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WO2016196822A1 - Exosomes d'urodèles comme agents thérapeutiques - Google Patents

Exosomes d'urodèles comme agents thérapeutiques Download PDF

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WO2016196822A1
WO2016196822A1 PCT/US2016/035561 US2016035561W WO2016196822A1 WO 2016196822 A1 WO2016196822 A1 WO 2016196822A1 US 2016035561 W US2016035561 W US 2016035561W WO 2016196822 A1 WO2016196822 A1 WO 2016196822A1
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mir
exosomes
cells
composition
exosome
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Eduardo Marban
Eleni TSELIOU
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Cedars Sinai Medical Center
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Cedars Sinai Medical Center
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/56—Materials from animals other than mammals
    • A61K35/65—Amphibians, e.g. toads, frogs, salamanders or newts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions

Definitions

  • compositions and techniques allowing for protection against and/or reversal of disease pathology in heart and vascular disease.
  • cardiac myocytes The death of cardiac myocytes is a major cause of myocardial infarct and heart failure.
  • the potential of cardiac regeneration in adult mammals has been suggested by the emerging possibility of endogenous regeneration persisting into later development, contrary to existing views that such mechanisms do not persist after birth. Additionally, it is now established that what had been considered terminally differentiated cells actually possess significant plasticity, as demonstrated by studies reporting dedifferentiation of somatic cells into pluripotent stem cells, or transdifferentiation into cardiac myocytes.
  • Other types of stem cells such as cardiosphere-derived cells (CDCs) have shown a proven therapeutic benefit by possibly tapping into the aforementioned repair and regeneration mechanisms. Current understanding of their salutary benefit indicates that indirect mechanisms are responsible.
  • exosomes the lipid bilayer nanovesicles secreted by cells when multivesicular endosomes fuse with the plasma membrane
  • endogenous cardiac regeneration mechanisms may similarly be governed, and could be exploited by, therapeutic approaches involving exosomes.
  • compositions and methods involving exosomes derived from urodeles which are demonstrated as capable of improving heart function in mammals.
  • Secreted exosomes from a newt cell line are bioactive in mammals, as shown to promote rat cardiomyocyte proliferation, increase SDF-1 secretion by human dermal fibroblasts, and improve functional recovery after myocardial infarct.
  • compositions including a plurality of exosomes isolated from urodele cells.
  • the plurality of exosomes comprise one or more exosomes with a diameter of about 30 nm to 300 nm and are about 2-5 kDa.
  • the plurality of exosomes comprise one or more exosomes with a diameter of about 40 nm to 100 nm and are about 3 kDa.
  • the plurality of exosomes comprise one or more exosomes including one or more microRNAs selected from the group consisting of: miR-1469, miR-762, miR-574-3p, miR-574-5p, miR-3197, miR-4281, miR- 1976, miR-1307, miR-1224-3p, miR-187, miR-3141, miR-1268, miR-155, miR-122, miR- 638, miR-3196, miR-223, miR-4267, miR-1281, miR-885-5p, miR-663, miR-let-7b, miR- 29d, miR-144, miR-let-7e 143, miR-lrt-7g, miR-17a, miR-96, miR-125a-5p, miR-128, miR- 720, miR-21, miR-9, miR-26b, miR-29b, miR-30c, miR-30b, miR-191, and miR-lb
  • the plurality of exosomes comprise one or more exosomes including miR-96, miR-29b, and miR-191.
  • the plurality of exosomes comprise one or more exosomes including one or more messenger RNAs selected from the group consisting of: PGM5-AS1, KD2, SLC43A1, PHF20L1, DLNZ, AMH, ARHGEF15, SCARF1, CPSF2, CEP170B, C16orfl l/PRR35, CPSF6, RNF39, C22orf26/PRR34, TMEM64, PRDM16, CHST7, ADCYAPl, VSIG10L, MUC22, SRMS, SAP30L, GAS6-AS1, ZFAND5, GAGE13, LCN12, R5A1, HLA6, LY6E, and SPIN4.
  • the urodele cells comprise Notophthalmus viridescens cells. In other embodiments, the Notophthalmus viridescens cells comprise Al cell line. In other embodiments, the urodele cells are dedifferentiating, transdifferentiating and/or proliferating when isolating the plurality of exosomes. In other embodiments, the composition includes a pharmaceutically acceptable carrier.
  • a method of treating a heart related disease including administering a composition including a plurality of exosomes isolated from urodele cells to a subject, thereby treating the subject.
  • the plurality of exosomes comprise one or more exosomes with a diameter of about 30 nm to 300 nm and are about 2-5 kDa.
  • the plurality of exosomes comprise one or more exosomes with a diameter of about 40 nm to 100 nm, and are about 3 kDa.
  • administering a composition includes 1 x 10 8 or more exosomes in a single dose.
  • the single dose is administered multiple times to the subject.
  • the subject has a chronic disease.
  • administering a composition includes one or more of intra-arterial infusion, intravenous infusion, and injection.
  • the subject is diagnosed as afflicted with a heart related disease prior to administering the composition.
  • the urodele cells comprise Notophthalmus viridescens cells.
  • the Notophthalmus viridescens cells comprise Al
  • FIG. IB Venn diagram showing the variable microRNA profile between CDC and NHDF exosomes. Font size reflects the magnitude of differential expression of each microRNA.
  • FIG. 2A Graphical representation of exosome isolation and purification for exosomes.
  • FIG. 2B Cell viability (calcein) and cell death (Ethidium homodimer-1) assay performed on CDCs over the 15 day serum-free conditioning period.
  • FIG. 2C Representative images of CDCs before and after serum-free conditioning.
  • Figure 3. Heat Map or microRNA PCR Array Identifies Mir-146a as a Highly
  • MicroRNAs reported to be upregulated following cardiac injury. Heat map showing urodele microRNAs conserved with human, frog, pipid frog, zebrafish microRNAs that are enriched 7 and 21 days post-injury (dpi). From Witman et al., "miR-128 regulates non-myocyte hyperplasia, deposition of extracellular matrix and Isletl expression during newt cardiac regeneration.” Dev Biol. 2013 Nov 15;383(2):253-63.
  • FIG. 5B Nanosight histogram of Al exosome diameter and number
  • FIG. 5C Graphical representation of exosome size and concentration per milliliter
  • RNA per microvesicle Amount of isolated total RNA per microvesicle as determined by Nanodrop (RNA Concentration) and Nanosight (particle number) for Al, hCDC (OD35220) and Normal Human Dermal Fibroblasts (NHDF) exosomes following three days of serum-starvation of cells in culture. Values given in picograms per microliter.
  • FIG. 6A Profile of RNA types identified by MAVERIX Biomics of all RNA isolated from Al exosomes and mapped to the Human genome.
  • Fig. 6B Proteomic profile of total protein of Al exosomes; analyzed by FUNRICH Functional Enrichment Analysis Tool software displaying relative protein abundances by cellular function.
