WO2020190672A1

WO2020190672A1 - Cardiomyocyte-derived exosomes inducing regeneration of damaged heart tissue

Info

Publication number: WO2020190672A1
Application number: PCT/US2020/022524
Authority: WO
Inventors: Ke CHENG
Original assignee: North Carolina State University
Priority date: 2019-03-15
Filing date: 2020-03-13
Publication date: 2020-09-24

Abstract

Exosome compositions of the disclosure comprise the miRNA species hsa-miR-21-5p but may also include a least one other miRNA species over-expressed in normal cardiac stromal cells compared to stromal cells from injured cardiac tissue. Further provided are methods of regenerating damaged cardiac tissue by administering to the site of injury a composition comprising exosomes that deliver at least one miRNA species that reduces the expression of phosphatase and tensin homolog (PTEN) thereby initiating new tissue formation.

Description

CARDIOMYOCYTE-DERIVED EXOSOMES INDUCING REGENERATION OF DAMAGED

HEART TISSUE

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent Application Serial No.:

62/818,945, entitled“microRNA-21-5P DYSREGULATION IN EXOSOMES DERIVED FROM HEART-FAILURE PATIENTS IMPAIRS REGENERATIVE POTENTIAL” filed on March 15, 2019, the entirety of which is hereby incorporated by reference.

SEQUENCE LISTING

This application contains a sequence listing filed in electronic form as an ASCII.txt file entitled“2214042370_ST25” created on March 12, 2020. The content of the sequence listing is incorporated herein in its entirety.

TECHNICAL FIELD

The present disclosure is generally related to compositions comprising cardiomyocyte exosomes having an miRNA down-regulating PTEN. The present disclosure is also generally related to methods of using cardiomyocyte-derived exosomes to induce regeneration of damaged heart tissue.

BACKGROUND

Mounting lines of evidence (Luo et ai, (2017) Circ. Res. 120: 1768-1775; Tang et ai, (2018) Nat. Biomed. Engineer. 2: 17-26; Tang et ai., (2017) ACS Nano 11 : 9738-9749; Tang et al., (2017) Nat. Commun. 8: 13724), have demonstrated that cell-based therapy holds great promise for the regeneration of injured heart muscle (Kreke et al., (2012) Expert Rev.

Cardiovasc. Therapy ' O. 1185-1194; Makkar et al., (2012) Lancet 379: 895-904; Chugh et ai., (2012) Circulation 126: S54-S64; Rhee et ai., (2018) Am Heart Assoc:, Qi et ai., (2008) Chin. Med. J. Beijing 121 : 544; Bellamy et ai., (2015) J. Heart Lung Transplant. 34: 1198-1207; Kim et ai., (2014) J. Am. Coll. Cardiol. 64: 1681-1694; Joladarashi etai., (2015) J. Am. Coll. Cardiol. 66: 2214-2226). However, cell-based products must be carefully preserved to maintain their viability and activity until transplantation and there are also some risks involved in cell transplantation. The modes of action for cell therapy products remain elusive, making it difficult to standardize each cell lot. Recent meta-analyses indicate that cardiac cell therapies are overwhelmingly safe but only show none-to-marginal efficacy (Tang et ai., (2018) Stem Cells Transl. Med. 7: 354-359). The development of cell-free and non-living therapeutics (e.g. proteins, RNAs) has the potential to revolutionize cardiovascular regenerative medicine. These therapeutics have compounded the evidence showing that the benefits of stem cell therapies mainly come from paracrine mechanisms instead of the direct differentiation of injected stem cells into cardiomyocytes (Chimenti et ai., (2010) Circ. Res. 106: 971-980; Malliaras et ai., (2011) Circulation 111.042598). The injected cells secrete proteins and nucleic acids to promote endogenous repair (Raposo and Stoorvogel (2013) J. Cell Biol. 200: 373-383).

Exosomes are nanosized membrane vesicles secreted by most cell types, including stem cells and cancer cells (Kalra et al., (2016) Int. J. Mol. Sci 17: 170; Melo et al., (2018) Frontiers Immunol. 9: 730). They are extracellular nanoshuttles that facilitate cell-cell communications and are crucial for maintaining the normal physiological functions of cells. It has been reported that exosomes, as functional paracrine units of stem cells, can partially recapitulate the regenerative activities of their parent cells, suggesting that they may provide an alternative, cell-free therapeutic option (Gallet et al., (2016) Euro. Heart J. 38: 201-211 ; Mathiyalagan et al., (2017) Circ. Res. 116.310557; Lai et al., (2011) Regen. Med. 6: 481-492; Sahoo and Losordo (2014) Circ. Res. 114: 333). The mechanisms that drive the

exosome-mediated repair processes rely largely on the transferring of exosomal cargos, including microRNAs (miR), mRNA, and proteins, to the recipient cells (Prathipati et al., (2017) Stem Cell Revs. Reports 13: 79-91). Accordingly, the constitution of exosomes, as well as their biological activity is largely dependent on the physiological state of their parent cells (Wang et al., (2015) Int. J. Cardiol. 192: 61-69; Yue et al., (2017) Tissue Eng Part A 23: 1241-1250).

SUMMARY

The present disclosure encompasses methods of regenerating damaged cardiac tissue by delivering to the site of injury a therapeutic composition comprising at least one miRNA that reduces the expression of phosphatase and tensin homolog (PTEN) and is over-expressed in normal cardiac stromal cells compared to stromal cells from injured cardiac tissue. The exosome compositions of the disclosure comprise the miRNA species hsa-miR-21-5p (SEQ ID NO: 1) but may also include a least one other miRNA species over-expressed in normal cardiac stromal cells compared to stromal cells from injured cardiac tissue.

One aspect of the disclosure, therefore, encompasses a composition comprising a population of cardiac stromal cell/fibroblast-derived exosomes, wherein the exosomes comprise an miRNA species can have at least 90% sequence similarity to cardiac stromal cell/fibroblast-derived miRNA species miRNA-21-5p (SEQ ID NO: 1) or a heterologous nucleic acid that expresses the miRNA species when in recipient cardiac stromal cells or fibroblasts cells.

In some embodiments of this aspect of the disclosure, the composition can further comprise a pharmaceutically acceptable carrier.

In some embodiments of this aspect of the disclosure, the composition can be formulated for delivery to a site of cardiac injury. In some embodiments of this aspect of the disclosure, the population of cardiac stromal cell/fibroblast-derived exosomes further comprises a plurality of miRNA species expressed in at least one of cardiac stromal cells or fibroblasts derived from a normal heart tissue.

In some embodiments of this aspect of the disclosure, at least one of the plurality of miRNA species can be over-expressed in cardiac stromal cells or fibroblasts cells derived from a normal heart tissue compared to a heart tissue having a pathological injury.

In some embodiments of this aspect of the disclosure, each miRNA species of the plurality of miRNA species can be selected from and can have at least 90% sequence similarity to one of the group consisting of hsa-let-7b-5p (SEQ ID NO: 2), hsa-miR-125a-5p (SEQ ID NO: 3), hsa-miR146a-5p (SEQ ID NO: 4), hsa-miR-125b-5p (SEQ ID NO: 5), hsa-miR-126-3p (SEQ ID NO: 6), hsa-miR-16-1-3p (SEQ ID NO: 7), hsa-miR-23a-5p (SEQ ID NO: 8), hsa-miR-21-3p (SEQ ID NO: 9), hsa-miR-26a-5p (SEQ ID NO: 10), hsa-miR-320a (SEQ ID NO: 1 1), hsa-miR-29a-3p (SEQ ID NO: 12), hsa-miR-16-5p (SEQ ID NO: 13), hsa-miR-23a-3p (SEQ ID NO: 14), and hsa-miR-16-5p (SEQ ID NO: 15).

Another aspect of the disclosure encompasses embodiments of a method of modulating the activity of PTEN in the cardiac tissue of an animal or human subject, the method comprising administering to a cardiac tissue of an animal or human subject a population of cardiac stromal cell/fibroblast-derived exosomes and a pharmaceutically acceptable carrier, wherein the population of cardiac stromal cell/fibroblast-derived exosomes can comprise an miRNA species having at least 90% sequence similarity to cardiac stromal cell/fibroblast-derived miRNA species miRNA-21-5p (SEQ ID NO: 1) that when delivered to recipient cardiac tissue of the animal or human subject reduces the activity of PTEN in the recipient cells thereof.

In some embodiments of this aspect of the disclosure, the population of cardiac stromal cell/fibroblast-derived exosomes can further comprise a plurality of miRNA species.

In some embodiments of this aspect of the disclosure, the population of cardiac stromal cell/fibroblast-derived exosomes can further comprise a plurality of miRNA species expressed in at least one of cardiac stromal cells or fibroblasts derived from a normal heart tissue.

In some embodiments of this aspect of the disclosure the population of cardiac stromal cell/fibroblast-derived exosomes can further comprise at least one nucleic acid species not derived from cardiac stromal cells or fibroblasts cells.

In some embodiments of this aspect of the disclosure the population of cardiac stromal cell/fibroblast-derived exosomes can further comprise a heterologous nucleic acid that expresses at least one miRNA species when in recipient cardiac tissue.

In some embodiments of this aspect of the disclosure the composition can be administered to the cardiac tissue of the animal or human subject by a percutaneous method.

In some embodiments of this aspect of the disclosure the percutaneous method is by injection into the cardiac tissue.

Yet another aspect of the disclosure encompasses embodiments of a method of repairing cardiac tissue damage, the method comprising administering to a cardiac tissue of an animal or human subject a therapeutic composition comprising a population of cardiac stromal cell/fibroblast-derived exosomes and a pharmaceutically acceptable carrier, wherein the population of cardiac stromal cell/fibroblast-derived exosomes comprise an miRNA species having at least 90% sequence similarity to cardiac stromal cell/fibroblast-derived miRNA species miRNA-21-5p (SEQ ID NO: 1) that when delivered to recipient cardiac tissue of the animal or human subject reduces the activity of PTEN in the recipient cells thereof.

In some embodiments of this aspect of the disclosure the population of cardiac stromal cell/fibroblast-derived exosomes can further comprise a plurality of miRNA species.

In some embodiments of this aspect of the disclosure the population of cardiac stromal cell/fibroblast-derived exosomes can further comprise a plurality of miRNA species expressed in cardiac stromal cells or fibroblasts cells derived from a normal heart tissue.

In some embodiments of this aspect of the disclosure at least some of the plurality of miRNA species can be over-expressed in cardiac stromal cells or fibroblasts cells derived from a normal heart tissue compared to a heart tissue having a pathological injury.

In some embodiments of this aspect of the disclosure each miRNA species of the plurality of miRNA species can be selected from and can have at least 90% sequence similarity to one of the group consisting of hsa-let-7b-5p (SEQ ID NO: 2), hsa-miR-125a-5p (SEQ ID NO: 3), hsa-miR146a-5p (SEQ ID NO: 4), hsa-miR-125b-5p (SEQ ID NO: 5), hsa-miR-126-3p (SEQ ID NO: 6), hsa-miR-16-1-3p (SEQ ID NO: 7), hsa-miR-23a-5p (SEQ ID NO: 8), hsa-miR-21-3p (SEQ ID NO: 9), hsa-miR-26a-5p (SEQ ID NO: 10), hsa-miR-320a (SEQ ID NO: 1 1), hsa-miR-29a-3p (SEQ ID NO: 12), hsa-miR-16-5p (SEQ ID NO: 13), hsa-miR-23a-3p (SEQ ID NO: 14), and hsa-miR-16-5p (SEQ ID NO: 15).

In some embodiments of this aspect of the disclosure the therapeutic composition can be administered to the cardiac tissue of the animal or human subject by a percutaneous method.

BRIEF DESCRIPTION OF THE DRAWINGS

Further aspects of the present disclosure will be more readily appreciated upon review of the detailed description of its various embodiments, described below, when taken in conjunction with the accompanying drawings.

Figs. 1A-1 J illustrate the effects of exosome treatment on cardiomyocytes, endothelial cells, and cardiac fibroblasts in vitro.

Fig. 1A shows representative fluorescent micrographs showing uptake of Dil-labeled exosomes derived from the cardiac cells of normal heart donors (NEXO) and exosomes derived from the cardiac cells of heart-failure patients (FEXO) by neonatal rat cardiomyocytes (NRCMs). Endocytosed exosomes can be seen within the cytoplasm of cardiomyocytes. Scale bar: 10pm.

Fig. 1 B shows quantitation of exosomes uptake (n=10). Two-tailed t-test.

Fig. 1C shows NRCM proliferation in response to NEXO, FEXO, or

phosphate-buffered saline (PBS) treatment. White arrows indicate Ki677a-SA⁺ cells. Scale bar: 20pm.

Fig. 1 D shows quantitation of proliferating cardiomyocytes (n=6).

