Extracellular vesicle comprising adiponectin or cargo induced by adiponectin signaling and methods of use thereof

Abstract

The present disclosure describes an extracellular vesicle (EV) derived from a cell over -expressing adiponectin, a cell treated with adiponectin, a cell treated with an adiponectin receptor agonist, and / or a cell obtained from a donor subject that has been treated with an adiponectin receptor agonist. Also provided is a method of treating an aging-related disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an EV derived from a cell over-expressing adiponectin, a cell treated with adiponectin, a cell treated with an adiponectin receptor agonist, and / or a cell obtained from a donor subject that has been treated with an adiponectin receptor agonist.

Description

EXTRACELLULAR VESICLE COMPRISING ADIPONECTIN OR CARGO INDUCED BY ADIPONECTIN SIGNALING AND METHODS OF USE THEREOFRELATED APPLICATION

[0001] This disclosure claims benefit of United States Provisional Patent Application serial no.63 / 755401 filed February 7, 2025, incorporated herein by reference in its entirety.INCORPORATION OF SEQUENCE LISTING

[0002] A computer readable form of the Sequence Listing “P93250282WO01_96665564_SequenceListing.xml” (4,463 bytes), is filed herewith by electronic submission and is incorporated by reference herein.FIELD

[0003] The present disclosure relates to the field of extracellular vesicles (EVs) involving adiponectin and / or adiponectin signaling, and methods of use thereof, for example, for promoting healthspan and longevity, or treating an aging-related disease.BACKGROUND

[0004] Aging is a natural biological process marked by functional declines across multiple organ systems and an increased risk of chronic diseases as individuals grow older. Despite advances in extending life expectancy, improvements in healthspan have lagged, leading to prolonged periods of illness and rising healthcare costs. Common aging-related diseases include diabetes, Alzheimer’s disease, chronic kidney disease, osteoarthritis, and cardiovascular disease. Multimorbidity among seniors often requires combinatorial treatments, highlighting the need for therapies targeting aging itself to promote healthy longevity. Adiponectin (a.k.a. AdipoQ and Acrp30) is a circulating hormone abundantly present in healthy individuals that is involved in regulating metabolic health and the aging process. Extracellular vesicles (EVs) are being investigated for their potential role in intercellular communication and regulation of processes associated with aging, such as metabolism and tissue maintenance.SUMMARY

[0001] The present disclosure relates to an extracellular vesicle (EV) derived from a cell treated with an adiponectin receptor agonist, a cell over-expressing adiponectin, a cell treated with adiponectin, and / or a cell obtained from a donor subject that has been treated with an adiponectin receptor agonist. In some embodiments, the EV is derived from a cell treated with an adiponectin receptor agonist. In some embodiments, the EV is derived from a cell treated with adiponectin. In some embodiments, the EV is derived from a cell over-expressing adiponectin. In some embodiments, the EV is derived from a cell obtained from a donor subject that has been treated with an adiponectin receptor agonist. In someembodiments, the adiponectin comprises an amino acid sequence having 75%, 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to the full length of SEQ ID NO: 1. In some embodiments, the adiponectin comprises an amino acid sequence having 100% sequence identity to the full length of SEQ ID NO: 1. In some embodiments, the adiponectin receptor agonist comprises ALY688 (D-Asn-Ile-Pro-Nva-Leu-Tyr-D-Ser-Phe-Ala-D-Ser; SEQ ID NO: 2) or adiporon. In some embodiments, the adiponectin receptor agonist is ALY688. In some embodiments, the EV is derived from an adipocyte, a cardiomyocyte, a hepatocyte, a platelet, an immune cell, an endothelial cell, an erythrocyte, a stem cell, a fibroblast, a smooth muscle cell, a neuronal cell, a glial cell, an astrocyte, a Schwann cell, a keratinocyte, a chondrocyte, an osteoblast, an osteocyte, or any combination thereof. In some embodiments, the EV is isolated from plasma. In some embodiments, the donor subject from which the cell is obtained is a mammal. In some embodiments, the mammal is mouse. In some embodiments, the mammal is human. In some embodiments, the EV is enriched with adiponectin relative to an EV isolated from plasma of a subject administered vehicle. In some embodiments, the EV is capable of reducing cardiac injury associated with myocardial infarction. In some embodiments, the EV is capable of suppressing an increase in plasma troponin I in a recipient subject experiencing myocardial infarction. In some embodiments, the EV is capable of suppressing an increase in plasma lactate dehydrogenase (LDH) in a recipient subject experiencing myocardial infarction. In some embodiments, the adiponectin receptor agonist used to treat the donor subject comprises ALY688.

[0002] In an aspect, the present disclosure relates to a pharmaceutical composition comprising an EV described herein and a pharmaceutically acceptable carrier.

[0003] In an aspect, the present disclosure relates to a method involving adiponectin and / or EV for aging-related disease treatment. In an aspect, provided herewith is a method of promoting healthspan or treating an aging-related disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an EV derived from a cell over -expressing adiponectin, a cell treated with adiponectin, a cell treated with an adiponectin receptor agonist, and / or a cell obtained from a donor subject that has been treated with an adiponectin receptor agonist.

[0005] In some embodiments, the EV is derived from an adipocyte, a cardiomyocyte, a hepatocyte, a platelet, an immune cell, an endothelial cell, an erythrocyte, a stem cell, a fibroblast, a smooth muscle cell, a neuronal cell, a glial cell, an astrocyte, a Schwann cell, a keratinocyte, a chondrocyte, an osteoblast, an osteocyte, or any combination thereof. In some embodiments, the EV is isolated from plasma. In some embodiments, the immune cell is a T cell, a B cell, a macrophage, a dendritic cell, or a natural killer cell. In some embodiments, the endothelial cell is a progenitor cell. In some embodiments, the cardiomyocyte is derived from a cardiosphere. In some embodiments, the EV is derived from a stem cell. In some embodiments, the stem cell is a mesenchymal stem cell or an induced pluripotent stem cell. In some embodiments, the stem cell is an induced pluripotent stem cell.

[0006] In some embodiments, the EV for treating the aging-related disease is derived from a cell overexpressing adiponectin. In some embodiments, the EV for treating the aging-related disease is derived from a cell treated with adiponectin. In some embodiments, the adiponectin for generating the EV comprises an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to the full length of SEQ ID NO: 1. In some embodiments, the EV for treating the aging-related disease is derived from a cell treated with an adiponectin receptor agonist. In some embodiments, the adiponectin receptor agonist for generating the EV comprises ALY688 (also known as ADP355) or adiporon. In some embodiments, the adiponectin receptor agonist is ALY688.

[0007] In some embodiments, the aging-related disease comprises a neurodegenerative disorder, a metabolic disorder, a cardiovascular disease, a musculoskeletal disorder, a renal or urological disease, a cancer, an eye disorder, a respiratory disorder, an endocrine disorder, and / or an immune system disorder. In some embodiments, the neurodegenerative disorder comprises Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, or amyotrophic lateral sclerosis (ALS). In some embodiments, the metabolic disorder comprises type 2 diabetes, metabolic associated steatohepatitis (MASH), or obesity. In some embodiments, the cardiovascular disease comprises hypertension, atherosclerosis, coronary artery disease, myocardial infarction, heart failure, arrhythmias, peripheral artery disease, or stroke. In some embodiments, the musculoskeletal disorder comprises osteoarthritis, sarcopenia, or osteoporosis. In some embodiments, the renal or urological disease comprises chronic kidney disease, diabetic nephropathy, or benign prostatic hyperplasia. In some embodiments, the cancer comprises breast cancer, prostate cancer, colorectal cancer, lung cancer, leukemia, or bladder cancer. In some embodiments, the eye disorder comprises age-related macular degeneration, cataracts, or glaucoma. In some embodiments, the respiratory disorder comprises chronic obstructive pulmonary disease or pulmonary fibrosis. In some embodiments, the endocrine disorder comprises hypothyroidism, hyperparathyroidism, adrenal insufficiency, hypercortisolism, a pituitary disorder, an alteration in metabolic hormone production, or an alteration in sex hormone production. In some embodiments, the immune system disorder comprises immunosenescence, chronic inflammation, or an autoimmune disease. In some embodiments, the autoimmune disease comprises rheumatoid arthritis, systemic lupus erythematosus, Hashimoto’s thyroiditis, Sjogren’s syndrome, or hypogammaglobulinemia. In some embodiments, the subject treated in the method is a mammal. In some embodiments, the subject is a human.

[0008] Other features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating embodiments of the disclosure, are given by way of illustration only and the scope of the claims should not be limited by these embodiments, but should be given the broadest interpretation consistent with the description as a whole.BRIEF DESCRIPTION OF THE DRAWINGS

[0009] The embodiments of the disclosure will now be described in greater detail with reference to the attached drawings in which:

[0010] FIG. 1A shows isolation of plasma EV using size exclusion chromatography from mice treated for 28 days with either PBS or ALY688 (10 mg / kg), in an exemplary embodiment of the disclosure. Western immunoblotting revealed significantly increased expression of well-established EV markers CD36 and Alix, in ALY688-treated mice. Adiponectin was detected within plasma EVs, indicating that EV functions as a carrier of adiponectin in circulation.

[0011] FIG. IB shows cryo-electron microscopy (cryo-EM) of isolated EV, in an exemplary embodiment of the disclosure. FIG. IB shows quality control of EV isolation by identifying the distinct ultrastructure of EV from an ALY688-treated mouse.

[0012] FIG. 1C shows a representative nanoparticle tracking analysis (NT A) of ALY688-treated mice compared to controls, in an exemplary embodiment of the disclosure. FIG. 1C shows a significant increase in EV particle concentration in ALY688-treated mice compared to controls.

[0013] FIG. ID shows NT A of ALY688-treated mice compared to controls, in an exemplary embodiment of the disclosure. FIG. ID shows a significant increase in EV particle concentration in ALY688-treated mice compared to controls. Data are presented as mean ± SD; N = 3; * indicates P<0.05 . Unpaired T-test.

[0014] FIG. 2A shows adiponectin-associated EV dynamics, in an exemplary embodiment of the disclosure. FIG. 2A shows Western immunoblot of EV from wild type (WT) mice contained adiponectin and EV markers CD63, Flotillin-1, and Alix, while EV from adiponectin-knock out (KO) mice lacked adiponectin and retained EV markers.

[0015] FIG.2B shows cryo-EM of isolated EV, in an exemplary embodiment of the disclosure. FIG. 2B shows quality control of EV isolation by identifying the vesicular structures in isolated EV of adiponectin-KO male mouse.

[0016] FIG.2C shows a representative NT A of adiponectin-KO mice compared to WT, in an exemplary embodiment of the disclosure. FIG. 2C shows an increase in particle number in EV from adiponectin-KO mice plasma compared to WT.

[0017] FIG.2D shows NT A of adiponectin-KO mice compared to WT, in an exemplary embodiment of the disclosure. FIG. 2D shows an increase in particle number in EV from adiponectin-KO mice plasma compared to WT. Data are presented as mean ± SD; N = 5; *** indicates PO.OOl. Unpaired T-test.

[0018] FIG.2E shows NT A of adiponectin-KO mice compared to WT, in an exemplary embodiment ofthe disclosure. FIG. 2E shows an increase in particle size in EV from adiponectin-KO mice plasma compared to WT. Data are presented as mean ± SD; N = 5; ** indicates PO.Ol. Unpaired T-test.

[0019] FIG. 2F shows the direct functional effect of the isolated plasma EV in skeletal muscle cells expressing an Akt biosensor for examination of insulin sensitivity, in an exemplary embodiment of the disclosure. FIG. 2F shows images of a time-course analysis after 10 nM insulin stimulation. In the presence of EV from adiponectin-KO mice, the skeletal muscle cells did not respond normally to insulin as compared to in the presence of EV from WT mice.