  • FIG. 7 qPCR, protein profiles Al cargo contents. Comparison of (Fig. 7A) RNA and (Fig. 7B) Protein profiles from Al, hCDC, and NHDF exosomes following 3 days of serum-starvation of cell culture.
  • FIG. 8 qPCR validation of Al exosome-unique messenger RNAs identified by RNA sequencing.
  • Al exosome RNA was polyadenylated and converted to cDNA using a universal 3' adapter. The genes were detected using a forward primer specific for the gene and a universal 3' reverse primer for the end of the transcript.
  • the forward primer sequences used in the qPCR validation experiments are shown in Table 1.
  • Total RNA isolation from the Al exosome was performed using the miRNeasy kit (Qiagen) as per manufacturer's instructions.
  • a total amount of lOOng RNA was converted to cDNA using the Quantimir kit (SBI) and PCR was performed using SYBR Green on ABI 7900HT detection system. The data were analyzed using the AACt method.
  • Figure 9 Newt exosome surface protein characterization. Percentage of exosomes positive for protein surface markers as determine by MACSQuant Flow Cytometer (Miltenyi).
  • Fig. 9A Secondary antibody-Alexa 488nm-treated of exosome (background control).
  • Fig. 9B Positive Thrombospondin antibody -treated exosome counts (-79% of total exosome counts)
  • Fig. 9C Postive Penostin antibody-treated exosome counts (-60% of total exosome counts)
  • Fig. 9D Positive Fibronectin antibody-treated exosome counts (-52% of total exosome counts)
  • Fig. 9E Positive CD81 antibody -treated exosome counts (-22% of total exosome counts)
  • FIG. 9F Graphical representation of MACSQuant Flow Cytometry data.
  • FIG. 10A PonceauS staining of PVDF membrane containing transferred total protein (40 ⁇ g/lane) of Al, CDC and NHDF cells and exosomes.
  • FIG. 10B PVDF membrane treated with rabbit polyclonal anti-periostin antibody (1 : 1000) overnight followed by goat anti-rabbit secondary antibody conjugated to horseradish peroxidase (1 :5000). Blot was developed using enhanced chemiluminescence substrate (ECS) and bioluminescence captured using a Biorad Gel Dock Imager.
  • ECS enhanced chemiluminescence substrate
  • FIG. 11A Representative confocal images of EdU-positive Neonatal rat ventricular cardiomyocytes (NRVMs) following Al exosome treatment.
  • the green channel is EdU (5-ethynyl-2 ' -deoxyuridine; 10 ⁇ ), a thymidine analog incorporated into DNA during DNA synthesis .
  • the red channel is actin staining and the blue channel is DAPI nuclear stain.
  • FIG. 11B Quantification of EdU-positive NRVMs after exposure to NHDF exosomes or Al exosomes. Experiments were performed in triplicate.
  • Fig. 11C Fold changes in expression of genes related to cell proliferation in NRVMs following treatment of Al exosomes versus NHDF exosomes. Experiments were performed in triplicate.
  • FIG. 12A Flow Cytometry plots of Annexin V-stained NRVMs following 24hr treatment with Al exosomes or NHDF exosomes and then 24hrs of ischemic injury ( ⁇ H 2 0 2 ).
  • FIG. 12B Graphical representation of Annexin V- positive NRVMs treated with NHDF exosomes or Al exosomes
  • Figure 13 In vitro studies: secreted factors. Expressed or Secreted Factors following Exosome exposure (Fig. 13A) ELISA performed from the supernatant collected from NHDFs in culture exposed to NHDF exosomes or Al exosomes for the cell proliferation-associated marker, SDF-1. Experiments were performed in triplicate. (Fig. 13B) ELISA performed from the supernatant collected from NHDFs in culture exposed to NHDF exosomes or Al exosomes for the angiogenesis marker, VEGF. Experiments were performed in triplicate. (Fig. 13C) Western blot of collagen expression in Al exosome- treated NHDFs versus NHDF exosome- treated cells (left panel) and pooled data of triplicate experiments (right panel).
  • FIG. 14 In vivo studies: heart function ejection fraction as measured by echocardiography. Functional analysis of ejection fraction in female Wistar-Kyoto rat hearts three weeks following MI and 25C ⁇ g or 50C ⁇ g Al exosome or 25C ⁇ g NHDF exosome treatments. For ejection fraction evaluation 2D parasternal views were used to measure end- systolic and end-diastolic volumes. All images were analyzed on an offline computer with the appropriate VEVO 7 VisualSonics Software installed.
  • FIG. 15A Representative brightfield images of stained rat heart sections (Masson's trichrome) three weeks after MI and NHDF or Al exosome treatments
  • FIG. 15B Analysis of the percent viable area three weeks following MI and exosome treatment
  • Fig. 15C Analysis of the percent scar area three weeks following MI and exosome treatment
  • Fig. 15D Analysis of the infarct wall thickness three weeks following MI and exosome treatment
  • FIG. 16A Representative confocal images of serial sections taken from paraffin-fixed left ventricle of hearts treated with 250 ⁇ g or 500 ⁇ g Al exosome or 250 ⁇ g NHDF exosome treatments. The heart tissue is stained with wheat germ agglutinin.
  • FIG. 16B Graphical representation of cardiomyocyte size in microns; four animals per group.
  • FIG. 19 Immunology: monocyte infiltration. Representative brightfield images of heart tissue stained with Hematoxylin and eosin from each of the three treatment groups, three weeks after MI and treatment. Arrows indicate infiltrating monocyte clusters within the tissue. Graphical data shows the average number of monocytes counted per image field; 5 slides per heart, 4 animals per treatment group.
  • FIG. 20 Immunology: macrophage infiltration. Representative confocal images of heart tissue stained with the macrophage marker CD68 from each of the three treatment groups, three weeks after MI and treatment. Graphical data shows the average number of monocytes counted per image field; 5 slides per heart, 4 animals per treatment group. The difference in macrophage number between groups was not statistically significant.
  • FIG. 21 Immunology: T-cell infiltration. Representative confocal images of heart tissue stained with antibodies against CD8+ (green) and CD4+ (red) T-cell markers, from each of the three treatment groups, three weeks after MI and treatment. Graphical data shows the average number of CD8+ and CD4+ T-cells counted per image field; 5 slides per heart, 4 animals per treatment group. The difference in T-cell infiltration (CD8+ and CD4+) following 500 ⁇ g of Al exosome treatment from the NHDF exosome treatment was not statistically significant.
  • FIG. 22 miR-9-5-p induced cell proliferation. Cardiomyocyte proliferation following treatment with miR-9-5p mimic or control microRNA
  • FIG. 22B Graphical representation of the percentage of EdU-positive cardiomyocytes in culture, treated with the 25nM miR-9-5p mimic or 25nM control microRNA. A total of 10 images were acquired and analyzed in each of the groups. Experiments were repeated in triplicate.
