Cardiosphere-derived cells and exosomes secreted by such cells in the treatment of muscular dystrophy

Abstract

To provide a composition used for a method for treating heart failure secondary to muscular dystrophy.SOLUTION: Compositions and techniques relating to the production and therapeutic applications of cardiosphere-derived cells (CDCs) and CDC-derived exosomes are provided. These cells and their secreted vesicles contain a unique milieu of biological factors, including cytokines, growth factors, transcription factors, nucleic acids (including non-coding nucleic acids such as microRNAs), that serve to initiate and promote numerous therapeutic effects. Exosomes and their "cargo" contents, such as microRNAs, may advantageously modulate apoptosis, inflammation and fibrosis in the injured heart. Thus, CDC-derived exosomes are novel "cell-free" therapeutic candidates for tissue repair.SELECTED DRAWING: None

Description

[Technical Field]

[0001] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH This invention was made with government support under R01 HL083109 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0002] FIELD OF THE INVENTION The present invention relates to the use of cells and their extracts, particularly cellular exosomes, for therapeutic uses, including the treatment of cardiac disease. [Background technology]

[0003] Background: Duchenne muscular dystrophy (DMD) affects approximately 20,000 boys and young adults in the United States. The primary cause is a genetic abnormality in the dystrophin complex, with secondary damage to skeletal muscle and cardiac tissue. While virtually all patients are treated with corticosteroids, no treatment has proven effective. Heart failure (HF) secondary to DMD affects virtually all DMD patients over the age of 15 and is frequently the cause of death. DMD-related HF progresses aggressively from early onset (genetic abnormalities in the dystrophin complex) to asymptomatic abnormalities in cardiac structure and function (Stage B), overt symptomatic HF (Stage C), progressive HF (Stage D), and death. HF progression is associated with a high risk of hospitalization and a depletion of overall healthcare resources. Mortality during the course of DMD-related HF includes sudden cardiac death (which increases as HF worsens) or progressive HF leading to circulatory collapse. Furthermore, much of the disability in later stages of DMD is due to HF rather than skeletal muscle disease. Therefore, DMD HF represents an important unmet target for innovative treatments.

[0004] A highly promising avenue for the treatment of cardiac-related diseases and conditions includes cardiosphere-derived cells (CDCs), which can stimulate regeneration, angiogenesis, and functional improvement in infarcted myocardium. Previous and ongoing trials using CDCs target adult patients with stage B disease (cardiac function is declining but symptoms of HF have not yet appeared). In DMD-associated HF, therapeutic approaches may be most dramatic for stages C and D. These later stages of the disease are associated with high mortality rates (>20% per year) despite optimal medical treatment (which, again, has not been shown to actually slow disease progression in DMD patients). Due to exclusive comorbidities, heart transplantation is not an option for DMD patients. These patients are also not candidates for mechanical circulatory support devices. In summary, currently available treatments do not address the underlying pathophysiology of DMD-associated HF, which is the loss of functional myocardium and the conversion of viable myocardium to scar.

[0005] Interestingly, increasing evidence suggests that the positive therapeutic benefits of CDCs occur through indirect mechanisms, with most of the newly regenerated myocardium and vasculature being of endogenous origin. Perhaps due to the fact that CDCs are rich biological factories secreting numerous growth factors and cytokines, the beneficial therapeutic effects of CDCs persist long after the injected cells are removed. Understanding whether these favorable factors may be present in cellular exosomes (lipid bilayer nanovesicles secreted by cells when multivesicular endosomes fuse with the plasma membrane) produced by CDCs is of key interest. The role of secreted exosomes in these processes remains to be confirmed, and understanding these processes governing CDC-initiated regeneration may pave the way for new therapeutic approaches. Because existing treatments are unable to halt the progression of DMD HF, CDC-derived exosomes may effectively address a major unmet medical need by recruiting various synergistic mechanisms observed in animal models of HF. This includes the ability to attract endogenous stem cells to sites of myocardial injury and promote their differentiation into myocardium and blood vessels, potentially reversing the pathophysiology of HF. Given that conventional treatments are unavailable for late-stage patients, the potential benefits of exosome-based approaches as an alternative to cell therapy are particularly attractive. There is a possibility that CDC-derived exosomes could fundamentally alter the natural history of the disease.

[0006] Described herein are compositions and technologies for the production and therapeutic application of CDC-derived exosomes. These biological molecules contain a unique milieu of biological factors, including cytokines, growth factors, transcription factors, and nucleic acids (including non-coding nucleic acids such as microRNAs), that act to initiate and promote many of the therapeutic effects of CDCs. Our research demonstrates that exosomes and their constituent microRNAs favorably regulate apoptosis, inflammation, and fibrosis in the injured heart, leading to functional recovery and increased tissue survival. Thus, CDC-derived exosomes represent a novel "cell-free" therapeutic candidate for tissue repair. Summary of the Invention

[0007] Described herein are methods of treatment, comprising selecting a subject in need of treatment for heart failure secondary to a chronic degenerative muscle disease, and administering to the subject a composition comprising a plurality of exosomes, wherein the plurality of exosomes are isolated from cardiosphere-derived cells (CDCs) grown in serum-free medium, comprise exosomes about 90 nm to about 200 nm in diameter, and are CD81+, CD63+, or both, and further comprising administering the composition to treat the subject. In another embodiment, the chronic degenerative muscle disease is Duchenne muscular dystrophy. In another embodiment, the administration of the composition comprises about 1 to about 100 mg of exosome protein in a single dose. In another embodiment, the single dose is administered multiple times to the subject. In another embodiment, the administration of the composition comprises injection. In another embodiment, the injection comprises percutaneous injection. In another embodiment, the injection is directly into the myocardium. In another embodiment, the administration of the composition comprises intramyocardial injection. In another embodiment, the intramyocardial injection is intra-arterial or intravenous. In another embodiment, treatment of a subject results in reduced fibrosis, reduced inflammation, increased mitochondrial function, and / or increased cardiomyogenesis. In another embodiment, the reduced fibrosis comprises reduced collagen accumulation. In another embodiment, the collagen comprises collagen I and / or collagen III. In another embodiment, the reduced inflammation comprises increased cytoplasmic nuclear factor (erythroid-derived 2)-like 2 (Nrf2), reduced fatty acid peroxidation end products, reduced inflammatory cell numbers, and / or upregulated antioxidant expression. In another embodiment, antioxidants comprise heme oxygenase-1 (HO-1), catalase, superoxide dismutase-2 (SOD-2), and glutamate-cysteine ​​ligase catalytic (GCLC) subunits. In another embodiment, the inflammatory cells comprise CD68+ macrophages and CD3+ T cells. In another embodiment, the increased mitochondrial function comprises increased mitochondrial ultrastructure and / or increased mitochondrial biogenesis. In another embodiment, the increased mitochondrial function comprises increased nuclear PPAR-γ coactivator-1 (PGC-1) expression.In other embodiments, the exosomes comprise one or more microRNAs selected from the group consisting of microRNAs miR-146a, miR148a, miR22, miR-24, miR-210, miR-150, miR-140-3p, miR-19a, miR-27b, miR-19b, miR-27a, miR-376c, miR-128, miR-320a, miR-143, miR-21, miR-130a, miR-9, miR-185, and miR-23a.

[0008] Further described herein is a method of treatment, comprising selecting a subject in need of treatment for heart failure secondary to a chronic muscle disease, and administering a composition comprising cardiosphere-derived cells (CDCs), wherein administering the composition treats the subject. In another embodiment, the chronic muscle disease is Duchenne muscular dystrophy. In another embodiment, administering the composition is a single dose of about 1 x 10 5 pieces~approx. 1×10 8In another embodiment, the administration of the composition comprises one or more CDCs. In another embodiment, the administration of the composition comprises intramyocardial injection. In another embodiment, the intramyocardial injection is intracoronary. In another embodiment, the intramyocardial injection is intraarterial or intravenous. In another embodiment, treatment of a subject results in reduced fibrosis, reduced inflammation, increased mitochondrial function, and / or increased cardiomyogenesis. In another embodiment, the reduced fibrosis comprises reduced collagen accumulation. In another embodiment, the collagen comprises collagen I and / or collagen III. In another embodiment, the reduced inflammation comprises increased cytoplasmic nuclear factor (erythroid-derived 2)-like 2 (Nrf2), reduced fatty acid peroxidation end products, reduced number of inflammatory cells, and / or upregulated expression of antioxidants. In another embodiment, antioxidants comprise heme oxygenase-1 (HO-1), catalase, superoxide dismutase-2 (SOD-2), and glutamate-cysteine ​​ligase catalytic (GCLC) subunits. In another embodiment, the inflammatory cells comprise CD68+ macrophages and CD3+ T cells. In other embodiments, increased mitochondrial function comprises increased mitochondrial ultrastructure and / or increased mitochondrial biogenesis, hi other embodiments, increased mitochondrial function comprises increased nuclear PPAR-γ coactivator-1 (PGC-1) expression. [Brief explanation of the drawings]

