Login| Sign Up| Help| Contact|

Patent Searching and Data


Title:
TREATMENT OF GENETIC MYOPATHIES USING BIOENGINEERED EXOSOMES
Document Type and Number:
WIPO Patent Application WO/2017/054086
Kind Code:
A1
Abstract:
A method of treating myopathy in a mammal is provided. The method includes administering to the mammal exosomes genetically modified to incorporate a muscle protein useful to treat the myopathy or nucleic acid encoding the protein.

Inventors:
TARNOPOLSKY, Mark (Room 2H261200 Main Street Wes, Hamilton Ontario L8N 3Z5, L8N 3Z5, CA)
SAFDAR, Adeel (Room 2H261200 Main Street Wes, Hamilton Ontario L8N 3Z5, L8N 3Z5, CA)
Application Number:
CA2016/051141
Publication Date:
April 06, 2017
Filing Date:
September 30, 2016
Export Citation:
Click for automatic bibliography generation   Help
Assignee:
EXERKINE CORPORATION (Room 2H261200 Main Street Wes, Hamilton Ontario L8N 3Z5, L8N 3Z5, CA)
International Classes:
C12N5/10; A61K9/00; A61K31/7088; A61K38/46; A61K48/00; A61P3/00; A61P21/00; C07K14/47; C07K14/705; C07K19/00; C12N15/12; C12N15/62; C12N15/85; C12N15/87
Domestic Patent References:
2013-04-04
2007-11-08
2009-08-27
2014-10-16
Attorney, Agent or Firm:
TANDAN, Susan (Gowling WLG LLP, One Main Street WestHamilton, Ontario L8P 4Z5, L8P 4Z5, CA)
Download PDF:
Claims:
CLAIMS 1. Exosomes genetically modified to incorporate a functional muscle protein and/or nucleic acid encoding a functional muscle protein. 2. The exosomes of claim 1 , essentially free from particles having a diameter less than 20 nm or greater than 140 nm. 3. The exosomes of claim 1, which exhibit a zeta potential having a magnitude of at least about 30 mV, and preferably 40 mV or greater.

4. The exosomes of claim 1, wherein the muscle protein is a native muscle protein or a modified muscle protein. 5. The exosomes of claim 1 , which are mammalian exosomes.

6. The exosomes of claim 1, wherein the muscle protein is encoded by a gene selected from the group consisting of Myotilin (MYOT), Lamin A/C (LMNA), Caveolin 3 (CAV3), DnaJ heat shock protein family (Hsp40) member B6 (DNAJB6), Spectrin repeat containing, nuclear envelope 1 (SYNE1), Spectrin repeat containing, nuclear envelope 2 (SYNE2), Desmin (DES), Crystallin alpha B (CRYAB), LIM domain binding 3 (LDB3), Filamin C (FLNC), BCL2 associated athanogene 3 (BAG3), Dystrophia myotonica protein kinase (DMPK), CCHC-type zinc finger, nucleic acid binding protein (CNBP), Poly(A) binding protein, nuclear 1 (PABPN1), Titin (TTN), Double homeobox 4 (DUX4), Structural maintenance of chromosomes flexible hinge domain containing 1 (SMCHD1), Calpain 3 (CAPN3), Dysferlin (DYSF), Sarcoglycan gamma (SGCO), Sarcoglycan alpha (SGCA), Sarcoglycan beta (SGCB), Sarcoglycan delta (SGCD), Titin-cap (TCAP), Tripartite motif containing 32 (TRIM32), Fukutin related protein (FKRP), TTN, Protein O-mannosyltransferase 1 (POMT1), Anoctamin 5, Fukutin (FKTN), Protein O- mannosyltransferase 2 (POMT2), Protein O-linked mannose N-acetylglucosaminyltransferase 1 (beta 1,2) (POMGnTl), Dystroglycan 1 (DAG1), Plectin (PLEC), DES, Trafficking protein particle complex 11 (TRAPPC 11), GDP-mannose pyrophosphorylase B (GMPPB), Tafazzin (TAZ), Dystrophin (DMD), Emerin (EMD), Four and a half LIM domains 1 Ly (FHL1), Lysosome-associated membrane protein 2 (LAMP-2), VMA21 vacuolar H+-ATPase homoiog (S. cerevisiae) (VMA21), Laminin subunit alpha 2 (LAMA2), Integrin subunit alpha 7 (ITGA7), Selenoprotein N, 1 (SEPN1), Collagen type VI alpha 1 (COL6A1), Collagen type VI alpha 2 (COL6A2), Collagen type VI alpha 3 (COL6A3), Docking protein 7 (DOK7), Protein O- mannosyltransferase 1 (POMT1), Protein O-mannosyltransferase 2 (POMT2), Protein O-linked mannose N-acetylglucosaminyltransferase 1 (beta 1,2-) (POMGNT1), FKTN, FKRP, Like- glycosyltransferase (LARGE), Isoprenoid synthase domain containing (ISPD), Protein O-linked mannose N-acetylglucosaminyltransferase 2 (beta 1,4-) (POMGNT2), Transmembrane protein 5 (TMEM5), GDP-mannose pyrophosphorylase B (GMPPB), Choline kinase beta (CHKB), Ryanodine receptor 1 (RYR1), Collagen type VI alpha 1 (COL6A1), Collagen type VI alpha 2 (COL6A2), Collagen type VI alpha 3 (COL6A3), Selenoprotein N, 1 (SEPNl), DES, Myosin, heavy chain 2, skeletal muscle, adult (MYH2), Valosin containing protein (VCP), Heterogeneous nuclear ribonucleoprotein A2/B1 (HNRNPA2B1), Heterogeneous nuclear ribonucleoprotein Al (HNRNPA1), Glucosamine (UDP-N-acetyl)-2-epimerase/N-acetylmannosamine kinase (GNE), Glucan (1,4-aIpha-), branching enzyme 1 (GBE1), Amylo-alpha-1, 6-glucosidase, 4-alpha- glucanotransferase (AGL), Enolase 3 (beta, muscle) (EN03), Glycogen synthase 1 (GYS1), Glycogenin 1 (GYG1), Hexokinase 1 (HK1), Lactate dehydrogenase A (LDHA), Phosphofructokinase, muscle (PFKM), Phosphoglucomutase 1 (PGM1), Phosphoglyceratemutase 2 (PGAM2), Phosphorylase, glycogen, muscle (PYGM), LAMP2, Phosphoglycerate kinase 1 (PGK1), Phosphorylase kinase, alpha 2 (liver) (PHKA2), Phosphorylase kinase, alpha 1 (muscle) (PHKA1), Carnitine palmitoyltransferase 1 A (CPT1 A), Carnitine palmitoyltransferase 2 (CPT2), Acyl-CoA dehydrogenase, C-4 to C-12 straight chain (ACADM), Acyl-CoA dehydrogenase, very long chain (ACADVL), Hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase/enoyl-CoA hydratase (Afunctional protein), alpha subunit (HADHA), Hydroxyacyl-CoA dehydrogenase/3- ketoacyl-CoA thiolase/enoyl-CoA hydratase (trifunctional protein), beta subunit (HADHB), Electron transfer flavoprotein alpha subunit (ETFA), Solute carrier family 25 (carnitine/acylcarnitine translocase), member 20 (SLC25A20), Solute carrier family 22 (organic cation/carnitine transporter), member 5 (SLC22AS) and Glucosidase, alpha; acid (GAA).