  • FIG. 22C Gene expression changes related to cell proliferation in NRVMs following treatment with miR-9-5p mimic or control microRNA. Data shows fold change of gene expression in miR-9-5p-treated NRVMs relative to control microRNA-treated NRVMs.
  • FIG. 23 miR-9-5p-induced secretion in NHDFs.
  • FIG. 23A Graphical representation of SDF-1 secretion by NHDFs into the supernatant in picograms per milliliter. The graphs show secretion by NHDFs treated with 25nM and 50nM concentrations of the miR-9-5p mimic or control microRNA. The experiment was repeated in triplicate.
  • FIG. 23B Graphical representation of VEGF secretion by NHDFs into the supernatant in picograms per milliliter. The graphs show secretion by NHDFs treated with 25nM and 50nM concentrations of the miR-9-5p mimic or control microRNA. The experiment was repeated in triplicate.
  • Figure 24 Quantification of EdU-positive NRVMs after exposure to NHDF exosomes or hCDC exosomes (OD35220). Experiments were performed in triplicate.
  • FIG. 25 NHDF microRNAs expression post Al exosome treatment.
  • miRNA isolation from the Al exosome-treated NHDFs was performed using the miRNeasy kit (Qiagen) as per manufacturer's instructions.
  • a total amount of lOOng RNA was converted to cDNA using the mi Script II RT Kit and PCR was performed using SYBR Green on ABI 7900HT detection system.
  • the mi Script RNA plates used were purchased from SA Biosciences. The data were analyzed using the AACt method. Fold change of microRNA expression in NRVMs is shown relative to untreated NHDFs.
  • FIG. 26 Tgfp/smad pathway gene expression following Al or NHDF exosome treatment in NHDFs Fibrosis and the Tgfp/smad pathway.
  • a total amount of 10-20 ⁇ g of protein was separated on 4-12% SDS-polyacrylamide gels and transferred onto nitrocellulose blotting membranes.
  • Blocking of the membranes was performed using 5% nonfat milk in PBS containing 0.5% Tween 20 followed by incubation with TGFBRI (1 :500 Novus), TGFBRII (1 :500 Novus), phosphorylated smad4 (1 :500 Sigma Aldrich), phosphorylated smad2/3 (1 :500 Sigma Aldrich) and total smad2 (1 :500 Abeam). Following washing with TBS-Tween buffer and incubation with appropriate HRP-labeled secondary antibodies protein detections was performed using ECL kit (Thermo Scientific). Image J was used for quantification of the bands. Fold protein changes was normalized to values of control cells. Pooled data showed no differences in p-smad2/3 and p-smad4 (Fig.
  • FIG. 26A and Tgfp-receptor 2 expression (Fig. 26B) between treatments. Data are mean ⁇ SEM (the experiments were performed in triplicate).
  • FIG. 27A Representative echocardiographic long axis images from each of the 3 groups highlighting the infarct.
  • FIG. 27B Graphical representation of left ventricle end-diastolic volumes of infarcted rat hearts following NHDF exosome or Al exosome treatments
  • FIG. 27C Graphical representation of left ventricle end-systolic volumes of infarcted rat hearts following NIFDF exosome or Al exosome treatments
  • FIG. 27D Graphical representation of fractional shoertening of infarcted rat hearts following NIFDF exosome or Al exosome treatments.
  • Figure 28 Native miR-9-5p expression levels and downstream target gene expression.
  • Fig. 28A Quantitative PCR of miR-9-5p from neonatal (PI) and 2 month-old rat hearts. microRNA was extracted from neonatal Sprague Dawley rat hearts at PI and at 2 months of age using miRNEasy minikit (Qiagen) according to manufacturer's instructions. Data shows fold difference in miR-9-5p expression in adult hearts relative to neonatal rat hearts.
  • Fig. 28B PCR array of miR-9-5p target genes in NFIDFs following Al exosome- treatment
  • Fig. 28C PCR array of miR-9-5p target genes in NHDFs following miR-9-5p treatment. All experiments described above were performed in triplicate.
  • microRNA miR-1 is down-regulated when hyperplasia is elevated and later returns to homeostatic levels when remodelling is occurring, suggesting a role in controlling cardiac cell fate.
  • microRNA miR-21 has been described as being upregulated in expression during the hyperplastic and remodelling phases of cardiac regeneration, possibly indicating elevated microRNA miR-21 expression within cardiac fibroblasts as inducing production of an extracellular matrix scaffold, which is required for reconstitution of the myocardium.
  • CDCs human cardiosphere-derived cells
  • exosomes the secreted lipid vesicles containing a rich milieu of biological factors, provide powerful paracrine signals by which stem cells effectuate 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 effectuate therapeutic benefits is suggested by 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), immune cells (dendritic and CD34+) and human neural stem cells (hNSCs).
  • MSC mesenchymal stromal
  • MNC bone marrow stem cells
  • immune cells dendritic and CD34+
  • hNSCs human neural stem cells
  • human cardiosphere-derived cells CDCs are known to improve myocardium and vasculature, for which exosomes and their microRNA cargo content appear to be important cellular actors.
  • Exosome-based, "cell-free” therapies in contrast to cell therapy, provide distinct advantages in regenerative medicine. Generally, their production under defined conditions allows for easier manufacture and scale-up opportunity. They further obviate safety issues as non-viable entities, with reduced or non-existent immunogenic or tumorigenic potential. For example, 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.
  • 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.
  • exosomes are likely to be 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.
  • exosomes are lipid bilayer vesicles that are enriched in a variety of biological factors, including cytokines, growth factors, transcription factors, 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 30-300 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 when formed.
  • Exosomes contain a biological milieu of different proteins, including cytokines and growth factors, coding and noncoding RNA molecules, all necessarily derived from their parental cells.
  • exosomes further express the extracellular domain of membrane-bound receptors at the surface of the membrane.
  • exosomes are involved in intercellular communication between different cell types, but 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. Because exosomes contain cargo contents reflecting the parental cell type and its cellular regulatory state at time of production, the resulting composition of exosomes play a critical role in determining their function.
  • exosomes secreted by cells such as cardiosphere-derived cells (CDCs)
  • CDCs cardiosphere-derived cells
  • the remarkable regenerative potential of urodeles suggests the possibility of isolating and deploying their potent biological factors to spur cardiac regeneration for human therapies.
  • some measure of urodele regeneration may occur through differentiated cells re-entering the cell cycle, recruitment of progenitor cells, or some combination of the two, and in this regard, isolating exosomes from urodele cells undergoing dedifferentiation, transdifferentiation, and/or proliferation, may serve to enrich those particular factors (e.g., microRNAs) critical to regeneration and repair processes.
  • 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 clearly that RNA molecules are selectively, not randomly incorporated into exosomes, as revealed by studies report 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 effecting 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 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.