Fig. 1 E shows apoptotic NRCMs in response to NEXO, FEXO, or PBS treatment. White arrows indicate TUNEL7a-SA⁺ cells. Scale bar: 20pm.

Fig. 1 F shows quantitation of apoptotic cardiomyocytes (n=6).

Fig. 1G shows measurement of tube formation in human umbilical vascular endothelial cells (HUVECs) co-cultured with NEXO, FEXO, or PBS. Scale bar: 100pm.

Fig. 1 H shows quantitation of average HUVEC tube length (n=20).

Fig. 11 shows neonatal rat fibroblasts (NRCFs) underwent phenotypic transition to myofibroblasts in response to NEXO, FEXO, or PBS treatment. Scale bar: 20pm. Fig. 1J shows quantitation of myofibroblasts (n=12). *, p<0.05. ***, p<0.001. N.S., no significance. (D, F, H, J) One-way ANOVA with Bonferroni post hoc correction. All values are mean ± S.D.

Figs. 2A-2G illustrate the effects of NEXO and FEXO treatment in a mouse model of acute Ml.

Figs. 2A and 2B show left ventricular ejection fraction (LVEF) measured by echocardiography at baseline (one day before Ml) (Fig. 2A) and endpoint (3 weeks after Ml) (Fig. 2B).

Fig. 2C shows the treatment effects (changes in LVEF at 3 weeks relative to baseline) in each group (A-C, n=9 animals per treatment group, n=3 animals for the sham group).

Fig. 2D shows representative Masson’s trichrome staining of myocardial section 3 weeks after treatment with NEXO, FEXO, or PBS. Scale bar: 0.5 mm.

Figs. 2E-2G show quantitative analyses of infarct size (Fig. 2E), infarct wall thickness (Fig. 2F), and viable tissue from Masson’s trichrome-stained heart sections (Fig. 2G) (n = 8 animals per treatment group). *, p<0.05. ***, p<0.001. One-way ANOVA with Bonferroni post hoc correction.

Figs. 3A-3F show mechanisms of exosome-mediated cardiac repair.

Fig. 3A shows representative images of post-myocardial infarction (Ml) heart sections stained with Ki67, a-SA, and DAPI. White boundaries show infarct area, and white arrows indicate Ki67⁺ cells in the peri-infarct zone. Scale bar: 10pm

Fig. 3B shows heart sections stained with vWF, a-SA in response to NEXO, FEXO or PBS treatment. White arrows indicate capillary structures in the peri-infarct zone. Scale bar: 50pm.

Fig. 3C shows heart sections stained with TUNEL, a-SA, and DAPI. White arrows indicate apoptotic cardiomyocytes in the peri-infarct zone. Scale bar: 50pm.

Fig. 3D shows quantification of cycling cardiomyocytes (Ki67⁺/a-SA⁺)

Fig. 3E shows quantification of capillary density (vWF⁺).

Fig. 3F shows quantification of cardiomyocyte apoptosis (TUNEL7a-SA⁺). n=6 animals per group, 3 heart sections for each animal. *, p<0.05. **, p<0.01. ***, p<0.001. One-way ANOVA with Bonferroni post hoc correction.

Figs. 4A-4C illustrate dysregulation of hsa-miR-21-5p (SEQ ID NO: 1) in heart failure exosomes.

Fig. 4A shows an miRNA array showing fold changes of miRNA abundance in NEXO or FEXO (n = 3 biological replicates, 3 technical replicates for each biological replicate).

Fig. 4B shows a Venn diagram showing the variable miRNA profile between NEXO and

FEXO.

Fig. 4C shows a qRT-PCR analysis validated that hsa-miR-21-5p (SEQ ID NO: 1) was highly enriched in NEXO (n = 3 biological replicates, 3 technical replicates for each biological replicate). *, p<0.05. N.S., no significance. Two-tailed t-test.

Figs. 5A-5H illustrate that manipulation of hsa-miR-21-5p (SEQ ID NO: 1) in exosomes modulates their pro-angiogenic and anti-apoptosis activities in vitro. FEXO+miR-scr, exosomes derived from the cardiac cells of heart-failure patients transfected with scrambled miRNA oligo. FEXO+miR-21 , exosomes derived from the cardiac cells of heart-failure patients transfected with hsa-miR-21-5p (SEQ ID NO: 1) oligo. NEXO+miR-scr, exosomes derived from the cardiac cells of the normal hearts transfected with scrambled miRNA oligo. NEXO+anti-miR-21 , exosomes derived from the cardiac cells of the normal hearts transfected with anti-miR-21-5p (SEQ ID NO: 1) oligo. FEXO, exosomes derived from the cardiac cells of heart-failure patients. NEXO, exosomes derived from the cardiac cells of normal heart donors.

Fig. 5A shows anti-apoptotic effects were diminished after hsa-miR-21-5p (SEQ ID NO: 1) knockdown in NEXOs. White arrows indicate TUNEL⁺ cells. Scale bar: 50pm.

Fig. 5B shows quantitation of apoptotic cells (n = 6).

Fig. 5C shows the pro-angiogenic effects of NEXO were diminished by hsa-miR-21-5p (SEQ ID NO: 1) knockdown. Scale bar: 50pm.

Fig. 5D shows quantitation of average tube length (n = 10).

Fig. 5E shows enhancing hsa-miR-21-5p (SEQ ID NO: 1) expression in FEXO partly rescued its ability to promote cardiomyocyte proliferation. Scale bar: 50pm.

Fig. 5F shows quantitation of apoptotic cells (n = 6).

Fig. 5G shows tube formation assay showing enhanced pro-angiogenic activity of FEXO with restored hsa-miR-21-5p (SEQ ID NO: 1) expression. Scale bar: 50pm.

Fig. 5H shows quantitation of average tube length (n = 10). *, p<0.05. Two-tailed t-test. All values are mean ± S.D.

Figs. 6A-6G illustrate the manipulation of miR21 in exosomes modulates their therapeutic potency. hsa-miR-21-5p (SEQ ID NO: 1)-deficient exosomes were produced by transfecting healthy cardiac cells with anti-miR-21-5p (SEQ ID NO: 1) oligo

(NEXO+anti-miR-21). hsa-miR-21-5p (SEQ ID NO: 1)-restored exosomes were engineered by transfecting heart failure cardiac cells with hsa-miR-21-5p (SEQ ID NO: 1) oligo

(FEXO+miR-21), followed by media conditioning and exosome isolation, as previously described. Scrambled miRNA oligo was used as a control (NEXO/FEXO+miR-scr).

Fig. 6A shows left ventricular ejection fraction (LVEF) was measured by

echocardiography 3 weeks after treatment.

Fig. 6B shows treatment effects (changes in LVEF at 3 weeks relative to baseline) were calculated for each group.

Figs. 6C and 6D show representative Masson’s trichrome staining of myocardial section 3 weeks after treatments. Scale bar: 1 m .

Figs. 6E-6H show quantitative analysis of infarct size, infarct wall thickness, and viable tissue from Masson’s trichrome-stained images n = 6 animals per group. *, p<0.05. **, p<0.01. ***, p<0.001. One-way ANOVA with Bonferroni post hoc correction. All values are mean ± S.D.

Figs. 7A-7H illustrate manipulation of miR21 in exosomes modulates their

pro-angiogenic and anti-apoptotic activities in vivo.

Fig. 7 A shows representative images of post-MI heart sections stained with vWF, a-SA, and DAPI. White circles indicate capillaries in the peri-infarct zone. Scale bar: 100pm.

Fig. 7B shows quantitation of capillary density (vWF⁺).

Fig. 7C shows heart sections stained with TUNEL, a-SA, and DAPI. White squares indicate apoptotic cardiomyocytes in the peri-infarct zone. Scale bar: 50pm.

Fig. 7D shows quantitation of cardiomyocyte apoptosis (TUNEL7a-SA⁺).

Fig. 7E shows representative images of post-MI heart sections stained with vWF, a-SA, and DAPI. White circles indicate capillaries in the peri-infarct zone. Scale bar: 50pm.

Fig. 7F shows quantitation of capillary density (vWF⁺).

Fig. 7G shows heart sections stained with TUNEL, a-SA, and DAPI. White squares indicate apoptotic cardiomyocytes in the peri-infarct zone. Scale bar: 50pm.

Fig. 7H shows quantitation of cardiomyocyte apoptosis (TUNEL7a-SA⁺). (B, D, F, H) n = 6 animals for each group, and 3 heart sections for each animal. **, p<0.01. Two-tailed t-test.

Figs. 8A-8J illustrate that hsa-miR-21-5p (SEQ ID NO: 1) targets the PTEN pathway in post-MI pathology.

Fig. 8A shows representative western blot images showing the expression of various PTEN/Akt pathway components.

Fig. 8B-8E show quantitation of the levels of PTEN (Fig. 8B), p-Akt (Fig. 8C), t-Akt (Fig. 8D), Bcl-2 (Fig. 8E), and caspase-3 (Fig. 8F) (n = 3).

Fig. 8F shows representative western blot images showing the expression of PCNA (proliferation marker), VEGF, and PDCD4 (miR-21 target).

Figs. 8G-8I show quantitation of the levels of PCNA (Fig. 8G), VEGF (Fig. 8H), and PDCD4 (Fig. 8I) (n = 3). *, p<0.05. **, p<0.01. ***, p<0.001. N.S., no significance. Two-tailed t-test. All values are mean ± S.D.

Fig. 8J shows a schematic showing the working model of our study.

CM/H9C2/EC/CF+miR-scr, human cardiomyocytes/H9C2 cells/HUVECs/human cardiac fibroblasts transfected with scrambled miRNA. CM/H9C2/EC/CF+miR-21 , human

cardiomyocytes/H9C2 cells/HUVECs/human cardiac fibroblasts transfected with

hsa-miR-21-5p (SEQ ID NO: 1) mimic.

Figs. 9A-9C illustrate the isolation of exosomes from cardiac explant-derived cells. Fig. 9A shows the derivation of cardiac cells and exosomes.

Fig. 9B shows representative images of explant culture, outgrowth cells from explant tissues, and the cardiac cells before and after 14-day conditioning. Scale bar: 100pm.

Fig. 9C shows a Live/Dead assay showing cell viability during the conditioning period

(n=3).

Figs. 10A and 10B illustrate a phenotypic analysis of explant-derived cardiac cells.

Fig. 10A shows representative flow cytometry histogram of cardiac cells derived from heart-failure patients (FEDC) or healthy heart donors (NEDC). Black lines in the histograms are the unstained or isotype controls. Lighter lines show the particular markers.

Fig. 10B shows quantitative analyses of CD90, CD105, CD31 , CD34, CD45, and c-kit expressions (n=3-4). N.S., no significance. Two-tailed t-test. All values are mean ± S.D.

Figs. 11A-11 E illustrate the characterization of exosomes from normal and failing hearts.

Fig. 11 A shows exosome quantitation using NanoSight particle tracking images (n = 3 biological replicates, 5 technical replicates for each biological replicate).

Fig. 11 B shows size distribution of NEXO and FEXO by NanoSight showing that the exosomes are within the expected size range.

Fig. 11C shows NEXO and FEXO express exosomal markers Alix, TSG 101 , and CD81 as revealed by western blot analysis.

Fig. 11 D shows transmission electron microscopy (TEM) and NanoSight showing exosome morphology and motion. Scale bar: 100 pm.

Fig. 11 E shows total RNA content measured in NEXO and FEXO by Nanodrop (n = 3 biological replicates). N.S., no significance. Two-tailed t-test. All values are mean ± S.D.

Figs. 12A-12H illustrate the effects of NEXO or FEXO treatment on cardiomyocytes in vitro.

Figs. 12A-12C show representative fluorescent micrographs showing adult human cardiomyocyte proliferation in response to NEXO, FEXO, or PBS treatment. Scale bar: 20 pm.

Figs. 12D-12F show the quantitation of cardiomyocyte proliferation, as detected by three proliferation markers: AURKB, Ki67, and PH3 (n=6).

Fig. 12G shows representative fluorescent micrographs showing adult human cardiomyocyte apoptosis in response to NEXO, FEXO, or PBS treatment. Scale bar: 20 pm.

Fig. 12H shows quantitation of apoptotic cardiomyocytes detected by TUNEL (n=6). *, p<0.05. **, p<0.01. ***, p<0.001. One-way ANOVA with Bonferroni post correction.

Figs. 13A-13D illustrate the effects of NEXO or FEXO therapy on cardiac function and morphometry.

Figs. 13A-13C show echocardiographic measurements of fractional shortening (FS) (Fig. 13), left ventricular end systolic volume (Fig. 13B), and left ventricular end diastolic volume (Fig. 13C) of Ml hearts treated with NEXO, FEXO, or PBS.

