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  • C12N2320/32—Special delivery means, e.g. tissue-specific

Definitions

• This invention relates to the use of cells and their extracts, specifically cellular exosomes, for therapeutic use, including treatment of heart disease.
• Duchenne muscular dystrophy 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.
• HF 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.
• CDCs cardiosphere-derived cells
• 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.
• 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.
• heart transplantation is not an option for DMD patients. These patients are also not candidates for mechanical circulatory support devices.
• 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.
• 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.
• CDC-derived exosomes may fundamentally alter the natural history of the disease.
• compositions and techniques related to generation and therapeutic application of CDC-derived exosomes 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.
• cytokines include 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.
• 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.
• CDC-derived exosomes represent a novel "cell-free" therapeutic candidate for tissue repair.
• 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.
• the chronic degenerative muscular disease is Duchenne muscular dystrophy.
• administering a composition includes about 1 to about 100 mg exosome protein in a single dose.
• administering a composition includes injection.
• the injection includes percutaneous injection.
• the injection is directly into heart muscle.
• administering a composition includes myocardial infusion.
• myocardial infusion is intra-arterial or intravenous.
• treatment of the subject results in decreased fibrosis, decreased inflammation, increased mitochondrial function and/or increased cardiomyogenesis.
• decreased fibrosis includes a reduction in collagen accumulation.
• collagen includes collagen I and/or collagen III.
• 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.
• antioxidants include heme oxygenase- 1 (HO-1), catalase, superoxide dismutase-2 (SOD-2), and glutamate-cystein ligase catalytic (GCLC) subunit.
• inflammatory cells include CD68+ macrophages and CD3+ T-cells.
• increased mitochondrial function includes increased mitochondrial ultrastructure and/or increased mitochondrial biogenesis.
• increased mitochondrial function includes increased nuclear PPAR- ⁇ co- activator- 1 (PGC-1) expression.
• 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.
• administering a composition includes about 1 x 10 5 to about 1 x 10 8 or more CDCs in a single dose.
• administering a composition includes myocardial infusion.
• myocardial infusion is intracoronary.
• myocardial infusion is intraarterial or intravenous.
• treatment of the subject results in decreased fibrosis, decreased inflammation, increased mitochondrial function and/or increased cardiomyogenesis.
• decreased fibrosis includes a reduction in collagen accumulation.
• collagen includes collagen I and/or collagen III.
• 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.
• antioxidants include heme oxygenase- 1 (HO-1), catalase, superoxide dismutase-2 (SOD-2), and glutamate-cystein ligase catalytic (GCLC) subunit.
• inflammatory cells include CD68+ macrophages and CD3+ T-cells.
• increased mitochondrial function includes increased mitochondrial ultrastructure and/or increased mitochondrial biogenesis.
• increased mitochondrial function includes increased nuclear PPAR- ⁇ co-activator- 1 (PGC-1) expression.
• FIG. 1 Characterization of Cardiosphere-Derived Cells Exosomes.
• A RNA content measured in exosome pellets derived from cardiosphere-derived cells (CDCs) and normal human dermal fibroblasts (NHDF) compared to conditioned media from both samples.
• C CDC and NHDF exosomes express ubiquitous exosome markers as revealed by mass spectrometry.
• FIG. 3 Exosome Inhibition Attenuates CDC-Mediated Benefits.
• RNA(including microRNAs) was isolated from CDC exosomes and NHDF exosomes. qRT- PCR was performed on an microRNA array.
• B Venn diagram showing the variable microRNA profile between CDC and NHDF exosomes. Font size reflects the magnitude of differential expression of each microRNA.
• (F) miR-146a- deficient animals have severely impaired cardiac function and structure following acute MI.
• miR-146a Improves Systolic Function in Acute and Chronic Mouse Models of ML
• CDCs were transfected with either a miR-146a inhibitor or a hairpin control with a sequence based on Caenorhabditis elegans microRNAs (HP-CTRL).
• 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 of miR-146a or mimic control.
• B Densitometric analysis of blot in (A) normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH).
• C Schematic of the Inventors' working hypothesis.
• CDCs promote functional and structural benefits in the injured myocardium in a primarily paracrine manner. CDCs secrete exosomes that contain microRNAs that mediate benefits in the injured myocardium. These microRNAs target transcripts in the various compartments of the myocardium, which ultimately leads to increased cardiac function, increases in viable tissue, and decrease of scar after MI.
• Figure 7 Isolation of Exosomes from CDCs.
• A Graphical representation of exosome isolation and purification for exosomes.
• B Cell viability (calcein) and cell death (Ethidium homodimer-1) assay performed on CDCs over the 15 day serum- free conditioning period.
• C Representative images of CDCs before and after serum-free conditioning.
• FIG. 8 CDC Exosomes Reduce Inflammation In A Mouse Model Of Acute MI.
• A Representative protein arrays for 40 pro-inflammatory markers.
• CDC-exosomes stimulate functional improvement and attenuate adverse remodeling and cardiac hypertrophy in a mouse model of chronic MI.
• Animals treated with CDCexosomes also showed structural improvements as noted as seen in percent of the circumference of tissue sections that are scar (D), decreased cardiomyocyte hypertrophy (E) as measured by staining with wheat germ agglutinin and DAPI (F) and increased angiogenesis in the infarct zone (G).
• FIG. 10 Inhibition Of Exosome Secretion In CDCs Diminishes The Protective Effects Of CDCs In Vitro. Neonatal rat ventricular myocytes were stressed with 50 ⁇ H202 for 15 minutes followed by trans-well treatment with CDCs pre-treated with 5 ⁇ of Spiroepoxide, 20 ⁇ of GW4869, or vehicle (DMSO). (A) Cell death was measured using TUNEL staining (red), Phalloidin (green), and DAPI (blue).
• Heat Map Of Mir PCR Array Identifies Mir-146a As The Most Differentially Expressed microRNA. Heat map showing fold regulation differential abundance data for transcripts between CDC exosomes and NHDF exosomes overlaid onto the PCR Array plate layout.
• FIG. 12 miR-146a Protects Stressed Neonatal Rat Cardiomyocytes.
• B, C CDC exosomes derived from CDCs transfected with mir-146a hairpin inhibitor. Exosomes were derived from conditioned media and mirl46a knockdown confirmed by qPCR in exosomes.
• miR-146a Reproduces Some But Not All The Effects Of CDC- Exosomes. miR-146a attenuates adverse remodeling and cardiac hypertrophy in a mouse model of chronic MI.
• FIG. 14 CDC Treatment Heightened Activity Of Nrf2 Antioxidative Pathway And Increased Expression Of Nrf2 Downstream Gene Products.
• A Representative immunohistochemical images depicting Nrf2 in the mdx mouse hearts three weeks after treatment with vehicle (Mdx+Vehicle) or CDC (Mdx+CDC). Age-matched wild type mice (CTL) served as control.
• FIG. 15 CDC Treatment Markedly Restored Mitochondrial Structure And Content And, Enhanced Expression Of Respiratory Chain Subunits In The Heart Tissue Of Mdx Mice.
• A Representative images of transmission electron microscopy of cardiomyocyte mitochondria in mdx mice 3 weeks after treatment with vehicle (Mdx+Vehicle) or CDC (Mdx+CDC). Elongated mitochondria with altered (rounded/tubular) crista were predominant in the cardiomyocyte of mdx mice at 10 months of age. CDC treatment significantly restored cardiomyocyte mitochondrial size and crista structure (lamellar crista).
• C Bar graph demonstrating mitochondrial DNA copy numbers per cell in the heart tissue of experimental animals 3 weeks after treatment.
• D Representative western blots and pool data showing protein content of mitochondrial respiratory chain subunits in the heart tissue of Mdx mice 3 weeks after treatment with vehicle (Mdx+vehicle) and CDC (Mdx+CDC).
• FIG. 1 Ultrastructeral Degenerative Alterations In The Heart Of 10-Month- Old Mdx Mice Diminished Markedly 3 Weeks After Treatment With CDC.
• A Representative images of transmission electron microscopy of cardiomyocytes illustrating intracellular accumulation of amorphous proteins, extensive sarcomeric disruption and irregularities (Z streaming) and disorganized altered interfibrillar mitochondria in the 10- month-old Mdx mice. CDC markedly decreased cardiomyocyte degenerative alterations 3 weeks after intramyocardial injection.
• FIG. 18 CDC Treatment Increased Cardiomyocyte Cycling And Proliferation And Augmented Number Of C-Kit Positive Cells Differentiating Into Cardiac Lineage (C-Kit+Nkx2.5+).
• Representative immunohistochemical images and pooled data ((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)) from Mdx mice treated at 10 months of age. Arrows point to Ki67+ (A) and aurora B+ (B) cardiomyocytes and the cells positive for both c-kit and Nkx2.5 (C).
• Fractions of cycling (Ki67+) and proliferating (Aurora B+) cardiomyocytes are expressed as the number of Ki67+ and aurora B+ cardiomyocytes divided by the total number of cardiomyocytes per high-power field (HPF), respectively (Pooled data (A), (B)).
• the portion 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)).
• FIG. 19 Functional Benefits After Cardiosphere-Derived Cell (CDC) Transplantation.
• EF left ventricular ejection fraction
• LV EDV LV end-diastolic
• LV ESV end-systolic
• FIG. 20 Enhanced Maximal Exercise Capacity With CDC Treatment.
• 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 (stepwise increase in average speed every two minutes until exhaustion), starting 3 weeks after CDC/vehicle treatment.
• Sustained improvement of exercise capacity was observed in CDC-treated mdx mice relative to vehicle-treated mice.
• FIG. 23 Function, survival and antioxidant pathways improved by CDC transplantation in mdx mice.
• C Kaplan-Meier analysis of survival in the same animals as B shows lower survival in vehicle-treated mdx mice than in CDC-treated mdx mice or wild-type controls (p ⁇ 0.001, log rank test); the latter two groups, however, were statistically comparable.
• D Immunohistochemical images of Nrf2 in mdx mouse hearts 3 weeks after administration of vehicle or CDCs. Age-matched wild- type mice (CTL) served as control. Scale bars: 10 ⁇ .
• F Pooled data and representative western blot of myocardial malondialdehyde protein adducts 3 weeks after injections as indicated, showing attenuation of oxidative stress in Mdx+CDC.
• FIG. 24 Mitochondrial dysfunction and inflammation attenuated by CDC transplantation in mdx mouse hearts.
• A Transmission electron microscopy (TEM) images from mdx mouse hearts 3 weeks after administration of vehicle (Mdx+Vehicle) or CDC (Mdx+CDC). Age-matched wild-type mice (CTL) served as control.
• B Numbers of mitochondria from TEM images.
• C Mitochondrial DNA copy numbers (per nuclear genome) in the heart tissue 3 weeks after treatment.