  • the net result of exosome-cell interactions is 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.
  • embryonic stem cell (ESC)-derived exosomes 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.
  • 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 supernatants 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 floatation velocity further allows for separation of differentially sized exosomes. In general, exosome sizes will possess a diameter ranging from 30-300 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 homogeneously sized particles and preserve their biological activity as the preparation is maintained at a physiological pH and salt concentration.
  • F1FFF Flow field-flow fractionation
  • focused techniques may be applied to isolated specific exosomes of interest. This includes relying on antibody immunoaffinity to recognizing certain exosome-associated antigens. Conjugation to magnetic beads, chromatography matrices, plates or microfluidic 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 Important goals of developing exosome-based therapy are creation of "cell-free" therapies, wherein the benefits of cell therapeutics can be provided with reduced risks or in scenarios in which cell therapy would be unavailable, or capturing the potential regenerative potential of urodeles for applications in mammals.
  • exosomes Without being bound by any particular theory, the Inventors believe that the therapeutic effects of stem cells or regenerative potential of urodeles can be reproduced by exosomes. In fact, focused application of exosomes may actually provide superior results for the following reasons. Firstly, the retention of delivered stem cells has been shown to be short lived. Second, the quantity of local release of exosomes from a stem cell is limited and occurs only as long as the cell is retained. Thirdly, the quantity of exosomes delivered can be much higher (i.e., high dosing of its contents). Fourth, exosomes can be readily taken up by the cells in the local tissue milieu. Fifth, issues of immunogenicity are avoided.
  • stem cell therapy for heart disease and related conditions has long been a promising concept for addressing such issues, they depend highly on successful delivery into the myocardial area of need.
  • General principles from such techniques e.g., concentration, timing of delivery, and sustained bioavailability
  • exosome-based therapy e.g., concentration, timing of delivery, and sustained bioavailability
  • a key benefit of exosome based therapy is that the central challenges limiting cellular transplants are largely obviated (e.g., cell engraftment of cells and prolonged survival of the transplanted cells).
  • a key limitation of cell delivery is providing a sufficient number of cells to maximize therapeutic effect, such cells being susceptible to clearance and washout.
  • the regenerative effects of delivered cells may further rely on migration and homing mechanisms to potentiate their stem cell activity at the site of injury.
  • Physiological or biochemical barriers may effectively eliminate administered cells moving to sites of repair.
  • the Inventors believe higher concentrations of biological agents to the local tissue milieu is possible via exosomes, and that repeated administration of such exosomes may maximize tissue regeneration and repair in a manner that would be infeasible for cell therapy.
  • exosome based therapy can delivered via a number of routes: intravenous, intracoronary, and intramyocardial. Exosomes, also allow for new delivery routes that were previously infeasible for cell therapy, such as inhalation. Benefits and drawbacks of these various approaches are described below.
  • Intravenous delivery technique can occur through a peripheral or central venous catheter. As the simplest delivery mode, this technique avoids the risk of an invasive procedure. However, intravenous may be regarded as a comparatively inefficient and less localized delivery method, as a high percentage of infused cell exosomes may become sequestered in organs such as the lung, liver, or spleen. Such sequestration may results in few or no cellular exosomes reaching coronary circulation or have unintended systemic effects following their distribution. Exosomes reaching the site of injury may also face additional obstacles when migrating across or effectuating signaling across cells in the arterial or capillary wall. Importantly, this route is unlikely to exist as an option for patients with occluded arteries, unless there are sufficient routes of collateral coronary artery circulation exist.
  • an approach that may be preferential involves intracoronary cell infusion.
  • exosomes can be administered with coronary flow.
  • balloon occlusion is used to introduce flow interruption as a means to minimize washout of the therapeutic.
  • a key advantage of the intracoronary approach is selective, local delivery of cells to the myocardial area of interest, thereby limiting risks of systemic administration.
  • Coronary delivery requires that the target myocardium be subtended by a patent coronary artery or identifiable collateral vessel and therefore performed following percutaneous coronary intervention (PCI).
  • PCI percutaneous coronary intervention
  • the relative ease of delivery following standard catheter intervention to re-establish coronary flow is a highly attractive opportunity for intracoronary delivery.
  • An alternative intravenous mode may be retrograde coronary sinus delivery.
  • This approach relies on catheter placement into the coronary sinus, inflation of the balloon, and exosome administered by infusion at pressures higher than coronary sinus pressure (e.g., 20 mL), thereby allowing for retrograde perfusion of cells into the myocardium.
  • coronary sinus pressure e.g. 20 mL
  • exosomes could be required to migrate across or effectuating their signaling across the arterial or capillary wall.
  • compositions including a plurality of exosomes.
  • the plurality of exosomes is generated by a method including providing a population of cells, and isolating a plurality of exosomes from the population of cells.
  • the cells are from amphibians within the order Caudata (also described as urodeles). For example, this includes species within the family Salamandridae (also known as newts). Various non-limiting examples include Notophthalmus viridescens and Ambystoma mexicanum.
  • the cells are cultured as a cell line capable of serial passaging. This includes, for example, the Al cell line of Notophthalmus viridescens.
  • the cells are stem cells, progenitors, precursors, and/or mesenchyme.
  • 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 a 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 cells are genetically modified to express endothelial nitric oxide synthase (eNOS), vascular endothelial growth factor (VEGF), SDF-1 (stromal derived factor), IGF-1 (insulin-like growth factor 1), HGF (hepatocyte growth factor).
  • eNOS endothelial nitric oxide synthase
  • VEGF vascular endothelial growth factor
  • SDF-1 stromal derived factor
  • IGF-1 insulin-like growth factor 1
  • HGF hepatocyte growth factor
  • 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. This includes, for example, isolating a plurality of exosomes from cells, such as urodele cells, undergoing dedifferentiation, transdifferentiation, and/or proliferation.
  • the plurality of exosomes includes 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 proteins (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 a pathway related to vasodilation, such as prostacyclin and nitric oxide, and/or vasoconstrictors such as thromboxane and endothelin-1 (ET-1).
  • 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- ⁇ . In other embodiments, the biological protein is capable of mediating Ml and/or M2 immune responses in macrophages. In other embodiments, the biological protein related to exosome formation and packaging of cytosolic proteins such as Hsp70, Hsp90, 14-3-3 epsilon, PKM2, GW182 and AG02. In certain embodiments, the exosomes express CD63, HSP70, CD 105 or combinations thereof. In other embodiments, the exosomes do not express CD9 or CD81, or express neither. For example, plurality of exosomes can include one or more exosomes that are CD63+, HSP+, CD105+, CD9-, and CD81-.
  • 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- 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., urodele-derived cells, 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.
  • the plurality of exosomes can include one or more exosomes containing microRNAs.