Fig. 13D shows quantitative analysis of circumference of scar tissue. n=6 animals per group. *, p<0.05. **, p<0.01. ***, p<0.001. N.S., no significance. One-way ANOVA with Bonferroni post correction. All values are mean ± S.D.

Figs. 14A-14C illustrate the effects of NEXO or FEXO treatment on cardiomyocyte proliferation in vivo.

Fig. 14A shows representative images of post-MI heart sections stained with mitosis marker PH3 or cytokinesis marker ARUKB. Scale bar: 20pm.

Figs. 14A and 14B shows the quantitation of proliferated cardiomyocytes (n=6). *, p<0.05. **, p<0.01. ***, p<0.001. One-way ANOVA with Bonferroni post correction. All values are mean ± S.D.

Figs. 15A-15C illustrate the heat map of microRNA PCR array identifies

hsa-miR-21-5p (SEQ ID NO: 1) as the most differentially expressed microRNA between NEXO and FEXO.

Figs. 15A and 15B show a heat map showing miRNA fold changes between NEXO and FEXO.

Fig. 15C shows that infarcted mouse hearts treated with NEXO have elevated levels of hsa-miR-21 compared to FEXO-treated hearts n = 3 animals per group. *, p<0.05. Two-tailed t-test. All values are mean ± S.D.

Figs. 16A-16C illustrate the direct manipulation of hsa-miR-21 in NEXO and FEXO. FEXO+miR-scr, exosomes derived from the cardiac cells of heart-failure patients transfected with scrambled miRNA oligo. FEXO+miR-21 , exosomes derived from the cardiac cells of heart-failure patients transfected with hsa-miR-21-5p (SEQ ID NO: 1) oligo. NEXO+miR-scr, exosomes derived from the cardiac cells of the normal hearts transfected with scrambled miRNA oligo. NEXO+anti-miR-21 , exosomes derived from the cardiac cells of the normal hearts transfected with anti-miR-21-5p (SEQ ID NO: 1) oligo.

Fig. 16A shows transfection of cardiac cells from heart-failure patients/healthy heart donors with hsa-miR-21/ anti-miR-21 oligo or scrambled miRNA oligo (as a negative control). Scale bar: 100pm.

Figs. 16B and 16C show the transfection efficiency of mi-R21/anti-miR-21 oligo was determined by qRT-PCR. n=3 biological replicates. ***, p<0.001. Two-tailed t-test. All values are mean ± S.D.

Figs. 17A and 17B show the effects of hsa-miR-21 on adult human cardiomyocytes in vitro. Adult human cardiomyocytes were transfected with hsa-miR-21-5p (SEQ ID NO: 1) mimic or scrambled control. CM+miR-scr: human cardiomyocytes transfected with scrambled miRNA. CM+miR-21 : human cardiomyocytes transfected with hsa-miR-21-5p (SEQ ID NO: 1) mimic. Fig. 17A shows representative images of transfected human cardiomyocytes stained with TUNEL. Scale bar: 20pm.

Fig. 17B shows quantitation of cardiomyocyte apoptosis (n=6). ***, p<0.001.

Two-tailed t-test. All values are mean ± S.D.

Fig. 18 illustrates pro-angiogenic effects of hsa-miR-21 on post-MI heart. Functional vessels were confirmed by co-staining of the endothelial cell marker von Willebrand factor (vWF) and the red blood cell (RBC) marker. RBCs were detected in the blood vessels in the Ml border zone of the heart treated with hsa-miR-21 -over-expressed FEXO.

Figs. 19A-19G illustrate the effects of various microRNA treatments on cardiomyocytes, endothelial cells, and cardiac fibroblasts in vitro. Human cardiomyocytes (CM), human cardiac fibroblasts (CF), and human umbilical vein endothelial cells (HUVECs) were treated with let-7b-5p (SEQ ID NO: 2), hsa-miR-125a-5p (SEQ ID NO: 3), hsa-miR-146a-5p (SEQ ID NO: 4), hsa-miR-21-5p (SEQ ID NO: 1) mimics or scrambled control, followed by

immunocytochemistry staining of proliferation or apoptotic markers.

Figs. 19A-19C show cardiomyocyte proliferation in response to let-7b-5p (SEQ ID NO: 2), hsa-miR-125a-5p (SEQ ID NO: 3), hsa-miR-146a-5p (SEQ ID NO: 4) or hsa-miR-21-5p (SEQ ID NO: 1), as calculated by three proliferation markers: Ki67, phospho-histone H3 (PH3, mitosis marker), and aurora kinase B (AURKB, cytokinesis marker) (n=6).

Fig. 19D shows apoptotic cardiomyocytes transfected with indicated miRNAs (n=6).

Fig. 19D shows measurement of tube length in HUVECs in the transfection of indicated miRNAs (n=22).

Figs. 19F and 19G show quantitation of apoptosis of HUVECs (Fig. 19F) and cardiac fibroblasts (Fig. 19G) after transfection with indicated miRNAs (n=6). *, p<0.05. **, p<0.01. ***, p<0.001. N.S., no significance. One-way ANOVA with Bonferroni post correction. All values are mean ± S.D.

Figs. 20A-20C illustrate that a signaling pathway phosphorylation array identifies PTEN/Akt as the hsa-miR-21-regulate genes in cardiomyocytes and endothelial cells. Three major cell types in the heart (cardiomyocytes (CM), endothelial cells (EC), and cardiac fibroblasts (CF)) were treated with hsa-miR-21 mimic or scrambled control, followed by the examination of 18 phosphorylated proteins with a signaling pathway phosphorylation array.

Fig. 20A shows a heat map showing expression level changes between cells transfected with hsa-miR-21 mimic or scrambled control.

Fig. 20A shows quantitation of expression changes in hsa-miR-21-upregulated cells relative to scrambled control (n = 2 biological replicates, 2 technical replicates for each biological replicate). All values are mean ± S.D. Cardiomyocytes (using both human induced pluripotent stem cell-derived cardiomyocytes (iCM) and H9C2 cells), cardiac fibroblasts (using human cardiac fibroblasts, HCF) (Fig. 20B), and endothelial cells (using human umbilical vein endothelial cells, HUVECs) (Fig. 20C).

DETAILED DESCRIPTION

This disclosure is not limited to particular embodiments described, and as such may, of course, vary. The terminology used herein serves the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

Where a range of values is provided, each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of medicine, organic chemistry, biochemistry, molecular biology, pharmacology, and the like, which are within the skill of the art. Such techniques are explained fully in the literature.

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to perform the methods and use the compositions and compounds disclosed and claimed herein. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C, and pressure is at or near atmospheric. Standard temperature and pressure are defined as 20 °C and 1 atmosphere.

Before the embodiments of the present disclosure are described in detail, it is to be understood that, unless otherwise indicated, the present disclosure is not limited to particular materials, reagents, reaction materials, manufacturing processes, dimensions, frequency ranges, applications, or the like, as such can vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting. It is also possible in the present disclosure that steps can be executed in different sequence, where this is logically possible. It is also possible that the embodiments of the present disclosure can be applied to additional embodiments involving measurements beyond the examples described herein, which are not intended to be limiting. It is furthermore possible that the embodiments of the present disclosure can be combined or integrated with other measurement techniques beyond the examples described herein, which are not intended to be limiting. It should be noted that, as used in the specification and the appended claims, the singular forms“a,”“an,” and“the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to“a support” includes a plurality of supports. In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings unless a contrary intention is apparent.

Each of the applications and patents cited in this text, as well as each document or reference cited in each of the applications and patents (including during the prosecution of each issued patent;“application cited documents”), and each of the PCT and foreign applications or patents corresponding to and/or claiming priority from any of these applications and patents, and each of the documents cited or referenced in each of the application cited documents, are hereby expressly incorporated herein by reference. Further, documents or references cited in this text, in a Reference List before the claims, or in the text itself; and each of these documents or references (“herein cited references”), as well as each document or reference cited in each of the herein-cited references (including any manufacturer’s specifications, instructions, etc.) are hereby expressly incorporated herein by reference.

Prior to describing the various embodiments, the following definitions are provided and should be used unless otherwise indicated.

Definitions

The term“cell” as used herein refers to an animal or human cell. The engineered cells of the disclosure can be removed (isolated) from a tissue and mixed with platelet-derived membrane vesicles without culturing of the cells, or after removal from a tissue or fluid of an animal or human may be cultured to increase the population size of the cells. If not immediately required for incorporation of the platelet-derived membrane vesicles, the animal or human cells may be maintained in a viable state either by culturing by serial passage in a culture medium or cryopreserved by methods well-known in the art. The cells may be obtained from the animal or human individual that has received an injury desired to be repaired by administration of the engineered cells of the disclosure or from a different individual.

Cells, and their extracellular vesicle such as an exosomes that are contemplated for use in the methods of the present disclosure may be derived from the same subject to be treated (autologous to the subject) or they may be derived from a different subject, preferably of the same species, (allogeneic to the subject).

Commercially available media may be used for the growth, culture and maintenance of mesenchymal stem cells. Such media include but are not limited to Dulbecco's modified Eagle's medium (DMEM). Components in such media that are useful for the growth, culture and maintenance of mesenchymal stem cells include but are not limited to amino acids, vitamins, a carbon source (natural and non-natural), salts, sugars, plant derived hydrolysates, sodium pyruvate, surfactants, ammonia, lipids, hormones or growth factors, buffers, non-natural amino acids, sugar precursors, indicators, nucleosides and/or nucleotides, butyrate or organics, DMSO, animal derived products, gene inducers, non-natural sugars, regulators of intracellular pH, betaine or osmoprotectant, trace elements, minerals, non-natural vitamins. Additional components that can be used to supplement a commercially available tissue culture medium include, for example, animal serum (e.g., fetal bovine serum (FBS), fetal calf serum (FCS), horse serum (HS)), antibiotics (e.g., including but not limited to, penicillin, streptomycin, neomycin sulfate, amphotericin B, blasticidin, chloramphenicol, amoxicillin, bacitracin, bleomycin, cephalosporin, chlortetracycline, zeocin, and puromycin), and glutamine (e.g., L-glutamine). Mesenchymal stem cell survival and growth also depends on the maintenance of an appropriate aerobic environment, pH, and temperature.

Mesenchymal stem cells can be maintained using methods known in the art (see for example Pittenger et ai, (1999) Science 284:143-147).

The term "extracellular vesicle" as used herein can refers to a membrane vesicle secreted by cells that may have a larger diameter than that referred to as an“exosome”. Extracellular vesicles (alternatively named“microvesicle” or“membrane vesicle”) may have a diameter (or largest dimension where the particle is not spheroid) of between about 10 nm to about 5000 nm (e.g., between about 50 nm and 1500 nm, between about 75 nm and 1500 nm, between about 75 nm and 1250 nm, between about 50 nm and 1250 nm, between about 30 nm and 1000 nm, between about 50 nm and 1000 nm, between about 100 nm and 1000 nm, between about 50 nm and 750 nm, etc.). Typically, at least part of the membrane of the extracellular vesicle is directly obtained from a cell (also known as a donor cell). Extracellular vesicles suitable for use in the compositions and methods of the present disclosure may originate from cells by membrane inversion, exocytosis, shedding, blebbing, and/or budding. Extracellular vesicles may originate from the same population of donor cells yet different subpopulations of extracellular vesicles may exhibit different surface/lipid characteristics. Alternative names for extracellular vesicles include, but are not limited to, exosomes, ectosomes, membrane particles, exosome-like particles, and apoptotic vesicles. Depending on the manner of generation (e.g., membrane inversion, exocytosis, shedding, or budding), the extracellular vesicles contemplated herein may exhibit different surface/lipid

characteristics.

The term“exosomes” as used herein refers to small secreted vesicles (typically about 30 nm to about 150 nm(or largest dimension where the particle is not spheroid)) that may contain, or have present in their membrane, nucleic acid, protein, or other biomolecules and may serve as carriers of this cargo between diverse locations in a body or biological system. The term“exosomes” as used herein advantageously refers to extracellular vesicles that can have therapeutic properties, including, but not limited to stem cell exosomes such as cardiac stem cell or mesenchymal stem cells.

Exosomes may be isolated from a variety of biological sources including mammals such as mice, rats, guinea pigs, rabbits, dogs, cats, bovine, horses, goats, sheep, primates or humans. Exosomes can be isolated from biological fluids such as serum, plasma, whole blood, urine, saliva, breast milk, tears, sweat, joint fluid, cerebrospinal fluid, semen, vaginal fluid, ascetic fluid and amniotic fluid. Exosomes may also be isolated from experimental samples such as media taken from cultured cells ("conditioned media", cell media, and cell culture media).

Exosomes may also be isolated from tissue samples such as surgical samples, biopsy samples, and cultured cells. When isolating exosomes from tissue sources it may be necessary to homogenize the tissue in order to obtain a single cell suspension followed by lysis of the cells to release the exosomes. When isolating exosomes from tissue samples it is important to select homogenization and lysis procedures that do not result in disruption of the exosomes.