[0009] [Figure 1]Characterization of cardiosphere-derived cell exosomes. (A) RNA content measured in exosome pellets derived from cardiosphere-derived cells (CDCs) and normal human dermal fibroblasts (NHDFs) compared with conditioned media from both samples. (B) Exosomal RNA is protected from RNase degradation by the exosome's lipid bilayer membrane. Exosome pellets were treated with RNase A in the presence or absence of triton to assess protection from RNase-mediated degradation. All samples were treated with proteinase K to dissociate complexes that might otherwise mask the RNA (n = 4 technical replicates). (C) CDC and NHDF exosomes express universal exosome markers, as revealed by mass spectrometry. (D) Exosome quantification from CDC- and NHDF-conditioned media based on expression of the conserved CD63 marker (n = 3 technical replicates). (E) Visualization of exosomes isolated from CDCs by transmission electron microscopy. Three populations (by size) are shown. (F) Size distribution of CDC-derived exosomes measured from transmission electron microscopy images; n = 100 counted exosomes. CDC exosomes promote angiogenesis and enhance the survival and proliferation of neonatal rat cardiomyocytes (NRCMs) in vitro. [Figure 2]CDC-exosomes confer structural and functional benefits in post-MI mouse hearts. (A) In the acute model, SCID Beige mice underwent MI and were injected intracardially with CDC-exosomes, NHDF-exosomes, or vehicle (control). Animals (n = 8 animals / group) underwent echocardiography on days 1, 15, and 30 and were then sacrificed for histological analysis. CDC-exosomes increase left ventricular ejection fraction (LVEF). (B-E) Structural benefits of CDC-exosomes. Masson's trichrome-stained sections (B) and pooled morphological analysis (C-E; n = 3 hearts / group) of hearts from each of the three groups reveal reduced scarring and increased viable myocardium mass in CDC-exosome-injected hearts. (F) In the chronic model, 3-month-old SCID Beige mice (n = 6 animals / group) underwent MI. Three weeks later, animals were intramyocardially injected with CDC-exosomes or control. Functional measurements were performed 24 hours before injection (day 21) and 3 weeks after injection (day 42), after which the animals were sacrificed for histological analysis. (G-J) Similar to the acute MI model, CDC-exosomes confer functional and structural benefits in mouse hearts (n = 4 hearts / group) in a model of chronic MI. *p<0.05, **p<0.01, and ***p<0.001 using one-way ANOVA with Tukey's post-hoc test and two-tailed Student's t-test. Data are presented as mean and SEM. See also Figures 8 and 9. [Figure 3]Exosome inhibition reduces the benefits of CDC. (A) GW4869 dose-dependently inhibited exosome production in CDCs (n = 3 technical replicates). (B) GW4869 does not affect CDC viability, as shown by calcein assay of CDCs treated with GW4869 or its solvent, DMSO (n = 4 technical replicates). (C and D) Newborn rat cardiomyocytes (NRCMs) were cultured on chamber slides and treated with medium conditioned by CDCs exposed to either GW4869 or DMSO. NRCMs were then treated with culture medium, and after 5 days, slides were stained for Ki67 and TUNEL to assess proliferation and apoptosis (n = 4 technical replicates / group). (E) Pooled data for left ventricular ejection fraction (n = 8 animals / group). (F-I) Representative Masson's Trichrome-stained cardiac sections (F) and pooled morphological analysis (G-I; n = 4 hearts / group) of the two groups reveal impaired CDC benefit as evidenced by pooled data for scar volume, viable myocardium volume, and infarct wall thickness (IWT) in GW869-treated CDC-injected hearts. *p<0.05 and **p<0.01 using Student's t-test. Data are presented as mean and SEM. See also Figure 10. [Figure 4]miR-146a is highly enriched in CDC exosomes and confers therapeutic benefit in vitro and in vivo. (A) Fold change in microRNA abundance in CDC exosomes compared to NHDF exosomes (n = 4 independent experiments). Total RNA (including microRNAs) was isolated from CDC and NHDF exosomes. qRT-PCR was performed by microRNA array. (B) Venn diagram showing variable microRNA profiles between CDC and NHDF exosomes. Font size reflects the magnitude of differential expression of each microRNA. (C) Infarcted mouse hearts treated with CDC-derived exosomes have higher levels of miR-146a compared to NHDF exosome-treated hearts (n = 2 animals / group, 3 technical replicates / group). (D) miR-146a protects stressed neonatal rat cardiomyocytes. Cardiomyocytes were pretreated with 80 nM miR-146a mimic or mimic control and then exposed to 100 mM hydrogen peroxide in serum-free medium for 2.5 hours (n = 4 technical replicates; neonatal rat cardiomyocytes studied were derived from 20-30 pups from three different mothers). (E) Microarray data showing the fold difference in mRNA abundance between miR-146a-treated and mimic control-treated cardiomyocytes. miR-146a suppresses Irak1 and Traf6 in stress-loaded neonatal rat cardiomyocytes. (F) After acute MI, miR-146a-deficient animals have severely impaired cardiac function and structure. Pooled data for left ventricular ejection fraction (n = 8 animals / group). (G-J) Representative Masson's Trichrome-stained cardiac sections (G) and pooled morphological analysis (H-J; n = 4 hearts / group) of the three groups reveal impaired CDC benefit as evidenced by pooled data for scar volume, viable myocardium volume, and infarct wall thickness (IWT) in GW869-treated CDC-injected hearts. *p<0.05, {p<0.05; **p<0.01, and {p<0.01 using Student's t-test (*KO vs. WT; {KO vs. KO-R). Data are presented as mean and SEM. See also Figures 11 and 12. [Figure 5]miR-146A improves contractile function in acute and chronic mouse models of MI. (A) Knockdown of miR-146a in CDC exosomes reduces their ability to protect stress-loaded NRVMs in vitro (n = 3 technical replicates; neonatal rat cardiomyocytes were derived from 20–30 rat pups from three different mothers). CDCs were transfected with either a miR-146a inhibitor or a hairpin control (HP-CTRL) carrying a Caenorhabditis elegans microRNA-based sequence. (B–F) Acute MI protocol data. Time course of left ventricular ejection fraction (n = 6 animals / group; B). Representative Masson's Trichrome-stained heart sections from each of the two groups (C) and pooled morphological analysis (n = 4 hearts / group) reveal reduced scar volume, increased viable myocardium, and increased infarct wall thickness in animals treated with miR-146a compared to the microRNA control (D–F). (G-L) miR-146a recapitulates some of the structural and functional benefits seen in CDC-exosome-treated hearts in a mouse model of chronic MI (miR-146a mimic or mimic control injected 21 days post-MI; n = 6 animals / group). Three weeks later (day 42), miR-146a-treated animals exhibited cardiac function comparable to controls (G), but adverse remodeling was significantly reduced (H). Scar volume was also similar (I). Viability and infarct wall thickness showed significant structural benefits (J and K), but scar volume was not reduced (I). Analysis was performed using Student's t-test; *p<0.05, **p<0.01, and ***p<0.001. Data are presented as mean and SEM. See also Figures 12 and 13. [Figure 6]miR-146a targets genes involved in MI pathology. (A and B) Downregulation of known miR-146a targets in chronic MI mouse hearts 7 days after injection with miR-146A or mimic control. (A) Western blots for IRAK, TRAF6, SMAD4, NOX4, and MPO (a marker of neutrophil infiltration). Pooled protein lysates from two hearts per group were loaded into each well, and blots represent pooled samples from two animals (n = 4 technical replicates). (B) Densitometric analysis of the blot in (A) normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). (C) Schematic representation of our research hypothesis. CDCs promote functional and structural benefits in injured myocardium, primarily in a paracrine manner. CDCs secrete exosomes containing microRNAs that mediate these benefits in injured myocardium. These microRNAs target transcripts in various myocardial compartments, ultimately resulting in increased cardiac function, increased viable tissue, and reduced scarring after MI. [Figure 7] Isolation of exosomes from CDCs. (A) Graphical representation of exosome isolation and exosome purification. (B) Cell viability (calcein) and cell death (ethidium homodimer-1) assays performed on CDCs over a 15-day serum-free acclimation period. (C) Representative images of CDCs before and after serum-free acclimation. [Figure 8] CDC-exosomes reduce inflammation in a mouse model of acute MI. (A) Representative protein array of 40 pro-inflammatory markers. (B) Quantification of inflammatory proteins in mouse hearts treated with CDC-exosomes, NHDF-exosomes, or control. Data are from three mouse hearts per group. Analysis was performed using one-way ANOVA (95% CI) (n = 3 hearts / group). Data are presented as mean and standard error of the mean. [Figure 9]CDC-exosomes confer structural and functional benefits in post-MI mouse hearts. CDC-exosomes stimulate functional improvement and attenuate adverse remodeling and cardiac hypertrophy in a mouse model of chronic MI. Animals treated with CDC-exosomes showed significant functional improvement compared to controls, as indicated by fractional area change (A), end-systolic volume (B), and end-diastolic volume (C) (A-C, n = 6 animals per group). CDC-exosome-treated animals also showed structural improvement, as seen in the percentage of the periphery of tissue sections that were scar (D), reduced cardiomyocyte hypertrophy measured by staining with wheat germ agglutinin and DAPI (F) (E), and increased angiogenesis in the infarcted area (G). Less cardiomyocyte death was observed in the border zone of CDC-exosome-treated animals compared to controls. (H, I) (DI n = 4 hearts / group) *P<0.05, **P<0.01, ***P<0.001 using Student's t-test. All scale bars represent 50 μm. Data are presented as the mean and standard error of the mean. [Figure 10] Inhibition of exosome secretion in CDCs reduces the protective effect of CDCs in vitro. Neonatal rat ventricular myocytes were stressed with 50 μM H2O2 for 15 minutes, followed by transwell treatment with CDCs pretreated with 5 μM spiroepoxide, 20 μM GW4869, or vehicle (DMSO). (A) Cell death was measured using TUNEL staining (red), phalloidin (green), and DAPI (blue). (B) Pooled data from four groups (n = 3 technical replicates; neonatal rat cardiomyocytes were derived from 20–30 pups from three different mothers) are presented as the percentage of TUNEL-positive cardiomyocyte nuclei in total cells counted. (B) Using Student's t-test, *P<0.05, **P<0.01, ***P<0.001. All scale bars represent 50 μm. Data are presented as the mean and standard error of the mean. [Figure 11]Heatmap of the Mir PCR array identifies Mir-146a as the most differentially expressed microRNA. Heatmap showing data on fold regulation of differential abundance of transcripts between CDC and NHDF exosomes overlaid on the PCR array plate layout. [Figure 12] miR-146a protects neonatal rat cardiomyocytes from stress. (A) Cardiomyocytes were pretreated with 80 nM miR-146a mimic or mimic control and then exposed to 5 mM cobalt chloride for 2 hours (n = 4 technical replicates / group; neonatal rat cardiomyocytes were derived from 20–30 rat pups from three different mothers). (B, C) CDC exosomes from CDCs transfected with miR-146a hairpin inhibitor. Exosomes were derived from conditioned medium, and miR-146a knockdown was confirmed by qPCR on exosomes. (C) Decreased miR-146a levels in NRVMs treated with miR-146a-free exosomes compared to controls (n = 3 technical replicates / group; neonatal rat cardiomyocytes were derived from 20–30 rat pups from three different mothers). (B) Pathway analysis derived from transcriptome data showing affected pathways and (B) pathway diagram showing MYC activation as a putative hub based on microarray data analysis. [Figure 13]miR-146a recapitulates some, but not all, of the effects of CDC-exosomes. miR-146a attenuates adverse remodeling and cardiac hypertrophy in a mouse model of chronic MI. (A, C) Animals treated with CDC-exosomes did not show significant functional improvement compared to controls, as indicated by fractional area change (A), end-systolic volume (B), and end-diastolic volume (A-C, n = 6 animals / group). However, structural improvement was observed, as seen in the percentage of the periphery of tissue sections that were scarred (D) and reduced cardiomyocyte hypertrophy measured by staining with wheat germ agglutinin and DAPI (E). No differences in angiogenesis were observed between the two groups (G). Less cardiomyocyte death was observed in the border zone of mir-146a-treated animals compared to controls. (H, I) (D-I n = 4 hearts / group) *P<0.05, **P<0.01, ***P<0.001 using Student's t-test. All scale bars represent 50 μm. Data are presented as the mean and standard error of the mean. [Figure 14] CDC treatment enhanced the activity of the Nrf2 antioxidant pathway and increased the expression of Nrf2 downstream gene products. (A) Representative immunohistochemical images showing Nrf2 in mdx mouse hearts after 3 weeks of treatment with vehicle (Mdx + Vehicle) or CDC (Mdx + CDC). Age-matched wild-type mice (CTL) served as controls. (B) and (C): Representative Western blot and pooled data demonstrating cytoplasmic and nuclear Nrf2 content (B) and protein abundance of Nrf2 downstream gene products (HO-1, glutamate-cysteine ​​ligase regulatory (GCLM) and catalytic (GCLC) subunits, SOD-1, catalase, and SOD-2) (C) in mdx mouse hearts after 3 weeks of treatment with vehicle or CDC. Experimental mice were recruited at 10 months of age. In CDC-treated mdx mice, a significant increase in cytoplasmic phosphorylated Nrf2 (Nrf2-ps40) was accompanied by increased nuclear Nrf2 content and increased expression of Nrf2 downstream gene products (B, C). Data are means ± SEM; n = 7 per group. †P < 0.05 compared with Mdx + vehicle and control (CTL; ​​wild-type); Scale bar: 5 μm. [Figure 15] In cardiac tissue from Mdx mice, CDC treatment significantly restored mitochondrial structure and content and enhanced the expression of respiratory chain subunits. (A): Representative transmission electron microscopy images of cardiomyocyte mitochondria in mdx mice treated with vehicle (Mdx + Vehicle) or CDC (Mdx + CDC) 3 weeks later. Elongated mitochondria with altered cristae (round / tubular) predominated in cardiomyocytes from 10-month-old mdx mice. CDC treatment significantly restored cardiomyocyte mitochondrial size and cristae structure (lamellar cristae). (B): Representative Western blot and pooled data showing nuclear Nrfl protein content and cytoplasmic and nuclear mitochondrial transcription factor A (mtTFA) protein abundance in cardiac tissue from vehicle / CDC-treated Mdx mice and age-matched wild-type mice (CTL) 3 weeks after treatment. (C): Bar graph demonstrating mitochondrial DNA copy number / cell in cardiac tissue from experimental animals 3 weeks after treatment. (D): Representative Western blot and pooled data showing the protein content of mitochondrial respiratory chain subunits in cardiac tissue from Mdx mice 3 weeks after treatment with vehicle (Mdx + Vehicle) and CDC (Mdx + CDC). Concomitant upregulation of Nrfl and mtTFA was associated with increased mitochondrial DNA copy number and was accompanied by restored expression of mitochondrial respiratory chain subunits. PC*: positive control. Data are mean ± SEM; n = 7 per group. †P < 0.05 vs. Mdx + Vehicle and control (CTL; ​​wild-type); ††P < 0.001 vs. Mdx + CDC and control (CTL; ​​wild-type). [Figure 16]Ultrastructural degenerative changes in the hearts of 10-month-old Mdx mice were significantly reduced after 3 weeks of treatment with CDC. (A): Representative transmission electron microscopy images of cardiomyocytes from 10-month-old Mdx mice show intracellular accumulation of amorphous protein, widespread disruption and irregularity of sarcomeres (Z-streaming), and disorganized changes in interfibrillar mitochondria. Three weeks after intramyocardial injection, CDC significantly reduced the degenerative changes in cardiomyocytes. (B): Bar graphs showing the average mitochondrial length and the total number and percentage (%) of round cristae in mitochondria from wild-type control mice (CTL) and vehicle- (Mdx + Vehicle) and CDC-treated Mdx mice (Mdx + CDC) after 3 weeks of treatment. Data are means ± SEM; †P<0.005 for Mdx + CDC vs. control (CTL; ​​wild-type); ††P<0.005 for Mdx + Vehicle vs. control (CTL; ​​wild-type). [Figure 17] CDC treatment reduced cardiac collagen content and fibrosis. Representative Masson's Trichrome images (A) and Western blot and pooled data (B) show fibrosis and collagen content in mdx mouse hearts after 3 weeks of treatment with vehicle (Mdx + Vehicle) or CDC (Mdx + CDC). Age-matched wild-type mice (CTL) served as controls. Collagen band size: 90–150 kDa. Data are mean ± SEM; n = 7 per group; † P < 0.01 vs. Mdx + CDC and control (CTL; ​​wild-type). Scale bar: 1 mm. [Figure 18]CDC treatment increased cardiomyocyte cycling and proliferation and enhanced the number of c-Kit-positive cells (c-Kit+Nkx2.5+) that differentiated into cardiac lineages. Representative immunohistochemical images and pooled data from Mdx mice treated at 10 months of age ((A)-(C); CTL [wild-type], vehicle, and CDC-treated Mdx mouse hearts stained for Ki67 (A), aurora B (B), c-Kit, and Nkx2.5 (C)). Arrows point to Ki67+ (A) and aurora B+ (B) cardiomyocytes, as well as cells positive for both c-Kit and Nkx2.5 (C). The ratio of cycling (Ki67+) and proliferating (Aurora B+) cardiomyocytes is expressed as the number of Ki67+ and aurora B+ cardiomyocytes divided by the total number of cardiomyocytes per high-power field (HPF) (pooled data (A), (B)). The percentage of c-kit+Nkx2.5+ cells was calculated as the number of c-kit+Nkx2.5+ cells divided by the total number of cardiomyocytes per HPF (pooled data (C)). WGA (wheat germ agglutinin) was applied for staining and delineation of cell membranes. Data are mean ± SEM; n = 7 per group. †P < 0.01 vs. Mdx+ vehicle and control (CTL; ​​wild type). Scale bar: 10 μm. [Figure 19] Functional benefits after cardiosphere-derived cell (CDC) transplantation. Pooled data for left ventricular ejection fraction (EF) and LV end-diastolic (LV EDV) and end-systolic (LV ESV) volumes show that in Mdx mice administered CDC at 10 months of age, CDC transplantation resulted in sustained improvements in EF, LV EDV, and LV ESV over a 3-month period. Data are mean ± SEM; n = 5 (control wild-type) and n = 12 (Mdx + vehicle, Mdx + CDC). *P < 0.05 vs. Gq + CDC; ***P < 0.001 vs. Gq + CDC. [Figure 20]CDC treatment improves maximal exercise capacity. Age-matched wild-type mice (CTL) and 10-month-old Mdx mice treated with vehicle (Mdx + Vehicle) or CDC (Mdx + CDC) were subjected to weekly high-intensity exercise (mean speed incrementally increased every 2 min until fatigue) beginning 3 weeks after CDC / vehicle treatment. A sustained improvement in exercise capacity was observed in CDC-treated mdx mice compared with vehicle-treated mice. Data are mean ± SEM; n = 6 (control wild-type) and n = 11 (Mdx + Vehicle, Mdx + CDC). *P < 0.05 vs. Gq + Vehicle. [Figure 21] Functional benefits after transplantation of human CDC-derived exosomes. Pooled data for left ventricular ejection fraction (EF) and LV end-diastolic (LV EDV) and end-systolic (LV ESV) volumes show that in 10-month-old Mdx mice, exosome transplantation resulted in improved EF, LV EDV, and LV ESV 3 weeks after intramyocardial injection. Data are mean ± SEM; n = 11 in each group. *P < 0.05 compared to Gq + CDC. [Figure 22] CDC-derived exosomes reduce cardiac collagen content and fibrosis. Representative Western blot and pooled data showing collagen I and III protein content in mdx mouse hearts after 3 weeks of treatment with vehicle (Mdx+Vehicle) or exosomes (Mdx+XO). Age-matched wild-type mice (CTL) served as controls. Collagen band size: 90–150 kDa. Data are mean ± SEM; n = 7 per group. †P<0.01 vs. Mdx+XO and control (CTL; ​​wild-type). [Figure 23]CDC transplantation in mdx mice improved function, survival, and antioxidant pathways. A: Ejection fraction (EF) in CDC-injected mdx mice (Mdx + CDC) and vehicle-injected mdx mice (Mdx + Vehicle) in response to injections at baseline (10 months of age) and 3 months later (n = 12 each). B: Exercise performance in mice subjected to weekly high-intensity treadmill exercise starting 3 weeks after CDC or vehicle administration (CTL: n = 7; Mdx + Vehicle and Mdx + CDC: n = 11 each). C: Kaplan-Meier analysis of survival in the same animals as in B shows that vehicle-treated mdx mice had lower survival rates than CDC-treated mdx mice or wild-type controls (p < 0.001, log-rank test); however, the latter two groups were statistically comparable. D: Immunohistochemical images of Nrf2 in mdx mouse hearts 3 weeks after vehicle or CDC administration. Age-matched wild-type mice (CTL) served as controls. Scale bar: 10 μm. E: Western blot and pooled data (n = 4–6) of protein abundance of phosphorylated Akt (Akt-pT308), cytoplasmic phosphorylated Nrf2 (Nrf2-pS40), nuclear Nrf2, and downstream gene products (heme oxygenase-1 (HO-1), catalase, superoxide dismutase 2 (SOD-2), and catalytic subunit of glutamate-cysteine ​​ligase (GCLC)) in mdx mouse hearts 3 weeks after administration of vehicle or CDC. F: Notes. Pooled data and representative Western blots of myocardial malondialdehyde protein adducts 3 weeks after injection demonstrate reduced oxidative stress in Mdx + CDC. Pooled data are mean ± SEM. *P < 0.05 vs. Mdx + CDC; #P < 0.005 vs. Mdx + CDC; †P < 0.05 vs. Mdx + vehicle and CTL (WT, wild-type mice); ‡P < 0.002 vs. Mdx + CDC and CTL (WT, wild-type mice). [Figure 24]Mitochondrial dysfunction and inflammation were alleviated by CDC transplantation in mdx mouse hearts. A: Transmission electron microscopy (TEM) images of mdx mouse hearts 3 weeks after treatment with vehicle (Mdx + Vehicle) or CDC (Mdx + CDC). Age-matched wild-type mice (CTL) served as controls. B: Number of mitochondria in TEM images. C: Mitochondrial DNA copy number (per nuclear genome) in cardiac tissue 3 weeks after treatment. D: Representative Western blot and pooled data of mitochondrial respiratory chain subunits in cardiac tissue from CTL and mdx mice 3 weeks after treatment (n = 4–6 per group). E: Oxygen consumption rate (OCR) of mitochondria isolated from CTL and CDC- or vehicle-treated mdx mouse hearts 3 weeks after treatment (CTL: n = 3; Mdx + Vehicle and Mdx + CDC: n = 8 each). Where indicated, substrates of oxidative phosphorylation (pyruvate, malate, and ADP), selective uncouplers (FCCP), and blockers (oligomycin [Olig.]; antimycin and rotenone [Anti. & Rot.]) were applied. F: Western blots and pooled data showing the protein abundance of mitochondrial PINK1 and nuclear PPARγ coactivator-1 (PGC-1) in mdx mouse hearts 3 days and 3 weeks after CDC administration (n = 4–6). G: Immunohistochemical images of hearts stained for inflammatory cell markers CD68, CD20, and CD3. H: Western blots, pooled data, and bar graphs (bottom right) showing the average number of indicated inflammatory cells in mdx mouse hearts. In CDC-treated mice, the accumulation of CD68+ macrophages (top row) and CD3+ T cells (bottom row) was reduced in association with inhibition of the NF-κB pathway. Data are means ± SEM. †P<0.05 compared with Mdx+vehicle and CTL (WT, wild-type mice); ‡P<0.003 compared with Mdx+CDC and CTL (WT, wild-type mice); *P<0.05 compared with Mdx+CDC. Scale bars: 5 μm (A); 10 μm (G). [Figure 25]CDC exosomes recapitulate the benefits of CDC in mdx mice. A: Sustained functional benefits for at least 3 months following two sequential injections of each CDC exosome in mdx mice (n = 11). B and C: Reduction in cardiac collagen content (B) and enhancement of cardiomyogenesis (C) 3 weeks after CDC exosome injection. Western blot and pooled data for cardiac collagens I and IIIA (B), and immunohistochemistry and pooled data (C: CTL [wild-type], vehicle-, and CDC exosome-treated [Mdx+XO] mdx mouse hearts stained for Ki67 [C1] and Aurora B [C2]; n = 4–6 per group). Arrows indicate Ki67+ (C1) and Aurora B+ (C2) cardiomyocytes. Wheat germ agglutinin (WGA) was applied for staining and delineation of cell membranes. Data are means ± SEM; *P<0.05 vs. Mdx+XO; †P<0.02 vs. Mdx+Vehicle and CTL (WT, wild-type mice); ‡P<0.01 vs. Mdx+XO and CTL (WT, wild-type mice). Scale bar: 10 μm. [Figure 26]CDC exosomes in human Duchenne cardiomyocytes and miR-148 in mdx mice. A: Calcium transients measured during 1 Hz burst pacing in normal and Duchenne human iPS-derived cardiomyocytes. Duchenne cardiomyocytes were primed with vehicle (DMD CM) or CDC exosomes (DMD CM+XO) one week prior to evaluation. Bar graphs of calcium transients: time to peak and alternans (variation in calcium transient amplitude between beats). B: Oxygen consumption rate (OCR) measurements in human Duchenne cardiomyocytes primed with CDC exosomes [DMD CM (CDC-XO)] or normal human dermal fibroblast-derived exosomes [NHDF, as a control; DMD CM (NHDF-XO+)] one week prior to OCR measurement. Normal (normal CM) and untreated Duchenne cardiomyocytes (DMD CM) were studied in parallel. For abbreviations, see the legend to Figure 2. C: Differential expression of microRNAs (including only miRs with >20 sequence hits) in CDC exosomes isolated from hypoxic (2% O2) conditioned medium compared to CDC exosomes isolated from normoxic conditioned medium (n=2). D: Intramyocardial injection of miR-148 mimic partially restored cardiac function in mdx mouse hearts after 3 weeks of treatment. E: Western blot and pooled data for nuclear p65 (left) and phosphorylated Akt (right) in mdx mouse hearts after 3 weeks of miR-148 treatment. F: Schematic of the pathophysiological mechanisms operating in Duchenne cardiomyopathy and the cellular machinery recruited by CDCs and their exosomes (XOs). All data are mean ± SEM (mean ± SD) except for box plots. Detailed Description of the Invention

[0010] All references cited herein are incorporated by reference in their entirety as if fully set forth. Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Allen et al., Remington: The Science and Practice of Pharmacy 22 nded.,Pharmaceutical Press(September 15,2012);Hornyak et al.,Introduction to Nanoscience and Nanotechnology,CRC Press(2008);Singleton and Sainsbury,Dictionary of Microbiology and Molecular Biology 3 rd ed.,revised ed.,J.Wiley&Sons(New York,NY 2006);Smith,March's Advanced Organic Chemistry Reactions,Mechanisms and Structure 7 th ed., J. Wiley & Sons (New York, NY 2013); Singleton, Dictionary of DNA and Genome Technology 3 rd ed., Wiley-Blackwell (November 28, 2012); and Green and Sambrook, Molecular Cloning: A Laboratory Manual 4th ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY 2012) provide those of skill in the art with a general guide to many of the terms used in this application. For references on methods for preparing antibodies, see Greenfield, Antibodies A Laboratory Manual 2 nded., Cold Spring Harbor Press (Cold Spring Harbor, NY, 2013); Kohler and Milstein, Derivation of specific antibody-producing tissue culture and tumor lines by cell fusion, Eur. J. Immunol. 1976 July, 6(7):511-9; Queen and Selick, Humanized immunoglobulins, U.S. Pat. No. 5,585,089 (1996 December); and Riechmann et al., Reshaping human antibodies for therapy, Nature 1988 March 24, 332(6162):323-7.

[0011] One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described. For purposes of the present invention, the following terms are defined below.

[0012] As used throughout this description and the claims that follow, the meanings of "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Also, as used throughout this description, the meaning of "in" includes "in" and "on" unless the context clearly dictates otherwise.

[0013] Duchenne muscular dystrophy, a debilitating genetic disease leading to premature death, affects the heart and skeletal muscles. Indeed, cardiomyopathy is the leading cause of death in Duchenne patients. There are no approved treatments for cardiomyopathy, and novel Duchenne-specific experimental approaches, such as exon skipping, have not shown cardiac benefit. Herein, we demonstrate that cardiosphere-derived cells (CDCs) rescue key pathophysiological features of Duchenne cardiomyopathy (oxidative stress, inflammation, fibrosis, and mitochondrial dysfunction) in mdx mice in advanced clinical trials for therapeutic regeneration after myocardial infarction. Exosomes secreted by human CDCs recapitulate the benefits of CDCs in mdx mice and rescue abnormalities in calcium cycling and mitochondrial respiration in human Duchenne cardiomyocytes.

