WO2021178514A1

WO2021178514A1 - Cardiosphere-derived cells, exosomes derived therefrom, and methods of using same to treat volumetric muscle loss

Info

Publication number: WO2021178514A1
Application number: PCT/US2021/020640
Authority: WO
Inventors: Eduardo MARBÁN; Russell Rogers
Original assignee: Cedars-Sinai Medical Center
Priority date: 2020-03-04
Filing date: 2021-03-03
Publication date: 2021-09-10

Abstract

Provided herein are methods of treating a traumatic muscle injury in a subject in need thereof, by administering a therapeutically effective amount of cardiosphere-derived cells (CDCs) or CDC-derived exosomes to the subject. In certain embodiments, CDCs or CDC-derived exosomes are administered intravenously or intramuscularly. In certain embodiments, the CDC-derived exosomes are derived from immortalized CDCs or primary human CDCs.

Description

CARDIOSPHERE-DERIVED CELLS, EXOSOMES DERIVED THEREFROM, AND METHODS OF USING SAME TO TREAT VOLUMETRIC MUSCLE LOSS

CROSS-REFERENCE TO RELATED APPLICATION [0001] This application claims priority to U.S. Provisional Application No. 62/984958, filed March 4, 2020, the entirety of which is incorporated herein by reference.

REFERENCE TO SEQUENCE LISTING

[0002] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled SEQLIST_CSMC011WO.txt created on March 2, 2021, which is 1.46 KB in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED R&D [0003] This invention was made with government support under U.S. Army Medical Research and Materiel Command (USAMRMC) prototype Other Transaction Agreement W81XWH-15-9-0001, awarded to Advanced Technology International (ATI) as the Consortium Manager of the Medical Technology Enterprise Consortium (MTEC). The U.S. Government has certain rights in the invention.

BACKGROUND

Field

[0004] The present disclosure generally relates to cardiosphere-derived cells (CDCs), exosomes derived therefrom, and methods of treating a traumatic muscle injury.

[0005] Traumatic muscle injury includes volumetric muscle loss (VML) in which a significant portion of muscle is lost by sudden trauma, and contributes to disability in military and civilian victims of trauma. VML injuries can result in chronic loss of muscle strength, reduced range of motion, and permanent disability. Thus, there is a need to identify therapeutic agents to treat traumatic muscle injury, such as VML. SUMMARY

[0006] Provided herein are methods of treating a traumatic muscle injury, e.g., volumetric muscle loss, by administering cardiosphere-derived cells (CDCs) or CDC-derived exosomes to a subject with the traumatic muscle injury. The present disclosure provides, a method of treating a traumatic muscle injury, comprising: identifying a subject having a traumatic injury to a muscle, wherein the muscle is a skeletal muscle, and wherein the traumatic injury comprises a volumetric muscle loss from about 10% to about 30% as compared to the muscle pre-injury; and administering a therapeutically effective amount of cardiosphere- derived cells (CDCs) to the subject, wherein the therapeutically effective amount ranges from or includes about 1 x 10⁴ to about 1 x 10⁷ CDCs, thereby treating the traumatic muscle injury. Optionally, the method includes administering the CDCs to the subject via one or more intravenous injections.

[0007] In some embodiments, the method includes administering the CDCs to the subject via one or more intramuscular injections. Optionally, the method includes administering a dose of CDCs at a first site adjacent a site of the traumatic injury. Optionally the method includes administering a second dose of CDCs at a second site adjacent the site of the traumatic injury, wherein the first and second sites are different.

[0008] In some embodiments, the CDCs are administered topically. Optionally, the CDCs are disposed in a biocompatible substrate, and wherein the method further comprises applying the substrate to a site of the traumatic injury. In some embodiments, the substrate is configured to release the CDC-derived exosomes upon application of the substrate to a site of the traumatic injury. Optionally, the biocompatible substrate comprises a biocompatible matrix. In some embodiments, the method further comprises administering the therapeutic amount in a single dose of the CDCs. In some embodiments, the administering comprises administering two or more doses of the CDCs at a dosing interval from about 3 days to about 6 months.

[0009] Also provided herein is a cell-free method of treating a traumatic muscle injury, comprising: identifying a subject having a traumatic injury to a muscle, wherein the muscle is a skeletal muscle, and wherein the traumatic injury comprises a volumetric muscle loss from about 10% to about 30% as compared to the muscle pre-injury; and administering a therapeutically effective amount of CDC-derived exosomes to the subject, wherein the therapeu tic ally effective amount comprises about 1 x 10⁸ to about 1 x 10¹¹ exosomes, thereby treating the traumatic muscle injury. Optionally, the method further includes administering the CDC-derived exosomes to the subject via one or more intravenous injections and/or one or more intramuscular injections. In some embodiments, the CDC-derived exosomes are disposed in a biocompatible substrate configured to release the CDC-derived exosomes upon application of the substrate to a site of the traumatic injury, and wherein the method further comprises applying the substrate to the site of the traumatic injury. In some embodiments, the biocompatible substrate comprises a biocompatible matrix. In some embodiments, the method further includes reconstituting preserved CDC-derived exosomes into a composition comprising the therapeutically effective amount of the CDC-derived exosomes; and administering the composition to the subject. In some embodiments, the preserved CDC- derived exosomes are frozen or lyophilized.

[0010] In some embodiments, the method further comprises administering the therapeutic amount in a single dose of the CDC-derived exosomes. In some embodiments, the administering comprises administering two or more doses of the CDC-derived exosomes at a dosing interval from about 3 days to about 6 months.

[0011] In some embodiments, the traumatic injury reduces muscle strength by 50% or more. In some embodiments, the muscle is comprised in a fascial compartment, and wherein the traumatic injury reduces muscle strength of the fascial compartment by 50% or more. In some embodiments, an extent of recovery of muscle strength after the administering is about 1.5 times or greater than a reference level of recovery of muscle strength. In some embodiments, a rate recovery of muscle strength is about 2 times or more faster relative to a reference rate of recovery of muscle strength. In some embodiments, the muscle strength is torque of the muscle, e.g., a fascial compartment comprising the muscle.

[0012] In some embodiments, muscle strength recovers about 25% or more of a pre-injury muscle strength, within 2 weeks after the administering. In some embodiments, the muscle is comprised in a fascial compartment, and wherein muscle strength of the fascial compartment recovers about 25% or more of a pre-injury muscle strength of the fascial compartment, in or within 2 weeks after the administering. In some embodiments, muscle strength increases by about 4 fold or more within 2 weeks after the administering. In some embodiments, the muscle is comprised in a fascial compartment, and wherein muscle strength of the fascial compartment increases by about 4 fold or more in or within 2 weeks after the administering. Optionally, the muscle strength is represented by application of torque by the muscle.

[0013] In some embodiments, a volume of the muscle increases by about 15% or more within 6 weeks after the administering. In some embodiments, a volume of the muscle increases by about 15% or about 25% or more in or within 2 weeks after the administering.

[0014] In some embodiments, the CDCs are allogeneic CDCs. In some embodiments, the CDCs are primary CDCs. In some embodiments, the CDCs and/or CDC- derived exosomes are administered within 3 hours of the subject suffering the traumatic injury to the muscle. In some embodiments, the CDCs and/or CDC-derived exosomes are administered within 3 hours to 3 days of the subject suffering the traumatic injury to the muscle. In some embodiments, the therapeutic amount of the CDCs and/or CDC-derived exosomes is administered in a single dose of CDCs and/or CDC-derived exosomes.

[0015] In some embodiments, the administering comprises administering two or more doses of CDCs and/or CDC-derived exosomes at a dosing interval in a range of 3 days to 6 months.

[0016] In some embodiments, the method further includes measuring a muscle function after administering the CDCs and/or CDC-derived exosomes to the subject. In some embodiments, the method further includes administering an analgesic to the subject.

[0017] Provided herein is a method of treating a traumatic muscle injury, comprising administering to a muscle having a traumatic injury comprising a volumetric muscle loss a therapeutically effective amount of cardiosphere-derived cells (CDCs) and/or CDC-derived exosomes to the muscle.

[0018] Also provided herein is a method of treating a traumatic muscle injury, comprising: identifying a subject having a traumatic injury to a muscle, wherein the muscle is a skeletal muscle, and wherein the traumatic injury comprises a volumetric muscle loss from about 10% to about 30%; and administering a therapeutically effective amount of cardiosphere-derived cells (CDCs) and/or CDC-derived exosomes to the subject, thereby treating the traumatic muscle injury. Optionally, the method includes administering a therapeutically effective amount of CDC-derived exosomes to the subject. Optionally, the therapeutically effective amount of CDC-derived exosomes comprises from about 1 x 10⁸ to about 1 x 10¹¹ exosomes. In some embodiments, the CDC-derived exosomes are primary human-derived exosomes. Optionally, the CDC-derived exosomes are immortalized CDC- derived exosomes.

[0019] In some embodiments, the method is a cell-free method of treating a traumatic muscle injury. In some embodiments, the method includes administering the CDC- derived exosomes to the subject via one or more intravenous injections and/or one or more intramuscular injections.

[0020] In some embodiments, the method includes reconstituting preserved CDC- derived exosomes into a composition comprising the therapeutically effective amount of the CDC-derived exosomes; and administering the composition to the subject. Optionally, the preserved CDC-derived exosomes are frozen or lyophilized.

[0021] Also provided is a kit for treating a traumatic muscle injury, comprising: preserved CDC-derived exosomes and/or preserved CDCs; and a biocompatible substrate. In some embodiments, the preserved CDC-derived exosomes and/or preserved CDCs provide a therapeutically effective amount of CDC-derived exosomes and/or CDCs upon reconstitution with the biocompatible substrate. In some embodiments, the therapeutically effective amount of CDC-derived exosomes comprises from about 1 x 10⁸ to about 1 x 10¹¹ exosomes. In some embodiments, the therapeutically effective amount of CDCs comprises from about 1 x 10⁴ to about 1 x 10⁷ CDCs. Optionally, the kit includes the preserved CDC-derived exosomes. Optionally, the preserved CDC-derived exosomes are frozen or lyophilized.

[0022] In some embodiments, the exosomes are primary human CDC-derived exosomes. In some embodiments, the exosomes are immortalized CDC-derived exosomes.

[0023] In some embodiments, the kit further includes an analgesic.

[0024] In some embodiments, the kit contains a syringe configured to administer to a subject a composition made by combining: the preserved CDC-derived exosomes and/or preserved CDCs, with the biocompatible substrate. Optionally, the syringe is configured to intramuscularly administer the composition. Optionally, the syringe is configured to intravenously administer the composition.

[0025] In some embodiments, the kit further includes a wound dressing configured to: receive a composition made by combining: the preserved CDC-derived exosomes and/or preserved CDCs, with the biocompatible substrate; and position the composition at a site of a traumatic muscle injury.

[0026] In some embodiments, the biocompatible substrate comprises a biocompatible matrix.

[0027] Also provided herein is a use of cardiosphere-derived cells (CDCs) and/or CDC-derived exosomes for the treatment of traumatic muscle injury. Further provided herein is a use of cardiosphere-derived cells (CDCs) and/or CDC-derived exosomes in the preparation of a medicament for the treatment of traumatic muscle injury. Optionally, the treatment improves one or more of the muscle strength, mass, volume, and/or endurance of the injured muscle. Also provided is a use of cardiosphere-derived cells (CDCs) and/or CDC-derived exosomes for the treatment of a wound, lesion or tissue damage.

BRIEF DESCRIPTION OF THE DRAWINGS

[0028] FIG. 1A shows changes in torque-frequency relationship of the anterior crural muscle before and after intravenous administration of cardiosphere-derived cells (CDCs) to animals with volumetric muscle loss (VML) in the tibialis anterior muscle, according to embodiments of the present disclosure.

[0029] FIG. IB shows changes in tetanic torque deficit of the anterior crural muscle before and after intravenous administration of CDCs to animals with volumetric muscle loss in the tibialis anterior muscle, according to embodiments of the present disclosure.

[0030] FIG. 1C shows changes in tetanic torque of the anterior crural muscle before and after intravenous administration of CDCs to animals with volumetric muscle loss in the tibialis anterior muscle, according to embodiments of the present disclosure.

[0031] FIG. ID shows the percentage recovery of tetanic torque in the anterior crural muscle in animals with volumetric muscle loss in the tibialis anterior muscle, after intravenous administration of CDCs, according to embodiments of the present disclosure.

[0032] FIG. IE shows the mass of muscle removed from the tibialis anterior to produce volumetric muscle loss in CDC-treated and control animals.

[0033] FIG. IF shows changes in tetanic torque of the anterior crural muscle before and after intramuscular administration of CDCs to animals with volumetric muscle loss in the tibialis anterior muscle, according to embodiments of the present disclosure. [0034] FIG. 2 is a flow chart of a method of treating a traumatic muscle injury, according to embodiments of the present disclosure.

