lipid carriers. "Viral vector" is defined herein in the ordinary and conventional sense as understood by those skilled in the art in conjunction with this disclosure. Viral vectors include adenovirus vectors, retrovirus vectors, lentivirus vectors, adeno-associated virus vectors, herpes simplex virus vectors, poxvirus vectors, baculovirus vectors, alphavirus vectors, rabies virus vectors, vaccinia virus vectors, foamy virus vectors, measles virus vectors, Sindbis virus vectors, and mumps virus vectors. "Cell-penetrating peptide" (CPP) has the ordinary and conventional sense as understood by those skilled in the art in conjunction with this disclosure. CPPs include, but are not limited to, TAT peptide, penetrantin, transport peptide, polyarginine, VP22, SynB peptide, MPG peptide, pVEC, R9, FHV peptide, MAP, CATY, Hph-1, KLA peptide, Pep-1, PTD-4, R8, GALA, HIV-1 Tat peptide, TP10, ANTP peptide, Buforin II, K-FGF, SV40 NLs, nonarginine, azurin, hCT(9-32)-k7, R7, LMWP, KALA, PepFect14, TP508, fusion peptide, M918, INF7, Pep-7, SynB3, Drosocin, SAP(1-8), LAH4, BP100, and hLF(1-11). The term “extracellular vesicle” or “EV” has the common and customary meaning as understood by those skilled in the art in conjunction with this disclosure.EVs comprise a lipid bilayer structure generated by cells, including exosomes, microvesicles, epididymal bodies, seminoma, exosome-like vesicles, microparticles, promininosomes, prostate bodies, dendritic exosomes, texosomes, dendritic vesicles (dex), tumor vesicles (tex), archeosomes, and carcinoma bodies.

[0062] As used in this article, "immunomodulation" refers to altering the activity of the adaptive or innate immune system in vertebrates. Compared to an unregulated immune response, immunomodulation can include the enhancement or suppression of one or more aspects of the immune response. Immunomodulation (e.g., targeted immunomodulation) can include enhancing or suppressing a particular aspect of the immune response without altering other aspects. Targeted immunomodulation can be tissue-specific (e.g., heart-specific) and / or targeted at a specific process (e.g., fibrosis).

[0063] As used herein, “immune cells” has the conventional and common meaning understood by one of ordinary skill in the art based on this disclosure, referring to specialized cells that mediate immune responses in vertebrates. For example, immune cells can recognize and neutralize pathogenic threats as well as dysfunctional or abnormal “self” cells. There are various 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 immune memory. These cell types typically work together and are essential for detecting, responding to, and eliminating exogenous substances and abnormal self-physiological states. This definition encompasses any modifications or variations of the standard biological functions or characteristics of immune cells that may be adopted, referenced, or understood by one of ordinary skill in the art based on this disclosure.

[0064] "Regulatory T cells" or "T cells" reg The term "cell" is used interchangeably herein and has the conventional and ordinary meaning as understood by one of ordinary skill in the art based on the content of this disclosure. reg The cells are anti-inflammatory T cells, characterized by expressing the ability to inflammatory T cells. reg Cell surface markers that distinguish cells from other immune cells (e.g., effector T cells, B cells, macrophages, NK cells, etc.). In some embodiments, T cells... reg The cell is CD4 + And it is CD25 + and FOXP3 + At least one of them. In some embodiments, T reg The cell is CD4+ / CD25 + / CD127 低 In some embodiments, T reg The cells are induced T cells reg Cell (iT) reg (Cells). In some embodiments, T reg Cells (e.g., iT) reg Cells) are formed in vitro from initial CD4 + They originate from T cell differentiation. In some embodiments, T cells... reg Cells are T cells in the body reg Cells. The "enhanced T" used in this article reg "Cell" refers to a T cell modified to express (e.g., overexpress) the Siglec-9 protein (as described in this article). reg Cells. In some embodiments, enhanced T cells. reg Cells express (e.g., overexpress) multiple membrane-bound Siglec-9 proteins, and according to this disclosure, interact with unmodified T cells. reg Compared to cells, it exhibits targeted anti-inflammatory activity.

[0065] As used herein, the term "effective amount" refers to the amount of a composition or agent required to provide a desired effect (e.g., cellular response, therapeutic effect, etc.). The term "therapeutic effective amount" refers to an amount of a composition or therapeutic agent sufficient to provide, for example, a specific anti-inflammatory effect when administered to a typical subject. As used herein, a therapeutic effective amount may, in various contexts, include an amount sufficient to delay the development of disease symptoms, alter the course of disease symptoms (e.g., but not limited to slowing the progression of disease symptoms), or reverse disease symptoms. A therapeutic effective amount may be administered in doses of one or more therapeutic agents. A therapeutic effective amount may be administered as a single dose or as multiple doses over a period of time.

[0066] When used in this document, "subject" refers to any vertebrate, including mammals and non-mammals. Subjects may include primates (including humans) and non-primate mammals such as rodents, livestock, or prey. Non-primate mammals may include mice, rats, hamsters, rabbits, dogs, foxes, wolves, cats, horses, cattle, pigs, sheep, goats, camels, deer, buffalo, bison, etc. Non-mammals may include birds (e.g., chickens, ostriches, emus, pigeons), reptiles (e.g., snakes, lizards, turtles), amphibians (e.g., frogs, salamanders), fish (e.g., salmon, cod, pufferfish, tuna), etc. "Individual," "patient," and "subject" are used interchangeably in this document.

[0067] As used herein, “treatment” means the beneficial alteration of one or more signs or symptoms of a condition (e.g., a disease) described herein; improvement of other clinically recognized symptoms, such as an increase of at least 2%, 3%, 4%, 5%, 10%, or more; or induction of a desired response by performing the methods described herein. The effectiveness of treatment can be assessed, for example, by measuring markers, indicators, symptoms, and / or the incidence of the condition treated according to the methods described herein, or any other suitable objective parameter (e.g., ventricular ejection fraction). The effectiveness of treatment can also be measured by the absence of deterioration in an individual, such as by hospitalization or the need for medical intervention (e.g., cessation of disease progression). Treatment includes any treatment of a disease in an individual or animal (some non-limiting examples include humans or animals) and includes: (1) suppressing the disease, such as preventing the worsening of symptoms (e.g., pain or inflammation); or (2) reducing the severity of the disease, such as causing symptom resolution. Therapeutic effective amount for treating a disease means an amount sufficient to achieve effective treatment of the disease as defined herein when administered to a subject in need. The efficacy of a pharmaceutical agent can be determined by assessing physical indicators of the condition or desired response (e.g., cardiac activity). Those skilled in the art can monitor the efficacy of administration and / or treatment by measuring any one or any combination of such parameters.

[0068] When used herein, “application” may include any suitable route of administration of the therapeutic agents or compositions disclosed herein. Suitable routes of administration include, but are not limited to, oral, parenteral, intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), pulmonary, skin, injection, or local application. Application may be local or systemic.

[0069] As used herein, “cardiac” or “heart-” refers to or specifically to the heart and is used to encompass cardiac-related structures, functions, pathologies, and treatments, including but not limited to cardiac tissues, cells (e.g., cardiomyocytes, cardiac stromal cells), physiological processes (e.g., heart rhythm, myocardial contraction), and related treatment methods. The scope of “cardiac” encompasses all modifications, innovations, or technological advancements aimed at diagnosing, treating, or researching cardiac-related conditions and functions.

[0070] As used herein, "fibrosis" has the common and general meaning as understood by one of ordinary skill in the art based on this disclosure, referring to a pathological condition characterized by the excessive accumulation of extracellular matrix components in a tissue or organ. Fibrosis can include any tissue remodeling (e.g., pathological remodeling) (e.g., connective tissue, skeletal muscle, cardiac muscle, skin), such as, but not limited to, the deposition of fibrous tissue and / or adipose tissue, replacement of muscle tissue with fibrous tissue and / or adipose tissue, etc. Fibrosis can include, but is not limited to, inflammatory reactions or inflammatory tissue damage. 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 (cardial fibrosis), and kidneys. As used herein, "fibrosis-related diseases" has the common and general meaning as understood by one of ordinary skill in the art based on this disclosure, referring to medical conditions caused and / or resulting from fibrosis, or conditions leading to fibrosis. For example, some fibrosis-related diseases include idiopathic pulmonary fibrosis, cystic fibrosis, cirrhosis, systemic sclerosis, renal fibrosis, palmar aponeurosis contracture, renal systemic fibrosis, peritoneal fibrosis, retroperitoneal fibrosis, postoperative adhesions, scleroderma, endocardial myocardial fibrosis, radiation-induced fibrosis, Asherman syndrome, pancreatic fibrosis, and Peroni disease. Several cardiac fibrosis-related diseases exist, including but not limited to myocardial fibrosis, cardiac fibrosis, endocardial myocardial fibrosis, hypertensive heart disease, ischemic heart disease, dilated cardiomyopathy, valvular heart disease, congenital heart disease, and arrhythmogenic right ventricular cardiomyopathy. As used herein, "myocardial infarction" has the common and ordinary meaning as understood by one of ordinary skill in the art based on this disclosure, referring to a medical disorder commonly known as a "heart attack," where reduced or obstructed blood flow to a region of the heart restricts oxygen supply to that region, leading to tissue damage or death. Following a myocardial infarction, cardiac fibrosis may occur. Damaged cardiomyocytes are replaced by scar tissue composed primarily of collagen. Because scar tissue has less elasticity than normal cardiomyocytes, it can reduce and / or impair cardiac function.

[0071] As used herein, "fibroblast" has the common and ordinary meaning as understood by one of ordinary skill in the art based on the content of this disclosure, referring to a cell type associated with connective tissue. Fibroblasts most commonly synthesize components of the extracellular matrix, including collagen. Fibroblasts also play an important role in tissue regeneration. Fibroblasts differentiate in response to physiological or pathological conditions in the body, including but not limited to dermal fibroblasts, cardiac fibroblasts, lung fibroblasts, bone marrow fibroblasts, periodontal ligament fibroblasts, adipose-derived fibroblasts, pancreatic fibroblasts, hepatic stellate cells (hepatic fibroblasts), intestinal fibroblasts, and corneal fibroblasts. As used herein, "myofibroblast" has the common and ordinary meaning as understood by one of ordinary skill in the art based on the content of this disclosure, referring to a specialized, contractile fibroblast characterized by expressing α-smooth muscle actin (α-SMA). Myofibroblasts primarily develop during wound healing and other fibrotic responses. As part of the body's innate tissue regeneration process, myofibroblasts secrete extracellular components, including but not limited to collagen and fibronectin. Persistent activation is associated with pathological fibrosis in various organs, including the heart.

[0072] As used herein, "gene expression" has the conventional and common meaning as understood by one of ordinary skill in the art based on this disclosure, referring 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 the production of a gene product from nucleic acids encoding the gene product, including but not limited to the production of mRNA from DNA encoding mRNA, the production of a protein from DNA (e.g., by producing mRNA), and the production of a protein from mRNA encoding the protein. This process includes, but is not limited to, one or more of the following: 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 modification, ribosome binding, ribonucleoprotein complex assembly, protein folding, protein sorting, post-translational modification, phosphorylation, glycosylation, acetylation, ubiquitination, signal peptide cleavage, membrane targeting, protein complex formation, protein localization, protein degradation, lysosome targeting, autophagy, secretion, and subcellular compartmentalization. In some implementations, gene expression can encompass both the natural expression of genes in an organism and the artificial manipulation of gene expression through recombinant DNA technology, gene editing tools, or the use of inducible promoters. This definition includes both quantitative and qualitative aspects of gene expression, as well as its use in a variety of applications, including but not limited to therapeutic protein production, gene therapy, functional genomics research, and synthetic biology.

[0073] As used herein, "overexpression" or "gene overexpression" refers to a gene expression level exceeding its reference level. The reference level can be, but is not limited to, the average or steady-state level of a gene in a specific organism or cell. Overexpression can be achieved through any suitable means, such as (but not limited to) increasing transcription and / or translation levels. Overexpression can be induced or engineered through various means, such as introducing additional gene copies, promoter modifications, and / or gene regulatory interventions. Overexpression encompasses both transient and stable overexpression and may include the upregulation of endogenous genes or the introduction of exogenous genetic elements, with applications ranging from gene function analysis to disease treatment. In some embodiments, overexpression can be achieved through gene modification of cells. In some embodiments, overexpression can be achieved through epigenetic modification of cells.

[0074] As used herein, "Siglecs" refers to sialic acid-binding immunoglobulin-like lectins. Siglecs are a family of cell surface proteins primarily found on the surface of immune cells, involved in cell-cell interactions and signal transduction. As used herein, "Siglec-9" refers to sialic acid-binding immunoglobulin-like lectin 9. In some embodiments, Siglec-9 includes mammalian Siglec-9. In some embodiments, Siglec-9 includes human Siglec-9 (gene ID: 27180). In some embodiments, the Siglec-9 protein has the following amino acid sequence (signal peptide is underlined), regardless of whether a signal peptide is included.

[0075] As used herein, "AOC3" refers to copper-containing amino oxidase 3. AOC3 is interchangeable with vascular adhesion protein-1 (VAP-1). AOC3 is an enzyme crucial to the immune system, for example, by transporting certain immune cell types to areas of inflammation or infection. In some embodiments, AOC3 is mammalian AOC3. In some embodiments, AOC3 includes human AOC3 (gene ID: 8639).

[0076] As used herein, the term "nucleic acid" or "oligonucleotide" refers to a molecule comprising multiple nucleotides (e.g., a molecule containing a sugar (e.g., ribose or deoxyribose) linked to a phosphate group and an exchangeable organic base, wherein the organic base is 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 polynucleotides (i.e., polynucleotides lacking a phosphate group) and any other polymers containing an organic base. 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 occurring and non-natural nucleobases, substituted and unsubstituted aromatic moieties. Nucleic acids may contain any other suitable modifications. Therefore, the term nucleic acid also encompasses nucleic acids with substitutions or modifications (e.g., in the bases and / or sugars).

[0077] The polypeptide or nucleic acid molecules disclosed herein may share a degree of sequence similarity or identity with reference molecules (e.g., reference polypeptides or reference polynucleotides), such as with molecules described in the prior art (e.g., engineered or designed molecules or wild-type molecules). The term "identity," as known in the prior art, refers to the relationship between two or more polypeptide or polynucleotide sequences determined by comparing sequences. In the prior art, identity also refers to the degree of sequence correlation between two or more amino acid residues or nucleic acid residue strings, determined by the number of matches between them. Identity measures the percentage of identical matches between the smaller of two or more sequences, including vacancy alignments (if any) processed by a specific mathematical model or computer program (e.g., an "algorithm"). The identity of related peptides can be readily calculated by known methods. "%identity," when applied 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 to residues in the amino acid or nucleic acid sequence of a second sequence after aligning the sequences and introducing vacancy as needed to achieve the maximum percentage identity. Any suitable alignment method and computer program can be used. It should be understood that identity depends on the calculation of percentage identity, but the value may vary due to gaps and penalties introduced in the calculation. Typically, a variant of a particular polynucleotide or polypeptide has at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% but less than 100% sequence identity relative to that particular reference polynucleotide or polypeptide, determined by sequence alignment procedures and parameters described herein and known to those skilled in the art. Such alignment tools include BLAST suites (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 commonly used local alignment technique is based on the Smith-Waterman algorithm (Smith, TF & Waterman, MS (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, SB & Wunsch. CD (1970) "A general method applicable to the search for similarities in the amino acid sequences of two proteins." J. Mol. Biol. 48:443-453.). Recently, a Fast Optimal Global Sequence Alignment Algorithm (FOGSAA) has been developed, which is claimed to generate global alignments of nucleotide and protein sequences faster than other optimal global alignment methods, including the Needleman-Wunsch algorithm. Other tools are described in this paper, particularly in the definition of "identity" below.

[0078] The term "identity" refers to the overall correlation between aggregated molecules, such as between polynucleotide molecules (e.g., DNA and / or RNA molecules) and / or polypeptide molecules. For example, the percentage identity of two nucleic acid sequences can be calculated by aligning the two sequences for optimal comparison purposes (e.g., vacancies can be introduced in one or both of the first and second nucleic acid sequences for optimal alignment, and non-identical sequences can be ignored for comparison purposes). In some embodiments, the length of the 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. 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, the molecules are identical at that position. The percentage identity between two sequences is a function of the number of common positions shared by the sequences, taking into account the number of vacancies introduced for optimal alignment of the two sequences and the length of each vacancy. Sequence comparison and the determination of the percentage identity between two sequences can be performed using suitable mathematical algorithms. For example, the percentage identity between two nucleic acid sequences can be determined using methods described in the following literature: *Computational Molecular Biology* (edited by Lesk, AM, Oxford University Press, New York, 1988); *Biocomputation: Informatics and the Genome Project* (edited by Smith, DW, Academic Press, New York, 1993); *Sequence Analysis in Molecular Biology* (edited by von Heinje, G., Academic Press, 1987); *Computer Analysis of Sequence Data, Part I* (edited by Griffin, AM and Griffin, HG, Humana Publishers, New Jersey, 1994); and *Introduction to Sequence Analysis* (edited by Gribskov, M. and Devereux, J., M Stockton Publishers, New York, 1991); all of which are incorporated herein by reference. For example, the percentage identity between two nucleic acid sequences can be determined using the algorithm of Meyers and Miller (CABIOS, 1989, 4:11-17), which has been integrated into the ALIGN program (version 2.0) using a PAM120 weighted residue table, a vacancy length penalty of 12, and a vacancy penalty of 4. Alternatively, the percentage identity between two nucleic acid sequences can be determined using the GAP program in the GCG software package and the NWSgapdna.CMP matrix. Methods commonly used to determine the percentage identity between sequences include, but are not limited to, the method disclosed in Carillo, H. and Lipman, D., SIAM J Applied Math., 48:1073 (1988), which is incorporated herein by reference.Techniques for determining homology have been compiled in publicly available computer programs. Exemplary computer software for determining homology between two sequences includes, but is not limited to, the GCG package; Devereux, J. et al., Nucleic Acid Research, 12(1), 387 (1984); BLASTP; BLASTN; and FASTA (Altschul, SF et al., J. Molec. Biol., 215, 403 (1990)).

[0079] The term "isolated" as used herein with respect to isolated biomolecules (e.g., nucleic acids) has the common and conventional meaning as understood by one of ordinary skill in the art when referenced to this disclosure. Isolated biomolecules (e.g., isolated nucleic acids) are typically in unnatural environments or in environments in which they would not otherwise exist without human intervention. In some embodiments, the isolated biomolecules are not located within cells or organisms.