• Substrates pyruvate, malate and ADP
• FCCP selective uncoupler
• blockers Oligomycin [Olig.]; Antimycin & Rotenone [Anti. & Rot.]
• G Immunohistochemical images of hearts stained for inflammatory cell markers CD68, CD20 and CD3.
• H Western blots, pooled data and bar graph (lower right) representing average number of indicated inflammatory cells in mdx mouse hearts. In CDC-treated mice, accumulation of CD68 + macrophages (upper row) and CD3 + T cells (lower row) was reduced in association with inhibition of NF- ⁇ pathway. Data are means ⁇ SEM. ⁇ P ⁇ 0.05 vs.
• FIG. 26 CDC exosomes in human Duchenne cardiomyocytes and miR-148 in mdx mice.
• A Calcium transients from normal and Duchenne human iPS-derived cardiomyocytes measured during lHz burst pacing.
• OCR Oxygen consumption rate in human Duchenne cardiomyocytes primed with CDC exosomes [DMD CM (CDC-XO )] or exosomes from normal human dermal fibroblasts [NHDF, as control; DMD CM (NHDF-XO + )] 1 week before OCR measurement.
• Normal (Normal CM) and non-treated Duchenne cardiomyocytes (DMD CM) were studied in parallel. See Fig. 2 legend for abbreviations.
• D Injection of miR-148 mimic intramyocardially partially restored cardiac function in mdx mouse hearts 3 weeks after treatment.
• E Western blots and pooled data for nuclear p65 (left) and phosphorylated Akt (right) in mdx mouse hearts 3 weeks after miR-148 treatment.
• F Schematic of pathophysiological mechanisms operative in Duchenne cardiomyopathy and the cellular mechanisms recruited by CDCs and their exosomes (XO). All data are means ⁇ SEM except for the box plot (means ⁇ SD).
• FIG. 27 LV end-diastolic (LV EDV) and end-systolic (LV ESV) volumes after cardiospherederived cell (CDC), CDC exosome (CDC-XO) and miR-148 administrations.
• CDC and CDC-XO transplantations resulted in a sustained improvement of LV EDV and LV ESV for 3 months after both first and second (3 months interval) injections in mdx mice, relative to placebo.
• Three weeks after miR148 injection, LV EDV and LV ESV were partially improved.
• FIG. 29 Cardiomyogenesis and diminished fibrosis with CDC treatment in mdx mice.
• Figure 30 Fold changes of microRNAs in CDC exosomes isolated from hypoxic conditioned media. Under hypoxic conditions (2% 0 2 ) compared to CDC exosomes isolated from normoxic conditioned media; fold change >10 and ⁇ 20 were included. NEBNext Small RNA Library Prep kit (New England BioLabs, Ipswich, MA) was used for miRNA-seq library preparation of extracted small RNAs from the exosomes. RNAs were extracted from exosomes using miRNeasy Serum/Plasma Kit (QIAGEN, Germantown, MD).
• Figure 31 Isolated exosomes by ultracentrifugation were analyzed by nanoparticle tracking.
• NTAsoftware version 2.3
• Camera shutter speed was fixed at 30.01 ms and camera gain was set to 500.
• Camera sensitivity and detection threshold were set close to maximum (15 or 16) and minimum (3 or 4), respectively, to reveal small particles.
• Ambient temperature was recorded manually, ranging from 24 to 27°C.
• five videos of 60 seconds duration were recorded, with a 10-second delay between recordings, generating five replicate histograms that were averaged. Representative five replicate histograms depicting size/concentration. Standard error of the mean concentration, calculated from 5 replicates, is shown in red in right graph. DETAILED DESCRIPTION
• Duchenne muscular dystrophy a crippling genetic disease leading to premature death, affects the heart as well as skeletal muscle. Indeed, cardiomyopathy is the leading cause of death in Duchenne patients. There are no approved treatments for the cardiomyopathy, and novel Duchenne-specific experimental approaches such as exon skipping do not benefit the heart.
• cardiosphere-derived cells CDCs
• Exosomes secreted by human CDCs reproduce the benefits of CDCs in mdx mice, and reverse abnormalities of calcium cycling and mitochondrial respiration in human Duchenne cardiomyocytes.
• DMD Duchenne muscular dystrophy
• CDCs Cardiosphere-derived cells
• CDCs are not only regenerative, but also anti-inflammatory and anti-fibrotic; they work indirectly via the secretion of exosomes laden with noncoding RNA including microRNAs (miRs).
• miRs microRNAs
• CDC-exosomes mimic the functional and structural benefits of CDCs, while blockade of exosome biosynthesis renders CDCs ineffective.
• the Inventors reasoned that CDCs might be useful in treating Duchenne cardiomyopathy. The goal is not to replace dystrophin, but rather to offset the pathophysiological consequences of dystrophin deletion, by recruiting regeneration, reversing fibrosis and targeting inflammation.
• Exosomes secreted lipid vesicles containing a rich milieu of biological factors, provide powerful paracrine signals by which stem cells potentiate their biological effects to neighboring cells, including diseased or injured cells.
• bio-active lipid and nucleic acid "cargo"
• these natural delivery devices are capable of inducing significant phenotypic and functional changes in recipient cells that lead to activation of regenerative programs.
• the role of such indirect mechanisms to in stem cell initiated regeneration is strongly suggested by growing evidence that after stem cell administration and clearance from delivery sites in tissue and organs, regeneration processes nevertheless persist and arise from endogenous tissues.
• Stem cell-derived exosomes have been identified and isolated from supernatants of several cell types with demonstrated therapeutic potential, including mesenchymal stromal (MSC), (bone marrow stem cells) mononuclear (MNC) cells, immune cells (dendritic and CD34+), human neural stem cells (hNSCs), among others.
• MSC mesenchymal stromal
• MNC mononuclear
• hNSCs human neural stem cells
• human cardiosphere derived cells CDCs
• Stem cell-derived exosomes including those produced by CDCs, may provide a potent and rich source for developing "cell-free" therapies.
• exosome-based, "cell-free” therapies in contrast to cell therapy, provide distinct advantages in regenerative medicine.
• these features significantly obviate safety issues.
• stem cell-derived exosomes are less immunogenic than parental cells, as a result of a lower content of membrane-bound proteins, including MHC complex molecules. Replacing the administration of live cells with their secreted exosomes, mitigates many of the safety concerns and limitations associated with the transplantation of viable replicating cells.
• exosome encapsulation of bioactive components in lipid vesicles allows protection of contents from degradation in vivo, thereby potentially negating obstacles often associated with delivery of soluble molecules such as cytokines, growth factors, transcription factors and RNAs. This comparative 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 tissue.
• exosome production under defined conditions allows for easier manufacture and scale-up opportunity.
• Manufacture of exosomes is akin to conventional biopharmacological product manufacture, allowing for standardization in production and quality control for dosage and biological activity testing.
• the durability of exosomes in culture allows for the acquisition of large quantities of exosomes through their collection from a culture medium in which the exosomes are secreted over periods of time.
• Exosomes are involved in intercellular communication between different cell types, much remains to be discovered in regard to the mechanisms of their production within parental cells of origin and effects on target recipient cells. Exosomes have been reported to be involved in numerous cellular, tissue and physiological processes, including immune modulating processes, angiogenesis, migration of endothelial cells in connection with tumor growth, or reducing damage in ischemia reperfusion injury. Of critical scientific interest in establishing whether exosomes secreted by cells, such as cardiosphere-derived cells (CDCs), are capable of reproducing the therapeutic benefits of their parental cells, or possibly, are indispensable in effectuating such therapeutic benefits.
• CDCs cardiosphere-derived cells
• exosomes are lipid bilayer vesicles that are enriched in a variety of 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 and extracellular spaces. These exocytosed vesicles of endosomal origin can range in size between 30-200 nm, including sizes of 40-100 nm, and possess a cup-shaped morphology, as revealed by electron microscopy.
• MVB multivesicular bodies
• exosomes reflect their parental cellular origin, as containing distinct subsets of biological factors in connection with their parent cellular origin, including the cell regulatory state of the parental cells when formed.
• the rich biological milieu of different proteins, including cytokines and growth factors, lipids, coding and noncoding RNA molecules, within exosomes are all necessarily derived from their parental cells.
• exosomes further express the extracellular domain of membrane -bound receptors at the surface of the membrane.
• endosome-associated proteins e.g., Rab GTPase, SNAREs, Annexins, and flotillin
• proteins that are known to cluster into microdomains at the plasma membrane or at endosomes four transmembrane domain tetraspanins, e.g., CD63, CD81, CD82, CD53, and CD37
• lipid raft associated proteins e.g., glycosylphosphatidylinositol- anchored proteins and flotillin
• cholesterol sphingomyelin
• hexosylceramides as examples.
• exosomes In addition to these core components reflecting their vesicle origin, a critical property of exosomes is a demonstrated capability to contain both mRNA and microRNA associated with signaling processes, with both cargo mRNA being capable to translation in recipient cells, or microRNA functionally degrading target mRNA in recipient cells. Other noncoding RNAs, capable for influencing gene expression, may also be present in exosomes. While the processes governing the selective incorporation of mRNA or microRNA populations into exosomes is not entirely understood, it is clear that RNA molecules are selectively, not randomly incorporated into exosomes, as revealed by studies reporting enrichment of exosome cargo RNAs when compared to the RNA profiles of the originating cells. Given the growing understanding of how such RNA molecules play a role in disease pathogenesis and regenerative processes, the presence of RNA molecules in exosomes and apparent potency in affecting target recipient cells suggests critical features that can be deployed in therapeutic approaches.
• the natural bilayer membrane encapsulation of exosomes provides a protected and controlled internal microenvironment that allows cargo contents to persist or migrate in the bloodstream or within tissues without degradation.
• Their release into the extracellular environment allows for interaction with recipient cells via adhesion to the cell surface mediated by lipid-ligand receptor interactions, internalization via endocytic uptake, or by direct fusion of the vesicles and cell membrane. These processes lead to the release of exosome cargo content into the target cell.
• exosome-cell interactions modulation of genetic pathways in the target recipient cell, as induced through any of several different mechanisms including antigen presentation, the transfer of transcription factors, cytokines, growth factors, nucleic acid such as mRNA and microRNAs.
• ESC embryonic stem cell
• mRNA and microRNAs have been demonstrated to shuttle/transfer mRNA and proteins to hematopoietic progenitors.
• Other studies have shown that adult stem cell-derived exosomes also shuttle selected patterns of mRNA, microRNA and pre-microRNA associated with several cellular functions involved in the control of transcription, proliferation and cell immune regulation. Isolation and Preparation of Exosomes.