[0035] FIG. 3 shows the nucleotide sequences of human microRNAs miR-92a, miR-146a, miR-148a, miR-181b, and miR-199b.

[0036] FIGS. 4A-4I show the effect of intravenous and intramuscular CDC infusion in VML-injured mice. FIG. 4A is a schematic diagram showing an experimental protocol for showing the therapeutic effect of CDCs in VML. FIG. 4B is a graph showing tibialis anterior (TA) muscle biopsy mass. FIG. 4C is a collection of graphs showing torque- frequency relationship in animals administered vehicle control (left panel) or CDCs (right panel) intravenously. FIG. 4D is a graph showing tetanic torque during recovery in animals administered vehicle control or CDCs intravenously. FIG. 4E is a collection of graphs showing torque-frequency relationship in animals administered vehicle control (left panel) or CDCs (right panel) intramuscularly. FIG. 4F is a graph showing tetanic torque during recovery in animals administered vehicle control or CDCs intramuscularly. FIG. 4G is a graph showing torque deficit during recovery in animals administered vehicle control or CDCs intravenously. FIG. 4H is is a graph showing torque deficit during recovery in animals administered vehicle control or CDCs intramuscularly. FIG. 41 is a graph showing percentage (%) recovery of tetanic torque. Bar graphs represent mean ± SEM. N=7-9 per group. Statistical significance was determined by a two-way analysis of variance (ANOVA) with p < 0.05. When appropriate, a Newman-Keuls correction for multiple comparisons was applied. *p<0.05, **p<0.01, ***p<0.001.

[0037] FIGS. 5A-5J show dose optimization for intravenously delivered CDCs in VML-injured mice. FIG. 5A is a schematic diagram showing an experimental protocol for showing the therapeutic effect of CDCs in VML. FIG. 5B is a graph showing TA muscle biopsy mass. FIG. 5C is a graph showing tetanic torque during recovery. FIG. 5D is a graph showing torque deficit during recovery. FIG. 5E is a graph showing percentage (%) recovery of tetanic torque. FIG. 5F is a schematic diagram showing an experimental protocol for showing the therapeutic effect of CDCs in VML. FIG. 5G is a graph showing TA muscle biopsy mass. FIG. 5H is a graph showing tetanic torque during recovery. FIG. 51 is a graph showing torque deficit during recovery. FIG. 5J is a graph showing percentage (%) recovery of tetanic torque. Bar graphs represent mean ± SEM. N=7-9 per group. Statistical significance was determined by a two-way analysis of variance (ANOVA) with p < 0.05. When appropriate, a Newman-Keuls correction for multiple comparisons was applied. *p<0.05, **p<0.01.

[0038] FIGS. 6A-6G show histological analysis of VML- injured skeletal muscle treated with CDCs. FIG. 6A is collection of images showing representative H&E stained micrographs of TA muscle tissue sections. FIG. 6B is a graph showing TA muscle mass. FIG. 6C is a graph showing TA muscle myofiber cross-sectional area. FIG. 6D is a graph showing TA muscle myofiber count. FIG. 6E is a graph showing size distribution of TA muscle myofibers. FIG. 6F is a collection of images showing representative immunohistochemical micrographs of innervated motor endplates, stained for WGA, nAChR and NF-M. FIG. 6G is a graph showing fraction of innervated motor endplates estimated based on the immunohistochemical staining data. Bar graphs represent mean ± SEM. N=3-5 per group (histology) and n=8-10 per group (immunohistochemistry). Statistical significance was determined by a two-way analysis of variance (ANOVA) or independent t test with p < 0.05. When appropriate, a Newman-Keuls correction for multiple comparisons was applied. *p<0.05, **p<0.01, ***p<0.001. Magnification bar represents 400 pm in Fig. 6A and 75 pm in Fig. 6B.

[0039] FIGS. 7A-7C show small animal MRI analysis of VML- injured skeletal muscle treated with CDCs. FIG. 7A is a collection of images showing magnetic resonance imaging (MRI) scans of mouse hindlimb muscles. FIG. 7B is a graph showing the total muscle volume of the VML-injured anterior compartment. FIG. 7C is a graph showing the muscle volume along the anterior muscle compartment. Bar graphs represent mean ± SEM. N=4-5 per group. Statistical significance was determined by a two-way analysis of variance (ANOVA) with p < 0.05. When appropriate, a Newman-Keuls correction for multiple comparisons was applied. ***p<0.001. Yellow dashed lines represent the anterior muscle compartment. White arrows indicate the VML-injured muscle compartment.

[0040] FIG. 8 is a collection of graphs showing therapeutic efficacy after delayed CDC administration.

[0041] FIGS. 9A-9F show CDC-derived exosomes improve recovery from VML injury in mice. FIG. 9A is is a schematic diagram showing the experimental protocol for showing the therapeutic effect of CDC-derived exosomes in VML. FIG. 9B is a graph showing TA muscle biopsy mass. FIG. 9C is a graph showing torque-frequency relationship in vehicle control animals. FIG. 9D is a graph showing torque-frequency relationship in CDC-derived exosome-treated animals. FIG. 9E is a graph showing tetanic torque during recovery. FIG. 9F is a graph showing torque deficit during recovery. N=8-10 per group. *P<0.05, **P<0.01, ***P<0.001. Where appropriate, a two-way ANOVA was used to determine statistical significance or an independent t-test.

DETAILED DESCRIPTION

[0042] Methods of treating a traumatic muscle injury are provided. In general terms, a non-limiting example of a method of the present disclosure includes administering a therapeutically effective amount of cardiosphere-derived cells (CDCs) and/or CDC-derived exosomes (including, but not limited to, primary human CDC-derived exosomes or immortalized CDC-derived exosomes) to a subject suffering from a traumatic muscle injury, to thereby promote and/or accelerate recovery of muscle function and/or muscle mass that was lost due to the traumatic muscle injury. In several embodiments, a single dose of CDCs or CDC-derived exosomes is sufficient to improve muscle function in the subject. In some embodiments, the method provides greater recovery of muscle function and/or muscle mass than otherwise would have occurred spontaneously, or through other treatment options (e.g., administration of myogenic stem cells, extracellular matrix scaffolds, etc.).

[0043] Also provided are kits that include CDCs and/or CDC-derived exosomes (e.g., primary human CDC-derived exosomes or immortalized CDC-derived exosomes) for performing the methods of the present disclosure. In some cases, the kit allows the CDCs and/or CDC-derived exosomes to be made available to promptly treat a trauma victim in situations where there are limited medical resources and/or rapidly providing treatment is important in the overall outcome for the trauma victim.

Definitions

[0044] “Traumatic injury” as used herein has its ordinary meaning as understood by one of ordinary skill in the art and in view of the present disclosure. Traumatic injury may include direct or indirect physical damage to a subject’s body part caused by a physical impact. The physical impact may be any form of physical interaction with the body that causes tissue damage, including those caused by mechanical, thermal, electromagnetic, and acoustic impact

-Si- to the tissue. A traumatic injury can be caused suddenly by a single impact or a series of physical impacts that occurs within a short period of time. A traumatic injury can cause physical loss of tissue. Traumatic injury can be severe enough that the body’s natural repair mechanisms (e.g., regeneration of tissue without medical intervention directed to promoting such tissue regeneration) is inadequate to restore some or all function of the injured tissue. A traumatic injury may be caused accidentally, inadvertently, or intentionally.

[0045] “Volumetric muscle loss” as used herein has its ordinary meaning as understood by one of ordinary skill in the art and in view of the present disclosure. Volumetric muscle loss can include loss of skeletal muscle through trauma or surgery. Volumetric muscle loss can result in impairment of muscle function. Volumetric muscle loss can be characterized interchangeably in terms of a physical loss of volume or mass of the muscle.

[0046] “Skeletal muscle” as used herein has its ordinary meaning as understood by one of ordinary skill in the art and in view of the present disclosure. Skeletal muscle can mediate voluntary movement of one or more body parts, and is typically attached, directly or indirectly (e.g., via a tendon), to the subject’s skeletal system (e.g., bone). Skeletal muscle may be distinguished from cardiac muscle and smooth muscle. Skeletal muscle includes, without limitation, muscles of the head, face, neck, shoulder, arm, back, torso, hands, hip, thigh, legs and feet.

[0047] “Fascial compartment” as used herein has its ordinary meaning as understood by one of ordinary skill in the art and in view of the present disclosure. A fascial compartment may include muscle fibers that are associated with connective tissue to form a functional unit. In some embodiments, a fascial compartment may refer to a muscle group.

[0048] As used herein, “exosome” has its ordinary meaning as understood by one of ordinary skill in the art and in view of the present disclosure. Exosomes may also include microvesicles, epididimosomes, argosomes, exosome-like vesicles, microparticles, promininosomes, prostasomes, dexosomes, texosomes, dex, tex, archeosomes and oncosomes. Unless otherwise indicated herein, each of the foregoing terms shall also be understood to include engineered high-potency varieties of each type of membrane-bound vesicle.

[0049] Wnt signaling pathways are a group of signal transduction pathways which begin with proteins that pass signals into a cell through cell surface receptors. Canonical and non-canonical Wnt signaling pathways are known. Both canonical and noncanonical Wnt signaling pathways are activated by the binding of a Wnt-protein ligand to a Frizzled family receptor, with biological signals passing to the Dishevelled protein inside the cell. The canonical Wnt pathway leads to regulation of gene transcription, while noncanonical pathways regulate the cytoskeleton and intracellular calcium, for example. Canonical Wnt signaling pathways involve b-catenin. By contrast, non-canonical Wnt signaling operates independent of b-catenin.

[0050] “Subject,” as used herein refers to any vertebrate animal, including mammals and non-mammals. A subject can include primates, including humans, and non primate mammals, such as rodents, domestic animals or game animals. Non-primate mammals can include mouse, rat, hamster, rabbit, dog, fox, wolf, cat, horse, cow, pig, sheep, goat, camel, deer, buffalo, bison, etc. Non-mammals can include bird (e.g., chicken, ostrich, emu, pigeon), reptile (e.g., snake, lizard, turtle), amphibian (e.g., frog, salamander), fish (e.g., salmon, cod, pufferfish, tuna), etc. The terms, “individual,” “patient,” and “subject” are used interchangeably herein.

[0051] A treatment can be considered “effective,” as used herein, if one or more of the signs or symptoms of a condition described herein are altered in a beneficial manner, other clinically accepted symptoms are improved, or even ameliorated, or a desired response is induced e.g., by at least 10% following treatment according to the methods described herein. Efficacy can be assessed, for example, by measuring a marker, indicator, symptom, and/or the incidence of a condition treated according to the methods described herein or any other measurable parameter appropriate, e.g. muscle torque or force. Efficacy can also be measured by a failure of an individual to worsen as assessed by hospitalization, or need for medical interventions (e.g., progression of the disease is halted). Treatment includes any treatment of a disease or condition in an individual or an animal (some non-limiting examples include a human or an animal) and includes: (1) inhibiting the disease or condition, e.g., preventing a worsening of symptoms (e.g. pain or inflammation); or (2) relieving the severity of the disease or condition, e.g., causing regression of symptoms. An effective amount for the treatment of a disease or condition means that amount which, when administered to a subject in need thereof, is sufficient to result in effective treatment as that term is defined herein, for that disease or condition. Efficacy of an agent can be determined by assessing physical indicators of a condition or desired response, (e.g. muscle function, mass or volume). One skilled in the art can monitor efficacy of administration and/or treatment by measuring any one of such parameters, or any combination of parameters.

[0052] The term “effective amount” as used herein refers to the amount of a composition or an agent needed to alleviate at least one or more symptom of the disease or condition, and relates to a sufficient amount of therapeutic composition to provide the desired effect. The term “therapeutically effective amount” refers to an amount of a composition or therapeutic agent that is sufficient to provide a particular muscle function recovery when administered to a typical subject. An effective amount as used herein, in various contexts, can include an amount sufficient to delay the development of a symptom of the disease or condition, alter the course of a symptom disease or condition (for example but not limited to, slowing the progression of a symptom of the disease or condition), or reverse a symptom of the disease or condition. The therapeutically effective amount may be administered in one or more doses of the therapeutic agent. The therapeutically effective amount may be administered in a single administration, or over a period of time in a plurality of doses.

[0053] “Administering” as used herein can include any suitable routes of administering a therapeutic agent or composition as disclosed herein. Suitable routes of administration include, without limitation, oral, parenteral, intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), pulmonary, cutaneous, injection or topical administration. Administration can be local or systemic.

[0054] As used herein, the term “pharmaceutical composition” refers to the active agent in combination with a pharmaceutically acceptable carrier e.g. a carrier commonly used in the pharmaceutical industry. The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

[0055] The singular terms “a,” “an,” and “the” include plural referents unless context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The abbreviation, “e.g.” is used herein to indicate a non- limiting example. Thus, the abbreviation “e.g.” is synonymous with the term “for example.”