[0014] The lack of dystrophin in Duchenne muscular dystrophy (DMD) results in membrane fragility and secondary muscle damage (both skeletal and cardiac). Early disability is primarily due to skeletal muscle damage, but heart failure is the most common cause of death. Currently available treatment modalities do not address the pathophysiology underlying DMD-associated heart failure, loss of functional myocardium, and the conversion of viable myocardium to scar. Cardiosphere-derived cells (CDCs) may be a viable therapeutic option. In the first-in-human CADUCEUS trial of CDCs in myocardial infarction, healthy myocardium was regenerated and scar was reduced; these findings are currently being further tested in a randomized, placebo-controlled, multicenter clinical trial of allogeneic CDCs. Preclinical studies have shown that CDCs are not only regenerative but also anti-inflammatory and anti-fibrotic; they act indirectly through the secretion of exosomes loaded with non-coding RNAs, including microRNAs (miRs). In a mouse model of myocardial infarction, CDC exosomes mimic the functional and structural benefits of CDC, while blocking exosome biogenesis renders CDC ineffective. Given the clinical data and mechanism of action, the inventors reasoned that CDC may be useful for treating Duchenne cardiomyopathy. The goal is not to replace dystrophin, but to compensate for the pathophysiological consequences of dystrophin deficiency by recruiting regeneration, reversing fibrosis, and targeting inflammation.

[0015] Exosomes, secreted lipid vesicles containing a rich milieu of biological factors, provide powerful paracrine signals for stem cells to enhance their biological effects on neighboring cells, including diseased or injured cells. There is growing recognition that, by encapsulating and transporting proteins, bioactive lipids, and nucleic acid "cargo," these natural delivery devices can induce significant phenotypic and functional changes in recipient cells, leading to the activation of regenerative programs. The role of such indirect mechanisms in stem cell-initiated regeneration is strongly suggested by growing evidence that the regenerative process persists and arises from endogenous tissues even after stem cells are administered and removed from the delivery site in tissues and organs.

[0016] The "paracrine hypothesis" of stem cell regenerative activity has led to a paradigm shift: clinical applications based on exosomes secreted by stem cells may offer superior or distinct advantages compared to transplantation and delivery of stem cells themselves. In particular, stem cell-derived exosomes have been identified and isolated from the supernatants of several cell types with demonstrated therapeutic potential, including mesenchymal stromal cells (MSCs), (bone marrow stem cells), mononuclear cells (MNCs), immune cells (dendritic cells and CD34+ cells), and human neural cells (hNSCs). In the context of cardiac disease, human cardiomyocyte-derived cells (CDCs) are known to improve myocardial and vasculature health. Stem cell-derived exosomes, including those produced by CDCs, may provide a potent and abundant source for developing "cell-free" therapies.

[0017] Additionally, exosome-based "cell-free" therapies offer distinct advantages in regenerative medicine compared to cell therapies. As non-living entities with reduced or no immunogenicity or tumorigenic potential, these characteristics significantly eliminate safety concerns. For example, stem cell-derived exosomes are less immunogenic than parental cells as a result of their low content of membrane-bound proteins, including MHC complex molecules. Replacing the administration of live cells with their secreted exosomes alleviates many of the safety concerns and limitations associated with the transplantation of viable replicating cells. Additionally, exosomes encapsulate bioactive components within lipid vesicles, potentially protecting the contents from in vivo degradation, thereby eliminating obstacles often associated with the delivery of soluble molecules such as cytokines, growth factors, transcription factors, and RNA. This relative ease of administration may ultimately allow for repeated and sustained delivery to patients, thereby maximizing the potential for regeneration and repair of diseased and / or dysfunctional tissues.

[0018] Furthermore, producing exosomes under defined conditions allows for easier manufacturing and scale-up opportunities. Because exosome production resembles that of conventional biopharmaceutical products, standardization of production and quality control for dosage and biological activity testing is possible. Furthermore, the durability of exosomes in culture medium allows for the acquisition of large quantities of exosomes by recovering them from the culture medium into which they are secreted over a period of time.

[0019] Although it is now well established that exosomes are involved in intercellular communication between different cell types, much remains to be discovered regarding their mechanisms of production within the original parent cells and their effects on target recipient cells. Exosomes have been reported to be involved in numerous cellular, tissue, and physiological processes, including immunomodulatory processes, angiogenesis, endothelial cell migration associated with tumor growth, and the reduction of damage in ischemia-reperfusion injury. It is of significant scientific interest to establish whether exosomes secreted by cells, such as cardiosphere-derived cells (CDCs), can recapitulate the therapeutic benefits of their parent cells, or, in some cases, are essential for achieving such therapeutic benefits.

[0020] General Characteristics of Exosomes. Secreted by a wide range of cell types, exosomes are lipid bilayer vesicles enriched in various biological factors, including cytokines, growth factors, transcription factors, lipids, and coding and non-coding nucleic acids. Exosomes are found in blood, urine, amniotic fluid, interstitial spaces, and the extracellular space. These exocytic vesicles of endosomal origin can range in size from 30 to 200 nm, including 40 to 100 nm, and have a cup-like morphology when viewed by electron microscopy. Their initial formation begins with inward budding of the plasma membrane to form endosomes, followed by invagination of the limiting membrane of late endosomes to form multivesicular bodies (MVBs). Fusion of the MVB with the plasma membrane results in the release of the internal vesicles into the extracellular space through the formation of vesicles known as exosomes.

[0021] As described, the "cargo" contents of exosomes reflect their parental cellular origin, as they contain distinct subsets of biological factors related to their parental cellular origin (including the cellular regulatory state of the parental cell at the time of formation). The rich biological milieu of different proteins (including cytokines and growth factors), lipids, coding and non-coding RNA molecules within exosomes all necessarily originates from their parental cells. In addition to containing an abundance of many cytosolic derivatives, exosomes also express the extracellular domains of membrane-bound receptors on their membrane surface.

[0022] The described encapsulation and formation processes necessarily result in heterogeneity in exosome composition based on the parent cell origin and regulatory state at the time of formation. Nevertheless, the general budding and release mechanisms establish a set of common features that result from their origin, such as endosome-associated proteins (e.g., Rab GTPases, SNAREs, annexins, and flotillins), proteins known to cluster into microdomains at the plasma membrane or endosomes (four-transmembrane domain tetraspanins, e.g., CD63, CD81, CD82, CD53, and CD37), lipid raft-associated proteins (e.g., glycosylphosphatidylinositol-anchored proteins and flotillins), cholesterol, sphingomyelin, and hexosylceramide.

[0023] In addition to these core components reflecting their vesicular origin, an important property of exosomes is their demonstrated ability to contain both mRNAs and microRNAs related to signaling processes; both cargo mRNAs can be translated in recipient cells, and microRNAs functionally degrade target mRNAs in recipient cells. Other non-coding RNAs capable of influencing gene expression may also be present in exosomes. While the processes controlling the selective uptake of mRNA or microRNA populations into exosomes are not fully understood, it is clear that RNA molecules are selectively, rather than randomly, incorporated into exosomes, as evidenced by studies reporting enrichment of exosomal cargo RNAs relative to the RNA profile of the cell of origin. Given our growing understanding of the role of such RNA molecules in disease pathogenesis and regenerative processes, the presence of RNA molecules in exosomes and their distinct potency in influencing target recipient cells suggests important features that could be deployed in therapeutic approaches.

[0024] Importantly, the natural bilayer membrane encapsulation of exosomes provides a protected and controlled internal microenvironment that allows their cargo contents to persist or migrate within the bloodstream or tissues without degradation. Their release into the extracellular environment allows interaction with recipient cells via adhesion to the cell surface mediated by lipid-ligand-receptor interactions (endocytic internalization) or direct fusion of the vesicle with the plasma membrane. These processes result in the release of exosomal cargo contents into target cells.

[0025] The end result of exosome-cell interactions is the modulation of gene pathways in target recipient cells, as induced by any of several different mechanisms, including antigen presentation, delivery of transcription factors, cytokines, growth factors, and nucleic acids such as mRNA and microRNA. In the context of stem cells, embryonic stem cell (ESC)-derived exosomes have been demonstrated to shuttle / transport mRNA and proteins to hematopoietic progenitor cells. Other studies have shown that adult stem cell-derived exosomes also shuttle selective patterns of mRNA, microRNA, and pre-microRNA associated with several cellular functions involved in the control of transcription, proliferation, and cellular immune regulation.

[0026] Exosome Isolation and Preparation. Exosome isolation relies on the use of common biochemical and biophysical characteristics for their separation and analysis. For example, differential ultracentrifugation has become the primary technique for isolating secreted exosomes from the supernatant of cultured cells. This approach allows for the separation of exosomes from non-membranous particles by utilizing their relatively low buoyant density. Size exclusion allows for the separation of exosomes from biochemically similar but biophysically distinct microvesicles with larger diameters of up to 1,000 nm. Differences in buoyant velocity further allow for the separation of exosomes of different sizes. Exosomes generally range in diameter from 30 to 200 nm, including sizes between 40 and 100 nm. Further purification can depend on the specific properties of the particular exosome of interest. This can involve, for example, immunoadsorption with a protein of interest to select for specific vesicles with extracytoplasmic or extracytoplasmic orientation.

[0027] Among current methods (differential centrifugation, discontinuous density gradient, immunoaffinity, ultrafiltration, and high-performance liquid chromatography (HPLC)), differential ultracentrifugation is the most commonly used for exosome isolation. This technique utilizes centrifugal forces increasing from 2,000 × g to 10,000 × g to separate medium- and large-sized particles and cellular debris from the exosome pellet at 100,000 × g. Centrifugation alone allows for significant isolation / collection of exosomes from conditioned medium, but is insufficient to remove common contaminants such as various protein aggregates, genetic material, medium-derived particulates, and cellular debris. High specificity in exosome purification can be achieved by deploying continuous centrifugation combined with ultrafiltration, or equilibrium density gradient centrifugation of a sucrose density gradient to provide higher purity exosome preparations (buoyant density 1.1–1.2 g / ml) or by applying a separate sugar cushion in the preparation.

[0028] Importantly, ultrafiltration can be used to purify exosomes without compromising their biological activity. To avoid the use of non-neutral pH or non-physiological salt concentrations, membranes with different pore sizes (e.g., 100 kDa molecular weight cutoff (MWCO) and gel filtration to exclude smaller particles) have been used. Currently available tangential flow filtration (TFF) systems are scalable (to over 10,000 L) and can not only purify but also concentrate exosome fractions, an approach that requires less time than differential centrifugation. Because the preparation is maintained at physiological pH and salt concentrations, HPLC can also be used to purify exosomes into uniformly sized particles and preserve their biological activity.

[0029] Other chemical methods, in addition to volume-exclusion polymers (e.g., polyethylene glycol (PEG)), optionally combined with further rounds of centrifugation or filtration, exploit the differential solubility of exosomes for precipitation techniques. For example, the precipitation reagent ExoQuick® can be added to conditioned cell media to rapidly and easily precipitate exosome populations, although resuspension of pellets prepared by this technique can be difficult. Flow field-flow fractionation (FlFFF), an elution-based technique used to separate and characterize macromolecules (e.g., proteins) and nano- to micro-sized particles (e.g., organelles and cells), has been successfully applied to fractionate exosomes from culture media.

[0030] In addition to these techniques that rely on common biochemical and biophysical characteristics, focused techniques can be applied to isolate specific exosomes of interest. This involves relying on antibody immunoaffinity to recognize specific exosome-associated antigens. As described, exosomes also express the extracellular domains of membrane-bound receptors on their membrane surface. This provides a convenient opportunity to isolate and separate exosomes related to their parent cell origin based on a common antigen profile. The combined use of magnetic beads, chromatography matrices, plates, or microfluidic devices makes it possible to isolate specific exosome populations of interest that may be related to their production from the parent cell of interest or related cellular regulatory states. Other affinity capture methods use lectins that bind to specific sugar residues on the exosome surface.

[0031] Exosome-Based Therapy. The primary goal of developing exosome-based therapies is to create "cell-free" therapies that can provide the benefits of cell therapy with low risk or in situations where cell therapy is unavailable. For example, Duchenne muscular dystrophy (DMD)-associated heart failure (HF), particularly in late-stage disease, presents significant and exclusive comorbidities, and in cases of late-stage disease C and D, cell, tissue, cardiac, or mechanical transplantation is not an option. As described, the therapeutic benefits of cell-based therapies, such as cardiosphere-derived cells (CDCs), appear to arise through an indirect mechanism involving regenerating myocardium and vasculature arising from endogenous sources. Cellular exosomes produced by CDCs enable the production and delivery of growth factors, transcription factors, cytokines, and nucleic acids for novel therapeutic approaches, not only ameliorating disease progression but also repairing and regenerating diseased and / or dysfunctional tissues. In this regard, CDC-derived exosomes may effectively address a major unmet medical need by recruiting synergistic mechanisms to attract endogenous stem cells to the site of myocardial injury and promote their differentiation into myocardium and blood vessels, thereby reversing the pathophysiology of HF.

[0032] More specifically, DMD is an X-linked recessive disorder characterized by myopathy (cell membrane damage in muscle fibers), as exemplified by various pathological features. This includes skeletal muscle weakness beginning 3–5 years after onset, progressive weakness at approximately 13 years after onset, and wheelchair dependency. Importantly, cardiomyopathy has been observed to develop in one-third of patients before 13 years, increasing to one-half of patients before 18 years after onset, and occurring in all patients after 18 years. Dilated cardiomyopathy includes fibrosis of the left ventricular posterobasal branch; conduction abnormalities are primarily SVT with abnormal intraatrial:AV nodal conduction. Patients may also suffer from smooth muscle dysfunction, including vascular dysfunction, including GI and urinary system involvement. The typical prognosis is respiratory failure or death from cardiomyopathy. The basis for these clinical features is that loss of dystrophin leads to cell membrane damage and the transfer of extracellular Ca into cells. 2+The cause is a dystrophin gene mutation (deletion) that results in leakage of dystrophin. High intracellular levels ultimately lead to oxidative and / or nitrosative stress, increased inflammation, and activation of calpains. The combination of these effects leads to muscle protein degradation and apoptosis, leading to the degradative characteristics described above.

[0033] Based on this pathophysiology of DMD patients (including increased oxidative and / or nitrosative stress, enhanced inflammation, and pro-apoptotic and remodeling states), therapeutic approaches involving CDCs may offer significant benefits in reversing the disease course. CDCs have been demonstrated to promote antioxidant, anti-inflammatory, anti-apoptotic, and anti-remodeling effects in addition to enhancing regenerative capacity. In this regard, CDC administration is beneficial for delaying / reversing DMD, and CDC-derived exosome populations may be able to provide these benefits while avoiding the obstacles associated with cell-based therapies.

[0034] In particular, stem cell-derived exosomes may be less immunogenic than parental cells. The potential replacement of live cell administration with secreted exosomes alleviates many of the safety concerns and limitations associated with viable cell transplantation. Additionally, exosomes encapsulate bioactive components in lipid vesicles, potentially protecting their contents from in vivo degradation, potentially enabling increased concentrations while eliminating obstacles often associated with the delivery of soluble molecules such as cytokines, growth factors, transcription factors, and RNA. Particularly in the case of chronic diseases such as DMD, repeated and sustained delivery to patients may maximize the potential for regeneration and repair of diseased and / or dysfunctional tissues in a manner that would be difficult or risky with cell-based therapies. Full realization of these benefits requires improved understanding of whether exosomes secreted by stem cells, such as CDCs, can independently recapitulate the therapeutic benefits of their parental cells or may be essential in these processes. Confirmation of the role of exosomes in such processes would allow for their application in new therapeutic approaches (e.g., "cell-free" use in subjects where cell transplantation or administration is unavailable (e.g., late-stage cardiac disease)), as device-based pharmacological intervention or surgery may not be a prudent treatment modality for such subjects. There is a great need in the art to identify means of providing the benefits of stem cell regeneration without mechanisms involving the administration or transplantation of cells themselves.

[0035] Described herein are compositions and methods that provide significant benefits in the repair or regeneration of damaged or diseased tissues through a "cell-free" method involving exosomes. Specifically, human cardiomyocyte-derived cell (CDC)-derived exosomes are demonstrated to be effective in reducing scar size and regenerating viable myocardium. These results support the main benefit of CDC cell therapy is mediated by exosomes containing specific microRNAs that the inventors have identified as being abundant in CDCs.

[0036] Described herein are methods of treatment, comprising selecting a subject in need of treatment for heart failure secondary to a chronic degenerative muscle disease, and administering to the subject a composition comprising a plurality of exosomes, wherein the plurality of exosomes are isolated from cardiosphere-derived cells (CDCs) grown in serum-free medium, comprise exosomes about 90 nm to about 200 nm in diameter, and are CD81+, CD63+, or both, and further comprising administering the composition to treat the subject. In another embodiment, the chronic degenerative muscle disease is Duchenne muscular dystrophy. In another embodiment, the administration of the composition comprises about 1 to about 100 mg of exosome protein in a single dose. In another embodiment, the single dose is administered multiple times to the subject. In another embodiment, the administration of the composition comprises injection. In another embodiment, the injection comprises percutaneous injection. In another embodiment, the injection is directly into the myocardium. In another embodiment, the administration of the composition comprises intramyocardial injection. In another embodiment, the intramyocardial injection is intra-arterial or intravenous. In another embodiment, treatment of a subject results in reduced fibrosis, reduced inflammation, increased mitochondrial function, and / or increased cardiomyogenesis. In another embodiment, the reduced fibrosis comprises reduced collagen accumulation. In another embodiment, the collagen comprises collagen I and / or collagen III. In another embodiment, the reduced inflammation comprises increased cytoplasmic nuclear factor (erythroid-derived 2)-like 2 (Nrf2), reduced fatty acid peroxidation end products, reduced inflammatory cell numbers, and / or upregulated antioxidant expression. In another embodiment, antioxidants comprise heme oxygenase-1 (HO-1), catalase, superoxide dismutase-2 (SOD-2), and glutamate-cysteine ​​ligase catalytic (GCLC) subunits. In another embodiment, the inflammatory cells comprise CD68+ macrophages and CD3+ T cells. In another embodiment, the increased mitochondrial function comprises increased mitochondrial ultrastructure and / or increased mitochondrial biogenesis. In another embodiment, the increased mitochondrial function comprises increased nuclear PPAR-γ coactivator-1 (PGC-1) expression.In other embodiments, the exosomes comprise one or more microRNAs selected from the group consisting of microRNAs miR-146a, miR148a, miR22, miR-24, miR-210, miR-150, miR-140-3p, miR-19a, miR-27b, miR-19b, miR-27a, miR-376c, miR-128, miR-320a, miR-143, miR-21, miR-130a, miR-9, miR-185, and miR-23a.