• Exosome isolation relies on exploiting their generic biochemical and biophysical features for separation and analysis. For example, differential ultracentrifugation has become a leading technique wherein secreted exosomes are isolated from the supematants of cultured cells. This approach allows for separation of exosomes from nonmembranous particles, by exploiting their relatively low buoyant density. Size exclusion allows for their separation from biochemically similar, but biophysically different microvesicles, which possess larger diameters of up to 1,000 nm. Differences in flotation velocity further allows for separation of differentially sized exosomes. In general, exosome sizes will possess a diameter ranging from 30-200 nm, including sizes of 40-100 nm. Further purification may rely on specific properties of the particular exosomes of interest. This includes, for example, use of immunoadsorption with a protein of interest to select specific vesicles with exoplasmic or outward orientations.
• differential ultracentrifugation is the most commonly used for exosome isolation. This technique utilizes increasing centrifugal force from 2000xg to 10,000xg to separate the medium- and larger-sized particles and cell debris from the exosome pellet at 100,000xg. Centrifugation alone allows for significant separation/collection of exosomes from a conditioned medium, although it is insufficient to remove various protein aggregates, genetic materials, particulates from media and cell debris that are common contaminants.
• Enhanced specificity of exosome purification may deploy sequential centrifugation in combination with ultrafiltration, or equilibrium density gradient centrifugation in a sucrose density gradient, to provide for the greater purity of the exosome preparation (flotation density l .l-1.2g/ml) or application of a discrete sugar cushion in preparation.
• ultrafiltration can be used to purify exosomes without compromising their biological activity.
• Membranes with different pore sizes - such as 100 kDa molecular weight cut-off (MWCO) and gel filtration to eliminate smaller particles - have been used to avoid the use of a nonneutral pH or non-physiological salt concentration.
• MWCO molecular weight cut-off
• THF tangential flow filtration
• HPLC can also be used to purify exosomes to homogeneouslysized particles and preserve their biological activity as the preparation is maintained at a physiological pH and salt concentration.
• F1FFF Flow field-flow fractionation
• exosomes may be applied to isolated specific exosomes of interest. This includes relying on antibody immunoaffinity to recognizing certain exosome-associated antigens. As described, exosomes further express the extracellular domain of membrane-bound receptors at the surface of the membrane. This presents a ripe opportunity for isolating and segregating exosomes in connections with their parental cellular origin, based on a shared antigenic profile. Conjugation to magnetic beads, chromatography matrices, plates or micro fluidic devices allows isolating of specific exosome populations of interest as may be related to their production from a parent cell of interest or associated cellular regulatory state. Other affinity-capture methods use lectins which bind to specific saccharide residues on the exosome surface.
• Exosome-Based Therapies A chief goal of developing exosome-based therapy is the creation of "cell-free" therapies, wherein the benefits of cellular therapeutics can be provided with reduced risks or in scenarios in which cell therapy would be unavailable.
• DMD Duchenne muscular dystrophy
• HF heart failure
• the therapeutic benefits of cell-based therapies such as cardiosphere-derived cells (CDCs) appear to occur through indirect mechanisms involving regenerated myocardium and vasculature arising from endogenous origin.
• CDCs cardiosphere-derived cells
• CDC- derived exosomes may effectively address a major unmet medical need, by recruiting synergistic mechanisms to attract endogenous stem cells to sites of myocardial injury, promote differentiation into heart muscle and vessels, thereby reversing the pathophysiology of HF.
• DMD is an X-linked recessive disorder characterized by myopathy (cell membrane damage in muscle fiber) as exemplified by a variety of pathological features. This includes skeletal muscle weakness starting 3-5 years from onset, progressive weakness, wheelchair dependency at approximately 13 years from onset. Importantly, cardiomyopathy is observed to take hold in 1/3 of patients from less than 13 years from onset, increasing to 1/2 of patients less than 18 years from onset, and in all patients after 18 years. Dilated cardiomyopathy includes left ventricle posterobasal fibrosis; conduction abnormalities are mainly intra-atrial: SVT with abnormal AV nodal conduction. Patients may further suffer from smooth muscle myopathy including vascular dysfunction, further including GI and urinary tract systems involvement.
• dystrophin gene mutation deletion
• loss of dystrophin results in cellular membrane damage and leakage of extracellular Ca 2+ into the cell.
• Elevated intracellular levels ultimately result in increased oxidative and/or nitrosative stress and inflammation, and activation of calpain.
• the combination of these effects results in muscle proteolysis and apoptosis, leading to the degradative features described above.
• CDCs have been demonstrated as promoting anti- oxidative, anti-inflammatory, anti-apoptotic, anti-remodeling effects, in addition to enhancing regenerative capacity.
• CDC administration is beneficial in retarding/reversing DMD, and exosome populations derived from CDCs may allow for these benefits to be delivered, while avoiding obstacles associated with cell-based therapy.
• stem cell-derived exosomes are likely to be less immunogenic than parental cells.
• exosome encapsulation of bioactive components in lipid vesicles allows protection of contents from degradation in vivo, thereby potentially negating obstacles often associated with delivery of soluble molecules such as cytokines, growth factors, transcription factors and R As, while potentially allowing for increased concentrations to be provided.
• soluble molecules such as cytokines, growth factors, transcription factors and R As
• compositions and methods and compositions providing significant benefits in the repair or regeneration of damaged or diseased tissues via "cell-free” methods involving exosomes.
• human cardiosphere-derived cells (CDC)-derived exosomes are demonstrated as effective in reducing scar size and regenerating viable myocardium.
• CDC cardiosphere-derived cells
• 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.
• the chronic degenerative muscular disease is Duchenne muscular dystrophy.
• administering a composition includes about 1 to about 100 mg exosome protein in a single dose.
• administering a composition includes injection.
• the injection includes percutaneous injection.
• the injection is directly into heart muscle.
• administering a composition includes myocardial infusion.
• myocardial infusion is intra-arterial or intravenous.
• treatment of the subject results in decreased fibrosis, decreased inflammation, increased mitochondrial function and/or increased cardiomyogenesis.
• decreased fibrosis includes a reduction in collagen accumulation.
• collagen includes collagen I and/or collagen III.
• 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.
• antioxidants include heme oxygenase- 1 (HO-1), catalase, superoxide dismutase-2 (SOD-2), and glutamate-cystein ligase catalytic (GCLC) subunit.
• inflammatory cells include CD68+ macrophages and CD3+ T-cells.
• increased mitochondrial function includes increased mitochondrial ultrastructure and/or increased mitochondrial biogenesis.
• increased mitochondrial function includes increased nuclear PPAR- ⁇ co- activator- 1 (PGC-1) expression.
• 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.
• a composition including cardiosphere-derived cells including cardiosphere-derived cells (CDCs), wherein administration of the composition treats the subject.
• the chronic muscular disease is Duchenne muscular dystrophy.
• administering a composition includes about 1 x 10 5 to about 1 x 10 8 or more CDCs in a single dose.
• the number of administered CDCs includes intracoronary 25 million CDCs per coronary artery (i.e., 75 million CDCs total) as another baseline for exosome dosage quantity.
• the numbers of CDCs includes 1 x 10 5 , 1 x 10 6 , 1 x 10 7 , 1 x 10 8 , 1 x 10 9 CDCs in a single dose as another baseline for exosome dosage quantity. In certain instances, this may be prorated to body weight (range 100,000- 1M CDCs/kg body weight total CDC dose).
• administering a composition includes myocardial infusion.
• myocardial infusion is intracoronary.
• myocardial infusion is intra-arterial or intravenous.
• treatment of the subject results in decreased fibrosis, decreased inflammation, increased mitochondrial function and/or increased cardiomyogenesis.
• decreased fibrosis includes a reduction in collagen accumulation.
• collagen includes collagen I and/or collagen III.
• 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.
• antioxidants include heme oxygenase- 1 (HO-1), catalase, superoxide dismutase-2 (SOD-2), and glutamate-cystein ligase catalytic (GCLC) subunit.
• inflammatory cells include CD68+ macrophages and CD3+ T-cells.
• increased mitochondrial function includes increased mitochondrial ultrastructure and/or increased mitochondrial biogenesis.
• increased mitochondrial function includes increased nuclear PPAR- ⁇ co- activator-1 (PGC-1) expression. Further examples are found in U.S. App. No. 11/666,685, 12/622,143, and 12/622,106, which are herein incorporated by reference.
• compositions including a plurality of exosomes.
• the plurality of exosomes are generated by a method including providing a population of cells, and isolating a plurality of exosomes from the population of cells.
• the cells are stem cells, progenitors and/or precursors.
• the stem cells, progenitors and/or precursors are cardiosphere-derived cells (CDCs).
• the stem cells, progenitors and/or precursors are pluripotent stem cells (pSCs), such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) derived from any one of various somatic sources in the body such as fibroblasts, blood and hematopoietic stem cells (hSCs), immune cells, bone and bone marrow, neural tissue, among others.
• pSCs pluripotent stem cells
• ESCs embryonic stem cells
• iPSCs induced pluripotent stem cells derived from any one of various somatic sources in the body
• fibroblasts fibroblasts
• hSCs hematopoietic stem cells
• immune cells bone and bone marrow, neural tissue, among others.
• the stem cells, progenitors and/or precursors include hSCs, mesenchymal stem cells (MSCs), or endothelial precursor cells (EPCs).
• the cells are stem cells, progenitors and/or precursors derived from human biopsy tissue.
• the cells are stem cells, progenitors and/or precursors are a primary culture.
• the cells are stem cells, progenitors and/or precursors which may constitute a cell line capable of serial passaging.
• the plurality of exosomes is isolated from the supernatants of the population of cells. This includes, for example, exosomes secreted into media as conditioned by a population of cells in culture, further including cell lines capable of serial passaging.
• the cells are cultured in serum-free media.
• the cells in culture are grown to 10, 20, 30, 40, 50, 60, 70, 80, 90, or 90% or more confluency when exosomes are isolated.
• the population of cells has been genetically manipulated. This includes, for example, knockout (KO) or transgenic (TG) cell lines, wherein an endogenous gene has been removed and/or an exogenous introduced in a stable, persistent manner.
• the population of cells has been altered by exposure to environmental conditions (e.g., hypoxia), small molecule addition, presence/absence of exogenous factors (e.g., growth factors, cytokines) at the time, or substantially contemporaneous with, isolating the plurality of exosomes in a manner altering the regulatory state of the cell.
• environmental conditions e.g., hypoxia
• exogenous factors e.g., growth factors, cytokines
• altering the regulatory state of the cell changes composition of one or more exosomes in the plurality of exosomes.
• the plurality of exosomes include one or more exosomes that are about 10 nm to about 250 nm in diameter, including those 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 nm3 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
• the plurality of exosomes includes one or more exosomes expressing a biomarker.
• the biomarkers are tetraspanins.
• the tetraspanins are one or more selected from the group including CD63, CD81, CD82, CD53, and CD37.