[0080] The phrases “physiologically compatible” and “pharmaceutically acceptable” used in this article are used interchangeably to refer to compounds, materials, compositions and / or dosage forms that, to a reasonable extent of medical judgment, are suitable for contact with human and animal tissues without causing excessive toxicity, irritation, allergic reactions or other problems or complications, and that are commensurate with a reasonable benefit / risk ratio.

[0081] Definitions of commonly used terms in cell biology and molecular biology can be found in: Merck Diagnostics & Therapeutics Manual, 19th edition, Merck Research Laboratories, 2006 (ISBN 0-91 1910-19-0); Robert S. Porter et al. (eds.), Encyclopedia of Molecular Biology, Blackwell Scientific, Ltd., 1994 (ISBN 0-632-02182-9); Benjamin Lewin, Gene X, Jones & Bartlett, 2009 (ISBN-10:0763766321); Kendrew et al. (eds.), Molecular Biology and Biotechnology: A Comprehensive Desk Reference, VCH Publishing Ltd., 1995 (ISBN 1-56081-569-8); and Current Protocols for Protein Science, 2009, Wiley Interdisciplinary Press, edited by Coligan et al.

[0082] The term “and / or” as used herein refers to and covers any and all possible combinations of one or more of the listed related items, as well as cases where no combination is available when interpreted selectively (“or”).

[0083] The singular terms “an,” “a,” and “the” include plural referents unless the context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise. The abbreviation “e.g.” (eg) is used herein to indicate non-limiting examples. Therefore, the abbreviation “eg” is synonymous with the term “e.g.” The term “about” used herein to define molecular weight values ​​and ranges (e.g.) means that the indicated value and / or range limit can vary within ±20%, such as within ±15%, ±10%, or including ±5%. The use of “about” before a number includes the number itself. For example, “about 5” provides explicit support for “5.” Numbers provided in ranges include overlapping ranges and integers within them; for example, ranges of 1–4 and 5–7 include, for example, 1–7, 1–6, 1–5, 2–5, 2–7, 4–7, 1, 2, 3, 4, 5, 6, and 7.

[0084] method This article provides a method for treating conditions associated with inflammation and / or fibrosis (e.g., cardiac fibrosis), comprising administering to a subject in need of treatment for conditions associated with inflammation and / or fibrosis (e.g., cardiac fibrosis) a nucleic acid (e.g., a therapeutically effective amount of nucleic acid) encoding a sialic acid-binding immunoglobulin-like lectin 9 (Siglec-9) protein, wherein the Siglec-9 protein is generated by the subject's regulatory T (T) reg Nucleic acid expression in cells. In some embodiments, administering nucleic acid encoding the Siglec-9 protein includes administering a T cell engineered (e.g., genetically modified) to express (e.g., overexpress) the Siglec-9 protein as described herein. reg Cell populations (e.g., containing T cells) reg (Therapeutic effective amount of the cell population composition). In some embodiments, administering nucleic acid encoding the Siglec-9 protein includes administering to a subject a carrier (e.g., lipid nanoparticles (LNPs)) containing nucleic acid encoding the Siglec-9 protein, wherein the carrier is configured to deliver the nucleic acid encoding the Siglec-9 protein to the subject's T cells. reg Cells, as described herein. In some embodiments, a method of treating fibrosis includes administering to a subject in need of treatment for fibrosis a nucleic acid (e.g., a therapeutically effective amount of nucleic acid) encoding a sialic acid-binding immunoglobulin-like lectin 9 (Siglec-9) protein, wherein the Siglec-9 protein is generated by regulatory T cells in the subject's body. regNucleic acid expression in cells. As used herein, “encoding” means a nucleic acid (e.g., DNA or RNA) that has sequence information for the transcription (e.g., RNA gene product) and / or translation (e.g., protein gene product) of a specified gene product, unless otherwise stated. For example, a DNA molecule “encoding” a protein gene product means that the DNA molecule has nucleotide sequence information for the specified protein gene product (through the sequence information of the RNA gene product (e.g., mRNA) encoded by the DNA molecule).

[0085] The present invention also provides a method for 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 nucleic acid) to a subject in need of treatment for a condition associated with inflammation and / or fibrosis (e.g., cardiac fibrosis), said nucleic acid being configured to induce sialic acid-binding immunoglobulin-like lectin 9 (Siglec-9) protein in the subject's regulatory T(T) reg Expression in cells. In some embodiments, administering the nucleic acid comprises administering a therapeutically effective amount of a composition comprising a T protein engineered (e.g., genetically modified) to express (e.g., overexpress) the Siglec-9 protein as described herein. reg Cell population. In some embodiments, administering the nucleic acid includes administering a carrier containing the nucleic acid (e.g., lipid nanoparticles (LNPs)) to a subject, wherein the carrier is configured to deliver the nucleic acid into the subject's T cells as described herein. reg Cells. In some embodiments, a method of treating fibrosis includes administering to a subject in need of treatment for fibrosis a regulatory T (T) cell configured to induce fibrosis in the subject. reg Nucleic acid (e.g., a therapeutically effective amount of nucleic acid) encoding sialic acid-binding immunoglobulin-like lectin 9 (Siglec-9) protein expressed in cells. In some embodiments, the method includes administering nucleic acid (e.g., a therapeutically effective amount of nucleic acid) encoding sialic acid-binding immunoglobulin-like lectin 9 (Siglec-9) protein to a subject in need of treatment for fibrosis.

[0086] Fibrosis treatable by this method includes, but is not limited to, cardiac, pulmonary, hepatic, and colorectal fibrosis. In some embodiments, the fibrosis is cardiac fibrosis. In some embodiments, cardiac fibrosis is associated with the progression of ischemic cardiomyopathy (ICM).

[0087] In some implementations, in T regThe Siglec-9 protein expressed (e.g., overexpressed) in cells is encoded by a nucleotide sequence that is identical to, or approximately identical to, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 99%, or approximately 100% of SEQ ID NO: 1, or by any two of the aforementioned values ​​within a range (e.g., 75% to 100%, 80% to 99%, 85% to 95%, 87% to 97%, etc.), as provided below: In some embodiments, the Siglec-9 protein is encoded by the nucleotide sequence of SEQ ID NO: 1 (with or without a 5' or 3' UTR). The 5' UTR is a single underscore, and the 3' UTR is a double underscore.

[0088] In some implementations, in T reg The Siglec-9 protein expressed (e.g., overexpressed) in cells has an amino acid sequence that is identical, or approximately identical, or at least 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99%, or approximately 100%, or within a range defined by any two of the foregoing values ​​(e.g., 75% to 100%, 80% to 99%, 85% to 95%, 87% to 97%, etc.) to the SEQ ID NO: 2 (with or without the signal peptide).

[0089] In some embodiments, the Siglec-9 protein has an amino acid sequence that is at least 90% identical to SEQ ID NO: 2 (excluding the signal peptide). In some embodiments, the Siglec-9 protein has an amino acid sequence that is at least 95% identical to SEQ ID NO: 2 (excluding the signal peptide). In some embodiments, the Siglec-9 protein has an amino acid sequence that is at least 98% identical to SEQ ID NO: 2 (excluding the signal peptide). In some embodiments, the Siglec-9 protein has an amino acid sequence that is at least 99% identical to SEQ ID NO: 2 (excluding the signal peptide). In some embodiments, the Siglec-9 protein has the amino acid sequence of residues 18-463 of SEQ ID NO: 2.

[0090] In some implementations, the method includes from relative to T regIn cellular terms, Siglec-9 is expressed or overexpressed in exogenous nucleic acids. In some embodiments, the nucleic acid encodes the Siglec-9 protein or contains a nucleotide sequence encoding the Siglec-9 protein. The nucleic acid encoding the Siglec-9 protein can be any suitable nucleic acid. In some embodiments, the nucleic acid contains RNA (e.g., mRNA) encoding the Siglec-9 protein. In some embodiments, the nucleic acid contains DNA encoding the Siglec-9 protein.

[0091] In some implementations, the method includes starting from T reg The cell's endogenous (e.g., genomic) sites express or overexpress siglec-9. In some embodiments, the nucleic acid encodes an epigenetic regulatory factor configured to induce T... reg Endogenous siglec-9 sites in cells (e.g., genomic loci of the siglec-9 gene) inhibit the expression (e.g., overexpression) of the siglec-9 protein. Any suitable epigenetic regulator can be used to induce T... reg Expression of the Siglec-9 protein at the endogenous siglec-9 site in the cell (e.g., overexpression). Suitable options include, but are not limited to, CRISPR activation systems configured to target the siglec-9 locus.

[0092] In some embodiments, the nucleic acid is contained in a vector (e.g., a plasmid, a viral vector, etc.). In some embodiments, the vector is a plasmid (e.g., a lentiviral vector). For example, the plasmid may contain a lentiviral vector (e.g., pLenti-GIII-CMV). In some alternative embodiments, the vector may be an adenovirus vector, an adeno-associated virus (AAV) vector, a retroviral vector, a transposon vector, and similar vectors known to those skilled in the art.

[0093] Reference Figure 1 This provides a non-limiting embodiment of the present disclosure. A method 100 for treating fibrosis (e.g., cardiac fibrosis) includes delivering a nucleic acid 120 encoding a sialic acid-binding immunoglobulin-like lectin 9 (Siglec-9) protein to a population of immune cells (e.g., T cells). reg The method includes obtaining at least one vector 110 containing a nucleic acid encoding Siglec9; and obtaining at least one vector containing a regulatory T (T) reg ) Immune cells of cell line 101. The nucleic acid encoding Siglec9 can be found in T cells. reg The Siglec9 protein is expressed in cells. In some embodiments, the method includes delivering the vector to T cells. reg Cell population. In some embodiments, the T regThe cell population is located within the subject's body, and delivery of the carrier includes administering the carrier to the subject. In some embodiments, the T... reg The cell population is provided in vitro. In some embodiments, the method includes attaching at least a portion of the at least one carrier 110 to the at least one T cell. reg Cells 101 are contacted (e.g., in vivo or in vitro) for a period of time, wherein the contact-inducing carrier 110 enters T cells. reg Cytoplasm 102 of cell 101. In some embodiments, the vector 110 enters T... reg After the cytoplasm of cell 101, the vector can be derived from T reg The cell membrane 130 encapsulates the carrier 110. Subsequently, the membrane 130 encapsulating the carrier 110 may degrade, followed by the degradation of the carrier 115, releasing the encapsulated nucleic acid 121 into the cytoplasm 102. At least one nucleic acid 121 in the cytoplasm can interact with at least one ribosome 140. A complex of nucleic acid 122 and ribosome 140 is then formed, followed by translation of nucleic acid 122, producing at least one polypeptide precursor 150, including but not limited to a Siglec-9 polypeptide precursor. Post-translational modifications of the polypeptide precursor 150 may then occur, leading to further modifications and T... reg Cellular remodeling leads to, for example, at least one Siglec-9 protein 160 in T cells. reg Expression on the cell membrane. In some embodiments, nucleic acids induce Siglec-9 protein expression (e.g., functional expression) at levels higher than T. reg The expression level of native Siglec-9 protein in cells. In some embodiments, Siglec-9 is expressed in modified T cells. reg Overexpression in cells. 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 fibroblasts, cardiac fibroblasts, or cardiomyocytes, wherein the enhanced targeting of the target cell 170 is due to the specificity of Siglec-9 160 for a copper-containing amine oxidase 3 (AOC3) protein 180 that modifies the membrane of the target cell 170. In some embodiments, the target cells express AOC3 at higher levels compared to non-target cells and / or cells that have not been induced to upregulate fibrosis genes.

[0094] In addition, a method for subject-targeted immune modulation is provided, including: obtaining regulatory T cells (T cells). reg ) group; to the T reg Cell population delivery of at least one vector (e.g., delivery vector), wherein at least one vector contains nucleic acid encoding sialic acid-binding immunoglobulin-type lectin-9 (Siglec-9) protein, and Siglec-9 is generated by the T regNucleic acid expression in the cell population, resulting in enhanced T cells overexpressing Siglec-9. reg Cell populations; and administration of the enhanced T to the subject reg Cell population. Additionally, a method for subject-targeted immune modulation is provided, including: obtaining regulatory T cells (T cells). reg ) group; to the T reg Cell population delivery of at least one vector, wherein the at least one vector contains nucleic acid configured to induce the expression of sialic acid-binding immunoglobulin-type lectin-9 (Siglec-9) protein, thereby generating enhanced T cells overexpressing Siglec-9. reg Cell populations; and administration of the enhanced T to the subject reg Cell population. In some embodiments, the nucleic acid encodes T cells. reg The Siglec-9 protein expressed in cells. In some embodiments, the nucleic acid encodes a T-cell induction protein. reg Epigenetic regulators of expression at the endogenous Siglec-9 locus in cells (e.g., CRISPR activation system).

[0095] In some embodiments, targeted immunomodulation includes selective immunosuppression of any suitable target tissue with upregulated expression of fibrosis genes (e.g., due to inflammation, tissue damage, etc.). In some embodiments, enhanced T cells... reg Cell-targeted tissues include, but are not limited to, heart, lung, liver, and / or colorectal tissue. In some embodiments, targeting immune regulation includes by... reg Cells target cardiomyocytes expressing AOC3 membrane-binding protein, thereby selectively suppressing the heart tissue.

[0096] In any of the methods disclosed herein, in some embodiments, the vector (or delivery vector) is used to deliver nucleic acids to T. regAny suitable carrier for cells. In some embodiments, the carrier includes, but is not limited to, liposomes, nanoparticles, lipid nanoparticles (LNPs), viral vectors, cell-penetrating peptides (CPPs), and / or extracellular vesicles (EVs). In some embodiments, the carrier comprises a polyethylene glycol (PEG)-cationic lipid complex (PCLC). In some embodiments, the viral vector includes adenovirus vectors, retroviral vectors, or lentiviral vectors. In some embodiments, the carrier is a transfection reagent. In some embodiments, the transfection reagent comprises one or more of liposomes, lipid nanoparticles (LNPs), extracellular vesicles (EVs), and polyethylene glycol (PEG)-cationic lipid complex (PCLCs). In some embodiments, the transfection reagent comprises EVs derived from cardioglobulin-derived cells (CDCs). In some embodiments, the nucleic acid comprises a promoter operatively linked to a nucleotide sequence encoding Siglec-9, wherein the promoter upregulates T... reg Expression of Siglec-9 in cells. In some embodiments, the nucleic acid contains a promoter operatively linked to a nucleotide sequence encoding an epigenetic regulator (e.g., a CRISPR activation system) configured to induce T... reg Siglec-9 expression from an endogenous locus in cells. When nucleic acids are integrated into cells (e.g., as described herein), this promoter can be used for expression in T cells. reg Any suitable promoter for expressing the Siglec-9 protein in the cell. In some embodiments, the promoter is a regulatory T(T) promoter. reg Cell-specific promoters. In some implementations, T... reg The cell-specific promoter is selected from the Foxp3 promoter and the 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 tetracycline-responsive promoter).

[0097] In any method of this disclosure, in some embodiments, the method includes engineered T reg Cells can be engineered or enhanced T cells by expressing (e.g., overexpressing) the siglec-9 protein. reg Cells. T reg Cells can be engineered using any suitable method. In some embodiments, the method includes delivering the nucleic acid of this disclosure to T cells. reg Cells. Nucleic acids can be delivered to T cells using any suitable delivery vector protocol. reg Cells. In some implementations, engineered T cells. reg Cells include those that, under suitable conditions, deliver the nucleic acids of this disclosure to T cells. reg Cells, to induce T regExpression of the siglec-9 protein in cells. In some implementations, nucleic acids are delivered to T... reg Cells include, under suitable conditions, allowing an effective amount of carrier to interact with T cells. reg Cell population contact. At least one carrier is brought into contact with at least one T cell. reg Cell contact can be accomplished using any suitable method. In some embodiments, contact is performed in vitro or ex vivo. In some embodiments, contact includes maintaining or intending to maintain T cells. reg Various carriers are provided in the cell culture medium. In some embodiments, contact may involve supplying or maintaining T cells. reg A certain amount of carrier is added to the cell culture medium; this amount can be expressed as concentration, molar concentration, or quantity. In some embodiments, the carrier can be added to the culture medium of T cells. reg The vector is provided in the culture medium at the start of cell culture. In some embodiments, the vector may be added periodically during culture. In some embodiments, the method includes, but is not limited to, the manipulation of T cells via transfection, transduction, electroporation, gene editing (e.g., CRISPR Cas9), etc. reg Cells undergo gene modification to enable Siglec-9 to function in T cells. reg Overexpression in cells. In some embodiments, the method includes transducing T cells with a viral vector containing nucleic acid encoding Siglec9. reg Cells. In some embodiments, the method includes using cells configured to induce T cells. reg Viral vector transduction of Siglec-9 protein-expressing nucleic acids (as provided in this article) in cells reg cell.

[0098] T can be made at any suitable time period reg Cells come into contact with the carrier. In some implementations, the contact time period can be T. reg The amount of time cells are cultured in the presence of a carrier in the culture medium. In some implementations, the contact time is T. reg The amount of time the cells are cultured in a medium containing the carrier. In some embodiments, the time period 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 within the range defined by any two of the foregoing values.

[0099] In some implementations, co-cultivation may include T reg Multiple carriers are provided in the culture medium in which the cells are being or will be maintained. In some embodiments, co-culture may involve introducing T... regA certain amount of carrier is added to the culture medium in which the cells are being or will be maintained. In some embodiments, this carrier is added at the beginning of T cell culture. reg When cells are cultured, a carrier is provided in the culture medium. In some embodiments, the carrier may be added at fixed time intervals during culture.

[0100] The carrier can be combined with T reg Cells are co-cultured for a suitable period of time. In some implementations, the co-culture period is T. reg The length of time cells are cultured in a medium containing the carrier. In some embodiments, the co-culture period can be T. reg The amount of time the cells are cultured in a medium with the carrier added. In some embodiments, the time period may 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 duration within the range defined by any two of the foregoing values.