[0020] FIG. 2G shows the direct functional effect of the isolated plasma EV in skeletal muscle cells expressing an Akt biosensor for examination of insulin sensitivity, in an exemplary embodiment of the disclosure. FIG. 2G shows quantification of fluorescence signal in a time-course analysis after 10 nM insulin stimulation. In the presence of EV from KO mice, the skeletal muscle cells did not respond normally to insulin (as seen in the skeletal muscle cells in the presence of EV from WT mice). Data are presented as mean ± SEM; N = 3; * indicates P<0.05; ** indicates P<0.01; *** indicates PO.OOl. One Way ANOVA.

[0021] FIG. 3 shows representative Western immunoblot images of EV markers in plasma EV isolated from human subjects with Type 2 Diabetes (T2D), T2D and cardiomyopathy (T2D-CMP), and healthy controls, in an exemplary embodiment of the disclosure. Plasma EVs were isolated from the human subjects and analyzed by Western immunoblotting. A significant decrease is seen in the expression of adiponectin in EV isolated from T2D and T2D-CMP patients compared to healthy controls. Each lane shows the results from a human subject.

[0022] FIG.4A shows proteomics analysis of plasma EV, in an exemplary embodiment of the disclosure. FIG. 4A shows principal component analysis (PCA) of all proteins detected in EV, indicating clear separation between PBS- and ALY688-injected mouse plasma EV.

[0023] FIG.4B shows proteomics analysis of plasma EV, in an exemplary embodiment of the disclosure. FIG. 4B shows a heat map of combined data from 4 mice in each group of plasma EV from PBS- and ALY688-injected mice, indicating the differential expression (black = increase; gray = decrease) of the top 50 individual proteins in ALY688-injected (left column) versus PBS-injected mouse plasma EV (right column).

[0024] FIG.5A shows EV isolated from mouse plasma and primary mouse adipocytes after injection of adiponectin receptor agonist having beneficial functional effects on cells, in an exemplary embodiment of the disclosure. Rat cardiomyocyte H9c2 cell line was treated with EV or subjected to regular media (control) for 30 minutes, and then the cell line was used as a target and incubated under 24-hour hypoxia (1% O2) to induce cell damage or normoxia (20% O2) as a control. Cell viability data showed that cell death induced by hypoxia was significantly reduced by the administration of Plasma-EV-ALY and Ad-EV-ALY, but notEV from mice subjected only to PBS injection. Data are presented as mean ± SEM; N = 5; * indicates P<0.05; ** indicates PO.Ol. One Way ANOVA.

[0025] FIG. 5B shows representative images of EV isolated from mouse plasma and primary mouse adipocytes after injection of adiponectin receptor agonist having beneficial functional effects on cells, in an exemplary embodiment of the disclosure. H9c2 cells from FIG. 5A were assessed for cellular oxidative stress by CellRox fluorescent probe. Data demonstrated a beneficial effect of EV isolated from plasma or adipocytes from ALY688-injected but not PBS-injected mice.

[0026] FIG.5C shows EV isolated from mouse plasma and primary mouse adipocytes after injection of adiponectin receptor agonist having beneficial functional effects on cells, in an exemplary embodiment of the disclosure. FIG. 5C shows the quantified results of FIG. 5B. Data are presented as mean ± SEM; N = 3; *** indicates P<0.005. One Way ANOVA.

[0027] FIG.5D shows EV isolated from mouse plasma and primary mouse adipocytes after injection of adiponectin receptor agonist having beneficial functional effects on cells, in an exemplary embodiment of the disclosure. A macrophage cell line (RAW) engineered with an interferon (IFN) reporter was used to elucidate effects on inflammation. RAW cells pretreated with Plasma-EV-ALY or Ad-EV-ALY show attenuation of hypoxia triggered IFN activation. Data are presented as mean ± SEM; N = 3; * indicates P<0.05; ** indicates PO.Ol; *** indicates P<0.001. One Way ANOVA.

[0028] FIG.5E shows EV isolated from mouse plasma and primary mouse adipocytes after injection of adiponectin receptor agonist having beneficial functional effects on cells, in an exemplary embodiment of the disclosure. RAW engineered with a nuclear factor KB (NFKB) reporter was used to elucidate effects on inflammation. Protective effect of combating hypoxia-induced NFKB was shown in Ad-EV ALY. Data are presented as mean ± SEM; N = 3; * indicates PO.05; *** indicates P<0.001. One Way ANOVA.

[0029] FIG.6A shows a histogram from NTA of plasma EVs isolated from mice administered ALY688 at (15 mg / kg) for 3 days, in an exemplary embodiment of the disclosure. Results are presented as mean ± SEM.

[0030] FIG. 6B shows particle number from NTA in ALY688 treated mice compared to controls, in an exemplary embodiment of the disclosure. FIG. 6B shows an increase in particle number with ALY688 treatment. Results are presented as mean ± SEM and N = 3 per group. *P<0.05 versus the indicated group. Two-way ANOVA.

[0031] FIG. 6C shows particle size from NTA in ALY688 treated mice compared to controls, in an exemplary embodiment of the disclosure. FIG. 6C shows that particle size remained unchanged. Results are presented as mean ± SEM and N = 3 group. Two-way ANOVA.

[0032] FIG. 6D shows SDS-PAGE and immunoblot analysis of plasma derived EVs using Alix, CD63,CD81, and adiponectin, in an exemplary embodiment of the disclosure.

[0033] FIG. 6E shows quantification of Alix abundance in EV isolates normalized by particle number, in an exemplary embodiment of the disclosure. FIG. 6E shows increased Alix with ALY688 where indicated. Results are presented as mean ± SEM and N = 3 per group. **P<0.01 versus the indicated group. Two-way ANOVA.

[0034] FIG.6F shows quantification of CD63 abundance in EV isolates normalized by particle number, in an exemplary embodiment of the disclosure. FIG. 6F shows increased CD63 with ALY688 where indicated. Results are presented as mean ± SEM and N = 3 per group. Two-way ANOVA.

[0035] FIG.6G shows quantification of CD81 abundance in EV isolates normalized by particle number, in an exemplary embodiment of the disclosure. FIG. 6G shows CD81 levels where indicated. Results are presented as mean ± SEM and N = 3 per group. One-way ANOVA.

[0036] FIG. 6H shows quantification of adiponectin abundance in EV isolates normalized by particle number, in an exemplary embodiment of the disclosure. FIG. 6H shows increased adiponectin loading after ALY688 treatment. Results are presented as mean ± SEM and N = 3 per group. **P<0.01 versus the indicated group. One-way ANOVA.

[0037] FIG. 61 shows SDS-PAGE and immunoblot analysis of H9c2 cells treated with EVs from PBS donors or ALY688 donors using p-AMPK, total AMPK, p-p38, and total p38 with GAPDH as a loading control, in an exemplary embodiment of the disclosure.

[0038] FIG. 6J shows quantification of p-AMPK expression in H9c2 cells treated as in FIG. 61, in an exemplary embodiment of the disclosure. Results are presented as mean ± SEM and N = 3 per group. **P<0.01 versus the indicated group. One-way ANOVA.

[0039] FIG. 6K shows quantification of p-p38 expression in H9c2 cells treated as in FIG. 61, in an exemplary embodiment of the disclosure. Results are presented as mean ± SEM and N = 3 per group. ***P<0.001 versus the indicated group. One-way ANOVA.

[0040] FIG. 7A shows LDH analyses in H9c2 cells subjected to 24 hours hypoxia after pretreatment with 10 pg / mL EVs from PBS donors or ALY688 donors, in an exemplary embodiment of the disclosure. FIG. 7A shows reduced cell death with EVALYcompared to EVPBS. Results are presented as mean ± SEM andN = 3 to 6 per group. *P<0.05, ***P<0.001 versus the indicated group. Two-way ANOVA.

[0041] FIG. 7B shows LDH analyses in induced pluripotent stem cell derived cardiomyocytes (iPSC-CM) subjected to 24 hours hypoxia after pretreatment with 10 pg / mL EVs from PBS donors or ALY688 donors, in an exemplary embodiment of the disclosure. FIG. 7B shows reduced cell death with EVALYcompared to EVPBS. Results are presented as mean ± SEM and N = 3 to 6 per group. **P<0.01, ***P<0.001versus the indicated group. Two-way ANOVA. iPSC = iPSC-CM.

[0042] FIG. 7C shows representative MitoTracker images of H9c2 cells and iPSC-CM subjected to 24 hours hypoxia after pretreatment with 10 pg / mL EVPBSor EVALY, in an exemplary embodiment of the disclosure. FIG. 7C shows improved mitochondrial morphology with EVAI,Y. Scale bar: 10 pm. iPSC = iPSC-CM.

[0043] FIG. 7D shows quantification of mitochondrial networking in H9c2 cells based on the images of FIG. 7C, in an exemplary embodiment of the disclosure. FIG. 7D shows that EVAI,Ymitigated hypoxia induced loss of mitochondrial networking. Results are presented as mean ± SEM and N = 3 to 6 per group. *P<0.05, **P<0.01 versus the indicated group. Two-way ANOVA.

[0044] FIG. 7E shows quantification of mitochondrial networking iPSC-CM based on the images of FIG. 7C, in an exemplary embodiment of the disclosure. FIG. 7E shows that EVALYmitigated hypoxia induced loss of mitochondrial networking. Results are presented as mean ± SEM and N = 3 to 6 per group. *P<0.05 versus the indicated group. Two-way ANOVA. iPSC = iPSC-CM.

[0045] FIG. 7F shows representative IMARIS analysis of mitochondrial sphericity in H9c2 cells subjected to 24 hours hypoxia after pretreatment with 10 pg / mL EVPBSor EVALY, in an exemplary embodiment of the disclosure. IMARIS sphericity analysis is an index of mitochondrial fission. Scale bar: 10 pm.

[0046] FIG. 7G shows IMARIS based sphericity quantification in H9c2 cells corresponding to FIG. 7F, in an exemplary embodiment of the disclosure. FIG. 7G shows increased mitochondrial fragmentation during hypoxia was reduced by EVALYin H9c2 cells. Results are presented as mean ± SEM and N = 3 biological replicates per group (the dots represent a total of 150 mitochondria for each bar). ***P<0.001 versus the indicated group Two-way ANOVA.

[0047] FIG. 7H shows representative CellROX deep red images from H9c2 cells and iPSC-CM subjected to 3 hours hypoxia after pretreatment with 10 pg / mL EVPBSor EVALY, in an exemplary embodiment of the disclosure. iPSC = iPSC-CM.

[0048] FIG. 71 shows quantification of mean fluorescence intensity from H9c2 cells corresponding to FIG. 7H, in an exemplary embodiment of the disclosure. FIG. 71 shows a reduction of reactive oxygen species with EVALYcompared to EVPBS. Results are presented as mean ± SEM and N = 3 to 6 per group. ***P<0.001 versus the indicated group Two-way ANOVA.

[0049] FIG. 7J shows quantification of mean fluorescence intensity from iPSC-CM corresponding to FIG. 7H, in an exemplary embodiment of the disclosure. FIG. 71 shows a reduction of reactive oxygen species with EVALYcompared to EVPBS. Results are presented as mean ± SEM and N = 3 to 6 per group. **P<0.01, ***P<0.001 versus the indicated group Two-way ANOVA. iPSC = iPSC-CM.

[0050] FIG. 7K shows a representative image of Hibit-LC3 H9c2 cells subjected to 24 hours hypoxia treated with 10 pg / mL EVPBSand EVALY, in an exemplary embodiment of the disclosure. Scale bar: 10 pm.

[0051] FIG. 7L shows quantification of Hibit-LC3 positive MFI corresponding to FIG. 7K, in an exemplary embodiment of the disclosure. ***P<0.001 versus the indicated group. Two-way ANOVA.

[0052] FIG.7M shows a representative image of MagicRed cathepsin B staining in H9c2 cells subjected to 24 hours hypoxia treated with 10 pg / mL EVPBSand EVALY, in an exemplary embodiment of the disclosure. FIG. 7M shows increased lysosomal activity with EVALY. Scale bar: 10 pm.