[0056] Definitions of common terms in cell biology and molecular biology can be found in “The Merck Manual of Diagnosis and Therapy”, 19th Edition, published by Merck Research Laboratories, 2006 (ISBN 0-91 1910-19-0); Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632- 02182-9); Benjamin Lewin, Genes X, published by Jones & Bartlett Publishing, 2009 (ISBN- 10: 0763766321); Kendrew et al. (eds.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081- 569-8) and Current Protocols in Protein Sciences 2009, Wiley Intersciences, Coligan et al., eds.

METHODS

[0057] Methods of treating a traumatic muscle injury by administering cardiosphere-derived cells (CDCs) and/or CDC-derived exosomes to a subject suffering from a traumatic muscle injury are provided. With reference to Figure 2, an embodiment of a method 200 of treating a traumatic muscle injury is described. The method can include identifying 210 a subject having a traumatic injury to a muscle, such as a skeletal muscle, where the traumatic injury causes a volumetric muscle loss from about 10% to about 30%. Then the method can include administering 220 a therapeutically effective amount of CDCs to the subject, where the therapeutically effective amount ranges or includes from about 1 x 10³ to about 1 x 10⁹ CDCs, including, for example, from about 1 x 10⁴ to about 1 x 10⁷ CDCs.

[0058] In some embodiments, the method includes administering a therapeutically effective amount of CDC-derived exosomes to the subject. In some embodiments, the method is a cell-free method of treating a traumatic muscle injury. “Cell-free” as used herein refers to a substantial or complete lack of cells, e.g., CDCs. A therapeutically effective amount of CDC- derived exosomes in several embodiments is from about 1 x 10⁵ to about 1 x 10¹² exosomes, including, for example about 1 x 10⁸ to about 1 x 10¹¹ exosomes. In some embodiments, the exosomes administered to the subject are engineered high-potency exosomes, as described herein. In some embodiments, the exosomes administered to the subject are derived from primary human CDCs, as described herein. In some embodiments, the exosomes administered to the subject are derived from i mortalized CDCs, as described herein. In some embodiments, the exosomes administered to the subject are engineered high-potency exosomes derived from immortalized CDCs, as described herein.

[0059] The subject may be identified as having a traumatic injury to a muscle by any suitable option(s). In some embodiments, identifying the subject may include, determining that a subject has suffered a traumatic injury based on, e.g., the result of one or more clinical tests, visual assessment of a physical trauma suffered by the subject, visual assessment of the bodily site of physical impact, imaging of the bodily site of physical impact (e.g., by magnetic resonance imaging (MRI)), the nature of the events that caused the physical trauma, and/or the loss of mobility suffered by the subject due to a physical impact, etc.

[0060] The subject to which the CDCs and/or CDC-derived exosomes (e.g., primary human CDC-derived exosomes or immortalized CDC-derived exosomes), are administered may be any suitable subject who has suffered a traumatic injury to a muscle. In some embodiments, the muscle is skeletal muscle. Volumetric muscle loss to a variety of skeletal muscle can be treated by the present methods. In some embodiments, the skeletal muscle is a muscle of the face, neck, shoulder, arm, back torso, hand, hip, thigh, leg, and/or feet. In some embodiments, the subject has a volumetric muscle loss from about 10% to about 35%, e.g., about 12% to about 35%, about 15% to about 35%, about 15% to about 30%, including about 15% to about 25%, or any percentage between the values listed. In some embodiments, the subject has a volumetric muscle loss of about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or about 35%, or any percentage between the values listed. The volumetric muscle loss may be measured using any suitable method. In some embodiments, volumetric muscle loss is measured by, e.g., visual assessment, or by magnetic resonance imaging (MRI). In some embodiments, the volumetric muscle loss is based on a comparison of the muscle mass before the injury and after the injury. In some embodiments, the volumetric muscle loss is an estimate of muscle lost due to the injury based on an MRI image of the muscle after the injury, without a measure of the muscle mass for the subject before the injury.

[0061] The traumatic muscle injury, e.g., volumetric muscle loss, treated by the present methods typically causes a functional impairment of the muscle or group of muscles. In some embodiments, the function of the muscle injured by trauma is the force or torque generated by contraction of the muscle, or group of muscles to which the injured muscle belongs. In some embodiments, where the injured muscle is part of a muscle compartment, e.g., a fascial compartment or muscle group, the traumatic injury reduces the muscle strength of the muscle compartment. In some embodiments, muscle function, e.g., muscle strength, is reduced by about 30% or more, e.g., about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, including about 95% or more, or by any percentage in between the values listed. In some embodiments, the traumatic muscle injury reduces muscle function, e.g., muscle strength, by about 30 to about 100%, e.g., about 40 to about 100%, about 50 to about 100%, about 50 to about 95%, about 60 to about 95%, about 70 to about 95%, including about 80 to about 95%.

[0062] The regimen for administering the CDCs may vary, depending on the embodiment. The amount of CDCs administered to a subject in a single injection or dose may vary, and may be in a range of about 1 x 10⁴ to about 1 x 10⁸ cells per dose, e.g., about 1 x 10⁴ to about 1 x 10⁷ cells per dose, about 2 x 10⁴ to about 1 x 10⁷ cells per dose, about 5 x 10⁴ to about 1 x 10⁷ cells per dose, about 1 x 10⁵ to about 1 x 10⁷ cells per dose, about 5 x 10⁵ to about 1 x 10⁷ cells per dose, about 1 x 10⁶ to about 1 x 10⁷ cells per dose, including about 1 x 10⁶ to about 5 x 10⁶ cells per dose, or any dose in between those listed. In some embodiments, the amount of CDCs administered to a subject in a single injection or dose is about 1 x 10⁴, about 2 x 10⁴, about 5 x 10⁴, about 1 x 10⁵, about 2 x 10⁵, about 5 x 10⁵, about 1 x 10⁶, about 2 x 10⁶, about 5 x 10⁶, about 1 x 10⁷, about 2 x 10⁷, about 5 x 10⁷, or about 1 x 10⁸ cells per dose, or any dose in between those listed. In certain embodiments, the CDC dose is administered on a per kilogram basis of the subject’s body mass, for example, about 1 x 10⁵ cells/kg to about 1 x 10⁹ cells/kg, e.g., about 5 x 10⁵ cells/kg to about 1 x 10⁹ cells/kg, about 5 x 10⁵ cells/kg to about 5 x 10⁸ cells/kg, about 1 x 10⁶ cells/kg to about 5 x 10⁸ cells/kg, including about 1 x 10⁶ cells/kg to about 1 x 10⁸ cells/kg, or any dose in between those listed. In certain embodiments, the amount of CDCs administered to a subject in a single injection or dose is about 1 x 10⁵ cells/kg, about 2 x 10⁵ cells/kg, about 5 x 10⁵ cells/kg, about 1 x 10⁶ cells/kg, about 2 x 10⁶ cells/kg, about 5 x 10⁶ cells/kg, about 1 x 10⁷ cells/kg, about 2 x 10⁷ cells/kg, about 5 x 10⁷ cells/kg, about 1 x 10⁸ cells/kg, about 2 x 10⁸ cells/kg, about 5 x 10⁸ cells/kg, about 1 x 10⁹ cells/kg of the subject’s body mass, or any dose in between those listed. In certain embodiments, the CDC dose is administered per gram of volumetric tissue loss, for example, about 1 x 10⁵ cells/g to about 1 x 10⁹ cells/g, e.g., about 5 x 10⁵ cells/g to about 1 x 10⁹ cells/g, about 5 x 10⁵ cells/g to about 5 x 10⁸ cells/g, about 1 x 10⁶ cells/g to about 5 x 10⁸ cells/g, including about 1 x 10⁶ cells/g to about 1 x 10⁸ cells/g of volumetric muscle loss, or any dose in between those listed. In certain embodiments, the amount of CDCs administered to a subject in a single injection or dose is about 1 x 10⁵ cells/g, about 2 x 10⁵ cells/g, about 5 x 10⁵ cells/g, about 1 x 10⁶ cells/g, about 2 x 10⁶ cells/g, about 5 x 10⁶ cells/g, about 1 x 10⁷ cells/g, about 2 x 10⁷ cells/g, about 5 x 10⁷ cells/g, about 1 x 10⁸ cells/g, about 2 x 10⁸ cells/g, about 5 x 10⁸ cells/g, or about 1 x 10⁹ cells/g of volumetric muscle loss, or any dose in between those listed.

[0063] The regimen for administering the CDC-derived exosomes, e.g., primary human CDC-derived exosomes or immortalized CDC-derived exosomes, may vary, depending on the embodiment. The amount of CDC-derived exosomes administered to a subject in a single injection or dose may vary, and may be in a range of about 1 x 10⁷ to about 1 x 10¹¹ particles per dose, e.g., about 5 x 10⁷ to about 5 x 10¹⁰ particles per dose, about 1 x 10⁸ to about

5 x 10¹⁰ particles per dose, about 2 x 10⁸ to about 2 x 10¹⁰ particles per dose, about 2 x 10⁸ to about 1 x 10¹⁰ particles per dose, about 5 x 10⁸ to about 1 x 10¹⁰ particles per dose, about 5 x 10⁸ to about 5 x 10⁹ particles per dose, about 7 x 10⁸ to about 5 x 10⁹ particles per dose, about

1 x 10⁹ to about 5 x 10⁹ particles per dose, including about 1 x 10⁹ to about 3 x 10⁹ particles per dose, or any dose in between those listed. In some embodiments, the amount of CDC- derived exosomes administered to a subject in a single injection or dose is about 1 x 10⁸, about

2 x 10⁸, about 3 x 10⁸, about 4 x 10⁸, about 5 x 10⁸, about 6 x 10⁸, about 7 x 10⁸, about 8 x 10⁸, about 9 x 10⁸, about 1 x 10⁹, about 2 x 10⁹, about 3 x 10⁹, about 4 x 10⁹, about 5 x 10⁹, about

6 x 10⁹, about 7 x 10⁹, about 8 x 10⁹, about 9 x 10⁹, about 1 x 10¹⁰, about 2 x 10¹⁰, about 5 x 10¹⁰, or about 1 x 10¹¹ particles per dose, or any dose in between those listed. In certain embodiments, the exosome dose is administered on a per kilogram basis of the subject’s body mass, for example, about 1 x 10⁶ exosomes/kg to about 1 x 10¹¹ exosomes/kg, e.g., about 5 x 10⁶ exosomes/kg to about 1 x 10¹¹ exosomes/kg, about 5 x 10⁶ exosomes/kg to about 5 x 10¹⁰ exosomes/kg, about 1 x 10⁷ exosomes/kg to about 5 x 10¹⁰ exosomes/kg, including about 1 x 10⁶ exosomes/kg to about 1 x 10¹⁰ exosomes/kg of the subject’s body mass, or any dose in between those listed. In certain embodiments, the amount of CDC-derived exosomes administered to a subject in a single injection or dose is about 1 x 10⁶ exosomes/kg, about 2 x 10⁶ exosomes/kg, about 5 x 10⁶ exosomes/kg, about 1 x 10⁷ exosomes/kg, about 2 x 10⁷ exosomes/kg, about 5 x 10⁷ exosomes/kg, about 1 x 10⁸ exosomes/kg, about 2 x 10⁸ exosomes/kg, about 5 x 10⁸ exosomes/kg, about 1 x 10⁹ exosomes/kg, about 2 x 10⁹ exosomes/kg, about 5 x 10⁹ exosomes/kg, about 1 x 10¹⁰ exosomes/kg, about 2 x 10¹⁰ exosomes/kg, about 5 x 10¹⁰ exosomes/kg, about 1 x 10¹¹ exosomes/kg, or any dose in between those listed. In certain embodiments, the exosome dose is administered per gram of volumetric tissue loss, for example, about 1 x 10⁶ exosomes/g to about 1 x 10¹¹ exosomes/g, e.g., about 5 x 10⁶ exosomes/g to about 1 x 10¹¹ exosomes/g, about 5 x 10⁶ exosomes/g to about 5 x 10¹⁰ exosomes/g, about 1 x 10⁷ exosomes/g to about 5 x 10¹⁰ exosomes/g, including about 1 x 10⁶ exosomes/g to about 1 x 10¹⁰ exosomes/g of volumetric muscle loss, or any dose in between those listed. In certain embodiments, the amount of CDC-derived exosomes administered to a subject in a single injection or dose is about 1 x 10⁶ exosomes/g, about 2 x 10⁶ exosomes/g, about 5 x 10⁶ exosomes/g, about 1 x 10⁷ exosomes/g, about 2 x 10⁷ exosomes/g, about 5 x 10⁷ exosomes/g, about 1 x 10⁸ exosomes/g, about 2 x 10⁸ exosomes/g, about 5 x 10⁸ exosomes/g, about 1 x 10⁹ exosomes/g, about 2 x 10⁹ exosomes/g, about 5 x 10⁹ exosomes/g, about 1 x 10¹⁰ exosomes/g, about 2 x 10¹⁰ exosomes/g, about 5 x 10¹⁰ exosomes/g, or about 1 x 10¹¹ exosomes/g of volumetric muscle loss, or any dose in between those listed.