7. The exosomes of claim 1 , further modified to incorporate or express a target-specific fusion product comprising a skeletal muscle targeting sequence linked to an exosomal membrane marker. 8. The exosomes of claim 7, wherein the exosomal membrane marker is selected from the group consisting of CD9, CD37, CD53, CD63, CD81, CD82, CD151, an integral, ICAM-1, CDD31, an annexin, TSG101, ALIX, lysosome-associated membrane protein 1, lysosome- associated membrane protein 2, lysosomal integral membrane protein and a fragment of any exosomal membrane marker that comprises at least one intact transmembrane domain. 9. The exosomes of claim 7, wherein the skeletal muscle targeting sequence is selected from the group consisting of SERCA2, acetylcholine receptor epsilon, SCN4A, muscle specific creatine kinase (CK-MM) and TARGEHKEEELI. 10. The exosomes of claim 1, wherein the muscle protein and nucleic acid encoding the functional muscle protein are exogenous. 11. A composition comprising genetically modified exosomes as defined in claim 1 combined with a pharmaceutically acceptable carrier. 12. The composition of claim 11, comprising exosomal protein in an amount of about 100- 2000 13. A method of treating myopathy in a mammal comprising administering to the mammal exosomes genetically modified to incorporate a gene-editing system, selected from the group consisting of CRISPR, TALEN and zinc finger nuclease systems, that corrects mutation leading to the myopathy. 14. A method of treating myopathy in a mammal comprising administering to the mammal exosomes genetically modified to incorporate gene-silencing systems such as siRNA to reduce the expression of a mutated gene that results in the myopathy followed by administering to the mammal exosomes genetically modified to incorporate a functional protein useful to treat the myopathy and/or nucleic acid encoding the functional protein. 15. A method of increasing the level or amount of a muscle protein in mammalian muscle comprising administering to the mammal exosomes genetically modified to incorporate the functional muscle protein and/or nucleic acid encoding the functional protein. 16. A method of treating myopathy in a mammal comprising administering to the mammal exosomes genetically modified to incorporate a functional muscle protein useful to treat the myopathy and/or nucleic acid encoding the functional protein.