[0101] In some implementations, a suitable amount of carrier may be provided to T reg Cells exposed to or co-cultured with multiple vectors induced Siglec-9 overexpression and / or increased proliferation. The T provided in the culture medium... reg The cell-to-vector ratio can be any suitable ratio. In some embodiments, the number of vectors can be T. reg The number of cells is at least about 30 times, about 40 times, about 50 times, about 75 times, about 100 times, about 125 times, about 150 times, about 200 times, about 300 times, about 400 times, about 500 times, about 600 times, about 700 times, about 800 times, about 900 times, about 1000 times, about 1200 times, about 1500 times, about 2000 times, about 3000 times, about 4000 times, about 5000 times, about 7500 times, about 10000 times, or more, or a multiple within the range defined by any two of the foregoing values. In some embodiments, the provided vector and T reg The cell ratio is approximately 1:1000. In some embodiments, this ratio is approximately 1:100, approximately 1:200, approximately 1:300, approximately 1:400, approximately 1:500, approximately 1:600, approximately 1:700, approximately 1:800, approximately 1:900, approximately 1:1000, approximately 1000:1, approximately 900:1, approximately 800:1, approximately 700:1, approximately 600:1, approximately 500:1, approximately 400:1, approximately 300:1, approximately 200:1, approximately 100:1, or any ratio between the listed values. In some embodiments, contact involves mixing an effective amount of vector (e.g., a viral vector) with T cells at a suitable multiplicity of infection (MOI). regCell contact. 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 alternatively, the MOI is within a range defined by any two of the foregoing values ​​(e.g., 0.1-100, 0.1-10, 0.2-20, 1-5, 1-3, etc.).

[0102] In some implementations, contact is performed within the body. In other implementations, contact is achieved within the subject, for example, by administering multiple carriers to the subject, wherein the carriers contain T molecules that bind to them. reg Cell-targeting component. In some embodiments, the method includes administering a vector containing nucleic acid encoding the Siglec-9 protein to a subject, wherein the vector is configured to deliver the nucleic acid encoding the Siglec-9 protein to the subject's T cells. reg Cells. In some embodiments, the vector includes a nucleic acid encoding the Siglec-9 protein, which, in the T cells of the subject... reg The Siglec-9 protein is specifically expressed in cells. Any suitable option can be used for T in the subject. reg The cell specifically expresses the Siglec-9 protein encoded by a nucleic acid. In some embodiments, the nucleic acid encoding the Siglec-9 protein is operatively linked to T... reg Cell-specific promoters (e.g., Foxp3 or IL-2 promoters). In some embodiments, the method includes administering a vector containing nucleic acids to a subject, the nucleic acids being configured to induce T cells. reg Expression of Siglec-9 protein in cells (e.g., endogenous or exogenous expression), wherein the vector is configured to deliver nucleic acids to the T cells in the subject. reg Cells. In some embodiments, the vector comprises a nucleic acid configured to specifically induce Siglec-9 protein expression (e.g., endogenous or exogenous expression). Any suitable option may be used via T cells in the subject. reg Nucleic acids in cells specifically induce the expression of the Siglec-9 protein (e.g., endogenous or exogenous expression). In some embodiments, the nucleic acid configured to specifically induce Siglec-9 protein expression (e.g., endogenous or exogenous expression) is operatively linked to T... reg Cell-specific promoters (e.g., Foxp3 or IL-2 promoters).

[0103] In some embodiments, the carrier comprises: lipid nanoparticles (LNPs); and T molecules bound to the LNPs. regCell-targeting component. In some implementations, T reg Cell-targeting components include binding to T reg Antigen-binding proteins of cell markers. Any suitable T reg Cell markers can be used to target vectors to T cells in subjects. reg Cells, such as, but not limited to, anti-CD25 monoclonal antibodies and / or anti-Foxp3 monoclonal antibodies. In some embodiments, the antigen-binding protein is a T-cell binding protein. reg Antibodies or antigen-binding fragments of cell markers. In some embodiments, the vector (e.g., LNP) binds to T... reg The vector is conjugated to an antibody or its antigen-binding fragment that is a cell marker, thereby targeting the T cells in the subject's body. reg cell.

[0104] In some embodiments, the method includes obtaining T reg The cell's immune cell population; delivery of nucleic acid encoding sialic acid-binding immunoglobulin-like lectin 9 (Siglec-9) protein to T cells. reg Cells, thereby enabling the Siglec-9 protein to function in T cells. reg Overexpression in cells; causing T cells to overexpress Siglec-9 protein reg Cells are contacted with at least one fibroblast and / or myofibroblast in the subject's body, wherein the at least one fibroblast and / or myofibroblast expresses copper-containing amino oxidase 3 (AOC3), thereby treating conditions associated with inflammation and / or fibrosis. In some embodiments, nucleic acids encoding the Siglec-9 protein are configured to cause the Siglec-9 protein to... reg Overexpression in cells. In some embodiments, this population of immune cells is obtained from the subject (e.g., for subsequent administration of autologous T cells). reg (Cells). In some embodiments, the immune cell population is allogeneic relative to the subject.

[0105] Engineering T reg Cells and / or vectors containing the said nucleic acid can be administered to the subject in any suitable manner. Administration can be local or systemic. In some embodiments, administration is parenteral. Suitable routes of administration include, but are not limited to, intravenous, intramuscular, subcutaneous, intra-arterial, intraperitoneal, or oral administration. In some embodiments, engineered T cells... reg Cells and / or vectors containing the said nucleic acid can be administered intravenously. In some embodiments, engineered T cells... reg Cells and / or vectors containing the said nucleic acids can be administered by infusion. In some embodiments, administration includes intravenous or oral administration.

[0106] Any appropriate amount of engineered T can be administered to the subject. reg Cells. In some embodiments, the method includes administering a therapeutically effective amount of engineered T cells to a subject. reg Cells. In some embodiments, the method includes administering approximately 10 [units of something] to the subject. 5 10 6 10 7 10 8 10 9 10 10 10 11 10 12 One or more engineered T reg Cells, or the number of cells within a range defined by any two of the aforementioned values ​​(e.g., approximately 10). 5 One to 10 12 10 cells, 10 6 One to 10 11 10 cells, 10 7 One to 10 10 (e.g., cells). In some embodiments, the method includes administering approximately 10 (cells, etc.) to the subject. 3 cells / kg, 10 4 cells / kg, 10 5 cells / kg, 10 6 cells / kg, 10 7 cells / kg, 10 8 cells / kg, 10 9 cells / kg, or 10 10 engineered T cells / kg body weight reg Cells, or a body weight-based quantity defined by any two of the aforementioned values ​​(e.g., approximately 10). 3 One to 10 10 cells / kg, 10 4 One to 10 9 cells / kg, 10 5 One to 10 8 (cells / kg, etc.). In some embodiments, the method includes using 10 6 One to 10 9 Engineered T cells were administered to the subject at a dose of 1 cell / kg body weight. reg Cells. In some embodiments, the method includes using 10 6 One to 10 8 Engineered T cells were administered to the subject at a dose of 1 cell / kg body weight. reg Cells. In some embodiments, the method includes using about 10 7 Engineered T cells were administered to the subject at a dose of 1 cell / kg body weight. reg cell.

[0107] In some embodiments, the method includes administering a therapeutically effective amount of nucleic acid (e.g., as a cell-free composition) to a subject. In some embodiments, a therapeutically effective amount of 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 amounts within the range defined by any two of the foregoing values ​​(e.g., 0.01 µg to 0.1 µg, 0.1 µg to 1 µg, 1 µg to 10 µg, 10 µg to 100 µg, 100 µg to 1 mg, 1 mg to 10 mg, 10 mg to 100 mg). In some embodiments, the therapeutically effective amount of nucleic acid includes approximately 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 body weight or more, or amounts within a range defined by any two of the foregoing values ​​(e.g., 0.001 mg / kg). mg / kg to 0.01 mg / kg, 0.01 mg / kg to 0.1 mg / kg, 0.1 mg / kg to 1 mg / kg, 1 mg / kg to 10 mg / kg, 10 mg / kg to 100 mg / kg).

[0108] According to some embodiments of this disclosure, the Siglec-9 protein can be used in engineered T cells. reg It is expressed in cells at any level suitable for the treatment subject, and typically at a level higher than unmodified T. reg The level of Siglec-9 protein expression in cells (e.g., cells not modified to express or overexpress the Siglec-9 protein described herein). In some embodiments, compared with a suitable reference (e.g., T cells not modified with nucleic acids). reg Compared to cells, through engineered T cells reg The expression of Siglec-9 protein in cells increases by approximately or at least 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2.0-fold, 2.1-fold, 2.2-fold, 2.3-fold, 2.4-fold, 2.5-fold, 3-fold, 3.2-fold, 3.5-fold, 4-fold, 4.2-fold, 4.5-fold, 5-fold, 5.2-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more, or the increase is within the range defined by any two of the foregoing values ​​(e.g., 1.5-fold to 10-fold, 2-fold to 8-fold, 3-fold to 6-fold, 4-fold to 6-fold, 1.5-fold to 2.5-fold, 1.6-fold to 2.4-fold, 1.7-fold to 2.3-fold, 1.8-fold to 2.2-fold, etc.). In some embodiments, with a suitable reference (e.g., unmodified T... reg Compared to cells, through engineered T cells reg Nucleic acid expression in cells increases Siglec-9 protein expression by approximately 1.5–2.5 times. In some embodiments, compared with a suitable reference (e.g., unmodified T...),... reg Compared to cells, through T reg Nucleic acid expression in cells increases Siglec-9 protein expression by 2 to 8 times. In some embodiments, compared with a suitable reference (e.g., unmodified T... reg Compared to cells, through engineered T cells reg Nucleic acid expression in cells increases Siglec-9 protein expression by approximately 2-fold (e.g., approximately 1.9-fold). In some embodiments, compared with a suitable reference (e.g., unmodified T... reg Compared to cells, through T reg Nucleic acid expression in cells increases Siglec-9 protein expression by approximately 5-fold. In some embodiments, the method disclosed herein can promote T... reg Cells express the Siglec-9 protein. As described herein, T cells can be contacted with or co-cultured with a delivery vector containing the nucleic acid disclosed herein. reg Cellular expression levels, and appropriate T reg The expression levels of Siglec-9 protein in cell control groups were compared. In some embodiments, T regThe control group of cells consisted of T cells that had not been exposed to or co-cultured with the vector. reg Cells. In some embodiments, engineered T cells. reg An increase in Siglec-9 protein expression in cells of approximately or 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 an increase within the range defined by any two of the above values, compared to a suitable T... reg Cell control groups (e.g., T cells not exposed to or co-cultured with the vector) reg (cells). In some embodiments, engineered T reg The expression level of Siglec-9 protein in cells was lower than that in immune cell populations that were not exposed to or co-cultured with the vector (e.g., T cells). reg The production of Siglec-9 protein in cells was at least 1.5 times higher. In some embodiments, engineered T cells... reg The expression level of Siglec-9 protein in cells was lower than that in immune cell populations that were not exposed to or co-cultured with the vector (e.g., T cells). reg The expression level of Siglec-9 protein in the cells was at least 2-fold higher. In some embodiments, engineered T cells... reg The expression level of Siglec-9 protein in cells was lower than that in immune cell populations that were not exposed to or co-cultured with the vector (e.g., T cells). reg The expression level of Siglec-9 protein in the cells was 4 to 6 times higher. In some embodiments, engineered T cells... reg The expression level of Siglec-9 protein in cells was lower than that in immune cell populations that were not exposed to or co-cultured with the vector (e.g., T cells). reg The expression level of Siglec-9 protein in cells is approximately 5-fold higher. Expression levels can be determined using any suitable method as needed. In some embodiments, expression levels are based on whole-cell expression levels. In some embodiments, expression levels are based on cell surface expression levels. In some embodiments, expression levels are determined by methods including, but not limited to, Western blotting, ELISA, flow cytometry, and fluorescence microscopy.

[0109] Also provided is a method for treating conditions associated with inflammation and / or fibrosis, comprising administering to a subject in need of treatment for conditions associated with inflammation and / or fibrosis a substance configured to induce a subject's regulatory T (T) reg The nucleic acid expresses a membrane-bound fibroblast-specific binding protein in the cell. In some embodiments, the nucleic acid encodes a membrane-bound fibroblast-specific binding protein. In some embodiments, the nucleic acid encodes an epigenetic regulator (e.g., a CRISPR activation system) that induces the expression of the membrane-bound fibroblast-specific binding protein from T cells. regEndogenous gene loci expression in cells. In some embodiments, a method of treating conditions 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 treatment for conditions associated with inflammation and / or fibrosis, wherein the membrane-bound fibroblast-specific binding protein is expressed by the nucleic acid in the subject's regulated T(T) reg It is expressed in cells. As used in this article, "membrane-bound fibroblast-specific binding protein" refers to a protein that can be expressed by T cells. reg Cellular expression, usually located on the plasma membrane, and can promote T reg Protein constructs for cell-fibroblast interactions. In some embodiments, the protein constructs are known to bind to fibroblast-specific cell surface markers or target proteins. In some embodiments, subject-regulated T(T) reg The expression or overexpression of membrane-bound fibroblast-specific binding proteins in cells can promote T cells. reg Anti-inflammatory activity of cells at target tissues designated by membrane-bound fibroblast-specific binding proteins. These membrane-bound fibroblast-specific binding proteins can bind any suitable fibroblast-specific target protein, which is a marker of inflammation and / or fibrosis-related conditions in the target tissue. In some embodiments, the fibroblast-specific target protein is a gene or protein 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 identical, approximately identical, or at least 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99%, or approximately 100% identical to SEQ ID NO: 2 (with or without the signal peptide), or alternatively, the percentage of identity of the amino acid sequence to SEQ ID NO: 2 is within a range defined by any two of the above values ​​(e.g., 75% to 100%, 80% to 99%, 85% to 95%, 87% to 97%, etc.). In some implementations, the membrane-bound fibroblast-specific binding protein is Siglec-9.

[0110] The target tissue can be any suitable tissue of the subject. In some embodiments, the target tissue includes, but is not limited to, heart, lung, brain, peripheral nervous system, liver, spleen, kidney, pancreas, bladder, muscle, stomach, intestine, colon, testis, prostate, ovary, uterus, and skin tissue. In some embodiments, the target tissue includes heart, lung, liver, or colorectal tissue. In some embodiments, the tissue is heart tissue. In some embodiments, the fibrosis gene is expressed (or upregulated) in the target tissue. In some embodiments, the expression of the fibrosis gene in the target tissue is caused by (or associated with) an autoimmune response or disease. In some embodiments, the target tissue is heart tissue affected by the proliferation of fibroblasts and / or cardiomyocytes. In some embodiments, the target tissue is affected by 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 expression levels of one or more fibroblast-specific markers are elevated in the cells of the target tissue compared to non-pathological or healthy tissue. In some embodiments, the target tissue contains a greater proportion of cells expressing one or more fibroblast-specific markers compared to non-pathological or healthy tissue. In some embodiments, the target tissue exhibits pathological expression of AOC3 membrane-binding protein (e.g., overexpression at the cellular level and / or expression by a greater proportion of cells in the target tissue). In some embodiments, the target tissue comprises cells expressing (e.g., overexpressing) AOC3 membrane-binding protein. In some embodiments, the target tissue includes cardiac myofibroblasts exhibiting pathological AOC3 expression (e.g., overexpression at the cellular level and / or expression by a greater proportion of cells in the target tissue). In some embodiments, the target tissue includes cardiac myofibroblasts expressing (e.g., overexpressing) AOC3. In some embodiments, the target tissue includes lung myofibroblasts exhibiting pathological AOC3 expression (e.g., overexpression at the cellular level and / or expression by a greater proportion of cells in the target tissue). In some embodiments, the target tissue includes lung 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 with pathological expression of AOC3 and NKX2-3 (e.g., overexpression at the cellular level and / or expression in a larger proportion of cells within the target tissue). In some embodiments, the target tissue includes colorectal myofibroblasts expressing (e.g., overexpressing) AOC3 and NKX2-3.

[0111] In some embodiments, fibroblast-specific target proteins include, but are not limited to, type I collagen, α-smooth muscle actin (α-SMA), and copper-containing amine oxidase 3 (AOC3). In some embodiments, membrane-bound fibroblast-specific binding proteins include sialic acid-binding immunoglobulin-like lectin 9 (Siglec-9). In some embodiments, diseases associated with inflammation and / or fibrosis include inflammatory tissue (e.g., inflamed cardiac tissue) containing fibroblasts (e.g., cardiac myofibroblasts) with elevated copper-containing amine oxidase 3 (AOC3) expression. AOC3 may be generated by enhanced or modified T... reg The cells or tissues targeted by the cell express AOC3 at any suitable level. In some embodiments, AOC3 expression levels in target cells or tissues are approximately 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, 10-fold, 10.5-fold, 11-fold, 11.5-fold, 12-fold, or higher, or higher folds within the range defined by any two of the foregoing values ​​(e.g., 4-fold to 12-fold, 5-fold to 11-fold, 6-fold to 10-fold, 7-fold to 9-fold, etc.). In some embodiments, AOC3 expression levels in target cells or tissues are approximately 9.0-fold to 9.5-fold (e.g., 9.3-fold higher) higher than a suitable reference (e.g., non-fibrotic cardiac myofibroblasts or cardiac tissue).

[0112] A method for inhibiting fibrosis gene expression is also provided, including: obtaining T reg Cell population; engineered T reg Cells overexpress Siglec-9, thereby producing enhanced T cells. reg Cells; and administration of this enhanced T to subjects requiring inhibition of fibrosis gene expression in cardiac myofibroblasts. reg Cells (or an effective amount of this enhanced T) reg (Cells). In some embodiments, the fibrosis gene is at least one of type I collagen, α-smooth muscle actin (α-SMA), and / or copper-containing amino oxidase 3 (AOC3). In some embodiments, the subject requiring inhibition of fibrosis gene expression in cardiac myofibroblasts is a subject requiring treatment for cardiac inflammation and / or cardiac fibrosis. In some embodiments, the cardiac inflammation and / or cardiac fibrosis is secondary to myocardial infarction. In some embodiments, the cardiac inflammation and / or cardiac fibrosis is associated with ICM. In some embodiments, the subject requiring inhibition of fibrosis gene expression in cardiac myofibroblasts is a subject requiring treatment for myocardial infarction, hypertension, myocarditis, transplant rejection, cardiomyopathy, heart failure, and amyloidosis.

[0113] In some embodiments, the engineering includes delivering at least one carrier containing substances configured to induce the T reg The nucleic acid expressing the Siglec-9 protein in a cell population. In some embodiments, engineering includes introducing nucleic acids into T cells. reg Cell population delivery of at least one vector containing a nucleic acid encoding the Siglec-9 protein. In some embodiments, engineering includes delivering at least one vector containing a nucleic acid encoding an epigenetic regulator (e.g., a CRISPR activation system), said nucleic acid being configured to be derived from T cells. reg The cell's endogenous gene locus induces the expression of the Siglec-9 protein. In some embodiments, engineering includes methods sufficient to deliver nucleic acids to T cells. reg Under cellular conditions, an effective amount of the at least one carrier is reacted with T reg Cellular contact. In some embodiments, the at least one carrier comprises at least one of liposomes, nanoparticles, lipid nanoparticles (LNPs), viral vectors, cell-penetrating peptides (CPPs), and / or extracellular vesicles (EVs). In some embodiments, overexpression of Siglec-9 includes upregulating the expression of mRNA encoding Siglec-9. In some embodiments, the subject requires treatment for fibrosis (e.g., cardiac fibrosis). In some embodiments, the subject needs treatment for cardiac fibrosis due to or secondary to myocardial infarction. In some embodiments, the subject needs treatment for cardiac fibrosis associated with ICM.