[0064] The CDCs and/or CDC-derived exosomes (e.g., primary human CDC- derived exosomes or immortalized CDC-derived exosomes) may be administered to the subject at any suitable time after the traumatic muscle injury. In some embodiments, the method includes administering a first dose (or an initial dose) of the CDCs and/or CDC-derived exosomes within about 1 minute, 2 minutes, 5 minutes, 10 minutes, 20 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 5 hours, 12 hours, 1 day, 2 days, 3 days, 5 days, 1 week, 2 weeks, or one month, or within any time point in between the time periods listed, after the subject suffers the traumatic injury. In some embodiments, the method includes administering a first dose (or an initial dose) of the CDCs and/or CDC-derived exosomes within 3 hours to 3 days, e.g., within 12 hours to 3 days, including within 1 to 3 days after the subject suffers the traumatic injury. In some embodiments, the method provides a therapeutic effect for treating a traumatic muscle injury when the therapeutic amount of CDCs and/or CDC-derived exosomes is administered no sooner than 1, 2, or 3 days after the subject suffers the traumatic injury. In some embodiments, the method includes administering the first dose (or initial dose) of the CDCs and/or CDC-derived exosomes at least 1, 2 or 3 days after the subject suffers the traumatic injury.

[0065] In several embodiments, the methods include administering any suitable number of doses of the CDCs and/or CDC-derived exosomes (e.g., primary human CDC- derived exosomes or immortalized CDC-derived exosomes) to the subject. In some embodiments, the CDCs and/or CDC-derived exosomes are administered once, e.g., in a single dose or in a single treatment episode, to the subject. In some embodiments, the CDC-derived exosomes are administered chronically. In some embodiments, the CDCs and/or CDC-derived exosomes are administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 40 or 50 times or more, or any number of times in between those listed, to the subject. The CDCs and/or CDC- derived exosomes can be administered at any suitable interval between consecutive doses. In some embodiments, the interval between administering consecutive doses of CDCs and/or CDC-derived exosomes is about 3 days, about 4 days, about 5 days, about 6 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, about 12 weeks, or about 6 months, or any interval defined by any two of the aforementioned lengths of time. In some embodiments, the CDCs and/or CDC-derived exosomes are administered to the subject weekly, bi-weekly, monthly, bi-monthly, semi-annually or annually. In some embodiments, the CDCs and/or CDC-derived exosomes are administered continuously. In certain embodiments, the CDCs and/or CDC-derived exosomes are administered continuously to the subject for 1 hour or more, e.g., 3 hours or more, 6 hours or more, 12 hours or more, 1 day or more, 5 days or more, 2 weeks or more, including 1 month or more, or any period of time in between the values listed.

[0066] In some embodiments, the CDCs and/or CDC-derived exosomes (e.g., primary human CDC-derived exosomes or immortalized CDC-derived exosomes) are administered to the subject parenterally, e.g., by injection. Any suitable route of injection (or other administration route disclosed herein) may be used to administer the CDCs and/or CDC- derived exosomes. In some embodiments, the CDCs and/or CDC-derived exosomes are administered to the subject via intravenous injection. Any suitable means of intravenous injection may be used to administer the CDCs and/or CDC-derived exosomes. In some embodiments, the CDCs and/or CDC-derived exosomes are administered to the subject via intramuscular injection. The intramuscular injection may be administered at any suitable location in the subject’s body. In some embodiments one or more intramuscular injections of the CDCs and/or CDC-derived exosomes is administered intramuscularly to a site adjacent the site of the traumatic injury. In some embodiments, the site of the traumatic injury is a wound or exposes tissue that is normally not exposed to an external environment (e.g., exposes tissue normally covered by skin). In some embodiments, the site of traumatic injury is internal to the subject’s body. In some embodiments, the site of administration is outside the margin of the traumatic injury, e.g., a wound. In certain embodiments, one or more doses of the CDCs and/or CDC-derived exosomes are administered at a plurality of different sites adjacent the site of the traumatic injury. In several embodiments, one or more doses of the CDCs and/or CDC-derived exosomes is administered at sites opposite each other across the site of the traumatic injury. In several embodiments, one or more doses of the CDCs and/or CDC-derived exosomes are administered adjacent the site of traumatic injury, and optionally at a plurality of sites that are at substantially opposite ends of the force-generating axis of the muscle to be treated. In several embodiments, one or more doses of the CDCs and/or CDC-derived exosomes are administered at a plurality of sites adjacent the site of the traumatic injury, where at least one site is proximal to the subject’s body relative to the site of the traumatic injury. In several embodiments, one or more doses of the CDCs and/or CDC-derived exosomes are administered at a plurality of sites adjacent the site of the traumatic injury, where at least one site is distal to the subject’s body relative to the site of the traumatic injury. In some embodiments, one or more doses of the CDCs and/or CDC-derived exosomes are administered at a first site adjacent the site of the traumatic injury and distal to the subject’s body relative to the site of the traumatic injury, and one or more doses of the CDCs and/or CDC-derived exosomes are administered at a second site adjacent the site of the traumatic injury and proximal to the subject’s body relative to the site of the traumatic injury.

[0067] In some embodiments, the CDCs and/or CDC-derived exosomes (e.g., primary human CDC-derived exosomes or immortalized CDC-derived exosomes) are administered to the subject topically. Any suitable means of topically administering the CDCs and/or CDC-derived exosomes may be used. In some embodiments, the CDCs and/or CDC- derived exosomes are disposed in a biocompatible substrate which can be applied to the site of the traumatic muscle injury to thereby administer the CDCs and/or CDC-derived exosomes. As used herein, the term “biocompatible” is given its ordinary meaning as understood by one of ordinary skill in the art and in view of the present disclosure, and includes the ability of biomaterial to perform its desired function without eliciting any undesirable local or systemic effects in the recipient. The biocompatible substrate may be any suitable substrate for containing the therapeutic agents (e.g., CDCs and/or CDC-derived exosomes) of the present disclosure and releasing the therapeutic agents at the site of the traumatic muscle injury when applied thereto. In some embodiments, the CDCs and/or CDC-derived exosomes are formulated into a liquid or fluid substrate, such as a cream, which can be topically applied to the site of the traumatic muscle injury.

[0068] In several embodiments, the method includes administering to the subject CDCs and/or CDC-derived exosomes that are disposed in a matrix. As used herein, “matrix” is given its ordinary meaning as understood by one of ordinary skill in the art and in view of the present disclosure, and includes, but not be limited to, biological and synthetic materials that can support living cells. A matrix may include, for example, hyaluronan, alginate, fibrin or combinations thereof. In some embodiments, a matrix includes biograft material or synthetic graft material. A matrix can be liquid, gelatinous or solid. In several embodiments, a matrix is embedded or seeded with, for example, cardiospheres, cardiosphere-derived cells, cardiosphere-forming cells, phase bright cells, stem cells, or other cells, or combinations thereof. In several embodiments, a matrix is embedded or seeded with CDC-derived exosomes, e.g., primary human CDC-derived exosomes or immortalized CDC-derived exosomes. In some embodiments, a matrix includes a scaffold or platform. In some embodiments, a matrix includes a hydrogel. In some embodiments, the biocompatible matrix includes extracellular matrix components, such as glycosaminoglycans, such as, but not limited to, hyaluronan, proteoglycans, and proteins, such as, but not limited to, collagen, elastin, fibronectin, fibrin, gelatin and laminin. In some embodiments, the biocompatible matrix includes naturally occurring biopolymers and their derivatives, such as but not limited to chitin, chitosan and alginate. In some embodiments, the biocompatible matrix includes biodegradable polymers, such as, but not limited to, polylactic acid (PLA), polyglycolic acid (PGA), poly(lactic-co-glycolic acid) (PLGA), polycaprolactone (PCL) and a variety of polycarbonate derivatives, and combinations thereof. In some embodiments, the biocompatible matrix includes non-biodegradable polymers, such as, but not limited to, poly(ethylene oxide) (PEO), poly(ethylene glycol) (PEG) and poly(ethylene-co-vinyl acetate) (EVA). Suitable biocompatible matrices are described in, e.g., U.S. Application Publication No. 20120039857, which disclosure is incorporated herein by reference in its entirety. In some embodiments, the CDCs and/or CDC-derived exosomes is disposed in a matrix that is a part of a wound dressing configured to position the matrix at, or adjacent to, the site of the traumatic muscle injury.

[0069] The methods of the present disclosure can promote and/or accelerate recovery of muscle function (e.g., muscle strength) from the traumatic muscle injury. In some embodiments, administering the therapeutic amount of the CDCs and/or CDC-derived exosomes (e.g., primary human CDC-derived exosomes or immortalized CDC-derived exosomes) to the subject promotes greater recovery of muscle strength (or other function or characteristic, such as muscle mass, volume, or endurance) compared to a reference level of recovery. In some embodiments, the level or rate of recovery of muscle strength is measured by measuring a force or torque that can be exerted by the muscle, or a group of muscles (e.g., fascial compartment) to which the injured muscle belongs.

[0070] In several embodiments, administering the therapeutic amount of the CDCs and/or CDC-derived exosomes results in an extent of recovery (e.g., of muscle strength) that is greater by at least about 1.5 times, e.g., at least about 1.6 times, at least about 1.7 times, at least about 1.8 times, at least about 1.9 times, at least about 2 times, at least about 2.2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 4.5 times, including at least about 5 times, or by any multiple in between the values listed, than a reference level of recovery (e.g., of muscle strength). The reference level of recovery in some embodiments is the level of spontaneous recovery (e.g., without receiving any active intervention for the injury) exhibited on average by an individual having a traumatic muscle injury similar to the subject’ s injury (e.g., similar in severity and/or location). In certain embodiments, the reference level of recovery is the level of recovery exhibited on average by an individual having a traumatic muscle injury similar to the subject’s injury (e.g., similar in severity and/or location) without the individual receiving the therapeutically effective dose of the CDCs and/or CDC-derived exosomes. In some embodiments, the reference level of recovery is the level of recovery by the subject before being administered the CDCs and/or CDC-derived exosomes. [0071] In some embodiments, administering the therapeutic amount of the CDCs and/or CDC-derived exosomes (e.g., primary human CDC-derived exosomes or immortalized CDC-derived exosomes) to the subject accelerates the rate of recovery (e.g., of muscle strength) compared to a reference rate of recovery. In several embodiments, administering the therapeutic amount of the CDCs and/or CDC-derived exosomes results in a recovery (e.g., of muscle strength) that is faster by at least about 1.5 times, e.g., at least about 2 times, at least about 2.2 times, at least about 2.5 times, at least about 3 times, at least about 3.2 times, at least about 3.5 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, including at least about 10 times, or by any multiple in between the values listed, than a reference rate of recovery (e.g., of muscle strength). The reference rate of recovery in some embodiments is the rate of recovery achieved spontaneously (e.g., without receiving any active intervention for the injury) on average by an individual having a traumatic muscle injury similar to the subject’s injury (e.g., similar in severity and/or location). In certain embodiments, the reference rate of recovery is the rate of recovery achieved on average by an individual having a traumatic muscle injury similar to the subject’s injury (e.g., similar in severity and/or location) without the individual receiving the therapeutically effective dose of the CDCs and/or CDC-derived exosomes (e.g., primary human CDC-derived exosomes or immortalized CDC-derived exosomes). In some embodiments, the reference rate of recovery is the rate of recovery by the subject before being administered the CDCs and/or CDC-derived exosomes.

[0072] In some embodiments, administering the therapeutic amount of the CDCs and/or CDC-derived exosomes (e.g., primary human CDC-derived exosomes or immortalized CDC-derived exosomes) to the subject results in recovery of about 25% or more, e.g., about 30% or more, about 35% or more, about 45% or more, about 50% or more, about 55% or more, including about 60% or more, or any percentage in between the values listed, of the pre-injury muscle strength, e.g., the pre-injury strength of a fascial compartment to which the injured muscle belongs, or other pre-injury characteristic of the muscle (e.g., mass or function). In some embodiments, the pre-injury muscle strength is estimated as the average muscle strength of healthy individuals, e.g., a cohort of individuals having similar physical fitness and without a traumatic muscle injury. In several embodiments, administering the therapeutic amount of the CDCs and/or CDC-derived exosomes to the subject results in an increase in the muscle strength, e.g., the strength of a fascial compartment to which the injured muscle belongs, of about 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.8, 8.5, 9, 9.5, 10 fold, or any multiple in between the values listed, compared to the muscle strength of the subject before the administering. In certain embodiments, muscle strength recovers or increases within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 40, 50 weeks, or any time period within a range defined by any two of the preceding time points, after the administering.