17. The method of claim 16, wherein the genetic myopathy is selected from the group consisting of Muscular dystrophy, limb-girdle, type IA, Muscular dystrophy, limb-girdle, type IB, Muscular dystrophy, limb-girdle, type IC, Muscular dystrophy, limb-girdle, type IE, Emery- Dreifuss muscular dystrophy 4, Emery-Dreifuss muscular dystrophy 5, Myopathy, myofibrillar, 1, Myopathy, myofibrillar, 2, Myopathy, myofibrillar, 4, Myopathy, myofibrillar, S, Myopathy, myofibrillar, 6, Myotonic dystrophy 1, Myotonic dystrophy 2, Oculopharyngeal muscular dystrophy, Tibial muscular dystrophy, tardive, Facioscapulohumeral muscular dystrophy 1, Facioscapulohumeral muscular dystrophy 2, digenic, Muscular dystrophy, limb-girdle, type 2A, Muscular dystrophy, limb-girdle, type 2B, Muscular dystrophy, limb-girdle, type 2C, Muscular dystrophy, limb-girdle, type 2D, Muscular dystrophy, limb-girdle, type 2E, Muscular dystrophy, limb-girdle, type 2F, Muscular dystrophy, limb-girdle, type 2G, Muscular dystrophy, limb-girdle, type 2H, Muscular dystrophy, limb-girdle, type 21 / Muscular dystrophy-dystroglycanopathy (limb-girdle), type C, S, Muscular dystrophy, limb-girdle, type 2J, Muscular dystrophy, limb- girdle, type 2K / Muscular dystrophy-dystroglycanopathy (limb-girdle), type C, 1, Muscular dystrophy, limb-girdle, type 2L, Muscular dystrophy, limb-girdle, type 2M / Muscular dystrophy- dystroglycanopathy (limb-girdle), type C, 4, Muscular dystrophy, limb-girdle, type 2N / Muscular dystrophy-dystroglycanopathy (limb-girdle), type C, 2, Muscular dystrophy, limb-girdle, type 20 / Muscular dystrophy-dystroglycanopathy (limb-girdle), type C, 3, Muscular dystrophy, limb- girdle, type 2P / Muscular dystrophy-dystroglycanopathy (limb-girdle), type C, 9, Muscular dystrophy, limb-girdle, type 2Q, Muscular dystrophy, limb-girdle, type 2R, Muscular dystrophy, limb-girdle, type 2S, Muscular dystrophy, limb-girdle, type 2T / Muscular dystrophy- dystroglycanopathy (limb-girdle), type C, 14, Barth syndrome, Muscular dystrophy, duchenne type, Muscular dystrophy, becker type, Emery-Dreifuss muscular dystrophy 1, X-linked, Emery- Dreifuss muscular dystrophy 6, X-linked, Danon disease, Myopathy, X-linked, with excessive autophagy, Muscular dystrophy, congenital merosin-deficient, 1A, Muscular dystrophy, congenital, due to ITGA7 deficiency, Myopathy, congenital, with fiber-type disproportion, Ullrich congenital muscular dystrophy 1, Ullrich congenital muscular dystrophy 1, Ullrich congenital muscular dystrophy 1, Myasthenic syndrome, congenital, 10, Muscular dystrophy- dystroglycanopathy (congenital with brain and eye anomalies), type A, 1 / Muscular dystrophy- dystroglycanopathy (congenital with mental retardation), type B, 1, Muscular dystrophy- dystroglycanopathy (congenital with brain and eye anomalies), type A, 2 / Muscular dystrophy- dystroglycanopathy (congenital with mental retardation), type B, 2, Muscular dystrophy- dystroglycanopathy (congenital with brain end eye anomalies), type A, 3 / Muscular dystrophy- dystroglycanopathy (congenital with mental retardation), type B, 3, Muscular dystrophy- dystroglycanopathy (congenital with brain and eye anomalies), type A, 4 / Muscular dystrophy- dystroglycanopathy (congenital without mental retardation), type B, 4, Muscular dystrophy- dystroglycanopathy (congenital with brain and eye anomalies), type A, 5 / Muscular dystrophy- dystroglycanopathy (congenital with or without mental retardation), type B, 5, Muscular dystrophy-dystroglycanopathy (congenital with brain and eye anomalies), type A, 6, Muscular dystrophy-dystroglycanopathy (congenital with brain and eye anomalies), type A, 7, Muscular dystrophy-dystroglycanopathy (congenital with brain and eye anomalies, type A, 8, Muscular dystrophy-dystroglycanopathy (congenital with brain and eye anomalies), type A, 10, Muscular dystrophy-dystroglycanopathy (congenital with brain and eye anomalies), type A, 14 / Muscular dystrophy-dystroglycanopathy (congenital with mental retardation), type B, 14, Muscular dystrophy, congenital, megaconial type, Central core disease, Bethlem myopathy 1, Bethlem myopathy 1, Bethlem myopathy 1, Muscular dystrophy, rigid spine, 1, Inclusion body myopathy 1 / Myopathy, myofibrillar, 1, Inclusion body myopathy 3 / Proximal myopathy and ophthalmoplegia, Inclusion body myopathy with early-onset Paget disease and frontotemporal dementia 1, Inclusion body myopathy with early-onset Paget disease with or without frontotemporal dementia 2, Inclusion body myopathy with early-onset Paget disease without frontotemporal dementia 3, Inclusion body myopathy 2 / Nonaka myopathy, Glycogen storage disease IV, Glycogen storage disease Ilia / Illb, Glycogen storage disease XIII, Glycogen storage disease 0, muscle, Glycogen storage disease XV, Glycogen storage disease XI, Glycogen storage disease VII, Glycogen storage disease X, Glycogen storage disease V / McArdle disease, Danon disease, Phosphoglycerate kinase 1 deficiency, Glycogen storage disease, type IXal / Glycogen storage disease, type IXa2, Muscle glycogenosis, CPT deficiency, hepatic, type IA, CPT deficiency, hepatic, type II, Acyl-CoA dehydrogenase, medium chain, deficiency of, VLCAD deficiency, Mitochondrial trifunctional protein deficiency, Mitochondrial trifunctional protein deficiency, Multiple acyl-CoA dehydrogenase deficiency (MADD) / Glutaric acidemia IIA, Carnitine-acylcarnitine translocase deficiency, Carnitine deficiency, systemic primary and Glycogen storage disease II.