[0114] The method of the present invention, as described herein, can effectively address the generation of fibroblasts and myofibroblasts caused by tissue trauma (e.g., cardiac tissue). In some embodiments, cardiac tissue trauma may be caused by myocardial infarction. In some embodiments, the proliferation of fibroblasts and myofibroblasts (e.g., cardiac myofibroblasts) may occur after or concurrently with myocardial infarction. As used herein, the generation of fibroblasts and myofibroblasts after myocardial infarction refers to a process that occurs or continues after the initial cardiac event. In some embodiments, the increased activity of fibroblasts and myofibroblasts may be a response to damage caused by myocardial infarction, which may lead to decreased cardiac function or fibrosis.

[0115] Various Ts can be used in the methods and compositions disclosed herein. reg Cells. In some implementations, T reg Cells are human T cells reg Cells. In some implementations, T reg Cells can be obtained from individuals (e.g., donors (e.g., for allogeneic use) or patients (e.g., for autologous use)). T cells can be obtained from individuals using any suitable method. regCells. Immune cells (e.g., T cells) used in the methods disclosed herein. reg Immune cells can be isolated from biological samples taken from mammalian subjects (e.g., human subjects), and their sources are diverse, including, for example, peripheral blood (e.g., peripheral blood mononuclear cells), bone marrow, thymus, tissue biopsy, tumors, lymph node tissue, intestinal-associated lymphoid tissue, mucosa-associated lymphoid tissue, spleen tissue, or any other lymphoid tissue and tumor. In some embodiments, immune cells can be isolated as peripheral blood mononuclear cells (PBMCs) from blood samples obtained from the peripheral blood of the subject. Immune cells (e.g., T cells) reg Cells can also be obtained from blood units obtained through apheresis or leukapheresis procedures.

[0116] In some implementations, T cells can be enriched from a population of immune cells (e.g., immune cells from a peripheral blood sample of a subject). reg Cells to obtain T reg Cell population. T cells can be obtained using any suitable method. reg Cells (e.g., enriched T cells) reg Cell populations), such as those described herein, use one or more markers to sort T cells. reg Cells. Any suitable method can be used to enrich T cells in the immune cell population. reg Cells. In some embodiments, T cells can be isolated from an immune cell population. reg Cells. In some implementations, T cells can be removed from the immune cell population. reg Extracellular immune cells (e.g., CD8) + T cells). In some embodiments, enrichment includes cell sorting based on the expression of cell markers (e.g., cell surface markers) within the immune cell population to differentiate between different types of immune cells. In some embodiments, T cells may be enriched based on the expression of at least CD4 and CD25. reg Cells are sorted. This sorting may include, but is not limited to, flow cytometry or magnetic sorting. In some embodiments, T... reg The cells are CD4 + CD25 + In some implementations, T reg The cells are CD4 + CD25 + CD127 低 In some implementations, CD4 in the cluster can be enriched. + FOXP3 + Cells. In some implementations, T reg Cells can be obtained or isolated from immune cell populations (e.g., immune cells from a subject's peripheral blood sample) and expanded in vitro.

[0117] As used herein, a "+" signifies a positive result for a specific biomarker, which, according to some embodiments, indicates that the expression level of that biomarker is above a detectable threshold. In several embodiments, the expression is upregulated compared to T cells that have not undergone the methods disclosed herein.

[0118] The subject can be any suitable individual or patient who needs treatment for a disease related to inflammation and / or fibrosis. In some embodiments, the subject has the disease or is suffering from the consequences of the disease. In some embodiments, the subject has been diagnosed with a disease related to inflammation and / or fibrosis. In some embodiments, the subject is at risk of developing the disease. In some embodiments, the subject is at risk of developing the disease based on one or more of their family history, genetic susceptibility, lifestyle, and medical history. In some embodiments, the subject has had or is at risk of having a myocardial infarction. In some embodiments, the subject needs treatment for cardiac fibrosis following a myocardial infarction. In some embodiments, the subject needs treatment for cardiac fibrosis associated with ICM. In some embodiments, the subject has at least one myofibroblast with elevated AOC3 expression compared to other cell types (and / or compared to myofibroblasts without upregulated fibrosis gene expression).

[0119] In some embodiments, as disclosed herein, T cells are contacted or co-cultured with at least one vector and / or multiple vectors. reg Overexpression and / or enhanced production of Siglec-9 in cells can promote the regulation of fibroblast and myofibroblast proliferation. When T cells overexpress Siglec-9... reg Cells (i.e., enhanced T cells) reg When these T cells are administered to subjects, reg Cells can influence the behavior of fibroblasts and myofibroblasts in target tissues. In some implementations, enhanced T cells... reg Cells can reduce the expression of fibrosis genes, thereby inhibiting myofibroblast transdifferentiation and reducing fibrosis. This regulatory effect can be compared with a suitable control, such as after administration of T... reg The proliferation levels of fibroblasts and myofibroblasts in the target tissue prior to cell exposure, or those not treated with enhanced T cells. reg The expected or average level of such cell proliferation in patients with these cells.

[0120] In some implementations, enhanced T is administered to the subject. reg Cells can reduce the proliferation of fibroblasts and / or myofibroblasts in target tissues, which can be a response to tissue damage or stress. In some further embodiments, enhanced T cells... regFollowing application, the cells can improve the function of target tissues impaired by fibroblast and myofibroblast proliferation. This improvement in tissue function can be assessed by comparison with appropriate controls, such as those administered with enhanced T cells. reg The average function of the target tissue prior to cell proliferation, or the average function of tissue impaired by excessive proliferation of fibroblasts and myofibroblasts.

[0121] In some further embodiments, the enhanced T reg Cellular administration can restore or improve cardiac function in patients with diseases involving abnormal fibroblast and myofibroblast activity, such as cardiac remodeling after myocardial infarction. Cardiac function can be assessed by parameters including, but not limited to, the patient's ejection fraction (e.g., left ventricular ejection fraction).

[0122] In some embodiments, the production of fibroblasts may be secondary to or accompanied by wound healing, tissue repair, inflammatory responses, fibrotic diseases, autoimmune diseases, chronic infections, radiation exposure, genetic diseases, and / or aging. In some embodiments, the production of fibroblasts may lead to the production of myofibroblasts, which may be secondary to or accompanied by myocardial infarction, heart failure, chronic hypertension, cardiomyopathy, valvular heart disease, chronic stress, neurohormonal activation, cardiac tissue aging, ischemic heart disease, arrhythmias, or amyloidosis.

[0123] In some embodiments, fibroblast proliferation may be associated with an autoimmune response or disease. In some embodiments, fibroblast proliferation may be associated with 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, autoimmune myocarditis, Crohn's disease, ulcerative colitis, pemphigus vulgaris, ankylosing spondylitis, Addison's disease, alopecia areata, autoimmune hemolytic anemia, celiac disease, and pulmonary diseases. 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, Chug-Strauss syndrome, mixed connective tissue disease, autoimmune pancreatitis, primary sclerosing cholangitis, and autoimmune encephalitis. In some embodiments, the autoimmune disease may be a cardiac autoimmune disease, such as autoimmune myocarditis.

[0124] In some embodiments, T is administered to the patient according to the method of this disclosure. reg Cells (e.g., enhanced T cells) regCells can restore or improve the function of target tissues (such as the heart, lungs, kidneys, liver, etc.) damaged by cardiac fibrosis in patients. This is in contrast to appropriate baseline values ​​(e.g., when administering enhanced T cells). reg The function of the target tissue may be improved compared to the function of the pre-cell target tissue, the average function of the target tissue damaged by cardiac fibrosis, etc. In some embodiments, compared to a suitable baseline value (e.g., after administration of enhanced T), the function of the target tissue may be improved. reg Compared to the function of the pre-target tissue, the function of the target tissue can be improved 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 within the range defined by any two of the foregoing values. In some embodiments, the enhanced T3 is administered to the subject according to the method of this disclosure. reg Cells can restore or reduce damage to target tissues. In some implementations, enhanced T cells are administered to the patient. reg Cells can reduce damage to 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 within the range defined by any two of the foregoing values.

[0125] In some implementations, when enhanced T is administered to subjects with cardiac fibrosis... reg When cells are involved, cardiac function can be restored or improved in patients. In some embodiments, 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 with cardiac fibrosis. In some embodiments, it is compared with an appropriate baseline value (e.g., when T is applied). reg Compared to the precellular ejection fraction, 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 within the range defined by any two of the foregoing values.

[0126] In some embodiments, the method of the present invention includes treating conditions associated with inflammation and / or fibrosis. In some embodiments, conditions associated with inflammation and / or fibrosis include, but are not limited to, inflammation and / or fibrosis of the heart, skeletal muscle, or skin. In some embodiments, conditions associated with inflammation and / or fibrosis include aging. In some embodiments, the condition treated by this treatment method is a symptom and / or sequela of infection. In some embodiments, the inflammatory condition is a chronic condition.

[0127] In some embodiments, the treatment methods of this disclosure can treat one or more of a variety of inflammatory diseases. In some embodiments, the inflammatory disease is a disease that responds to the anti-inflammatory effects of IL-10. In some embodiments, the inflammatory disease includes viral infections, sepsis, arthritis (rheumatoid arthritis, juvenile rheumatoid arthritis, psoriatic arthritis), multiple sclerosis, pemphigus, and type 1 diabetes (also known as insulin-dependent diabetes mellitus (IDDM)).

[0128] In some embodiments, the inflammation may be secondary to or concurrent with an infectious disease. The inflammation can be associated with a variety of infectious diseases. In some embodiments, the infectious disease is associated with myocardial injury. In some embodiments, the cardiac disease includes acute myocarditis associated with an infectious disease. In some embodiments, the inflammatory disease includes a cytokine storm or excessive inflammation associated with an infectious disease. In some embodiments, the inflammatory disease includes acute lung injury or acute respiratory distress syndrome (ARDS).

[0129] In some embodiments, the infectious disease may be caused by (but is not limited to) one or more of the following pathogens: viruses (including but not limited to coronaviruses, human immunodeficiency virus, herpes simplex virus, papillomavirus, parainfluenza virus, influenza virus, hepatitis virus, Coxsackie virus, varicella-zoster virus, measles virus, mumps virus, rubella, rabies virus, viral hemorrhagic fever, H1N1, etc.), prions, parasites, fungi, molds, yeasts, and bacteria (Gram-positive and Gram-negative). In some embodiments, the pathogens include, but are not limited to, Candida albicans, Aspergillus niger, Escherichia coli (… E. coli ), Pseudomonas aeruginosa ( P. aeruginosa ), and Staphylococcus aureus ( S.aureus Group A streptococci, Streptococcus pneumoniae, Mycobacterium tuberculosis, Campylobacter jejuni, Salmonella, Shigella, and various drug-resistant bacteria.

[0130] In some embodiments, the inflammation is secondary to or concurrent with a viral infection, such as a DNA or RNA virus. In some embodiments, the virus is an RNA virus, such as a single-stranded 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 order Nidovirales. In some embodiments, the virus belongs to the family Coronaviridae. In some embodiments, the virus belongs to the genera α-coronavirus, β-coronavirus, γ-coronavirus, or δ-coronavirus. In some embodiments, α-coronaviruses include, but are not limited to, human coronavirus 229E, human coronavirus NL63, or transmissible gastroenteritis virus (TGEV). In some embodiments, β-coronaviruses include, but are not limited to, severe acute respiratory syndrome coronavirus (SARS-CoV), SARS-CoV-2 (COVID-19), Middle East respiratory syndrome coronavirus (MERS-CoV), human coronavirus HKU1, or human coronavirus OC43. In some embodiments, the γ-coronavirus is infectious bronchitis virus (IBV).

[0131] In some embodiments, the inflammation and / or fibrosis are associated with autoimmune diseases, graft-versus-host disease (GVHD), or organ transplantation. In some embodiments, the inflammation is associated with one or more of the following diseases or conditions: Behçet's disease, polymyositis / dermatomyositis, autoimmune cytopenia, autoimmune myocarditis, primary cirrhosis, Goodpassuia syndrome, autoimmune meningitis, Sjögren's syndrome, systemic lupus erythematosus, Addison's disease, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune mumps, Crohn's disease, insulin-dependent diabetes mellitus, malnutrition-related 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, spondyloarthritis, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, and ulcerative colitis. In some embodiments, the autoimmune disease is scleroderma or systemic sclerosis.

[0132] In some embodiments, the inflammation is associated with bone marrow transplantation. In some embodiments, the inflammation is associated with allogeneic graft rejection after tissue transplantation. In some embodiments, the inflammation is associated with cardiomyopathy, heart failure, and / or amyloidosis. In some embodiments, the autoimmune disease is a cardiac autoimmune disease, such as autoimmune myocarditis. Non-limiting examples of the autoimmune disease or disorder include autoimmune myocarditis, conditions involving T-cell infiltration and chronic inflammatory responses, arthritis (rheumatoid arthritis, juvenile rheumatoid arthritis, psoriatic arthritis), multiple sclerosis, pemphigus, and type 1 diabetes (also known as insulin-dependent diabetes mellitus (IDDM)).

[0133] Composition This document provides compositions suitable for use in the treatment of subjects described herein. This document also provides 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, said nucleotide sequence being operatively linked to a regulatory T(T) protein. reg The promoter for expressing the Siglec-9 protein in T cells. When the nucleic acid is integrated into the cell (e.g., as described herein), the promoter can be any suitable for expression in T cells. reg The promoter for expressing the Siglec-9 protein in cells. In some embodiments, the promoter is a regulatory T(T) promoter. reg Cell-specific promoters. In some embodiments, the T... reg The cell-specific promoter is selected from the Foxp3 promoter and the 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 tetracycline-responsive promoter.

[0134] The composition contains any suitable amount of nucleic acid. In some embodiments, the composition contains a therapeutically effective amount of nucleic acid (e.g., for treating conditions associated with inflammation and / or fibrosis when administered to a subject in need). In some embodiments, the composition comprises, about comprises, or at least comprises 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 Nucleic acid in mg, 75 mg, 100 mg, or more, or in amounts within the range defined by any two of the foregoing values ​​(e.g., 0.01 µg to 0.1 µg, 0.1 µg to 1 µg, 1 µg to 10 µg, 10 µg to 100 µg, 100 µg to 1 mg, 1 mg to 10 mg, 10 mg to 100 mg). In some embodiments, the composition comprises, about comprises, or at least comprises 0.01 µg / mL, 0.02 µg / mL, 0.05 µg / mL, 0.1 µg / 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. Nucleic acid in amounts of µg / mL, 600 µg / mL, 700 µg / mL, 800 µg / mL, 900 µg / mL, 1 mg / mL, or amounts within the range defined by any two of the foregoing values ​​(e.g., 0.01 µg / mL to 1 mg / mL, 0.05 µg / mL to 100 µg / mL, 0.1 µg / mL to 10 µg / mL, 0.02 µg / mL to 200 µg / mL, etc.).

[0135] This article also provides a vector (e.g., plasmid, viral vector) containing a nucleic acid (with or without a promoter) encoding the Siglec-9 protein (e.g., having the sequence SEQ ID NO: 2).

[0136] This article also provides a regulatory T(T) comprising the aforementioned nucleic acid and / or vector. reg ) cells (e.g., isolated T cells) reg (cells). In some embodiments, the T reg The cells have been genetically modified (e.g., using nucleic acids or vectors disclosed herein) to express (e.g., overexpress) the Siglec-9 protein.

[0137] Furthermore, a composition comprising an enhanced (or engineered) T cell overexpressing sialic acid-binding immunoglobulin-like lectin 9 (Siglec-9) is provided. reg Cell population. In some implementations, this is compared with a suitable reference object (e.g., a T cell not engineered to overexpress Siglec-9 protein). reg Compared to cells, enhanced or engineered T cells reg The expression level of Siglec-9 protein in cells increases by approximately or 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 the increase is within the range defined by any two of the above values ​​(e.g., 1.5-fold to 10-fold, 2-fold to 8-fold, 3-fold to 7-fold, 4-fold to 6-fold, etc.). In some embodiments, compared with a suitable reference object (e.g., a T cell not engineered to overexpress Siglec-9 protein). reg Compared to cells, enhanced or engineered T cells reg The expression level of Siglec-9 in the cells was at least 1.5 times higher. In some embodiments, compared with a suitable reference object (e.g., a T cell not engineered to overexpress Siglec-9 protein), the expression level was increased. reg Compared to cells, enhanced or engineered T cells reg The expression level of Siglec-9 in the cells was at least 2-fold higher. In some embodiments, compared with a suitable reference object (e.g., a T cell not engineered to overexpress Siglec-9 protein). reg Compared to cells, enhanced or engineered T cells reg The expression level of Siglec-9 in cells was 4 to 6 times higher. In some embodiments, compared with a suitable reference object (e.g., a T cell not engineered to overexpress Siglec-9 protein). reg Compared to cells, enhanced or engineered T cells reg The expression level of Siglec-9 in cells was approximately 5-fold higher. In some implementations, enhanced T... regCells have been genetically modified (e.g., using nucleic acids or vectors) to express (e.g., overexpress) the Siglec-9 protein. In some embodiments, enhanced T... reg Endocytosis involves a vector containing multiple nucleic acids encoding sialic acid-binding immunoglobulin-like lectin 9 (Siglec-9). In some embodiments, enhanced T... reg The cell contains at least one endocytic carrier, said at least one endocytic carrier comprising a plurality of nucleic acids encoding sialic acid-binding immunoglobulin-like lectin 9 (Siglec-9). In some embodiments, said endocytic carrier comprises at least one of liposomes, nanoparticles, lipid nanoparticles (LNPs), viral vectors, cell-penetrating peptides (CPPs), and / or extracellular vesicles (EVs). In some embodiments, said plurality of nucleic acids comprises a plurality of mRNA transcripts encoding Siglec-9.

[0138] In some embodiments, the enhanced T reg Cells exhibit Siglec-9-dependent interactions with cells expressing copper-containing amine oxidase 3 (AOC3). In some embodiments, enhanced T cells... reg The interaction between Siglec-9-mediated cells and AOC-3-expressing cells is a functional interaction (e.g., a mechanism that enhances T cells). reg The cells can suppress the interaction of immune responses in tissues containing AOC-3 cells.