Exosomes may be isolated from freshly collected samples or from samples that have been stored frozen or refrigerated. Although not necessary, higher purity exosomes may be obtained if fluid samples are clarified before precipitation with a volume-excluding polymer, to remove any debris from the sample. Methods of clarification include centrifugation, ultracentrifugation, filtration or ultrafiltration.

The genetic information within the extracellular vesicle such as an exosome may easily be transmitted by fusing to the membranes of recipient cells, and releasing the genetic information into the cell intracellularly. Though exosomes as a general class of compounds represent great therapeutic potential, the general population of exosomes are a combination of several class of nucleic acids and proteins which have a constellation of biologic effects both advantageous and deleterious. In fact, there are over 1000 different types of exosomes.

The terms“microRNA” and“miRNA” as used herein refer to a small non-coding RNA molecule (containing about 22 nucleotides) found in plants, animals and some viruses, and which function in RNA silencing and post-transcriptional regulation of gene expression.

miRNAs function via base-pairing with complementary sequences within mRNA molecules. As a result, these mRNA molecules are silenced, by one or more of cleavage of the mRNA strand into two pieces, destabilization of the mRNA through shortening of its poly(A) tail, and less efficient translation of the mRNA into proteins by ribosomes. miRNAs are derived from regions of RNA transcripts that fold back on themselves to form short hairpins.

The term "stem cell" as used herein refers to cells that have the capacity to self-renew and to generate differentiated progeny. The term "pluripotent stem cells" refers to stem cells that have complete differentiation versatility, i.e. , the capacity to grow into any of the fetal or adult mammalian body's approximately 260 cell types. For example, pluripotent stem cells have the potential to differentiate into three germ layers: endoderm (e.g., blood vessels), mesoderm (e.g., muscle, bone and blood) and ectoderm (e.g., epidermal tissues and nervous system), and therefore, can give rise to any fetal or adult cell type. The term "cardiac stem cells" refers to stem cells obtained from or derived from cardiac tissue. The term

"cardiosphere-derived cells (CDCs)" as used herein refers to undifferentiated cells that grow as self-adherent clusters from subcultures of postnatal cardiac surgical biopsy specimens. CDCs can express stem cell as well as endothelial progenitor cell markers, and typically possess properties of adult cardiac stem cells. For example, human CDCs can be distinguished from human cardiac stem cells in that human CDCs typically do not express multidrug resistance protein 1 (MDR1 ; also known as ABCB1), CD45 and CD133 (also known as PROM1). CDCs are capable of long-term self-renewal, and can differentiate in vitro to yield cardiomyocytes or vascular cells after ectopic (dorsal subcutaneous connective tissue) or orthotopic (myocardial infarction) transplantation in SCID beige mouse.

The terms“cardiac progenitor cells” and“cardiac stem cells” as used herein can refer to a population of progenitor cells derived from human heart tissue. In some aspects the present disclosure, of the cardiac progenitor (stem) cells, at least 3%, 5%, 7%, 10%, 12%, or 15%, e.g., 3-50%, 3-20%, 3-10%, 5-30%, 5-10%, etc., of the cells express Isl1. In some aspects, CPCs comprise about 10%, 15%, 20%, 30%, 40%, or 50%, e.g., 10-50%, 10-40%, 10-30%, 15-40%, etc., GATA4 expressing cells. In some aspects, cardiac stem cells comprise about 5%, 8%, 10%, 13%, or 15%, e.g., 8-15%, 5-15%, 5-13%, etc., NKX2.5 expressing cells. Before fusion with platelet membrane vesicles by the methods of the present disclosure, cardiac stem cells are unmodified cells in that recombinant nucleic acids or proteins have not been introduced into them or the Sca-1 ⁺, CD45- cell from which it is derived. As such, cardiac stem cells as isolated from cardiac tissue are non-transgenic, or in other words have not been genetically modified. For example, expression of genes such as Isl 1 , GATA4, and NKX2.5 in CPCs is from the endogenous gene. Cardiac stem cells may comprise Sca-1+, CD45- cells, c-kit⁺ cells, CD90⁺ cells, CD133⁺ cells, CD31⁺ cells, Flk1⁺ cells, GATA4⁺ cells, or NKX2.5+ cells, or combinations thereof. In some aspects, cardiac stem cells comprise about 50% GATA4 expressing cells. In some aspects, cardiac stem cells comprise about 15% NKX2.5 expressing cells. Cardiac stem cells can replicate and are capable of differentiating into endothelial cells, cardiomyocytes, smooth muscle cells, and the like.

The term "cardiac cells" as used herein refers to any cells present in the heart that provide a cardiac function, such as heart contraction or blood supply, or otherwise serve to maintain the structure of the heart. Cardiac cells as used herein encompass cells that exist in the epicardium, myocardium or endocardium of the heart. Cardiac cells also include, for example, cardiac muscle cells or cardiomyocytes; cells of the cardiac vasculatures, such as cells of a coronary artery or vein. Other non-limiting examples of cardiac cells include epithelial cells, endothelial cells, fibroblasts, cardiac conducting cells and cardiac pacemaking cells that constitute the cardiac muscle, blood vessels and cardiac cell supporting structure.

The term "cardiac function" as used herein refers to the function of the heart, including global and regional functions of the heart. The term "global" cardiac function as used herein refers to function of the heart as a whole. Such function can be measured by, for example, stroke volume, ejection fraction, cardiac output, cardiac contractility, etc. The term "regional cardiac function" refers to the function of a portion or region of the heart. Such regional function can be measured, for example, by wall thickening, wall motion, myocardial mass, segmental shortening, ventricular remodeling, new muscle formation, the percentage of cardiac cell proliferation and programmed cell death, angiogenesis and the size of fibrous and infarct tissue. Techniques for assessing global and regional cardiac function are known in the art. For example, techniques that can be used to measure regional and global cardiac function include, but are not limited to, echocardiography (e.g., transthoracic echocardiogram, transesophageal echocardiogram or 3D echocardiography), cardiac angiography and hemodynamics, radionuclide imaging, magnetic resonance imaging (MRI), sonomicrometry and histological techniques.

The term "cardiac tissue" as used herein refers to tissue of the heart, for example, the epicardium, myocardium or endocardium, or portion thereof, of the heart. The term "injured" cardiac tissue as used herein refers to a cardiac tissue that is, for example, ischemic, infarcted, reperfused, or otherwise focally or diffusely injured or diseased. Injuries associated with a cardiac tissue include any areas of abnormal tissue in the heart, including any areas caused by a disease, disorder or injury and includes damage to the epicardium, endocardium and/or myocardium. Non-limiting examples of causes of cardiac tissue injuries include acute or chronic stress (e.g., systemic hypertension, pulmonary hypertension or valve dysfunction), atheromatous disorders of blood vessels (e.g., coronary artery disease), ischemia, infarction, inflammatory disease and cardiomyopathies or myocarditis.

The terms "generate", "generation", and "generating" as used herein shall be given their ordinary meaning and shall refer to the production of new cells in a subject and optionally the further differentiation into mature, functioning cells. Generation of cells may comprise regeneration of the cells. Generation of cells comprises improving survival, engraftment and/or proliferation of the cells.

The terms "regenerate," "regeneration" and "regenerating" as used herein refer to the process of growing and/or developing new cardiac tissue in a heart or cardiac tissue that has been injured, for example, injured due to ischemia, infarction, reperfusion, or other disease. Tissue regeneration may comprise activation and/or enhancement of cell proliferation.

Cardiac tissue regeneration comprises activation and/or enhancement of cell migration. The term“cell therapy” as used herein refers to the introduction of new cells into a tissue in order to treat a disease and represents a method for repairing or replacing diseased tissue with healthy tissue.

The term "derived from" as used herein refers to cells or a biological sample (e.g., blood, tissue, bodily fluids, etc.) and indicates that the cells or the biological sample were obtained from the stated source at some point in time. For example, a cell derived from an individual can represent a primary cell obtained directly from the individual (i.e., unmodified). In some instances, a cell derived from a given source undergoes one or more rounds of cell division and/or cell differentiation such that the original cell no longer exists, but the continuing cell (e.g., daughter cells from all generations) will be understood to be derived from the same source. The term includes directly obtained from, isolated and cultured, or obtained, frozen, and thawed. The term“derived from” may also refer to a component or fragment of a cell obtained from a tissue or cell.

The term "isolating" or "isolated" when referring to a cell or a molecule (e.g., nucleic acids or protein) indicates that the cell or molecule is or has been separated from its natural, original or previous environment. For example, an isolated cell can be removed from a tissue derived from its host individual, but can exist in the presence of other cells (e.g., in culture), or be reintroduced into its host individual.

The term "culturing" as used herein refers to growing cells or tissue under controlled conditions suitable for survival, generally outside the body (e.g., ex vivo or in vitro). The term includes "expanding," "passaging," "maintaining," etc. when referring to cell culture of the process of culturing. Culturing cells can result in cell growth, differentiation, and/or division.

The term "disaggregating" includes separating, dislodging, or dissociating cells or tissue using mechanical or enzymatic disruption to isolate single cells or small clusters of cells. In some instances, enzymatic disruption can be replaced with one of more enzyme alternatives having substantially the same effect as the enzyme.

The term“tissue injury” as used herein refers to damage to a vascularized tissue of an animal or human, wherein the damage is adjacent to, or in close proximity to, a blood vessel that has also undergone injury, and in particular loss of endothelial cells lining the lumen of the blood vessel. For example, but not intended to be limiting, vascular ischemia can result in both loss of vascular endothelial cells to expose the underlying subendothelial matrix. The loss of adequate blood flow can result in loss of cell viability in such as cardiac tissue, brain or neurological tissue that is in contact with the occluded blood vessel.

The term "endothelial cell" refers to a cell necessary for the formation and

development of new blood vessel from pre-existing vessels (e.g., angiogenesis). Typically, endothelial cells are the thin layer of cells that line the interior surface of blood vessels and lymphatic vessels. Endothelial cells are involved in various aspects of vascular biology, including atherosclerosis, blood clotting, inflammation, angiogenesis, and control of blood pressure.

The term "smooth muscle cell" refers to a cell comprising non-striated muscle (e.g., smooth muscle). Smooth muscle is present within the walls of blood vessels, lymphatic vessels, cardiac muscle, urinary bladder, uterus, reproductive tracts, gastrointestinal tract, respiratory tract, and iris of the eye.

The term "cardiomyocyte cell" refers to a cell comprising striated muscle of the walls of the heart. Cardiomyocytes can contain one or more nuclei.

The term "cardiosphere" refers to a cluster of cells derived from heart tissue or heart cells. In some instances, a cardiosphere includes cells that express stem cell markers (e.g., c-Kit, Sca-1 , and the like) and differentiating cells expressing myocyte proteins and the gap protein (connexin 43).

The term "autologous" refers to deriving from or originating in the same subject or patient. An“autologous transplant" refers to collection (e.g., isolation) and re-transplantation of a subject's own cells or organs. In some instances, an "autologous transplant" includes cells grown or cultured from a subject's own cells. For example, in the methods of the present disclosure, the cardiac stem cells may be derived from a cardiac tissue sample excised from the heart of the patient to be treated, cultured, engineered to be fused with platelet membrane vesicles according to the methods of the disclosure and then administered to the same patient for the treatment of a cardiovascular injury therein.

The term "allogeneic" refers to deriving from or originating in another subject or patient. An "allogeneic transplant" refers to collection (e.g., isolation) and transplantation of the cells or organs from one subject into the body of another. In some instances, an "allogeneic transplant" includes cells grown or cultured from another subject's cells.

The term "transplant" as used herein refers to cells, e.g., cardiac progenitor cells, introduced into a subject. The source of the transplanted material can include cultured cells, cells from another individual, or cells from the same individual (e.g., after the cells are cultured, enriched, or expanded ex vivo or in vitro).

The terms "treatment," "therapy," "amelioration" and the like refer to any reduction in the severity of symptoms. As used herein, the terms "treat" and "prevent" are not intended to be absolute terms. Treatment can refer to any delay in onset, amelioration of symptoms, improvement in patient survival, repair/regeneration of heart tissue or blood vessels, increase in survival time or rate, etc. The effect of treatment can be compared to an individual or pool of individuals not receiving the treatment. In some instances, the effect can be the same patient prior to treatment or at a different time during the course of therapy. In some aspects, the severity of disease, disorder or injury is reduced by at least 10%, as compared, e.g., to the individual before administration or to a control individual (e.g., healthy individual or an individual no longer having the disease, disorder or injury) not undergoing treatment. In some instances, the severity of disease, disorder or injury is reduced by at least 20%, 25%, 50%, 75%, 80%, or 90%. In some cases, the symptoms or severity of disease are no longer detectable using standard diagnostic techniques.