  • microRNAs known in the art, such as miR- 23a, miR-23b, miR-24, miR-26a, miR27-a, miR-30c, let-7e, mir- 19b, miR-125b, mir-27b, let-7a, miR-19a, let-7c, miR-140-3p, miR-125a-5p, miR-132, miR-150, miR-155, mir-210, let-7b, miR-24, miR-423-5p, miR-22, let-7f, and/or miR-146a, further including microRNAs evolutionarily conserved with the aforementioned microRNAs.
  • 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, further including microRNAs evolutionarily conserved with the aforementioned microRNAs.
  • 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, 5000-6000xg, 6000- 7000xg, 7000-8000xg, 8000-9000xg, 9000-10,000xg, to 10,000xg or more to separate larger- sized particles from a plurality of exosomes 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 (UPLC) 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 immunoaffinity 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 cells are from amphibians within the order Caudata (also described as urodeles). For example, this includes species within the family Salamandridae (also known as newts). Various non-limiting examples include Notophthalmus viridescens and Ambystoma mexicanum.
  • the cells are cultured as a cell line capable of serial passaging. This includes, for example, the Al cell line of Notophthalmus viridescens.
  • the cells are stem cells, progenitors, precursors, and/or mesenchyme.
  • synthetic exosomes are generated, which can be isolated by similar mechanisms as those above.
  • 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 cells are from amphibians within the order Caudata (also described as urodeles). In certain embodiments, the plurality of exosomes includes one or more exosomes that are CD63+, CD105+, or both.
  • 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 plurality of exosomes can include one or more exosomes containing microRNAs.
  • the one or more microRNAs comprise one or more exosomes including miR-96, miR-29b, and miR-191.
  • 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.
  • the method is a method of treating heart related disease, including administering a composition including a plurality of exosomes isolated from urodele cells to a subject, thereby treating the subject.
  • the method includes selecting a subject in need of treatment, administering a composition including a plurality of exosomes to the individual, wherein administration of the composition treat 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 pulmonary disease.
  • 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 subject is diagnosed as afflicted with a heart related disease prior to administering the composition.
  • the plurality of exosomes is generated by a method including providing a population of cells, and isolating a plurality of exosomes from the population of cells.
  • the cells are from amphibians within the order Caudata (also described as urodeles). For example, this includes species within the family Salamandridae (also known as newts). Various non-limiting examples include Notophthalmus viridescens and Ambystoma mexicanum.
  • the cells are cultured as a cell line capable of serial passaging. This includes, for example, the Al cell line of Notophthalmus viridescens.
  • the cells are stem cells, progenitors, precursors, and/or mesenchyme.
  • the exosomes are synthetic.
  • the plurality of exosomes is derived from urodele cells. In other embodiments, the plurality of exosomes includes exosomes including one or more biological molecules. In other embodiments, the plurality of exosomes including exosomes enriched for one or more biological molecules when derived from urodele compared to exosome derived from non-urodele sources. In various embodiments, the one or more biological molecules are proteins, growth factors, cytokines, transcription factors and/or morphogenic factors.
  • the plurality of exosomes including exosomes enriched for one or more biological molecules includes microRNAs, further including microRNAs that are enriched when derived from urodele compared to exosome derived from non- urodele 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- 23a.
  • the plurality of exosomes includes one or more exosomes enriched in at least one of miR-146a, miR-22, miR-24. In other embodiments, the plurality of exosomes can include one or more exosomes containing microRNAs.
  • microRNAs known in the art such as miR-1469, miR-762, miR-574-3p, miR-574-5p, miR-3197, miR-4281, miR-1976, miR-1307, miR-1224-3p, miR-187, miR-3141, miR-1268, miR-155, miR-122, miR-638, miR-3196, miR-223, miR-4267, miR-1281, miR-885-5p, miR- 663, miR-let-7b, miR-29d, miR-144, miR-let-7e 143, miR-lrt-7g, miR-17a, miR-96, miR- 125a-5p, miR-128, miR-720, miR-21, miR-9, miR-26b, miR-29b, miR-30c, miR-30b, miR- 191, and miR-lb.
  • the one or more microRNAs comprise one or more exosomes including
  • 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.
  • CDCs human cardiac-derived cells
  • administration can be in repeated doses.
  • 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 assessed 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 and/or injection into heart or skeletal muscle. 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 myocardium, associated arterial, venous, and/or ventricular locations. In certain embodiments, administration can include delivery to a tissue or organ site that is the same as the site of diseased and/or dysfunctional tissue.
  • administration can include delivery to a tissue or organ site that is different from the site or diseased and/or dysfunctional tissue.
  • the delivery is via inhalation or oral administration.
  • administration of exosomes can include combinations of multiple delivery techniques, such as intravenous, intracoronary, and intramyocardial delivery.
  • 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 tissue is pulmonary, arterial or capillary tissue. In several embodiments, the damaged or dysfunctional tissue includes cardiac tissue.
  • the plurality of exosomes modulate smad pathway activity, including for example, smad4 and smad 2/3. In various embodiments, the plurality of exosomes increase cardiomyocyte proliferation. In various embodiments, the plurality of exosomes are capable of modulating SDF-1, VEGF and/or collagen expression. In various embodiments, the plurality of exosomes is capable of enhancing infiltration of monocytes, macrophages, and T-cells.
  • functional improvement may comprise increased cardiac output, contractility, ventricular function and/or reduction in arrhythmia (among other functional improvements). For example, this may include a decrease in right ventricle systolic pressure.
  • 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.
  • the disease and/or condition involving tissue damage or dysfunction is pulmonary tissue, including pulmonary, arterial or capillary tissue, such as the endothelial lining of distal pulmonary arteries.
  • the disease and/or condition involving tissue damage or dysfunction is heart disease.
  • 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 pulmonary, arterial or capillary tissue, such as the endothelial lining of distal pulmonary arteries.
  • 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.
  • damaged or dysfunctional tissue is due to chronic disease, such as for example congestive heart failure, including as conditions secondary to diseases such as emphysema, ischemic heart disease, hypertension, valvular heart disease, connective tissue diseases, HIV infection, liver disease, sickle cell disease, dilated cardiomyopathy, infection such as Schistosomiasis, 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.
  • damaged or dysfunctional tissue is in the brain and due to an acute event or chronic disease, such as traumatic head injury, stroke or neurodegenerative conditions such as Alzheimer's, Parkinson's, Huntington's, ALS or similar conditions, wherein exosomes, including urodele-derived exosomes are capable of to delivering microRNAs and other exosome cargo by crossing the blood-brain barrier.
  • an acute event or chronic disease such as traumatic head injury, stroke or neurodegenerative conditions such as Alzheimer's, Parkinson's, Huntington's, ALS or similar conditions
  • exosomes, including urodele-derived exosomes are capable of to delivering microRNAs and other exosome cargo by crossing the blood-brain barrier.
  • exosomes including urodele-derived exosomes are capable of to delivering microRNAs and other exosome cargo by crossing the blood-brain barrier.