[0139] The composition may contain any suitable amount of reinforced T. reg Cells. In some embodiments, the composition comprises a therapeutically effective amount of enhanced T cells. reg Cells (e.g., for treating conditions associated with inflammation and / or fibrosis when administered to a subject in need). In some embodiments, the composition comprises, at least comprises, or comprises about 10 3 10 4 10 5 10 6 10 7 10 8 10 9 10 10 10 11 10 12 Or a quantity within the range defined by the two values ​​mentioned above (e.g., approximately 10). 3 -10 12 10 4 -10 11 10 5 -10 10 10 6 -10 9Enhanced T (etc.) reg Cells. This composition may contain any suitable concentration of enhanced T cells. reg Cells. In some embodiments, the composition comprises, at least comprises, or comprises about 10 3 / mL, 10 4 / mL, 10 5 / mL, 10 6 / mL, 10 7 / mL, 10 8 / mL, 10 9 / mL, 10 10 / mL, 10 11 / mL, 10 12 / mL or a concentration within the range defined by the two values ​​mentioned above (e.g., about 10). 3 Up to 10 12 / mL, 10 4 Up to 10 11 / mL, 10 5 Up to 10 10 / mL, 10 6 Up to 10 9 Enhanced T (e.g., / mL) reg Cells. In some embodiments, the composition comprises substantially pure enhanced T cells compared to other cells. reg Cell population.

[0140] A composition is also provided comprising a group of vectors, each vector comprising a nucleic acid encoding sialic acid-binding immunoglobulin-like lectin 9 (Siglec-9); and configured to deliver the nucleic acid encoding Siglec-9 to a T-cell receptor. reg Regulatory T cells (T) reg Cell-targeting component. In some embodiments, the carrier comprises lipid nanoparticles (LNPs). In some embodiments, the carrier is a transfection reagent (e.g., liposomes). In some embodiments, the T... reg The cell-targeting component includes binding to T reg Antigen-binding proteins of cell markers. In some embodiments, the antigen-binding protein is selected from: anti-CD25 monoclonal antibodies and / or anti-Foxp3 monoclonal antibodies. In some embodiments, the vector is a viral vector.

[0141] In some embodiments, the composition is a pharmaceutical or therapeutic composition. In some embodiments, the composition comprises a pharmaceutically acceptable excipient. In some embodiments, the composition is a cell-free composition, for example, the composition is substantially cell-free, such as T... regCells. Some non-limiting examples of substances that can be used 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, methyl cellulose, ethyl cellulose, microcrystalline cellulose, and cellulose acetate; (4) powdered astragalus gum; (5) malt; (6) gelatin; (7) lubricants, 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. Alcohols, such as glycerol, sorbitol, mannitol, and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethanol; (20) pH buffer solution; (21) polyesters, polycarbonates and / or polyanhydrides; (22) fillers, such as peptides and amino acids; (23) serum components, such as serum albumin, HDL, and LDL; (22) C2-C12 alcohols, such as ethanol; and (23) other non-toxic and compatible substances used in the formulation.

[0142] Other implementation methods In addition, non-limiting embodiments of this disclosure are provided below.

[0143] In some implementations, engineered T cells that overexpress Siglec-9 reg Cells can be used as a therapeutic approach for diseases where inflammation and tissue fibrosis are the main pathological drivers, including myocardial infarction (heart attack), hypertension, transplant rejection, myocarditis, cardiomyopathy, heart failure, and amyloidosis. In some embodiments, T cells overexpressing Siglec-9... reg Cell-specific targeting of fibroblasts expressing AOC3 inhibits cardiac fibrosis. In some embodiments, the method can be extended to target fibrosis in other organs.

[0144] Cardiac fibrosis and related diseases, such as myocardial infarction (MI), pose serious health challenges. Cardiac fibrosis manifests as an excessive buildup of fibrous connective tissue in the heart, leading to decreased cardiac function and other heart-related diseases. While inflammation often precedes fibrosis, treatments using immunosuppression to reduce inflammation can be a double-edged sword. While nonspecific immunosuppression is effective in reducing inflammation, it can inadvertently impair the body's ability to repair and regenerate tissues. In some cases, this impairment can exacerbate fibrosis. The key issue is the lack of targeted therapies that can modulate the immune response—specifically preventing or reducing fibrosis without inducing widespread nonspecific immunosuppression. Regulatory T cells (T cells) reg While these therapies are potential candidates for achieving this goal, they often lack specificity, leading to the aforementioned nonspecific immunosuppressive effects.

[0145] In some embodiments, this document provides a targeted and effective solution for cardiac fibrosis and related conditions. In some embodiments, this disclosure focuses on enhancing T... reg Cell specificity. Through engineered T cells... reg Cells overexpressing Siglec-9 can have their targeting specificity against cardiac myofibroblasts (which are highly AOC3-expressing) enhanced by the method of this invention. In some embodiments, this targeting method ensures that: 1. Enhanced specificity: Engineered T reg The cells can specifically target cardiac myofibroblasts, reducing the risk of nonspecific 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 T cells of this disclosure... reg Cells provide more precise regulation of the immune response, focusing on the root causes of fibrosis without causing widespread immunosuppression.

[0146] 2. Effective fibrosis relief: By targeting the root cause of fibrosis, this method and composition provide a treatment for myocardial infarction (MI) and other fibrosis-related diseases.

[0147] 3. Broad therapeutic potential: Although this method and composition are primarily focused on MI, they can be applied to other fibrosis-related diseases.

[0148] In some embodiments, key findings supporting the method and composition include: 1. Angiotensin II induces upregulation of fibrosis genes (type I collagen and α-SMA) and AOC3 in MCF in both dose- and time-dependent manner.

[0149] 2. The binding of Siglec-9 and AOC3 promotes T reg Cells precisely target and target cardiac fibroblasts that express AOC3.

[0150] 3. T cells overexpressing Siglec-9 reg It enhances the inhibitory effect on the expression of fibrosis genes in cardiac fibroblasts.

[0151] 4. Through AOC3 interaction, engineered T with OE-Siglec-9 reg It exhibits superior anti-fibrotic effects on cardiac myofibroblasts.

[0152] 5. Upregulation of AOC3 and fibrosis genes was observed after MI in vivo.

[0153] 6. Compared with traditional T reg In comparison, the Siglec-9 engineered T reg It has a higher efficacy in downregulating fibrosis gene expression after MI.

[0154] In some embodiments, this disclosure provides: 1. Targeted Immune Regulation: This disclosure provides a targeted treatment method that addresses the issue of natural T cells. reg The challenge of nonspecific immunosuppression exhibited by cells.

[0155] 2. Siglec-9 overexpression: T reg Overexpression of Siglec-9 in cells enhances their specificity, directly targeting the root cause of cardiac fibrosis (cardiac myofibroblasts).

[0156] 3. Broad therapeutic potential: In addition to myocardial infarction, this disclosure also covers the treatment of other fibrosis-related diseases.

[0157] Other non-limiting embodiments of this disclosure are provided by embodiments numbered as follows.

[0158] 1. A method of treating fibrosis, comprising administering to a subject requiring treatment for fibrosis a nucleic acid encoding a sialic acid-binding immunoglobulin-like lectin 9 (Siglec-9) protein, wherein the Siglec-9 protein is generated by the nucleic acid in the subject's regulatory T(T) receptor. reg It is expressed in cells.

[0159] 2. The method as described in Embodiment 1, wherein the subject has a need for treatment of cardiac fibrosis.

[0160] 3. A method of treating a condition associated with inflammation and / or fibrosis, comprising administering to a subject in need of treatment for a condition associated with inflammation and / or fibrosis a nucleic acid encoding a sialic acid-binding immunoglobulin-like lectin 9 (Siglec-9) protein, wherein the Siglec-9 protein is generated by the nucleic acid in the subject's regulatory T(T) receptor. reg It is expressed in cells.

[0161] 3a. A method of treating a condition associated with inflammation and / or fibrosis, comprising administering to a subject in need of treatment for a condition associated with inflammation and / or fibrosis a substance configured to induce sialic acid binding of immunoglobulin-like lectin 9 (Siglec-9) protein in the subject's regulatory T (T) receptor. reg Nucleic acid expressed in cells.

[0162] 4. The method as described in embodiment 3 or 3a, wherein the condition associated with inflammation and / or fibrosis includes cardiac inflammation and / or cardiac fibrosis.

[0163] 5. The method as described in embodiments 3, 3a, or 4, wherein the conditions associated with inflammation and / or fibrosis include myocardial infarction, hypertension, myocarditis, transplant rejection, cardiomyopathy, heart failure, and amyloidosis.

[0164] 6. The method of any one of embodiments 3-5, wherein the condition associated with inflammation and / or fibrosis is angiotensin II induced.

[0165] 7. The method as described in any of the foregoing embodiments, wherein administering the nucleic acid or nucleic acid encoding the Siglec-9 protein comprises administering to the subject a T-cell containing nucleic acid encoding the Siglec-9 protein. reg Cell population.

[0166] 8. The method as described in Embodiment 7, wherein the T cells in the cell population reg The cell contains a plasmid or viral vector containing the nucleic acid or a nucleic acid encoding the Siglec-9 protein.

[0167] 9. The method as described in Embodiment 8, comprising gene-modifying T using a plasmid or viral vector containing the said nucleic acid or a nucleic acid encoding the Siglec-9 protein. reg cell.

[0168] 10. The method as described in embodiment 8 or 9, wherein the viral vector comprises an adenovirus vector, a retrovirus vector, or a lentivirus vector.

[0169] 11. The method as described in any one of embodiments 7-10, wherein the T regThe cell population was derived from the subject's own cells.

[0170] 12. The method as described in any one of embodiments 7-10, wherein the T reg The cell population was derived from allogeneic cells of the subject.

[0171] 13. The method of any one of embodiments 1-6, comprising administering to the subject a vector containing the nucleic acid or nucleic acid encoding the Siglec-9 protein, wherein the vector is configured to deliver the nucleic acid or nucleic acid encoding the Siglec-9 protein to the T cells in the subject. reg cell.

[0172] 14. The method of embodiment 1, wherein the carrier comprises: lipid nanoparticles (LNPs); and a T associated with the LNPs. reg Cell-targeting component.

[0173] 15. The method as described in embodiment 13 or 14, wherein the T reg The cell-targeting component includes binding to T reg Antigen-binding proteins of cell markers.

[0174] 16. The method as described in embodiment 15, wherein the T reg Cellular markers were selected from CD25 and / or Foxp3.

[0175] 17. The method as described in embodiment 15 or 16, wherein the antigen-binding protein is an antibody.

[0176] 18. The method of any one of embodiments 15-17, wherein the antigen-binding protein is selected from: anti-CD25 monoclonal antibody and / or anti-Foxp3 monoclonal antibody.

[0177] 19. The method of any one of embodiments 12-17, wherein the nucleic acid comprises mRNA encoding the Siglec-9 protein.

[0178] 20. The method as described in any of the foregoing embodiments, wherein the subject has or is at risk of having a myocardial infarction.

[0179] 21. The method as described in any of the foregoing embodiments, wherein the subject has a need for treatment of angiotensin II-induced cardiac fibrosis.

[0180] 22. The method as described in any of the foregoing embodiments, wherein the subject has a need for treatment of cardiac fibrosis following myocardial infarction.

[0181] 23. The method of any of the foregoing embodiments, wherein the Siglec-9 protein comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 2.

[0182] 24. A method of treating a condition associated with inflammation and / or fibrosis, comprising administering to a subject in need of treatment for a condition associated with inflammation and / or fibrosis a nucleic acid encoding a membrane-bound fibroblast-specific binding protein, wherein the membrane-bound fibroblast-specific binding protein is expressed by the nucleic acid in the subject's regulatory T(T) level. reg It is expressed in cells.

[0183] 24a. A method of treating a condition associated with inflammation and / or fibrosis, comprising administering to a subject in need of treatment for a condition associated with inflammation and / or fibrosis a protein configured to induce membrane-bound fibroblast-specific binding in the subject's regulatory T (T) receptor. reg Nucleic acid expressed in cells.

[0184] 25. The method of embodiment 24 or 24a, wherein the membrane-bound fibroblast-specific binding protein includes sialic acid-binding immunoglobulin-like lectin 9 (Siglec-9).

[0185] 26. The method of embodiment 24 or 24a, wherein the membrane-bound fibroblast-specific binding protein comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 2.

[0186] 27. The method of any one of embodiments 24-26, wherein the condition associated with inflammation and / or fibrosis includes inflammatory tissue comprising fibroblasts having elevated expression of copper-containing amine oxidase 3 (AOC3).

[0187] 28. The method of embodiment 27, wherein the fibroblasts having elevated AOC3 expression include myofibroblasts.

[0188] 29. The method of 8 as described in Embodiment 2, wherein the myofibroblasts include cardiac myofibroblasts.

[0189] 30. The method of any one of embodiments 24-29, further comprising: Obtain the regulation T(T) reg ) immune cell populations; The nucleic acid or nucleic acid encoding sialic acid-binding immunoglobulin-like lectin 9 (Siglec-9) protein is delivered to the T reg Cells, thereby allowing the Siglec-9 protein to be present in the T cells.reg Overexpression in cells; T cells overexpressing Siglec-9 protein reg The cells are in contact with at least one fibroblast and / or myofibroblast of the subject, wherein the at least one fibroblast and / or myofibroblast expresses copper-containing amino oxidase 3 (AOC3). This can treat conditions associated with the inflammation and / or fibrosis.

[0190] 31. A method for targeted immune modulation in subjects, comprising: Obtain regulatory T cells (T cells) reg )group; To the T reg Cell population delivery of at least one vector, wherein the at least one vector contains nucleic acid encoding sialic acid-binding immunoglobulin-type lectin-9 (Siglec-9) and is located in the T cell population. reg Cells express Siglec-9, thereby producing enhanced T cells that overexpress Siglec-9. reg Cell population; and Administer the enhanced T to the subject reg Cell population.

[0191] 31a. A method for targeted immune modulation in a subject, comprising: Obtain regulatory T cells (T cells) reg )group; To the T reg Cell population delivery of at least one vector, wherein the at least one vector comprises a protein configured to induce sialic acid binding of immunoglobulin-type lectin-9 (Siglec-9) protein in the T cells. reg Nucleic acid expressed in the cell population leads to the production of enhanced T cells overexpressing Siglec-9. reg Cell population; and Administer the enhanced T to the subject reg Cell population Optionally, the nucleic acid encodes the Siglec-9 protein.

[0192] 32. The method of embodiment 31 or 31a, wherein the at least one carrier comprises liposomes or lipid nanoparticles (LNPs).

[0193] 33. The method as described in embodiment 31 or 31a, wherein the at least one vector comprises a viral vector.

[0194] 34. The method of embodiment 33, wherein the viral vector includes an adenovirus vector, a reverse transcription vector, or a lentivirus vector.

[0195] 35. The method of any one of embodiments 31-34, wherein the nucleic acid comprises a promoter operatively linked to a nucleotide sequence encoding Siglec9, wherein the promoter upregulates the T reg Expression of Siglec9 in cells.

[0196] 36. The method of any one of embodiments 31-35, wherein the subject comprises at least one myofibroblast with elevated AOC3 expression compared to other cell types.

[0197] 37. The method of embodiment 36, wherein the at least one myofibroblast is at least one cardiac myofibroblast.

[0198] 38. The method of any one of embodiments 31-37, wherein the subject has a need for treatment of fibrosis.

[0199] 39. The method as described in embodiment 38, wherein the fibrosis is cardiac fibrosis.

[0200] 40. The method of embodiment 39, wherein the cardiac fibrosis is secondary to myocardial infarction.

[0201] 41. A method for inhibiting the expression of fibrosis genes, comprising: Get T reg Cell population; For the T reg Cells were engineered to overexpress Siglec-9, thereby producing enhanced T cells. reg Cells; and Administer the enhanced T to subjects who require inhibition of fibrosis gene expression. reg cell.

[0202] 42. The method of embodiment 41, wherein the fibrotic gene is at least one of type I collagen, α-smooth muscle actin (α-SMA), and / or copper-containing amine oxidase 3 (AOC3).

[0203] 43. The method as described in embodiment 41 or 42, wherein the engineering includes: To the T reg Cell population delivery of at least one vector containing a nucleic acid encoding Siglec-9, optionally, said nucleic acid comprising mRNA; or Delivery of at least one carrier containing a substance configured to induce the T reg Nucleic acid expression of Siglec-9 protein in cell population.

[0204] 44. The method of embodiment 43, wherein the at least one carrier comprises at least one of liposomes, nanoparticles, lipid nanoparticles (LNP), viral vectors, cell-penetrating peptides (CPP), and / or extracellular vesicles (EV).

[0205] 45. The method as described in embodiment 43 or 44, wherein the nucleic acid comprises mRNA.

[0206] 46. ​​The method of embodiment 45, wherein overexpression of Siglec-9 includes upregulating the expression of the mRNA.

[0207] 47. The method of any one of embodiments 41-46, wherein the subject has a need for treatment of fibrosis.

[0208] 48. The method as described in embodiment 47, wherein the fibrosis is cardiac fibrosis.

[0209] 49. The method of embodiment 48, wherein the cardiac fibrosis is secondary to myocardial infarction.

[0210] 50. The method as described in any of the preceding embodiments, wherein the subject has upregulated expression of copper-containing amine oxidase 3 (AOC3) in cardiac myofibroblasts.

[0211] 51. The method as described in any of the preceding embodiments, wherein the administration includes intravenous administration or oral administration.

[0212] 52. An isolated nucleic acid comprising a nucleotide sequence encoding a sialic acid-binding immunoglobulin-like lectin 9 (Siglec-9) protein, operably linked to a protein configured to be used in a regulatory T(T) reg The promoter for expressing the Siglec-9 protein in cells.

[0213] 53. The isolation of nucleic acids as described in Embodiment 52, wherein the promoter is a regulatory T(T) promoter selected from the Foxp3 promoter and the interleukin-2 (IL-2) promoter. reg Cell-specific promoters.

[0214] 54. A vector comprising the nucleic acid described in embodiment 52 or 53.

[0215] 55. A regulatory T (T reg Cells comprising the isolated nucleic acid as described in embodiment 52 or 53, or the vector as described in embodiment 54, optionally wherein the T reg Cells are human T cells reg cell.

[0216] 56. A composition comprising a group of carriers, each carrier comprising: Nucleic acid encoding sialic acid-binding immunoglobulin-like lectin 9 (Siglec-9); and Configured to deliver nucleic acids encoding Siglec-9 to T reg Regulatory T cells (T) reg ) cell-targeting portion, optionally, wherein the T reg Cells are human T cells reg cell.