[0037] Further described herein is a method of treatment, comprising selecting a subject in need of treatment for heart failure secondary to a chronic muscle disease, and administering a composition comprising cardiosphere-derived cells (CDCs), wherein administering the composition treats the subject. In another embodiment, the chronic muscle disease is Duchenne muscular dystrophy. In another embodiment, administering the composition is a single dose of about 1 x 10 5 pieces~approx. 1×10 8 In another example, the number of CDCs administered comprises 25 million intracoronary CDCs per coronary artery (i.e., a total of 75 million CDCs) as another baseline for exosome administration. In various embodiments, the number of CDCs administered comprises 1 x 10 in a single dose as another baseline for exosome administration. 5 pieces, 1×10 6 pieces, 1×10 7 pieces, 1×10 8 pieces, 1×10 9In certain cases, this can be proportional to body weight (ranging from 100,000 to 1 million CDCs per kg of body weight for a total CDC dose). In other embodiments, administration of the composition comprises myocardial injection. In other embodiments, the myocardial injection is intracoronary. In other embodiments, the myocardial injection is intraarterial or intravenous. In other embodiments, treatment of a subject results in reduced fibrosis, reduced inflammation, increased mitochondrial function, and / or increased cardiomyogenesis. In other embodiments, the reduced fibrosis comprises reduced collagen accumulation. In other embodiments, the collagen comprises collagen I and / or collagen III. In other embodiments, the reduced inflammation comprises increased cytoplasmic nuclear factor (erythroid-derived 2)-like 2 (Nrf2), reduced fatty acid peroxidation end products, reduced number of inflammatory cells, and / or upregulated expression of antioxidants. In other embodiments, the antioxidants include heme oxygenase-1 (HO-1), catalase, superoxide dismutase-2 (SOD-2), and glutamate-cysteine ​​ligase catalytic (GCLC) subunits. In other embodiments, the inflammatory cells include CD68+ macrophages and CD3+ T cells. In other embodiments, the increased mitochondrial function includes increased mitochondrial ultrastructure and / or increased mitochondrial biogenesis. In other embodiments, the increased mitochondrial function includes increased nuclear PPAR-γ coactivator-1 (PGC-1) expression. Further examples can be found in U.S. Patent Application Nos. 11 / 666,685, 12 / 622,143, and 12 / 622,106, which are incorporated herein by reference.

[0038] Described herein are compositions comprising a plurality of exosomes. In certain embodiments, the plurality of exosomes is produced by a method comprising providing a cell population and isolating a plurality of exosomes from the cell population.

[0039] In various embodiments, the cells are stem, progenitor, and / or progenitor cells. In other embodiments, the stem, progenitor, and / or progenitor cells are cardiosphere-derived cells (CDCs). In other embodiments, the stem, progenitor, and / or progenitor cells are pluripotent stem cells (pSCs), such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), derived from any one of a variety of somatic cell sources within the body, such as fibroblasts, blood and hematopoietic stem cells (hSCs), immune cells, bone and bone marrow, neural tissue, and the like. In other embodiments, the stem, progenitor, and / or progenitor cells comprise hSCs, mesenchymal stem cells (MSCs), or endothelial progenitor cells (EPCs). In various embodiments, the cells are stem, progenitor, and / or progenitor cells derived from human biopsied tissue. In various embodiments, the cells are stem cells and progenitor and / or progenitor cells are primary cultures. In various embodiments, the cells are stem, progenitor, and / or progenitor cells capable of constituting a cell line capable of serial passage.

[0040] In various embodiments, the plurality of exosomes are isolated from the supernatant of a cell population. This includes, for example, exosomes secreted into a medium conditioned by a cell population in a culture medium further comprising a subcultured cell line. In certain embodiments, the cells are cultured in a serum-free medium. In certain embodiments, when exosomes are isolated, the cells in the culture medium are grown to 10, 20, 30, 40, 50, 60, 70, 80, 90, or 90% or more confluence. In certain embodiments, the cell population is genetically engineered. This includes, for example, knockout (KO) or transgenic (TG) cell lines in which endogenous genes have been removed and / or exogenous genes have been introduced in a stable and persistent manner. This further includes transient knockdown of one or more genes and related coding and non-coding transcripts in the cell population by any of several methods known in the art, such as the introduction of dsRNA, siRNA, microRNA, etc. This further includes transient expression of one or more genes and associated coding and non-coding transcripts in a cell population via any of several methods known in the art, such as the introduction of a vector, a plasmid, an artificial plasmid, a replicating and / or non-replicating virus, etc. In other embodiments, the cell population is altered at the time of isolating the plurality of exosomes, or substantially simultaneously therewith, by exposure to an environmental condition (e.g., hypoxia), the addition of a small molecule, or the presence / absence of an exogenous factor (e.g., growth factor, cytokine), in a manner that alters the regulatory state of the cells. For example, a differentiation agent may be added to a population of stem, progenitor, and / or precursor cells to promote partial or complete differentiation of the cells and then generate a plurality of exosomes. In various embodiments, the alteration of the regulatory state of the cells alters the composition of one or more exosomes in the plurality of exosomes.

[0041] In various embodiments, the plurality of exosomes have a diameter of about 10 nm to about 250 nm, e.g., a diameter of about 10 nm to about 15 nm, about 15 nm to about 20 nm, about 20 nm to about 25 nm, about 25 nm to about 30 nm, about 30 nm to about 35 nm, about 35 nm to about 40 nm, about 40 nm to about 50 nm, about 50 nm to about 60 nm, The exosomes may have a diameter of about 60 nm to about 70 nm, about 70 nm to about 80 nm, about 80 nm to about 90 nm, about 90 nm to about 95 nm, about 95 nm to about 100 nm, about 100 nm to about 105 nm, about 105 nm to about 110 nm, about 110 nm to about 115 nm, about 115 nm to about 120 nm, about 120 nm to about 125 nm, about 125 nm to about 130 nm, about 130 nm to about 135 nm, about 135 nm to about 140 nm, about 140 nm to about 145 nm, about 145 nm to about 150 nm, about 150 to about 200 nm, about 200 nm to about 250 nm, or about 250 nm or greater.

[0042] In various embodiments, the plurality of exosomes comprises one or more exosomes expressing a biomarker. In certain embodiments, the biomarker is a tetraspanin. In other embodiments, the tetraspanin is one or more selected from the group including CD63, CD81, CD82, CD53, and CD37. In other embodiments, the exosome expresses one or more lipid raft-associated proteins (e.g., glycosylphosphatidylinositol-anchored proteins and flotillins), cholesterol, sphingomyelin, and / or hexosylceramide.

[0043] In some embodiments, the plurality of exosomes comprises one or more exosomes containing a biological protein. In various embodiments, the biological proteins include transcription factors, cytokines, growth factors, and similar proteins capable of regulating signaling pathways in target cells. In various embodiments, the biological proteins can promote tissue regeneration and / or functional improvement. In various embodiments, the biological proteins can regulate pathways related to Irak1, Traf6, toll-like receptor (TLR) signaling pathways, NOX-4, SMAD-4, and / or TGF-β. In other embodiments, the biological proteins are involved in exosome formation and packaging of cytosolic proteins (e.g., Hsp70, Hsp90, 14-3-3ε, PKM2, GW182, and AG02).

[0044] In other embodiments, the plurality of exosomes comprises one or more exosomes containing signaling lipids, including ceramides and derivatives. In other embodiments, the plurality of exosomes comprises one or more exosomes containing coding and / or non-coding nucleic acids.

[0045] In embodiments, the plurality of exosomes comprises one or more exosomes containing microRNAs. In various embodiments, these microRNAs may include miR-146a, miR-22, miR-24, miR-210, miR-150, miR-140-3p, miR-19a, miR-27b, miR-19b, miR-27a, miR-376c, miR-128, miR-320a, miR-143, miR-21, miR-130a, miR-9, miR-185, and / or miR-23a. In some embodiments, the plurality of exosomes comprises one or more exosomes enriched in at least one of miR-146a, miR-22, miR-24, miR-210, miR-150, miR-140-3p, miR-19a, miR-27b, miR-19b, miR-27a, miR-376c, miR-128, miR-320a, miR-143, miR-21, miR-130a, miR-9, miR-185, and / or miR-23a. In some embodiments, the plurality of exosomes comprises one or more exosomes enriched in at least one of miR-146a, miR-22, and miR-24. Abundance can be measured, for example, by comparing the amount of one or more of the described microRNAs when produced from cells that provide a beneficial benefit in a therapeutic setting (e.g., cardiosphere-derived cells (CDCs)) compared to cells that do not (e.g., fibroblasts). Abundance can also be measured in absolute amounts or relative amounts, for example, compared to a standardized dilution series.

[0046] In other embodiments, the plurality of exosomes may include one or more exosomes containing microRNAs, including various microRNAs known in the art, such as miR-23a, miR-23b, miR-24, miR-26a, miR27-a, miR-30c, let-7e, mir-19b, miR-125b, mir-27b, let-7a, miR-19a, let-7c, miR-140-3p, miR-125a-5p, miR-132, miR-150, miR-155, mir-210, let-7b, miR-24, miR-423-5p, miR-22, let-7f, and / or miR-146a.

[0047] In other embodiments, the plurality of exosomes may include one or more exosomes containing microRNAs, such as various microRNAs known in the art, including miR-17, miR-21, miR-92, miR92a, miR-29, miR-29a, miR-29b, miR-29c, miR-34, miR34a, miR-150, miR-451, miR-145, miR-143, miR-144, miR-193a-3p, miR-133a, miR-155, miR-181a, miR-214, miR-199b, miR-199a, miR-210, miR-126, miR-378, miR-363, and miR-30b and miR-499. Other microRNAs known in the art include miR-92, miR-17, miR-21, miR-92, miR92a, miR-29, miR-29a, miR-29b, miR-29c, miR-34, mi-R34a, miR-150, miR-451, miR-145, miR-143, miR-144, miR-193a-3p, miR-133a, miR-155, miR-181a, miR-214, miR-199b, miR-199a, miR-126, miR-378, miR-363, and miR-30b and / or miR-499.

[0048] In some embodiments, isolating the exosomes from the cell population comprises centrifugation of the cells and / or medium conditioned by the cells. In some embodiments, ultracentrifugation is used. In some embodiments, isolating the exosomes from the cell population is by size exclusion filtration. In other embodiments, isolating the exosomes from the cell population comprises the use of discontinuous density gradients, immunoaffinity, ultrafiltration, and / or high performance liquid chromatography (HPLC).

[0049] In certain embodiments, differential ultracentrifugation comprises separating larger sized particles from a plurality of exosomes derived from a cell using centrifugal forces of 1000-2000×g, 2000-3000×g, 3000-4000×g, 4000-5000×g, 5000×g-6000×g, 6000-7000×g, 7000-8000×g, 8000-9000×g, 9000-10,000×g, up to or exceeding 10,000×g. In certain embodiments, differential ultracentrifugation comprises separating larger sized particles from a plurality of exosomes derived from a cell using centrifugal forces of 10,000 to 20,000 x g, 20,000 to 30,000 x g, 30,000 to 40,000 x g, 40,000 to 50,000 x g, 50,000 to 60,000 x g, 60,000 to 70,000 x g, 70,000 to 80,000 x g, 80,000 to 90,000 x g, 90,000 to 100,000 x g, or up to 10,000 x g or more.

[0050] In other embodiments, isolation of multiple exosomes from a cell population involves the use of filtration or ultrafiltration. In certain embodiments, size-exclusion membranes with different pore sizes are used. For example, size-exclusion membranes may include filters with pore sizes of 0.1-0.5 μM, 0.5-1.0 μM, 1-2.5 μM, 2.5-5 μM, 5 μM, or greater. In certain embodiments, the pore size is approximately 0.2 μM. In certain embodiments, filtration or ultrafiltration involves size exclusion in the ranges of 100-500 Daltons (Da), 500-1 kDa, 1-2 kDa, 2-5 kDa, 5-10 kDa, 10-25 kDa, 25-50 kDa, 50-100 kDa, 100-250 kDa, 250-500 kDa, or 500 kDa or greater. In certain embodiments, size exclusion is approximately 2-5 kDa. In certain embodiments, the size exclusion is about 3 kDa. In other embodiments, filtration or ultrafiltration involves the use of hollow fiber membranes capable of isolating particles in the range of 100-500 Daltons (Da), 500-1 kDa, 1-2 kDa, 2-5 kDa, 5-10 kDa, 10-25 kDa, 25-50 kDa, 50-100 kDa, 100-250 kDa, 250-500 kDa, or 500 kDa or greater. In certain embodiments, the size exclusion is about 2-5 kDa. In certain embodiments, the size exclusion is about 3 kDa. In other embodiments, molecular weight cutoff (MWCO) gel filtration can isolate particles in the range of 100-500 Daltons (Da), 500-1 kDa, 1-2 kDa, 2-5 kDa, 5-10 kDa, 10-25 kDa, 25-50 kDa, 50-100 kDa, 100-250 kDa, 250-500 kDa, or even larger than 500 kDa. In certain embodiments, the size exclusion is about 2-5 kDa. In certain embodiments, the size exclusion is about 3 kDa. In various embodiments, such systems are used in combination with a variable fluid flow system.

[0051] In other embodiments, isolating a plurality of exosomes from a cell population involves the use of a tangential flow filtration (TFF) system, which is used to purify and / or concentrate the exosome fraction. In other embodiments, isolating a plurality of exosomes from a cell population involves the use of HPLC, which can also be used to purify exosomes into uniformly sized particles. In various embodiments, the density gradient used is, for example, centrifugation in a sucrose density gradient, or the application of a separate sugar cushion in the preparation.

[0052] In other embodiments, isolating a plurality of exosomes from a cell population involves the use of a precipitation reagent. For example, the precipitation reagent ExoQuick® can be added to conditioned cell medium to quickly and easily precipitate the exosome population. In other embodiments, isolating a plurality of exosomes from a cell population involves the use of a volume-excluding polymer (e.g., polyethylene glycol (PEG)). In another embodiment, isolating a plurality of exosomes from a cell population involves the use of flow field-flow fractionation (FIFFF), an elution technique.

[0053] In certain embodiments, isolating a plurality of exosomes from a cell population involves the use of one or more capture agents to isolate one or more exosomes bearing a specific biomarker or containing a specific biological molecule. In one embodiment, the one or more capture agents include at least one antibody. For example, an antibody immunoaffinity that recognizes an exosome-associated antigen is used to capture a specific exosome. In other embodiments, at least one antibody is conjugated to an immobilized surface, such as a magnetic bead, a chromatography matrix, a plate, or a microfluidic device, thereby enabling the isolation of a specific exosome population of interest. In other embodiments, isolating a plurality of exosomes from a cell population involves the use of one or more capture agents that are not antibodies. This includes, for example, the use of a "bait" molecule that displays antigenic features complementary to a corresponding molecule of interest on the exosome surface, such as a receptor or other coupling molecule. In one embodiment, the non-antibody capture agent is a lectin that can bind to polysaccharide residues on the exosome surface.

[0054] In various embodiments, the CDCs are mammalian. In other embodiments, the CDCs are human. As disclosed above, in some embodiments, synthetic exosomes are produced that can be isolated by mechanisms similar to those described above. In various embodiments, the composition that is a plurality of exosomes is a pharmaceutical composition that further comprises a pharmaceutically acceptable carrier.

[0055] In various embodiments, the plurality of exosomes ranges in size from 30 to 300 nm. In various embodiments, the plurality of exosomes ranges in size from 40 to 100 nm. In certain embodiments, the plurality of exosomes are cardiosphere-derived cell (CDC) exosomes. In certain embodiments, the plurality of exosomes comprises exosomes that are CD63+. In various embodiments, the exosomes comprise microRNAs miR-146a, miR-22, miR-24, miR-210, miR-150, miR-140-3p, miR-19a, miR-27b, miR-19b, miR-27a, miR-376c, miR-128, miR-320a, miR-143, miR-21, miR-130a, miR-9, miR-185, and / or miR-23a. In other embodiments, the exosomes are 2-5 kDa, for example 3 kDa. Other examples or embodiments of compositions and techniques involving exosomes are provided in WO 2014 / 028,493, which is incorporated herein by reference in its entirety.

[0056] Described herein are methods of treatment, comprising selecting a subject in need of treatment and administering to the individual a composition comprising a plurality of exosomes, wherein administering the composition treats the subject. In certain embodiments, the subject is in need of treatment for a disease and / or condition involving tissue damage or dysfunction. In other embodiments, the disease and / or condition involving tissue damage or dysfunction is cardiac disease. In other embodiments, the plurality of exosomes comprises exosomes comprising one or more microRNAs.

[0057] In certain embodiments, the plurality of exosomes is produced by a method comprising providing a cell population and isolating the plurality of exosomes from the cell population. In various embodiments, the cells are stem cells, progenitor cells, and / or progenitor cells. In other embodiments, the stem cells, progenitor cells, and / or progenitor cells are cardiosphere-derived cells (CDCs). In other embodiments, the stem cells, progenitor cells, and / or progenitor cells are pluripotent stem cells (pSCs), such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), derived from any one of a variety of somatic cell sources within the body, such as fibroblasts, blood and hematopoietic stem cells (hSCs), immune cells, bone and bone marrow, neural tissue, etc. In other embodiments, the stem cells, progenitor cells, and / or progenitor cells comprise hSCs, mesenchymal stem cells (MSCs), or endothelial progenitor cells (EPCs). In various embodiments, the cells are stem cells, progenitor cells, and / or progenitor cells derived from human biopsied tissue. In various embodiments, the cells are stem cells, and the progenitor cells and / or precursor cells are primary cultures.In various embodiments, the cells are stem cells, progenitor cells and / or precursor cells that can be serially subcultured to form cell lines.In certain embodiments, the exosomes are synthetic.

[0058] In various embodiments, the plurality of exosomes is derived from cardiosphere-derived cells (CDCs). In other embodiments, the plurality of exosomes comprises exosomes comprising one or more biological molecules. In other embodiments, the plurality of exosomes, when derived from CDCs, comprises exosomes that are enriched for one or more biological molecules compared to exosomes derived from a non-CDC source. In various embodiments, the one or more biological molecules are proteins, growth factors, cytokines, transcription factors, and / or morphogenetic factors. In other embodiments, the plurality of exosomes comprises exosomes enriched for one or more biological molecules, and, when derived from CDCs, comprises exosomes comprising microRNAs that further comprise enriched microRNAs compared to exosomes derived from a non-CDC source. In various embodiments, these microRNAs may include miR-146a, miR-22, miR-24, miR-210, miR-150, miR-140-3p, miR-19a, miR-27b, miR-19b, miR-27a, miR-376c, miR-128, miR-320a, miR-143, miR-21, miR-130a, miR-9, miR-185, and / or miR-23a. In several embodiments, the plurality of exosomes comprises one or more exosomes enriched in at least one of miR-146a, miR-22, and miR-24.

[0059] In various embodiments, the CDCs are mammalian. In other embodiments, the CDCs are human. In certain embodiments, the exosomes are synthetic. In certain embodiments, synthetic exosomes have substantially similar contents (e.g., microRNAs, biological molecules) as CDC-derived exosomes.