The terms "subject," "patient," "individual" and the like are used interchangeably and refer to, except where indicated, mammals such as humans and non-human primates, as well as rabbits, rats, mice, dogs, cats, goats, pigs, cows, and other mammalian species. The term does not necessarily indicate that the subject has been diagnosed with a particular disease, but typically refers to an individual under medical supervision.

Abbreviations

PTEN, phosphatase and tensin homolog; NEXO, normal heart donor exosomes; FEXO, heart-failure patient exomes; NRCM, neonatal rat cardiomyocyte; PBS,

phosphate-buffered saline; LVEF, left ventricular ejection fraction; Ml, myocardial infarction; vWF, von Willebrand factor; CM, cardiomyocyte; CF, cardiac fibroblast (CF); EC, endothelial cell; DMEM, Dulbecco's modified Eagle's medium

Discussion

The present disclosure encompasses methods of regenerating damaged cardiac tissue by delivering to the site of injury a therapeutic composition comprising at least one miRNA or modified miRNA that reduces the expression of PTEN that is over-expressed in normal cardiac stromal cells compared to stromal cells from injured cardiac tissue. The exosome compositions of the disclosure comprise the miRNA species hsa-miR-21-5p (SEQ ID NO: 1) but may also include a least one other miRNA species over-expressed in normal cardiac stromal cells compared to stromal cells from injured cardiac tissue and which may induce the expression of genes promoting the regeneration of cardiac cells, inhibit the expression of such genes or induce directly or indirectly the differentiation of cardiac stem cells to provide new and healthy cardiac tissue and/or vascular formation. For example, but not intended to be limiting, the exosome compositions of the disclosure may induce angiogenesis to restore blood flow to damaged heart tissue.

It has been reported that stem cells derived from animals or patients with physiological stresses, such as systemic inflammation, have impaired regenerative activity (Pang et al., (2013) Eur. Rev. Med. Pharmacol. Sci. 17:.3169-3177; Desouza et al., (2011) Diabetes 60: 1286-1294; Hong et al., (2015) Arthritis Res. Therapy 17: 292). Since many patients with cardiac diseases, especially heart failure, suffer from these physiological stresses, it was hypothesized that cardiac-cell-secreted exosomes derived from these patients may have impaired therapeutic activity, or possibly make the condition worse. The treatment effects of cardiac exosomes from healthy donor hearts (normal exosomes, or NEXO) were compared to those of cardiac exosomes from failing hearts (failure exosomes, or FEXO). Exosomes, as functional paracrine units of therapeutic cells, can partially reproduce the reparative properties of their parental cells. The constitution of exosomes, as well as their biological activity, is largely dependent on the cells which secrete them. Exosomes from explant-derived cardiac stromal cells from heart-failure patients (FEXO), or from normal donor hearts (NEXO) were isolated and their regenerative activities compared in vitro and in vivo.

FEXO exhibited impaired ability to promote endothelial tube formation and

cardiomyocyte proliferation in vitro. Intramyocardial injection of NEXO resulted in structural and functional improvements in a murine model of acute myocardial infarction. In contrast, FEXO therapy exacerbated cardiac dysfunction and left ventricular remodeling

MicroRNA array and PCR analysis has shown dysregulation of hsa-miR-21-5p (SEQ

ID NO: 1) in exosomes from heart-failure patients. Restoring hsa-miR-21-5p (SEQ ID NO: 1) expression rescued FEXO’s reparative function, while blunting hsa-miR-21-5p (SEQ ID NO: 1) expression in NEXO diminished its therapeutic benefits. Other mechanistic studies showed that hsa-miR-21-5p (SEQ ID NO: 1) augmented Akt kinase activity through the inhibition of PTEN. Taken together, the heart failure pathological condition altered the miRNA cargos of cardiac-derived exosomes and impaired their regenerative activities. hsa-miR-21-5p (SEQ ID NO: 1) contributes to exosome-mediated heart repair by enhancing angiogenesis and cardiomyocyte survival through the PTEN/Akt pathway.

Generation and characterization of NEXO and FEXO: Information regarding patients and healthy donors are presented in Table 1.

Table 1: Patient and Healthy Heart donors

A: NYHA = New York Heart Association

There were no significant differences in age or body mass index between the two groups.

Explant-derived cardiac stromal cells were harvested from the donors’ hearts as shown in Figs. 9A and 9B. Flow cytometry revealed that explant-derived cells contain a negligible subpopulation of c-kit⁺ cells and endothelial cells (CD31⁺, CD34⁺), along with a dominating subpopulation that phenotypically resembles mesenchymal cells or fibroblasts (CD90⁺, CD105⁺) (Fig. 10A). NEXO and FEXO were isolated from the 14-day conditioned media of cardiac cells from normal heart donors or heart-failure patients, respectively (Fig. 9A). Cell viability was confirmed at the end of the conditioning period (Figs. 9B and 9C). Cardiac cells from heart-failure patients, and those from healthy heart donors, had comparable exosome yields (Fig. 11 A) and exosome sizes (Fig. 11 B), as measured by NanoSight nanoparticle tracking analysis. Transmission electron microscopy (TEM) revealed the vesicular morphology of exosomes and confirmed their size did not exceed 200 nm (34) (Fig. 11 D). In addition, western blot analysis indicated the presence of signature exosomal markers, such as protein CD81 , Alix and TSG101 (Fig. 11 C). Both types of exosomes were abundant in RNA, and the amount of RNA contents in NEXO and FEXO was indistinguishable (Fig. 11 E). Effects of NEXO or FEXO on cardiomyocytes, endothelial cells, and cardiac fibroblasts in vitro: Cardiomyocytes, endothelial cells, and cardiac fibroblasts are the three major cell types in the heart. The uptake efficiency of NEXO and FEXO by those cells was first measured.

Dil-labeled exosomes were readily internalized by neonatal rat cardiomyocytes (NRCMs) (Fig. 1A). There was no significant difference in uptake efficiency between NEXO and FEXO by NRCMs (Fig. 1 B).

NEXO significantly promoted cardiomyocyte proliferation, as evidenced by the higher percentage of Ki67-positive nuclei, while treatment with FEXO suppressed cardiomyocyte proliferation (Figs. 1 C and 1 D). In addition, cardiomyocytes treated with NEXO exhibited fewer TUNEL-positive nuclei, indicating less apoptosis. In contrast, FEXO did not show any anti-apoptotic effects (Figs. 1 E and 1 F).

To further validate the effects of NEXO and FEXO on adult cardiomyocytes, human cardiomyocytes were cultured on human cardiomyocyte cell culture extracellular matrix, then treated with NEXO or FEXO for 24 hours. NEXO significantly promoted cardiomyocyte proliferation, as indicated by the higher percentage of Ki67, while treatment with FEXO suppressed cardiomyocyte proliferation (Figs. 12A and 12D). This result was supported by the assessment of two additional proliferation markers: phospho-histone H3 (PH3), a characteristic marker of mitosis, and aurora kinase B (AURKB), a cytokinesis marker, (Figs. 12B-12F). Furthermore, adult cardiomyocytes treated with NEXO exhibited fewer

TUNEL-positive nuclei, suggesting less apoptosis. In contrast, FEXO did not show any anti-apoptotic effects (Figs. 12G-12H).

A tube formation assay using HUVECs, was used to evaluate the pro-angiogenic effects of exosomes. Treatment with FEXO inhibited tube formation of HUVECs, while NEXO promoted tube formation, compared to normal saline control (Figs. 1 G and H). Alpha-smooth muscle actin (a-SMA) was employed as an indicator of transition from fibroblasts to myofibroblasts. The expression of a-SMA was up-regulated in FEXO-treated cardiac fibroblasts (Figs. 11 and 1 J), suggesting a phenotype transition to myofibroblasts. Taken together, the data indicate that cardiac cells in the failing heart secreted exosomes with an impaired ability to promote cardiomyocyte proliferation, decrease programmed cell death, and stimulate angiogenesis in vitro. Moreover, such diseased exosomes drive the differentiation of fibroblasts to myofibroblasts.

Effects of NEXO and FEXO therapy on cardiac function and morphometry in a mouse model of acute Ml: An acute myocardial infarction (Ml) was induced in CD1 mice by coronary vessel ligation, then intramyocardially injected NEXO, FEXO, or PBS into the Ml border zone. Left ventricular ejection fraction (LVEF) was used to assess the enhancement or preservation of heart function.

There was no significant difference in LVEF between the three treatment groups at baseline (Fig. 2A). LVEF progressively decreased in the control group over the next 3 weeks, whereas hearts injected with NEXO showed greater LVEFs, compared to those treated with FEXO or PBS control. FEXO-treated hearts exhibited a worsening of LVEFs when compared to PBS-treated control hearts (Fig. 2B).

To facilitate head-to-head comparisons, treatment effect was evaluated by calculating the changes in endpoint LVEF relative to baseline LVEF. Control treatment had a negative effect, as LVEF declined over time, while NEXO treatment robustly prevented the decline of LVEF post-MI (Fig. 2C). Likewise, the animals that received NEXO treatment showed a recovery in fractional shortening (FS), whereas the ones injected with FEXO, suffered from FS deterioration (Fig. 13A). These results demonstrate that exosomes from heart-failure patients have reduced reparative activity, and may exacerbate LV dysfunction post-MI. At the histological level, Masson’s trichrome staining was performed to simultaneously explore infarcted and viable cardiac tissues (Fig. 2D).

NEXO-treated hearts exhibited decreased infarct size (Fig. 2E), increased infarcted wall thickness (Fig. 2F), and increased viable tissue (Fig. 2G) compared to FEXO and PBS controls. Likewise, injured hearts treated with NEXO displayed less chamber dilation (Figs. 13B and 13C) and smaller infarct circumference (Fig. 13D) when compared to control or FEXO treated hearts. These data indicated that exosomes from healthy hearts attenuated LV remodeling after Ml, while exosomes from failing hearts exacerbate it.

Effects of NEXO and FEXO treatment on post-MI heart angiomyogenesis and apoptosis: It has been well established that adult stem cells exert their therapeutic benefits though indirect regenerative mechanisms. The secretome from the injected cells enhances cardiomyocyte proliferation, promotes neovascularization, and inhibits cardiomyocyte apoptosis (Chimenti et ai, (2010) Circ. Res. 106: 971-980; Gnecchi et ai, (2005) Nat. Med 11 : 367). Three weeks after Ml and exosome injections, the percentage of cycling cardiomyocytes (Ki677a-SA⁺, Figs. 3A and 3D) in the peri-infarct zone of NEXO-treated hearts was significantly higher than in those that received FEXO or PBS control injections. This was further confirmed by mitosis marker PH3 and cytokinesis marker AURKB (Figs. 14A-14C). A similar trend was detected in capillary density (vWF⁺ capillaries, Figs. 3B and 3E), indicating the proangiogenic role of NEXO but not of FEXO. Hearts injected with NEXO displayed significantly fewer apoptotic cardiomyocytes (TUNEL7a-SA⁺, Figs. 3C and F), suggesting NEXO therapy led to tissue preservation. Overall, FEXO treatment is detrimental to post-MI healing, as it inhibits angiogenesis and cardiomyocyte proliferation.

Dysregulation of hsa-miR-21-5p (SEQ ID NO: 1) in heart failure exosomes: Several studies imply that the mechanism of exosome-mediated repair involves exosomal microRNAs, which target specific signaling pathways in the recipient cells (e.g. injured cardiomyocytes) (Ibrahim et ai., (2014) Stem Cell Report. 2: 606-619; Barile et ai, (2014) Cardiovascular Res. 103: 530-541). It was possible, therefore, that the functional impairment of FEXO might result from altered miRNA cargos.

The miRNA components of NEXO to that of FEXO were compared using PCR microarrays for 84 well-reported miRNAs (Figs. 15A and 15B). Twenty-three miRNAs were differentially expressed in the two groups of exosomes. Among those, hsa-miR-21-5p (SEQ ID NO: 1) was the most dysregulated in FEXO, as compared to the expression in NEXO (Figs. 4A and 4B). qRT-PCR was then performed to verify the results. hsa-miR-21-5p (SEQ ID NO: 1), but not hsa-miR-21-3p (SEQ ID NO: 9), was dysregulated in FEXO (Figs. 4C). The myocardial tissue levels of hsa-miR-21-5p (SEQ ID NO: 1) were decreased in the post-MI hearts injected with FEXO (Fig. 15C), as compared to the ones injected with NEXO.

