FIELD OF THE INVENTION

This invention relates to the use of cells and their extracts, specifically cellular exosomes, for therapeutic use, including treatment of heart disease.

BACKGROUND

Duchenne muscular dystrophy (DMD) afflicts ˜20,000 boys and young men in the USA. The central cause is a genetic abnormality in the dystrophin complex, with secondary damage to skeletal muscle and heart tissue. Although virtually all patients are treated with corticosteroids, no treatment has been proven effective. Heart failure (HF) secondary to DMD afflicts virtually all DMD patients aged >15 years, and is often the cause of death. DMD-associated HF aggressively progresses from the initial insult (a genetic abnormality in the dystrophin complex), to asymptomatic abnormalities in cardiac structure and function (stage B), to overt symptomatic HF (stage C), to advanced HF (stage D) and death. Progression of HF is associated with high risk of hospitalization and intense overall health care resource utilization. Modes of death during the course of DMD-associated HF include sudden cardiac death (which increases as HF worsens), or progressive HF culminating in circulatory collapse. Moreover, much of the disability in the later years of DMD is due to HF rather than to skeletal muscle disease. Thus, DMD HF represents an important, neglected target for innovative therapy.

A highly promising avenue of therapy for cardiac-related diseases and conditions includes cardiosphere-derived cells (CDCs) that are capable of stimulating regeneration, angiogenesis, and functional improvement in infarcted myocardium. Previous or ongoing trials involving CDCs target adult patients in stage B; wherein heart function is depressed, but symptoms of HF have yet to appear. In DMD-associated HF, therapeutic approaches may be most dramatic for stages C and D. These later stages of disease are associated with high mortality (>20% per year) despite optimal medical therapy, which have also never been shown to actually slow the progression of disease in DMD patients. Because of exclusionary comorbidities, heart transplantation is not an option for DMD patients. These patients are also not candidates for mechanical circulatory support devices. In summary, no treatment modality currently available addresses the underlying pathophysiology of DMD-associated HF, which is a loss of functional heart muscle and conversion of living heart muscle to scar.

Interestingly, growing 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 that secrete many growth factors and cytokines, the beneficial therapeutic effects of CDCs manage to persist long after the injected cells have been cleared. Of critical interest is understanding whether these positive factors may exist in cellular exosomes produced by CDCs, the lipid bilayer nanovesicles secreted by cells when multivesicular endosomes fuse with the plasma membrane. Confirming a role for secreted exosomes in these processes has yet to be considered, and understanding these processes governing CDC-initiated regeneration may open new avenues therapeutic approaches. Because no existing therapy can reverse the progression of DMD HF, CDC-derived exosomes may effectively address a major unmet medical need, by recruiting various synergistic mechanisms of benefit that have been observed in animal models of HF. This includes the ability to attract endogenous stem cells to sites of myocardial injury, promote differentiation into heart muscle and vessels, and potentially reversing the pathophysiology of HF. The potential benefits of an exosome-based approach as an alternative to cell therapy is particularly compelling given the unavailability of conventional therapy to late stage patients. The possibility exists that CDC-derived exosomes may fundamentally alter the natural history of the disease.

Described herein are compositions and techniques related to generation and therapeutic application of CDC-derived exosomes. These biological molecules 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 many of the therapeutic effects of CDCs. The Inventors' work demonstrates that exosomes and their constituent microRNAs favorably modulate apoptosis, inflammation and fibrosis in the injured heart, leading to functional recovery and increase tissue viability. Thus, CDC-derived exosomes represent a novel “cell-free” therapeutic candidate for tissue repair.

SUMMARY OF THE INVENTION

Described herein is a method of treatment, including selecting a subject in need of treatment for heart failure secondary to a chronic degenerative muscular disease and administering a composition including a plurality of exosomes to the subject, wherein the plurality of the exosomes are isolated from cardiosphere-derived cells (CDCs) grown in serum-free media, include exosomes with a diameter of about 90 nm to about 200 nm and are CD81+, CD63+, or both, and further wherein administration of the composition treats the subject. In other embodiments, the chronic degenerative muscular disease is Duchenne muscular dystrophy. In other embodiments, administering a composition includes about 1 to about 100 mg exosome protein in a single dose. In other embodiments, a single dose is administered multiple times to the subject. In other embodiments, administering a composition includes injection. In other embodiments, the injection includes percutaneous injection. In other embodiments, the injection is directly into heart muscle. In other embodiments, administering a composition includes myocardial infusion. In other embodiments, myocardial infusion is intra-arterial or intravenous. In other embodiments, treatment of the subject results in decreased fibrosis, decreased inflammation, increased mitochondrial function and/or increased cardiomyogenesis. In other embodiments, decreased fibrosis includes a reduction in collagen accumulation. In other embodiments, collagen includes collagen I and/or collagen III. In other embodiments, decreased inflammation includes an increase in cytoplasmic nuclear factor (erythroid-derived 2)-like 2 (Nrf2), reduction in fatty acid peroxidation end products, reduced numbers of inflammatory cells, and/or upregulated expression of antioxidants. In other embodiments, antioxidants include heme oxygenase-1 (HO-1), catalase, superoxide dismutase-2 (SOD-2), and glutamate-cystein ligase catalytic (GCLC) subunit. In other embodiments, inflammatory cells include CD68+ macrophages and CD3+ T-cells. In other embodiments, increased mitochondrial function includes increased mitochondrial ultrastructure and/or increased mitochondrial biogenesis. In other embodiments, increased mitochondrial function includes increased nuclear PPAR-γ co-activator-1 (PGC-1) expression. In other embodiments, the exosomes include 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.

Further described herein is method of treatment, including selecting a subject in need of treatment for heart failure secondary to a chronic muscular disease and administering a composition including cardiosphere-derived cells (CDCs), wherein administration of the composition treats the subject. In other embodiments, the chronic muscular disease is Duchenne muscular dystrophy. In other embodiments, administering a composition includes about 1×105to about 1×108or more CDCs in a single dose. In other embodiments, administering a composition includes myocardial infusion. In other embodiments, myocardial infusion is intracoronary. In other embodiments, myocardial infusion is intra-arterial or intravenous. In other embodiments, treatment of the subject results in decreased fibrosis, decreased inflammation, increased mitochondrial function and/or increased cardiomyogenesis. In other embodiments, decreased fibrosis includes a reduction in collagen accumulation. In other embodiments, collagen includes collagen I and/or collagen III. In other embodiments, decreased inflammation includes an increase in cytoplasmic nuclear factor (erythroid-derived 2)-like 2 (Nrf2), reduction in fatty acid peroxidation end products, reduced numbers of inflammatory cells, and/or upregulated expression of antioxidants. In other embodiments, antioxidants include heme oxygenase-1 (HO-1), catalase, superoxide dismutase-2 (SOD-2), and glutamate-cystein ligase catalytic (GCLC) subunit. In other embodiments, inflammatory cells include CD68+ macrophages and CD3+ T-cells. In other embodiments, increased mitochondrial function includes increased mitochondrial ultrastructure and/or increased mitochondrial biogenesis. In other embodiments, increased mitochondrial function includes increased nuclear PPAR-γ co-activator-1 (PGC-1) expression.