  • exosomes including urodele-derived exosomes are capable of to delivering microRNAs and other exosome cargo by crossing the
  • 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 treating a heart related disease includes administering a composition including a plurality of exosomes isolated from urodele cells to a subject, thereby treating the subject.
  • the method of treatment includes, selecting a subject in need of treatment for a pulmonary disease and/or condition, administering a composition including a plurality of exosomes to the individual, wherein administration of the composition treat the subject.
  • 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 administration of the composition treat the subject.
  • the heart related disease and/or condition includes 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 urodele-derived cell exosomes. In certain embodiments, the plurality of exosomes includes one or more exosomes that are CD63+, CD105+, or both.
  • 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 plurality of exosomes can include one or more exosomes containing microRNAs.
  • microRNAs known in the art such as miR-1469, miR-762, miR-574-3p, miR-574-5p, miR-3197, miR-4281, miR-1976, miR-1307, miR-1224-3p, miR-187, miR- 3141, miR-1268, miR-155, miR-122, miR-638, miR-3196, miR-223, miR-4267, miR-1281, miR-885-5p, miR-663, miR-let-7b, miR-29d, miR-144, miR-let-7e 143, miR-lrt-7g, miR-17a, miR-96, miR-125a-5p, miR-128, miR-720, miR-21, miR-9, miR-26b, miR-29b, miR-30c, miR-30b, miR-191, and miR-lb.
  • the one or more microRNAs comprise one or more exosomes including miR-96, miR-29b, and miR-191.
  • 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.
  • the numbers of exosomes is relative to the number of cells used in a clinically relevant dose for a cell-therapy method.
  • 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 myocardial infusion.
  • administering a composition includes use of an intracoronary catheter.
  • administration of a composition includes intra-arterial infusion. In other embodiments, administration of a composition includes intravenous infusion. In other embodiments, administering a composition includes percutaneous injection. In other embodiments, injection includes injection into heart or skeletal muscle. In other embodiments, administration of a composition includes inhalation. In other embodiments, exosome therapy is provided in combination with standard therapy for a disease and/or condition. This may include co-administration of the exosomes with a therapeutic agent.
  • Described herein is a method of administering a plurality of exosomes including selecting a subject and administering a composition including a plurality of exosomes to the subject, wherein administration consists of one or more of: intra-arterial infusion, intravenous infusion, and injection.
  • injection includes percutaneous injection.
  • injection includes injection into heart or skeletal muscle.
  • administering a composition includesl x 10 8 or more exosomes in a single dose. In other embodiments, 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. In other embodiments, 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.
  • 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.
  • 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 assessed for safety and bioactivity (e.g., using MRI endpoints: scar size, global and regional function).
  • a single dose is administered multiple times to the subject.
  • the multiple administrations to the subject includes of two or more of intra-arterial infusion, intravenous infusion, and injection.
  • injection includes percutaneous injection.
  • injection includes injection into heart or skeletal muscle.
  • the plurality of exosomes include one or more exosomes with a diameter of about 40 nm to 100 nm and at least about 3 kDa.
  • the cells are from amphibians within the order Caudata (also described as urodeles). For example, this includes species within the family Salamandridae (also known as newts). Various non-limiting examples include Notophthalmus viridescens and Ambystoma mexicanum.
  • the cells are cultured as a cell line capable of serial passaging. This includes, for example, the Al cell line of Notophthalmus viridescens.
  • the cells are stem cells, progenitors, precursors, and/or mesenchyme.
  • the plurality of exosomes includes one or more exosomes including one or more microRNAs selected from the group consisting 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 miR-23a.
  • the one or more microRNAs include miR-146a, miR22, and miR-24.
  • the plurality of exosomes can include one or more exosomes containing microRNAs. This includes various microRNAs known in the art, such as miR-1469, miR- 762, miR-574-3p, miR-574-5p, miR-3197, miR-4281, miR-1976, miR-1307, miR-1224-3p, miR-187, miR-3141, miR-1268, miR-155, miR-122, miR-638, miR-3196, miR-223, miR- 4267, miR-1281, miR-885-5p, miR-663, miR-let-7b, miR-29d, miR-144, miR-let-7e 143, miR-lrt-7g, miR-17a, miR-96, miR-125a-5p, miR-128, miR-720, miR-21, miR-9,
  • the one or more microRNAs comprise one or more exosomes including miR-96, miR-29b, and miR-191.
  • the plurality of exosomes includes one or more exosomes that are CD63+, CD105+, or both.
  • the subject has a heart related disease and/or condition.
  • the heart related disease and/or condition includes myocardial infarct.
  • the heart related disease and/or condition includes heart failure.
  • the heart failure is associated with Duchenne muscular dystrophy.
  • the subject has a brain related disease and/or condition, such as damaged or dysfunctional brain or other neural tissue.
  • the brain related disease and/or condition is due to an acute event such as traumatic head injury or stroke.
  • the brain related disease and/or condition is due to chronic disease, such as neurodegenerative diseases including such as Alzheimer's, Parkinson's, Huntington's, ALS or similar conditions.
  • the intra-arterial infusion, intravenous infusion, and/or injection of a plurality of exosomes, including urodele-derived exosomes is capable of delivering microRNAs and other exosome cargo by crossing the blood-brain barrier.
  • administration is at the site of diseased and/or dysfunctional tissue. In certain embodiments, administration is not at the site of diseased and/or dysfunctional tissue.
  • 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 administration of the composition improves cardiac performance in the subject. In some embodiments, this includes a decrease in right ventricle systolic pressure. In other embodiments, there is a reduction in arteriolar narrowing, or pulmonary vascular resistance. In other embodiments, 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 administration of the composition treat the subject.
  • the heart related disease and/or condition includes 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 urodele cell derived exosomes. In certain embodiments, the plurality of exosomes includes one or more exosomes that are CD63+, CD105+, or both.
  • 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 include microRNAs miR-1469, miR-762, miR-574-3p, miR-574-5p, miR- 3197, miR-4281, miR-1976, miR-1307, miR-1224-3p, miR-187, miR-3141, miR-1268, miR- 155, miR-122, miR-638, miR-3196, miR-223, miR-4267, miR-1281, miR-885-5p, miR-663, miR-let-7b, miR-29d, miR-144, miR-let-7e 143, miR-lrt-7g, miR-17a, miR-96, miR-125a-5p, miR-128, miR-720, miR-21, miR-9, miR-26b, miR-29b, miR-30c, miR-30b, miR-191, and miR-lb.
  • the one or more microRNAs comprise one or more exosomes including miR-96, miR-29b, and miR-191.
  • 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.
  • the numbers of exosomes is relative to the number of cells used in a clinically relevant dose for a cell-therapy method.
  • 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 injection into heart or skeletal muscle.