[0060] In various embodiments, administration of multiple exosomes alters gene expression in damaged or dysfunctional tissue, improves the viability of damaged tissue, and / or enhances the regeneration or production of new tissue in an individual. In various embodiments, the amount of exosomes administered to achieve these effects is greater than or equal to 1×10 6 pieces~1×10 7 pieces, 1×107 pieces~1×10 8 pieces, 1×10 8 pieces~1×10 9 pieces, 1×10 9 pieces~1×10 10 pieces, 1×10 10 pieces~1×10 11 pieces, 1×10 11 pieces~1×10 12 Range of 1×10 12 In other embodiments, the number of exosomes is related to the number of cells used in a clinically relevant dose for cell therapy methods, e.g., 3 mL / 3 x 10 5 Because it has been demonstrated that CDCs can provide therapeutic benefit in intracoronary administration, the number of exosomes is determined from the number of cells in a clinically relevant dose for cell therapy methods. In various embodiments, the administration can be repeated. In another example, the number of CDCs administered includes 25 million intracoronary CDCs per coronary artery (i.e., a total of 75 million CDCs) as another baseline for exosome administration. In various embodiments, the number of CDCs administered includes 1 x 10 in a single dose as another baseline for exosome administration. 5 pieces, 1×10 6 pieces, 1×10 7 pieces, 1×10 8 pieces, 1×10 9 In certain cases, this may be proportional to body weight (ranging from 100,000 to 1 million CDCs per kg of body weight for a total CDC dose). In various embodiments, the exosome amount may be defined by the amount of protein, for example, 1-10 mg, 10-25 mg, 25-50 mg, 50-75 mg, 75-100 mg, or a dose containing 100 mg or more of exosomal protein.

[0061] Defining effective dose ranges, dosing regimens, and routes of administration can be guided by studies using fluorescently labeled exosomes and measuring target tissue retention (which can be greater than 10-fold, greater than 50-fold, or greater than 100-fold above background when measured at 5, 10, 15, 30, or more minutes as a screening criterion). In certain embodiments, greater than 100-fold above background measured at 30 minutes is the baseline measurement for low and high doses, which are then evaluated for safety and bioactivity (e.g., using MRI endpoints: scar size, global and regional function). In various embodiments, a single dose is compared to two, three, four, or more sequential doses. In various embodiments, administration can be repeated. In various embodiments, repeated or sequential administration is provided for the treatment of acute diseases and / or conditions. In various embodiments, repeated or sequential administration is provided for the treatment of chronic diseases and / or conditions.

[0062] In various embodiments, administration of exosomes to a subject is carried out by any of the techniques known in the art. In some embodiments, this includes transdermal delivery. In other embodiments, myocardial injection is used, for example, using an intracoronary catheter. In various embodiments, delivery can be intra-arterial or intravenous. Additional delivery sites include any one or more cardiac compartments, such as arterial, venous, and / or ventricular sites. In certain embodiments, administration can include delivery to a tissue or organ site different from the site or the diseased and / or dysfunctional tissue. In certain embodiments, delivery is by inhalation or oral administration.

[0063] In various embodiments, administration of a plurality of exosomes alters gene expression in damaged or dysfunctional tissue, improves the viability of the damaged tissue, and / or enhances the regeneration or production of new tissue in an individual. In various embodiments, administration of the exosomes results in functional improvement in the tissue. In several embodiments, the damaged or dysfunctional tissue comprises cardiac tissue.

[0064] For example, in certain embodiments where cardiac tissue is damaged or dysfunctional, functional improvement may include (among other functional improvements) increased cardiac output, contractility, ventricular function, and / or reduced arrhythmias. Functional improvements may also be achieved in other tissues, such as enhanced cognition in response to treatment of neuronal injury, improved blood oxygen transfer in response to treatment of pulmonary injury, and improved immune function in response to treatment of damaged immunologically relevant tissue.

[0065] In various embodiments, administration of a plurality of exosomes alters gene expression in damaged or dysfunctional tissue, improves the viability of the damaged tissue, and / or enhances the regeneration or production of new tissue in an individual. In various embodiments, administration of the exosomes results in functional improvement in the tissue. In several embodiments, the damaged or dysfunctional tissue comprises skeletal muscle tissue.

[0066] For example, in certain embodiments where skeletal muscle tissue is damaged or dysfunctional, functional improvement may include increased contractile force, improved walking ability (e.g., increased 6-minute walk test results), improved ability to rise from a sitting position, improved ability to sit from a supine or lying position, or improved manual dexterity (pointing and / or clicking a mouse).

[0067] In various embodiments, the damaged or dysfunctional tissue requires repair, regeneration, or functional improvement due to an acute event. Acute events include, but are not limited to, trauma, such as laceration, contusion, or crush injury, shock, loss of blood or oxygen flow, infection, chemical or heat exposure, toxic or toxin exposure, drug overuse or overexposure, etc. In other embodiments, the damaged tissue is cardiac tissue, and the acute event comprises myocardial infarction. In some embodiments, administration of exosomes results in an increase in cardiac wall thickness in the infarcted region.

[0068] In other embodiments, tissues are also damaged by chronic diseases, such as congestive heart failure (including conditions secondary to diseases such as Duchenne muscular dystrophy, ischemic heart disease, hypertension, valvular heart disease, dilated cardiomyopathy, infection, diabetes, etc.). In various embodiments, administration can be repeated, e.g., two, three, four, or more sequential administrations. In various embodiments, repeated or sequential administrations are provided for the treatment of acute diseases and / or conditions. In various embodiments, repeated or sequential administrations are provided for the treatment of chronic diseases and / or conditions.

[0069] Other sources of damage include, but are not limited to, injury, age-related degeneration, cancer, and infection. In some embodiments, the regenerative cells are derived from the same tissue type as that requiring repair or regeneration. In other embodiments, the regenerative cells are derived from a tissue type other than the tissue requiring repair or regeneration.

[0070] In certain embodiments, the method of treatment comprises selecting a subject in need of treatment for a cardiac-related disease and / or condition and administering to the individual a composition comprising a plurality of exosomes, wherein administering the composition treats the subject. In various embodiments, the cardiac-related disease and / or condition comprises heart failure, further comprising Duchenne muscular dystrophy-associated heart failure. In various embodiments, the plurality of exosomes ranges in size from 30 to 300 nm. In various embodiments, the plurality of exosomes ranges in size from 40 to 100 nm. In certain embodiments, the plurality of exosomes are cardiosphere-derived cell (CDC) exosomes. In certain embodiments, the plurality of exosomes comprises exosomes that are CD63+. In various embodiments, the exosomes comprise the microRNAs miR-146a, miR-22, miR-24, miR-210, miR-150, miR-140-3p, miR-19a, miR-27b, miR-19b, miR-27a, miR-376c, miR-128, miR-320a, miR-143, miR-21, miR-130a, miR-9, miR-185, and / or miR-23a. In other embodiments, the exosomes are 2-5 kDa, e.g., 3 kDa. In other embodiments, administration of the composition is at least 1 x 10 8 pieces, 1×10 8 pieces~1×10 9 pieces, 1×10 9 pieces~1×10 10 pieces, 1×10 10 pieces~1×10 11 pieces, 1×10 11 pieces~1×10 12 pieces, 1×10 12 For example, 3 mL / 3 x 10 exosomes. 5Because it has been demonstrated that CDCs can provide therapeutic benefit in intracoronary administration, the number of exosomes is determined from the number of cells in a clinically relevant dose for cell therapy methods. In various embodiments, the administration can be repeated. In another example, the number of CDCs administered includes 25 million intracoronary CDCs per coronary artery (i.e., a total of 75 million CDCs) as another baseline for exosome administration. In various embodiments, the number of CDCs is 1 x 10 in a single dose. 5 pieces, 1×10 6 pieces, 1×10 7 pieces, 1×10 8 pieces, 1×10 9 In certain cases, this may be proportional to body weight (ranging from 100,000 to 1 million CDCs per kg of body weight for a total CDC dose). In various embodiments, the exosome amount may be defined by the amount of protein, e.g., a dosage containing 1-10 mg, 10-25 mg, 25-50 mg, 50-75 mg, 75-100 mg, or 100 mg or more of exosome protein. In various embodiments, administration of the composition comprises multiple administrations of exosomes. In various embodiments, repeated or sequential administrations are provided for the treatment of acute diseases and / or conditions. In various embodiments, repeated or sequential administrations are provided for the treatment of chronic diseases and / or conditions. In other embodiments, administration of the composition comprises transdermal injection. In other embodiments, administration of the composition comprises intramyocardial injection. In other embodiments, administration of the composition comprises use of an intracoronary catheter. In other embodiments, administration of the composition comprises intra-arterial or intravenous delivery.

[0071] Further described herein are methods for improving cardiac function in a subject, comprising selecting a subject and administering to the individual a composition comprising a plurality of exosomes, wherein administering the composition improves cardiac function in the subject. In other embodiments, improved cardiac function may be demonstrated, for example, by an improvement in baseline ejection volume. In other embodiments, improved cardiac function is related to increased viable tissue, reduced scar volume, improved wall thickness, regenerative remodeling of the injured site, enhanced angiogenesis, improved cardiomyogenic effects, reduced apoptosis, and / or reduced levels of pro-inflammatory cytokines.

[0072] In certain embodiments, a method for improving cardiac function comprises selecting a subject in need of treatment for a cardiac-related disease and / or condition and administering to the individual a composition comprising a plurality of exosomes, wherein administering the composition treats the subject. In various embodiments, the cardiac-related disease and / or condition comprises heart failure, further comprising Duchenne muscular dystrophy-associated heart failure. In various embodiments, the plurality of exosomes ranges in size from 30 to 300 nm. In various embodiments, the plurality of exosomes ranges in size from 40 to 100 nm. In certain embodiments, the plurality of exosomes are cardiosphere-derived cell (CDC) exosomes. In certain embodiments, the plurality of exosomes comprises exosomes that are CD63+. In various embodiments, the exosomes comprise the microRNAs miR-146a, miR-22, miR-24, miR-210, miR-150, miR-140-3p, miR-19a, miR-27b, miR-19b, miR-27a, miR-376c, miR-128, miR-320a, miR-143, miR-21, miR-130a, miR-9, miR-185, and / or miR-23a. In other embodiments, the exosomes are 2-5 kDa, e.g., 3 kDa. In other embodiments, administration of the composition is at least 1 x 10 8 pieces, 1×10 8 pieces~1×10 9 pieces, 1×10 9 pieces~1×10 10 pieces, 1×10 10pieces~1×10 11 pieces, 1×10 11 pieces~1×10 12 pieces, 1×10 12 For example, 3 mL / 3 x 10 exosomes. 5 Because it has been demonstrated that CDCs can provide therapeutic benefit in intracoronary administration, the number of exosomes is determined from the number of cells in a clinically relevant dose for cell therapy methods. In various embodiments, the administration can be repeated. In another example, the number of CDCs administered includes 25 million intracoronary CDCs per coronary artery (i.e., a total of 75 million CDCs) as another baseline for exosome administration. In various embodiments, the number of CDCs is 1 x 10 in a single dose. 5 pieces, 1×10 6 pieces, 1×10 7 pieces, 1×10 8 pieces, 1×10 9 In certain cases, this may be proportional to body weight (ranging from 100,000 to 1 million CDCs per kg of body weight for a total CDC dose). In various embodiments, the exosome amount may be defined by the amount of protein, e.g., a dosage containing 1-10 mg, 10-25 mg, 25-50 mg, 50-75 mg, 75-100 mg, or 100 mg or more of exosomal protein. In various embodiments, administration of the composition comprises multiple administrations of exosomes. In various embodiments, repeated or sequential administrations are provided for the treatment of acute diseases and / or conditions. In various embodiments, repeated or sequential administrations are provided for the treatment of chronic diseases and / or conditions. In other embodiments, administration of the composition comprises transdermal injection. In other embodiments, administration of the composition comprises intramyocardial injection. In other embodiments, administration of the composition comprises use of an intracoronary catheter. In other embodiments, administration of the composition comprises intra-arterial or intravenous delivery.

[0073] Herein, we demonstrate that cardiosphere-derived cells (CDCs) reverse key pathophysiological features of Duchenne cardiomyopathy (oxidative stress, inflammation, fibrosis, and mitochondrial dysfunction) in mdx mice in advanced clinical trials for therapeutic regeneration after myocardial infarction. Exosomes secreted by human CDCs recapitulate the benefits of CDCs in mdx mice and reverse abnormalities in calcium cycling and mitochondrial respiration in human Duchenne cardiomyocytes. Both CDCs and their exosomes improve cardiac function in mdx mice; a single injection of CDCs is sufficient to increase maximal exercise capacity and improve survival. Delivery of a microRNA (miR-148a) enriched in CDC exosomes mimics the key effects of CDCs and CDC exosomes. Thus, CDCs ameliorate Duchenne cardiomyopathy via exosome-mediated transport of signaling molecules, including miR-148a. These findings motivate clinical trials of CDCs in patients with Duchenne cardiomyopathy. [Example]

[0074] Example 1 CDC culture Endocardial biopsies from the right ventricular surface of the interventricular septum were obtained from healthy hearts of deceased tissue donors. Cardiosphere-derived cells were derived as previously described. See Makkar et al., (2012) "Intracoronary cardiosphere-derived cells for heart regeneration after myocardial infarction (CADUCEUS): a prospective, randomized phase 1 trial." Lancet 379, 895-904 (2012), which is incorporated herein by reference in its entirety.

[0075] Briefly, cardiac biopsies were minced into small fragments and briefly digested with collagenase. The explants were then cultured on 20 mg / ml fibronectin-coated dishes. Interstitial-like squamous cells and luminal round cells spontaneously grew from the tissue fragments and reached confluence within 2–3 weeks. These cells were harvested using 0.25% trypsin and cultured in suspension on 20 mg / ml poly-d-lysine to form self-aggregating cardiomyosphers. Cardiomyospheric-derived cells (CDCs) were obtained by seeding and subculturing the cardiomyosphers on fibronectin-coated dishes. All cultures were maintained at 5% CO2 and 37°C using IMDM basal medium supplemented with 20% FBS, 1% penicillin / streptomycin, and 0.1 ml of 2-mercaptoethanol.

[0076] Example 2 Media conditioning and exosome purification Exosomes are harvested from CDCs at passage 4. Exosomes can also be isolated from normal human dermal fibroblasts (NHDFs), cells previously used as a control that do not provide any beneficial effects.

[0077] CDCs and NHDFs were conditioned for 15 days at 100% confluence in serum-free medium. The aspirated medium was then centrifuged at 3,000 × g for 15 minutes to remove cellular debris. Exosomes were then isolated using Exoquick exosome precipitation solution.

[0078] The exosome pellet is resuspended in the appropriate medium and used in the assay. Expression of the stored exosome marker CD63 is verified using ELISA. The RNA content of the exosome pellet can also be quantified using a Nanodrop spectrophotometer. To generate miR-146a-deficient exosomes, CDCs are transfected in suspension with miRIDIAN miR-146a hairpin inhibitor or miRIDIAN hairpin control and plated onto fibronectin-coated flasks. Exosomes are isolated from serum-free conditioned medium (48 h conditioned).

[0079] Example 3 Exosomal RNA degradation Exosomal RNA degradation is performed by suspending the exosome pellet in 2 ml of PBS. To one sample, 100 ml of Triton X-100 (Sigma Aldrich) is added to achieve a 5% triton concentration. Exosomes are treated with 0.4 mg / ml RNase A for 10 minutes at 37°C. Samples are further treated with 0.1 mg / ml proteinase K for 20 minutes at 37°C. RNA is purified from samples using a microRNA isolation kit. RNA levels are measured using a Nanodrop.

[0080] Example 4 Mass spectrometry analysis of exosome pellets Proteins were prepared for digestion using the filter-aided sample preparation (FASP) method. Concentration was measured using a Qubitfluorometer. Trypsin was added at a 1:40 enzyme-to-substrate ratio and the sample was incubated overnight on a 37°C heat block. The device was centrifuged and the filtrate was collected. The digested peptides were desalted using a C18 stop-and-go extraction (STAGE) tip. Peptides were fractionated by strong anion-exchange STAGE tip chromatography. Peptides were eluted from the C18 STAGE tip and dried. Each fraction was analyzed using liquid chromatography-tandem mass spectrometry. The sample was loaded onto a 2 cm x 100 mm trap column. The analytical column was a 13 cm x 75 mm fused silica column with a pulled-tip emitter. The mass spectrometer was programmed to acquire tandem mass spectra from the top 15 ions in a full scan from 400 to 1,400 m / z using data-dependent acquisition. Use msconvert to convert the mass spectrometer raw data files to MGF format. Use X!Hunter to search the MGF files against the latest spectral library available in GPM at the time. Also, use X!!Tandem, which uses both native and k-score scoring algorithms, to search the MGF files by OMSSA. Proteins must have at least one unique peptide with a peptide E-value score of 0.01 or less in X!!Tandem, 0.01 or less, 0.001 or less in OMSSA, and a theta value of 0.5 or greater in X!Hunter searches, and a protein E-value score of 0.0001 or less in X!!Tandem and X!Hunter.

[0081] Example 5 Muscle cell isolation and angiogenesis assay Various cell types can be used for studies demonstrating the effects of exosome application. For example, neonatal rat cardiomyocytes (NRCMs) can be isolated from 1- to 2-day-old Sprague-Dawley rat pups and cultured as monolayers. Another useful source includes human umbilical vein endothelial cells plated on growth factor-deficient Matrigel (BD Biosciences) to assay angiogenesis.

[0082] The cells were then diluted to 7 x 10 8 and 4.0 x 10 8 The cells were incubated with CDC exosomes or NHDF exosomes. The difference in dose reflects the different exosome production from the cells during acclimation. Because the cells were able to produce exosomes under similar conditions, the relative doses may represent the relative exosome production in vivo. After 4 hours, tube formation was measured.

[0083] Example 6 In vitro cardiomyocyte assay, exosome treatment The inventors used 1.5 × 10 4 NRCMs were plated onto fibronectin-coated 8-chamber slides. After 5 days, the medium was replaced with 3.5 × 10 8 pcs or 2 x 10 8The medium was replaced with fresh new medium containing CDCs or NHDF exosomes. The cells were then fixed with 4% paraformaldehyde for 30 minutes at 4°C. The chambers were washed three times with cold PBS, then blocked and permeabilized with Dako / 0.1% saponin (Invitrogen) for 1 hour at 37°C. The cells were incubated overnight with rabbit anti-Ki-67 (1:100) primary antibody and mouse anti-α-sarcomeric actinin (Abeam) at 4°C. The cells were then washed three times with PBS and incubated with goat anti-mouse (Cy5) and goat anti-rabbit (FITC) in TUNEL staining solution for 1 hour at 37°C. The slides were then washed three times with PBS, stained with 1:8,000 40,6-diamidino-2-phenylindole staining solution, and mounted using Prolong antifade solution (Invitrogen). The slides were imaged using confocal microscopy.