[0053] FIG. 8A shows a schematic diagram of the animal study in which EVs from ALY688-treated donors or PBS treated donors were administered by tail vein one hour after myocardial ischemia induced by LAD occlusion with sacrifice at two days post myocardial infarction, in an exemplary embodiment of the disclosure.

[0054] FIG. 8B shows plasma troponin I levels measured six hours post myocardial infarction in mice subjected to sham or acute myocardial infarction and injected with EVs from PBS donors or ALY688 donors, in an exemplary embodiment of the disclosure. FIG. 8B shows that EVALYsuppressed the myocardial infarction induced increase in troponin I. Results are presented as mean ± SEM and N = 6 to 8 per group. *P<0.05, **P<0.01, ***P<0.001 versus the indicated group. Two-way ANOVA.

[0055] FIG. 8C shows terminal LDH levels measured in plasma from the mice of FIG. 8B, in an exemplary embodiment of the disclosure. FIG. 8C shows that EVAI,Ysuppressed the myocardial infarction induced increase in LDH. Results are presented as mean ± SEM and N = 6 to 8 per group. *P<0.05, **P<0.01, ***P<0.001 versus the indicated group. Two-way ANOVA.

[0056] FIG. 8D shows SDS-PAGE and immunoblot analysis of infarcted heart tissue using cleaved caspase 3 with [3-tubulin as a loading control, in an exemplary embodiment of the disclosure.

[0057] FIG. 8E shows quantification of cleaved caspase 3 from FIG. 8D, in an exemplary embodiment of the disclosure. FIG. 8E shows reduced apoptosis with E VAI,Ytreatment. Results are presented as mean ± SEM and N = 6 to 8 per group. *P<0.05, **P<0.01, ***P<0.001 versus the indicated group. Two-way ANOVA.

[0058] FIG.8F shows SDS-PAGE and immunoblot analysis of infarcted heart tissue using MFF, p-Drp 1 s637, p-Drpl s616, and OPA1 with (3-tubulin as a loading control, in an exemplary embodiment of the disclosure.

[0059] FIG. 8G shows quantification of MFF corresponding to FIG. 8F, in an exemplary embodiment of the disclosure. FIG. 8G shows a decrease with EVALYtreatment. Results are presented as mean ± SEM and N = 6 to 8 per group. **P<0.01, ***P<0.001 versus the indicated group. Two-way ANOVA.

[0060] FIG. 8H shows quantification of p-Drpl serine 637 corresponding to FIG. 8F, in an exemplary embodiment of the disclosure. FIG. 8H shows an increase with EVALYtreatment. Results are presented as mean ± SEM and N = 6 to 8 per group. **P<0.01, ***P<0.001 versus the indicated group. Two-way ANOVA.

[0061] FIG. 81 shows quantification of p-Drpl serine 616 corresponding to FIG. 8F, in an exemplary embodiment of the disclosure. FIG. 81 shows a decrease with EVAI,Ytreatment. Results are presented as mean ± SEM and N = 6 to 8 per group. *P<0.05, **P<0.01 versus the indicated group. Two-way ANOVA.

[0062] FIG. 8J shows quantification of OPA1 corresponding to FIG. 8F, in an exemplary embodiment of the disclosure. FIG. 8J shows an increase with EVALYtreatment. Results are presented as mean ± SEM and N = 6 to 8 per group. *P<0.05, **P<0.01, ***P<0.001 versus the indicated group. Two-way ANOVA.

[0063] FIG. 8K shows Sodl, Sod2, and Sod3 mRNA expression in infarcted heart tissue from the mice of FIG. 8A, in an exemplary embodiment of the disclosure. FIG. 8K shows restoration of antioxidant gene expression with EVAI,Y. Results are presented as mean ± SEM and N = 6 to 8 per group. *P<0.05, ***P<0.001 versus the indicated group. Two-way ANOVA.

[0064] FIG. 8L shows SDS-PAGE and immunoblot analysis of infarcted heart tissue using LC3, p62, and ATG3 with (3-tubulin as a loading control, in an exemplary embodiment of the disclosure.

[0065] FIG. 8M shows quantification of LC3 corresponding to FIG. 8L, in an exemplary embodiment of the disclosure. FIG. 8M shows no change in LC3 II (normalized with (3-tubulin) with EVALYtreatment. Results are presented as mean ± SEM and N = 6 to 8 per group. ***P<0.001 versus the indicated group. Two-way ANOVA.

[0066] FIG. 8N shows quantification of p62 corresponding to FIG. 8L, in an exemplary embodiment of the disclosure. FIG. 8N shows enhanced p62 (normalized with (3-tubulin) degradation with EVALYtreatment. Results are presented as mean ± SEM and N = 6 to 8 per group. **P<0.01, ***P<0.001 versus the indicated group. Two-way ANOVA.

[0067] FIG. 80 shows quantification of ATG3 corresponding to FIG. 8L, in an exemplary embodiment of the disclosure. FIG. 80 shows increased ATG3 (normalized with (3-tubulin) with EVALYtreatment. Results are presented as mean ± SEM and N = 6 to 8 per group. ***P<0.001 versus the indicated group. Two-way ANOVA.

[0068] FIG. 9A shows a histogram demonstrating particle size distribution from NTA of plasma EVs isolated from rats administered ALY688 at 15 mg / kg for three consecutive days or PBS, in an exemplary embodiment of the disclosure.

[0069] FIG. 9B shows quantification of average particle concentration from nanoparticle trackinganalysis in the rats of FIG. 9 A, in an exemplary embodiment of the disclosure. FIG. 9B shows increased particle concentration with ALY688 treatment. Data are presented as mean ± SEM and N = 7. Unpaired T-test.

[0070] FIG. 9C shows quantification of average particle size from nanoparticle tracking analysis in the rats of FIG. 9A, in an exemplary embodiment of the disclosure. FIG. 9C shows that particle size remained consistent with ALY688 treatment. Data are presented as mean ± SEM and N = 7. Unpaired T-test.

[0071] FIG. 10A shows representative images of a dose response analysis with 3, 6, 12, 25, 50, or 100 nM insulin stimulation in skeletal muscle cells after 24 hour pretreatment with rat derived plasma EVs from ALY688 donors or PBS donors, in an exemplary embodiment of the disclosure. FIG. 10A shows an insulin sensitizing effect with ALY688 vesicles.

[0072] FIG. 10B shows quantification of the half maximal effective concentration of insulin calibrated from fluorescence signal in the dose response analysis of FIG. 10A, in an exemplary embodiment of the disclosure. FIG. 10B shows a reduced insulin EC50 after pretreatment with vesicles from ALY688 treated rats compared to PBS controls. Data are presented as mean ± SEM and N = 3. ** indicates P<0.01. Oneway ANOVA.

[0073] FIG. 11A shows NT A quantification of average particle concentration for EVs isolated from adipocyte derived mesenchymal stem cells treated with ALY688 compared to PBS treated controls, in an exemplary embodiment of the disclosure.

[0074] FIG. 11B shows representative images of a dose response analysis with 3, 6, 12, 25, 50, or 100 nM insulin stimulation in skeletal muscle cells after 24 hour pretreatment with EVs from ALY688-treated or PBS treated adipocyte derived mesenchymal stem cells in the presence of 250 pM iron sulfate, in an exemplary embodiment of the disclosure.

[0075] FIG. 11C shows quantification of insulin EC50 from the dose response analysis of FIG. 1 IB, in an exemplary embodiment of the disclosure. FIG. 11C shows a lower insulin EC50 with ALY688 ADSC EV compared to PBS ADSC EV in the iron challenge. Data are presented as mean ± SEM and N = 3. DETAILED DESCRIPTION

[0076] The following is a detailed description provided to aid those skilled in the art in practicing the present disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The terminology used in the description herein is for describing particular embodiments only and is not intended to be limiting of the disclosure.I, Definitions

[0077] Unless otherwise indicated, the definitions and embodiments described in this and other sections are intended to be applicable to all embodiments and aspects of the present disclosure herein described for which they are suitable as would be understood by a person skilled in the art.

[0078] In understanding the scope of the present disclosure, the term “comprising” and its derivatives, as used herein, are intended to be open ended terms that specify the presence of the stated features, elements, components, groups, integers, and / or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and / or steps. The foregoing also applies to words having similar meanings such as the terms, “including”, “having” and their derivatives. The term “consisting” and its derivatives, as used herein, are intended to be closed terms that specify the presence of the stated features, elements, components, groups, integers, and / or steps, but exclude the presence of other unstated features, elements, components, groups, integers and / or steps. The term “consisting essentially of’, as used herein, is intended to specify the presence of the stated features, elements, components, groups, integers, and / or steps as well as those that do not materially affect the basic and novel characteristic(s) of features, elements, components, groups, integers, and / or steps.

[0079] Terms of degree such as “substantially”, “about” and “approximately” as used herein mean a reasonable amount of deviation of the modified term such that the end result is not significantly changed. These terms of degree should be construed as including a deviation of at least ±5% of the modified term if this deviation would not negate the meaning of the word it modifies.

[0080] As used in this disclosure, the singular forms “a”, “an” and “the” include plural references unless the content clearly dictates otherwise.

[0081] In embodiments comprising an “additional” or “second” component, the second component as used herein is chemically different from the other components or first component. A “third” component is different from the other, first, and second components, and further enumerated or “additional” components are similarly different.

[0082] The term “and / or” as used herein means that the listed items are present, or used, individually or in combination. In effect, this term means that “at least one of’ or “one or more” of the listed items is used or present.

[0083] The recitation of numerical ranges by endpoints herein includes all numbers and fractions subsumed within that range (e.g. 1 to 5 includes for example 1, 1.5, 2, 2.75, 3, 3.90, 4, and 5). It is also to be understood that all numbers and fractions thereof are presumed to be modified by the term “about”.

[0084] As used herein, “derived from a cell treated with adiponectin” refers to adiponectin having been applied to the source cell under conditions effective to, for example, activate adiponectin signaling in thesource cell prior to extracellular vesicle collection.

[0085] As used herein, “derived from a cell treated with an adiponectin receptor agonist” refers to the source cell having been contacted with an agonist of adiponectin receptor signaling, including without limitation ALY688 (ADP355) or adiporon, under conditions effective to, for example, activate adiponectin receptor signaling, prior to extracellular vesicle collection.

[0086] As used herein, “derived from a cell treated with an adiponectin receptor agonist” refers to the source cell having been contacted with an agonist of adiponectin receptor signaling, including without limitation ALY688 (ADP355) or adiporon, prior to extracellular vesicle collection.

[0087] As used herein, “cargo induced by adiponectin signaling” refers to a proteome, transcriptome, metabolome, and / or lipidome pattern present in extracellular vesicles following activation of adiponectin signaling in the source cell, as exemplified in this disclosure.

[0088] The term "aging", as used herein, refers to the complex biological process involving the gradual decline of physical, cognitive, and physiological functions over time. Characterized by a decrease in the body's ability to maintain homeostasis, repair tissue damage, and respond to environmental stresses, aging manifests through various signs such as the appearance of wrinkles, loss of skin elasticity, reduction in muscle mass and bone density, and a decline in cognitive abilities. Additionally, aging is associated with an increased risk of chronic diseases, including cardiovascular diseases, diabetes, and cancer. Genome organization and stability are pivotal in the aging process.

[0089] The term "aging-related disease", as used herein, refers to a medical condition whose onset, progression, or severity is closely associated with the aging process and becomes increasingly prevalent with advancing age, which includes aging-related chronic disease. Such diseases arise due to age-related molecular and cellular changes, including oxidative stress, inflammation, genomic instability, telomere shortening, and reduced regenerative capacity. Examples of aging-related diseases include neurodegenerative disorders such as Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis (ALS); metabolic disorders such as type 2 diabetes and obesity; cardiovascular diseases such as hypertension, atherosclerosis, and coronary artery disease; musculoskeletal disorders such as osteoarthritis, osteoporosis, and sarcopenia; chronic kidney disease; cancers including breast, prostate, and colorectal cancers; respiratory disorders such as chronic obstructive pulmonary disease (COPD); endocrine disorders such as hypothyroidism and hyperparathyroidism; and immune system dysfunctions, including immunosenescence and autoimmune diseases. Aging-related diseases are distinct from acute illnesses and are often progressive, contributing to functional decline and increased morbidity in older populations. The person skilled in the art is able to determine the status of a subject as it relates to aging-related diseases. For example, this may involve assessing cardiac function (e.g., echocardiography) relevant to heart disease, strength / endurance relevant to muscle loss or sarcopenia, and glucose tolerance tests relevant to metabolicdisease.