• the exosomes express one or more lipid raft associated protiens (e.g., glycosylphosphatidylinositol-anchored proteins and flotillin), cholesterol, sphingomyelin, and/or hexosylceramides.
• the plurality of exosomes includes one or more exosomes containing a biological protein.
• the biological protein includes transcription factors, cytokines, growth factors, and similar proteins capable of modulating signaling pathways in a target cell.
• the biological protein is capable of facilitating regeneration and/or improved function of a tissue.
• the biological protein is capable of modulating pathways related to Iraki, Traf6, toll-like receptor (TLR) signaling pathway, NOX-4, SMAD-4, and/or TGF- ⁇ .
• TLR Traf6, toll-like receptor
• the biological protein related to exosome formation and packaging of cytosolic proteins such as Hsp70, Hsp90, 14-3-3 epsilon, PKM2, GW182 and AG02.
• the plurality of exosomes includes one or more exosomes containing a signaling lipid. This includes ceramide and derivatives. In other embodiments, the plurality of exosomes includes one or more exosomes containing a coding and/or non- coding nucleic acid.
• the plurality of exosomes includes one or more exosomes containing microRNAs.
• these microRNAs can include miR-146a, 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/or miR- 23 a.
• the plurality of exosomes include one or more exosomes enriched in at least one of miR-146a, 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/or miR-23a.
• the plurality of exosomes includes one or more exosomes enriched in at least one of miR-146a, miR-22, miR-24.
• Enrichment can be measured by, for example, comparing the amount of one or more of the described microRNAs when derived from cells providing salutary benefit in a therapeutic setting (e.g., cardiosphere-derived cells (CDCs) compared to cells that do not provide such a salutary benefit (e.g., fibroblasts). Enrichment may also be measured in absolute or relative quantities, such as when compared to a standardized dilution series.
• a therapeutic setting e.g., cardiosphere-derived cells (CDCs)
• fibroblasts e.g., fibroblasts
• the plurality of exosomes can include one or more exosomes containing microRNAs.
• the plurality of exosomes can include one or more exosomes containing microRNAs.
• microRNAs known in the art, such as 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-210, miR-126, miR-378, miR-363 and miR-30b, and miR-499.
• 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.
• isolating a plurality of exosomes from the population of cells includes centrifugation of the cells and/or media conditioned by the cells. In several embodiments, ultracentrifugation is used. In several embodiments, isolating a plurality of exosomes from the population of cells is via size-exclusion filtration. In other embodiments, isolating a plurality of exosomes from the population of cells includes use of discontinuous density gradients, immunoaffinity, ultrafiltration and/or high performance liquid chromatography (HPLC).
• HPLC high performance liquid chromatography
• differential ultracentrifugation includes using centrifugal force from 1000-2000xg, 2000-3000xg, 3000-4000xg, 4000-5000xg, 5000xg-6000xg, 6000- 7000xg, 7000-8000xg, 8000-9000xg, 9000-10,000xg, to 10,000xg or more to separate larger- sized particles from a plurality of exosome derived from the cells.
• differential ultracentrifugation includes using centrifugal force from 10, 000-20, OOOxg, 20,000-30,000xg, 30,000-40,000xg, 40,000-50,000xg, 50,000xg-60,000xg, 60,000-70,000xg, 70,000-80,000xg, 80,000-90,000xg, 90,000- 100,000xg, to 10,000xg or more to separate larger-sized particles from a plurality of exosome derived from the cells.
• isolating a plurality of exosomes from the population of cells includes use of filtration or ultrafiltration.
• a size exclusion membrane with different pore sizes is used.
• a size exclusion membrane can include use of a filter with a pore size of 0.1-0.5 ⁇ , 0.5-1.0 ⁇ , 1-2.5 ⁇ , 2.5-5 ⁇ , 5 or more ⁇ . In certain embodiments, the pore size is about 0.2 ⁇ .
• filtration or ultrafiltration includes size exclusion ranging from 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, 500 or more kDa.
• the size exclusion is for about 2-5 kDa. In certain embodiments, the size exclusion is for about 3 kDa.
• filtration or ultrafiltration includes size exclusion includes use of hollow fiber membranes capable of isolating particles ranging from 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, 500 or more kDa.
• the size exclusion is for about 2-5 kDa. In certain embodiments, the size exclusion is for about 3 kDa.
• a molecular weight cut-off (MWCO) gel filtration capable of isolating particles ranging from 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, 500 or more kDa.
• the size exclusion is for about 2-5 kDa. In certain embodiments, the size exclusion is for about 3 kDa. In various embodiments, such systems are used in combination with variable fluid flow systems.
• isolating a plurality of exosomes from the population of cells includes use of tangential flow filtration (TFF) systems are used purify and/or concentrate the exosome fractions.
• isolating a plurality of exosomes from the population of cells includes use of (HPLC) can also be used to purify exosomes to homogeneously sized particles.
• density gradients as used such as centrifugation in a sucrose density gradient or application of a discrete sugar cushion in preparation.
• isolating a plurality of exosomes from the population of cells includes use of a precipitation reagent.
• a precipitation reagent can be added to conditioned cell media to quickly and rapidly precipitate a population of exosomes.
• isolating a plurality of exosomes from the population of cells includes use of volume-excluding polymers (e.g., polyethylene glycols (PEGs)) are used.
• isolating a plurality of exosomes from the population of cells includes use of flow field-flow fractionation (F1FFF), an elution-based technique.
• F1FFF flow field-flow fractionation
• isolating a plurality of exosomes from the population of cells includes use of one or more capture agents to isolate one or more exosomes possessing specific biomarkers or containing particular biological molecules.
• one or more capture agents include at least one antibody.
• antibody immunoaffmity recognizing exosome-associated antigens is used to capture specific exosomes.
• the at least one antibody are conjugated to a fixed surface, such as magnetic beads, chromatography matrices, plates or microfluidic devices, thereby allowing isolation of the specific exosome populations of interest.
• isolating a plurality of exosomes from the population of cells includes use of one or more capture agents that is not an antibody.
• the non-antibody capture agent is a lectin capable of binding to polysaccharide residues on the exosome surface.
• the CDCs are mammalian. In other embodiments, the CDCs are human. As disclosed above, in some embodiments, synthetic exosomes are generated, which can be isolated by similar mechanisms as those above. In various embodiments, the composition that is a plurality of exosomes is a pharmaceutical composition further including a pharmaceutically acceptable carrier.
• the plurality of exosomes range in size from 30 to 300 nm. In various embodiments, the plurality of exosomes range in size from 40 to 100 nm. In certain embodiments, the plurality of exosomes is cardiosphere-derived cell (CDC) exosomes. In certain embodiments, the plurality of exosomes includes exosomes that are CD63+.
• CDC cardiosphere-derived cell
• the exosomes include microRNAs miR-146a, 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/or miR-23a.
• the exosomes are 2-5 kDa, such as 3 kDa.
• Other examples or embodiments relating to the composition and techniques involving exosomes are presented, in PCT Pub. No. WO 2014/028,493, which is fully incorporated herein by reference.
• Described herein is a method for treatment including, selecting a subject in need of treatment, administering a composition including a plurality of exosomes to the individual, wherein the administration of the composition treats the subject.
• the subject is in need to treatment for a disease and/or condition involving tissue damage or dysfunction.
• the disease and/or condition involving tissue damage or dysfunction is heart disease.
• the plurality of exosomes includes exosomes including one or more microRNAs.
• the plurality of exosomes are generated by a method including providing a population of cells, and isolating a plurality of exosomes from the population of cells.
• the cells are stem cells, progenitors and/or precursors.
• the stem cells, progenitors and/or precursors are cardiosphere-derived cells (CDCs).
• the stem cells, progenitors and/or precursors are pluripotent stem cells (pSCs), such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) derived from any one of various somatic sources in the body such as fibroblasts, blood and hematopoietic stem cells (hSCs), immune cells, bone and bone marrow, neural tissue, among others.
• pSCs pluripotent stem cells
• ESCs embryonic stem cells
• iPSCs induced pluripotent stem cells
• the stem cells, progenitors and/or precursors includes hSCs, mesenchymal stem cells (MSCs), or endothelial precursor cells (EPCs).
• the cells are stem cells, progenitors and/or precursors derived from human biopsy tissue.
• the cells are stem cells, progenitors and/or precursors are a primary culture. In various embodiments, the cells are stem cells, progenitors and/or precursors are a cell line capable of serial passaging. In certain embodiments, the exosomes are synthetic. In various embodiments, the plurality of exosomes is derived from cardiosphere- derived cells (CDCs). In other embodiments, the plurality of exosomes includes exosomes including one or more biological molecules. In other embodiments, the plurality of exosomes includes exosomes enriched for one or more biological molecules when derived from CDCs compared to exosome derived from non-CDC sources.
• CDCs cardiosphere- derived cells
• the one or more biological molecules are proteins, growth factors, cytokines, transcription factors and/or morphogenic factors.
• the plurality of exosomes includes exosomes enriched for one or more biological molecules includes microRNAs, further including microRNAs that are enriched when derived from CDCs compared to exosome derived from non-CDC sources.
• these microRNAs can include miR-146a, 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/or miR- 23 a.
• the plurality of exosomes includes one or more exosomes enriched in at least one of miR-146a, miR-22, miR-24.
• the CDCs are mammalian. In other embodiments, the CDCs are human.
• the exosomes are synthetic. In certain embodiments, the synthetic exosomes possess substantially similar content (e.g., microRNAs, biological molecules) as exosomes derived from CDCs.
• administration of the plurality of exosomes alters gene expression in the damaged or dysfunctional tissue, improves viability of the damaged tissue, and/or enhances regeneration or production of new tissue in the individual.
• the quantities of exosomes that are administered to achieved these effects range from 1 x 10 6 to 1 x 10 7 , 1 x 10 7 to 1 x 10 8 , 1 x 10 8 to 1 x 10 9 , 1 x 10 9 to 1 x 10 10 , 1 x 10 10 to 1 x 10 11 , 1 x 10 11 to 1 x 10 12 , 1 x 10 12 or more.
• the numbers of exosomes is relative to the number of cells used in a clinically relevant dose for a cell-therapy method.
• 3mL / 3 x 10 5 CDCs is capable of providing therapeutic benefit in intracoronary administration, and therefore, a plurality of exosomes as derived from that number of cells in a clinically relevant dose for a cell-therapy method.
• administration can be in repeated doses.
• the number of administered CDCs includes intracoronary 25 million CDCs per coronary artery (i.e., 75 million CDCs total) as another baseline for exosome dosage quantity.
• the numbers of CDCs includes 1 x 10 5 , 1 x 10 6 , 1 x 10 7 , 1 x 10 8 , 1 x 10 9 CDCs in a single dose as another baseline for exosome dosage quantity. In certain instances, this may be prorated to body weight (range 100,000- 1M CDCs/kg body weight total CDC dose).