18. The method of claim 17, wherein the muscle protein is encoded by a gene selected from the group consisting of Myotilin (MYOT), Lamin A/C (LMNA), Caveolin 3 (CAV3), DnaJ heat shock protein family (Hsp40) member B6 (DNAJB6), Spectrin repeat containing, nuclear envelope I (SYNE1), Spectrin repeat containing, nuclear envelope 2 (SYNE2), Desmin (DES), Crystallin alpha B (CRYAB), LIM domain binding 3 (LDB3), Filamin C (FLNC), BCL2 associated athanogene 3 (BAG3), Dystrophia myotonica protein kinase (DMPK), CCHC-type zinc finger, nucleic acid binding protein (CNBP), Poty(A) binding protein, nuclear 1 (PABPN1), Titin (TTN), Double homeobox 4 (DUX4), Structural maintenance of chromosomes flexible hinge domain containing 1 (SMCHD1), Calpain 3 (CAPN3), Dysferlin (DYSF), Sarcoglycan gamma (SGCO), Sarcoglycan alpha (SGCA), Sarcoglycan beta (SGCB), Sarcoglycan delta (SGCD), Thin-cap (TCAP), Tripartite motif containing 32 (TRIM32), Fukutin related protein (FKRP), TTN, Protein O-mannosyltransferase 1 (POMT1), Anoctamtn 5, Fukutin (FKTN), Protein O-mannosyltransferase 2 (POMT2), Protein O-linked mannose N-acetylglucosaininyltransferase 1 (beta 1,2) (POMGnTl), Dystroglycan 1 (DAG1), Plectin (PLEC), DES, Trafficking protein particle complex 11 (TRAPPC11), GDP-mannose pyrophosphorylase B (GMPPB), Tafazzin (TAZ), Dystrophin (DMD), Emerin (EMD), Four and a half LIM domains 1 Ly (FHL1), Lysosome-associated membrane protein 2 (LAMP-2), VMA21 vacuolar H+-ATPase homolog (S. cerevisiae) (VMA21), Laminin subunit alpha 2 (LAMA2), Integrin subunit alpha 7 (ITGA7), Selenoprotein N, 1 (SEPN1), Collagen type VI alpha 1 (COL6A1), Collagen type VI alpha 2 (COL6A2), Collagen type VI alpha 3 (COL6A3), Docking protein 7 (DOK7), Protein O-mannosyltransferase 1 (POMT1), Protein O-mannosyltransferase 2 (POMT2), Protein O-linked mannose N-acetylglucosaminyltransferase 1 (beta 1,2-) (POMGNT1), FKTN, FKRP, Like-glycosyltransferase (LARGE), Isoprenoid synthase domain containing (ISPD), Protein O-linked mannose N-acetylglucosaminyltransferase 2 (beta 1,4-) (POMGNT2), Transmembrane protein 5 (TMEM5), GDP-mannose pyrophosphorylase B (GMPPB), Choline kinase beta (CHKB), Ryanodine receptor 1 (RYRl), Collagen type VI alpha 1 (COL6A1), Collagen type VI alpha 2 (COL6A2), Collagen type VI alpha 3 (COL6A3), Selenoprotein N, 1 (SEPN1), DES, Myosin, heavy chain 2, skeletal muscle, adult (MYH2), Valosin containing protein (VCP), Heterogeneous nuclear ribonucleoprotem A2/B1 (HNRNPA2B1), Heterogeneous nuclear ribonucleoprotem Al (HNRNPAl), Glucosamine (UDP-N- acetyl)-2-epitnerase/N-acetylmannosamine kinase (GNE), Glucan (1,4-alpha-), branching enzyme 1 (GBE1), Amylo-alpha-1, 6-glucosidase, 4-alpha-glucanotransferase (AGL), Enolase 3 (beta, muscle) (EN03), Glycogen synthase 1 (GYS1), Glycogenin 1 (GYG1), Hexokinase 1 (HK1), Lactate dehydrogenase A (LDHA), Phosphofhictokinase, muscle (PFKM), Phosphogtucomutase 1 (PGM1), Phosphoglycerate mutase 2 (PGAM2), Phosphorylase, glycogen, muscle (PYGM), LAMP2, Phosphoglycerate kinase 1 (PGK1), Phosphorylase kinase, alpha 2 (liver) (PHKA2), Phosphorylase kinase, alpha 1 (muscle) (PHKA1), Carnitine palmitoyltransferase 1A (CPT1A), Carnitine palmitoyltransferase 2 (CPT2), Acyl-CoA dehydrogenase, C-4 to C-12 straight chain (ACADM), Acyl-CoA dehydrogenase, very long chain (ACADVL), Hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase/enoyl-CoA hydratase (trifunctional protein), alpha subunit (HADHA), Hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase/enoyl-CoA hydratase (trifunctional protein), beta subunit (HADHB), Electron transfer flavoprotein alpha subunit (ETFA), Solute carrier family 25 (carnitine/acylcarnitine translocase), member 20 (SLC25A20), Solute carrier family 22 (organic cation/carnitine transporter), member S (SLC22A5) and Olucosidase, alpha; acid (GAA). 19. The method of claim 17, wherein the genetic myopathy is creatine transporter deficiency 1 and the protein is acid creatine transporter protein. 20. The method of claim 17, wherein the myopathy is Duchenne Muscular Dystrophy and the protein is dystrophin. 21. The method of claim 17, wherein the myopathy is a glycogen storage disease. 22. The method of claim 17, wherein a dosage of exosomes sufficient to deliver about 0.1 mg/kg to about 100 mg/kg of the muscle protein, or an exosome dosage that delivers an amount of nucleic acid to yield about 0.1 mg/kg to about 100 mg/kg of the muscle protein, is

administered to the mammal. 23. A method of increasing the activity of a target muscle protein in a mammal, comprising administering to the mammal exosomes which are genetically modified to incorporate the functional muscle protein and/or nucleic acid encoding the functional protein. 24. The method of claim 23, wherein the exosomes are essentially free from particles having a diameter less than 20 nm or greater than 140 nm. 25. The method of claim 23, wherein the exosomes exhibit a zeta potential having a magnitude of at least about 30 mV, and preferably 40 mV or greater. 26. The method of claim 23, wherein the exosomes are isolated from a biological sample using a method comprising the following steps: i) exposing the biological sample to a first centrifugation to remove cellular debris greater than about 7-10 microns in size from the sample and obtaining the supernatant following centrifugation; ii) subjecting the supernatant from step i) to centrifugation to remove microvesicles therefrom; iii) microfiltering the supernatant from step ii) and collecting the microfiltered supernatant; iv) subjecting the microfiltered supernatant from step iii) to at least one round of ultracentrifugation to obtain an exosome pellet; and v) re-suspending the exosome pellet from step iv) in a physiological solution and conducting a second ultracentrifugation in a density gradient and removing the exosome pellet fraction therefrom.

Description:
TREATMENT OF GENETIC MYOPATHIES USING BIOENGINEERED EXOSOMES Field of the Invention

[0001] The present invention generally relates to the treatment of genetic myopathies (muscular dystrophy, congenital myopathy, and metabolic myopathies), and more particularly, relates to a method of treating genetic myopathies using bioengineered exosomes.

Background of the Invention

[0002] Myopathy refers to disease in muscle tissue, and generally results in dysfunctional muscle fibers. The myopathies represent a broad category of acquired and genetic entities. The genetic myopathies are broadly categorized as muscular dystrophy, congenital myopathy and metabolic myopathies. Muscular dystrophy is a broad term relating to muscle diseases that occur due to a gene mutation usually in a structural gene (e.g. dystrophin, sarcoglycan, dystroglycan) or other muscle protein (e.g. calpain 3). The muscle eventually shows evidence of centralized nuclei, fibrosis, necrosis and sarcolemmal damage as reflected by an elevation of creatine kinase (CK) in the serum. The congenital myopathies are genetic disorders that often present in the first few years of life, are often less progressive than the muscular dystrophies, may have a characteristic pattern on histology (e.g. central cores, central nuclear myopathy, inclusion bodies, abnormal protein accumulation), and may have a normal serum CK level. The metabolic myopathies include glycogen storage diseases (e.g. McArdle disease, Tarui disease), fatty acid oxidation defects (VLCAD deficiency, trifunctional protein deficiency, CPT2 deficiency) and the mitochondrial cytopatbies.