[0217] 57. The composition of embodiment 56, wherein the carrier of the cell population comprises lipid nanoparticles (LNP).

[0218] 58. The composition as described in embodiment 56 or 57, wherein the nucleic acid comprises mRNA.

[0219] 59. The composition according to any one of embodiments 56-58, wherein the T reg Cell-targeting components include binding to T reg Antigen-binding proteins of cell markers.

[0220] 60. The composition as described in embodiment 59, wherein the T reg Cellular markers were selected from CD25 and / or Foxp3.

[0221] 61. The composition as described in embodiment 59 or 60, wherein the antigen-binding protein is an antibody.

[0222] 62. The composition of any one of embodiments 59-61, wherein the antigen-binding protein is selected from: anti-CD25 monoclonal antibody and / or anti-Foxp3 monoclonal antibody.

[0223] 63. A composition comprising an enhanced regulatory T (T) cell overexpressing sialic acid-binding immunoglobulin-like lectin 9 (Siglec-9). reg ) cell population, optionally, wherein the T reg Cells are human T cells reg cell.

[0224] 64. The composition as described in embodiment 63, wherein the enhanced T reg The cell contains at least one endocytic carrier, which contains multiple nucleic acids encoding sialic acid-binding immunoglobulin-like lectin 9 (Siglec-9).

[0225] 65. The composition of embodiment 64, wherein the at least one endocytic carrier comprises at least one of liposomes, nanoparticles, lipid nanoparticles (LNPs), viral vectors, cell-penetrating peptides (CPPs), and / or extracellular vesicles (EVs).

[0226] 66. The composition of embodiment 64 or 65, wherein the plurality of nucleic acids comprises a plurality of mRNA transcripts encoding Siglec-9.

[0227] 67. The composition of any one of embodiments 63-66, wherein the enhanced T reg The cells exhibited Siglec-9-dependent interactions with cells expressing copper-containing amine oxidase 3 (AOC3).

[0228] 68. The composition as described in embodiment 67, wherein the enhanced T reg The cells exhibited Siglec-9-dependent interactions with AOC3-expressing cardiac myofibroblasts.

[0229] 69. The composition of any one of embodiments 56-68 is used to treat conditions associated with inflammation and / or fibrosis in subjects in need.

[0230] 70. T as described in embodiment 55 reg Use of the cell or the composition of any one of embodiments 56-68 for treating conditions associated with inflammation and / or fibrosis in subjects in need.

[0231] 71. T as described in embodiment 55 reg Use of the cell or the composition of any one of embodiments 56-68 in the preparation of a medicament for treating a condition associated with inflammation and / or fibrosis in a subject in need.

[0232] 72. The method, nucleic acid isolation, vector, and T method described in any of the foregoing embodiments. reg Cells, or compositions, wherein the Siglec-9 is human Siglec-9.

[0233] The techniques described herein are further illustrated by the following embodiments, which should in no way be construed as further limitations.

[0234] Example Example 1: This non-limiting example demonstrates that angiotensin II can induce fibrosis genes in primary mouse cardiac fibroblasts.

[0235] Angiotensin II dose-dependently induces upregulation of fibrosis genes in mouse cardiac fibroblasts. To determine whether angiotensin II induces dose-dependent upregulation of AOC3 and other fibrosis genes in mouse cardiac cells, angiotensin II was administered at different concentrations to isolated primary mouse cardiac fibroblasts (MCFs). Subsequently, after administering 10 μM angiotensin II to MCF cells, the expression levels of fibrosis-related genes (specifically type I collagen and α-SMA) were monitored over time.

[0236] Figure 2 A graphical representation of data obtained from in vitro experiments on the effects of angiotensin II concentration on fibrosis gene expression is shown 200. The data show three concentrations of angiotensin II administered to MCF cells: 100 nM, 1 μM, and 10 μM. The control treatment, in which no angiotensin II was administered to MCF cells, is also shown in the figure. After culturing MCF cells for 24 hours, mRNA was isolated from the MCF cells to quantify the fold increase in type I collagen 210 and α-SMA220 mRNA transcripts relative to the control group. The data indicate that the fold increase in the fold of type I collagen 210 and α-SMA220 mRNA expression was positively correlated with increasing angiotensin II concentration administered to MCF cells.

[0237] Angiotensin II time-dependently induces upregulation of fibrosis genes in mouse cardiomyocytes. To determine whether angiotensin II induces time-dependent upregulation of fibrosis genes in mouse cardiac cells, primary mouse cardiac fibroblasts (MCFs) were exposed to angiotensin II and changes over time were monitored. The expression levels of fibrosis-related genes (specifically type I collagen and α-SMA) were then measured to assess their correlation with increased angiotensin II concentrations.

[0238] Figure 3 A graphical representation of data obtained from an in vitro experiment demonstrating the effect of 10 μM angiotensin II on the expression of fibrosis genes over time is shown

[300] . The data show expression levels measured at three time points: 6, 12, and 24 hours after administration of angiotensin II to MCF cells. The control treatment (Ctrl), where no angiotensin II was administered to MCF cells, is also shown. At each time point, mRNA was isolated from MCF cells to quantify the fold increase in type I collagen 310 and α-SMA320 mRNA transcripts relative to the control group. The data indicate that the fold increase in the expression of both type I collagen 310 and α-SMA320 mRNA was positively correlated with time when 10 μM angiotensin II was administered to MCF cells.

[0239] Angiotensin II dose- and time-dependently induced AOC3 expression in mouse cardiac fibroblasts. To determine whether angiotensin II induces dose- and time-dependent upregulation of AOC3 in mouse cardiac cells, primary mouse cardiac fibroblasts (MCFs) were isolated and exposed to angiotensin II. Angiotensin II was then administered to the MCFs, and AOC3 expression levels were measured at each dose or over time.

[0240] Figure 4 Graphical representations of data obtained from in vitro experiments demonstrating the effects of angiotensin II concentration and time on AOC3 gene expression are shown 400. 410 shows three concentrations of angiotensin II administered to MCF cells: 100 nM, 1 μM, and 10 μM. A control treatment (Ctrl) is also shown, in which no angiotensin II was administered to MCF cells. 420 shows expression levels measured at three time points: 6, 12, and 24 hours after administration of 10 μM angiotensin II to MCF cells. A control treatment (Ctrl) is also shown, in which no angiotensin II was administered to MCF cells. In both experiments, mRNA was isolated from MCF cells to quantify the fold increase in AOC3 mRNA transcripts relative to the control. The data showed a positive correlation between the fold increase in AOC3 mRNA expression and both dose and time.

[0241] To confirm the positive correlation between AOC3 upregulation and angiotensin II, Western blot analysis was performed. The results showed that AOC3 was upregulated compared to both the negative control (no angiotensin II administration) and the positive control (angiotensin II administration had no effect on β-actin (Act) expression levels).

[0242] Example 2: This non-limiting embodiment demonstrates that Siglec-9 binds to AOC3 and in T reg Overexpression in cells promotes T reg Fibroblasts that target and express AOC3.

[0243] The combination of Siglec-9 and AOC3 promotes T reg Fibroblasts that target AOC3 expression.

[0244] To determine whether the binding of Siglec-9 to AOC3 promotes T reg Enhanced T cells overexpressing Siglec-9 were prepared by targeting cardiac fibroblasts expressing AOC3. regCells. A lentiviral vector (pLenti-GIII-CMV) containing the SIGLEC9 gene was obtained from Applied Biological Materials Inc. (catalog number 43776061). The SIGLEC9 gene, human sialic acid-binding immunoglobulin-like lectin 9, is represented as a 1,707-base-pair mRNA sequence (NCBI accession number BC035365; SEQ ID NO: 1). The SIGLEC9 protein encoded by this lentiviral vector has the predicted amino acid sequence shown in SEQ ID NO: 2, as shown below (predicted signal peptide underlined): Obtain mouse T reg Cells were transduced using lentiviral vectors to generate enhanced T cells. reg cell. Figure 5 Western blot analysis 500 demonstrates the generation of T cells overexpressing Siglec-9. reg Cellular capacity. 510 shows T reg Increased expression of Siglec-9 in cells leads to the generation of enhanced T cells. reg Cells. 520 shows the β-actin control. Western blotting confirmed a 5-fold increase in expression levels (…). Figure 26 ).

[0245] Use CD4+CD25+T reg Cell isolation kit (catalog number: 130-091-041, from MILTENYIBIOTEC) TM Regulatory T cells (T cells) were obtained from the spleen of mice. reg The spleen was placed in a sterile culture dish containing phosphate-buffered saline (PBS) + 1 mM EDTA. Excess tissue was removed, and the spleen was ground using a syringe plunger to release spleen cells. Next, a single-cell suspension was prepared by passing the ground tissue through a 70 μm cell filter into a tube. After filtration, the cells were washed with PBS, centrifuged at 300 × g for 10 min to form a cell pellet, and the supernatant was carefully removed.

[0246] For effective magnetic labeling, cells were passed through a 30 µm nylon mesh to remove any clumps. The total cell count was determined, and then counted at 10⁻⁶ cells per 10⁻⁶ cells. 7 Resuspend the cell pellet in 40 μL of buffer per cell. Repeat every 10... 7 Each cell was labeled with 10 µL of CD4+CD25+CD127 dim / -T cell biotin-antibody mixture II. The solution was thoroughly mixed and incubated at 2°C to 8°C for 10 minutes. Next, 10 μL of the solution was added to the mixture. 710 µL of buffer, 20 µL of avidin beads, and 20 µL of CD20 beads per cell were mixed and incubated at 2°C to 8°C for 15 minutes. Then, the mixture was incubated at 10 μL of buffer per cell. 7 Wash cells with 1 to 2 mL of buffer per cell and centrifuge at 300 × g for 10 minutes. Then completely aspirate the supernatant and resuspend the cells to approximately 10 mL of buffer. 8 Cells were placed in 500 µL of buffer. Magnetic separation / depletion was performed using an LD sorting column in a MACS separator. The sorting column was washed with 2 mL of buffer, the cell suspension was added, and the eluent containing the pre-enriched CD4+ cell fraction was collected. The sorting column was washed with 2 × 1 mL of buffer.

[0247] To perform CD4+CD25+CD127 dim / -T reg Positive cell sorting was performed using an MS sorting column. The sorting column was washed with 500 μL buffer, cell suspension was added, and the eluent was collected. The sorting column was then washed with 3 × 500 µL buffer. The sorting column was then removed from the separator, and the magnetically labeled cells were washed out with 1 mL buffer. For higher purity, the eluted fraction was passed through a second new MS sorting column.

[0248] Throughout the process, there were no pauses between steps, and both cells and solutions were maintained at low temperatures (e.g., 2°C to 8°C) to preserve cell viability and prevent non-specific labeling. As those skilled in the art will understand, the volumes of the reagents and buffers described above can be adjusted according to the number of cells being isolated and prepared. The above protocol yields purified T cells for further analysis or experiments. reg Cell sample.

[0249] Figure 6 Immunoprecipitation analysis 600 was shown, displaying the results of T... reg Siglec-9 expressed by cells binds to AOC3 expressed by cardiomyocytes (CF). This enhances T... reg Cells were co-cultured with cardiomyocytes overexpressing AOC3. After co-culture, cells were lysed and immunoprecipitated against the Siglec-9 protein. Western blot analysis of the immunoprecipitate was performed to determine the targeted binding of Siglec-9 to AOC3. Western blot analysis of the immunoprecipitate 600 and 610 confirmed that the binding was caused by T… regCellular expression of Siglec-9 binds to AOC3 expressed by CF. For example, this binding can be observed by adding a sequence encoding GFP to a lentiviral vector, allowing GFP to be co-expressed with Siglec-9 in lentivirally transduced cells. Analysis of the input samples confirmed CF overexpression of AOC3620, while expression of Siglec-9 protein 630 or β-actin 640 remained consistent.

[0250] To determine whether the binding of Siglec-9 to AOC3 promotes T reg Cell adhesion experiments were conducted on cardiomyocytes that target AOC3 expression. Figure 7 This demonstrates the method used to study the interaction between Siglec-9 and AOC3 in enhanced T reg Cell adhesion assays in cells 700. MCF monolayers were prepared and exposed to angiotensin II (Ang II) solution 720 to induce MCF fibrosis. Control MCF cells were not exposed to Ang II. Enhanced T cells overexpressing Siglec-9 and engineered to express GFP were prepared. reg Cells 730. Control T reg Cells express only GFP. Enhanced T cells expressing GFP will be... reg Cells (or control T cells) reg Cells were spread on the surface of an MCF monolayer (whether or not exposed to Ang II) 740. Next, T... reg Cells adhered to the MCF monolayer 750. The MCF monolayer was washed to remove any unbound cells 760. The number of cells bound to the MCF monolayer was determined by fluorescence microscopy 770.

[0251] MCF cell exposure to Ang II increased the binding of control T cells to the MCF monolayer. reg Cell number. Compared with control T cells bound to Ang II-exposed MCF monolayers. reg Compared to the number of cells, there are a greater number of enhanced T cells. reg Cells bound to an AngII-exposed MCF monolayer. These results support a model, namely T... reg Siglec-9 expressed on cells promotes T reg Cardiac fibroblasts that target and express AOC3.

[0252] Siglec-9 in T reg Overexpression in cells enhances the inhibition of fibrosis gene expression in cardiac fibroblasts. To determine Siglec-9 in T reg Does overexpression in cells enhance the inhibition of fibrosis gene expression in cardiac fibroblasts, and does it reduce the overexpression of T... regCells were co-cultured with cardiac fibroblasts. After co-culture, the cells were lysed and mRNA was collected.

[0253] Figure 8 Figure 800 shows data obtained from an in vitro experiment using Treg cells overexpressing Siglec-9, demonstrating enhanced suppression of fibrosis gene expression in cardiac fibroblasts. The fold change in mRNA transcription relative to controls of the fibrosis genes type I collagen 810 and α-SMA 820 was measured. Angiotensin II increased the expression of fibrosis genes in cardiac fibroblasts. The Treg cells were then subjected to further testing. reg Co-culturing cells with cardiac fibroblasts exposed to angiotensin II eliminated the increased expression of fibrosis genes (type I collagen and α-SMA) induced by angiotensin II. In T reg Overexpression of Siglec-9 in cells enhances T reg Cellular inhibition of fibrosis gene expression.

[0254] Example 3: This non-limiting embodiment demonstrates the interaction between Siglec-9 and AOC3 in enhanced T reg Its role in cell-mediated anti-cardiac fibrosis effects.

[0255] Cell adhesion and co-culture assays were performed to determine the effect of the Siglec-9-AOC3 interaction on enhanced T cells. reg The cell-targeting effect on cardiac fibroblasts and its anti-fibrotic effect.

[0256] Figure 9 This demonstrates the method used to study the interaction between Siglec-9 and AOC3 in enhanced T reg Cell adhesion assay 900, demonstrating the role of cells in targeting cardiac fibroblasts and their anti-fibrotic effects. As described above, MCF monolayers were prepared and exposed to angiotensin II solution 920 to induce MCF fibrosis. Control group MCF cells were not exposed to Ang II. Mouse T cells were prepared as described in Example 2. reg Cells. Enhanced T cells. reg Cells were overexpressing Siglec-9 and engineered to express GFP930. Enhanced T cells were then treated with either an IgG control group or an anti-Siglec-9 antibody (“anti-Siglec-9”). reg Cells. Enhanced T cells expressing GFP. reg Cells were spread on the surface of an MCF monolayer (exposed or not exposed to Ang II) at 940°C. Next, T... regCells adhere to the MCF monolayer 950. The MCF monolayer is washed to remove any unbound cells 960. The number of cells bound to the MCF monolayer is determined by fluorescence microscopy 970.

[0257] For T reg Cells were treated with anti-Siglec-9 antibody to eliminate enhanced T cells. reg Increased binding between cells and the MCF monolayer exposed to Ang II, while no such effect was observed in the IgG control group. Therefore, enhanced T cells... reg Cell targeting of AOC3-expressing cardiac fibroblasts depends on Siglec-9 binding.

[0258] Figure 10 The diagram illustrates data obtained from in vitro cell adhesion experiments used to investigate the effect of Siglec-9 / AOC3 interaction on fibrosis gene expression. The data show that, compared to the IgG control group, anti-Siglec-9 antibody reduced enhanced T cell expression. reg Cellular inhibition of fibrosis gene expression (type I collagen 1010 and α-SMA 1020). Therefore, enhanced T... reg The targeting of cells to AOC3-expressing cardiac fibroblasts depends on Siglec-9 binding.

[0259] In relation to T reg Prior to cell binding, MCF monolayers (regardless of exposure to Ang II) were treated with anti-AOC3 antibody for cell adhesion assays and co-culture assays. Figure 11 and Figure 12 MCF monolayers were prepared and exposed to angiotensin II solution 1120 to induce MCF fibrosis. Control MCF cells were not exposed to Ang II. Subsequently, MCF cells treated with Ang II or as controls were treated with either IgG control or anti-AOC3 antibody (“anti-AOC3”). Enhanced T cells reg Cells were overexpressing Siglec-9 and engineered to express GFP1130. Enhanced T cells expressing GFP were then... reg Cells were spread on the surface of an MCF monolayer (whether or not exposed to Ang II, and treated with anti-AOC3 antibody or IgG) at 1140°. Then, T cells were... reg Cells adhered 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 fluorescence microscopy 1170.

[0260] Similar to blocking Siglec-9, treatment of MCF monolayers with anti-AOC3 antibody eliminated enhanced T cells. regIncreased binding of cells to Ang II-exposed MCF monolayers, while no such effect was observed in the IgG control group. Therefore, enhanced T cells... reg Cardiac fibroblasts that target AOC3 expression depend on AOC3 binding.

[0261] Figure 12 A graphical representation of data obtained from in vitro cell adhesion assays is shown, used to probe the effect of Siglec-9 / AOC3 interaction on fibrosis gene expression. The data indicate that, compared to the IgG control group, the anti-AOC3 antibody reduced enhanced T cell expression. reg Cellular inhibition of fibrosis gene expression (type I collagen 1210 and α-SMA 1220). Therefore, enhanced T... reg Fibroblasts that target AOC3 expression depend on AOC3 binding.

[0262] These results indicate that enhanced T reg Fibroblasts that target AOC3 expression depend on the interaction between Siglec-9 and AOC3.

[0263] Example 4: This non-limiting embodiment illustrates the role of upregulation of AOC3 and fibrosis genes in vivo after myocardial infarction.