• exosome quantity may be defined by protein quantity, such as dosages including 1-10, 10-25, 25-50, 50-75, 75-100, or 100 or more mg exosome protein.
• Defining an effective dose range, dosing regimen and route of administration may be guided by studies using fluorescently labeled exosomes, and measuring target tissue retention, which can be >10X, >50X, or >100X background, as measured 5, 10, 15, 30, or 30 or more min as a screening criterion.
• >100X background measured at 30 mins is a baseline measurement for a low and high dose that is then assess for safety and bioactivity (e.g., using MRI endpoints: scar size, global and regional function).
• single doses are compared to two, three, four, four or more sequentially-applied doses.
• the repeated or sequentially-applied doses are provided for treatment of an acute disease and/or condition.
• the repeated or sequentially-applied doses are provided for treatment of a chronic disease and/or condition.
• administration of exosomes to the subject occurs through any of known techniques in the art. In some embodiments, this includes percutaneous delivery. In other embodiments, myocardial infusion is used, for example, the use of intracoronary catheters. In various embodiments, delivery can be intra-arterial or intravenous. Additional delivery sites include any one or more compartments of the heart, such as arterial, venous, and/or ventricular locations. In certain embodiments, administration can include delivery to a tissue or organ site that is different from the site or diseased and/or dysfunctional tissue. In certain embodiments, the delivery is via inhalation or oral administration.
• administration of the plurality of exosomes alters gene expression in the damaged or dysfunctional tissue, improves viability of the damaged tissue, and/or enhances regeneration or production of new tissue in the individual. In various embodiments, administration of the exosomes results in functional improvement in the tissue. In several embodiments, the damaged or dysfunctional tissue includes cardiac tissue.
• functional improvement may include increased cardiac output, contractility, ventricular function and/or reduction in arrhythmia (among other functional improvements).
• improved function may be realized as well, such as enhanced cognition in response to treatment of neural damage, improved blood-oxygen transfer in response to treatment of lung damage, improved immune function in response to treatment of damaged immunological-related tissues.
• administration of the plurality of exosomes alters gene expression in the damaged or dysfunctional tissue, improves viability of the damaged tissue, and/or enhances regeneration or production of new tissue in the individual. In various embodiments, administration of the exosomes results in functional improvement in the tissue.
• the damaged or dysfunctional tissue includes skeletal muscle tissue.
• functional improvement may include increased contractile strength, improved ability to walk (for example, and increase in the six-minute walk test results), improved ability to stand from a seated position, improved ability to sit from a recumbent or supine position, or improved manual dexterity such as pointing and/or clicking a mouse.
• the damaged or dysfunctional tissue is in need of repair, regeneration, or improved function due to an acute event.
• Acute events include, but are not limited to, trauma such as laceration, crush or impact injury, shock, loss of blood or oxygen flow, infection, chemical or heat exposure, poison or venom exposure, drug overuse or overexposure, and the like.
• the damaged tissue is cardiac tissue and the acute event includes a myocardial infarction.
• administration of the exosomes results in an increase in cardiac wall thickness in the area subjected to the infarction.
• tissue is also subject to damage due to chronic disease, such as for example congestive heart failure, including as conditions secondary to diseases such as Duchenne muscular dystrophy, ischemic heart disease, hypertension, valvular heart disease, dilated cardiomyopathy, infection, diabetes, and the like.
• the administration can be in repeated doses, such as two, three, four, four or more sequentially- applied doses.
• the repeated or sequentially-applied doses are provided for treatment of an acute disease and/or condition.
• the repeated or sequentially-applied doses are provided for treatment of a chronic disease and/or condition.
• Other sources of damage also include, but are not limited to, injury, age-related degeneration, cancer, and infection.
• the regenerative cells are from the same tissue type as is in need of repair or regeneration. In several other embodiments, the regenerative cells are from a tissue type other than the tissue in need of repair or regeneration.
• the method of treatment includes, selecting a subject in need of treatment for a heart related disease and/or condition, administering a composition including a plurality of exosomes to the individual, wherein the administration of the composition treats thesubject.
• the heart related disease and/or condition includes heart failure, further including Duchenne muscular dystrophy related heart failure.
• the plurality of exosomes range in size from 30 to 300 nm. In various embodiments, the plurality of exosomes range in size from 40 to 100 nm.
• the plurality of exosomes are cardiosphere-derived cell (CDC) exosomes.
• the plurality of exosomes include exosomes that are CD63+.
• the exosomes include microRNAs miR-146a, 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/or miR-23a.
• the exosomes are 2-5 kDa, such as 3 kDa.
• administering a composition includes a dosage of 1 x 10 8 , 1 x 10 8 to 1 x 10 9 , 1 x 10 9 to 1 x 10 10 , 1 x 10 10 to 1 x 10 11 , 1 x 10 11 to 1 x 10 12 , 1 x 10 12 or more exosomes.
• 3mL / 3 x 10 5 CDCs is capable of providing therapeutic benefit in intracoronary administration, and therefore, a plurality of exosomes as derived from that number of cells in a clinically relevant dose for a cell-therapy method.
• administration can be in repeated doses.
• the number of administered CDCs includes intracoronary 25 million CDCs per coronary artery (i.e., 75 million CDCs total) as another baseline for exosome dosage quantity.
• the numbers of CDCs includes 1 x 10 5 , 1 x 10 6 , 1 x 10 7 , 1 x 10 8 , 1 x 10 9 CDCs in a single dose. In certain instances, this may be prorated to body weight (range 100,000- 1M CDCs/kg body weight total CDC dose).
• exosome quantity may be defined by protein quantity, such as dosages including 1-10, 10-25, 25-50, 50-75, 75-100, or 100 ore more mg exosome protein.
• administering a composition includes multiple dosages of the exosomes.
• the repeated or sequentially-applied doses are provided for treatment of an acute disease and/or condition.
• the repeated or sequentially-applied doses are provided for treatment of a chronic disease and/or condition.
• administering a composition includes percutaneous injection.
• administering a composition includes myocardial infusion.
• administering a composition includes use of a intracoronary catheter.
• administration a composition includes intraarterial or intravenous delivery. Further described herein is a method of improving cardiac performance in a subject including, selecting a subject, administering a composition including a plurality of exosomes to the individual, wherein the administration of the composition improves cardiac performance in the subject.
• improving cardiac performance can be demonstrated, by for example, improvements in baseline ejection volume.
• improving cardiac performance relates to increases in viable tissue, reduction in scar mass, improvements in wall thickness, regenerative remodeling of injury sites, enhanced angiogenesis, improvements in cardiomyogenic effects, reduction in apoptosis, and/or decrease in levels of pro-inflammatory cytokines.
• the method of improving cardiac performance includes, selecting a subject in need of treatment for a heart related disease and/or condition, administering a composition including a plurality of exosomes to the individual, wherein the administration of the composition treats thesubject.
• the heart related disease and/or condition includes heart failure, further including Duchenne muscular dystrophy related heart failure.
• the plurality of exosomes range in size from 30 to 300 nm. In various embodiments, the plurality of exosomes range in size from 40 to 100 nm. In certain embodiments, the plurality of exosomes are cardiosphere- derived cell (CDC) exosomes.
• the plurality of exosomes include exosomes that are CD63+.
• the exosomes include microRNAs miR-146a, 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/or miR-23a.
• the exosomes are 2-5 kDa, such as 3 kDa.
• administering a composition includes a dosage of 1 x 10 8 , 1 x 10 8 to 1 x 10 9 , 1 x 10 9 to 1 x 10 10 , 1 x 10 10 to 1 x 10 11 , 1 x 10 11 to 1 x 10 12 , 1 x 10 12 or more exosomes.
• 3mL / 3 x 10 5 CDCs is capable of providing therapeutic benefit in intracoronary administration, and therefore, a plurality of exosomes as derived from that number of cells in a clinically relevant dose for a cell-therapy method.
• administration can be in repeated doses.
• the number of administered CDCs includes intracoronary 25 million CDCs per coronary artery (i.e., 75 million CDCs total) as another baseline for exosome dosage quantity.
• the numbers of CDCs includes 1 x 10 5 , 1 x 10 6 , 1 x 10 7 , 1 x 10 8 , 1 x 10 9 CDCs in a single dose. In certain instances, this may be prorated to body weight (range 100,000- 1M CDCs/kg body weight total CDC dose).
• exosome quantity may be defined by protein quantity, such as dosages including 1-10, 10-25, 25-50, 50-75, 75-100, or 100 or more mg exosome protein.
• administering a composition includes multiple dosages of the exosomes.
• the repeated or sequentially-applied doses are provided for treatment of an acute disease and/or condition.
• the repeated or sequentially-applied doses are provided for treatment of a chronic disease and/or condition.
• administering a composition includes percutaneous injection.
• administering a composition includes myocardial infusion.
• administering a composition includes use of a intracoronary catheter.
• administration a composition includes intraarterial or intravenous delivery.
• CDCs cardiosphere-derived cells
• Exosomes secreted by human CDCs reproduce the benefits of CDCs in mdx mice, and reverse abnormalities of calcium cycling and mitochondrial respiration in human Duchenne cardiomyocytes.
• Both CDCs and their exosomes improve heart function in mdx mice; a single injection of CDCs suffices to increase maximal exercise capacity and improve survival.
• CDCs ameliorate Duchenne cardiomyopathy via exosome -mediated transfer of signaling molecules including miR-148a.
• the present findings motivate clinical testing of CDCs in patients with Duchenne cardiomyopathy.
• Endomyocardial biopsies from the right ventricular aspect of the interventricular septum are obtained from healthy hearts of deceased tissue donors. Cardiosphere-derived cells were derived as described previously. 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 fully incorporated by reference herein.
• CADUCEUS myocardial infarction
• heart biopsies are minced into small fragments and briefly digested with collagenase. Explants were then cultured on 20 mg/ml fibronectin-coated dishes. Stromal- like flat cells and phase-bright round cells grow out spontaneously from tissue fragments and reach confluence by 2-3 weeks. These cells are harvested using 0.25% trypsin and cultured in suspension on 20 mg/ml poly d-lysine to form self-aggregating cardiospheres.
• cardiosphere-derived cells CDCs
• All cultures are maintained at 5% C02 at 37°C, using IMDM basic medium supplemented with 20%> FBS, 1% penicillin/streptomycin, and 0.1 ml 2- mercaptoethanol.
• Exosomes are harvested from CDCs at passage 4.
• NHDF normal human dermal fibroblasts
• CDCs and NHDFs are conditioned in serum- free media for 15 days at 100% confluence. Aspirated media is then centrifuged at 3,000xg for 15 min to remove cellular debris. Exosomes were then isolated using Exoquick Exosome Precipitation Solution.