  • administering a composition includes myocardial infusion. In other embodiments, administering a composition includes use of an intracoronary catheter. In other embodiments, administration a composition includes intra-arterial or intravenous delivery. Additional delivery sites include any one or more compartments of the heart, such as myocardium, associated arterial, venous, and/or ventricular locations. In certain embodiments, administration can include delivery to a tissue or organ site that is the same as the site of diseased and/or dysfunctional tissue. 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 exosomes can include combinations of multiple delivery techniques, such as intravenous, intracoronary, and intramyocardial delivery.
  • exosome therapy is provided in combination with standard therapy for a disease and/or condition. This may include coadministration of the exosomes with a therapeutic agent.
  • stem cells might be helpful in not only preventing or ameliorating disease and/or conditions, but actually capable of treating heart disease and related conditions via regeneration and repair of damaged cells and promotion of vascular cell growth. It is suggested that therapeutic effects of stem cells via regeneration can be significantly enhanced by directly delivering exosomes produced by such stem cells as an alternative to delivering the cell themselves. There is increasing evidence that, for example, cardiosphere derived cellular exosomes are indeed capable of delivering therapeutic benefits of their parental cell type.
  • Urodele cell line such as Al cell line isolated from mesenchyme
  • NHDF normal human dermal fibroblasts
  • CDCs cardiosphere derived cells
  • Cells can be 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 ( Figure 2).
  • Exosome pellets are resuspended in the appropriate media and used for assays. Expression of the conserved exosome marker CD63 can be verified using ELISA. RNA content of exosome pellets can also be quantified using a Nanodrop spectrophotometer. 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.
  • Proteins are prepared for digestion using the filter-assisted sample preparation (FASP) method. Concentrations were measured using a Qubit fluorometer (Invitrogen). 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 is centrifuged and the filtrate collected. Digested peptides are desalted using CI 8 stop-and-go extraction (STAGE) tips. Peptides are fractionated by strong anion exchange STAGE tip chromatography. Peptides are eluted from the CI 8 STAGE tip and dried. Each fraction can be analyzed with liquid chromatography-tandem mass spectrometry. Samples are loaded to a 2 cm 3 100 mm ID.
  • FASP filter-assisted sample preparation
  • 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 are converted to MGF format using msconvert.
  • MGF files are 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 are required to have one or more unique peptides with peptide E-value scores of 0.01 or less from X!
  • NRCMs Neonatal rat cardiomyoctes
  • CDC exosomes Towards investigating the basis of the therapeutic benefit of CDC exosomes, the Inventors previously compared their microRNA repertoire to that of NHDF exosomes using a PCR microarray of the 88 best-defined microRNAs, and it was shown that the microRNA content of the two cell types differed dramatically. Forty-three 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 1 A, IB, and 3).
  • microRNA microarray screen with detection probes complementary to Xenopus, zebrafish and human miRNAs, such as ⁇ ParafloTM, is capable of gauging differentially expressed microRNAs to identify those microRNAs involved in cardiac regeneration.
  • miRNAs such as newts
  • miR-146a leads to thicker infarct wall thickness and increased viable tissue in a mouse model of myocardial infarct.
  • FIG. 5 shows that miR-128 regulates non-myocyte hyperplasia via Isletl expression during newt cardiac regeneration.
  • exosomes are isolated from human CDCs as described using a technique such as ExoQuick® precipitation in order to generate a composition
  • a technique such as ExoQuick® precipitation in order to generate a composition
  • a single dose such as 3mL / 3 x 10 5 CDCs, can be delivered to a subject in need of treatment for a heart related diseases and/or conditions, which can include both acute and chronic diseases and/or conditions.
  • exosomes provide both cardioprotective and regenerative effects, thereby providing multiple timepoints for administration ranging from immediately after an acute event (e.g., myocardial infarct) or at much later timepoints such as weeks and//or months during the progression of chronic disease (e.g., congestive heart disease).
  • an acute event e.g., myocardial infarct
  • chronic disease e.g., congestive heart disease
  • Administration may occur as a single dose or a series of repeated doses, and it understood that dosages may be provided by variable routes of administration combined together.
  • Administration may be via intracoronary infusion as delivered through the central lumen of a balloon catheter positioned in the coronary artery, such as via over-the-wire balloon catheter, with a subtended by a patent coronary artery.
  • Subsequent repeat doses can also be via intracoronary infusion, but may rely on other methods of administration (e.g., intravenous infusion).
  • a variety of techniques may be relied upon to evaluate the therapeutic effects of exosome therapy. This includes echocardiographic assessment, wherein wall thickness, ejection volume or a variety of other parameters may indicate cardiac improvement. Other examples include hemodynamic measurement.
  • exosomes from the Al cell line of Notophthalmus viridescens are bioactive in mammals.
  • exosomes isolated via the above described methods are capable of promoting rat cardiomyocyte proliferation, increase SDF-1 secretion by human dermal fibroblasts, and improve functional recovery after myocardial infarct in rats.
  • Al ceils, derived from the amputated limb buds of Notopthalmus viridescense were expanded in culture. Further description can be found for example, in Ferreti and Brockes, "Culture of newt cells from different tissues and their expression of a regeneration-associated antigen" J Exp ⁇ ' , ⁇ . 1988 Jul;247(l):77-91.Culture of newt cells from different tissues and their expression of a regeneration-associated antigen, which is fully incorporated by reference herein.
  • Exosomes were isolated by polyethylene glycol precipitation of A 1 -conditioned serum-free media (or media conditioned by human dermal fibroblasts (DF) as a control) followed by centrifugation. Bioactivity was tested in vitro on neonatal rat ventricular myocytes (NRVM), and in vivo on acute myocardial infarction in Wi star-Kyoto rats (250_jig or 50( g of A 1 -exosomes or vehicle (placebo) injected intramyocardially). Functional and histological analyses were performed 3 weeks after therapy.
  • Al -conditioned media yielded ⁇ 2.8 ⁇ lBillion particles/ml of 129+1.1 nm diameter.
  • Donor-specific antibodies were present at barely detectable levels in the serum of animals that had been injected with Al -exosomes.
  • Newt exosomes stimulate rat cardiomyocyte proliferation and improve functional and structural outcomes in rats with myocardial infarction. Characterization of the RNA and protein content of newt exosomes, provides clues regarding conserved (or newt-unique) molecular mediators of therapeutic benefi t.
  • exosomes reproduce cardiosphere derived therapeutic regeneration, via paracrine mechanisms involving exosomes, for which their cargo content of microRNAs, have the ability to alter cell behavior.
  • the Inventors have extended these studies by demonstrated urodele derived cellular exosomes are bioactive in mammals, and may contain microRNAs similarly capable of delivering a salutary benefit.
  • microRNAs such as miR-146a appear to play an important part in mediating the effects of CDC exosomes, but alone may 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) modulates cardiac fibrosis by targeting furin, a member of the profibrotic TGF-b signaling pathway; overexpression of miR-24 in a model of MI decreased myocardial scar formation.