[0084] Example 7 Cardiomyocyte stress assay The first injury model involves the use of NRCMs plated in monolayer on fibronectin-coated 12-well plates and treated with either 40 nM miR-146a or a mimic for 24 hours. The medium was then replaced, and the cells were washed three times with PBS. Cells were then stressed using hydrogen peroxide (100 mM H2O2 in serum-free medium for 2 hours) or cobalt chloride (5 mM CoCl2 in serum-free medium for 2 hours). Cells were washed with PBS and viability was measured by treating them with 20 mM calcein in PBS for 20 minutes at 37°C in the dark. Fluorescence was read using a Soft Max Pro 5 Plate Reader (Molecular Devices). Data per well is the average of nine consecutive measurements.

[0085] The second model involves plating cardiomyocytes on 25 mm pre-coated glass coverslips (Fischer Scientific) in 6-well plates. Cells are stressed with 50 mM H2O2 for 15 minutes, followed by incubation with transwell membrane inserts containing CDC or with CDC exosomes for 4 hours. Cells are then washed, fixed with paraformaldehyde, and stained for analysis as described above.

[0086] Example 8 Exosome inhibition in CDC CDCs were grown to confluence in T175 flasks. For in vitro studies, CDCs were conditioned for 15 days in serum-free medium containing 20 mM GW4869 (Sigma Aldrich) or an equal volume of DMSO. For in vitro transwell insert assays of cardiomyocyte stress, CDCs could be treated with 20 mM GW4869 (Sigma Aldrich) or 5 mM spiroepoxide (Santa Cruz Biotechnology) for 12 hours. CDCs were washed three times with PBS and replaced with serum-free medium. The inserts containing treated CDCs were added to 6-well plates containing cardiomyocytes. For in vivo studies, CDCs were treated with 20 mM GW4869 or an equal volume of DMSO for 12 hours. Before injection, CDC flasks were washed twice with PBS, trypsinized, and counted; 10 per animal. 5 CDC was injected.

[0087] Example 9 Acute and chronic myocardial infarction models Three-month-old male severe combined immunodeficiency (SCID) beige mice were anesthetized with isoflurane. After surgical preparation, a 2 cm vertical incision was made at the midclavicular line for lateral thoracotomy. The left anterior descending vein was ligated using 7-0 silk. Exosomes, microRNA, CDC, or media control were injected into two peri-infarct sites in the animals at a volume of 40 ml per injection.

[0088] In the chronic model of MI, animals are infarcted as described above without any treatment. After 3 weeks, animals are treated as described above. For exosome treatment, the pellet is resuspended in Iscove's Modified Dulbecco's (IMDM) basal medium. 2.8 x 10 9 and 1.56 x 10 9 Animals are injected with CDC and NHDF exosomes. 80 ng of miPv-146a or microRNA mimic control is injected into microRNA-treated animals. Briefly, miRIDIAN miR-146a or miRIDIAN negative control is vortexed in Dharmafect (Thermo Scientific) transfection reagent and IMDM basal medium and incubated at room temperature for 10 minutes to allow complex formation. The microRNA complex is resuspended in IMDM for injection. For CDC treatment, 10 5 CDCs are injected into animals as described above.

[0089] Echocardiography. SCID beige mice were evaluated by echocardiography using a Vevo 770 imaging system (Visual Sonics) 24 hours after surgery (baseline), 14 days, and 4 weeks after surgery. After induction of light general anesthesia, hearts were imaged in 3D in the long axis view at the level of the maximum left ventricular diameter. Left ventricular ejection fraction (LVEF) was measured from 2D images of the LV end-diastolic and end-systolic areas using Visual Sonics version 1.3.8 software. Each animal was measured multiple times per time point, and the average was used for statistical analysis.

[0090] Example 10 histology Four weeks after MI, the animals are sacrificed. The hearts are harvested and a transverse cut is made slightly above the MI suture. The distal portion is then embedded in a base mold / embedding ring block in optimal cutting temperature solution. The blocks are flash-frozen by immersion in cold 2-methylbutane. The hearts are sectioned at a thickness of 5 mm.

[0091] Example 11 Masson's trichrome staining Two slides containing a total of four sections per heart were stained using Masson's trichrome stain. Briefly, the sections were treated overnight in Bouin's solution. The slides were then rinsed under running water for 10 minutes and stained with Weigert's hematoxylin for 5 minutes. The slides were then rinsed, stained with scarlet acid fuchsin for 5 minutes, and rinsed again. The slides were then further stained with phosphotungsten / phosphomolybdenum, aniline blue, and 2% acetic acid for 5 minutes each. The slides were then rinsed, dried, and mounted using DPX mounting medium.

[0092] Example 12 Morphometry Morphometric analysis of cardiac sections was performed using Image J software. Briefly, 2D images of stained sections were divided into blue, red, and green channels (only blue was used). Infarct size could be established by measuring the area and intensity of blue in each section to calculate infarct size. The percentage of viable myocardium and infarct volume was calculated by averaging the infarct fraction of the four sections analyzed per heart. The infarct volume and viable myocardium volume were calculated as the product of infarcted or viable tissue, the average mouse heart height (3 mm), and the specific gravity of cardiac tissue (1.05 g / ml). The infarct wall thickness was calculated by measuring the thinnest area of ​​the infarct. In chronic models of MI, where significant hypertrophy and adverse remodeling have occurred, the viable myocardium volume of each heart can be adjusted based on the amount of cardiomyocytes produced in the tissue.

[0093] The amount of myocytes was determined by measuring the cross-sectional area of ​​vertically sectioned cardiomyocytes (defined as round cells with red cytoplasm and a visible central nucleus). We measured at least 25 myocytes per heart. The volume of myocytes was quantified using a simple cylindrical assumption; mass was determined by multiplying the volume by the specific gravity of cardiomyocytes (1.15 mg / ml). The amount of viable myocardium in each mouse heart was divided by the amount of cardiomyocytes in that heart.

[0094] Example 13 CDC exosomes enhance angiogenesis and promote cardiomyocyte survival and proliferation Exosomes were isolated from serum-free medium conditioned by cultured human CDCs (or normal human dermal fibroblasts (NHDFs) as a therapeutically inactive control) for 15 days (Figure 7, available online). Despite not regularly changing the medium, most CDCs remained viable by the end of the conditioning period (Figures 7B and 7C). Purified exosome pellets were enriched in RNA (Figure 1A). We confirmed the presence of RNA within exosomes by exposing the pellets to RNase A in the presence of 5% triton (Figure 1B) and adding proteinase K to dissociate protein complexes that may occlude the RNA. Mass spectrometry confirmed the presence of conserved exosome biogenesis proteins, including CD63 (Figure 1C), which we used to quantify exosome yield (Figure 1D). Transmission electron microscopy revealed that most exosomes were 30-90 nm in diameter, but smaller and larger particles were also present (Figures 1E and 1F), consistent with reports of vascular cell-derived exosomes. In vitro assays revealed the primary effects of CDC-exosomes on angiogenesis, cardiomyocyte proliferation, and apoptosis.

[0095] CDC exosomes, but not NHDF exosomes, promoted tube formation (an indicator of enhanced angiogenesis) in human umbilical endothelial cells (Figure 1G). CDC exosome-treated neocardiomyocytes proliferated more than those exposed to NHDF exosomes or medium alone, as evidenced by a higher percentage of Ki67-positive nuclei (Figure 1H). In addition, CDC exosome-treated cardiomyocytes showed fewer terminal deoxynucleotidyl transferase nick-end labeling (TUNEL)-positive nuclei (Figure 1I). Thus, CDC exosomes stimulate angiogenesis, promote cardiomyocyte proliferation, and reduce programmed cell death. These effects recapitulate those of the parent CDC.

[0096] Example 14 CDC exosomes improve cardiac function, confer structural benefits, and increase viable myocardial mass after MI Although CDCs are known to stimulate functional improvement and regeneration in infarcted myocardium in both animals and humans, whether CDC-derived exosomes can recapitulate or are essential for these processes remains a key question for this technology. To evaluate therapeutic efficacy in an established preclinical model, we induced acute MI in immunodeficient mice and then injected CDC exosomes, NHDF exosomes, or serum-free medium into the MI border zone.

[0097] At 15 and 30 days after injection, animals injected with CDC-exosomes showed superior overall cardiac function compared with NHDF-exosome or medium controls (Figure 2A). At the histological level, CDC-exosome-treated hearts showed reduced scar volume, increased viable myocardium, and increased infarct wall thickness compared with NHDF-exosome and medium controls (Figures 2B-2E). CDC-exosome-treated hearts also had lower pro-inflammatory cytokine levels (Figure 8). In all these respects, CDC-exosomes mimic the known benefits of CDC itself.

[0098] Although acute MI models are widely used to assess physiological activity, they cannot distinguish between cardioprotective effects and true regeneration. To distinguish between these two, we performed another series of experiments in which we injected exosomes 21 days after MI (at which point myocardial scarring was well established). Three weeks later, CDC-exosome-injected hearts demonstrated multiple structural and functional benefits: improved ejection fraction (Figure 2F; furthermore, improved functional area change, Figure 9A), reduced scar volume (representative images in Figure 2G and pooled data in Figure 2H), increased viable myocardium (Figure 2I), and thickened infarct walls (Figure 2J). Furthermore, compared with significantly deformed control hearts, CDC-exosome-treated hearts demonstrated reduced chamber enlargement (Figures 9B and 9C), reduced infarct perimeter (Figure 9D), and reduced compensatory myocyte hypertrophy (Figures 9E and 9F). CDC-exosome-treated hearts exhibited increased microvascular density (Figures 2K and 9G) and a low frequency of apoptotic cardiomyocyte nuclei (Figures 9H and 9I). Net growth of new myocardium in the setting of established scar fulfills the central criteria of therapeutic regeneration; improved function and reduced adverse remodeling are evidence of physiologically relevant tissue changes. We conclude that CDC-exosomes indeed mediate true cardiac regeneration, promoting angiogenesis and tissue preservation.

[0099] Example 15 Inhibition of exosome secretion reduces the benefits of CDC If exosomes mediate the therapeutic effects of CDC transplantation, it is logical to expect that inhibiting exosome secretion would block this benefit. To test this idea, we treated CDCs with GW4869, a reversible inhibitor of neutral sphingomyelinase that prevents exosome release. Exposure to GW4869 dose-dependently blocked exosome production (Figure 3A), with complete inhibition at 20 mM (a dose without apparent short-term cytotoxicity; e.g., no impairment of proliferation; Figure 3B).

[0100] Inhibition of exosome release abolished the indirect benefits of CDC in vitro, as medium conditioned by GW4869-treated CDCs did not enhance cardiomyocyte proliferation or reduce apoptosis (Figures 3C and 3D). Spiroepoxide, a specific, irreversible inhibitor of neutral sphingomyelinase, mimicked the anti-apoptotic effects of GW4869 on stressed cardiomyocytes (Figures 10A and 10B). In vivo, in contrast to vehicle-only (DMSO) controls, which conferred all the expected therapeutic effects of CDC, CDCs pretreated with GW4869 conferred no functional (Figure 3E) or structural (Figures 3F-3I) benefits in acute MI. Thus, exosome secretion by CDCs is required for CDC-mediated benefits in vitro and in vivo.

[0101] Example 16 CDC exosomes are enriched in miR-146a, which plays a key role in MI pathology To investigate the basis for the therapeutic benefit of CDC exosomes, we compared their microRNA repertoire with that of NHDF exosomes using PCR microarrays of the 88 best-defined microRNAs. The microRNA content of the two cell types differed dramatically. 43 microRNAs were differentially present in the two groups; among these, miR-146a was most abundant in CDC exosomes (262-fold more abundant than in NHDF exosomes; Figures 4A, 4B, and 11). Furthermore, tissue levels of miR-146a were increased in post-MI hearts from animals injected with CDC exosomes compared with those injected with NHDF exosomes (Figure 4C), plausibly supporting the idea that CDC exosomes may act via miR-146a transport. Exposure of neonatal rat cardiomyocytes to miR-146a mimics increased cardiomyocyte viability and protected them from oxidative stress (Figures 4D and 12A). Whole-transcriptome microarrays revealed downregulation of Irak1 and Traf6, two signaling mediators of the TLR-NFkB pathway that are known targets of miR-146a (Figure 4E). Our pathway analysis revealed alterations in pathways involved in cell survival, cell cycle, cell organization, and morphology, all of which are associated with ischemic injury (Figure 11D) and share a connection with the basal transcription factor Myc (Figure 11E). To demonstrate the biological role of miR-146a in myocardial injury, we induced acute MI in miR-146a knockout (146aKO) mice and compared them with the same strain of wild-type mice (WT) and 146aKO mice (146aKO-R) that were "rescued" by injection of a miR-146a mimic at the time of MI.

[0102] After MI, 146aKO mice exhibited significantly impaired cardiac function and adverse remodeling compared with WT or 146aKO-R mice (Figures 4F and 4G). Histological analysis revealed significantly increased scar volume (Figure 4H) and decreased infarct wall thickness (Figure 4J) in 146aKO, but not WT or 146aKO-R, mice. The 146aKO-R group had the greatest amount of viable myocardium (Figure 4I), likely indicating a supraphysiological effect of the injected miR-146a mimic. These findings indicate a critical role for miR-146a in MI and provide reason to speculate that miR-146a may mediate some of the therapeutic benefits of CDC-exosomes.

[0103] Importantly, we further demonstrated that miR-146a resulted in thickened infarct walls and increased tissue survival in a mouse model of myocardial infarction. To investigate the contribution of miR-146a to the larger exosome effect, we developed miR-146a-deficient exosomes by transfecting CDCs with a miR-146a hairpin inhibitor (or a control hairpin), followed by conditioning the medium and isolating exosomes. Successful miR-146a knockdown was confirmed by qPCR on the resulting exosomes and on NRVMs exposed to either control or miR-146a-deficient exosomes (Figures 12B and 12C). The anti-apoptotic effect of CDC exosomes was evident by comparing TUNEL positivity in untreated NRVMs (left column, Figure 5A) with that in NRVMs treated with control CDC exosomes (right column, Figure 5A). Although miR-146a-deficient exosomes conferred less protection from oxidative stress than control CDC exosomes, their suppression of apoptosis was still significant. These data suggest that miR-146a underlies some, but not all, of the beneficial effects of CDC exosomes. To further demonstrate this issue in vivo, we used the same MI model as in Figure 2, except that mice were injected with either miR-146a mimics or a microRNA mimic control. Mice injected with miR-146a mimics during acute MI showed improved pump function (Figure 5B), reduced scar volume, and increased viable cardiac tissue (Figures 5C-5F). In a chronic MI model, where regeneration can be studied more rigorously, animals treated with miR-146a showed only modest, statistically insignificant functional improvements (Figures 5G and 13A). Furthermore, histological analysis showed no difference in scar volume (Figures 5H and 5I). However, hearts treated with miR-146a mimics showed increased viable tissue, thickened infarct walls (Figures 5J and 5K), and reduced adverse remodeling (Figures 13B-13D) compared with controls. Assessment of angiogenesis revealed no significant differences between the two groups (Figures 5L and 13G).However, consistent with the in vitro data (Figure 5A), a reduced frequency of cardiomyocyte apoptosis was observed in miR-146a-injected hearts (Figures 13H and 13I). Thus, in a chronic MI model, miR-146a recapitulates the cardiomyogenic and anti-apoptotic effects but not the other functional and structural benefits of CDC-exosomes (see Figures 2F-2J and 9A-9C). Exogenous miR-146a is known to suppress ischemia / reperfusion injury through targeting Irak-1 and Traf6, both of which are involved in the toll-like receptor (TLR) signaling pathway. TLR signaling, which underlies innate immunity, plays a major role in the pathology of sterile inflammation, including MI.

[0104] The reduction of pro-inflammatory cytokines by CDC-exosomes (Figure 8) and the suppression of Irak1 and Traf6 by miR-146a (Figure 4C), which is increased in CDC-exosome-injected hearts, are consistent with blunted TLR signaling. Additionally, miR-146a suppresses NOX-4 (which has been shown to confer oxidative stress and promote cardiac injury) and SMAD4 (a member of the transforming growth factor b (TGF-b) fibrotic pathway). To confirm that these targets were indeed downregulated, we performed Western blot analysis of chronic MI hearts 7 days after treatment with miR-146a. Indeed, all of the above targets were silenced in miR-146a-treated hearts compared with mimic controls (Figures 6A and 6B). We also found lower levels of myeloperoxidase (a proxy for neutrophil infiltration).

[0105] Example 17 Differences in baseline ejection fraction between different mouse strains We observed significantly higher baseline ejection fractions in these animals. This difference was speculated to be due to differences in the background of the mouse strain (C57BL6) used for the knockout. In all other experiments in this paper, the mouse strain used was SCID-Beige. SCID-Beige mice lack mature B and T cells and natural killer (NK) cells. Because their responses to injury differ, this fundamental difference in immune competence is likely the primary cause of the differences in baseline measurements. For most of the experiments in this paper, we chose SCID-Beige mice because they are permissive to human cells (which are the source of CDCs and exosomes). However, an appropriate control for the 146aKO mice would have been a wild-type mouse of the same background strain as the BL6 background. This has been previously demonstrated. Strain has previously been shown to be an important determinant of wound healing after myocardial infarction.

[0106] Example 18 Effect of miR-146a on immune infiltration Attenuation of the inflammatory immune response does not necessarily abolish it completely. Innate immune cells, including macrophages, have been shown to play a role in promoting regeneration. Furthermore, unpublished data from our laboratory show that macrophage trafficking is unaffected by CDC treatment, but that CDC-treated macrophages switch from a pro-inflammatory M1 to an anti-inflammatory and pro-healing M2 phenotype.

[0107] Example 19 Consideration Cardiosphere-derived cells have been shown to induce therapeutic regeneration in infarcted human hearts. In a form of injury traditionally considered irreversible, CDC resulted in scar reduction and the growth of new functional myocardium. Similar effects have been confirmed in animal models. Herein, we show that exosomes recapitulate CDC-induced therapeutic regeneration and that inhibiting exosome production impairs the benefits of CDC. Exosomes contain microRNAs that have the ability to alter cellular behavior through paracrine mechanisms (Figure 6B). Among these, we identified miR-146a as being particularly abundant in CDC exosomes. When administered alone, miR-146a recapitulates some, but not all, of the remarkable benefits of CDC and CDC exosomes (Figure 3). Similarly, miR-146a-deficient exosomes were still able to suppress cardiomyocyte apoptosis, albeit more weakly than in the presence of miR-146a (Figure 5A). Treatment of hearts with miR-146a in chronic models of MI (where scarring is persistent) reproduces the increased amount of viable myocardium that characterizes therapeutic regeneration, but fails to mimic two important beneficial effects of CDC-exosomes (reduced scar volume and improved overall function): the increase in viable myocardium is not sufficient to enhance function, likely because miR-146a-induced angiogenesis is insufficient. We conclude that miR-146a plays a key role in mediating the effects of CDC-exosomes, but is not sufficient alone to confer a comprehensive therapeutic effect. Other microRNAs in the repertoire may exert synonymous or perhaps synergistic effects with miR-146a. For example, miR-22 (another microRNA highly enriched in CDC-exosomes) has been shown to be important for the adaptive response to cardiac stress. Similarly, miR-24 (also identified in CDC exosomes) regulates cardiac fibrosis by targeting furin, a member of the profibrotic TGF-β signaling pathway; overexpression of miR-24 in a model of MI reduced myocardial scar formation.The potential role of these microRNAs as mediators of the benefits of CDC exosomes is under investigation, either alone or in combination with miR-146a. While probing the active components within CDC exosomes would be beneficial, disassembly of the nanovesicles may be counterproductive from a therapeutic perspective. Because CDC exosomes are inherently cell-permeable and their lipid bilayer coat protects the payload from degradation as the particles shuttle between cells, the intact particles themselves may be suitable for disease applications.