[0073] In some embodiments, administering the therapeutic amount of the CDCs and/or CDC-derived exosomes (e.g., primary human CDC-derived exosomes or immortalized CDC-derived exosomes) to the subject promotes and/or accelerates recovery of muscle mass/volume. In some embodiments, administering the therapeutic amount of the CDCs and/or CDC-derived exosomes to the subject results in greater recovery of muscle mass compared to a reference level of recovery. In some embodiments, muscle volume is measured using MRI. In some embodiments, muscle mass is estimated based on the mass volume measured using MRI.

[0074] In some embodiments, administering the therapeutic amount of the CDCs and/or CDC-derived exosomes (e.g., primary human CDC-derived exosomes or immortalized CDC-derived exosomes) to the subject results in an increase of about 25% or more, e.g., about 30% or more, about 35% or more, about 45% or more, about 50% or more, about 55% or more, including about 60% or more, or any percentage in between the values listed, in muscle mass relative to the pretreatment mass. In certain embodiments, muscle mass recovers or increases within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 40, 50 weeks, or any time period within a range defined by any two of the preceding time points, after the administering. In some embodiments, administering the therapeutic amount of the CDCs and/or CDC-derived exosomes (e.g., primary human CDC-derived exosomes or immortalized CDC-derived exosomes) to the subject results in an increase of about 15% or more, e.g., about 20% or more, about 25% or more, about 30% or more, about 35% or more, about 45% or more, about 50% or more, about 55% or more, including about 60% or more, or any percentage in between the values listed, in muscle volume, e.g., total volume, relative to the pretreatment volume. In certain embodiments, muscle volume, e.g., total volume, recovers or increases within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 40, 50 weeks, or any time period within a range defined by any two of the preceding time points, after the administering. In some embodiments, administering the therapeutic amount of the CDCs and/or CDC-derived exosomes (e.g., primary human CDC-derived exosomes or immortalized CDC-derived exosomes) to the subject results in an increase of about 25% or more, e.g., about 30% or more, about 35% or more, about 45% or more, about 50% or more, about 55% or more, including about 60% or more, or any percentage in between the values listed, in muscle mass relative to the pretreatment mass. In certain embodiments, muscle mass recovers or increases within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 40, 50 weeks, or any time period within a range defined by any two of the preceding time points, after the administering.

[0075] In some embodiments, methods of the present disclosure include providing CDCs and/or CDC-derived exosomes (e.g., primary human CDC-derived exosomes or immortalized CDC-derived exosomes), that are preserved, and reconstituting the preserved CDCs and/or exosomes into a composition (e.g., a pharmaceutically acceptable composition) for administering to the subject. In certain embodiments, reconstituting the preserved CDCs and/or exosomes includes combining the preserved CDCs and/or exosomes with a biocompatible substrate, e.g., a pharmaceutically acceptable excipient, a biocompatible matrix, etc., as described herein. The CDCs and/or CDC-derived exosomes may be preserved in any suitable manner, as disclosed herein.

[0076] In some embodiments, methods of the present disclosure includes administering one or more additional therapeutic agents to the subject. The additional therapeutic agent can include, without limitation, an analgesic, a local anesthetic, or an anti inflammatory drug. In some embodiments, the method includes administering an analgesic, e.g., an opioid analgesic, to the subject. Suitable analgesics include, without limitation, morphine, codeine, fentanyl, fentanyl analogs, pentazocine, buprenorphine, methadone, enkephalins, dynorphins, endorphins, and similarly acting opioid alkaloids and opioid peptides. In some embodiments, the method includes administering a local anesthetic, including without limitation, lidocaine/lignocaine hydrochloride, xylocaine (adrenaline, lidocaine), bupivacaine, ropivacaine, prilocaine, chinchocaine, etidocaine, tetracaine, trimecaine, procaine, and benzocaine, proxymetacaine, chloroprocaine, piperocaine, cyclomethycaine, dimethocaine, propxycaine. In some embodiments, the method includes administering an anti-inflammatory drug. A suitable anti-inflammatory drug includes, steroidal anti-inflammatory drugs, for example, betamethasone, prednisone, dexamethasone, cortisone, hydrocortisone, methylprednisolone, and prednisolone, or nonsteroidal anti-inflammatory drugs such as aspirin, ibuprofen, and naproxen.

[0077] Also provided herein is a method of treating a tissue damage, a wound or a lesion by administering a therapeutically effective amount of the cardiosphere-derived cells (CDCs) and/or CDC-derived exosomes, as provided herein, to a subject suffering from the tissue damage, wound or lesion. The tissue damage, wound or lesion can be, without limitation, a traumatic injury, a pressure ulcer, an infection, etc. Any of the options for treating a traumatic muscle injury provided herein can be used in the method of treating a tissue damage, a wound or a lesion.

CARDIOSPHERE-DERIVED CELLS AND CDC-DERIVED EXOSOMES

[0078] The CDCs used in the several methods of the present disclosure can be derived from cardiac stem cells from various regions of the heart, including but not limited to the atria, septum, ventricles, auricola, and combinations thereof (e.g., a partial or whole heart may be used to obtain cardiac stem cells in some embodiments). In several embodiments, CDCs are derived from cells (or groups of cells) that comprise cardiac stem cells or can be manipulated in culture to give rise to cardiac stem cells (e.g., cardiospheres and/or cardiosphere derived cells (CDCs)). The CDCs may be derived from cardiac stem cells of any suitable organism (e.g., vertebrate organism). In some embodiments, the CDCs are derived from human cardiac cells (e.g., human cardiac stem cells). Suitable means of generating cardiospheres are described, e.g., in U.S. Patent No. 8,268,619, which disclosure is incorporated herein by reference in its entirety. Suitable means of generating CDCs are described, e.g., in U.S. Application Publication Nos. 20080267921; Smith et al., Circulation. 2007. 115:896-908; and Ibrahim et al., Stem Cell Reports. 2014 May 8;2(5):606-19, which disclosures are incorporated herein by reference in their entirety.

[0079] Depending on the embodiment, CDCs are derived from cardiac tissue from any suitable source. In some embodiments, the CDCs are primary CDCs, e.g., primary human CDCs. In some embodiments, the CDCs are derived from the same species of animal as the subject to which the CDCs or CDC-derived exosomes are administered. In some embodiments, the CDCs are derived from a different species of animal as the subject to which the CDCs or CDC-derived exosomes are administered. In some embodiments, the CDCs are derived from cells obtained from a source that is allogeneic, autologous, xenogeneic, or syngeneic with respect to the eventual recipient of the CDCs and/or CDC-derived exosomes. In some embodiments, the CDCs are derived from cardiac tissue from the subject to which the CDCs and/or CDC-derived exosomes are administered. In some embodiments, the CDCs are allogenic, and can be derived from cardiac tissue from individuals other than the subject to which the CDCs and/or CDC-derived exosomes are administered. The individual from which the CDCs are derived can have varying degrees of immunological compatibility with the subject to which the CDCs and/or CDC-derived exosomes are administered. In some embodiments, the CDCs are derived from cardiac tissue from individuals who are immunologically incompatible with the subject to which the CDCs and/or CDC-derived exosomes are administered. In some embodiments, the CDCs are derived from cardiac tissue from individuals who are immunologically compatible with the subject to which the CDCs and/or CDC-derived exosomes are administered. Regardless of a determination of compatibility, in several embodiments, the administered CDCs and/or CDC-derived exosomes are from a donor who is allogeneic with respect to the ultimate recipient. In several embodiments, the use of exosomes, as a cell-free composition, presents a reduced risk of a host immune response.

[0080] In some embodiments, the CDCs are immortalized CDCs. Immortalized CDCs can be used for deriving exosomes for use in the present methods. Advantageously, immortalized CDCs can be passaged more times than their non-immortalized counterpart. In some embodiments, immortalized CDCs can be passaged 8 times or more, e.g., 9 times or more, 10 times or more 11 times or more, 12 times or more, 15 times or more, 18 times or more, 20 times or more, 25 times or more, 30 times or more, 40 times or more, including 50 times or more after the cardiosphere formation stage. Any suitable means of immortalizing CDCs may be used. In some embodiments, CDCs are immortalized by transduction with simian virus 40 large and small T antigen (SV40 T+t). In some embodiments, the CDCs are immortalized (e.g., by SV40 T+t transduction) and treated with an agent that activates Wnt/b- catenin signaling (e.g., shRNA that targets MEST or Extll). Suitable means of generating high-potency exosomes, immortalized CDCs, and exosomes derived therefrom, are described, e.g., in U.S. Provisional Patent Application No. 62/845,228, filed May 8, 2019; and Ibrahim et al., Nat Biomed Eng. 2019 Sep;3(9):695-705, which disclosures are incorporated herein by reference in their entirety.

[0081] Exosomes, in several embodiments, are isolated from cellular preparations by methods comprising one or more of filtration, centrifugation, antigen-based capture and the like. For example, in several embodiments, a population of cells, e.g., cardiosphere-derived cells, grown in culture are collected and pooled. In several embodiments, monolayers of cells are used, in which case the cells are optionally treated in advance of pooling to improve cellular yield (e.g., dishes are scraped and/or enzymatically treated with an enzyme such as trypsin to liberate cells). In several embodiments, cells grown in suspension are used. The pooled population is then subject to one or more rounds of centrifugation (in several embodiments ultracentrifugation and/or density centrifugation is employed) in order to separate the exosome fraction from the remainder of the cellular contents and debris from the population of cells. In some embodiments, centrifugation need not be performed to harvest exosomes. In several embodiments, pre-treatment of the cells is used to improve the efficiency of exosome capture. For example, in several embodiments, agents that increase the rate of exosome secretion from cells are used to improve the overall yield of exosomes. In some embodiments, augmentation of exosome secretion is not performed. In some embodiments, size exclusion filtration is used in conjunction with, or in place of centrifugation, in order to collect a particular size (e.g., diameter) of exosome. In several embodiments, filtration need not be used. In still additional embodiments, exosomes (or subpopulations of exosomes) are captured by selective identification of unique markers on or in the exosomes (e.g., transmembrane proteins). In such embodiments, the unique markers can be used to selectively enrich a particular exosome population. In some embodiments, enrichment, selection, or filtration based on a particular marker or characteristic of exosomes is not performed. The exosomes used in the several methods of the present disclosure can be derived from CDCs. Suitable means of generating exosomes are described, e.g., in U.S. Application Publication Nos. 20160158291 and 20160160181; and Ibrahim et al., Stem Cell Reports. 2014 May 8;2(5):606-19, which disclosures are incorporated herein by reference in their entirety. The exosomes may be derived from any suitable CDCs, e.g., as provided herein. In some embodiments, the exosomes are derived from CDCs from the same species of animal as the subject to which the exosomes are administered. In some embodiments, the exosomes are derived from CDCs from a different species of animal as the subject to which the exosomes are administered.

[0082] Exosomes disclosed herein can vary in size, depending on the embodiment. Depending on the embodiment, the size of the exosomes ranges in diameter from about 15 nm to about 95 nm in diameter, including about 15 nm to about 20 nm, about 20 nm to about 30 nm, about 30 nm to about 40 nm, about 40 nm to about 50 nm, about 50 nm to about 60 nm, 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, and overlapping ranges thereof. In several embodiments, exosomes are larger (e.g., those ranging from about 140 to about 210 nm, including about 140 nm to about 150 nm, about 150 nm to about 160 nm, about 160 nm to about 170 nm, about 170 nm to about 180 nm, about 180 nm to about 190 nm, about 190 nm to about 200 nms, about 200 nm to about 210 nm, and overlapping ranges thereof). In some embodiments, the exosomes that are generated from the original cellular body are 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 5000, or 10,000 times smaller in at least one dimension (e.g., diameter) than the original cellular body.

[0083] In several embodiments, exosomes (e.g., exosomes engineered for high potency), can be manipulated, for example through gene editing using, for example CRISPR- Cas, zinc finger nucleases, and/or TALENs, to reduce their potential immunogenicity. Moreover, master banks of exosomes that have been characterized for their expression of certain miRNAs and/or proteins can be generated and stored long-term for subsequent use in defined subjects on an “off-the-shelf’ basis. In some embodiments, exosomes are isolated and then used without long-term or short-term storage (e.g., they are used as soon as practicable after their generation).

[0084] In some embodiments, the CDCs are treated to generate high-potency exosomes. Any suitable means for generating high-potency exosomes can be used. In some embodiments, the CDCs are treated with an agent that activates Wnt/p-catenin signaling. In some embodiments, an agent that activates Wnt/p-catenin signaling can repress or downregulate an inhibitor of Wnt/p-catenin signaling. In some embodiments, the agent that activates Wnt/p-catenin signaling is a GSK3P inhibitor. In some embodiments, the GSK3P inhibitor is 6-bromoindirubin-3’ -oxime (BIO) or tideglusib (or a combination thereof). In some embodiments, an agent that activates Wnt/p-catenin signaling represses expression of an inhibitor of Wnt/p-catenin signaling. In some embodiments, the agent inhibits expression of Mest or Extll. In some embodiments, the agent is a short hairpin (sh) RNA that targets Mest or Extll. In some embodiments, the CDCs are transduced with an shRNA that targets MEST or Extll.