[0003] The defining hallmark of muscular dystrophy and the congenital myopathies is skeletal muscle weakness that can progress and require gait assistive devices (canes, walkers, wheelchairs), and may affect respiratory function leading to progressive respiratory failure and the need for assistive ventilation. In contrast, the metabolic myopathies often show no weakness or hyperCKemia between episodes (except McArdle disease), yet show episodes of muscle breakdown (rhabdomyolysis) during exercise and other metabolic stressors. During bouts of rhabdomyolysis, CK is elevated and there is often muscle pain, cramps and weakness. [0004] Although the identification of the first dystrophin mutation associated with Duchenne muscular dystrophy was discovered in 1987 (nearly 30 years ago) the treatment of muscular dystrophies, congenital myopathies and the metabolic myopathies remain largely supportive. General supportive therapy for muscular dystrophies and the congenital myopathies include physiotherapy (bracing when needed), stretching (when needed for contractures), optimal nutrition, creatine monohydrate supplementation, therapeutic exercise and use of braces and mobility aids as needed. The mainstay of therapy for Duchenne muscular dystrophy has been the use of corticosteroids, although several case reports and case series have suggested mild improvements in other forms of muscular dystrophy. A variety of genetic manipulations including exon skipping, myoblast transfer, adeno-associated virus gene delivery, and stop codon read through have been tried predominately in Duchenne muscular dystrophy with no evidence of clinical benefits at this point and none of the genetic therapies are currently available clinically in North American or Europe. The treatment of the metabolic myopathies is primarily an avoidance of triggering episodes (high intensity activity for the glycogen storage disorders and fasting, concurring illness and prolonged endurance exercise for the fatty acid oxidation defects). Pre- activity carbohydrate supplementation has been shown to be a benefit for McArdle disease by improving exercise capacity. High protein diets have generally been recommended for the glycogen storage disorders but have not been proven to be effective. Variable symptomatic relief have been found using high carbohydrate intakes, MCT (medium chain triglyceride) supplementation, and triheptanoin for the fatty acid oxidation defects. Regular exercise is also helpful in all of the metabolic disorders and it is essential to avoid excessive and unaccustomed activity to avoid rhabdomyolysis.

[0005] Since all of the aforementioned therapies are supportive and may have only minimal functional impact on the life of the individual, there is a need to develop improved methods for treating myopathies.

Summary of the Invention

[0006] It has now been found that bioengineered exosomes are useful as a vehicle to deliver a protein and/or nucleic acid to muscle tissue to effectively treat myopathies such as muscular dystrophy, congenital myopathy and metabolic myopathies. [0007] Thus, in one aspect of the invention, a method of treating myopathy in a mammal is provided comprising administering to the mammal exosomes genetically modified to incorporate a functional muscle protein useful to treat the myopathy and/or nucleic acid encoding the functional protein.

[0008] In another aspect of the invention, a method of increasing the level or amount of a functional muscle protein in mammalian muscle is provided, comprising administering to the mammal exosomes genetically modified to incorporate the functional muscle protein or nucleic acid encoding the muscle protein.

[0009] In another aspect, exosomes genetically modified to incorporate the functional muscle protein and/or nucleic acid encoding the muscle protein are provided.

[0010] In another aspect of the invention, a method of treating a myopathy in a mammal resulting from expression of a mutated gene is provided comprising administering to the mammal exosomes genetically modified to incorporate gene-silencing systems (e.g., siRNA) to reduce the expression of the mutated gene followed by administering to the mammal exosomes genetically modified to incorporate a functional muscle protein useful to treat the myopathy and/or nucleic acid encoding the muscle protein.

[0011] In another aspect of the invention, a method of treating myopathy in a mammal is provided comprising administering to the mammal exosomes genetically modified to incorporate gene-editing (e.g., CRISPR-Cas9, TALEN, zinc finger nucleases) systems to correct the inherent primary mutation leading to the myopathy.

[0012] In another aspect, a method of increasing the activity of a muscle protein in a mammal is provided, comprising administering to the mammal exosomes which are genetically modified to incorporate a functional muscle protein and/or nucleic acid encoding the muscle protein.

[0013] Additional aspects of the invention include aspects and variations set forth in the following lettered paragraphs: [0014] A1. An exosome produced by a process that comprises: (a) isolating exosomes from a biological sample from an organism or from a conditioned medium from a cultured cell; and (b) introducing a modification into the exosome selected from the group consisting of: (i) at least one functional muscle protein or precursor thereof; (ii) at least one nucleic acid comprising a nucleotide sequence that encodes the functional muscle protein or precursor thereof; (iii) at least one fusion product comprising a skeletal muscle targeting sequence linked to an exosomal membrane marker; (iv) at least one nucleic acid comprising a nucleotide sequence that encodes the fusion product; and (v) two or more of (i), (ii,) (iii), and (iv).

[0015] A2. The exosome according to paragraph Al , wherein the isolating includes at least one density gradient centrifugation step ideally using Percoll or other colloidal silica product.

[0016] A2.1 The exosome according to paragraph Al or A2, wherein the isolating removes vesicles that are greater than 120 or 140 nm in diameter.

[0017] A3. The exosome according to paragraph Al or A2 or A2.1, wherein the biological sample is from a mammal, or the cell is from a mammal or a mammalian cell line.

[0018] A4. The exosome according to any one of paragraphs Al to A3, wherein the isolating removes vesicles and cellular debris less than 20 nm in diameter.

[0019] AS. An exosome that comprises a modification selected from the group consisting of: (i) at least one functional muscle protein or precursor thereof; (ii) at least one nucleic acid comprising a nucleotide sequence that encodes the functional muscle protein or precursor thereof; (iii) at least one fusion product comprising a skeletal muscle targeting sequence linked to an exosomal membrane marker; (iv) at least one nucleic acid comprising a nucleotide sequence that encodes the fusion product; and (v) two or more of (i), (ii,) (iii), and (iv).

[0020] Bl . The exosome according to any of paragraphs Al - AS, having a diameter of 20-140 nm, e.g. 20-120 nm.

[0021] B2. The exosome according to any of paragraphs Al - AS, that comprises a functional muscle protein or precursor thereof, wherein the protein is present in a lumen of the exosome.