[0090] The term "cargo", as used herein, with respect to EV, refers to the molecular contents that EV carries, playing roles in biological functions and interactions. The cargo of EV induced by adiponectin includes diverse biomolecules that can be determined through proteomic, metabolomic, transcriptomic, and lipidomic analyses. Proteomic profiling reveals the presence of specific proteins, such as signaling molecules or enzymes, that mediate processes such as cellular communication and metabolic regulation. Metabolomic studies identify small molecules and metabolites within the EV, which contribute to biochemical pathways and energy metabolism. Transcriptomic analyses uncover RNA species, including microRNAs and non-coding RNAs, which influence gene expression and cellular functions in recipient cells. Lipidomic analyses, for example, using mass spectrometry, nuclear magnetic resonance (NMR) spectroscopy, and / or gas chromatography (GC), can identify lipid components. The proteome, metabolome, transcriptome, and lipidome cargo profde of EV induced by adiponectin, or recombinantly and / or overexpressed components thereof, can provide therapeutic benefits in aging-related diseases. Exemplary EV cargoes carried by EV include ALB, Iglc2, Ctss, Serpincl, Itihl.l, Serpina6, Agt, Gm2663, Cpb2, Gpldl, Masp2, Igkv-124el, Igfals, Ighv5-16, Ctrbl, Cfd, C3, Clec3b, Myrfl, Ig heavy chain V r, Ighv5-4, Ighv5-9, Apod, Selenop, Ighv5-17, Serpingl, Apoc2, Igkv-91a3, Ighvl-18, Fblnl, Igkv8-24, Gm5629, Clqb, Proz, C6, Ig kappa chain V-I, Igkv6-17, Igkv9-124, Serpinalb, Lonpl, Ighv2-2, C9, Serpina3m, Colla2, C8a, Igkv4-58, Ncaml, Gm, Apoe, and Ighvl4-2. Exemplary EV cargoes induced by adiponectin include ALB, Iglc2, Ctss, Serpincl, Itihl.l, Serpina6, Agt, Gm2663, Cpb2, Gpldl, Masp2, Igkv-124el, Igfals, Ighv5-16, Ctrbl, Cfd, C3, Clec3b, Myrfl, Ig heavy chain V r, Ighv5-4, Ighv5-9, Apod, Selenop, Ighv5-17, Serpingl, and Apoc2.

[0091] The EV described herein is useful for treating aging-related diseases and promoting healthspan and longevity.II. Methods and Uses

[0092] The present disclosure describes methods and uses involving adiponectin and EV for aging-related disease treatment. EV plays a role in aging by promoting regeneration and exacerbating senescence. Adiponectin regulates EV biogenesis and is also carried by EV, serving as cargo that mediates functional effects. Adiponectin signaling alters the proteome cargo of circulating EV, influencing its biological activity. In diabetic human subjects, EV lacks adiponectin, which can impair its regenerative and antiinflammatory functions. Activation of adiponectin signaling enhances the beneficial effects of EV produced from cells or animals, including reduced oxidative stress and inflammation. Both preclinical and clinical data indicate that adiponectin enhances EV functionality, cooperatively providing beneficial effects against aging-related diseases.

[0093] Accordingly, provided herein is a method of treating an aging-related disease in a subject in needthereof, comprising administering or use of a therapeutically effective amount of an extracellular vesicle (EV) derived from a cell over-expressing adiponectin, a cell treated with adiponectin, and / or a cell treated with an adiponectin receptor agonist in the subject. In some embodiments, the method is for treating an aging-related disease. In some embodiments, the treatment comprises administering or use of an EV derived from a cell over-expressing adiponectin in a subject in need thereof. In some embodiments, the treatment comprises administering or use of an EV derived from a cell treated with adiponectin in a subject in need thereof. In some embodiments, the treatment comprises administering or use of an EV derived from a cell treated with an adiponectin receptor agonist in a subject in need thereof. In some embodiments, the treatment comprises administering or use of an EV derived from a cell obtained from a donor subject that has been treated with an adiponectin receptor agonist.

[0094] Also provided is use of an EV derived from a cell over-expressing adiponectin, a cell treated with adiponectin, cell treated with an adiponectin receptor agonist, and / or a cell obtained from a donor subject that has been treated with an adiponectin receptor agonist, for treating an aging-related disease in a subject in need thereof. In some embodiments, the use is for treating an aging-related disease. In some embodiments, the treatment comprises use of an EV derived from a cell over -expressing adiponectin in a subject in need thereof. In some embodiments, the treatment comprises use of an EV derived from a cell treated with adiponectin in a subject in need thereof. In some embodiments, the treatment comprises use of an EV derived from a cell treated with an adiponectin receptor agonist in a subject in need thereof. In some embodiments, the treatment comprises use of an EV derived from a cell obtained from a donor subject that has been treated with an adiponectin receptor agonist.

[0095] Further provided is use of an EV derived from a cell over-expressing adiponectin, a cell treated with adiponectin, a cell treated with an adiponectin receptor agonist, and / or a cell obtained from a donor subject that has been treated with an adiponectin receptor agonist, in the manufacture of a medicament for treating an aging-related disease in a subject in need thereof. In some embodiments, the medicament is for treating an aging-related disease. In some embodiments, the medicament comprises an EV derived from a cell over-expressing adiponectin for use in a subject in need thereof. In some embodiments, the medicament comprises an EV derived from a cell treated with adiponectin for use in a subject in need thereof. In some embodiments, the medicament comprises an EV derived from a cell treated with an adiponectin receptor agonist for use in a subject in need thereof. In some embodiments, the medicament comprises an EV derived from a cell obtained from a donor subject that has been treated with an adiponectin receptor agonist.

[0096] Even further provided is an EV derived from a cell over-expressing adiponectin, a cell treated with adiponectin, a cell treated with an adiponectin receptor agonist, and / or a cell obtained from a donor subject that has been treated with an adiponectin receptor agonist, for use in treating an aging-related disease in a subject in need thereof. In some embodiments, the EV is for use in treating an aging-related disease. Insome embodiments, the EV is derived from a cell over-expressing adiponectin. In some embodiments, the EV is derived from a cell treated with adiponectin. In some embodiments, the EV is derived from a cell treated with an adiponectin receptor agonist. In some embodiments, the EV is derived from a cell obtained from a donor subject that has been treated with an adiponectin receptor agonist.

[0097] EV can be derived or isolated using a variety of techniques that leverage differences in size, density, and surface markers, by methods that are known in the art in accordance with approved guidelines detailed in the Minimal Information for Studies of Extracellular Vesicles (MISEV) (Welsh J. A., etal 2024). For example, common methods include ultracentrifugation, which separates EV based on size and density through sequential centrifugation steps, and size -exclusion chromatography, which isolates EV based on particle size while preserving integrity. Additional approaches, such as polymer-based precipitation, filtration, and immunoaffinity capture, utilize chemical or antibody -based strategies to enrich specific EV populations. Isolation methods may be combined or optimized depending on the source, such as plasma, serum, or cell culture media, and the intended downstream applications, including proteomic, transcriptomic, and functional studies. The EV can originate from biological fluids and various cell types, reflecting diverse origins and potential applications. In some embodiments, the EV is derived from an adipocyte, a cardiomyocyte, a hepatocyte, a platelet, an immune cell, an endothelial cell, an erythrocyte, a stem cell, a fibroblast, a smooth muscle cell, a neuronal cell, a glial cell, an astrocyte, a Schwann cell, a keratinocyte, a chondrocyte, an osteoblast, an osteocyte, or any combination thereof. In some embodiments, the EV is derived from an adipocyte. In some embodiments, the EV is derived from a cardiomyocyte. In some embodiments, the EV is derived from a hepatocyte. In some embodiments, the EV is derived from a platelet. In some embodiments, the EV is derived from an immune cell. In some embodiments, the EV is derived from an endothelial cell. In some embodiments, the EV is derived from an erythrocyte. In some embodiments, the EV is derived from a stem cell. In some embodiments, the EV is derived from a fibroblast. In some embodiments, the EV is derived from a smooth muscle cell. In some embodiments, the EV is derived from a neuronal cell. In some embodiments, the EV is derived from a glial cell. In some embodiments, the EV is derived from an astrocyte. In some embodiments, the EV is derived from a Schwann cell. In some embodiments, the EV is derived from a keratinocyte. In some embodiments, the EV is derived from a chondrocyte. In some embodiments, the EV is derived from an osteoblast. In some embodiments, the EV is derived from an osteocyte. In some embodiments, the EV is isolated from plasma. In some embodiments, the EV is derived from any combination of the foregoing. In some embodiments, the immune cell is a T cell, a B cell, a macrophage, a dendritic cell, or a natural killer cell. In some embodiments, the immune cell is a T cell. In some embodiments, the immune cell is a B cell. In some embodiments, the immune cell is a macrophage. In some embodiments, the immune cell is a dendritic cell. In some embodiments, the immune cell is a natural killer cell. In some embodiments, the endothelial cell is a progenitor cell. In some embodiments, the cardiomyocyte is derived from a cardiosphere. In someembodiments, the stem cell is a mesenchymal stem cell or an induced pluripotent stem cell. In some embodiments, the stem cell is a mesenchymal stem cell. In some embodiments, the stem cell is an induced pluripotent stem cell. In some embodiments, the stem cell is an induced pluripotent stem cell. In some embodiments, the cell is an induced pluripotent stem cell -cardiomyocyte. In some embodiments, the cell is an adipocyte-derived mesenchymal stem cell. In some embodiments, the EV is derived from a cell overexpressing adiponectin. In some embodiments, the EV over-expresses adiponectin compared to EV derived from a cell of the same cell type without any treatment. In some embodiments, the EV is derived from a cell treated with adiponectin. In some embodiments, the EV is an endosomal-derived EV.

[0098] Sequence identity can be determined with standard sequence alignment algorithms, such as BLAST or Clustal Omega, by comparing the nucleotide or amino acid sequences of interest. It is typically expressed as a percentage that represents the number of identical residues shared between two sequences over a specified alignment length. Gaps introduced during alignment to optimize sequence matching are usually considered, and parameters such as gap penalties and substitution matrices can affect the calculated identity. Sequence identity provides a quantitative measure of similarity, which is useful for evaluating homology, predicting functional relationships and conservation. In some embodiments, the adiponectin comprises an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to the full length of SEQ ID NO: 1. In some embodiments, the adiponectin comprises an amino acid sequence having at least about 75% sequence identity to the full length of SEQ ID NO: 1. In some embodiments, the adiponectin comprises an amino acid sequence having at least about 80% sequence identity to the full length of SEQ ID NO: 1. In some embodiments, the adiponectin comprises an amino acid sequence having at least about 85% sequence identity to the full length of SEQ ID NO: 1. In some embodiments, the adiponectin comprises an amino acid sequence having at least about 90% sequence identity to the full length of SEQ ID NO: 1. In some embodiments, the adiponectin comprises an amino acid sequence having at least about 95% sequence identity to the full length of SEQ ID NO: 1. In some embodiments, the adiponectin comprises an amino acid sequence having at least about 96% sequence identity to the full length of SEQ ID NO: 1. In some embodiments, the adiponectin comprises an amino acid sequence having at least about 97% sequence identity to the full length of SEQ ID NO: 1. In some embodiments, the adiponectin comprises an amino acid sequence having at least about 98% sequence identity to the full length of SEQ ID NO: 1. In some embodiments, the adiponectin comprises an amino acid sequence having at least about 99% sequence identity to the full length of SEQ ID NO: 1. In some embodiments, the adiponectin comprises an amino acid sequence having at least about 99.5% sequence identity to the full length of SEQ ID NO: 1. In some embodiments, the adiponectin comprises an amino acid sequence having at least about 99.9% sequence identity to the full length of SEQ ID NO: 1. In some embodiments, the adiponectin comprises the amino acid sequence having 100% sequence identity to the full length of SEQ ID NO: 1. In some embodiments, the adiponectin consists of the amino acid sequencehaving 100% sequence identity to the full length of SEQ ID NO: 1.