[0108] Injection of CDC-derived exosomes into injured hearts mimics the structural and functional benefits of CDC transplantation; conversely, inhibition of exosome secretion by CDCs abolishes the therapeutic benefits of transplanted CDCs. Not all exosomes are beneficial: injection of exosomes derived from dermal fibroblasts (therapeutically inactive control cells) was not beneficial. DC exosomes reduced acute cardiomyocyte death and inflammatory cytokine release while attenuating left ventricular (LV) remodeling and fibrosis in injured hearts. MicroRNA arrays reveal several "signature microRNAs" that are highly upregulated in CDC exosomes. In contrast, mass spectrometry analysis indicates that the protein composition of CDC exosomes is conventional and comparable to that of fibroblast exosomes.

[0109] This study suggests that exosomes and the microRNAs they contain are key mediators of CDC-induced cardiac regeneration. CDCs exert diverse but coordinated effects: they recruit endogenous progenitor cells and promote the proliferation of surviving cardiac cells; whereas, injected CDCs suppress maladaptive LV remodeling, apoptosis, inflammation, and tissue fibrosis after MI. While CDCs may secrete a series of individual growth factors and cytokines that jointly exert diverse effects, the involvement of master regulator microRNAs within exosomes may help connect these various effects without the need for a complex mixture of numerous secreted protein factors. Furthermore, microRNAs, despite being short-lived in tissue, are known to confer long-term benefits and fundamental changes to the injury microenvironment, helping to justify the sustained benefits of CDC. CDC exosomes contain rich signaling information provided by the cell type first shown to be capable of conferring regeneration in the setting of "permanent" injury, conferring the same benefits as CDC without the need for live cell transplantation. For all these reasons, CDC exosomes merit further development as potential cell-free therapeutics.

[0110] Based on the results described herein, CDC-exosomes have been demonstrated to be capable of treating cardiac-related conditions, such as heart failure (HF) associated with Duchenne muscular dystrophy (DMD). Cell-secreted exosomes are able to recapitulate the therapeutic benefits of parent cells and, based on the described knockdown studies, appear to be essential for achieving such therapeutic benefits. Importantly, these results further revealed that, within a diverse range of protein- and RNA-rich biological cargoes, microRNAs play a central role in activating regenerative processes, suggesting potential applications in clinical treatments. Exosomes offer significant advantages over traditional cell-based therapies, including manufacturing advantages, relative ease of definition and characterization, lack of tumorigenicity and immunogenicity, and the potential for administration in therapeutic settings where cell, tissue, organ, or mechanical transplantation is unavailable. Therefore, CDC-exosomes represent a significant advancement in biological therapy.

[0111] Example 20 statistical analysis All results are presented as mean ± SEM; alternans results are presented as mean ± SD. Data sets were tested for normality and homogeneity of variance using the Kolmogorov-Smirnov test and Levene's test, respectively. If both were confirmed, a t-test or analysis of variance followed by a Bonferroni post-hoc test was used to determine statistical significance. If either normality or homogeneity of variance was not assured, a nonparametric test (Wilcoxon test or Kruskal-Wallis test followed by a Dunn post-hoc test) was applied (SPSS II, SPSS Inc., Chicago, Illinois). Preliminary data were not available for power analysis. The experiment was planned as an initial pilot project with a sample size of four animals per group. The results of the pilot project allowed us to conduct subsequent studies. This study complied with preclinical reporting standards, as described.

[0112] Example 21 Echocardiography Echocardiography studies were performed using a Vevo 770 imaging system (VisualSonics, Toronto, Canada) 2 days (baseline) and 3 weeks, 2 months, and 3 months after the first CDC / CDC-exosome (CDC-XO) injection, and 3 weeks, 2 months, and 3 months after the second CDC / CDC-XO injection. Using the same imaging system, echocardiography studies were performed at baseline (2 days) and 3 weeks after the miR148a mimic injection. After induction of light general anesthesia, hearts were imaged at the level of maximum LV diameter. LV ejection fraction (LVEF) was measured from two-dimensional long-axis images using VisualSonics version 1.3.8 software. Changes in left ventricular (LV) end-diastolic and end-systolic volumes: First and second CDC or CDC-XO implantations resulted in sustained improvements in LV end-diastolic (LV EDV) and end-systolic (LV ESV) volumes in mdx mice for at least 6 months compared with placebo. Delivery of miR-148a partially improved LV EDV and LV ESV (Figure 27). LV end-diastolic (LV EDV) and end-systolic (LV ESV) volumes after administration of cardiosphere-derived cells (CDCs), CDC-exosomes (CDC-XO), and miR-148. CDC and CDC-XO transplantation resulted in sustained improvement of LV EDV and LV ESV over 3 months after both the first and second injections (3 months apart) in mdx mice compared with placebo. Three weeks after miR148 injection, LV EDV and LV ESV were partially improved. Data are mean ± SEM; n = 12 in each group. #P < 0.05 vs. Mdx + vehicle.

[0113] Example 22 Treadmill exercise test Starting 3 weeks after CDC / vehicle injection, exercise capacity was assessed weekly using an Exer-3 / 6 open treadmill (Columbus Instruments, Columbus, OH). (Exercise capacity measured in a subset of mdx mice 1 week preoperatively was comparable to that measured in the Mdx + vehicle group 3 weeks postoperatively; data not shown). After an acclimation period (20 min at 10 m / min), a stepwise increase in average speed (1 m / min) was applied every 2 min during treadmill exercise until the mice fatigued (more than 10 s spent in the shocker; gentle pressure was maintained while treadmilling to help the mice stay on the track). Mice were then returned to their cages, and total distance traveled was recorded. After 3 months of weekly exercise, CDC / vehicle mdx mice were tracked alongside wild-type, age-matched mice for mortality assessment. The treadmill protocol was adapted from the American Physiological Society 3 was in accordance with the guidelines.

[0114] Example 23 CDC proliferation As stated 2 Murine CDCs were propagated from wild-type strain-matched mouse hearts (C57BL / 10ScSnJ wild-type mouse hearts). Briefly, ventricular tissue was minced into approximately 1 mm explants, partially enzymatically digested, and plated onto adherent (fibronectin-coated) culture dishes. Spontaneously produced proliferating cells (explant-derived cells) from these explants were harvested after confluence and plated in suspension culture (10 cells on poly-D-lysine-coated dishes). 5 Cells / mL) to allow self-assembly of three-dimensional cardiospheres. Subsequently, the cardiospheres were replated onto adherent culture dishes to obtain CDCs, which were used at passage 1 in all experiments.

[0115] Example 24 Assessment of CDC engraftment by real-time polymerase chain reaction Quantitative polymerase chain reaction (PCR) was performed at 1, 2, and 3 weeks after CDC injection to assess cell engraftment. Male CDCs were injected to allow for the detection of the SRY gene, located on the Y chromosome, as an engraftment marker using a TaqMan assay (Applied Biosystems, Foster City, CA). All mouse hearts were harvested, weighed, and homogenized. A standard curve was generated using multiple dilutions of genomic DNA isolated from the injected CDCs. All samples were spiked with an equal amount of genomic DNA from uninjected mouse hearts as a control. 50 ng of genomic DNA was used in each reaction. Real-time PCR was performed in triplicate. Engraftment was quantified from the standard curve. The engraftment rate of CDCs at 1 week was approximately 8% and at 2 weeks was less than 1%. At 3 weeks, no viable CDCs could be detected (Figure 28). CDC engraftment rates at 1, 2, and 3 weeks after transplantation. The survival rate of CDCs was approximately 8% at 1 week and >1% at 2 weeks. At 3 weeks, no viable CDCs could be detected (n=3 per time point).

[0116] Example 25 Cardiomyocyte proliferation and cardiac collagen content after CDC injection Paraffin-embedded sections from the apical, mid-, and basal portions of each heart were used for Masson's trichrome staining and immunostaining with antibodies against Ki67 and aurora B. Myocardial abundance of collagen IA1 and collagen IIIA1 was measured by Western blot analysis (Figure 29). CDC treatment in mdx mice reduces cardiomyogenesis and fibrosis. Enhanced cardiomyogenesis (A and B) and reduced myocardial fibrosis (C) and collagen content (D) 3 weeks after CDC injection in mdx mice. Representative immunohistochemical images and pooled data (A and B: CTL [wild-type], vehicle-, and CDC-treated [Mdx+CDC] mdx mouse hearts stained for Ki67 [A] and Aurora B [B]; n = 4–6 per group). Arrows indicate Ki67. + (A) and Aurora B +(B) Cardiomyocytes. Representative Masson's Trichrome image (C) and Western blot and pooled data (D) showing cardiac collagens IA and IIIA. Data are mean ± SEM; P < 0.05 vs. Mdx + vehicle and CTL (control); P < 0.05 vs. Mdx + CDC and CTL (control). Scale bar: 10 μm (A).

[0117] Example 26 Exosomes Exosomes were isolated from serum-free medium conditioned overnight (24 hours) by human CDC (CDC-XO) cells (or normal human dermal fibroblasts (NHDF) cells as a control) under hypoxic (2% O2, default condition) or normoxic (20% O2, for studies comparing exosomal RNA content alone). After sequential centrifugation at 300g (10 minutes) and 10,000g (30 minutes) and filtration through a 0.22 micron filter, exosomes were isolated from the conditioned medium using ultracentrifugation (100,000g, 1 hour). Isolated exosomes were subjected to RNA extraction and subsequent RNA sequencing (Figure 30). [Fold change of microRNAs in CDC exosomes isolated from hypoxic (2% O2) and normoxic (20% O2) conditioned medium; fold changes greater than 10-fold and less than 20-fold were included.] For miRNA-seq library preparation of small RNAs extracted from exosomes, we used the NEBNext Small RNA Library Prep kit (New England BioLabs, Ipswich, MA). RNA was extracted from exosomes using the miRNeasy Serum / Plasma Kit (QIAGEN, Germantown, MD). The exosomes were then either lysed or resuspended in PBS (for in vivo and in vitro experiments), and the exosome-to-protein ratio was measured using the MicroBCA Protein Assay Kit (Life Technologies, Grand Island, NY) and a Nanosight particle counter, respectively (Figure 31). Exosomes isolated by ultracentrifugation were analyzed by nanoparticle tracking. Videos were collected using a NanoSight NS300 system (NanoSight Ltd, UK) and analyzed using NTA software (version 2.3) (minimum expected particle size, minimum track length, and blur settings were all set to automatic). The camera shutter speed was fixed at 30.01 ms, and the camera gain was set to 500. The camera sensitivity and detection threshold were set to near maximum (15 or 16) and minimum (3 or 4), respectively, to display small particles. Ambient temperature was recorded manually, ranging from 24 to 27 °C.For each sample, five 60-second videos were recorded with a 10-second delay between recordings to generate five replicate histograms that were then averaged. Representative five replicate histograms show size / concentration. The standard error of the mean concentration calculated across the five replicates is shown in red in the graph on the right. A preliminary dose-response study demonstrated a 2 x 10 per gram of protein from hypoxic CDCs. 7 pcs and 1×10 9 This was found to be an effective dose for in vivo and in vitro experiments. Similar concentrations of exosomes were used in experiments using NHDF exosomes. Preliminary pilot in vivo experiments were performed using exosomes isolated by ultracentrifugation or the Exoquick kit (SBI, Mountain View, CA) as described, with similar results obtained for the two isolation methods.

[0118] Example 27 Injection of CDCs, CDC exosomes and miR-148 To optimize the process of CDC implantation, a preliminary dose-response experiment was performed, which showed that the first injection was 1 × 10 cells / mL, consistent with previous dose-ranging experiments in ischemic and non-ischemic mouse models. 5 cells and 1 × 10 for the second injection (3 months after the first injection). 4 A total of 1 x 10 cells was found to be an effective dose. 5 cells / 40 μL phosphate-buffered saline (PBS; first injection) or 1 × 10 cells 4 40 μL PBS (second injection) or PBS alone was injected into the left ventricular (LV) myocardium, equally divided among the four points indicated. The LV was visually divided into three regions (basal, mid, and apical), with one injection in the basal region, two injections in the midregion, and one injection in the apical region. CDC (Mdx + CDC, n = 12) or vehicle [placebo: Mdx + vehicle (PBS), n = 12] (3-month interval) was injected twice into 10-month-old CDC / mdx and vehicle / mdx mice, respectively. During thoracotomy, 28 1Injections were performed using a 1 / 2-gauge needle. All surgical procedures were performed while the animals were under general anesthesia (dexmedetomidine (0.5 mg / kg) / ketamine (75 mg / kg); IP; once preoperatively). A similar protocol was used for myocardial injection of CDC exosomes and miR-148. miR-148a mimic (hsa-miR-148a-3p, 2 μg; Sigma-Aldrich, St. Louis, MO) was mixed with RNAiMAX transfection reagent (Life Technologies, Grand Island, NY) in a total volume of 40 μl for 30 minutes at room temperature and injected into four sites per heart as described above.

[0119] Example 28 histology Mice were sacrificed 3 weeks (CTL: n = 4; Mdx + Vehicle: n = 6; Mdx + CDC / Mdx + CDC-XO: n = 6 each) or 3 months (CTL: n = 4; Mdx + Vehicle: n = 6; Mdx + CDC / Mdx + CDC-XO: n = 6) after the first CDC / CDC-XO injection and 3 weeks (n = 6) after miR-148 injection. Paraffin-embedded sections from the apical, mid-, and basal regions of each heart were used for histology. Masson's Trichrome Stain (HT15 Trichrome Stain [Masson] Kit; Sigma-Aldrich, St. Louis, MO) was used to assess fibrosis. T cells, B cells, and macrophages were assessed by immunostaining with antibodies against mouse CD3, CD20, and CD68, respectively. Cells were counted in 10 fields (20x magnification) from 10 randomly selected sections from the apical (3 sections; 50 μm intervals), central (4 sections; 50 μm intervals), and basal (3 sections; 50 μm intervals) regions of each heart, and the average cell number in each heart was calculated. Active cycling and proliferating (Ki67) cells were also counted. + and Aurora B + ) Cardiomyocytes were similarly counted and analyzed for Ki67 as described. + and Aurora B +The cycling and proliferative fractions were expressed as the number of cardiomyocytes divided by the total number of cardiomyocytes per high-power field (HPF), respectively. Measurements were averaged for each heart. Immunofluorescence staining: Heat-induced epitope retrieval was performed in low pH buffer (DAKO, Carpinteria, CA), followed by permeabilization / blocking for 2 hours with protein blocking solution (DAKO, Carpinteria, CA) containing 1% saponin (Sigma, St. Louis, MO; protein blocking solution containing 3% saponin was applied for immunofluorescence staining of Ki67). Subsequently, for immunofluorescence staining of 5 μm sections from the apical, mid, and basal regions of each heart, protein blocking solution for the primary antibody was applied overnight at 4°C. After three washes with PBS (10 min each), an Alexa Fluor secondary antibody (Life Technologies, Grand Island, NY) was used for detection. Images were captured using a Leica TCS SP5X confocal microscope system. Immunofluorescence staining was performed using antibodies against mouse Ki-67 (SP6; 1:50; Thermo Fisher Scientific, Fremont, CA), Wheat Germ Agglutinin (WGA; 1:200; Life Technologies, Grand Island, NY), Nrf2 (C20; 1:50; Santa Cruz Biotechnology, Santa Cruz, CA), and aurora B (1:250; BD Biosciences, San Jose, CA). Immunoperoxidase staining: Immunohistochemical detection of CD3, CD20, and CD68 was performed on 5-μm sections using prediluted rabbit monoclonal antibodies from Ventana Medical System (Tuscon, AZ; CD68) and Cell Marque (Rocklin, CA; CD3, CD20). Staining was performed with a Leica Bond-Max Ventana automated slide stainer (Chicago, IL) using an on-board heat-induced epitope retrieval method in high pH ER2 buffer (Leica Biosystems, Buffalo Grove, IL). +Staining was visualized using a rabbit detection system and Dako DAB (Carpinteria, CA). Slides were then counterstained with Mayer's hematoxylin for 1 minute and coverslipped. Electron microscopy: 1 mm 2 The apical (one cube), central (three cubes from the right, central, and left subsections), and basal (three cubes from the right, central, and left subsections) posterior wall sections from each heart (CTL: n = 3; Mdx + Vehicle: n = 3; Mdx + CDC: n = 3) were fixed by immersion in 2% glutaraldehyde, postfixed with osmium, and embedded in Epon. Sections were cut with silver foil, stained with uranyl acetate and lead citrate, and viewed using a JEOL 1010 with an AMT digital camera system.

[0120] Example 29 Western blot Western blot was performed to analyze Nrf2 signaling [Nrf2, phosphorylated Nrf2 (Nrf2-p s40 ) and Nrf2 downstream gene products: heme oxygenase-1 (HO-1), catalase, superoxide dismutase-2 (SOD-2), and the catalytic subunit of glutamate-cysteine ​​ligase (GCLC)], Nrf2 phosphorylation [phosphorylated Akt (Akt-p 308Myocardial abundance of target proteins contributing to oxidative phosphorylation [CI (NDUFB8 subunit), CII (SDHB subunit), CIV (MTCO1 subunit), CIII (UQCRC2 subunit), and CV (ATPSA subunit)], mitochondrial biogenesis (PGC-1), mitophagy (PINK1), inflammation (NF-κB and MCP-1), and fibrosis (collagen IA1 and collagen IIIA1) was compared. Myocardial density of malondialdehyde protein adducts (a marker of oxidative stress) was also measured by Western blotting (WB). Samples from the apical, mid, and basal sections of each heart (1 mm thick cross sections each) were mixed, homogenized, and nuclear and cytoplasmic fractions were extracted according to the manufacturer's instructions (CelLytic NuCLEAR Extraction Kit, Sigma-Aldrich, St. Louis, MO). Mitochondria were extracted from fresh whole hearts (CTL: n = 3; Mdx+vehicle: n = 8; Mdx+CDC: n = 8) as described in the respiration measurement section. Cytoplasmic, nuclear, and mitochondrial extracts for WB analysis were stored at -80°C. Protein concentrations in the extracts were determined using a microBCA protein assay kit (Life Technologies, Grand Island, NY). Target proteins in the cytoplasmic, nuclear, and mitochondrial fractions were measured by Western blot analysis using the following antibodies: mouse Nrf2, HO-1, catalase, SOD-2, GCLC, collagen IA1, collagen IIIA1, and PGC-1. Antibodies against phosphorylated Nrf2 (Nrf2-p) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). s40 Biorbyt, San Francisco, CA), respiratory chain subunits (pan-OXPHOS rodent WB antibody cocktail antibodies), malondialdehyde, citrate synthase and TBP (Abeam, Cambridge, MA), Akt and Akt-p T308Antibodies against κB-α, ρ-κB-α (Cell Signaling Technology, Denver, CO), PINK1, MCP-1, and NF-κB p65 (Sigma-Aldrich, St. Louis, MO) were purchased from the cited suppliers. For measurements of housekeeping proteins, including nuclear (TBP), cytosolic, and mitochondrial (citrate synthase) target proteins, antibodies against TBP (TATA-binding protein) and citrate synthase were used. Western blotting: Briefly, aliquots containing 20 μg of protein were fractionated on 8, 10, and 4–12% Bis-Tris gels (Life Technologies, Grand Island, NY) at 120 V for 2 h and transferred to PVDF membranes (Life Technologies, Grand Island, NY). Membranes were incubated for 1 hour in blocking buffer (1x TBS, 0.05% Tween-20, and 5% nonfat milk) and then incubated overnight in the same buffer containing the indicated antibodies at optimal dilutions as listed in Table 1.