[0022] B3. The exosome according to any of paragraphs Al - AS, that comprises a nucleic acid comprising a nucleotide sequence encoding a functional muscle protein or precursor thereof, wherein the nucleic acid is present in a lumen of the exosome.

[0023] B3.1. The exosome according to paragraph B3, wherein the nucleic acid comprises mRNA or modified mRNA (modRNA, e.g. S methyl cytosine, or N6 methyladenine) encoding for a protein set forth in Table 1 and/or Table 2 and/or Table 3.

[0024] B4. The exosome according to paragraph B2 or B3 or B3.1, wherein the protein comprises one or more of the proteins set forth in Table 1 and/or Table 2 and/or Table 3.

[0025] B5. The exosome according to paragraph B4, wherein the protein is an enzyme.

[0026] B6. The exosome according to any one of paragraphs B2 - B3.1, wherein the protein is encoded by a gene selected from Myotilin (MYOT), Lamin A/C (LMNA), Caveolin 3 (CAV3), DnaJ heat shock protein family (Hsp40) member B6 (DNAJB6), Spectrin repeat containing, nuclear envelope 1 (SYNE1 ), Spectrin repeat containing, nuclear envelope 2 (S YNE2), Desmin (DES), Crystallin alpha B (CRYAB), LIM domain binding 3 (LDB3), Filamin C (FLNC), BCL2 associated athanogene 3 (BAG3), Dystrophia myotonica protein kinase (DMPK), CCHC- type zinc finger, nucleic acid binding protein (CNBP), Poly(A) binding protein, nuclear 1 (PABPN1), Titin (TTN), Double homeobox 4 (DUX4), Structural maintenance of chromosomes flexible hinge domain containing 1 (SMCHDl), Calpain 3 (CAPN3), Dysferlin (DYSF), Sarcoglycan gamma (SGCG), Sarcoglycan alpha (SGCA), Sarcoglycan beta (SGCB), Sarcoglycan delta (SGCD), Titin-cap (TCAP), Tripartite motif containing 32 (TR1M32), Fukutin related protein (FKRP), TTN, Protein O-mannosyltransferase 1 (POMT1), Anoctamin 5, Fukutin (FKTN), Protein O-mannosyltransferase 2 (POMT2), Protein O-linked mannose N- acetylglucosaminyltransferase 1 (beta 1,2) (POMGnTl), Dystroglycan 1 (DAG1), Plectin (PLEC), DBS, Trafficking protein particle complex 11 (TRAPPCl 1), GDP-mannose pyrophosphorylase B (GMPPB), Tafazzin (TAZ), Dystrophin (DMD), Emerin (EMD), Four and a half LIM domains 1 Ly (FHL1), Lysosome-associated membrane protein 2 (LAMP-2), VMA21 vacuolar H+-ATPase homolog (S. cerevisiae) (VMA21), Laminin subunit alpha 2 (LAMA2), Integrin subunit alpha 7 (1TGA7), Selenoprotein N, 1 (SEPN1), Collagen type VI alpha 1 (COL6AI), Collagen type VI alpha 2 (COL6A2), Collagen type VI alpha 3 (COL6A3), Docking protein 7 (DOK7), Protein O- mannosyltransferase 1 (POMT1), Protein O-mannosyltransferase 2 (POMT2), Protein O-linked mannose N-acetylglucosaminyltransferase 1 (beta 1,2-) (POMGNT1), FKTN, FKRP, Like- glycosyltransferase (LARGE), Isoprenoid synthase domain containing (ISPD), Protein O-linked mannose N-acetylglucosaminyltransferase 2 (beta 1,4-) (POMGNT2), Transmembrane protein S (TMEM5), GDP-mannose pyrophosphorylase B (GMPPB), Choline kinase beta (CHKB), Ryanodine receptor 1 (RYR1), Collagen type VI alpha 1 (COL6A1), Collagen type VI alpha 2 (COL6A2), Collagen type VI alpha 3 (COL6A3), Selenoprotein N, 1 (SEPN1), DES, Myosin, heavy chain 2, skeletal muscle, adult (MYH2), Valosin containing protein (VCP), Heterogeneous nuclear ribonucleoprotein A2/B1 (HNRNPA2B1), Heterogeneous nuclear ribonucleoprotein Al (HNRNPAl), Glucosamine (UDP-N-acetyl)-2-epimerase/N-acetylmannosamine kinase (GNE), Glucan (1,4-alpha-), branching enzyme 1 (GBE1), Amylo-alpha-1, 6-glucosidase, 4-alpha- glucanotransferase (AGL), Enolase 3 (beta, muscle) (EN03), Glycogen synthase 1 (GYS1), Glycogenin 1 (GYG1), Hexokinase 1 (HK1), Lactate dehydrogenase A (LDHA), Phosphofructokinase, muscle (PFKM), Phosphoglucomutase 1 (PGM1), Phosphoglyceratemutase 2 (PGAM2), Phosphorylase, glycogen, muscle (PYGM), LAMP2, Phosphoglycerate kinase 1 (PGK1), Phosphorylase kinase, alpha 2 (liver) (PHKA2), Phosphorylase kinase, alpha 1 (muscle) (PHKA1), Carnitine palmitoyltransferase 1A (CPT1A), Carnitine palmitoyltransferase 2 (CPT2), Acyl-CoA dehydrogenase, C-4 to C-12 straight chain (ACADM), Acyl-CoA dehydrogenase, very long chain (ACADVL), Hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase/enoyl-CoA hydratase (trifunctional protein), alpha subunit (HADHA), Hydroxyacyl-CoA dehydrogenase/3- ketoacyl-CoA thiolase/enoyl-CoA hydratase (triftmctional protein), beta subunit (HADHB), Electron transfer flavoprotein alpha subunit (ETFA), Solute carrier family 25 (carnitine/acylcarnitine translocase), member 20 (SLC25A20), Solute carrier family 22 (organic cation/carnitine transporter), member 5 (SLC22A5) and Glucosidase, alpha; acid (OAA).