[0099] The cargo carried by EV induced by adiponectin can be useful in treating an aging-related disease described herein. Further, the EV can carry the cargo described herein without being induced by adiponectin, for example, through the recombinant expression of the constituents. In some embodiments, the EV described herein comprises or further comprises a cargo induced by adiponectin treatment. In some embodiments, the EV comprises or further comprises a cargo comprising ALB, Iglc2, Ctss, Serpincl, Itihl.l, Serpina6, Agt, Gm2663, Cpb2, Gpldl, Masp2, Igkv-124el, Igfals, Ighv5-16, Ctrbl, Cfd, C3, Clec3b, Myrfl, Ig heavy chain V r, Ighv5-4, Ighv5-9, Apod, Selenop, Ighv5-17, Serpingl, Apoc2, Igkv-91a3, Ighvl-18, Fblnl, Igkv8-24, Gm5629, Clqb, Proz, C6, Ig kappa chain V-I, Igkv6-17, Igkv9-124, Serpinalb, Lonpl, Ighv2-2, C9, Serpina3m, Colla2, C8a, Igkv4-58, Ncaml, Gm, Apoe, Ighvl4-2, or any combination thereof. In some embodiments, the EV comprises or further comprises a cargo comprising ALB, Iglc2, Ctss, Serpincl, Itihl.l, Serpina6, Agt, Gm2663, Cpb2, Gpldl, Masp2, Igkv-124el, Igfals, Ighv5-16, Ctrbl, Cfd, C3, Clec3b, Myrfl, Ig heavy chain V r, Ighv5-4, Ighv5-9, Apod, Selenop, Ighv5-17, Serpingl, Apoc2, or any combination thereof. In some embodiments, the cargo comprises at least three proteins selected from ALB, Serpincl, Gpldl, Masp2, Igfals, C3, Clec3b, Apod, Selenop, and Serpingl.

[0100] In some embodiments, a nucleic acid encoding a molecule described herein, such as adiponectin, is administered or introduced to the cell. The nucleic acid is administered or for use in the form of an expression vector, such as a viral expression vector. In some embodiments, the expression vector is a retroviral expression vector, an adenoviral expression vector, a DNA plasmid expression vector, or an AAV expression vector. In some embodiments, one or more polynucleotides encoding the molecule is delivered to the cell. In some embodiments, the delivery is by delivery of one or more vectors, one or more transcripts thereof, and / or one or more proteins transcribed therefrom, is delivered to the cell.

[0101] In some embodiments, the polypeptides are synthesized in situ in the cell as a result of the introduction of polynucleotides encoding the polypeptides into the cell. In some embodiments, the polypeptides could be produced outside the cell and then introduced thereto. Methods for introducing a polynucleotide construct into animal cells are known and include as non-limiting examples stable transformation methods wherein the polynucleotide construct is integrated into the genome of the cell. In some embodiments, a transient transformation method comprises polynucleotide construct that is not integrated into the genome of the cell, and virus mediated methods. In some embodiments, the polynucleotides can be introduced into the cell by for example, recombinant viral vectors (e.g. retroviruses, adenoviruses), liposome and the like. In some embodiments, transient transformation methods include microinjection, electroporation, or particle bombardment. In some embodiments, the polynucleotides are included in vectors, more particularly plasmids or virus, in view of being expressed in the cells.

[0102] In some embodiments, viral and non-viral based gene transfer methods can be used to introducenucleic acids in mammalian cells or target tissues. Such methods can be used to administer nucleic acids encoding components of a CRISPR, ZFP, ZFN, TALE, and / or TALEN system to cells in culture, or in a host organism. Non-viral vector delivery systems include DNA plasmids, RNA (e.g. a transcript of a vector described herein), naked nucleic acid, and nucleic acid complexed with a delivery vehicle, such as a liposome. Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell.

[0103] Methods of non-viral delivery of nucleic acids include lipofection, nucleofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipidmucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA. Lipofection is described in (e.g., US5049386 and US4897355) and lipofection reagents are sold commercially (e.g., Transfectam™ and Lipofectin™).

[0104] In some embodiments, delivery is via the use of RNA or DNA viral based systems for the delivery of nucleic acids. In some embodiments, viral vectors are administered directly to a subject in vivo or they can be used to treat cells in vitro or ex vivo, and then administered to a subject. Viral -based systems in some embodiments include retroviral, lentivirus, adenoviral, adeno-associated and herpes simplex virus vectors for gene transfer.

[0105] The EV can be obtained from cells subjected to treatments, such as an adiponectin receptor agonist, that enhance its functional properties. In some embodiments, the EV is derived from a cell treated with an adiponectin receptor agonist. In some embodiments, the adiponectin receptor agonist comprises ALY688 (a.k.a. ADP355), adiporon, or a variant of any of the foregoing. In some embodiments, the adiponectin receptor agonist comprises ALY688 or a variant thereof. In some embodiments, the adiponectin receptor agonist comprises ALY688. In some embodiments, ALY688 comprises D-Asn-Ile-Pro-Nva-Leu-Tyr-D-Ser-Phe-Ala-D-Ser (SEQ ID NO: 2), where Nva is norvaline. In some embodiments, the adiponectin receptor agonist comprises adiporon or a variant thereof. In some embodiments, the adiponectin receptor agonist comprises adiporon.

[0106] In a further aspect, provided is an EV isolated a cell, for example plasma, of a donor subject administered an adiponectin receptor agonist. In some embodiments, the EV comprises adiponectin. In some embodiments, the EV is enriched with adiponectin. In some embodiments, the donor subject is a mammal. In some embodiments, the donor subject is a mouse or a human. In some embodiments, the donor subject is a human. In some embodiments, the adiponectin receptor agonist comprises ALY688 or adiporon. In some embodiments, the donor subject is a normal subject. In some embodiments, the donor subject has diabetes. In some embodiments, the donor subject does not have diabetes.

[0107] Aging-related diseases encompass a wide range of conditions that arise or progress with advancing age, affecting multiple organ systems and physiological functions. The methods and usesdescribed herein are useful for treating aging-related diseases. In some embodiments, the aging-related disease comprises a neurodegenerative disorder, a metabolic disorder, a cardiovascular disease, a musculoskeletal disorder, a renal or urological disease, a cancer, an eye disorder, a respiratory disorder, an endocrine disorder, and / or an immune system disorder. In some embodiments, the subject is an animal. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human, a canine, a feline, a bovine, a porcine, a caprine, an equine, an ovine, a cervine, a murine, a leporine, a primate, a rodent, a lagomorph, a marsupial, a chiropteran, a cetacean, or a pinniped. In some embodiments, the subject is a human.

[0108] In some embodiments, the aging-related disease comprises a neurodegenerative disorder. In some embodiments, the neurodegenerative disorder comprises Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, or amyotrophic lateral sclerosis (ALS). In some embodiments, the neurodegenerative disorder comprises Alzheimer’s disease. In some embodiments, the neurodegenerative disorder comprises Parkinson’s disease. In some embodiments, the neurodegenerative disorder comprises Huntington’s disease. In some embodiments, the neurodegenerative disorder comprises ALS.

[0109] In some embodiments, the aging-related disease comprises a metabolic disorder. In some embodiments, the metabolic disorder comprises type 2 diabetes, metabolic associated steatohepatitis (MASH), or obesity. In some embodiments, the metabolic disorder comprises type 2 diabetes. In some embodiments, the metabolic disorder comprises MASH. In some embodiments, the metabolic disorder comprises obesity.

[0110] In some embodiments, the aging-related disease comprises a cardiovascular disease. In some embodiments, the cardiovascular disease comprises hypertension, atherosclerosis, coronary artery disease, myocardial infarction, heart failure, arrhythmias, peripheral artery disease, or stroke. In some embodiments, the cardiovascular disease comprises hypertension. In some embodiments, the cardiovascular disease comprises atherosclerosis. In some embodiments, the cardiovascular disease comprises coronary artery disease. In some embodiments, the cardiovascular disease comprises myocardial infarction. In some embodiments, the cardiovascular disease comprises heart failure. In some embodiments, the cardiovascular disease comprises arrhythmias. In some embodiments, the cardiovascular disease comprises peripheral artery disease. In some embodiments, the cardiovascular disease comprises stroke.

[0111] In some embodiments, the aging-related disease comprises a musculoskeletal disorder. In some embodiments, the musculoskeletal disorder comprises osteoarthritis, sarcopenia, or osteoporosis. In some embodiments, the musculoskeletal disorder comprises osteoarthritis. In some embodiments, the musculoskeletal disorder comprises sarcopenia. In some embodiments, the musculoskeletal disorder comprises osteoporosis.

[0112] In some embodiments, the aging-related disease comprises a renal or urological disease. In some embodiments, the renal or urological disease comprises chronic kidney disease, diabetic nephropathy, or benign prostatic hyperplasia. In some embodiments, the renal or urological disease comprises chronic kidney disease. In some embodiments, the renal or urological disease comprises diabetic nephropathy. In some embodiments, the renal or urological disease comprises benign prostatic hyperplasia.

[0113] In some embodiments, the aging-related disease comprises a cancer, an eye disorder. In some embodiments, the cancer comprises breast cancer, prostate cancer, colorectal cancer, lung cancer, leukemia, or bladder cancer. In some embodiments, the cancer comprises breast cancer. In some embodiments, the cancer comprises prostate cancer. In some embodiments, the cancer comprises colorectal cancer. In some embodiments, the cancer comprises lung cancer. In some embodiments, the cancer comprises leukemia. In some embodiments, the cancer comprises bladder cancer.

[0114] In some embodiments, the aging-related disease comprises an eye disorder. In some embodiments, the eye disorder comprises age-related macular degeneration, cataracts, or glaucoma. In some embodiments, the eye disorder comprises age-related macular degeneration. In some embodiments, the eye disorder comprises age-related cataracts. In some embodiments, the eye disorder comprises glaucoma.

[0115] In some embodiments, the aging-related disease comprises a respiratory disorder. In some embodiments, the respiratory disorder comprises chronic obstructive pulmonary disease or pulmonary fibrosis. In some embodiments, the respiratory disorder comprises chronic obstructive pulmonary disease. In some embodiments, the respiratory disorder comprises pulmonary fibrosis.

[0116] In some embodiments, the aging-related disease comprises an endocrine disorder. In some embodiments, the endocrine disorder comprises hypothyroidism, hyperparathyroidism, adrenal insufficiency, hypercortisolism, a pituitary disorder, an alteration in metabolic hormone production, or an alteration in sex hormone production. In some embodiments, the endocrine disorder comprises hypothyroidism. In some embodiments, the endocrine disorder comprises hyperparathyroidism. In some embodiments, the endocrine disorder comprises adrenal insufficiency. In some embodiments, the endocrine disorder comprises hypercortisolism. In some embodiments, the hypercortisolism comprises Cushing’s syndrome or ectopic ACTH secretion. In some embodiments, the endocrine disorder comprises Cushing’s syndrome. In some embodiments, the endocrine disorder comprises ectopic ACTH secretion. In some embodiments, the endocrine disorder comprises a pituitary disorder. In some embodiments, the pituitary disorder comprises acromegaly, hypopituitarism, or pituitary adenomas. In some embodiments, the endocrine disorder comprises acromegaly. In some embodiments, the endocrine disorder comprises hypopituitarism. In some embodiments, the endocrine disorder comprises pituitary adenomas. In someembodiments, the endocrine disorder comprises an alteration in metabolic hormone production. In some embodiments, the metabolic hormone is insulin, glucagon, leptin, ghrelin, adiponectin, cortisol, a thyroid hormone, amylin, an incretin, or a growth hormone. In some embodiments, the thyroid hormone is triiodothyronine (T3) or thyroxine (T4). In some embodiments, the incretin is glucagon-like peptide-1 (GLP-1) or glucose-dependent insulinotropic polypeptide (GIP). In some embodiments, the endocrine disorder comprises an alteration in sex hormone production. In some embodiments, the alteration in sex hormone production comprises menopause -related estrogen deficiency or andropause -related testosterone deficiency. In some embodiments, the endocrine disorder comprises menopause-related estrogen deficiency. In some embodiments, the endocrine disorder comprises andropause -related testosterone deficiency.