[0121] [Table 1]

[0122] The membranes were washed three times for 5 minutes with 1x TBS, 0.05% Tween-20, and then incubated for 2 hours in a buffer (1x TBS, 0.05% Tween-20, and 3% nonfat milk) containing horseradish peroxidase-conjugated anti-rabbit IgG, anti-mouse IgG (Cell Signaling Technology, Denver, CO), and anti-goat IgG (Sigma-Aldrich, St. Louis, MO) at 1:1000–3000 dilutions. The membranes were washed three times for 5 minutes with 1x TBS, 0.05% Tween-20, and developed by autoluminography using ECL chemiluminescent substrate (Super Signal West Pico Chemiluminescent Substrate; Life Technologies, Grand Island, NY). Citrate synthase and TBP were used as housekeeping proteins to normalize the expression of the proteins of interest. Phosphorylated Akt, Nrf2, and IκB-α were normalized to total Akt, Nrf2, and IκB-α. Western blot analysis of collagen I and collagen III was performed under non-reducing, non-denaturing conditions.

[0123] Example 30 mitochondrial DNA DNA extracted from whole heart tissue (QIAamp DNA Mini Kit, QIAGEN, Germantown, MD) was used to measure the ratio of mitochondrial DNA to nuclear DNA using a PCR format according to the manufacturer's instructions (NovaQUANT™ Mouse Mitochondrial to Nuclear Ratio kit, EMD Millipore, Billerica, MA).

[0124] Example 31 respiration measurement After isoflurane anesthesia, mice were sacrificed by cervical dislocation. The hearts were immediately removed, rinsed with PBS, and homogenized with a Polytron in 1 mL of ice-cold HES buffer (250 mM sucrose, 1 mM EDTA, 10 mM HEPES, pH 7.4). The lysate was spun down at 1000 g for 5 min at 4°C to remove unbroken cells and large debris. The supernatant was then spun down at 7000 g for 10 min at 4°C to separate the mitochondrial-rich fraction from the crude cytosol. The pellet was resuspended in 1 mL of HES buffer (a portion of the lysis buffer for WB). Protein quantification was performed and adjusted with HES buffer to obtain samples containing 10 μg of protein in 50 μL of buffer. This was loaded into a 24-well Seahorse cell culture plate and spun down at 2000 g for 20 min at 4°C to allow mitochondrial adhesion to the plate surface. Then, 450 μL MAS buffer (70 mM sucrose, 220 mM mannitol, 5 mM KH2PO4, 5 mM MgCl2, 1 mM EGTA, 0.2% fatty acid-free BSA, pH 7.4) was added prior to the Seahorse XF24 mitochondrial stress test. Mitochondrial oxidative phosphorylation was stimulated using 5 mM / 5 mM pyruvate / malate and 0.25 mM ADP, followed by a mixture of 1 μM oligomycin, 1 μM FCCP, 1 μM antimycin, and 500 nM rotenone. Citrate synthase activity was measured in sample lysates and normalized for the actual amount of mitochondria loaded for the test. Seahorse respiration measurements were performed on normal and human Duchenne iPs cell-derived cardiomyocytes using a Seahorse™ XF96 Extracellular Flux Analyzer as described.

[0125] Example 32 Intracellular Ca2+ recording iPS-derived cardiomyocytes were loaded with 5 μM of the fluorescent calcium-sensitive dye Cal-520 (AAT Bioquest, Sunnyvale, CA) for 30 min and paced by electrical field stimulation at a frequency of 1 Hz using an Ion-Optix Myopacer (IonOptix Corp) delivering 0.2 ms squared voltage pulses with an amplitude of 20 V via two platinum wires (approximately 1 cm apart) placed on either side of the chamber base. We measured intracellular Ca using the xyt mode (2D) of a Leica TCS-SP5-II (Leica Microsystems Inc.; Wetzlar, Germany). 2+ The images were taken using a 488 nm laser to excite Cal520, and its emission (>505 nm) was collected using a 10x objective (Leica: N PLAN 10x / 0.25) at a scanning speed of 36–7 ms / frame depending on the field size. 2+ The fluorescence intensity (F), which is proportional to the concentration, was normalized to the baseline fluorescence F (F / F). The time to peak and Ca concentration were calculated using the software Clampfit (ver. 10.2, Molecular Devices, Inc.). 2+ The transient amplitude (F / F0) was analyzed. Beat-to-beat alternans in each group was calculated at 5-10 second intervals during 1 Hz pacing. The transient amplitude of each cell (n = 10 cells in each group) was measured during pacing, and the mean and standard deviation were calculated and compared between groups.

[0126] Example 33 Improved function, survival and antioxidant pathways by CDC transplantation in mdx mice Intramyocardial injection of the first and second (lower) doses of CDC in mdx mice resulted in sustained improvements in left ventricular function and volume (as measured by ejection fraction [EF]) for at least 6 months compared with placebo (Figures 23A and 27). CDC-induced improvements in EF persisted beyond the time point at which viable CDC was undetectable in mdx hearts (3 weeks after CDC delivery; Figure 28). In addition to improving EF, CDC injection enhanced ambulatory function (Figure 23B). Age-matched wild-type mice (CTL) and 10-month-old mdx mice (different from the mdx mice studied for cardiac function assessment) were subjected to weekly high-intensity treadmill exercise beginning 3 weeks after CDC or vehicle administration. CDC-treated mdx mice showed a substantial increase in maximal exercise capacity over the 3-month period over which it was measured compared with vehicle-treated mdx mice; survival also differed between the two groups (Figure 23C). By approximately 23 months of age, all vehicle-treated mdx mice had died, whereas over 50% of CDC-treated mdx mice remained alive (Figure 23C). CDC injection resulted in activation of the Nrf2 antioxidant pathway and upregulation of downstream gene products (Figure 23E). Concomitantly, oxidative stress was alleviated (Figure 23F). In the cytoplasm, Nrf2 is normally sequestered via binding to its repressor molecule, Keap1. Oxidative stress (and Nrf2 phosphorylation by protein kinases such as Akt) causes dissociation of the Nrf2-Keap1 complex, resulting in nuclear translocation of Nrf2 and transcriptional activation of antioxidant enzymes. In mdx hearts, levels of phosphorylated Akt and cytoplasmic and nuclear Nrf2 were elevated (as expected in response to oxidative stress); CDC treatment further increased these protein levels (Figure 23E). Consequently, downstream effectors heme oxygenase-1 (HO-1), catalase, superoxide dismutase-2 (SOD-2), and the catalytic subunit of glutamate-cysteine ​​ligase (GCLC) were upregulated in CDC-treated mdx hearts (Figure ​(Figure23E),23E), and malondialdehyde adducts (a fatty acid peroxidation end product; Figure ​Figure23F)23F were significantly reduced.Histological analysis revealed that fibrosis was extensive in typical vehicle-treated mdx hearts but was significantly less in CDC-treated mdx hearts (similar to age-matched wild-type [WT] controls). Similarly, Western blot analysis showed that CDC treatment significantly restored collagen I and III accumulation in mdx cardiac tissue after 3 weeks of treatment (Figure 29).

[0127] Example 34 Mitochondrial dysfunction and inflammation attenuated by CDC transplantation in mdx mouse hearts Mitochondrial structure and function are abnormal in muscular dystrophy-associated heart failure. In mdx hearts, 3 weeks after CDC injection, mitochondrial integrity improved: CDC restored mitochondrial ultrastructure (Figure 24A), increased mitochondrial DNA copy number (but not mitochondrial number; Figures 24B and C), enhanced levels of respiratory chain subunits (Figure 24D), and normalized the insufficient respiratory capacity of isolated mdx mitochondria (Figure 24E).

[0128] PGC-1 (nuclear PPARγ coactivator 1) and PINK1, key regulators of mitochondrial biogenesis and mitophagy, respectively, were upregulated after 3 days of CDC treatment and downregulated after 3 weeks of CDC treatment (Figure 24F), consistent with the initial turnover of damaged mitochondria followed by repopulation with stable, competent mitochondria. Notably, the improved mitochondrial integrity and reduced mitochondrial turnover observed after 3 weeks of CDC treatment in mdx mouse hearts were associated with upregulation of antioxidant enzymes and reduced oxidative stress and inflammation (Figure 24G and H). In vehicle-treated mdx hearts, NFκB, a master regulator of pro-inflammatory cytokines and chemokines, was activated: increased phosphorylated ΙκB and nuclear p65 content were associated with a marked upregulation of MCP1 (monocyte chemoattractant protein 1) and CD68. + Macrophages and CD3 +This was accompanied by the accumulation of T cells. In mdx hearts 3 weeks after CDC injection, CDC treatment reversed NFκB activation and reduced the number of inflammatory cells (Figures 24G and H). We also demonstrated the effect of CDC on cardiomyogenesis. Vehicle-treated mdx hearts showed a significant increase in cycling (Ki67), possibly as a compensation for ongoing cardiomyocyte loss. + ) and proliferation (aurora B + ) showed a several-fold increase in the number of cardiomyocytes (Figure 29). CDC is known to increase endogenous cardiomyogenesis in ischemic and non-ischemic models. Similarly, in mdx hearts, CDC treatment increased Ki67 + and aurora B + It promoted cardiomyogenesis as evidenced by a significant increase in cardiomyocytes (FIG. 29).

[0129] Example 35 CDC-secreted exosomes recapitulate the benefits of CDC in mdx mice. Exosomes secreted by CDCs (CDC exosomes) mimic the functional and structural benefits of CDCs in a mouse model of myocardial infarction. Similarly, in the mdx mouse model of DMD, the functional, antifibrotic, and cardiomyogenic benefits of CDCs are recapitulated by administration of exosomes isolated from media conditioned by hypoxic CDCs. Intramyocardial injection of two repeated doses of human CDC exosomes (3 months apart) resulted in sustained improvement in EF in mdx mice compared with vehicle-treated mice (Figures 25A and 27). Meanwhile, CDC exosome-injected mdx hearts exhibited significant improvements in cycling (Ki67 + , Fig. 25C1) and proliferation (aurora B + , Fig. 25C2) There was a significant increase in the number of cardiomyocytes, along with a decrease in the amount of collagen I and III (Fig. 25B).

[0130] Example 36 CDC exosomes in human Duchenne cardiomyocytes and miR-148a in mdx mice. Duchenne human iPS-derived cardiomyocytes (DMD CMs) exhibit many of the phenotypic defects also seen in mdx mouse hearts. Decreased oxygen consumption rate (OCR), reminiscent of that observed in mdx cardiac mitochondria (Figure 24E), and abnormal calcium cycling are reported defects. 21 Priming DMD CM with CDC exosomes one week prior normalized OCR, whereas priming with exosomes derived from normal human dermal fibroblasts (NHDF exosomes) had no effect. Changes in interbeat calcium transients (a measure of arrhythmogenicity) during 1 Hz burst pacing were similarly suppressed by priming DMD CM with CDC exosomes (Figures 26A and B). Comparison of the microRNA (miR) content of CDC exosomes isolated from hypoxic and normoxic CDCs revealed differences in miR expression (Figure 26C), with a significant increase in miR-148a in hypoxia. Given that our CDC exosomes were grown under hypoxia, we tested the effects of miR-148a administration. Three weeks after intramyocardial injection of the miR-148a mimic, miR-148a partially restored EF rate and suppressed NFκB, but reduced phosphorylated Akt levels (Figures 26D and 26E). The change in phosphorylated Akt was in the opposite direction to that seen with CDC injection (Figure 23E), indicating that miR-148a mimics some, but not all, of the effects of CDC and CDC exosomes.

[0131] [Table 2]

[0132] Example 37 Consideration In patients with DMD, cardiac disease may not manifest for 10 years or more after the diagnosis of skeletal myopathy, but once evident, cardiomyopathy progresses rapidly. Serial cardiac magnetic resonance imaging studies reveal that fibrosis is often initially limited to a small portion of the heart but then spreads rapidly and relentlessly. 23 The result is impaired overall cardiac function and premature death. There are no effective treatments to halt, prevent, or slow the progression of DMD cardiomyopathy. Recognizing that CDC exerts potentially beneficial regenerative effects in DMD, we tested the effects of CDC injections in early DMD cardiomyopathy. We found that CDC reduced fibrosis and inflammation in mdx hearts while improving pump function, increasing exercise capacity, and enhancing survival. The remarkable benefits of CDC were recapitulated by CDC exosomes. Our findings support the hypothesis that CDC acts by secreting exosomes loaded with genetic signals, including (but not limited to) miR-148a. These exosomes are taken up by the surrounding myocardium and antagonize multiple pathophysiological pathways underlying DMD cardiomyopathy. The cascade of effects is synergistic: oxidative stress, inflammation, and fibrosis are slowed, while cardiomyogenesis and mitochondrial function are increased. These results are noteworthy in that CDCs and their exosomes not only prevent progression but actually reverse the core dysfunction of DMD cardiomyopathy. The significant improvements in mortality and exercise capacity occurred without targeting dystrophin, supporting the idea that DMD treatments are so effective that correcting the underlying genetic cause is unnecessary. Given that CDCs are already in advanced clinical trials, our results support the initiation of clinical trials of CDCs in patients with DMD cardiomyopathy. Indeed, based on these findings, the HOPE-Duchenne trial will soon investigate the safety and tolerability of allogeneic CDCs administered via multivessel intracoronary infusion in subjects with heart failure secondary to DMD.

[0133] The various methods and techniques described above provide many ways to implement the present invention. Of course, it should be understood that not all of the described objectives or advantages can necessarily be achieved in accordance with any particular embodiment described herein. Thus, for example, one skilled in the art will recognize that the method can be implemented to achieve or optimize one or more advantages taught herein without necessarily achieving other objectives or advantages that may be taught or suggested herein. Various advantageous and disadvantageous alternatives are mentioned herein. It should be understood that some preferred embodiments specifically incorporate one, another, or more advantageous features, others specifically exclude one, another, or more disadvantageous features, and still others specifically mitigate an adverse feature of the present invention by incorporating one, another, or more advantageous features.

[0134] Moreover, those skilled in the art will recognize the applicability of various features from different embodiments. Similarly, those skilled in the art may mix and match the various elements, features, and steps described above, as well as other known equivalents of each such element, feature, or step, to implement methods in accordance with the principles described herein. In various embodiments, among the various elements, features, and steps, some may be specifically incorporated and others may be specifically excluded.

[0135] Although the present invention has been disclosed in terms of particular embodiments and examples, those skilled in the art will appreciate that the embodiments of the present invention extend beyond the specifically disclosed embodiments to other alternative embodiments and / or uses and modifications thereof and equivalents.

[0136] Many variations and alternative elements are disclosed in the embodiments of the present invention. Still further variations and alternative elements will be apparent to those skilled in the art. These variations include, but are not limited to, the source of cardiosphere-derived cells, cells derived directly from cardiac biopsies (explant-derived cells) or from self-assembled clusters of cardiac-derived cells (cardiospheres), exosomes produced by such cells, methods for isolating, characterizing, or altering exosomes produced by such cells, and specific uses of the products made according to the teachings of the present invention. Various embodiments of the present invention may specifically incorporate or exclude any of these variations or elements.

[0137] In some embodiments, numbers expressing quantities, concentrations, reaction conditions, and other properties of ingredients used to describe and claim particular embodiments of the present invention should be understood to be modified in some instances by the term "about." Accordingly, in some embodiments, the numerical parameters set forth in the specification and appended claims are approximations that may vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the present invention are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible. The numerical values ​​set forth in some embodiments of the present invention may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0138] In some embodiments, the terms "a," "an," and "the" and similar references, as used in connection with describing specific embodiments of the invention (particularly as used in connection with the claims that follow), can be interpreted to encompass both the singular and the plural. The description of ranges herein is merely intended to serve as a shorthand method of referring individually to each individual value within that range. Unless otherwise indicated herein, each individual value is incorporated herein as if it were individually set forth herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or unless otherwise clearly contradicted by context. The use of any and all examples or exemplary language (e.g., "etc.") provided with respect to specific embodiments herein is merely to better describe the invention and does not limit the scope of the invention as otherwise claimed. No language in the specification should be construed as indicating any element described in a claim essential to the practice of the invention.

[0139] The categorization of alternative elements or embodiments of the invention disclosed herein should not be construed as limiting. Members of each group may be referenced and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group may be incorporated into or deleted from a group for reasons of convenience and / or patentability. When any such incorporation or deletion occurs, the specification is deemed to contain the modified group, thereby satisfying the recitation requirements of all Markush groups used in the appended claims.

[0140] Preferred embodiments of the present invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of these preferred embodiments will be apparent to those of ordinary skill in the art upon reading the foregoing description. Those skilled in the art will be able to employ such variations as appropriate, and it is contemplated that the present invention may be practiced otherwise than as specifically described herein. Accordingly, many embodiments of the present invention include all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Also, any combination of the above-described elements in all possible variations thereof is encompassed by the present invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0141] Additionally, throughout this specification, numerous references are made to patents and publications. Each of the above-cited references and publications is individually incorporated herein by reference in its entirety.

[0142] Finally, it is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the invention. Other modifications that may be employed may be within the scope of the invention. Thus, by way of example, and not of limitation, alternative configurations of the invention may be utilized in accordance with the teachings herein. Accordingly, embodiments of the invention are not limited to that precisely as shown and described.

Claims

1. A composition for use in a method for treating heart failure secondary to muscular dystrophy, comprising:

1. A composition comprising a therapeutically effective dose of cardiosphere-derived cells (CDCs) for treating a subject in need of treatment for heart failure secondary to muscular dystrophy, wherein said treatment of said subject results in improved diastolic or systolic function.

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