[0027] B7. The exosome according to any one of paragraphs B2 - B6, further comprising at least one fusion product comprising a skeletal muscle targeting sequence linked to an exosomal membrane marker.

[0028] B8. The exosome according to any one of paragraphs Al - AS or Bl, that comprises at least one fusion product comprising a skeletal muscle targeting sequence linked to an exosomal membrane marker.

[0029] B9. The exosome according to paragraph B7 or B8, wherein the exosomal membrane marker is selected from the group consisting of CD9, CD37, CDS3, CD63, CD81, CD82, CD151, an integrin, ICAM-1, CDD31, an annexin, TSG101, ALIX, lysosome-associated membrane protein 1, lysosome-associated membrane protein 2, lysosomal integral membrane protein and a fragment of any exosomal membrane marker that comprises at least one intact transmembrane domain.

[0030] B10. The exosome according to any one of paragraphs B7 - B9, wherein the skeletal muscle targeting sequence is selected from the group consisting of SERCA2, acetylcholine receptor epsilon, SCN4A, muscle specific creatine kinase (CK-MM) and fragments thereof, including the peptide sequence, TARGEHKEEELI (SEQ ID NO: 1).

[0031 ] B 11. The exosome according to any one of paragraphs B7 ~ B 10, wherein the fusion product is a fusion protein.

[0032] B12. The exosome according to paragraph Bl l, further wherein the fusion protein includes a peptide linker between the skeletal muscle targeting sequence and the exosomal membrane marker. [0033] B13. The exosome according to any one of paragraphs B7-B12, wherein the exosomal membrane marker of the fusion product includes a transmembrane domain and localizes in a membrane of the exosome.

[0034] CI. A composition comprising exosomes according to any one of paragraphs Al - A5, and a pharmaceutically acceptable carrier.

[003S] C2. The composition according to paragraph CI, wherein the composition is substantially free of vesicles having a diameter less than 20 nm.

[0036] C3. The composition according to paragraph CI or C2, wherein the composition is substantially free of vesicles having a diameter greater than 140 nm or greater than 120nm.

[0037] C4. The composition according to any one of claims CI - C3, which exhibits a zeta potential having a magnitude of at least 30 mV, or at least 40 mV, or at least SO mV, or at least 60 mV, or at least 70 mV, or at least 80 mV, e.g. 30-80 mV, or -80 to -30 mV.

[0038] CS. The composition according to claim C4, which exhibits a zeta potential having a magnitude of up to 200 mV, or up to 175 mV, or up to 150 mV, or up to 140 mV, or up to 130 mV, or up to 120 mV, or up to 110 mV, or up to 100 mV.

[0039] Dl . A method of increasing the amount of a muscle protein in muscles in a mammal, comprising administering to the mammal an exosome according to any one of paragraphs A1 - B13, or a composition according to any one of paragraphs CI- C5.

[0040] D2. Use of an exosome according to any one of paragraphs Al - B13, or a composition according to any one of paragraphs CI- C5, for increasing the amount of a muscle protein in muscle in a mammal.

[0041] D3. A method of treating a genetic myopathy in a mammal comprising administering to the mammal an exosome according to any one of paragraphs Al - B13, or a composition according to any one of paragraphs C1- C5. [0042] D4. Use of an exosome according to any one of paragraphs Al - B13, or a composition according to any one of paragraphs CI- C5, for treating a genetic myopathy in a mammal.

[0043] D5. The method or use according to any one of paragraphs Dl - D4, wherein the mammal is human.