[0117] In some embodiments, the aging-related disease comprises an immune system disorder. In some embodiments, the immune system disorder comprises immunosenescence, chronic inflammation, or an autoimmune disease. In some embodiments, the immune system disorder comprises immunosenescence. In some embodiments, the immune system disorder comprises chronic inflammation. In some embodiments, the immune system disorder comprises an autoimmune disease. In some embodiments, the autoimmune disease comprises rheumatoid arthritis, systemic lupus erythematosus, Hashimoto’s thyroiditis, Sjogren’s syndrome, or hypogammaglobulinemia. In some embodiments, the autoimmune disease comprises rheumatoid arthritis. In some embodiments, the autoimmune disease comprises systemic lupus erythematosus. In some embodiments, the autoimmune disease comprises Hashimoto’s thyroiditis. In some embodiments, the autoimmune disease comprises Sjogren’s syndrome. In some embodiments, the autoimmune disease comprises hypogammaglobulinemia.

[0118] In some embodiments, the EV described herein inhibits oxidative stress. In some embodiments, the EV described herein inhibits inflammation. In some embodiments, the EV described herein inhibits IFN activation. In some embodiments, the EV described herein is formulated for reducing oxidative stress and reducing inflammatory pathway activation in a hypoxia-challenged cell. In some embodiments, the EV described herein is capable of activating AMPK and p38 signaling in cardiomyocytes. In some embodiments, the EV described herein is capable of reducing hypoxia induced reactive oxygen species and preserving mitochondrial network integrity in cardiomyocytes. In some embodiments, the EV described herein is capable of improving myocardium remodeling. In some embodiments, the EV described herein is capable of improving insulin sensitization.

[0119] A pharmaceutical composition comprising EV can be formulated for therapeutic use in treating aging-related diseases. In some embodiments, the EV described herein is comprised in a pharmaceutical composition. In some embodiments, the pharmaceutical composition comprises the EV described herein, and a pharmaceutically acceptable carrier, excipient, or stabilizer.

[0120] In some embodiments, the EV or pharmaceutical composition described herein is administered or for use through a variety of routes including oral, rectal, transdermal, subcutaneous, intravenous, intramuscular, and intranasal, or via intratracheal instillation or aerosol inhalation. In some embodiments, the administration of adiponectin and / or an adiponectin receptor agonist in a donor or recipient subject comprises oral, rectal, transdermal, subcutaneous, intravenous, intramuscular, and intranasal, or via intratracheal instillation or aerosol inhalation, and by any methods known to a person skilled in the art.

[0121] In an aspect, also provided is an EV comprising over-expression of the adiponectin described herein. In another aspect, also provided is an EV derived from a cell over-expressing adiponectin. In another aspect, also provided is an EV derived from a cell treated with adiponectin. In another aspect, also provided is an EV derived from a cell having an activated adiponectin signaling. In another aspect, also provided is an EV derived from a cell treated with an adiponectin receptor agonist. In another aspect, also provided is an EV comprising an over-expression of the adiponectin described herein.

[0122] In an aspect, also provided is a method for producing EV for treating an aging-related disease in a subject in need thereof, comprising: i) isolating an EV from a cell over-expressing adiponectin, ii) isolating an EV from a cell treated with adiponectin, or iii) isolating an EV from a cell treated with an adiponectin receptor agonist. In some embodiments, the method comprises over-expressing adiponectin in a cell. In some embodiments, the method comprises treating a cell with adiponectin. In some embodiments, the method comprises treating a cell with an adiponectin receptor agonist. In some embodiments, the method comprises purifying the EV. In some embodiments, the method comprises collecting the isolated EV. In some embodiments, the method comprises collecting the purified EV.Table 1: Sequences described in this disclosure.

[0123] In an aspect, also provided is a method of diagnosing an aging-related disease or predicting the risk of developing an aging-related disease described herein, comprising obtaining an EV sample from a subject, identifying proteome, transcriptome and / or metabolome profiles of the EV sample, and determining the subject having the aging-related disease or the subject having the risk of developing the aging-related disease.

[0124] In an aspect, also provided is a method of diagnosing for treatment of an aging-related disease described herein, comprising obtaining an EV sample from a test subject, measuring the level of adiponectin, wherein if the level of adiponectin is low compared to the level of adiponectin of EV from a healthy subject, the test subject is a candidate for EV treatment. In some embodiments, the EV treatment comprises administering an EV described herein to the subject.

[0125] In an aspect, provided herein is an EV for use in treating an aging-related disease, wherein the EV is derived from a cell treated with adiponectin, or from a cell treated with an adiponectin receptor agonist, such that adiponectin signaling is activated in the source cell prior to vesicle isolation.

[0126] In another aspect, provided herein is a method of producing EVs for therapeutic use, comprising treating a cell with adiponectin or with an adiponectin receptor agonist to activate adiponectin signaling, and isolating EVs released by the treated cell.

[0127] In another aspect, provided herein is a method of treating an aging-related disease in a recipient subject in need thereof, comprising administering to the recipient subject a therapeutically effective amount of an EV isolated from plasma of a donor subject that has been administered an adiponectin receptor agonist. In another aspect, provided herein is a use of an EV isolated from plasma of a donor subject that has been administered an adiponectin receptor agonist for treating an aging-related disease in a recipient subject. In another aspect, provided herein is a use of an EV isolated from plasma of a donor subject that has been administered an adiponectin receptor agonist in the manufacture of a medicament for treating an aging-related disease in a recipient subject. In another aspect, provided herein is an EV isolated from plasma of a donor subject that has been administered an adiponectin receptor agonist for use in treating an aging-related disease in a recipient subject. In some embodiments, the adiponectin receptor agonist comprises ALY688. In some embodiments, the EV is administered intravenously. In some embodiments, the aging-related disease is a cardiovascular disease. In some embodiments, the cardiovascular disease is myocardial infarction (MI). In some embodiments, the EV reduces cardiac injury associated with MI. In some embodiments, the EV suppresses an increase in one or more cardiac injury markers following MI. In some embodiments, the cardiac injury marker comprises plasma troponin I. In some embodiments, the cardiac injury marker comprises plasma lactate dehydrogenase (LDH). In some embodiments, administration of the EV reduces apoptosis in infarcted heart tissue following MI. In some embodiments, apoptosis is assessed by cleaved caspase-3 in infarcted heart tissue. In some embodiments, administration of the EVmodulates mitochondrial dynamics in infarcted heart tissue following MI. In some embodiments, mitochondrial dynamics is assessed by one or more of mitochondrial fission factor (MFF), phospho-dynamin-related protein 1 (p-Drpl) serine 637, p-Drpl serine 616, and optic atrophy 1 (OPA1). In some embodiments, administration of the EV is associated with a decrease in MFF and p-Drpl s616, and an increase in p-Drpl s637 and OPA1, in infarcted heart tissue following MI. In some embodiments, administration of the EV restores antioxidant gene expression in infarcted heart tissue following MI. In some embodiments, the antioxidant gene expression comprises Sodl, Sod2, and Sod3 mRNA expression. In some embodiments, administration of the EV enhances autophagy flux in infarcted heart tissue following MI. In some embodiments, autophagy flux is assessed by one or more of LC3, p62, and ATG3. In some embodiments, administration of the EV increases LC3-II and ATG3 levels and enhances p62 degradation in infarcted heart tissue following MI.

[0128] In various embodiments, any of the features or components of any embodiments, examples, figures, or tables discussed above or herein can be combined, and such combinations are encompassed within the scope of the present disclosure. Any specific value discussed above or herein can be combined with another related value discussed above or herein to recite a range with the values representing the upper and lower ends of the range, and such ranges are encompassed within the scope of the present disclosure. A therapeutic agent for use in any of the methods discussed herein, or use of a therapeutic agent in the manufacture of a medicament for use in any of the methods discussed herein are also encompassed within the scope of this disclosure.

[0129] Hereinafter are provided examples of specific embodiments and implementations for performing the methods and uses of the present disclosure. The examples are provided for illustrative purposes only, and are not intended to limit the scope of the present disclosure in any way:EXAMPLESExample 1. Adiponectin-Associated Extracellular Vesicle (EV) Dynamics in Mice - Effects of ALY688 Preclinical Findings with ALY688 part AMethodsSize-exclusion chromatography isolation of Plasma-E V

[0130] Size-exclusion chromatography (SEC) using the qEV columns (IZON, Cambridge, MA) was the primary method to isolate EVs from plasma samples. 50pL of plasma sample from each animal was diluted in lOOpL of filtered (0.22 pm pore size) PBS and resuspended. The sample mixtures were centrifuged at lOOOOxg for 10 minutes to remove large debris. After centrifugation, sample was loaded on top of the qEV / 70 nm SEC column and eluted with filtered PBS. Each collected fraction contained 450pL of elutedPBS. In all experiments, fractions 3 to 6 were collected and used for subsequent EV characterization and downstream applications as per manufacturer’s recommendations. The isolated EVs were either used immediately or stored at -80°C until usage.Size and particle quantification by Nanoparticle Tracking Analysis (NT A)

[0131] To determine the number and size of particles in the samples, plasma EV were pooled by groups, diluted in PBS, and analyzed using the NanoSight Pro system (Malvern Instruments, Saint-Laurent, Canada). Thirty-second videos of EV were recorded at room temperature across 3-5 independent experiments. The videos were averaged and analyzed using NS Xplorer software. For size calibration, 100 nm polystyrene beads (Malvern Instruments, Saint-Laurent, Canada) were used as described previously (McVey, M.J. et al., 2018).Results

[0132] Plasma EV were isolated using size exclusion chromatography from C57BL / 6 mice treated daily for 28 days with either PBS or ALY688 (10 mg / kg) by subcutaneous administration. Characterization by Western immunoblotting revealed significantly increased expression of well-established EV markers, including CD36 and Alix, in ALY688-treated mice (FIG. 1A). Adiponectin was detected within plasma EVs, indicating that EV function as carriers of adiponectin in circulation. ALY688 treatment enhanced the adiponectin content of EVs (FIG. 1A).

[0133] The accuracy and quality of the EV isolation process was assessed by cryo-electron microscopy (cryo-EM). The distinct ultrastructure was shown under cryo-EM, indicating the integrity and purity of EV isolates (FIG. IB).

[0134] Further, nanoparticle tracking analysis (NTA) demonstrated a significant increase in EV particle concentration in ALY688-treated mice compared to controls (FIG. 1C and FIG. ID).Example 2. Adiponectin-Associated Extracellular Vesicle (EV) Dynamics in Mice: Adiponectin-KnockoutPreclinical Findings with ALY688 part B

[0135] Following Example 1, inventors then used adiponectin-knockout (KO) and wild-type (WT) mice and showed that plasma EV from WT mice (about 10 to 12 weeks of age) contained adiponectin and EV markers (CD63, Flotillin- 1 , and Alix), while EVs from adiponectin-KO mice lacked adiponectin, as verified, and retained EV markers (FIG. 2 A).

[0136] Cryo-EM was performed for quality control of EV isolation and demonstrated the vesicular structures of the isolated EVs (FIG. 2B).