  • miR-128 in the newt Notophthalmus viridescens has been reported as elevated when cardiac hyperplasia is at its peak following injury, with a localised expression pattern for miR-128 in the cardiomyocytes and non-cardiomyocytes in close proximity to the regeneration zone and a regulatory role for miR-128 in proliferating non-cardiomyocyte populations and extracellular matrix deposition, possibly via interaction with Isletl .
  • the bioactivity of urodeles in mammals provides compelling avenues for which the extraordinary regenerative potential of urodeles may find application in new therapeutic avenues for heart disease in mammals.
  • 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.
  • Newt exosomes are bioactive on mammalian heart, enhancing proliferation of rat
  • Exosomes are nanoparticles which mediate intercellular communication and play a critical role in therapeutic regeneration.
  • Al cells derived from the amputated limb buds of Notopthalmus viridescens, were expanded in culture. Exosomes from these cells were assayed for bioactivity both in vitro on neonatal rat ventricular myocytes (NRVMs), and in vivo on acute myocardial infarction in Wistar-Kyoto rats. Functional and histological analyses were performed 3 weeks after therapy. The Al exosomes were analyzed for both RNA using next-generation sequencing and protein cargo using mass spectrometry.
  • NRVMs neonatal rat ventricular myocytes
  • a 1 -conditioned media yielded exosomes that increased the proliferative capacity of NRVMs and significantly increased cardiac function and infarct wall thickness as compared to placebo controls.
  • Al exosome RNA included numerous miRNAs, IncRNAs and mRNAs that have homology to known mammalian exosome RNA cargo, as well as newt-specific sequences. Proteomic analysis revealed a plethora of contents, some of which are homologues of proteins known to be present in human exosomes.
  • newt exosomes are bioactive on the mammalian heart. Newt exosomes stimulate rat cardiomyocyte proliferation and improve functional and structural outcomes in rats with myocardial infarction. Characterization of the RNA and protein content of newt exosomes is beginning to provide clues regarding conserved (or newt-unique) molecular mediators of therapeutic benefit.
  • RNA profiling was validated using qRT-PCR as shown in Figures 7 and 8.
  • qRT-PCR primers used as shown in Table 1.
  • Protein profiling was validated using flow cytometery as shown in Figure 9, and western blot as shown in Figure 10.
  • Neonatal rat ventricular cardiomyocytes were treated with Al -derived exosomes or control, with analysis of gene expression as shown in Figure 11. Cardiomyocyte apoptosis was measured using flow cytometry and Annexin V staining, 24 hours after ischemic injury, as shown in Figure 12. Further analysis via ELISA analysis of expressed or secreted after application of exosomes, including supernatant collected from NFIDFs in culture exposed to NFIDF exosomes or Al exosomes for the cell proliferation-associated marker, SDF-1, and western blot detection of collagen, as shown in Figure 13.
  • Morphological analysis included cardiomyocyte diameter following MI and PBS or
  • Monocyte infiltration was measured including identification of infiltrating monocyte clusters as shown in Figure 19 with the numbers of monocytes increasing significantly with Al exosome treatment and escalating with increased dosage.
  • Macrophage infiltration was also measured, including heart tissue stained with the macrophage marker CD68, with graphical data shows the average number of monocytes counted per image field as shown in Figure 20.
  • T-cell infiltration was also measured using heart tissue stained with antibodies against CD8+ (green) and CD4+ (red) T-cell markers, with graphical data shows the average number of CD8+ and CD4+ T-cells counted per image field as shown in Figure 21. Notably, an increase in CD4+ cells was observed with Al exosome treatment.
  • NHDFs demonstrated enrichment of miR-9, a microRNA regulating cardiac hypertrophy also found to be enriched in CDCs ( Figure IB).
  • NHDF microRNAs expression was measured post Al exosome treatment. miRNA isolation from the Al exosome-treated NBFDFs was performed and measured using qRT-PCR as shown in Figure 25. Additionally, native miR-9-5p expression levels and downstream target gene expression was measured as shown in Figure 28.
  • miR-9-5-p specifically added to in vitro cultured cells.
  • cardiomyocyte proliferation was measured following treatment with miR-9-5p mimic or control microRNA.
  • NRVMs in culture were treated with 25nM miR-9-5p microRNA mimic and EdU (10 ⁇ ) for 24 hours, stained with sarcomeric actin and DAPI nuclei staining with cell proliferation increasing following miR-9-5p.
  • miR-9-5p was capable of inducing SDF-1 secretion in NHDFs, as shown in Figure 23.
  • EdU 5-ethynyl-2'-deoxyuridine
  • Echocardiography of infarcted rat hearts following A 1 and NHDF exosome treatments Echocardiography of infarcted rat hearts following A 1 and NHDF exosome treatments.
  • sources of urodele derived cells are sources of urodele derived cells, the use of alternative sources such as cells derived directly from urodeles or urodele cell lines, or from urodele derived cells undergoing dedifferentiation, transdifferentiation, and/or proliferation, 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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Abstract

L'invention concerne des compositions et des techniques associées à la production et à l'application thérapeutique d'exosomes dérivés de cellules d'urodèles. Les urodèles comme les tritonbs et les salamandres, sont des animaux hautement régénératifs. De plus en plus de signes montrent que les exosomes sont des acteurs vitaux dans la potentialisation de l'activité de réparation et de régénération. La présente invention indique que les exosomes cellulaires dérivés d'urodèles sont bioactifs chez les mammifères et favorisent la prolifération des cardiomyocytes chez le rat, augmentent la sécrétion de SDF-1 par les fibroblastes dermiques humains et améliorent la récupération fonctionnelle après infarctus du myocarde chez le rat. L'invention concerne des compositions et des techniques permettant une protection contre une cardiopathie et une maladie vasculaire et/ou une inversion de celles-ci.
PCT/US2016/035561 2015-06-02 2016-06-02 Exosomes d'urodèles comme agents thérapeutiques Ceased WO2016196822A1 (fr)

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WO2019198995A1 (fr) * 2018-04-10 2019-10-17 한국과학기술연구원 Procédé de conversion à base d'exosomes pour cellules immunitaires
CN109276575A (zh) * 2018-07-23 2019-01-29 海南医学院 miR-9在制备治疗急性冠脉综合症的药物中的用途
WO2020188433A1 (fr) * 2019-03-15 2020-09-24 Unicyte Ev Ag Procédé pour prédire le potentiel pro-angiogénique de vésicules extracellulaires (ve)
CN113574179A (zh) * 2019-03-15 2021-10-29 联合细胞Ev股份公司 用于预测胞外囊泡(ev)的促血管生成潜力的方法
CN115768284A (zh) * 2020-07-10 2023-03-07 雀巢产品有限公司 包含mir-3141的营养组合物
CN114099534A (zh) * 2021-11-29 2022-03-01 苏州大学附属第一医院 高表达miR-214的外泌体及其制备方法与应用
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