• Exosome pellets are resuspended in the appropriate media and used for assays. Expression of the conserved exosome marker CD63 is verified using ELISA. RNA content of exosome pellets can also be quantified using a Nanodrop spectrophotometer.
• CDC are transfected in suspension with miRIDIAN miR- 146a hairpin inhibitor or a miRIDIAN hairpin control and seeded on to fibronectin-coated flasks. Exosomes are isolated from serum-free conditioned media (48 hr conditioning).
• Exosomal RNA degradation is performed by suspending exosome pellets in 2 ml of PBS. To one sample, 100 ml of Triton X-100 (Sigma Aldrich) is added to achieve 5% triton concentration. Exosomes are treated with 0.4 mg/ml RNase A treatment for 10 min at 37°C. Samples are further treated with 0.1 mg/ml Proteinase K for 20 min at 37°C. RNA is purified from samples using an microRNA isolation kit. RNA levels are measured using Nanodrop. Example 4
• Proteins are prepared for digestion using the filter-assisted sample preparation (FASP) method. Concentrations re measured using a Qubitfluorometer. Trypsin is added at a 1 :40 enzyme-to-substrate ratio and the sample incubated overnight on a heat block at 37°C. The device was centrifuged and the filtrate collected. Digested peptides are desalted using C 18 stop-and-go extraction (STAGE) tips. Peptides are fractionated by strong anion exchange STAGE tip chromatography. Peptides re eluted from the CI 8 STAGE tip and dried. Each fraction is analyzed with liquid chromatography-tandem mass spectrometry. Samples are loaded to a 2 cm x 100 mm I.D. trap column.
• FASP filter-assisted sample preparation
• the analytical column is 13 cm x 75 mm I.D. fused silica with a pulled tip emitter.
• the mass spectrometer is programmed to acquire, by data-dependent acquisition, tandem mass spectra from the top 15 ions in the full scan from 400 to 1,400 m/z.
• Mass spectrometer RAW data files re converted to MGF format using msconvert.
• MGF files re searched using X! Hunter against the latest spectral library available on the GPM at the time.
• MGF files are also searched using X! ! Tandem using both the native and k-score scoring algorithms and by OMSSA. Proteins re required to have one or more unique peptides with peptide E-value scores of 0.01 or less from X!
• !Tandem 0.01 or less from OMSSA, 0.001 or less and theta values of 0.5 or greater from X!Hunter searches, and protein E-value scores of 0.0001 or less from X! !Tandem and X!Hunter.
• NRCMs neonatal rat cardiomyoctes
• NRCMs neonatal rat cardiomyoctes
• Another useful source includes human vein umbilical vein endothelial cells plated on growth factor-deprived Matrigel (BD Biosciences) to assay angiogenesis.
• the Inventors plated 1.5xl0 4 NRCMs in fibronectin-coated eight-chamber slides. After 5 days, media is replaced with new fresh media containing 3.5xl0 8 or 2xl0 8 CDC or NHDF exosomes, respectively. Cells are then fixed with 4% paraformaldehyde for 30 min at 4°C. Chambers are washed three times with cold PBS then blocked and permeabilized with Dako/0.1% Saponin (Invitrogen) for 1 hr at 37°C. Cells are incubated (overnight, 4°C) with rabbit anti-Ki-67 (1 : 100) primary antibody and mouse anti-a-sarcomericactinin (Abeam).
• One injury model can include use of NRCMs plated in a monolayer on fibronectin- coated 12-well plates and treated with either 40 nM of miR-146a or mimic for 24 hr. Media is then changed and cells were washed three times with PBS. Cells are then stressed using hydrogen peroxide (100 mM H202 in serum free media for 2 hr) or cobalt chloride (5mMCoC12 in serum-free media for 2 hr). Viability is measured by washing cells with PBS and treating with 20 mM Calcein PBS solution for 20 min at 37°C in dark conditions. Fluorescence is read using a Soft Max Pro 5 Plate Reader (Molecular Devices). Data per well is the average of nine consecutive measurements.
• a second model includes plating cardiomyocytes on 25 mm precoated glass coverslips (Fischer Scientific) in six-well plates. Cells are stressed using 50 mM H202 for 15min followed incubation with transwell membrane inserts containing CDCs or incubation with CDC exosomes for 4 hr. Cells are then washed, fixed with paraformaldehyde, and stained for analysis as explained above.
• Exosome Inhibition in CDCs are grown to confluence in T175 flasks.
• CDCs were conditioned for 15 days in 20 mM GW4869 (Sigma Aldrich), serum- free media, or serum- free media containing an equivalent volume of DMSO.
• For in vitro transwell insert assays of cardiomyocyte stress one can treat CDCs with 20 mM GW4869 (Sigma Aldrich), or 5 mM Spiroepoxide (Santa Cruz Biotechnology) for 12 hr. CDCs are washed three times in PBS and supplanted with serum-free media. Inserts containing treated CDCs are added into six- well plates containing cardiomyocytes.
• CDCs are treated with 20 mM GW4869 or an equivalent volume of DMSO for 12 hr. Prior to injection, CDC flasks are washed twice with PBS, trypsinized, and counted; 10 5 CDCs were injected per animal.
• mice Three-month-old male severe combined immunodeficient (SOD)- beige mice are anesthetized with isoflurane. Following surgical preparation, a 2 cm vertical incision is introduced in the midclavicular line for a lateral thoracotomy. The left anterior descending was ligated using 7-0 silk. Animals are injected with exosomes, microRNAs, CDCs, or media control at two peri-infarct sites with a volume of 40 ml per injection.
• SOD severe combined immunodeficient
• mice are infarcted as described above without any treatment administration. Three weeks later, the animals are given the treatment in the same manner as above.
• pellets are resuspended in Iscove's Modified Dulbecco's (IMDM) basal media. Animals are injected with 2.8xl0 9 and 1.56xl0 9 of CDC and NHDF exosomes, respectively.
• microRNA-treated animals are injected with 80 ng of miPv-146a or microRNA mimic control.
• miRIDIAN miR-146a or miRIDIAN negative control is vortexed in Dharmafect (Thermo Scientific) transfection reagent and IMDM basal media and incubated for 10 min at room temperature to allow complexes to form. microRNA complexes are resuspended in IMDM for injection. For CDC treatments, animals are injected with 10 5 CDCs as described.
• Echocardiography SCID beige mice are evaluated via echocardiography 24 hr (baseline), 14 days, and 4 weeks after surgery using Vevo 770 Imaging System (Visual Sonics). After induction of light general anesthesia, hearts are 3D imaged in the long axis view at the level of maximum left ventricular diameter. Left ventricular ejection fraction can be measured with Visual Sonics version 1.3.8 software from 2D views of LV end-diastolic and LV end-systolic area. Each animal/time point is measured multiple times and the average used for statistical analysis.
• Infarct size can be established by measuring area and intensity of blue in each section to calculate infarct size. Percent viable and infarct mass were calculated by averaging percent infarct across four sections analyzed per heart. Infarct and viable masses were calculated as the product of the infarct or viable tissue, the height of the average mouse heart (3 mm) and the specific gravity of heart tissue (1.05 g/ml). Infarct wall thickness is calculated by measuring the thinnest area of the infarct. In a chronic model of MI where significant hypertrophy and adverse remodeling took place, one can adjust the viable mass of each heart based on the derived mass of cardiomyocytes in the tissue.
• Myocyte mass is obtained by measuring the cross-sectional area of perpendicularly sectioned cardiomyocytes (defined as round cells with red cytoplasm and a visible nucleus in the center). The Inventors measured at least 25 myocytes per heart. Myocyte volume is quantified using the simplifying assumption of a cylindrical shape; mass was derived by multiplying volumes by the specific gravity of a cardiomyocyte (1.15 mg/ml). The viable mass of each mouse heart was divided by the mass of the cardiomyocytes in that heart.
• Exosomes are isolated from serum- free media conditioned over 15 days by cultured human CDCs (or normal human dermal fibroblasts (NHDFs) as a therapeutically inert control) (Figure 7 available online). By the end of the conditioning period, most of the CDCs remained alive despite the absence of regular media changes ( Figures 7B and 7C). Purified exosome pellets were enriched in RNA ( Figure 1A). The Inventors confirmed that the RNA resides within exosomes by exposing the pellet to RNase A in the presence of 5% triton (Figure IB), with proteinase K added to dissociate protein complexes that may shield RNA.
• Figure 1A The Inventors confirmed that the RNA resides within exosomes by exposing the pellet to RNase A in the presence of 5% triton ( Figure IB), with proteinase K added to dissociate protein complexes that may shield RNA.
• CDC exosomes but not NHDF exosomes, promoted tube formation in human umbilical cord endothelial cells, indicative of enhanced angiogenesis (Figure 1G).
• CDC- exosome-treated neonatal cardiomyocytes proliferated more than those exposed to NHDF exosomes or media only, as evidenced by higher proportions of Ki67-positive nuclei ( Figure 1H).
• CDC-exosome-treated cardiomyocytes exhibited fewer terminal deoxynucleotidyltransferase nick end labeling (TUNEL)-positive nuclei ( Figure II).
• TUNEL terminal deoxynucleotidyltransferase nick end labeling
• CDC-exosome-treated hearts exhibited decreased scar mass, increased viable mass, and increased infarcted wall thickness compared to NHDF exosome and media controls ( Figures 2B-2E). Proinflammatory cytokine levels were also lower in CDC- exosome-treated hearts ( Figure 8). In all these respects, CDC exosomes mimic the known benefits of CDCs themselves.
• microRNAs were differentially present in the two groups; among these, miR-146a was the most highly enriched in CDC exosomes (262-fold higher than in NHDF exosomes; Figures 4A, 4B, and 11).
• miR-146a tissue levels were increased in post-MI hearts from animals injected with CDC exosomes relative to those injected with NHDF exosomes (Figure 4C), rendering plausible the idea that CDC exosomes might act via miR-146a transfer.
• Exposure of neonatal rat cardiomyocytes to a miR-146a mimic increased cardiomyocyte viability and protected against oxidant stress ( Figures 4D and 12A).
• Whole-transcriptome microarrays revealed downregulation of Iraki and Traf6, two signaling mediators of the TLR-NFkB axis that are known targets of miR-146a ( Figure 4E).
• miR-146a leads to thicker infarct wall thickness and increased viable tissue in a mouse model of myocardial infarct.
• the Inventors developed miR-146a-deficient exosomes by trans fecting CDCs with a miR-146a hairpin inhibitor (or a control hairpin) followed by media conditioning and exosome isolation. Successful knockdown of miR-146a was confirmed by qPCR on resultant exosomes and on NRVMs exposed to either control or miR-146adepleted exosomes ( Figures 12B and 12C).