[0085] The CDC-derived exosomes, e.g., primary human CDC-derived exosomes or immortalized CDC-derived exosomes, of the present disclosure may include a variety of biomolecules, such as nucleic acids and proteins. In some embodiments, the exosomes contain DNA, DNA fragments, DNA plasmids, mRNA, tRNA, snRNA, saRNA, miRNA, rRNA, regulating RNA, other non-coding and coding RNA, etc. In some embodiments, the exosomes contain non-coding RNAs (ncRNAs), such as, but not limited to, long non-coding RNAs (IncRNAs), microRNAs (miRNAs) and Y RNA fragments.

[0086] In some embodiments, the CDC-derived exosomes, e.g., primary human CDC-derived exosomes or immortalized CDC-derived exosomes, are enriched in or depleted for one or more biomolecules, such as nucleic acids and proteins. In some embodiments, a biomolecule may be enriched (or depleted) in the exosomes relative to the level of a suitable reference biomolecule. In some embodiments, the CDC-derived exosomes are enriched for a miRNA relative to a reference miRNA. In some embodiments, the CDC-derived exosomes are depleted for a miRNA relative to a reference miRNA. In some embodiments, an miRNA is enriched if the amount of miRNA present in the exosomes is 5, 10, 15, 20, 25, 30, 40, 50, 75, 100, 200, 300, 500, 750, 1,000, 2,000, 5,000, 10,000 fold or more, or has a fold change in a range defined by any two of the preceding values, than the amount of the reference miRNA present in the exosomes. In some embodiments, an miRNA is depleted if the amount of miRNA present in the exosomes is 0.75, 0.5, 0.4, 0.3, 0.2, 0.1, 0.08, 0.06, 0.04, 0.02, 0.01, 0.005, 0.001 fold or less, or has a fold change in a range defined by any two of the preceding values, than the amount of the reference miRNA present in the exosomes. In some embodiments, the CDC-derived exosomes are enriched or depleted for a biomolecule (e.g., miRNA) relative to the level of the biomolecule in non-therapeutic exosomes (e.g., exosomes derived from human dermal fibroblasts (HDFs)).

[0087] In some embodiments, the CDC-derived exosomes, e.g., primary human CDC-derived exosomes or immortalized CDC-derived exosomes, contain or are enriched for miR-92a, miR-181b, miR-148a, and/or miR-146a. In some embodiments, the CDC-derived exosomes do not contain, or are depleted for miR-199b. In some embodiments, the CDC- derived exosomes are enriched for miR-92a, miR-181b, miR-148a, and/or miR-146a relative to the level of miR-199b in the exosomes. In some embodiments, the CDC-derived exosomes are enriched for miR-92a, miR-181b, miR-148a, and/or miR-146a relative to a reference miRNA, e.g., miR-23a. In some embodiments, the CDC-derived exosomes are depleted for miR-199b relative to a reference miRNA, e.g., miR-23a.

[0088] In some embodiments, the CDCs and/or CDC-derived exosomes, e.g., primary human CDC-derived exosomes or immortalized CDC-derived exosomes, are provided in a composition, e.g., a pharmaceutically acceptable composition. In some embodiments, the CDCs and/or CDC-derived exosome-containing composition is prepared in a pharmaceutically acceptable excipient, such as water or a buffer. Pharmaceutically acceptable excipients include, but not limited to, saline, aqueous buffer solutions, solvents and/or dispersion media. Some non-limiting examples of materials which can serve as pharmaceutically-acceptable excipients include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc; (8) cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and/or poly anhydrides; (22) bulking agents, such as polypeptides and amino acids (23) serum component, such as serum albumin, HDL and LDL; (22) C2-C12 alcohols, such as ethanol; and (23) other non-toxic compatible substances employed in pharmaceutical formulations. In some embodiments, the excipient inhibits the degradation of the active agent, e.g., CDC-derived exosomes and/or bioactive contents thereof.

[0089] In some embodiments, the composition is in a parenteral dose form. In some embodiments, parenteral dosage forms is sterile or capable of being sterilized before administering to a patient. Examples of parenteral dosage forms include, but are not limited to, solutions ready for injection, dry products ready to be dissolved or suspended in a pharmaceutically acceptable vehicle for injection, suspensions ready for injection, and emulsions. In addition, controlled-release parenteral dosage forms can be prepared for administration to a subject. Suitable excipients that can be used to provide parenteral dosage forms of CDC-derived exosomes include, without limitation: sterile water; water for injection USP; saline solution; glucose solution; aqueous vehicles such as but not limited to, sodium chloride injection, Ringer's injection, dextrose Injection, dextrose and sodium chloride injection, and lactated Ringer's injection; water-miscible vehicles such as, but not limited to, ethyl alcohol, polyethylene glycol, and propylene glycol; and non-aqueous vehicles such as, but not limited to, com oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.

[0090] In some embodiments, a composition that includes CDCs and/or CDC- derived exosomes (e.g., primary human CDC-derived exosomes or immortalized CDC-derived exosomes) is provided in a topical formulation. In certain embodiments, compositions may further include one or more alcohols, emollients, humectants, thickening and/or gelling agents, neutralizing agents, and surfactants. In some embodiments, the composition includes one or more of antipruritics, astringents, local anesthetics or anti-inflammatory agents (e.g., steroidal or non-steroidal anti-inflammatory drugs), dyes, preservatives, antioxidants, opacifiers, thickening agents or stabilizers. In some embodiments, a composition that includes CDCs and/or CDC-derived exosomes are provided in a biocompatible substrate, e.g., a biocompatible matrix, as described herein.

KITS

[0091] Also provided herein are kits for performing several methods of the present disclosure. Kits of the present disclosure can include CDCs and/or CDC-derived exosomes (e.g., primary human CDC-derived exosomes or immortalized CDC-derived exosomes) that are preserved, and a biocompatible substrate. “Preserved” as used herein, can describe a state of CDCs or CDC-derived exosomes in which the therapeutic activity of the CDCs or exosomes is retained for at least a defined period under standard storage conditions. In some embodiments, the preserved CDC-derived exosomes and/or preserved CDCs of the kit provide a therapeutically effective amount of CDC-derived exosomes and/or CDCs upon reconstitution with the biocompatible substrate. The preserved CDC-derived exosomes may retain 10% or more, e.g., 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or about 100% of the therapeutic activity when reconstituted after storage compared to the therapeutic activity before being preserved. The preserved CDCs or CDC-derived exosomes may be reconstituted into a composition (e.g., a pharmaceutically acceptable composition) for administering to the subject by combining with the biocompatible substrate.

[0092] The CDCs or CDC-derived exosomes (e.g., primary human CDC-derived exosomes or immortalized CDC-derived exosomes), may be preserved and stored under standard storage conditions for 1 day or more, e.g., 2 days or more, 5 days or more, 2 weeks or more, one month or more, 3 months or more, 6 months or more, 1 year or more, 3 years or more, 5 years or more, including 10 years or more. The CDCs or CDC-derived exosomes may be preserved and stored under standard storage conditions for a period of 1 day to 5 years, e.g., 5 days to 3 years, 10 days to 2 years, one month to 1 year, including 3 months to 6 months.

[0093] The preserved CDCs or CDC-derived exosomes (e.g., primary human CDC-derived exosomes or immortalized CDC-derived exosomes), may be stored under any suitable standard storage conditions. A standard storage condition may include a temperature of 25 °C or lower, e.g., 20 °C or lower, 15 °C or lower, 10 °C or lower, 5 °C or lower, 0 °C or lower, -10 °C or lower, -20 °C or lower, -30 °C or lower, -40 °C or lower, -50 °C or lower, -60 °C or lower, -70 °C or lower, including -80 °C or lower. In some embodiments, a standard storage condition has a temperature in the range of -90 °C to -80 °C, -80 °C to -70 °C, -70 °C to -60 °C, -60 °C to -50 °C, -50 °C to -40 °C, -40 °C to -30 °C, -30 °C to -20 °C, -20 °C to -10 °C, -10 °C to -5 °C, -5 °C to 0 °C, 0 °C to 5 °C, 5 °C to 10 °C, 10 °C to 15 °C, 15 °C to 20 °C, 20 °C to 25 °C, 25 °C to 30 °C, or 30 °C to 35 °C. In some embodiments, a standard storage condition is at room temperature and standard atmospheric pressure.

[0094] The CDCs or CDC-derived exosomes, e.g., primary human CDC-derived exosomes or immortalized CDC-derived exosomes, may be preserved in any suitable manner. In some embodiments, the CDCs or CDC-derived exosomes are frozen (e.g., cryopreserved). The CDCs may be frozen in any suitable manner, including suitable means for freezing cell lines. Means for freezing exosomes are described in, e.g., Bosch et al., Sci Rep. 2016 Nov 8;6:36162. In some embodiments, the CDC-derived exosomes are lyophilized. Means for lyophilizing exosomes are described in, e.g., PCT Publication No. W02018070939 (the entirety of which is incorporated by reference herein).

[0095] Kits of the present disclosure optionally further include a biocompatible substrate. The biocompatible substrate can be, for example, a pharmaceutically acceptable excipient, a biocompatible matrix, etc., as described herein. The biocompatible substrate can be suitable for reconstituting the preserved CDCs or preserved CDC-derived exosomes into a composition for administering to the subject, according to certain methods of the present disclosure. In some embodiments, the biocompatible substrate is suitable for reconstituting the preserved CDCs or preserved CDC-derived exosomes into a composition for parenteral administration to the subject, as described herein. In some embodiments, the biocompatible substrate is suitable for reconstituting the preserved CDCs or preserved CDC-derived exosomes into a composition for topical administration to the subject, as described herein. In some embodiments, the biocompatible substrate is a biocompatible matrix, as described herein.

[0096] In some embodiments, the kit includes one or more devices for administering the reconstituted CDCs or preserved CDC-derived exosomes. In some embodiments, the kit includes a syringe for administering a composition that includes CDCs or CDC-derived exosomes. In several embodiments, the syringe is configured to administer the composition intramuscularly or intravenously. In some embodiments, the kit includes a wound dressing configured to receive a composition that includes CDCs or CDC-derived exosomes and to position the composition at, or proximate to, a site of traumatic muscle injury. In certain embodiments, the wound dressing positions the CDC or CDC-derived exosome composition in a manner sufficient to deliver a therapeutic amount of the CDCs or CDC- derived exosomes to the subject. In certain embodiments, the wound dressing is configured to receive a CDC or CDC-derived exosome composition in a biocompatible matrix, as described herein.

[0097] Kits can include one or more containers (e.g., vials, ampoules, test tubes, flasks or bottles) for holding one or more components of the kits. In some embodiments, the kit includes one or more additional therapeutic agents for administering to the subject. The additional therapeutic agents can include, without limitation, analgesics, local anesthetics, and anti-inflammatory drugs, as described herein. The kits may further include instructions for using the kit to treat a traumatic muscle injury. The information and instructions may be in the form of words, pictures, or both, and the like.

[0098] All patents and other publications; including literature references, issued patents, published patent applications, and co-pending patent applications; cited throughout this application are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the technology described herein. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.

[0099] The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize. For example, while method steps or functions are presented in a given order, alternative embodiments may perform functions in a different order, or functions may be performed substantially concurrently. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the disclosure can be modified, if necessary, to employ the compositions, functions and concepts of the above references and application to provide yet further embodiments of the disclosure. Moreover, due to biological functional equivalency considerations, some changes can be made in protein structure without affecting the biological or chemical action in kind or amount. These and other changes can be made to the disclosure in light of the detailed description. All such modifications are intended to be included within the scope of the appended claims.

[0100] Specific elements of any of the foregoing embodiments can be combined or substituted for elements in other embodiments. Furthermore, while advantages associated with certain embodiments of the disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages to fall within the scope of the disclosure.

[0101] The technology described herein is further illustrated by the following examples which in no way should be construed as being further limiting.

EXAMPLES

Example 1: Therapeutic bioactivity of intravenously administered cardiosphere-derived cells (CDCs) in mice with volumetric muscle loss

[0102] Volumetric muscle loss was induced in mice by surgical resection of the tibialis anterior muscle. Approximately 20% of muscle, by weight, was resected from each animal. To determine the effect of volumetric muscle loss on muscle function, development of muscle torque of the anterior crural muscles was assessed. Immediately after surgery (“injury”), animals received either vehicle or 2.5 x 10⁵ murine CDCs by intravenous injection. Fig. 4A shows a schematic diagram of the experimental protocol.

[0103] Fig. 4A: Schematic of the experimental design. Mice underwent baseline testing of muscle function (pre-injury), then the TA muscle was injured and mice were immediately treated with CDCs IV (1 injection at 2.5 x 10⁵), CDCs IM (2 injections at 1.25 x 10⁵ each surrounding the muscle defect), or vehicle. Muscle function was retested immediately following treatment and biweekly until a predetermined study endpoint.