[0264] Compared with normal T reg Compared to other cells, engineered enhanced T cells overexpressing Siglec-9 reg Cells show activity after myocardial infarction It demonstrates a stronger ability to downregulate fibrosis gene expression. To determine the difference between ordinary T reg Compared to other cells, engineered T cells with enhanced Siglec-9 overexpression... reg To determine whether cells have a stronger ability to downregulate fibrosis gene expression after myocardial infarction, qPCR and Western blot analysis were used to quantify gene expression levels.

[0265] Figure 13Data on the upregulation of AOC3 and fibrosis genes observed in an in vivo experiment using a mouse model of myocardial infarction are shown

[1300] . Gene expression was measured relative to the control sham-operated group, showing a fold increase over time compared to the control group. For fibrosis-related genes AOC31310, type I collagen 1320, and α-SMA1330, gene expression increased over time following induced myocardial infarction in the mouse model. Expression levels were measured at 72 hours (day 3), 168 hours (day 7), and 504 hours (day 21). Western blot analysis was also performed to demonstrate the increased expression of the aforementioned fibrosis-related genes relative to the negative (sham-operated) control group and the positive control gene (β-actin).

[1340] illustrates the increase in fibrosis gene expression over time after myocardial infarction.

[0266] Figure 14 Experimental design 1400 and enhanced T are shown. reg Cells and normal T cells reg Compared to other cells, the study investigated data on enhanced therapeutic efficacy by downregulating fibrosis gene expression in vivo after myocardial infarction. The study also examined T cells. reg The role of cells during a 21-day in vivo administration. Following myocardial infarction, animals were administered 2 × 10⁻⁶ cells every 7 days. 5 Control T per dose reg Cells (not overexpressing Siglec-9) or enhanced T cells reg Cells (OE-sig9-T) reg The animals were administered the drug three times in total. Another group of animals received a carrier control (without T). reg (Cells). After 21 days, heart tissue was collected and subjected to Western blot analysis and qPCR to analyze the expression levels of fibrosis-related genes.

[0267] Gene expression was measured as a fold change relative to the control group. After 21 days, in the vector and normal T... reg Cells, and enhanced T reg Cells (Sig-T) reg In the presence of fibrosis-related genes (type I collagen 1440, α-SMA1450, and Foxp31460), expression levels were tested. These genes were found to be associated with fibrosis after myocardial infarction and in the presence of enhanced T cells. reg The expression level of AOC31430 was significantly reduced in the presence of cells. The expression level of AOC31430 was compared with that in the vector or normal (non-enhanced) T cells. reg There was no significant difference in AOC3 expression levels in the presence of cells. Foxp31460 expression levels were observed in enhanced T cells. regThe expression significantly increased in the presence of cells, suggesting a possible role in immune regulation and fibrosis development. Expression data were confirmed by Western blot analysis 1410, which further demonstrated the presence of enhanced T cells. reg The efficacy of cells in downregulating fibrosis-related genes (such as type I collagen and α-SMA) after myocardial infarction.

[0268] Example 5: This non-limiting embodiment illustrates an enhanced T reg In vivo therapeutic effects of cells on angiotensin II-induced cardiac fibrosis.

[0269] Figure 15 This shows the method used to study enhanced T reg In vivo experimental protocol for antifibrotic effects in cells 1500. Subjects (e.g., mice) received angiotensin II infusion at 1 mg / kg / day (day 0). Enhanced T3 was administered to subjects via infusion on days 3, 7, and 14. reg Cells. On day 21, assess one or more of the following: T reg Cell infiltration / accumulation in damaged myocardium; Siglec9-T reg Targeting AOC3+ myofibroblasts in the heart; T reg Expression levels of IL-10 / Foxp3 in cells; expression of fibrosis genes and proteins in myofibroblasts of the heart; cardiac function and pathology (e.g., ejection fraction (ΔEF); fibrosis area (PS red)).

[0270] Example 6: This non-limiting embodiment illustrates an enhanced T reg In vivo therapeutic effects of cells on myocardial infarction-induced cardiac fibrosis.

[0271] Administer 2 × 10⁻⁶ doses to animals following myocardial infarction every 7 days. 5 / Agent Enhanced T reg Cells (OE-sig9-T) reg (or refer to T) reg Cells (not overexpressing Siglec-9) were administered three times. Another group of animals received a vector control (without T cells). reg (Cells). After 21 days, assess one or more of the following: T reg Cell infiltration / accumulation in damaged myocardium; Siglec9-T reg Targeting AOC3+ myofibroblasts in the heart; T regExpression levels of IL-10 / Foxp3 in cells; expression of fibrosis genes and proteins in myofibroblasts of the heart; cardiac function and pathology (e.g., ejection fraction (ΔEF); fibrosis area (PS red)).

[0272] Example 7: This non-limiting embodiment provides a Siglec-9 mediated enhancement of T reg Functional model.

[0273] Siglec-9 can promote T cells by increasing the release of interleukin-10 (IL-10). reg Function.

[0274] Figure 16 This demonstrates that Siglec-9 promotes T cells by influencing immune cell signaling. reg Exemplary implementations of cellular processes 1600 for cellular function. In some implementations, this is achieved by administering at least one enhanced T to the subject. reg Cellular protection achieves cardiac protection 1610. Enhanced T cells. reg Cellular overexpression of Siglec-9 protein, when binding to AOC3 expressed in, for example, cardiomyocytes fibroblasts, can activate pathways such as tyrosine phosphatases, such as Src homology 2 domain phosphatase 1 (SHP-1). SHP-1 activation leads to the expression of cytokines such as interleukin-10 (IL-10). The SHP-1-Siglec-9 protein complex can promote enhanced T cells by increasing IL-10 release. reg Function. IL-10 can promote T reg This enhances function, thereby further reducing cardiac fibrosis. IL-10 can promote T... reg Cell lineage stability.

[0275] Example 8: This non-limiting embodiment evaluates the Siglec-9 engineered T reg Anti-fibrotic effects of targeting AOC3 expression in cardiac myofibroblasts.

[0276] Figure 17 Collagen shrinkage experiments 1710 showed that Siglec-9 engineered T reg Cells were more effective than non-engineered T cells in reversing Ang II-induced fibroblast contraction by 1720%. reg The cells are more effective, further validating their functional superiority in anti-fibrotic intervention. For example... Figure 18As shown in the collagen contraction assay 1810, blocking Siglec-9 with a neutralizing antibody significantly reduced myofibroblast contraction 1820, highlighting the crucial role of Siglec-9 in mediating these effects. Furthermore, Figure 19 The results showed that, in the collagen contraction experiment 1910, neutralizing AOC3 also reduced myofibroblast contraction 1920, confirming the importance of the Siglec-9-AOC3 interaction in targeting and attenuating the fibrosis process.

[0277] Example 9: This non-limiting embodiment evaluates the Siglec-9 engineered T reg In vivo anti-fibrotic effect of cells in a mouse model of myocardial infarction (MI). This example is related to Example 4.

[0278] In vivo studies have confirmed the efficacy of Siglec-9 engineered T reg Cells have the potential to target anti-fibrosis in a MI model with ligation of the left anterior descending artery (LAD). Figure 20 Intravenous injection of Siglec-9 engineered T cells identifiable by RFP tags. reg Cells that selectively locate in AOC3+ cells within the infarct area ( Figure 21 ), indicating that the engineered T reg The cells targeted and bound to myofibroblasts expressing AOC3 in the damaged heart. Quantitative PCR (qPCR) Figure 22 This further supports the upscaling of AOC32210 in MI, and the Siglec-9 engineering T reg Cells are most effective at reducing the expression of fibrosis genes, 2220 and 2230.

[0279] The Siglec-9 engineering T was discussed reg The functional effects of cells on cardiac tissue remodeling and recovery. For example... Figure 23 As shown, Sirius red staining of heart tissue 2310 revealed that, compared to the vector group, the staining with two T... reg Cell-treated myocardial fibrosis significantly reduced the fibrotic area in MI hearts. Notably, T cells engineered with Siglec-9 showed a significant reduction. reg Cell therapy for the heart showed better results in reducing fibrosis2320, highlighting its superior efficacy in reducing fibrotic tissue deposition.

[0280] Echocardiographic assessment provides quantitative evidence for improving cardiac function. Figure 24 The data shows that, compared to the unengineered T reg Compared to other cells, Siglec-9 engineered T cells... regCells more effectively increased ejection fraction 2410 and were better able to maintain cardiac function after myocardial infarction. These findings suggest that, in addition to targeting and modulating fibrosis pathways, Siglec-9 engineered T cells... reg Cells may also contribute to significantly improving heart function.

[0281] These findings indicate that the Siglec-9 engineered T reg The cells effectively reduced cardiac fibrosis by targeting myofibroblasts that express AOC3.

[0282] As shown in the above examples, the Siglec-9 engineered T reg Cells can selectively target AOC3-expressing cardiac myofibroblasts, a key component in the pathogenesis of ICM. Through targeted regulation of the AOC3 pathway, these engineered T cells... reg The cells effectively reduced the expression of key fibrosis markers such as type I collagen and α-SMA both in vitro and in vivo. Adhesion experiments confirmed that Siglec-9 engineered T cells... reg Cells exhibit significantly enhanced adhesion to fibrotic regions, primarily driven by their interaction with AOC3. Blocking this interaction weakens T cells. reg Cell adhesion and the ability to suppress fibrosis genes highlight the central role of the Siglec-9-AOC3 interaction in mediating these effects. Furthermore, in vivo experiments using a mouse MI model demonstrate that these engineered T cells... reg Cells not only reduce fibrosis, but also help improve heart function, as evidenced by the increase in left ventricular ejection fraction (LVEF).

[0283] Example 10 This non-limiting embodiment illustrates that, upon stimulation with AOC3 (a myofibroblast marker), T... reg Characterization of the functional effects and molecular mechanisms of siglec-9 overexpression in cells.

[0284] T was evaluated reg The effect of siglec-9 overexpression in cells on the cellular response to the myofibroblast marker AOC3.

[0285] Human cells: Human T cells purchased from IQ Biosciences reg Cells (hT) reg(Catalog number IQB-Hu1-iTr-1) was obtained from a healthy volunteer participating in an IRB-approved donor program. The supplier provided flow cytometry-certified purity and identity certificates (90% CD4+CD25+ (90% CD127, 85% FOXP3+)), as well as negative test reports for HIV, HCV, and hepatitis B.

[0286] Vector: Siglec-9 expression will be driven by the CMV promoter and co-expressed with a GFP or RFP reporter gene in a lentiviral vector, which can be used for non-viral plasmid transfection of Siglec-9 in target cells (e.g., Figure 5 , 7 Alternatively, the vector can be packaged into lentiviral particles to achieve efficient transduction and stable integration expression. Transient transfection is primarily used because human regulation of hT... reg These are primary cells and do not require long-term gene modification. The pLenti-CMV-GFP-2A-Puro-Blank vector and the Siglec-9 lentiviral vector were both purchased from Applied Biological Materials, Inc. (catalog numbers LV590, 43776061-GFP, and C129-RFP, respectively).

[0287] Testing the effect of siglec-9 protein overexpression on hT cells stimulated by the myofibroblast marker AOC3 reg Functional adjustment The impact, with a focus on IL-10 production and T reg Cell lineage stability, migration, and inhibition experiments.

[0288] The following experiments were used to investigate the effects of siglc-9 overexpression on key T cells. reg Impact on cell function.

[0289] a) IL-10 production: After AOC3 stimulation, the expression of Siglec-9 overexpressing T cells was quantitatively measured using enzyme-linked immunosorbent assay (ELISA) and qRT-PCR, respectively. reg Cells and their control T reg b) Protein levels and mRNA expression of IL-10 in cells; reg Cell lineage stability: Siglec-9 overexpressing T cells were assessed by Western blotting and flow cytometry after AOC3 stimulation. reg Cells and their control T reg T cells reg Expression of cell-specific lineage markers (FoxP3, CD25, CTLA-4); c) T reg Cell migration assay: Migration to AOC3 was assessed using a Trans-well migration apparatus. This assay will measure Siglec-9 overexpression of T cells. regThe ability of cells to migrate to the AOC3 gradient was used to characterize their targeting response; d) Inhibitory activity assessment: Siglec-9 overexpression of T cells was evaluated using co-culture proliferation assays. reg The ability of cell-inhibiting effector T cells (Teff) to proliferate was investigated, and CFSE staining and flow cytometry were used to track the division of Teff cells in the presence of AOC3.

[0290] Siglec-9 overexpression T reg Cells exhibited an enhanced anti-inflammatory response, primarily through increased IL-10 production and improved T cells. reg This targeting is achieved through the stability of cell lineage markers (FoxP3, CD25, CTLA-4) and more efficient migration to the AOC3 gradient. This targeting also leads to a significant inhibition of Teff cell proliferation. In summary, these effects highlight the role of siglec-9-modified Teff cells. reg The potential of T cells in effectively regulating fibrosis pathways. Different vector designs or promoters can be explored to enhance siglec-9 expression. Furthermore, comprehensive cytokine profiling can be performed to understand the broad spectrum of inflammatory or regulatory cytokines affected by siglec-9 overexpression, thus providing a more comprehensive understanding of engineered T cells. reg Changes in cell function.

[0291] Example 11: This non-limiting embodiment aims to elucidate the SHP-1 signaling pathway mediating T in response to AOC3 stimulation. reg Molecular mechanisms of Siglec-9 function in cells. This example is related to Example 10.

[0292] Preliminary data indicate that AOC3 interacts with Siglec-9, confirming the role of Siglec-9 as a functional ligand for AOC3. Figure 6 This interaction promotes downstream activation of the SHP-1 signaling pathway, and this response is enhanced in the presence of AOC3, which is achieved by siglec-9 engineered T. reg The increased phosphorylation levels of SHP-12510, Pyk22520, and Src2530 in cells confirmed this. Figure 25 The SHP-1 pathway, located downstream of Siglec-9, significantly affects IL-10 production and Foxp3 expression, which is a key factor in T... reg Key factors in cellular immune regulation. Based on these findings, AOC3-Siglec-9 interaction can directly regulate T cells. reg It can enhance cellular function and potentially improve its anti-fibrotic effects through precise molecular pathways.

[0293] Example 12 This non-limiting embodiment demonstrates that, upon AOC3 stimulation, the SHP-1 signaling pathway in siglec-9 mediated T... reg Characterization of its role in cellular function. This example is related to Examples 10 and 11.

[0294] The following experiments were used to investigate the effect of AOC3-siglec-9 interaction on the direct regulation of T. reg Impact on cell function.

[0295] a) SHP-1 pathway activation: After blocking AOC3 or siglec-9 interaction with neutralizing antibodies, the phosphorylation status of SHP-1, Pyk2, and Src was detected by Western blotting to assess the activation of SHP-1 pathway components. This indicator aims to evaluate the impact of disruption of AOC3-siglec-9 interaction on downstream signaling pathways. b) IL-10 cytokine release assay: After blocking AOC3-siglec-9 interaction or siglec-9 overexpression of T... reg After SHP-1 knockout in cells, IL-10 levels were quantified using ELISA and qRT-PCR. This experiment confirmed the reduction in the production of anti-inflammatory cytokines, thus demonstrating the role of this pathway in T cells. reg Role in cellular function. In some implementations, a 50% reduction in IL-10 compared to the control group indicates significant disruption of the pathway. c) Lineage stability assay: After blocking AOC3-siglec-9 interaction or knocking out SHP-1, T cells were examined using flow cytometry and Western blotting. reg The stability of cell lineage markers (Foxp3, CD25, CTLA-4). In some implementations, a reduction of at least 20% in marker expression is considered a successful result, indicating T... reg Cell stability has changed. d) Teff inhibition assay: Co-culture proliferation assays will be used to evaluate siglec-9 overexpression of Teff. reg The effectiveness of cell inhibition of Teff cell proliferation was assessed. This included using CFSE staining and flow cytometry to evaluate Teff cell division under AOC3 or siglec-9 blockade. A reduction in inhibition efficiency (e.g., at least 30%) compared to the control group demonstrated the role of AOC3-siglec-9 interaction in Teff cell proliferation. reg A key role in cell inhibition.

[0296] Blocking AOC3-siglec-9 interaction with neutralizing antibodies leads to reduced SHP-1 pathway activation, as evidenced by decreased phosphorylation levels of SHP-1, Pyk2, and Src. This disruption is expected to result in reduced IL-10 production, indicating that siglec-9 overexpression of T... regThe anti-inflammatory capacity of cells decreases. Similarly, T... reg The stability of cell lineage markers (such as Foxp3, CD25, and CTLA-4) may decrease, reflecting T... reg Cell function is impaired. Furthermore, these T cells... reg The effectiveness of cell-mediated inhibition of Teff cell proliferation may be reduced, further confirming the role of AOC3-siglec-9 interaction in maintaining Teff cell proliferation. reg SHP-1 plays a crucial role in cellular regulatory functions. Knocking out SHP-1 using siRNA may reflect these effects by similarly inhibiting the SHP-1 pathway, thus validating its role in mediating T cell activity through AOC3-siglec-9 interaction. reg It plays an important role in cell function. Alternative approaches include using other molecular tools (such as CRISPR-Cas9) to target T cells. reg Gene knockout of AOC3 or siglec-9 in cells was performed to investigate their effects on the SHP-1 pathway and T cells. reg The impact on cell function. Furthermore, expanding the scope of functional assessment to include additional regulatory and activation markers contributes to a more comprehensive understanding of the effects of pathway disruption on T cells. reg The impact on cellular capabilities.

[0297] Example 13 This non-limiting embodiment shows T reg Optimization of Siglec-9 overexpression levels in cells to enhance anti-fibrotic efficacy.

[0298] Optimize T using the following options reg Siglec-9 overexpression level in cells.

[0299] a) Vector Construction and Validation: A lentiviral vector with a controllable promoter will be constructed for siglec-9 overexpression. Efficiency and expression levels will be validated by Western blotting and flow cytometry. b) Expression Level Optimization: This will be achieved by titrating T... reg c) Functional experiments: Evaluate the effects of different siglec-9 expression levels on T cells. (c) The concentrations of the carrier and doxycycline in cell cultures were determined, and the resulting expression levels were quantitatively assessed to determine the optimal siglec-9 expression level. reg Effects of cell-mediated antifibrotic activity: 1) Inhibition of fibroblast proliferation: Evaluation of the effects of different siglec-9 expression levels (3-fold, 5-fold, and 10-fold) on T cells. regEffects of siglc-9 on the ability of angiotensin II to inhibit cardiac fibroblast proliferation. Quantitative experiments were used to establish a direct correlation between increased siglc-9 levels and reduced proliferation of stimulated fibroblasts. 2) Reduction of fibrosis markers: PCR and Western blotting were used to quantitatively assess the effect of different siglc-9 expression levels on the reduction of fibrosis markers (type I collagen and α-SMA) in cardiac fibroblasts. 3) Collagen gel shrinkage assay: The effects of different siglc-9 expression levels on the reduction of fibrosis markers (type I collagen and α-SMA) in cardiac fibroblasts were measured in cardiac fibroblasts exposed to angiotensin II and T cells with different levels of siglc-9 overexpression. reg The shrinkage of collagen gels co-cultured with cells. This experiment will demonstrate T... reg The ability of cells to effectively regulate fibrotic responses.