The role of hsa-miR-21-5p (SEQ ID NO: 1) in exosome-mediated heart repair: Various gain- and loss-of-function studies were designed to verify the role of hsa-miR-21-5p (SEQ ID NO: 1) in exosome-mediated, post-MI repair. The silencing of exosomal hsa-miR-21-5p (SEQ ID NO: 1) by transfecting healthy cardiac cells with anti-miR-21-5p (SEQ ID NO: 1) oligo

(NEXO+anti-miR-21) was confirmed by qRT-PCR on the exosome products (Figs. 16A and 16C). Knocking down hsa-miR-21-5p (SEQ ID NO: 1) in NEXO led to impaired anti-apoptotic effects of NEXO on cultured H9C2 cells (Figs. 5A and B). In contrast to the control NEXO, NEXO+anti-miR-21 lost the ability to promote tube formation (Figs. 5C and D).

miR-21-rescued exosomes were also engineered by transfecting heart failure cardiac cells with hsa-miR-21 oligo. These cells were engineered to produce exosomes with hsa-miR-21-5p (SEQ ID NO: 1) over-expression (FEXO+miR-21) (Figs. 16A and 16B). As expected, upregulation of hsa-miR-21-5p (SEQ ID NO: 1) in FEXO rescued their ability to inhibit apoptosis (Figs. 5E and 5F) and promote tube formation (Figs. 5G and H). To further validate the functions of hsa-miR-21 in adult cardiomyocytes, adult human cardiomyocytes were transfected with hsa-miR-21-mimic or scrambled control. Over-expression of hsa-miR-21 led to improved survival of adult cardiomyocytes, as indicated by less apoptotic cells (TUNEL-positive nuclei) (Figs. 17A and 17B).

To confirm those results in vivo, the same mouse Ml model was used to test the therapeutic effects of FEXO+miR-21 and NEXO+anti-miR-21. Mice injected with

NEXO+anti-miR-21 exhibited decreased pump function (Figs. 6A and 6B), increased scar mass, and decreased viable heart tissue, compared to the hearts injected with NEXO+miR-scr (Figs. 6C-6G). In contrast, hsa-miR-21-5p (SEQ ID NO: 1) enhancement rescued the therapeutic functions of heart failure exosomes. FEXO+miR-21 treatment led to improvement in cardiac function (Figs. 6A and 6B) and attenuation of LV remodeling (Figs. 6D-6G).

Histological analysis showed that hsa-miR-21-5p (SEQ ID NO: 1) inhibition abolished the ability of NEXO therapy to promote angiogenesis (Figs. 7 A and 7B) and inhibit apoptosis

(Figs. 7C and 7D). Vice versa, restoring hsa-miR-21-5p (SEQ ID NO: 1) expression in FEXO rescued those paracrine effects (Figs. 7E-7H). Moreover, the pro-angiogenic effects of hsa-miR-21-5p (SEQ ID NO: 1) was confirmed by the increased number of functional vessels.

The functionality of a blood vessel was confirmed by co-staining the endothelial cell marker, Von Willebrand factor (vWF), and the red blood cell (RBC) marker. As shown in Fig. 18, RBCs were detected in the blood vessels in the Ml border zone of the hearts treated with hsa-miR-21-over-expressed FEXO. Through those gain- and loss-of-function studies, the pivotal role of hsa-miR-21-5p (SEQ ID NO: 1) in exosome-mediated heart repair was shown, possibly through pro-angiogenic and anti-apoptotic mechanisms.

Alongside with hsa-miR-21 such as, but not limited to, let-7b-5p (SEQ ID NO: 2), miR125a-5p (SEQ ID NO: 3), and hsa-miR-146a-5p (SEQ ID NO: 4) were examined for their effects on cardiomyocytes, cardiac fibroblasts, and endothelial cells. The results indicated that hsa-miR-146a inhibited apoptosis in cardiomyocyte but promoted tube formation in endothelial cells (Fig. 19). This was consistent with previous studies (Ibrahim et ai, (2014) Stem Cell Report. 2: 606-619). In contrast, neither let-7b-5p (SEQ ID NO: 2) nor

hsa-miR-125a-5p (SEQ ID NO: 3) showed beneficial effects on cardiomyocyte survival or endothelial tube formation (Figs. 19A-19G).

Effects of hsa-miR-21-5p (SEQ ID NO: 1) on PTEN/Akt signaling: It has been established that hsa-miR-21-5p (SEQ ID NO: 1) promotes cancer cell proliferation, angiogenesis, migration, and invasion by targeting pathways involving phosphatase and tensin homolog (PTEN), Programmed Cell Death 4 (PDCD4), Forkhead Box 01 (FOX01), SMAD Family Member 7 (SMAD7), Tumor necrosis factor-a induced protein-8-like 2 (TIPE2), Sprouty2 (SPRY2) (Melnik, B.C. (2015) J. Translational Med. 13: 202). Recent studies have also shown that PTEN and SPRY1 are targeted by hsa-miR-21-5p (SEQ ID NO: 1) in cardiovascular diseases (Thum et al. (2008) Nature 456: 980-984; Wang et ai, (2017) Stem Cells Translational Med. 6: 209-222). To identify the potential target genes of hsa-miR-21 on three major cell types in the heart, cardiomyocytes (using human induced-pluripotent stem cell-derived cardiomyocytes (iCM) and H9C2 cells), cardiac fibroblasts (using human cardiac fibroblasts, CF), and endothelial cells (using human umbilical vein endothelial cells, HUVECs) were incubated with hsa-miR-21 mimic or scrambled control. Eighteen phosphorylated proteins were then examined with a signaling pathway phosphorylation array. The signaling pathway array pointed at PTEN, AKT, and BAD as the hsa-miR-21-regulated genes in cardiomyocytes and endothelial cells, but not in cardiac fibroblasts (Figs. 20A-20C).

Western blot studies further confirmed that PTEN expression was significantly attenuated in human cardiomyocytes (CMs) and HUVECs treated with hsa-miR-21 mimics (Figs. 8A and 8B). This coincides with enhanced Akt phosphorylation, as well as the alteration of other downstream targets such as caspase-3 and Bcl-2 (Figs. 8A and 8C-8E). Upregulation of hsa-miR-21 in cardiomyocytes and HUVECs also promoted PCNA (a proliferation marker) and vascular endothelial growth factor (VEGF) expressions (Figs. 8F-8H), which are consistent with the data in Figs. 5A-5H showing the pro-angiogenic effects of hsa-miR-21.

Previous studies indicated that programmed cell death 4 (PDCD4), a pro-apoptotic protein, is a known hsa-miR-21 target (Khan et ai, (2015) Circulation Res. 115. 305990), so PCDC4 expressions was checked in CMs, HCFs, and HUVECs treated with hsa-miR-21 mimics. Western blot analysis showed reduced PDCD4 expression in CMs only, but not in HCFs or HUVECs (Figs. 8F and 8I).

Taken together, the data indicate that hsa-miR-21 inhibits cardiomyocyte apoptosis by targeting PDCD4, promotes angiogenesis by activating PTEN/Akt signaling, and promotes the expression of VEGF in endothelial cells. Since the flow cytometry showed a significant proportion of mesenchymal cells or fibroblasts (CD90+, CD105+) in the cardiac cells (Figs. 10A and 10B), it is possible that those cardiac cells secreted exosomes containing hsa-miR-21 to communicate with nearby cardiomyocytes and endothelial cells under physiological and heart failure conditions.

The results show that exosomes derived from heart-failure patients have impaired therapeutic potency in an animal model of myocardial infarction and are incapable of promoting angiomyogenesis in vitro and in vivo. MicroRNA array and a series of gain- and loss-of-function experiments revealed that the impairment of regenerative activities of heart failure exosomes is related to dysregulation of hsa-miR-21-5p (SEQ ID NO: 1) (Fig. 8J).

Cell therapy has emerged as a promising therapeutic option for cardiac regeneration and protection. As a major paracrine component, exosomes have been revealed to play a vital role in tissue repair by packaging and delivering RNAs and proteins. It has been reported that injured mouse hearts treated with cardiac stem cell-secreted exosomes exhibited regenerative and functional improvements produced by the stem cells themselves (Gallet et ai, (2016) Euro. Heart J. 38: 201-211 ; Ibrahim et ai, (2014) Stem Cell Report. 2: 606-619). Exosomes derived from mouse embryonic stem cells promote neovascularization and cardiomyocyte survival, and reduce fibrosis after myocardial infarction, consistent with the resurgence of the cardiac proliferative response produced by embryonic stem cells (Kervadec et ai, (2016) J. Heart Lung Transplantation 35: 795-807). However, most of these exosome products are derived from young and healthy donors. Since most patients with cardiac diseases, especially heart failure, suffer from systemic inflammation and are of old age, it is possible that heart failure modifies exosome contents and further compromises their reparative properties. However, now it is found that exosomes derived from the cardiac cells of heart-failure patients lose their pro-angiogenic drive of HUVEC tube formations and fail to promote cardiomyocyte proliferation in injured mouse hearts, suggesting a dysfunction of heart failure exosomes in ischemic tissue repair. The finding can partly explain the compromised therapeutic effects in clinical trials using autologous stem cells. The advanced cardiomyopathy condition modifies exosome contents and renders the stem cells ineffective in therapeutic repair.

The mechanisms that drive exosome-mediated repair rely on the transfer of specific donor cell microRNAs and proteins to recipient cells (Dhanabal et ai., (2005) Cancer Biol. Therapy 4: 659-668). MicroRNA array analysis was performed to compare exosomes secreted from heart-failure patients with those from healthy donors. Exosomal hsa-miR-21-5p (SEQ ID NO: 1), secreted from healthy cardiac cells, can be delivered to recipient cardiac cells to regulate apoptosis and angiogenesis, and improve heart function in a mouse Ml model. The silencing of hsa-miR-21-5p (SEQ ID NO: 1) in normal exosomes (NEXO) eliminated these benefits, and the restoration of hsa-miR-21-5p (SEQ ID NO: 1) in heart failure exosomes (FEXO) rescued their therapeutic potency, further supporting the essential role of hsa-miR-21-5p (SEQ ID NO: 1) in exosome-mediated heart repair.

Although the studies focused on hsa-miR-21-5p (SEQ ID NO: 1), there are several other miRNAs dysregulated in FEXO, such as hsa-miR-146a, let-7b, hsa-miR-125a, and hsa-miR-23. Exosomal hsa-miR-146a leads to beneficial effects in post Ml hearts by targeting Irak-1 and Traf6, both associated with the toll-like receptor signaling pathway (Ibrahim et ai, (2014) Stem Cell Report. 2: 606-619). It has also been shown that hsa-miR-23 can promote angiogenesis in cardiac endothelial cells by activating proangiogenic signaling through the inhibition of Sprouty2 and Sema6A (Impagnatiello et ai, (2001) J. Cell Biol. 152: 1087-1098). The results in the present disclosure indicate that hsa-miR-146a inhibited apoptosis in cardiomyocytes but promoted tube formation in endothelial cells, which is consistent with previous studies, while neither let-7b-5p (SEQ ID NO: 2) nor hsa-miR-125a-5p (SEQ ID NO: 3) showed beneficial effects on cardiomyocyte survival or endothelial tube formation.

miR-21 expression has been associated with various disorders (Thum et ai (2008) Nature 456: 980-984, Thum et ai., (2008) Nature 456 980; Bang et ai., (2014) J. Clinical Invest. 124: 2136-2146; Patrick et at., (2010) J. Clinical Invest. 120: 3912-3916.). However, the precise functional role of hsa-miR-21 in cardiac diseases is still controversial. Thomas et al. reported that hsa-miR-21 expression is highly upregulated by cardiac stress, leading to activation of ERK/MAP kinase signaling through inhibition of Spryl , which in turn promoted cardiac fibrosis and heart dysfunction (Thum et al. (2008) Nature 456: 980-984). hsa-miR-21 also reportedly induced cardiac fibrosis by inhibiting PTEN expression in cardiac fibroblasts at the infarct zone after myocardial ischemia-reperfusion (Roy et ai., (2009) Cardiovascular Res. 82: 21-29). However, Patrick et al. reported that miR21 was not essential for cardiac fibrosis, as hsa-miR-21-null mice still display cardiac hypertrophy, fibrosis, and the loss of cardiac contractility (Patrick et ai., (2010) J. Clinical Invest. 120: 3912-3916).

In the present study, hsa-miR-21 did not affect the survival of cardiac fibroblasts (Fig. 19G). Moreover, hsa-miR-21 regulated the PTEN/Akt signaling pathway in cardiomyocytes and endothelial cells instead of fibroblasts (Figs. 8A-8J), indicating that hsa-miR-21 does not promote cardiac fibrosis, as reported in previous studies.

Exosomal hsa-miR-21 , secreted by induced pluripotent stem cells (iPSCs), promoted angiogenesis in transfected bronchial epithelial cells (Xu et al., (2015) Arch. Toxicol. 89:

1071-1082). Kristin et ai. recently reported that hsa-miR-21a-5p is the most abundant miRNA in murine mesenchymal stem cell (MSC) exosomes, and that it contributes to myocardial salvage via synergistic anti-apoptotic activity. hsa-miR-21-5p (SEQ ID NO: 1) plays a crucial role in the MSC-exosome-mediated effects on cardiac contractility through PI3K signaling (Mayourian et ai., (2018) Circulation Res. 122: 933-944).