[0137] Further, NT A revealed an increase in particle number and size in EV from adiponectin-KO mice plasma (FIG. 2C, FIG. 2D, FIG. 2E).

[0138] Akt activation provides a measurable indicator of insulin signaling efficiency, enabling evaluation of glucose metabolism and detection of insulin resistance. Rat L6 skeletal muscle cells stably expressing FoxOl fused to the green fluorescent clover protein (GFP) were used as an Akt biosensor based upon translocation of GFP-tagged FoxOl (Sung, H.K. et al., 2022). These engineered skeletal muscle cells were tested to evaluate the direct functional effect of the isolated plasma EV on insulin sensitivity. A time-course analysis measuring GFP (green fluorescent protein clover) signal in nuclear after 10 nM insulin stimulation showed that in the presence of EV from adiponectin-KO mice, the skeletal muscle cells did not respond normally to insulin, unlike the normal response of nuclear GFP signal seen in the presence of EV from WT mice (FIG. 2F and FIG. 2G).Example 3. Decrease in EV adiponectin content in diabetic as compared to control human subjects Clinical Findings in Control vs Diabetic human subjects

[0139] All sample collection was approved by Institutional Review Board at Keimyung University Dongsan Medical Center, South Korea, where type 2 diabetes (T2D) was defined by an HbAlc level exceeding 6.5%, and T2D with cardiomyopathy (CMP) was diagnosed if the E / e' ratio was greater than 15 or the NT-proBNP level was above 124. Human studies included three groups: 15 healthy controls, 15 individuals with T2D, and 15 individuals with T2D and CMP (T2D-CMP). Plasma EVs were isolated and randomly selected samples from each group were characterized by Western immunoblotting for EV markers (FIG. 3). Western blot analysis revealed a significant decrease in the expression of adiponectin in EV isolated from T2D and T2D-CMP patients compared to healthy controls (FIG. 3).Example 4. ALY688 induced adiponectin signaling leading to altered proteome cargo of circulating EV Methods: Proteomic analysis

[0140] Isolated EV were lysed in 5% SDS (Fisher Chemical), 200 mM TRIS (Fisher BioReagents™) pH 8.5 with 10 mM TCEP (60C x 25 minutes). Free disulfides were alkylated with 40 mM IAA (room temperature, 30 minutes) prior to proteolytic digestion by S-TRAP micro. Peptide containing eluates were lyophilized and rehydrated at 10 ng / uL prior to loading on EvoTips and analysis by library free DIA using an Evosep One system coupled to a Broker timsTOF HT MS operated in DIA-PASEF mode. Samples were separated on an EVI 137 column @ 30SPD.Results

[0141] WT mice were injected with PBS or adiponectin receptor agonist ALY688 to induce adiponectin signaling. Proteomics analysis of plasma EV was performed to elucidate the effect of adiponectin action onreshaping EV proteomic cargo. Principal Component Analysis (PCA) analysis of all proteins detected in isolated plasma EV indicated clear separation between PBS- and ALY688-injected samples (FIG. 4A). A heat map of combined data from 4 mice in each group indicated the differential expression (black = increase; gray = decrease) of the top 50 individual proteins in ALY688-injected versus PBS-injected mouse plasma EV (FIG. 4B).Example 5. Beneficial functional effects of plasma and primary adipocyte EV from mice treated with an adiponectin receptor agonist

[0142] C57 / B16 mice were injected subcutaneously with PBS or the adiponectin receptor agonist ALY688 daily at 15mg / kg. 3 days after the injection, plasma EV (Plasma-EVPBS or Plasma-EV-ALY) and primary adipocyte EV (Ad-EV-PBS or Ad-EV-ALY) were isolated, and subjected to functional analysis.

[0143] The rat cardiomyocyte H9c2 cell line was first used as a target in measuring response to hypoxia. Prior to testing under oxygenation conditions, this cell line was exposed to isolated EV for 30 minutes, and then the cell line was incubated under 24-hour hypoxia (1% O2) to induce cell damage or under normoxia (20% O2) as a control. Cell viability was then measured using the Ready Probe™ Cell Viability assay kit from ThermoFisher. Data showed that cell death induced by hypoxia was significantly reduced by the administration of Plasma-EV-ALY and Ad-EV-ALY, but not with EV from mice with PBS injection (FIG.5A). Cellular oxidative stress was then measured by the CellRox fluorescent probe from ThermoFisher and the results demonstrated a beneficial effect of EV isolated from plasma or adipocytes from ALY688-injected but not PBS-injected mice, in reducing oxidative stress in H9c2 cells in response to hypoxia (FIG. 5B and FIG. 5C).

[0144] Next, a macrophage cell line (RAW) engineered with interferon (IFN) and nuclear factor KB (NFKB) reporters were used to elucidate effects on inflammation. Upon growth under hypoxia conditions, there is as expected a significant induction of IFN (FIG. 5D) and NFKB (FIG. 5E). There was attenuation of hypoxia triggered IFN activation when RAW cells were pretreated with Plasma-EV-ALY or Ad-EV-ALY, and protective effect in repressing hypoxia-induced NFKB was also seen with Ad-EV-ALY.

[0145] Together, inventors showed that adiponectin signaling can regulate EV biogenesis and adiponectin is carried by EV, which is also an important cargo mediating functional effects of the EV. In addition, adiponectin signaling changed the proteome cargo of EV. Further, EV produced from animals or cells upon stimulation of adiponectin signaling had beneficial effects on cells, including reduced oxidative stress and inflammation which are contributing processes in aging and aging-related diseases. As well, the lack of adiponectin in EV was shown in human diabetic patients. These preclinical and clinical data underscore the utility of EV as a biomarker, and indicate that adiponectin enhances EV functionality, cooperatively providing beneficial effects against aging-related diseases.Example 6. Adiponectin-associated EV dynamics in mice after ALY688 administration

[0146] Inventors further investigated ALY688's capacity to enhance EV-adiponectin loading.

[0147] Methods: Mice were administered ALY688 at 15 mg / kg for 3 days, plasma EVs were isolated, and EVs were characterized by NTA and immunoblotting.

[0148] Result: EVs from ALY688-treated mice exhibited an increased particle number without a change in particle size (FIG. 6A, FIG. 6B, and FIG. 6C). EV marker analysis showed Alix, CD63, and CD81 positivity (FIG. 6D), and EV adiponectin content was increased when normalized by particle number (FIG. 6E, FIG.6F, FIG. 6G, and FIG. 6H), in particular, EV-loaded adiponectin was significantly enhanced (FIG. 6D and FIG. 6H).Example 7. EV-mediated protection of cardiomyocvtes under hypoxia via mitochondrial and autophagy mechanisms

[0149] Inventors next showed the functional cardioprotective potency of EV-carried adiponectin in cardiomyocytes. Inventors treated rat cardiomyoblast H9c2 cell lines with EVs isolated from mice injected with either PBS (EVPBS) or ALY (EVALY) and examined adiponectin signaling activation. As shown, phospho-AMPK and phospho-p38 were activated upon EVAI,Ytreatment, whereas no activation was observed with of EVPBStreatment (FIG. 61, FIG. 6 J, and FIG. 6K).

[0150] Together with the results from Example 6, these findings show that ALY688 enhances EV-carried adiponectin content, which maintains similar functional properties to native adiponectin. Because the subsequent mechanistic experiments including EVs functional assays and targeted validation required a consistent metabolic background, all follow-up studies were conducted in the Standard Chow (SC) cohort to minimize diet-driven confounding and allow clearer interpretation of ALY688- and Ml-related EVs changes.Example 8. ALY688 driven plasma EVs protective effect in hypoxia cellular model

[0151] To better show the functional effects of EVAI,Yin ischemic injury, inventors employed a hypoxia system in H9c2 and induced pluripotent stem cell -cardiomyocytes (iPSC-CM) to simulate in vivo ischemia. LDH analysis was carried out with H9c2 or iPSC-CM subjected to 24 hours hypoxia treated with 10 pg / mL EVPBSand E VAI, Y. Inventors first measured apoptosis and cell death in the hypoxia model, as these represent the major event during ischemic remodeling (Zhu et al., 2025). Caspase 3 / 7 activity was used to evaluate apoptosis and propidium iodide (PI) staining and LDH release were used to measure cell death as described before (Tang et al., 2024). In H9c2 cells, hypoxia induced apoptosis and cell death was significantly reduced by the EVAI Ytreatment compared to control (FIG. 7A). Similar protective effects against cell death were observed in iPSC-CM (FIG. 7B).

[0152] Mitochondrial dysfunction is the major cause of cellular ROS accumulation and subsequent cell death under ischemia conditions (Chouchani et al., 2014). Next, inventors tested whether EVALYreduced mitochondrial damage and the consequent ROS production in cardiomyocytes. Mitochondrial morphology was evaluated by the index of networking, demonstrating that hypoxia impaired mitochondrial networking and the impairment was improved by EVALYin both H9c2 cells and iPSC-CM (FIG. 7C, FIG. 7D, and FIG.7E). Furthermore, IM ARIS sphericity analysis, used as an index of mitochondrial fission, revealed increased mitochondrial fragmentation during hypoxia, which was reduced by EVALYin H9c2 cells (FIG.7F and FIG. 7G).

[0153] ROS accumulation assessed by CellRox staining demonstrated that E VAI,Ysignificantly decreased hypoxia-induced ROS level in H9c2 cells and iPSC-CM, whereas this protective effect was not observed with EVPBStreatment (FIG. 7H, FIG. 71, and FIG. 7J).

[0154] Autophagy, a well-characterized function of adiponectin, is an important regulatory mechanism for ischemic cardiac protection (Tang et al., 2024; Liu et al., 2021; Xing et al., 2022; Sung et al., 2024). Inventors then investigated how the EVALYmodulates autophagy under hypoxic conditions by performing quantitative analysis of autophagy flux using a genetically engineered H9c2 cell line expressing the LC3 HiBiT reporter (Tang et al., 2024). A significant increase of red fluorescent signal, indicating accumulation of LC3-II, was detected in cells subjected to hypoxia, while pretreatment with EVALYeffectively mitigated this response (FIG. 7K and FIG. 7L). To determine whether lysosome activity was altered in response to hypoxia ± EVALY, inventors used MagicRed (MR), a cathepsin B probe, to measure lysosomal enzyme activity. Inventors showed that in response to EVAI,Ytreatment increased lysosomal activity, whereas hypoxia alone produced no change in H9c2 cells (FIG. 7M).Example 9. ALY688 driven plasma EVs improved myocardium remodeling in MI mice

[0155] Circulating EVs have diagnostic, prognostic, and therapeutic potential in CVD (Bank et al., 2015). As such, inventors proceeded to determine whether ALY688 shaped-EVs mediate pro-autophagy and mitochondrial protective actions in cardiomyocytes under physiological conditions of acute myocardial infarction. Mice were injected with EVALYor EVPBS, both previously characterized for standard EVs markers and confirmed to be enriched with adiponectin in the EVALYpreparation (FIG. 6D and FIG. 6H). One hour following myocardial ischemia induced by LAD occlusion, EVALYor EVPBSwere administered to mice via tail vein injection, and mice were sacrificed 2 days post -MI (FIG. 8A). Plasma Troponin I and LDH levels were measured to determine cell death. Following MI, both troponin I and LDH were significantly elevated, while only EVALYtreatment suppressed the increase in these cardiac injury markers (FIG. 8B and FIG. 8C).

[0156] Apoptosis in infarcted hearts was then evaluated by caspase-3 cleavage, demonstrating more abundant cleaved caspase-3 in MI compared to sham conditions, which was reduced following EVALYtreatment (FIG. 8D and FIG. 8E). Collectively, these data evidenced that EVALYreduced apoptosis and cell death induced by acute MI.

[0157] Mitochondrial function was next assessed through analysis of fission and fusion proteins, EVALYtreated mice showed slight decrease in MFF (mitochondrial fission factor), p-Drpl (phospho-dynamin-related protein 1) s616, and increase in p-Drpl s637 and OPA1 (Optic Atrophy 1), showing a reduction in Mi-induced mitochondrial dynamic disruption (FIG. 8F, FIG. 8G, FIG. 8H, FIG. 81, and FIG. 8J).