[0104] Fig. 1A shows the torque-frequency relationship before injury compared to immediately after the injury (top panel), 2 weeks after the injury (upper-middle panel), 4 weeks after the injury (lower-middle panel), and 6 weeks after the injury (bottom panel) in animals treated with CDCs and vehicle. Volumetric muscle loss caused about 90% reduction in torque at all frequencies tested. Two weeks after the surgery, animals that were treated with CDCs showed greater recovery over most frequencies compared to vehicle-treated animals. After four weeks, animals treated with CDCs continued to show greater recovery compared to vehicle control. At 6 weeks post-injury, animals treated with CDCs continued to show greater recovery compared to vehicle control, and showed better recovery even at the lower frequencies (< 50 Hz). Fig. 4C shows an alternative representation of the data. [0105] Fig. 4C: Torque-frequency relationship in IV-treated mice at pre-injury, day 0 post-injury, and biweekly during the 6-week study duration.

[0106] In Fig. IB, the data was normalized to the pre-injury torque to obtain the percentage tetanic torque deficit at day 0, week 2, week 4 and week 6 post injury. Torque at 200 Hz, representing a physiological rate of stimulation, was used to compare tetanic torque. Both control and treatment group animals suffered a torque deficit of about 90% due to the volumetric muscle loss. Animals treated with CDCs reduced the torque deficit at a faster rate and greater extent than control animals treated with vehicle. CDC-treated animals achieved the same level of recovery in Week 2 as did vehicle-treated animals in Week 6, which indicates faster recovery is achieved by intravenous CDC administration. The data is represented in alternate form in Fig. 4G. Fig. 1C compares the time course of recovery of muscle function between CDC- and vehicle-treated animals. Tetanic torque measured at 200 Hz recovered at a faster rate and greater extent in animals treated with CDCs compared to vehicle control. The data is represented in alternate form in Fig. 4D.

[0107] Fig. 4D: Tetanic torque was not different at pre-injury, nor at day 0 or week 2 post-injury between IV-treated groups. By 4- and 6-weeks post-injury, mice treated with CDCs produced significantly greater torque than vehicle-treated mice.

[0108] Fig. 4G: Tetanic torque deficit revealed CDCs delivered IV greatly diminished the deficit relative to vehicle-treated counterparts.

[0109] Fig. ID shows the time course of recovery of muscle function as a percentage of the tetanic torque before injury. Recovery of tetanic torque occurred faster and to a greater extent in animals treated with CDCs than control animals. Muscle function continued to recover in CDC-treated animals at 6 weeks. The data is represented in alternate form in Fig. 41.

[0110] Fig. 41: CDC treatment, regardless of delivery route, resulted in 47% recovery of muscle function by 6 weeks post-injury. See also Example 2. Vehicle-treated counterparts only recovered -20% of muscle function at the same time.

[0111] Measurement of the mass removed from the tibialis anterior confirmed that the extent of volumetric muscle loss was similar between vehicle and treatment groups (Fig. IE; Fig. 4B (“IV”) shows an alternative representation of the data). There were no differences in biopsy mass between groups. [0112] These results demonstrate the therapeutic effect of intravenous administration of CDCs, according to some embodiments disclosed herein, in promoting recovery of muscle function after traumatic muscle injury, such as volumetric muscle loss. According to several embodiments, treatment with CDC-derived exosomes results in similar recovery after volumetric muscle loss.

Example 2: Therapeutic bioactivity of intramuscularly administered CDCs in mice with volumetric muscle loss

[0113] To determine whether the route of administration affected the therapeutic effect of CDCs on volumetric muscle loss, the experiment in Example 1 was repeated using the same protocol except the CDCs were administered intramuscularly. Each animal received two intramuscular injections of vehicle or 1.25 x 10⁵ CDCs each, one injection at a site proximal to the body from the injury site, and another at a site distal to the body from the injury site.

[0114] Fig. 4E shows the torque-frequency relationship before injury compared to immediately after the injury, and 2, 4, and 6 weeks after the injury in animals treated with CDCs (right panel) and vehicle (left panel). Volumetric muscle loss caused about 90% reduction in torque at all frequencies tested. Two weeks after the surgery, animals that were treated with CDCs showed greater recovery over most frequencies compared to vehicle-treated animals. After four weeks, animals treated with CDCs continued to show greater recovery compared to vehicle control. At 6 weeks post-injury, animals treated with CDCs continued to show greater recovery compared to vehicle control, and showed better recovery even at the lower frequencies (< 50 Hz).

[0115] Fig. 4E: Torque-frequency relationship in IM-treated mice at pre-injury, day 0 post-injury, and biweekly during the 6-week study duration.

[0116] Fig. IF shows data comparing the time course of recovery of muscle function between CDC- and vehicle-treated animals. Two weeks after the injury, animals that were treated with intramuscular CDCs showed greater recovery of tetanic torque compared to control animals. After four weeks, animals treated with CDCs continued to show greater recovery of tetanic torque compared to vehicle control. At six weeks, CDC-treated animals continued to show greater recovery compared to vehicle control. As with intravenous administration, local administration of CDCs to animals achieved the same level of recovery in Week 2 as did vehicle-treated animals in Week 6, which indicates faster recovery is achieved by local CDC administration. The data is represented in alternate form in Fig. 4F.

[0117] Fig. 4F: Tetanic torque was not different at pre-injury, nor at day 0 or week 2 post-injury between IV-treated groups. By 4- and 6-weeks post-injury, mice treated with CDCs produced significantly greater torque than vehicle-treated mice.

[0118] In Fig. 4H, the data was normalized to the pre-injury torque to obtain the percentage tetanic torque deficit at day 0, week 2, week 4 and week 6 post injury. Torque at 200 Hz, representing a physiological rate of stimulation, was used to compare tetanic torque. Both control and treatment group animals suffered a torque deficit of about 90% due to the volumetric muscle loss. Animals treated with CDCs reduced the torque deficit at a faster rate and greater extent than control animals treated with vehicle. CDC-treated animals achieved the same level of recovery in Week 2 as did vehicle-treated animals in Week 6, which indicates faster recovery is achieved by intravenous CDC administration.

[0119] Fig. 4H: Tetanic torque deficit revealed CDCs delivered IM greatly diminished the deficit relative to vehicle-treated counterparts.

[0120] There were no differences in biopsy mass between groups (Fig. 4B (“IM”)).

[0121] These results demonstrate the therapeutic effect of local, intramuscular administration of CDCs, according to embodiments disclosed herein, in promoting recovery of muscle function after traumatic muscle injury, such as volumetric muscle loss.

Example 2.1

[0122] This non-limiting example shows CDCs has therapeutic effect for treating Volumetric Muscle Loss when administered at various dosages and dosing frequencies.

[0123] The effect of CDC dosage on VML-injured mice was tested. Fig. 5A shows a schematic of the experimental design. Mice underwent baseline testing of muscle function (pre-injury), then the tibialis anterior (TA) muscle was injured and mice were immediately treated with CDCs intravenously (5 x 10⁵ or 1 x 10⁶). Muscle function was retested immediately following treatment and biweekly until a predetermined study endpoint. There were no differences in biopsy mass between groups (Fig. 5B). [0124] Fig. 5C shows that tetanic torque was not different at pre-injury, nor at day 0 or week 2 post-injury between groups. By 4- and 6-weeks post- injury, mice treated with either dose of CDCs produced significantly greater torque than vehicle-treated mice. Tetanic torque deficit revealed CDCs, regardless of dose, greatly diminish the deficit relative to vehicle treatment by 6 weeks post-injury (Fig. 5D). As shown in Fig. 5E, CDC treatment, regardless of dose, improved recovery of muscle function. However, there were no differences between groups.

[0125] The effect of dosing frequency on VML-injured mice was tested. Fig. 5F shows a schematic of the experimental design. Mice underwent baseline testing of muscle function (pre-injury), then the TA muscle was injured and mice were immediately treated with CDCs IV (2.5 x 10⁵) delivered either 2 or 3 times (indicated by “Tx” and either “Tx 2 doses” (3 weeks between doses) or “Tx 3 doses”(two weeks between doses)). Muscle function was retested immediately following treatment and biweekly until a predetermined study endpoint. Fig. 5G shows that there were no differences in biopsy mass between groups.

[0126] Fig. 5H shows that tetanic torque was not different at pre-injury, nor at day 0 or week 2 post-injury between groups. By 4- and 6-weeks post-injury, mice treated with either number of doses of CDCs produced significantly greater torque than vehicle-treated mice. Tetanic torque deficit revealed CDCs, regardless of the number of doses, greatly diminish the deficit relative to vehicle treatment by 6 weeks post-injury (Fig. 51). Fig. 5J shows that CDC treatment, regardless of the number of doses, improved recovery of muscle function. However, there were no differences between groups.

[0127] In some embodiments, CDCs promote recovery of muscle function after traumatic muscle injury, such as volumetric muscle loss, when administered under different dosing regimens. In some embodiments, the dosage (e.g., number of CDCs administered) and/or the frequency and/or the number of doses of CDCs administered to the subject with a traumatic muscle injury, such as volumetric muscle loss, can be adjusted to treat the subject. In some embodiments, intravenously administered CDCs promote recovery of muscle function after traumatic muscle injury, such as volumetric muscle loss, when administered at a dose of up to lxlO⁶ CDCs. In some embodiments, intravenously administered CDCs promote recovery of muscle function after traumatic muscle injury, such as volumetric muscle loss, when administered every two or three weeks. [0128] In some embodiments, CDC-derived exosomes promote recovery of muscle function after traumatic muscle injury, such as volumetric muscle loss, when administered under different dosing regimens. In some embodiments, the dosage (e.g., number of exosomes administered) and/or the frequency and/or the number of doses of CDC-derived exosomes administered to the subject with a traumatic muscle injury, such as volumetric muscle loss, can be adjusted to treat the subject.

Example 2.2:

[0129] This non-limiting example shows that CDC administration to VML-injured animals restores damage to muscle tissue.

[0130] To examine the effect of CDC administration on muscle tissue in VML- injured animals, tibialis anterior (TA) muscles were examined histologically. Fig. 6A shows representative H&E stained micrographs from CDC- (IV; 2.5 x 10⁵) and vehicle-treated mice 6 weeks post-injury. TA muscles from CDC-treated mice weighed significantly more than TA muscles from vehicle-treated mice (Fig. 6B). Pooled data indicate CDC treatment had no effect on average myofiber cross-sectional area (Fig. 6C). However, pooled data indicate a greater number of myofibers comprising the TA by CDC treatment (Fig. 6D). Fig. 6E shows that myofiber size distribution of pooled data demonstrate CDC treatment boosted the frequency of small and very large myofibers.

[0131] Fig. 6F shows a representative immunohistochemical micrographs of innervated motor endplates from uninjured (left panels), vehicle (middle panels), and CDC- treated (right panels) mice 6 weeks post-injury. Arrows indicate staining for neuromuscular junctions. Pooled data from the immunohistochemical staining indicate CDC treatment restored myofiber innervation to uninjured levels (Fig. 6G).

[0132] The effect of CDCs on muscle volume was tested using magnetic resonance imaging (MRI). Fig. 7A shows a representative MRI scan of mouse hindlimb muscles from CDC- (IV; 2.5 x 10⁵) and vehicle-treated mice 6 weeks post- injury. Pooled data indicate CDC treatment significantly increased total muscle volume of the VML-injured anterior compartment (Fig. 7B). Topographical analysis demonstrated CDC treatment boosted muscle volume throughout the entire length of the anterior muscle compartment (Fig. 7C). [0133] In some embodiment, CDCs restore damaged muscle tissue in traumatic muscle injury, such as volumetric muscle loss. In some embodiments, CDCs promote recovery of muscle mass after traumatic muscle injury, such as volumetric muscle loss. In some embodiments, CDCs promote recovery of muscle fibers after traumatic muscle injury, such as volumetric muscle loss. In some embodiments, CDCs restore myofiber innervation to uninjured levels after traumatic muscle injury, such as volumetric muscle loss. In some embodiments, CDCs promote recovery of muscle volume after traumatic muscle injury, such as volumetric muscle loss.

[0134] In some embodiment, CDC-derived exosomes restore damaged muscle tissue in traumatic muscle injury, such as volumetric muscle loss. In some embodiments, CDC-derived exosomes promote recovery of muscle mass after traumatic muscle injury, such as volumetric muscle loss. In some embodiments, CDC-derived exosomes promote recovery of muscle fibers after traumatic muscle injury, such as volumetric muscle loss. In some embodiments, CDC-derived exosomes restore myofiber innervation to uninjured levels after traumatic muscle injury, such as volumetric muscle loss. In some embodiments, CDC-derived exosomes promote recovery of muscle volume after traumatic muscle injury, such as volumetric muscle loss.