[0300] Example 14 This non-limiting embodiment shows a design for T reg Lentiviral vectors with controlled overexpression of Siglec-9 in cells.

[0301] A Siglec-9 lentiviral vector with a controllable promoter (Doxycycline is used to control gene expression in both the Tet-On and Tet-Off systems) was developed. Titration experiments to optimize the multiple of infection (MOI) were performed, and overexpression levels were validated by PCR and Western blotting. The vector showed that its targeting of Siglec-9 expression levels reached those of unengineered T... reg It is 3 to 10 times more potent than cells and does not affect cell viability.

[0302] 1. Carrier construction: A lentiviral vector containing the siglec-9 gene was constructed under the regulation of a tetracycline-responsive promoter. A reporter gene (such as GFP) linked to siglec-9 was integrated to facilitate the quantification of expression levels. 2. Initial titration of doxycycline: a) Transducing T cells using this lentiviral vector reg Cell population; b) Transduced T cells from different groups reg Cells were exposed to different concentrations of doxycycline (e.g., 0.1 µg / mL, 0.5 µg / mL, 1 µg / mL, and 2 µg / mL) to establish dose-response curves. c) Expression levels were measured using qPCR and Western blot at 24, 48, and 72 hours post-doxycycline exposure to determine the kinetics of siglec-9 expression. 3. Optimization of doxycycline dosing regimen: a) Select a doxycycline concentration that will initiate the expected baseline expression level (e.g., 3-fold); b) Gradually increase the doxycycline concentration, fine-tuning expression to achieve 5-fold and 10-fold levels; c) Confirm the results of multiple experiments and multiple batches of T. reg Reproducibility of expression levels between cells. 4. Verification of expression level:Western blotting and qPCR were performed to verify siglec-9 protein and mRNA levels, ensuring they were consistent with the expected baseline expression fold.

[0303] A lentiviral vector with a Tet-responsive promoter was constructed, allowing for controlled expression of Siglec-9 and ligation into a GFP reporter gene for monitoring. Preliminary titration of doxycycline established dose-response curves, demonstrating predictable Siglec-9 overexpression levels at different doxycycline concentrations. Furthermore, Siglec-9 expression could be fine-tuned to target levels (3-fold, 5-fold, 10-fold) by incrementally adjusting doxycycline concentration without impairing T... reg Cell viability and function. Alternative induction systems include inducible CRISPR activation systems.

[0304] Example 15 This non-limiting example demonstrates the determination of the optimal Siglec-9 expression level for effective inhibition of fibrosis.

[0305] The effects of different Siglec-9 expression levels on T were evaluated. reg Effects of Siglec-9 on the ability of cells to inhibit angiotensin II-induced cardiac fibroblast proliferation and reduce fibrosis markers. A correlation was observed between increased Siglec-9 expression levels (3-, 5-, and 10-fold) and reduced angiotensin II-stimulated cardiac fibroblast proliferation. Furthermore, in T cells overexpressing Siglec-9 (3-, 5-, and 10-fold)... reg Reduced collagen gel contraction was observed in cell cultures, indicating that it effectively inhibited the contractility of cardiac fibroblasts exposed to angiotensin II.

[0306] In some implementations, at the lowest effective Siglec-9 expression level, proliferation rate was reduced by at least 50% compared to the control group. Simultaneously, PCR and Western blot analyses showed a significant reduction (e.g., at least 30%) in the levels of fibrosis markers (such as type I collagen and α-SMA) at the lowest effective Siglec-9 level. In collagen gel shrinkage quantification, compared with the use of unengineered T... reg Compared to gels containing cells, gels with the lowest effective Siglec-9 expression showed reduced shrinkage (e.g., at least about 40%).

[0307] Example 16 This non-limiting embodiment shows the Siglec-9 engineered T reg Therapeutic applications of cells in preclinical ICM models.

[0308] The above results indicate that the Siglec-9 engineered Treg Cells effectively reduced cardiac fibrosis and improved cardiac function. To effectively mimic the ICM phenotype, a non-reperfusion MI model with more chronic characteristics was used. This mouse ICM model was also used for efficient and controlled dosing studies to determine the optimal therapeutic dose. Simultaneously, the optimal dose was tested in a pig model, which is highly similar to human cardiac physiology and disease. This model is crucial for translating preclinical findings into clinical applications. The optimal dose determined using the mouse model was converted to the pig model dose on a weight-based basis.

[0309] To determine the minimum effective dose and assess the recovery of cardiac function after treatment in the ICM mouse model, the following options were used.

[0310] a) Quantitative fibrosis: Sirius red staining was used to observe fibrotic areas, Western blotting was used to detect fibrosis protein markers (type I collagen and α-SMA), and qPCR was used to detect the gene expression levels of these markers. b) Cardiac function and structural analysis: Left ventricular ejection fraction (LVEF) and end-diastolic and end-systolic volumes were measured by echocardiography. c) Inflammation assessment: General inflammation was observed using H&E staining, specific inflammatory cells (CD68, CD3) were detected using immunohistochemistry (IHC), inflammatory cytokines (TNF-α, IL-6, IL-1β) were detected using ELISA, and inflammatory gene expression was detected using qPCR.

[0311] An engineered T that leads to a reduction in fibrosis (e.g., at least 30%) was identified. reg The minimum effective dose (optimal dose) for cells, and the corresponding reduction (e.g., 20% to 30%) in the expression levels of fibrosis-related proteins and genes. Determine the dose that will improve LVEF (e.g., at least 20%) and achieve a reduction (e.g., at least 25%) in inflammatory markers (including cell infiltration and cytokine levels). Additional markers or more sensitive assays may be used to better capture the anti-inflammatory effects of the treatment.

[0312] In a chronic ICM pig model, engineered T was evaluated using the following options. reg The therapeutic effects of cells on cardiac function, structure, and safety.

[0313] a) LVEF Measurement: LVEF was calculated using cine MRI by determining end-diastolic and end-systolic volumes. b) Scar Size Measurement: Myocardial scars were identified and measured using gadolinium-delayed enhancement (LGE) MRI. c) Inflammation Assessment: Histopathological Analysis: Cell infiltration was assessed using hematoxylin-eosin (H&E) staining. IHC: Specific inflammatory cells (macrophages and T cells) were detected using CD68 and CD3 antibodies. Cytokine Quantification: Inflammatory cytokines (TNF-α, IL-6, IL-1β) in cardiac tissue or serum were measured by ELISA. Gene Expression Analysis: mRNA levels of inflammatory markers were measured using qPCR.

[0314] Optimal dose of engineered T reg Cells significantly improved LVEF and reduced scar size via LGE, demonstrating effective cardiac recovery. Inflammatory markers may be reduced, for example, by at least 25%, indicating reduced cardiac inflammation. Doses can be examined at 3-fold lower and 3-fold higher levels to fine-tune the dosage, ensuring optimal efficacy and safety. Safety analyses confirmed no adverse effects on survival, blood parameters, or heart rhythm.

[0315] All patents and other publications cited throughout this application, including references, granted patents, published patent applications, and pending patent applications, are expressly incorporated herein by reference for the purpose of describing and disclosing methods, for example, those described in publications relating to the art described herein. These publications are provided solely for their publication prior to the filing date of this application. In this respect, nothing should be construed as an admission that the inventor was not entitled to place such disclosure prior to the filing date of this application by virtue of a prior invention or for any other reason. Any statement regarding the dates or contents of these documents is based on information available to the applicant and does not constitute any admission of the accuracy of such dates or contents.

[0316] The description of embodiments in this disclosure is not exhaustive and is not intended to limit this disclosure to the precise forms disclosed. While specific embodiments and examples of this disclosure have been described herein for illustrative purposes, those skilled in the art will recognize that various equivalent modifications are possible within the scope of this disclosure. For example, although method steps or functions are presented in a particular order, alternative embodiments may perform functions in a different order or substantially simultaneously. The teachings of this disclosure provided herein can be suitably applied to other procedures or methods. The various embodiments described herein can be combined to provide further embodiments. Where necessary, aspects of this disclosure may be modified to employ the compositions, functions, and concepts from the foregoing references and applications, thereby providing further embodiments of this disclosure. Furthermore, some changes to the protein structure may be made in consideration of biological functional equivalence without affecting the type or amount of biological or chemical action. These and other changes may be made to this disclosure based on the detailed description. All such modifications are intended to be included within the scope of the appended claims.

[0317] Specific elements of any of the above embodiments may be combined or substituted with elements in other embodiments. Furthermore, while advantages relating to certain embodiments of this disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments must exhibit such advantages to fall within the scope of this disclosure.

Claims

1. A method of treating fibrosis, comprising administering to a subject requiring treatment for fibrosis a nucleic acid encoding a sialic acid-binding immunoglobulin-like lectin 9 (Siglec-9) protein, wherein the Siglec-9 protein is generated by the nucleic acid in the subject's regulatory T(T) receptor. reg It is expressed in cells.

2. The method according to claim 1, wherein, The subjects had a need for treatment of cardiac fibrosis.

3. A method of treating a condition associated with inflammation and / or fibrosis, comprising administering to a subject in need of treatment for a condition associated with inflammation and / or fibrosis a substance configured to induce sialic acid binding of immunoglobulin-like lectin 9 (Siglec-9) protein in the subject's regulatory T (T) receptor. reg Nucleic acid expressed in cells.

4. The method according to claim 3, wherein, The nucleic acid encoding is derived from the T cells in the subject's body. reg The cell expresses the Siglec-9 protein, optionally wherein the nucleic acid comprises mRNA encoding the Siglec-9 protein.

5. The method according to claim 3, wherein, The conditions associated with inflammation and / or fibrosis include cardiac inflammation and / or cardiac fibrosis.

6. The method according to claim 3, wherein, The conditions associated with inflammation and / or fibrosis include myocardial infarction, hypertension, myocarditis, transplant rejection, cardiomyopathy, heart failure, and amyloidosis.

7. The method according to claim 3, wherein, The conditions associated with inflammation and / or fibrosis are angiotensin II-induced conditions.

8. The method according to claim 3, wherein, Administration of the nucleic acid includes administering a therapeutically effective amount of the composition to the subject, the composition comprising a T group containing the nucleic acid. reg Cell population, optionally, wherein the therapeutically effective amount of composition comprises 10 7 The T cells / kg body weight or more reg cell.

9. The method according to claim 8, wherein, T in the cell population reg The cell contains a plasmid or viral vector containing the nucleic acid, optionally wherein the viral vector comprises an adenovirus vector, a retrovirus vector, or a lentivirus vector.

10. The method of claim 9, comprising gene-modifying T cells using a plasmid or viral vector containing said nucleic acid. reg cell.

11. The method according to claim 3, wherein, The T reg The cell population was derived from the subject's own cells.

12. The method according to claim 3, wherein, The T reg The cell population was derived from allogeneic cells of the subject.

13. The method of claim 3, further comprising administering to the subject a vector containing the nucleic acid, wherein the vector is configured to deliver the nucleic acid to T cells within the subject. reg cell.

14. The method according to claim 13, wherein, The carrier comprises: lipid nanoparticles (LNPs); and T associated with the LNPs. reg Cell-targeting component.

15. The method according to claim 14, wherein, The T reg The cell-targeting component includes binding to T reg Antigen-binding proteins of cell markers, optionally, wherein the T reg Cellular markers were selected from CD25 and / or Foxp3.

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

17. The method according to claim 1, wherein, The subjects had or were at risk of having myocardial infarction.

18. The method according to claim 1, wherein, The subjects had a need for treatment of angiotensin II-induced cardiac fibrosis.

19. The method according to claim 1, wherein, The subjects had a need for treatment of cardiac fibrosis following myocardial infarction.

20. The method according to claim 3, wherein, The Siglec-9 protein contains an amino acid sequence that is 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 treatment for a condition associated with inflammation and / or fibrosis a protein configured to induce membrane-bound fibroblast-specific binding in the subject's regulatory T (T) receptor. reg Nucleic acid expressed in cells, 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 that is at least 80% identical to SEQ ID NO: 2, and optionally, the membrane-bound fibroblast-specific binding protein includes sialic acid-binding immunoglobulin-like lectin 9 (Siglec-9).

23. The method according to claim 21, wherein, The conditions associated with inflammation and / or fibrosis include inflammatory tissue containing fibroblasts with elevated expression of copper-containing amine oxidase 3 (AOC3).

24. The method according to claim 23, wherein, The fibroblasts with elevated AOC3 expression include myofibroblasts, and optionally, the myofibroblasts include cardiac myofibroblasts.

25. The method of claim 21, further comprising: Obtain the regulation T(T) reg ) immune cell populations; The nucleic acid was delivered to the T reg Cells, thereby allowing the Siglec-9 protein to be present in the T cells. reg Overexpression in cells; T cells overexpressing Siglec-9 protein reg The cells are in contact with at least one fibroblast and / or myofibroblast of the subject, wherein the at least one fibroblast and / or myofibroblast expresses copper-containing amino oxidase 3 (AOC3). This can treat conditions associated with the inflammation and / or fibrosis.

26. A method for targeted immune modulation in a subject, comprising: Obtain regulatory T cells (T cells) reg )group; To the T reg Cell population delivery of at least one vector, wherein the at least one vector comprises a protein configured to induce sialic acid binding of immunoglobulin-type lectin-9 (Siglec-9) protein in the T cells. reg Nucleic acid expressed in the cell population leads to the production of enhanced T cells overexpressing Siglec-9. reg Cell population; as well as Administer the enhanced T to the subject reg Cell population Optionally, the nucleic acid encodes the Siglec-9 protein.

27. The method according to claim 26, wherein, The at least one carrier comprises liposomes, lipid nanoparticles (LNPs), or viral vectors, optionally wherein the viral vector comprises adenovirus vectors, reverse transcription vectors, or lentivirus vectors.

28. The method according to claim 26, wherein, The nucleic acid includes a promoter operatively linked to a nucleotide sequence encoding the Siglec9 protein, wherein the promoter upregulates the T reg Expression of Siglec9 protein in cells.

29. The method according to claim 26, wherein, The subject includes at least one myofibroblast with elevated AOC3 expression compared to other cell types, optionally, wherein the at least one myofibroblast is at least one cardiac myofibroblast.

30. The method according to claim 26, wherein, The subjects had a need for treatment of fibrosis.

31. The method according to claim 30, wherein, The fibrosis is cardiac fibrosis, optionally wherein the cardiac fibrosis is secondary to myocardial infarction.

32. A method for inhibiting the expression of fibrosis genes, comprising: Get T reg Cell population; For the T reg Cells were engineered to overexpress the Siglec-9 protein, thereby producing enhanced T cells. reg cell; as well as Administer an effective amount of the enhanced T to subjects who require inhibition of fibrosis gene expression. reg Cells, optionally, wherein the fibrosis gene is at least one of type I collagen, α-smooth muscle actin (α-SMA), and / or copper-containing amine oxidase 3 (AOC3).

33. The method according to claim 32, wherein, Engineering includes: To the T reg A cell population delivers a vector containing at least one nucleic acid encoding the Siglec-9 protein, optionally, wherein the nucleic acid comprises mRNA; or Delivery of at least one carrier containing a substance configured to induce the T reg Nucleic acid expression of Siglec-9 protein in a cell population.

34. The method according to claim 33, wherein, The at least one carrier includes at least one of liposomes, nanoparticles, lipid nanoparticles (LNP), viral vectors, cell-penetrating peptides (CPP), and / or extracellular vesicles (EV).

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

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

37. The method of claim 36, wherein, The cardiac fibrosis is secondary to myocardial infarction.

38. The method according to claim 3, wherein, The subjects had upregulated expression of copper-containing amine oxidase 3 (AOC3) in cardiac myofibroblasts.

39. The method according to claim 1, wherein, The administration includes 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, operably linked to a regulatory T(T) protein. reg The promoter for expressing Siglec-9 protein in cells, optionally, wherein the promoter is a regulatory T (T) promoter selected from the Foxp3 promoter and the interleukin-2 (IL-2) promoter. reg Cell-specific promoters.

41. A vector comprising the isolated nucleic acid according to claim 40.

42. A regulatory T (T reg ) cells, comprising the isolated nucleic acid according to claim 41, optionally wherein the T reg Cells are human T reg cell.

43. A composition comprising a group of carriers, each carrier comprising: Nucleic acid encoding sialic acid-binding immunoglobulin-like lectin 9 (Siglec-9); as well as Configured to deliver nucleic acids encoding Siglec-9 to T reg Regulatory T cells (T) reg ) cell-targeting portion, optionally, wherein the T reg Cells are human T cells reg cell.

44. The composition according to claim 43, wherein, The carrier of the cell population contains lipid nanoparticles (LNPs).

45. The composition according to claim 43, wherein, The nucleic acid includes mRNA.

46. ​​The composition according to claim 43, wherein, The T reg The cell-targeting component includes binding to T reg Antigen-binding proteins of cell markers, optionally, wherein the T reg Cellular markers were selected from CD25 and / or Foxp3.

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

48. A composition comprising an enhanced regulatory T (T) cell overexpressing sialic acid-binding immunoglobulin-like lectin 9 (Siglec-9). reg ) cell population, optionally, wherein the T reg Cells are human T cells reg cell.

49. The composition according to claim 48, wherein, The enhanced T reg The cell contains at least one endocytic carrier, which contains multiple nucleic acids encoding sialic acid-binding immunoglobulin-like lectin 9 (Siglec-9).

50. The composition according to claim 49, wherein, The at least one endocytic carrier includes at least one of liposomes, nanoparticles, lipid nanoparticles (LNPs), viral vectors, cell-penetrating peptides (CPPs), and / or extracellular vesicles (EVs).

51. The composition according to claim 49, wherein, The multiple nucleic acids include multiple mRNA transcripts encoding Siglec-9.

52. The composition according to claim 48, wherein, The enhanced T reg Cells exhibit Siglec-9-dependent interactions with cells expressing copper-containing amine oxidase 3 (AOC3), optionally, wherein the enhanced T cells... reg The cells exhibited Siglec-9-dependent interactions with AOC3-expressing cardiac myofibroblasts.

53. The method, nucleic acid isolation, vector, and T according to any one of the preceding claims. reg Cells, or compositions thereof, wherein The Siglec-9 mentioned is human Siglec-9.

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