• mice injected with miR-146a mimic during acute MI exhibited improved pump function (Figure 5B), decreased scar mass, and increased viable heart tissue (Figures 5C-5F).
• animals treated with miR-146a showed only minor, statistically insignificant functional improvement ( Figures 5G and 13 A).
• histological analysis showed no difference in scar mass ( Figures 5H and 51).
• hearts treated with miR-146a mimic did show increased viable tissue, thicker infarcted walls ( Figures 5 J and 5K), and less adverse remodeling than controls ( Figures 13B-13D). Evaluation of angiogenesis showed no significant differences between the two groups ( Figures 5L and 13G).
• Cardiosphere-derived cells have been shown to induce therapeutic regeneration of the infarcted human heart.
• CDCs led to shrinkage of scar and growth of new, functional myocardium. Similar effects have been corroborated in animal models.
• the Inventors show that exosomes reproduce CDC- induced therapeutic regeneration, and that inhibition of exosome production undermines the benefits of CDCs.
• Exosomes contain microRNAs, which have the ability to alter cell behavior through paracrine mechanisms ( Figure 6B). Among these, the Inventors have identified miR-146a as being particularly enriched in CDC exosomes.
• miR-146a When administered alone, miR-146a reproduces some, but not all, of the salient benefits of CDCs and of CDC exosomes (Figure 3). Likewise, miR-146a-depleted exosomes were still able to suppress cardiomyocyte apoptosis ( Figure 5 A), albeit more weakly than when miR-146a is present. Treating hearts with miR-146a in a chronic model of MI (after the scar is permanent) does reproduce the increase in viable mass that is the signature of therapeutic regeneration, but fails to mimic two key beneficial effects of CDC exosomes: decreased scar mass and improved global function. The increase in viable myocardium does not suffice to increase function, possibly because of inadequate angiogenesis elicited by miR-146a.
• miR-146a plays an important part in mediating the effects of CDC exosomes, but alone does not suffice to confer comprehensive therapeutic benefit.
• Other microRNAs in the repertoire may exert synonymous or perhaps synergistic effects with miR-146a.
• miR-22 another microRNA highly enriched in CDC exosomes
• miR-24 also identified in CDC exosomes
• overexpression of miR-24 in a model of MI decreased myocardial scar formation.
• CDC exosomes are naturally cell permeant, and their lipid bilayer coat protects their payloads from degradation as particles shuttle from cell to cell, so that the intact particles themselves may be well suited for disease applications.
• Injection of CDC-derived exosomes into the injured heart mimics the structural and functional benefits of CDC transplantation; conversely, inhibition of exosome secretion by CDC s abrogates the therapeutic benefits of transplanted CDCs. Not all exosomes are salutary: Injection of exosomes from dermal fibroblasts, control cells which are therapeutically inert, had no benefit. DC -exosomes decreased acute cardiomyocyte death and inflammatory cytokine release, while attenuating left ventricular (LV) remodeling and fibrosis in the injured heart. MicroRNA arrays reveal several "signature microRNAs" that are highly up-regulated in CDC-exosomes. In contrast, mass spectrometry indicates that the protein composition of CDC-exosomes is conventional and comparable to that of fibroblast exosomes.
• CDCs exert diverse but coordinated effects: they recruit endogenous progenitor cells and coax surviving heart cells to proliferate; on the other hand, injected CDCs suppress maladaptive LV remodeling, apoptosis, inflammation, and tissue fibrosis after MI. While it is possible that CDCs secrete a medley of individual growth factors and cytokines that collectively produce diverse benefits, the involvement of master-regulator microRNAs within exosomes would help tie together the various effects without postulating complex mixtures of numerous secreted protein factors.
• CDC exosomes contain rich signaling information conferred by a cell type that is the first shown to be capable of producing regeneration in a setting of "permanent" injury, and confer the same benefits as CDCs without transplantation of living cells. For all these reasons, CDC exosomes merit further development as cell-free therapeutic candidates.
• CDC-exosomes are demonstrated as capable of treating heart-related conditions, such as treat heart failure (HF) associated with Duchenne muscular dystrophy (DMD).
• HF heart failure
• DMD Duchenne muscular dystrophy
• Exosomes secreted by cells are capable of reproducing therapeutic benefits of their parental cells and based on the described knockdown studies, appear to be indispensable in effectuating such therapeutic benefits.
• these results have further identified that within their rich biological cargo of various proteins and RNA, microRNAs play a central role in activating regenerative processes, suggesting compelling applications in clinical therapeutics.
• Exosomes have significant advantages over traditional cell-based therapies including manufacturing advantages, relative ease of definition and characterization, lack of tumorgenicity and immunogenicity, and possibility of administration in therapeutic scenarios for which cell, tissue, organ or mechanical transplant is not available.
• CDC-exosomes represent a significant advance in biologic therapy.
• results are presented as mean ⁇ SEM; results for alternans are presented as mean ⁇ SD.
• Normality and equality of variances of data sets were first tested using Kolmogorov- Smirnov and Levene's tests, respectively. If both were confirmed, t-test or analysis of variance followed by Bonferroni 's post hoc test were used for determination of statistical significance; if either normality or equality of variances was not assured, nonparametric tests (Wilcoxon test or Kruskal-Wallis test followed by Dunn's post-test) were applied (SPSS II, SPSS Inc., Chicago, Illinois). No preliminary data were available for a power analysis. Experiments were planned with a sample size of 4 animals per group as an initial pilot project. Results from the pilot project allowed us to power subsequent studies. The study followed preclinical reporting standards, as described.
• Echocardiographic studies were performed two days before (Baseline) and 3 weeks, 2 and 3 months after first CDC/CDC exosome (CDC-XO) injection and 3 weeks, 2 and 3 months after second CDC/CDC-XO injection using the Vevo 770 Imaging System (VisualSonics, Toronto, Canada). The same imaging system was used to perform echocardiographic studies at baseline (2 day before) and 3 weeks after miR148a mimic injection. After induction of light general anesthesia, the heart was imaged at the level of the greatest LV diameter. LV ejection fraction (LVEF) was measured with VisualSonics version 1.3.8 software from 2-dimensional long-axis views.
• LVEF LV ejection fraction
• Exercise capacity was assessed weekly with Exer-3/6 open treadmill (Columbus Instruments, Columbus, OH), beginning 3 weeks after CDC/vehicle injection (exercise capacity measured in a subset of mdx mice 1 week pre-operatively was equivalent to that measured 3 weeks post-operatively in the Mdx+Vehicle group; data not shown). After an acclimation period (10 m/min for 20 min) stepwise increases in average speed (1 m/min) were applied every two minutes during treadmill exercise until the mouse became exhausted (spending >10 seconds on shocker; continuous nudging was used during treadmill to help mice stay on the track). Subsequently, the mouse was returned to the cage and the total distance recorded. After 3 months of weekly exercise, CDC/vehicle mdx mice along with wild-type age-matched mice were followed for assessment of mortality. The treadmill protocol conformed to guidelines from the American Physiological Society 3 .
• Mouse CDCs were expanded from wild-type strain-matched mouse hearts (C57BL/10ScSnJ wild type mouse heart) as described 2 . Briefly, ventricular tissues were minced into ⁇ 1 mm explants, partially digested enzymatically and plated on adherent (fibronectin-coated) culture dishes. These explants spontaneously yield outgrowth cells (explant-derived cells) which were harvested once confluent and plated in suspension culture (10 5 cells/mL on poly-D-lysine-coated dishes) to enable self-assembly of three-dimensional cardiospheres. Subsequent replating of cardiospheres on adherent culture dishes yielded CDCs which were used in all experiments at passage one.
• PCR Quantitative polymerase chain reaction
• Paraffin-embedded sections from apical, middle and basal parts of each heart were used for Masson's trichrome staining and immuno staining with antibodies against Ki67 and aurora B.
• Myocardial abundance of collagen I Al and collagen III Al was measured by Western blot analysis (Fig. 29).
• Exosomes were isolated from serum-free media conditioned overnight (24 hr) by cultured human CDCs (CDC-XO) [or normal human dermal fibroblasts (NHDF) as a control] in hypoxia (2% 0 2 ; default condition) or normoxia (20% 0 2 , solely for studies comparing RNA content of exosomes).
• Ultracentrifugation 100,000g for 1 hr was used to isolate exosomes from conditioned media after sequential centrifugations at 300g (lOmin) and 10,000g (30min) and filtration with 0.22 micron filters. Isolated exosomes underwent RNA extraction and subsequently RNA sequencing (Fig.
• preliminary dose-response experiments were performed, which identified lxl 0 5 cells in first injection and lxl 0 4 cells in second injection (3 months after first injection) as effective doses, consistent with prior dose- ranging experiments in ischemic and nonischemic mouse models.
• PBS phosphate-buffered saline
• LV left ventricular
• the LV was visually divided into three zones: basal, middle, and apical, with one injection in the basal, two in the middle and one in the apical zone.
• a miR-148a mimic (hsa-miR- 148a-3p, 2 ⁇ g; Sigma-Aldrich, St. Louis, MO) was mixed with RNAiMAX transfection reagent (life technologies, Grand Island, NY) for 30 min at room temperature at a total volume of 40 ⁇ and injected into 4 points per heart as described above.
• RNAiMAX transfection reagent life technologies, Grand Island, NY
• Paraffin-embedded sections from apical, middle and basal parts of each heart were used for histology.
• Masson's trichrome staining HT15 Trichrome Stain [Masson] Kit; Sigma-Aldrich, St.
• T cells, B cells and macrophages were assessed by immunostaining with antibodies against mouse CD3, CD20 and CD68, respectively, and the average number of cells in each heart was calculated from counting cells in 10 fields (20x magnification) from each of 10 sections selected randomly from the apical (3 sections; 50 ⁇ interval), middle (4sections; 50 ⁇ interval) and basal (3 sections; 50 ⁇ interval) regions of each heart.
• Protein Block Solution contained 3% saponin was applied for immunofluorescence staining of Ki67). Subsequently, primary antibodies in Protein Block Solution were applied overnight in 4 C° for immunofluorescence staining of 5- ⁇ sections from apical, middle and basal parts of each heart. After 3x wash with PBS, each 10 minutes, Alexa Fluor secondary antibodies (Life Technologies, Grand Island, NY) were used for detection. Images were taken by a Leica TCS SP5 X confocal microscopy system.
• Immunofluorescence staining was conducted using antibodies against mouse Ki-67 (SP6; 1 :50; Thermo Fisher Scientific, Fremont, CA), WGA (Wheat germ agglutinin; 1 :200; Life Technologies, Grand Island, NY), Nrf2 (C20; 1 :50; Santa Cruz Biotechnology, Santa Cruz, CA), aurora B (1 :250; BD Biosciences, San Jose, CA).