A unique aspect of the methods and compositions of the present disclosure is the cell source. While therapeutic MSCs can be readily derived from bone marrow and adipose tissues, these cells do not naturally reside in the heart. Focusing on intrinsic cardiac cells in failing or normal hearts demonstrated that heart failure pathology leads to a severe dysregulation of hsa-miR-21-5p (SEQ ID NO: 1) in intrinsic cardiac cells, which further leads to the impaired regenerative function of the intrinsic heart exosomes. Therefore, the present findings demonstrate that hsa-miR-21 not only plays a key role in therapeutic interventions (e.g. in the case of MSCs), but also represents a crucial component in the naturally-occurring repair process post heart injury.

The regulation of PTEN by hsa-miR-21 was reported in carcinoma (Meng et al., (2007) Gastroenterology 133: 647-658) and cardiovascular cells (Roy et al., (2009) Cardiovascular Res. 82: 21-29). Consistent with this, upregulation of hsa-miR-21-5p (SEQ ID NO: 1) in exosomes augmented phosphorylation of Akt via the inhibition of PTEN in the recipient cells. These effects were eliminated by the knockdown of hsa-miR-21-5p (SEQ ID NO: 1), indicating a possible molecular mechanism for the positive role of hsa-miR-21 -5p (SEQ ID NO: 1) in post-MI repair. Based on the flow cytometry data, cardiac stromal cells/fibroblasts might be the main source of exosomes with altered hsa-miR-21.

It has been shown that strong hsa-miR-21 expression occurs primarily in cardiac fibroblasts but was low in cardiomyocytes. This is consistent with the present findings. There is every probability that cardiac fibroblasts released exosomal hsa-miR-21 to support survival of neighboring cardiomyocytes and endothelial cells under physiological conditions.

These findings have important clinical implications. Unlike cell-based therapy products, exosomes can offer an off-the-shelf and universal therapeutic option. The cryo-preservation of exosomes normally does not require the sophisticated steps associated with cellular products. In addition, injection of allogeneic or even xenogeneic exosomes is well tolerated by the host immune system (Vandergriff et al., (2018) Theranostics 8: 1869-1878; Vandergriff et al., (2015) Stem Cells Int. 2015: 960926). The present findings indicate that exosomes from heart-failure patients carry significantly altered miRNA signatures compared to those from healthy heart cells and are functionally deficient in their ischemic myocardial repair capabilities. This may explain why patients’ autologous cell therapies only show modest therapeutic effects in clinical trials. More importantly, the present data showed that the dysfunction of heart failure exosomes can be rescued by modulation of a specific miRNA in the exosomes. The study provides new mechanistic insights into the therapeutic potential of exosomes and new strategies to rescue patients’ defective exosomes through modulation of specific miRNA cargos.

In some embodiments of this aspect of the disclosure, the composition can further comprise a pharmaceutically acceptable carrier. In some embodiments of this aspect of the disclosure, the composition can be formulated for delivery to a site of cardiac injury.

In some embodiments of this aspect of the disclosure, the population of cardiac stromal cell/fibroblast-derived exosomes further comprises a plurality of miRNA species expressed in at least one of cardiac stromal cells or fibroblasts derived from a normal heart tissue.

Another aspect of the disclosure encompasses embodiments of a method of modulating the activity of phosphatase and tensin homolog (PTEN) in the cardiac tissue of an animal or human subject, the method comprising administering to a cardiac tissue of an animal or human subject a population of cardiac stromal cell/fibroblast-derived exosomes and a pharmaceutically acceptable carrier, wherein the population of cardiac stromal

cell/fibroblast-derived exosomes can comprise an miRNA species having at least 90% sequence similarity to cardiac stromal cell/fibroblast-derived miRNA species miRNA-21-5p (SEQ ID NO: 1) that when delivered to recipient cardiac tissue of the animal or human subject reduces the activity of PTEN in the recipient cells thereof.

In some embodiments of this aspect of the disclosure, each miRNA species of the plurality of miRNA species can be selected from and can have at least 90% sequence similarity to one of the group consisting of hsa-let-7b-5p (SEQ ID NO: 2), hsa-miR-125a-5p (SEQ ID NO: 3), hsa-miR146a-5p (SEQ ID NO: 4), hsa-miR-125b-5p (SEQ ID NO: 5), hsa-miR-126-3p (SEQ ID NO: 6), hsa-miR-16-1-3p (SEQ ID NO: 7), hsa-miR-23a-5p (SEQ ID NO: 8), hsa-miR-21-3p (SEQ ID NO: 9), hsa-miR-26a-5p (SEQ ID NO: 10), hsa-miR-320a (SEQ ID NO: 11), hsa-miR-29a-3p (SEQ ID NO: 12), hsa-miR-16-5p (SEQ ID NO: 13), hsa-miR-23a-3p (SEQ ID NO: 14), and hsa-miR-16-5p (SEQ ID NO: 15).

As mentioned above, compounds of the present disclosure and pharmaceutical compositions can be used in combination of one or more other therapeutic agents for treating viral infection and other diseases. For example, compounds of the present disclosure and pharmaceutical compositions provided herein can be employed in combination with other anti-viral agents to treat viral infection.

While embodiments of the present disclosure are described in connection with the Examples and the corresponding text and figures, there is no intent to limit the disclosure to the embodiments in these descriptions. On the contrary, the intent is to cover all alternatives, modifications, and equivalents included within the spirit and scope of embodiments of the present disclosure.

EXAMPLES

Example 1

Cell Culture and Exosome Isolation: Diseased heart tissues were received from patients who underwent heart transplantation at UNO Hospital after heart failure. Healthy heart tissues were received from normal, non-damaged, human donor hearts. Cardiac cells were separated and cultured as previously described (Makkar et ai, (2012) Lancet 379: 895-904). In brief, myocardial tissues harvested from donors’ hearts were separated and washed with PBS, then cut into smaller 2 mm³ fragments, and partially enzymatically digested at 37°C with collagenase type IV (C1889, Sigma-Aldrich). The tissue samples were plated onto fibronectin-coated petri dishes in Iscove’s modified Dulbecco’s medium (IMDM, Gibco) infused with 20% fetal bovine serum (FBS; Corning). After 3-5 days, cardiac cells began to grow from the explants. Exosomes were isolated from the conditioned medium of these cardiac cells. Passage 1-3 cardiac cells were cultured to 80% confluency. The medium was then switched to serum-free IMDM and conditioned for 14 days. Then, exosomes were isolated from the conditioned medium by ultrafiltration, as previously described (Vandergriff et al., (2018) Theranostics 8: 1869-1878; Vandergriff et al., (2015) Stem Cells Int. 2015: 960926). In brief, conditioned medium was filtered through 0.22 pm Steriflip filters to remove cellular debris and large vesicles. The filtrate was then added to Amicon Ultra-15 100 kDa filters (SCGP00525, Millipore) to centrifuge at 5,000 ^c g for 5min. The flow-through was discarded. The concentrated exosomes were collected and washed with PBS three times, and stored at - 80 °C. Labeling was performed using 10 pM Dil (V22889, Thermo Fisher Scientific)

Example 2

Flow Cytometry: Flow cytometry was performed to examine the antigenic phenotypes of cardiac cells. Cells were incubated with antibodies against CD90 (555595, BD), CD105 (ab11414, Abeam), CD31 (555445, BD), CD 34 (ab81289, Abeam), CD 45 (555482, BD), and c-kit (550412, BD) for 60 mins at 4 °C. Both unstained and isotype controls (555748, 559320, BD) were used as negative controls. Flow cytometry was conducted with a CytoFlex Flow Cytometer (Beckman Coulter) and data were analyzed with FCS Express software (De Novo). Example 3

Exosome Characterization : Nanoparticle tracking analysis (NanoSight NS300, Malvern) was used to measure the concentration and size of exosomes. Each sample was imaged five times for 60 secs and analyzed. Transmission electron microscopy was used to assess the morphology of exosomes. In preparation for TEM, exosomes were fixed with 4% PFA and 1 % glutaraldehyde. They were stored at room temperature. Microscopy was conducted with a transmission electron microscope (JEM-2000FX, JEOL).

Example 4

Immunocytochemistry: Neonatal rat cardiomyocytes (NRCMs) and cardiac fibroblasts were isolated and cultured as previously described (Vandergriff et al., (2015) J. Visualized Expts 98: 52726). The H9C2 cell line was purchased from Sigma-Aldrich (88092904, Sigma-Aldrich). Adult human cardiomyocytes were purchased from Celprogen and cultured on human cardiomyocyte cell culture extracellular matrix (36044-15, Celprogen). Human induced pluripotent stem cell-derived cardiomyocytes (iCM) were kindly provided by Dr. Freytes (UNC/NC State Joint Department of Biomedical Engineering, Chapel Hill/Raleigh). After 3-days of cultivation, the culture medium was supplemented with 7 ^c 10⁸ exosomes or PBS for an additional 24 hours. They were fixed with 4% paraformaldehyde (PFA) and blocked with Protein Block Solution (DAKO) with 1 % saponin (Sigma-Aldrich). Subsequently, the cells were stained with anti-a-sarcomeric actin (a-SA, a7811 , Sigma-Aldrich), anti-ki67 (ab15580, Abeam), anti-a-smooth muscle actin (ab5694, Abeam), or anti-von Willebrand factor (vWF, ab6994, Abeam) antibodies. Flour 488 or Texas-Red conjugated secondary antibodies (ab150117, ab6787, ab6719, ab150077, Abeam) were used for detection. Sections treated with terminal deoxynucleotidyl transferase nick end labeling (TUNEL, 12156792910, Roche) were incubated for 30 minutes after the secondary antibody incubation. Cell nuclei were counter-stained with 4’,6-diamidino-2-phenylindole (DAPI). Images were taken by a fluorescent microscope (Olympus 1X81 ; Olympus).

Example 5

Angiogenesis Assay: Human umbilical vein endothelial cells (HUVECs) were co-incubated with 1.5 x 10⁸ exosomes for 24 hours, then plated on growth factor-deprived Matrigel (356230, Corning) to evaluate angiogenesis (Manoussaki et al., (1996) Acta Biotheoretica 44: 271-282). Eight hours later, tube formation was examined with a white light microscope and analyzed with NIH Image J software.

Example 6

miR-21-5p (SEQ ID NO: 1) transfection: hsa-miR-21-5p (SEQ ID NO: 1) oligo, anti-miR-21-5p (SEQ ID NO: 1) or scrambled miRNA oligo transfection was performed according to the XMIR exosome RNA packaging protocol (XMIR-21 , AXMIR-21 , XMIR-POS, SBI System

Biosciences) with slight modifications. Briefly, cardiac cells were cultured to 70% confluency. hsa-miR-21-5p (SEQ ID NO: 1), anti-miR-21-5p (SEQ ID NO: 1), or scrambled miRNA oligo was mixed with a transfection reagent, then added to the cell culture at a final concentration of 20 nM. Cells were returned to the incubator for exosome production for 24 hours. Culture media was collected, and exosomes were isolated as previously described. Transfection efficiency was determined by performing qRT-PCR on exosomes derived from the transfected cells. Hs_miR-21_2 miScript Primer Assay (MS00009079, Qiagen) was performed according to the manufacturer’s protocol. qRT-PCR was performed using Roche Light Cycler 480 Instrument II (Roche) and relative exosome abundance levels were calculated using the delta-delta Ct method. hsa-miR-16 (MS00031493, Qiagen) was used as a reference control. Example 7

Animal Studies: The acute myocardial infarction (Ml) model was created as previously described (Luo et al., (2017) Circ. Res. 120: 1768-1775; Tang et al., (2018) Nat. Biomed. Engineer. 2: 17-26; Tang et al., (2017) ACS Nano 11 : 9738-9749; Tang et al., (2017) Nat. Commun. 8: 13724). Sixty 6-week-old female CD-1 mice (Charles River Labs, Wilmington, MA) underwent left thoracotomy under general anesthesia. The left anterior descending (LAD) coronary artery was permanently ligated. Subsequently, intramyocardial injections were performed at four sites in the peri-infarct zone. All mice were randomly assigned to the following groups. (1) NEXO: intramyocardial injection of 50 pi PBS containing 30 x 10⁹ NEXO; (2) FEXO: injection of 50 mI PBS containing 30 x 10⁹ FEXO; (3) control: injection of 50 mI PBS; and (4) Sham: mice with sham surgery underwent the same procedures except for the permanent ligation. To validate the role of hsa-miR-21-5p (SEQ ID NO: 1) in exosome-mediated heart repair, we created the same Ml models in mice. hsa-miR-21-5p (SEQ ID NO: 1)-deficient exosomes were engineered by transfecting healthy cardiac cells with anti-miR-21-5p (SEQ ID NO: 1) oligo (NEXO+anti-miR-21). We also produced hsa-miR-21 -rescued exosomes by transfecting heart failure cardiac cells with hsa-miR-21 RNA oligo (FEXO+miR-21) to harvest exosomes with hsa-miR-21-5p (SEQ ID NO: 1) over-expression. Scrambled miRNA oligo was used as a control (NEXO/FEXO+miR-scr): (1) NEXO+anti-miR-21 : injection of 50mI PBS containing 30 x 10⁹ NEXO+anti-miR-21 ; (2) NEXO+miR-scr: injection of 50mI PBS containing 30 x 10⁹ NEXO+miR-scr; (3) FEXO+miR-21 : injection of 50mI PBS containing 30 x 10⁹ FEXO+miR-21 ; (4) FEXO+miR-scr: injection of 50mI PBS containing 30 x 10⁹ FEXO+miR-scr.