Example 2.3

[0135] This non-limiting example shows the therapeutic efficacy of CDCs administered at various time periods after injury.

[0136] Fig. 8, left panel, shows in vivo tetanic force production before and after (Day zero [DO], and weeks 2, 4 and 6 [Wk2, Wk4, Wk6]) punch injury to the tibialis anterior muscle to study the effect of delaying CDC treatment by either 1, 2, or 3 days following VML injury. Values for four experimental groups are shown as means ± SEM. *= p<0.05, **= p<0.01 CDCs when comparing vehicle to CDCs; # =p<0.05 when comparing CDCs (1 Day vs 3 Days). N = 9-10 / group. The percentage improvement relative to vehicle control is shown in Fig. 8, right panel.

[0137] In some embodiments, CDCs administered within one to three days after traumatic muscle injury, such as volumetric muscle loss, provide a therapeutic effect to restore muscle function. In some embodiments, CDC-derived exosomes administered within one to three days after traumatic muscle injury, such as volumetric muscle loss, provide a therapeutic effect to restore muscle function.

Example 3: Therapeutic bioactivity of intravenously administered CDC-derived exosomes in mice with volumetric muscle loss

[0138] Volumetric muscle loss is induced in mice by surgically resecting about 20% of the tibialis anterior muscle. Loss of function of anterior crural muscles due to the volumetric muscle is tested by measuring development of muscle torque across a range of frequencies.

[0139] Animals are administered about 2 x 10⁸ immortalized CDC-derived exosomes intravenously immediately after the surgery. Two, four and six weeks after the surgery, animals are tested for recovery of muscle function.

Example 4: Therapeutic bioactivity of intramuscularly administered CDC-derived exosomes in mice with volumetric muscle loss

[0140] Volumetric muscle loss is induced in mice by surgically resecting about 20% of the tibialis anterior muscle. Loss of function of anterior crural muscles due to the volumetric muscle is tested by measuring development of muscle torque across a range of frequencies.

[0141] Animals are administered immortalized CDC-derived exosomes intramuscularly immediately after the surgery, one dose of about 1 x 10⁸ exosomes administered proximal to the body from the site of resection, and another dose of about 1 x 10⁸ exosomes administered distal to the body from the site of resection. Two, four and six weeks after the surgery, animals are tested for recovery of muscle function.

Example 5

[0142] This non-limiting example shows the therapeutic effect of CDC-derived exosomes in treating animals with volumetric muscle loss.

[0143] VML was induced as described in Example 1. Mice were treated with 4xl0⁹ primary human CDC-derived exosomes or vehicle by infusion into the femoral vein immediately following injury and allowed 6 weeks to recover. Fig. 9A shows a schematic of the Experimental design. There were no differences in biopsy mass between groups (Fig. 9B). Measurement of torque-frequency showed improved recovery in animals treated with CDC- derived exosomes at 2, 4 and 6 weeks after administration compared to vehicle control (Figs. 9C, 9D). Tetanic torque was significantly improved compared to vehicle control at 2, 4, and 6 weeks (Fig. 9E; for each time point, left bar: Vehicle, right bar: CDC_exos). This can also be seen in the recovery of torque deficit (Fig. 9F). C, D) Torque-frequency relationship. E) Tetanic torque during recovery. F) Torque deficit during recovery.

[0144] In some embodiments, administration of primary human CDC-derived exosomes promote recovery of muscle function after traumatic muscle injury, such as volumetric muscle loss. In some embodiments, intravenous administration of primary human CDC-derived exosomes promote recovery of muscle function after traumatic muscle injury, such as volumetric muscle loss.

Claims

WHAT IS CLAIMED IS:

1. A method of treating a traumatic muscle injury, comprising: identifying a subject having a traumatic injury to a muscle, wherein the muscle is a skeletal muscle, and wherein the traumatic injury comprises a volumetric muscle loss from about 10% to about 30% as compared to the muscle pre-injury; and administering a therapeutically effective amount of cardiosphere-derived cells

(CDCs) to the subject, wherein the therapeutically effective amount ranges from about 1 x 10⁴ to about

1 x 10⁷ CDCs, thereby treating the traumatic muscle injury.

2. The method of claim 1, comprising administering the CDCs to the subject via one or more intravenous injections.

3. The method of claim 1, comprising administering the CDCs to the subject via one or more intramuscular injections.

4. The method of claim 3, comprising administering a dose of CDCs at a first site adjacent a site of the traumatic injury.

5. The method of claim 4, further comprising administering a second dose of CDCs at a second site adjacent the site of the traumatic injury, wherein the first and second sites are different.

6. The method of claim 1, wherein the CDCs are administered topically.

7. The method of claim 6, wherein the CDCs are disposed in a biocompatible substrate configured to release the CDC-derived exosomes upon application of the substrate to a site of the traumatic injury, and wherein the method further comprises applying the substrate to the site of the traumatic injury.

8. The method of claim 7, wherein the biocompatible substrate comprises a biocompatible matrix.

9. The method of any one of the preceding claims, further comprising administering the therapeutic amount in a single dose of the CDCs.

10. The method of any one of claims 1 to 8, wherein the administering comprises administering two or more doses of the CDCs at a dosing interval from about 3 days to about 6 months.

11. The method of any one of the preceding claims, wherein the CDCs are allogeneic CDCs.

12. The method of any one of the preceding claims, wherein the CDCs are primary

CDCs.

13. A cell-free method of treating a traumatic muscle injury, comprising: identifying a subject having a traumatic injury to a muscle, wherein the muscle is a skeletal muscle, and wherein the traumatic injury comprises a volumetric muscle loss from about 10% to about 30% as compared to the muscle pre-injury; and administering a therapeutically effective amount of CDC-derived exosomes to the subject, wherein the therapeutically effective amount comprises about 1 x 10⁸ to about 1 x 10¹¹ exosomes, thereby treating the traumatic muscle injury.

14. The method of claim 13, comprising administering the CDC-derived exosomes to the subject via one or more intravenous injections and/or one or more intramuscular injections.

15. The method of claim 13, wherein the CDC-derived exosomes are disposed in a biocompatible substrate configured to release the CDC-derived exosomes upon application of the substrate to a site of the traumatic injury, and wherein the method further comprises applying the substrate to the site of the traumatic injury.

16. The method of claim 13, wherein the biocompatible substrate comprises a biocompatible matrix.

17. The method of claim 13, comprising: reconstituting preserved CDC-derived exosomes into a composition comprising the therapeutically effective amount of the CDC-derived exosomes; and administering the composition to the subject.

18. The method of claim 17, wherein the preserved CDC-derived exosomes are frozen or lyophilized.

19. The method of any one of claims 13 to 18, further comprising administering the therapeutic amount in a single dose of the CDC-derived exosomes.

20. The method of any one of claims 13 to 18, wherein the administering comprises administering two or more doses of the CDC-derived exosomes at a dosing interval from about 3 days to about 6 months.

21. The method of any one of the preceding claims or claim 62, wherein the traumatic injury reduces muscle strength by 50% or more.

22. The method of any one of the preceding claims or claim 62, wherein the muscle is comprised in a fascial compartment, and wherein the traumatic injury reduces muscle strength of the fascial compartment by 50% or more.

23. The method of any one of the preceding claims or claim 62, wherein an extent of recovery of muscle strength after the administering is about 1.5 times or greater than a reference level of recovery of muscle strength.

24. The method of any one of the preceding claims or claim 62, wherein a rate recovery of a muscle strength is about 2 times or more faster relative to a reference rate of recovery of the muscle strength.

25. The method of any one of claims 21-24, wherein the muscle strength is torque generated by the muscle.

26. The method of any one of claims 21-24, wherein the muscle strength is torque of a fascial compartment comprising the muscle.

27. The method of any one of the preceding claims or claim 62, wherein muscle strength recovers about 25% or more of a pre-injury muscle strength, within 2 weeks after the administering.

28. The method of any one of the preceding claims or claim 62, wherein the muscle is comprised in a fascial compartment, and wherein a muscle strength of the fascial compartment recovers about 25% or more of a pre-injury muscle strength of the fascial compartment, within 2 weeks after the administering.

29. The method of any one of the preceding claims or claim 62, wherein muscle strength increases by about 4 fold or more within 2 weeks after the administering.

30. The method of any one of the preceding claims or claim 62, wherein the muscle is comprised in a fascial compartment, and wherein a muscle strength of the fascial compartment increases by about 4 fold or more within 2 weeks after the administering.

31. The method of any one of claims 27-30, wherein the muscle strength is represented by application of torque by the muscle.

32. The method of any one of the preceding claims or claim 62, wherein a volume of the muscle increases by about 15% or more within 6 weeks after the administering.

33. The method of any one of the preceding claims or claim 62, wherein a volume of the muscle increases by about 15% or more within 2 weeks after the administering.

34. The method of any one of the preceding claims or claim 62, wherein the administering occurs within about 3 hours of the subject suffering the traumatic injury to the muscle.

35. The method of any one of the preceding claims or claim 62, wherein the administering occurs within about 3 hours to about 3 days of the subject suffering the traumatic injury to the muscle.

36. The method of any one of the preceding claims or claim 62, further comprising measuring a muscle function after the administering.

37. The method of any one of the preceding claims or claim 62, further comprising administering an analgesic to the subject.

38. A method of treating a traumatic muscle injury, comprising: identifying a subject having a traumatic injury to a muscle, wherein the muscle is a skeletal muscle, and wherein the traumatic injury comprises a volumetric muscle loss from about 10% to about 30%; and administering a therapeutically effective amount of cardiosphere-derived cells (CDCs) and/or CDC-derived exosomes to the subject, thereby treating the traumatic muscle injury.

39. The method of claim 38, comprising administering a therapeutically effective amount of CDC-derived exosomes to the subject, wherein the method is a cell-free method of treating a traumatic muscle injury.

40. The method of claim 39, wherein the therapeutically effective amount of CDC- derived exosomes comprises from about 1 x 10⁸ to about 1 x 10¹¹ exosomes.

41. The method of claim 39, wherein the CDC-derived exosomes are primary human CDC-derived exosomes.

42. The method of claim 39, wherein the CDC-derived exosomes are immortalized CDC-derived exosomes.

43. The method of claim 39, comprising: reconstituting preserved CDC-derived exosomes into a composition comprising the therapeutically effective amount of the CDC-derived exosomes; and administering the composition to the subject.

44. The method of claim 43, wherein the preserved CDC-derived exosomes are frozen or lyophilized.

45. A kit for treating a traumatic muscle injury, comprising: preserved CDC-derived exosomes and/or preserved CDCs; and a biocompatible substrate, wherein the preserved CDC-derived exosomes and/or preserved CDCs provide a therapeutically effective amount of CDC-derived exosomes and/or CDCs upon reconstitution with the biocompatible substrate.

46. The kit of claim 45, wherein the therapeutically effective amount of CDC- derived exosomes comprises from about 1 x 10⁸ to about 1 x 10¹¹ exosomes.

47. The kit of claim 45, wherein the therapeutically effective amount of CDCs comprises from about 1 x 10⁴ to about 1 x 10⁷ CDCs.

48. The kit of claim 45, wherein the kit comprises the preserved CDC-derived exosomes.

49. The kit of claim 48, wherein the preserved CDC-derived exosomes are frozen or lyophilized.

50. The kit of claim 45, wherein the exosomes are primary human CDC-derived exosomes.

51. The kit of claim 45, wherein the exosomes are immortalized CDC-derived exosomes.

52. The kit ofclaim 45, further comprising an analgesic.

53. The kit of claim 45, comprising a syringe configured to administer to a subject a composition made by combining: the preserved CDC-derived exosomes and/or preserved CDCs, with the biocompatible substrate.

54. The kit of claim 53, wherein the syringe is configured to intramuscularly administer the composition.

55. The kit of claim 53, wherein the syringe is configured to intravenously administer the composition.

56. The kit of claim 45, further comprising a wound dressing configured to: receive a composition made by combining: the preserved CDC-derived exosomes and/or preserved CDCs, with the biocompatible substrate; and position the composition at a site of a traumatic muscle injury.

57. The kit of any one of claims 45 to 56, wherein the biocompatible substrate comprises a biocompatible matrix.

58. Use of cardio sphere-derived cells (CDCs) and/or CDC-derived exosomes for the treatment of a wound, lesion or tissue damage.

59. Use of cardio sphere-derived cells (CDCs) and/or CDC-derived exosomes for the treatment of traumatic muscle injury.

60. Use of cardio sphere-derived cells (CDCs) and/or CDC-derived exosomes in the preparation of a medicament for the treatment of traumatic muscle injury.

61. The use of claim 59 or 60, wherein the treatment improves one or more of the muscle strength, mass, volume, and/or endurance of the injured muscle.

62. A method of treating a traumatic muscle injury, comprising administering to a muscle having a traumatic injury comprising a volumetric muscle loss a therapeutically effective amount of cardio sphere-derived cells (CDCs) and/or CDC-derived exosomes to the muscle.