[0158] Mice treated with EVALYalso displayed significant restoration of antioxidant gene expression, including Sodl, Sod2, and Sod3 (FIG. 8K).

[0159] To further correspond with the previous recipient cell proteomics findings, autophagy flux was also measured in infarcted heart. EVI,Ytreatment resulted in increased LC3-II and ATG3 levels, along with enhanced p62 degradation (FIG. 8L, FIG. 8M, FIG. 8N, and FIG. 80). As such, autophagic flux impaired by MI was relieved by EVALY. Taken together, these data indicate that the ALY688-shaped EVs enhance cardiac resilience in acute MI.

[0160] As such, the EVs described herein are useful for treating an age-related disease, for example, a cardiovascular disease, for example, MI.Example 10. The Effect of ALY688 Administration on the Dynamics of Plasma-Derived EVs in Rats

[0161] To investigate the effect of ALY688 on EV dynamics, inventors isolated plasma EVs using size exclusion chromatography (SEC) from a rat administered with either PBS or ALY688 (15mg / kg) for three consecutive days. The NT A data shows an increase in EV particle concentration from ALY688 treated rat compared to controls while the particle size remains consistent (FIG. 9 A, FIG. 9B, and FIG. 9C).Example 11. ALY688 driven plasma EVs mediated insulin sensitizing effect in skeletal muscle cell

[0162] To show the direct functional significance of the isolated plasma EVs in insulin sensitivity, inventors utilized the skeletal muscle cells expressing an Akt biosensor and examined the insulin response through the efficacy on translocation of nuclear fluorescence signal with insulin stimulation at varied doses. In the presence of 24-hr pretreatment with EVs isolated from an ALY688 treated rat the skeletal muscle cells show an insulin sensitizing effect as compared to in the presence of EV from PBS treated rat (FIG. lOA and FIG. 10B).Example 12: ALY688 modulates EV dynamic in adipocyte- derived mesenchymal stem cell (ADSC)

[0163] To show the functional significance of ALY688 treated ADSC EV in iron-induced insulin resistance, inventors employed the skeletal muscle cells expressing an Akt biosensor and examined the insulin response through the efficacy on translocation of nuclear fluorescence signal with insulin stimulation at varied doses. 24-hour pretreatment with EVs isolated from ALY688 treated ADSCeffectively alleviated the EC50 insulin dose in the skeletal muscle cells in the presence of 250 pM iron sulfate induced insulin resistance, as compared to in the presence of EV from PBS treated ADSC (FIG.11 A, FIG. 11B, and FIG. 11C).

[0164] Together, the present disclosure shows that the EV described herein is useful for promoting healthspan and longevity, or treating an aging-related disease.

[0165] While the present disclosure has been described with reference to examples, it is to be understood that the scope of the claims should not be limited by the embodiments set forth in the examples, but should be given the broadest interpretation consistent with the description as a whole.

[0166] All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety. Where a term in the present disclosure is found to be defined differently in a document incorporated herein by reference, the definition provided herein is to serve as the definition for the term.FULL CITATIONS FOR DOCUMENTS REFERRED TO IN THE DISCLOSUREBank, I.E. et al., 2015. The diagnostic and prognostic potential of plasma extracellular vesicles for cardiovascular disease. Expert RevMolDiagn 15, 1577-1588.Chouchani, E.T., et al., 2014. Ischaemic accumulation of succinate controls reperfusion injury through mitochondrial ROS. Nature 515, 431-435.Liu, B.Y., et al., 2021. Baicalein attenuates cardiac hypertrophy in mice via suppressing oxidative stress and activating autophagy in cardiomyocytes. Acta Pharmacol Sin 42, 701-714.McVey, M.J., Spring, C.M. and Kuebler, W.M., 2018. Improved resolution in extracellular vesicle populations using 405 instead of 488 nm side scatter. Journal of Extracellular Vesicles, 7(1), p.1454776. Sung, H.K., Mitchell, P.L., Gross, S., Marette, A. and Sweeney, G., 2022. ALY688 elicits adiponectin-mimetic signaling and improves insulin action in skeletal muscle cells. American Journal of Physiology-Cell Physiology, 322(2), pp.C151-C163.Sung, H.K., et al., 2024. Ischemia-induced cardiac dysfunction is exacerbated in adiponectin-knockout mice due to impaired autophagy flux. Clinical and Translational Science 17, el3758.Tang, J., et al., 2024. Impaired autophagy flux contributes to enhanced ischemia reperfusion injury in the diabetic heart. Autophagy Reports 3, 2330327.Welsh, J.A., Goberdhan, D.C., O'Driscoll, L., Buzas, E.I., Blenkiron, C., Bussolati, B., Cai, H., Di Vizio, D., Driedonks, T.A., Erdbriigger, U. and Falcon-Perez, J.M., 2024. Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches. Journal of Extracellular Vesicles, 13(2), p.e 12404.Xing, Y. et al., 2022. Blunting TRPML1 channels protects myocardial ischemia / reperfusion injury by restoring impaired cardiomyocyte autophagy. Basic Res Cardiol 117, 20.Zhu, L., et al, 2025. Regulated cell death in acute myocardial infarction: Molecular mechanisms and therapeutic implications. Ageing Res Rev 104, 102629.US5049386US4897355

Claims

Claims:

1. An extracellular vesicle (EV) derived from a cell treated with an adiponectin receptor agonist, a cell treated with adiponectin, a cell over-expressing adiponectin, and / or a cell obtained from a donor subject that has been treated with an adiponectin receptor agonist.

2. The EV of claim 1, wherein the EV is derived from a cell treated with an adiponectin receptor agonist.

3. The EV of claim 1, wherein the EV is derived from a cell treated with adiponectin.

4. The EV of claim 1, wherein the EV is derived from a cell over-expressing adiponectin.

5. The EV of claim 1, wherein the EV is derived from a cell obtained from a donor subject that has been treated with an adiponectin receptor agonist.

6. The EV of claim 3, wherein the adiponectin comprises an amino acid sequence having 75%, 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to the full length of SEQ ID NO: 1.

7. The EV of claim 6, wherein the adiponectin comprises an amino acid sequence having 100% sequence identity to the full length of SEQ ID NO: 1.

8. The EV of claim 2, wherein the adiponectin receptor agonist comprises ALY688 (D-Asn-Ile-Pro-Nva-Leu-Tyr-D-Ser-Phe-Ala-D-Ser; SEQ ID NO: 2) or adiporon.

9. The EV of claim 8, wherein the adiponectin receptor agonist is ALY688.

10. The EV of any one of claims 1-9, wherein the EV is derived from an adipocyte, a cardiomyocyte, a hepatocyte, a platelet, an immune cell, an endothelial cell, an erythrocyte, a stem cell, a fibroblast, a smooth muscle cell, a neuronal cell, a glial cell, an astrocyte, a Schwann cell, a keratinocyte, a chondrocyte, an osteoblast, an osteocyte, or any combination thereof.

11. The EV of any one of claims 1-10, wherein the EV is isolated from plasma.

12. The EV of claim 5, wherein the donor subject is a mammal.

13. The EV of claim 5 or 12, wherein the donor subject is a mouse or a human.

14. The EV of any one of claims 5, 12, and 13, wherein the EV is enriched with adiponectin relative to an EV isolated from plasma of a subject administered vehicle.

15. The EV of any one of claims5 and 12-14, wherein the EV is capable of reducing cardiac injury associated with myocardial infarction.

16. The EV of any one of claims 5 and 12-15, wherein the EV is capable of suppressing an increase in plasma troponin I in a recipient experiencing myocardial infarction.

17. The EV of any one of claims 5 and 12-16, wherein the EV is capable of suppressing an increase in plasma lactate dehydrogenase (LDH) in a recipient subject experiencing myocardial infarction.

18. The EV of any one of claims 5 and 12-17, wherein the adiponectin receptor agonist comprises ALY688.

19. A pharmaceutical composition comprising the EV of any one of claims 1-18 and a pharmaceutically acceptable carrier.

20. A method of treating an aging-related disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an extracellular vesicle (EV) derived from a cell overexpressing adiponectin, a cell treated with adiponectin, a cell treated with an adiponectin receptor agonist, and / or a cell obtained from a donor subject that has been treated with an adiponectin receptor agonist.

21. The method of claim 20, wherein the EV is derived from an adipocyte, a cardiomyocyte, a hepatocyte, a platelet, an immune cell, an endothelial cell, an erythrocyte, a stem cell, a fibroblast, a smooth muscle cell, a neuronal cell, a glial cell, an astrocyte, a Schwann cell, a keratinocyte, a chondrocyte, an osteoblast, an osteocyte, or any combination thereof.

22. The method of claim 20, wherein the EV is isolated from plasma.

23. The method of claim 22, wherein the immune cell is a T cell, a B cell, a macrophage, a dendritic cell, or a natural killer cell.

24. The method of claim 22, wherein the endothelial cell is a progenitor cell.

25. The method of claim 22, wherein the cardiomyocyte is derived from a cardiosphere.

26. The method of claim 22, wherein the EV is derived from a stem cell.

27. The method of claim 26, wherein the stem cell is a mesenchymal stem cell or an induced pluripotent stem cell.

28. The method of any one of claims 20-27, wherein the EV is derived from a cell over-expressing adiponectin.

29. The method of any one of claims 20-28, wherein the EV is derived from a cell treated with adiponectin.

30. The method of claim 28 or 29, wherein the adiponectin comprises an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, 99%, 99.9% sequence identity to the full length of SEQ ID NO: 1.

31. The method of any one of claims 20-27, wherein the EV is derived from a cell treated with an adiponectin receptor agonist.

32. The method of claim 31, wherein the adiponectin receptor agonist comprises ALY688 (a.k.a. ADP355) or adiporon.

33. The method of any one of claims 20-32, wherein the aging-related disease comprises a neurodegenerative disorder, a metabolic disorder, a cardiovascular disease, a musculoskeletal disorder, a renal or urological disease, a cancer, an eye disorder, a respiratory disorder, an endocrine disorder, and / or an immune system disorder.

34. The method of claim 33, wherein the neurodegenerative disorder comprises Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, or amyotrophic lateral sclerosis (ALS).

35. The method of claim 33, wherein the metabolic disorder comprises type 2 diabetes, metabolic associated steatohepatitis (MASH), or obesity.

36. The method of claim 33, wherein the cardiovascular disease comprises hypertension, atherosclerosis, coronary artery disease, myocardial infarction, heart failure, arrhythmias, peripheral artery disease, or stroke.

37. The method of claim 33, wherein the musculoskeletal disorder comprises osteoarthritis, sarcopenia, or osteoporosis.

38. The method of claim 33, wherein the renal or urological disease comprises chronic kidney disease, diabetic nephropathy, or benign prostatic hyperplasia.

39. The method of claim 33, wherein the cancer comprises breast cancer, prostate cancer, colorectal cancer, lung cancer, leukemia, or bladder cancer.

40. The method of claim 33, wherein the eye disorder comprises age-related macular degeneration, cataracts, or glaucoma.

41. The method of claim 33, wherein the respiratory disorder comprises chronic obstructive pulmonary disease or pulmonary fibrosis.

42. The method of claim 33, wherein the endocrine disorder comprises hypothyroidism, hyperparathyroidism, adrenal insufficiency, hypercortisolism, a pituitary disorder, an alteration inmetabolic hormone production, or an alteration in sex hormone production.

43. The method of claim 33, wherein the immune system disorder comprises immunosenescence, chronic inflammation, or an autoimmune disease.

44. The method of claim 43, wherein the autoimmune disease comprises rheumatoid arthritis, systemic lupus erythematosus, Hashimoto’s thyroiditis, Sjogren’s syndrome, or hypogammaglobulinemia.

45. The method of any one of claims 20-44, wherein the subject is a mammal.

46. The method of claim 45, wherein the subject is a human.

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