• Immunoperoxidase staining Immunohistochemical detection of CD3, CD20 and CD68 was performed on 5- ⁇ sections using prediluted rabbit monoclonal antibodies from Ventana Medical System (Tuscon, AZ; CD68) and Cell Marque (Rocklin, CA; CD3, CD20).
• Staining was conducted on the Leica Bond-Max Ventana automated slide stainer (Chicago, IL) using onboard heat-induced epitope retrieval method in high pH ER2 buffer (Leica Biosystems, Buffalo Grove, IL). The staining was visualized using the Dako Envision + rabbit detection System and Dako DAB (Carpinteria, CA). The slides were subsequently counterstained with mayer's hematoxylin for 1 minute and coverslipped.
• Example 29 Example 29
• Nrf2 signaling [Nrf2, phospho-Nrf2 (Nrf2-p s40 ) and Nrf2 downstream gene products: heme oxygenase- 1 (HO-1), catalase, superoxide dismutase-2 (SOD-2), and catalytic subunit of glutamate-cysteine ligase (GCLC)], Nrf2 phosphorylation [phospho-Akt(Akt-p 308 )], oxidative phosphorylation [CI (NDUFB8 subunit), CII (SDHB subunit), CIV (MTCOl subunit), CIII (UQCRC2 subunit) and CV (ATPSA subunit)], mitochondrial biogenesis (PGC-1), mitophagy (PINK1), inflammation (NF- ⁇ and MCP-1) and fibrosis (Collagen IA1 and collagen IIIAl).
• Nrf2 signaling [Nrf2, phospho-Nrf2 (Nrf2-p s40 ) and Nrf2 downstream gene products: heme oxygenas
• Myocardial density of malondialdehyde protein adducts was also measured by Western blotting (WB).
• WB Western blotting
• Samples from apical, middle and basal parts of each heart were mixed and homogenized, and nuclear and cytoplasmic fractions were extracted per manufacturer's instructions (CelLytic NuCLEAR Extraction Kit, Sigma- Aldrich, St. Louis, MO).
• Cytoplasmic, nuclear and mitochondrial extracts for WB analysis were stored at -80C°.
• the protein concentrations in extracts were determined by the Micro BCA 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: antibodies against mouse Nrf2, HO-1, catalase, SOD-2, GCLC, collagen IA1, and collagen IIIAl, and PGC-1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), phospho-Nrf2 (Nrf2-p s40 ; Biorbyt, San Francisco, CA), respiratory chain subunits (Total OXPHOS Rodent WB Antibody Cocktail antibody), malondialdehyde, citrate synthase and TBP (Abeam, Cambridge, MA), Akt and Akt-p T308 , ⁇ - ⁇ , ⁇ - ⁇ - ⁇ , (Cell Signaling Technology, Denver, CO), PINK1, MCP-1 and NF
• TBP TATA binding protein
• citrate synthase cytosolic and mitochondrial target proteins.
• Western blot methods Briefly, aliquots containing 20 ⁇ g proteins were fractionated on 8, 10 and 4-12% Bis-Tris gel (Life technologies, Grand Island, NY) at 120 V for 2 h and transferred to a PVDF membrane (Life technologies, Grand Island, NY). The membrane was incubated for 1 h in blocking buffer (l x TBS, 0.05% Tween-20 and 5% nonfat milk) and then overnight in the same buffer containing the given antibodies at optimal dilutions listed in Table 1.
• blocking buffer l x TBS, 0.05% Tween-20 and 5% nonfat milk
• the membrane was washed 3 times for 5 min in l x TBS, 0.05% Tween-20 before a 2- h incubation in a buffer (l x TBS, 0.05% Tween-20 and 3% nonfat milk) containing horseradish peroxidase-linked anti-rabbit IgG, anti-mouse IgG (Cell Signaling Technology, Denver, CO) and anti-goat IgG (Sigma-Aldrich, St. Louis, MO) at 1 :1000-3000 dilution.
• the membrane was washed 3 times for 5 min in 1 x TBS, 0.05%> Tween-20 and developed by autoluminography using the ECL chemiluminescent agents (Super Signal West Pico Chemiluminescent Substrate; Life Technologies, Grand Island, NY). Citrate synthase and TBP were used as housekeeping proteins against which expressions of the proteins of interest were normalized. Phosphorylated Akt, Nrf2 and ⁇ - ⁇ were normalized to total Akt, Nrf2 and ⁇ - ⁇ . Western blot analyses of collagen I and collagen III were conducted under non- reducing, non-denaturing condition.
• mice were sacrificed via cervical dislocation after isofluorane anesthesia.
• Hearts were immediately excised, rinsed in PBS and homogenized via polytron in lmL ice cold HES buffer (250mM sucrose, ImM EDTA, lOmM HEPES, pH 7.4). Lysates were spun down at lOOOg for 5min at 4°C to remove unbroken cells and large debris. Supernatant was then spun down at 7000g for lOmin at 4°C to separate mitochondria-enriched fraction from crude cytosol. Pellet was resuspended in lmL HES buffer (A subportion in lysis buffer for WB).
• iPS- derived cardiomyocytes were loaded for 30 min with 5 ⁇ of the fluorogenic calcium-sensitive dye, Cal-520 (AAT Bioquest, Sunnyvale, CA) and paced via field stimulation at a frequency of 1 Hz using an Ion-Optix Myopacer (IonOptix Corp) delivering 0.2 ms square voltage pulses with an amplitude of 20 V via two platinum wires placed on each side of the chamber base ( ⁇ 1 cm separation).
• the Inventors used the xyt mode (2D) of a Leica TCS-SP5-II (Leica Microsystems Inc.; Wetzlar, Germany) to image intracellular Ca 2+ .
• Cal 520 was excited with a 488 nm laser and its emission (>505 nm) was collected with a 10X objective (Leica: N PLAN 1 Ox/0.25) at scan speeds ranging from 36 to 7 ms per frame depending on the field size.
• the fluorescence intensity (F) proportional to Ca 2+ concentration was normalized to baseline fluorescence, F0 (F/F0).
• Time to peak and Ca 2+ transient amplitude (F/F0) were analyzed with the software Clampfit (ver. 10.2, Molecular Devices, Inc.). Beat-to-beat alternans in each group calculated over the 5-10 sec interval of pacing at 1 Hz.
• the amplitude of each transient from each cell was measured during pacing and mean and standard deviation were calculated and compared among groups.
• CDC injection enhanced ambulatory function (Fig. 23B).
• CDC-treated mdx mice showed a substantial increase in maximal exercise capacity, relative to vehicle-treated mdx mice, over the 3 mos that it was measured; survival also differed in the two groups (Fig. 23C).
• Fig. 23C By -23 mos of age all vehicle- treated mdx mice had died, whereas >50% of CDC-treated mdx mice remained alive (Fig. 23 C).
• Nrf2 is normally sequestered in the cytoplasm via binding to its repressor molecule, Keapl .
• Oxidative stress (as well as Nrf2 phosphorylation by protein kinases such as Akt) causes dissociation of the Nrf2 -Keapl complex, which culminates in nuclear translocation of Nrf2 and transcriptional activation of antioxidant enzymes.
• Akt and cytoplasmic and nuclear Nrf2 were high (as expected in response to oxidative stress); CDC treatment further increased their protein levels (Fig. 23E).
• heme oxygenase- 1 HO-1
• catalase catalase
• superoxide dismutase-2 SOD-2
• GCLC glutamate-cysteine ligase
• Mitochondrial structure and function are abnormal in muscular dystrophy-associated heart failure.
• mitochondrial integrity improved 3 weeks after CDC injection: CDCs restored mitochondrial ultrastructure (Fig. 24A), increased mitochondrial DNA copy numbers (but not mitochondrial number; Fig. 24B & C), augmented levels of respiratory chain subunits (Fig. 24D) and normalized the deficient respiratory capacity of isolated mdx mitochondria (Fig. 24E).
• NFKB the master regulator of pro-inflammatory cytokines and chemokines
• MCP1 monocyte chemoattractant proteinl
• CD68 + macrophages and CD3 + T cells Increases in phosphorylated ⁇ and nuclear p65 contents were accompanied by marked upregulation of MCP1 (monocyte chemoattractant proteinl) and accumulation of CD68 + macrophages and CD3 + T cells.
• CDC treatment reversed activation of NFKB and decreased the number of inflammatory cells in mdx hearts 3 weeks after CDC injection (Fig. 24G & H). The Inventors also probed the effects of CDCs on cardiomyogenesis.
• CDCs are known to increase endogenous cardiomyogenesis in ischemic and non-ischemic models. Likewise, CDC treatment promoted cardiomyogenesis in the mdx heart, as evidenced by a marked increase in Ki67 + and aurora B + cardiomyocytes (Fig. 29).
• CDC-secreted Exosomes Reproduce Benefits of CDCs in mdx Mice
• Exosomes secreted by CDCs (CDC-exosomes) mimic the functional and structural benefits of CDCs in a murine model of myocardial infarction.
• functional, anti-fibrotic, and cardiomyogenic benefits of CDCs are reproduced by administration of exosomes isolated from media conditioned by hypoxic CDCs.
• Intramyocardial injection of two repeat doses of human CDC-exosomes led to sustained improvement in EF in mdx mice, relative to vehicle -treated mice (Fig. 25A & Fig. 27).
• DMD CMs Duchenne human iPS-derived cardiomyocytes
• OCR oxygen consumption rate
• Fig. 24E mdx heart mitochondria
• Wild type 2 Mdx+Vehicle 3: Mdx+CDC or Mdx+CDC-exosome or Mdx+miR148
• CDCs attenuate fibrosis and inflammation in the mdx heart, while improving pump function, increasing exercise capacity and enhancing survival.
• the salient benefits of CDCs were reproduced by CDC-exosomes.
• the Inventors' findings support the hypothesis that CDCs act by secreting exosomes laden with genetic signals, including (but not limited to) miR-148a. These exosomes are taken up by the surrounding myocardium, where they antagonize multiple pathophysiological pathways that underlie DMD cardiomyopathy.
• the constellation of effects is synergistic: oxidative stress, inflammation and fibrosis are blunted, while cardiomyogenesis and mitochondrial function are augmented.
• cardiosphere derived cells are sources of cardiosphere derived cells, the use of alternative sources such as cells derived directly from heart biopsies (explant-derived cells), or from self-assembling clusters of heart-derived cells (cardiospheres), exosomes produced by such cells, method of isolating, characterizing or altering exosomes produced by such cells, and the particular use of the products created through the teachings of the invention.
• Various embodiments of the invention can specifically include or exclude any of these variations or elements.
• the numbers expressing quantities of ingredients, properties such as concentration, reaction conditions, and so forth, used to describe and claim certain embodiments of the invention are to be understood as being modified in some instances by the term "about.” Accordingly, in some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can 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 invention are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the invention may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

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