Example 8

Heart Function Assessment: Cardiac function was measured by blinded echocardiography analysis using a Philips Cx-70 Ultrasound System with an L15-7io high-frequency probe. Each measurement was performed 3 times. Long-axis views were measured at the greatest left ventricular diameter. Left ventricular ejection fraction was measured from views taken through the infarcted area.

Example 9

Heart Morphometry: Animals were sacrificed 3 weeks after treatments. Six cryo-sections from each heart, collected at 400-pm intervals, were stained with Masson’s trichrome. Infarct size, infarct circumference, infarct wall thickness, and viable tissue in the risk area were measured with NIH Image J software, as previously described (Luo et ai, (2017) Circ. Res. 120:

1768-1775; Tang et ai, (2018) Nat. Biomed. Engineer 2: 17-26; Tang et ai., (2017) ACS Nano 11 : 9738-9749; Tang et ai., (2017) Nat. Commun. 8: 13724).

Example 10

Antibody Array and Western Blot Analysis: Adult human cardiomyocytes (CM, 36044-15, Celprogen), human induced pluripotent stem cell-derived cardiomyocytes (iCM), H9C2 cells (88092904, Sigma), human cardiac fibroblasts (HCF, 306-05A, Sigma), and human umbilical vein endothelial cells (HUVECs, PCS-100-010, ATCC) were transfected with hsa-miR-21-5p (SEQ ID NO: 1) mimic or scrambled control (miScript miRNA Mimic, 219600, Qiagen), according to the manufacturer’s protocol with slight modifications. Briefly, cells were cultured to 70% confluency. hsa-miR-21-5p (SEQ ID NO: 1) mimic or scrambled control was mixed with HiPerFect Transfection Reagent and culture medium without serum, at a final concentration of 5 nM. Cells were returned to the incubator for 48 hours. Cells were collected, lysed by RIPA Lysis Buffer (89900, Thermo Fisher Scientific), and centrifuged at 14,000 ^c g for 15 minutes to pellet the cell debris. The protein concentration was quantified with a BCA Protein Assay (23227, Thermo Fisher Scientific).

Protein from transfected iCM, H9C2, HCF, and HUVEC cells was used on the signaling pathway phosphorylation array (AAH-AKT-1-2, RayBiotech). The protein array assay was performed according to the manufacturer’s protocol with slight modification. Briefly, the antibody membranes were blocked for 30 minutes at RT. The samples were then added and incubated overnight at 4°C. The detection antibody cocktail was prepared, pipetted onto the membranes, and incubated overnight at 4°C. Then, the samples were incubated with HRP-anti-rabbit IgG and visualized with the ChemiDoc Touch Imaging System (ChemiDoc, Bio-Rad). Quantitative analysis was performed with ImageJ or Image Lab software.

Protein from transfected CM, H9C2, HCF and HUVEC cells was used on western blot analysis. The equivalent of 25 mg of total protein per lane was loaded onto Mini-PROTEAN TGX Stain-Free Protein Gels (4568083, Bio-RAD) and transferred to Immun-Blot^® PVDF

Membranes (1620177, Bio-RAD). Membranes were blocked with 5% blocking buffer for 1 hour and incubated with primary antibodies against PTEN (ab31392, Abeam), p-akt (phospho T308) (ab38449, Abeam), t-akt (ab8805, Abeam), BCL-2 (ab59348, Abeam), caspase-3 (ab13847, Abeam), PDCD4 (ab51495, Abeam), PCNA (ab29, Abeam), or VEGF (PA5-16754,

Thermo Fisher Scientific) overnight, at 4°C on a rocker. Subsequently, the samples were incubated with appropriate horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 hour on a shaker. The blots were exposed with Clarity Western ECL Blotting Substrates (1705061 , Bio-Rad) and visualized with the ChemiDoc Touch Imaging System (ChemiDoc, Bio-Rad). Quantitative analysis was performed with ImageJ or Image Lab software, and expressions were normalized to GAPDH.

Example 11

Statistics: GraphPad Prism (GraphPad Software, La Jolla, CA) was used for statistical analysis. Results were presented as mean ± SD. All comparisons between two groups were performed with two-tailed unpaired Student’s t-test. One-way ANOVA analysis, with post hoc Bonferroni correction, was used to compare means among more than two groups.

Differences were considered statistically significant when p < 0.05.

Example 12

Table 2

amiRBase accession number

Claims

CLAIMS What is claimed:

1. A composition comprising a population of cardiac stromal cell/fibroblast-derived exosomes, wherein the exosomes comprise an miRNA species having at least 90% sequence similarity to cardiac stromal cell/fibroblast-derived miRNA species miRNA-21-5p (SEQ ID NO: 1) or a heterologous nucleic acid that expresses the miRNA species when in recipient cardiac stromal cells or fibroblasts cells.

2. The composition of claim 1 , wherein the composition further comprises a pharmaceutically acceptable carrier.

3. The composition of claim 2, wherein the composition is formulated for delivery to a site of cardiac injury.

4. The composition of claim 1 , wherein the population of cardiac stromal

cell/fibroblast-derived exosomes further comprises a plurality of miRNA species expressed in at least one of cardiac stromal cells or fibroblasts derived from a normal heart tissue.

5. The composition of claim 4, wherein at least one of the plurality of miRNA species is over-expressed in cardiac stromal cells or fibroblasts cells derived from a normal heart tissue compared to a heart tissue having a pathological injury.

6. The composition of claim 4, wherein each miRNA species of the plurality of miRNA species is selected from and has at least 90% sequence similarity to one of the group consisting of hsa-let-7b-5p (SEQ ID NO: 2), hsa-miR-125a-5p (SEQ ID NO: 3), hsa-miR146a-5p (SEQ ID NO: 4), hsa-miR-125b-5p (SEQ ID NO: 5), hsa-miR-126-3p (SEQ ID NO: 6), hsa-miR-16-1-3p (SEQ ID NO: 7), hsa-miR-23a-5p (SEQ ID NO: 8), hsa-miR-21-3p (SEQ ID NO: 9), hsa-miR-26a-5p (SEQ ID NO: 10), hsa-miR-320a (SEQ ID NO: 11), hsa-miR-29a-3p (SEQ ID NO: 12), hsa-miR-16-5p (SEQ ID NO: 13), hsa-miR-23a-3p (SEQ ID NO: 14), and hsa-miR-16-5p (SEQ ID NO: 15).

7. A method of modulating the activity of phosphatase and tensin homolog (PTEN) in the cardiac tissue of an animal or human subject, the method comprising administering to a cardiac tissue of an animal or human subject a population of cardiac stromal

cell/fibroblast-derived exosomes and a pharmaceutically acceptable carrier, wherein the population of cardiac stromal cell/fibroblast-derived exosomes comprise an miRNA species having at least 90% sequence similarity to cardiac stromal cell/fibroblast-derived miRNA species miRNA-21-5p (SEQ ID NO: 1) that when delivered to recipient cardiac tissue of the animal or human subject reduces the activity of PTEN in the recipient cells thereof.

8. The method of claim 7, wherein the population of cardiac stromal cell/fibroblast-derived exosomes further comprises a plurality of miRNA species.

9. The method of claim 7, wherein the population of cardiac stromal cell/fibroblast-derived exosomes further comprises a plurality of miRNA species expressed in at least one of cardiac stromal cells or fibroblasts derived from a normal heart tissue.

10. The method of claim 9, wherein at least one of the plurality of miRNA species is over-expressed in cardiac stromal cells or fibroblasts cells derived from a normal heart tissue compared to a heart tissue having a pathological injury.

11. The method of claim 9, wherein each miRNA species of the plurality of miRNA species is selected from and has at least 90% sequence similarity to one of the group consisting of hsa-let-7b-5p (SEQ ID NO: 2), hsa-miR-125a-5p (SEQ ID NO: 3), hsa-miR146a-5p (SEQ ID NO: 4), hsa-miR-125b-5p (SEQ ID NO: 5), hsa-miR-126-3p (SEQ ID NO: 6), hsa-miR-16-1-3p (SEQ ID NO: 7), hsa-miR-23a-5p (SEQ ID NO: 8), hsa-miR-21-3p (SEQ ID NO: 9), hsa-miR-26a-5p (SEQ ID NO: 10), hsa-miR-320a (SEQ ID NO: 11), hsa-miR-29a-3p (SEQ ID NO: 12), hsa-miR-16-5p (SEQ ID NO: 13), hsa-miR-23a-3p (SEQ ID NO: 14), and hsa-miR-16-5p (SEQ ID NO: 15).

12. The method of claim 7, wherein the population of cardiac stromal cell/fibroblast-derived exosomes further comprises at least one nucleic acid species not derived from cardiac stromal cells or fibroblasts cells.

13. The method of claim 8, wherein the population of cardiac stromal cell/fibroblast-derived exosomes further comprises a heterologous nucleic acid that expresses at least one miRNA species when in recipient cardiac tissue.

14. The method of claim 7, wherein the composition is administered to the cardiac tissue of the animal or human subject by a percutaneous method.

15. The method of claim 14, wherein the percutaneous method is by injection into the cardiac tissue.

16. A method of repairing cardiac tissue damage, the method comprising administering to a cardiac tissue of an animal or human subject a therapeutic composition comprising a population of cardiac stromal cell/fibroblast-derived exosomes and a pharmaceutically acceptable carrier, , wherein the population of cardiac stromal cell/fibroblast-derived exosomes comprise an miRNA species having at least 90% sequence similarity to cardiac stromal cell/fibroblast-derived miRNA species miRNA-21-5p (SEQ ID NO: 1) that when delivered to recipient cardiac tissue of the animal or human subject reduces the activity of PTEN in the recipient cells thereof.

17. The method of claim 16, wherein the population of cardiac stromal cell/fibroblast-derived exosomes further comprises a plurality of miRNA species.

18. The method of claim 16, wherein the population of cardiac stromal cell/fibroblast-derived exosomes further comprises a plurality of miRNA species expressed in cardiac stromal cells or fibroblasts cells derived from a normal heart tissue.

19. The method of claim 17, wherein at least some of the plurality of miRNA species are over-expressed in cardiac stromal cells or fibroblasts cells derived from a normal heart tissue compared to a heart tissue having a pathological injury.

20. The method of claim 17, wherein each miRNA species of the plurality of miRNA species is selected from and has at least 90% sequence similarity to one of the group consisting of hsa-let-7b-5p (SEQ ID NO: 2), hsa-miR-125a-5p (SEQ ID NO: 3), hsa-miR146a-5p (SEQ ID NO: 4), hsa-miR-125b-5p (SEQ ID NO: 5), hsa-miR-126-3p (SEQ ID NO: 6), hsa-miR-16-1-3p (SEQ ID NO: 7), hsa-miR-23a-5p (SEQ ID NO: 8), hsa-miR-21-3p (SEQ ID NO: 9), hsa-miR-26a-5p (SEQ ID NO: 10), hsa-miR-320a (SEQ ID NO: 1 1), hsa-miR-29a-3p (SEQ I D NO: 12), hsa-miR-16-5p (SEQ ID NO: 13), hsa-miR-23a-3p (SEQ ID NO: 14), and hsa-miR-16-5p (SEQ ID NO: 15).

21. The method of claim 17, wherein the population of cardiac stromal cell/fibroblast-derived exosomes further comprises at least one nucleic acid species not derived from cardiac stromal cells or fibroblasts cells.

22. The method of claim 17, wherein the population of cardiac stromal cell/fibroblast-derived exosomes further comprises a heterologous nucleic acid that expresses at least one miRNA species when in recipient cardiac tissue.

23. The method of claim 17, wherein the therapeutic composition is administered to the cardiac tissue of the animal or human subject by a percutaneous method.

24. The method of claim 17, wherein the percutaneous method is by injection into the cardiac tissue.