[0044] D6. The method or use according to paragraph DS, wherein the human has a genetic myopathy selected from Muscular dystrophy, limb-girdle, type IA, Muscular dystrophy, limb-girdle, type IB, Muscular dystrophy, limb-girdle, type IC, Muscular dystrophy, limb-girdle, type IE, Emery-Dreifiiss muscular dystrophy 4, Emery-Dreifuss muscular dystrophy S, Myopathy, myofibrillar, 1, Myopathy, myofibrillar, 2, Myopathy, myofibrillar, 4, Myopathy, myofibrillar, 5, Myopathy, myofibrillar, 6, Myotonic dystrophy 1, Myotonic dystrophy 2, Oculopharyngeal muscular dystrophy, Tibial muscular dystrophy, tardive, Facioscapulohumeral muscular dystrophy 1, Facioscapulohumeral muscular dystrophy 2, digenic, Muscular dystrophy, limb-girdle, type 2A, Muscular dystrophy, limb-girdle, type 2B, Muscular dystrophy, limb-girdle, type 2C, Muscular dystrophy, limb-girdle, type 2D, Muscular dystrophy, limb-girdle, type 2E, Muscular dystrophy, limb-girdle, type 2F, Muscular dystrophy, limb-girdle, type 20, Muscular dystrophy, limb-girdle, type 2H, Muscular dystrophy, limb-girdle, type 21 / Muscular dystrophy-dystroglycanopathy (limb-girdle), type C, 5, Muscular dystrophy, limb-girdle, type 2J, Muscular dystrophy, limb- girdle, type 2K / Muscular dystrophy-dystroglycanopathy (limb-girdle), type C, 1, Muscular dystrophy, limb-girdle, type 2L, Muscular dystrophy, limb-girdle, type 2M / Muscular dystrophy- dystroglycanopathy (limb-girdle), type C, 4, Muscular dystrophy, limb-girdle, type 2N / Muscular dystrophy-dystroglycanopathy (limb-girdle), type C, 2, Muscular dystrophy, limb-girdle, type 20 / Muscular dystrophy-dystroglycanopathy (limb-girdle), type C, 3, Muscular dystrophy, limb- girdle, type 2P / Muscular dystrophy-dystroglycanopathy (limb-girdle), type C, 9, Muscular dystrophy, limb-girdle, type 2Q, Muscular dystrophy, limb-girdle, type 2R, Muscular dystrophy, limb-girdle, type 2S, Muscular dystrophy, limb-girdle, type 2T / Muscular dystrophy- dystroglycanopathy (limb-girdle), type C, 14, Barth syndrome, Muscular dystrophy, duchenne type, Muscular dystrophy, becker type, Emery-Dreifuss muscular dystrophy 1, X-linked, Emery- Dreifuss muscular dystrophy 6, X-linked, Danon disease, Myopathy, X-linked, with excessive autophagy, Muscular dystrophy, congenital merosin-deficient, 1A, Muscular dystrophy, congenital, due to ITOA7 deficiency, Myopathy, congenital, with fiber-type disproportion, Ullrich congenital muscular dystrophy 1, Ullrich congenital muscular dystrophy 1, Ullrich congenital muscular dystrophy 1, Myasthenic syndrome, congenital, 10, Muscular dystrophy- dystroglycanopathy (congenital with brain and eye anomalies), type A, 1 / Muscular dystrophy- dystroglycanopathy (congenital with mental retardation), type B, 1, Muscular dystrophy- dystroglycanopathy (congenital with brain and eye anomalies), type A, 2 / Muscular dystrophy- dystroglycanopathy (congenital with mental retardation), type B, 2, Muscular dystrophy- dystroglycanopathy (congenital with brain and eye anomalies), type A, 3 / Muscular dystrophy- dystroglycanopathy (congenital with mental retardation), type B, 3, Muscular dystrophy- dystroglycanopathy (congenital with brain and eye anomalies), type A, 4 / Muscular dystrophy- dystroglycanopathy (congenital without mental retardation), type B, 4, Muscular dystrophy- dystroglycanopathy (congenital with brain and eye anomalies), type A, 5 / Muscular dystrophy- dystroglycanopathy (congenital with or without mental retardation), type B, 5, Muscular dystrophy-dystroglycanopathy (congenital with brain and eye anomalies), type A, 6, Muscular dystrophy-dystroglycanopathy (congenital with brain and eye anomalies), type A, 7, Muscular dystrophy-dystroglycanopathy (congenital with brain and eye anomalies, type A, 8, Muscular dystrophy-dystroglycanopathy (congenital with brain and eye anomalies), type A, 10, Muscular dystrophy-dystroglycanopathy (congenital with brain and eye anomalies), type A, 14 / Muscular dystrophy-dystroglycanopathy (congenital with mental retardation), type B, 14, Muscular dystrophy, congenital, megaconial type, Central core disease, Bethlem myopathy 1, Bethlem myopathy 1, Bethlem myopathy 1, Muscular dystrophy, rigid spine, 1, Inclusion body myopathy 1 / Myopathy, myofibrillar, 1, Inclusion body myopathy 3 / Proximal myopathy and ophthalmoplegia, Inclusion body myopathy with early-onset Paget disease and frontotemporal dementia 1, Inclusion body myopathy with early-onset Paget disease with or without frontotemporal dementia 2, Inclusion body myopathy with early-onset Paget disease without frontotemporal dementia 3, Inclusion body myopathy 2 / Nonaka myopathy, Glycogen storage disease IV, Glycogen storage disease Ilia / Illb, Glycogen storage disease XIII, Glycogen storage disease 0, muscle, Glycogen storage disease XV, Glycogen storage disease XI, Glycogen storage disease VII, Glycogen storage disease X, Glycogen storage disease V / McArdle disease, Danon disease, Phosphoglycerate kinase 1 deficiency, Glycogen storage disease, type IXal / Glycogen storage disease, type IXa2, Muscle glycogenosis, CPT deficiency, hepatic, type IA, CPT deficiency, hepatic, type II, Acyl-CoA dehydrogenase, medium chain, deficiency of, VLCAD deficiency, Mitochondrial trifunctional protein deficiency, Mitochondrial Afunctional protein deficiency, Multiple acyl-CoA dehydrogenase deficiency (MADD) / Glutaric acidemia IIA, Carnitine-acylcarnitine translocase deficiency, Carnitine deficiency, systemic primary and Glycogen storage disease II.

[0045] D7. The method or use according to any one of paragraphs Dl - D4, wherein the mammal is human and has a disease set forth in Table 1 and/or Table 2 and/or Table 3, and the exosome contains the corresponding protein in Table 1 and/or Table 2 and/or Table 3, or a nucleic acid encoding said protein.

[0046] These and other aspects of the invention will be described by reference to the following figures.

Brief Description of the Figures

[0047] Figure 1 graphically illustrates dystrophin protein content in primary dermal fibroblasts from control and DMD patients after treatment with exosomes loaded with dystrophin (Dmd) mRNA as determined by quantification of confocal microscopy images.

[0048] Figure 2 graphically illustrates dystrophin protein expression in primary dermal fibroblasts from control and DMD patients after treatment with exosomes loaded with dystrophin (Dmd) mRNA as determined by immunoblot analysis.

[0049] Figure 3 illustrates dystrophin content in tibialis anterior (TA) muscle (A), soleus (SOL) muscle (B), and heart (C) in dystrophin-deficient mdx mice after treatment with vehicle, empty exosomes and dystroph in-mRN A loaded exosomes.

[0050] Figure 4 graphically illustrates body (A), fast-twitch muscle (tibialis anterior (TA) (B), extensor digitorum longus (EDL) (C)) and slow-twitch (soleus (SOL)) muscle (D) weights in mdx mice after treatment with vehicle, empty exosomes, dystrophin mRNA loaded exosomes and prednisolone.

[0051] Figure 5 graphically illustrates maximum grip strength relative to TA muscle weight in mdx mice after treatment with vehicle, empty exosomes, dystrophin mRNA loaded exosomes and prednisolone. [0052] Figure 6 graphically illustrates serum creatine kinase (CK) levels in mdx mice after treatment with vehicle, empty exosomes, dystrophin mRNA loaded exosomes and prednisolone.

[0053] Figure 7 graphically illustrates acid alpha glucosidase (OAA) activity in primary myotubes isolated from GAA wild-type (GA A + ^ and knock-out (GA A" 7 ') mice treated with naked GAA protein, empty exosomes (exosome control) or GAA protein-loaded exosomes.

[0054] Figure 8 graphically illustrates GAPDH gene expression in muscle cells treated with GAPDH siRNA or scRNA (scrambled RN A; control for siRNA) by delivery of naked scRNA or siRNA, scRNA or siRNA transfected with lipofectamine 2000 (LF2000), scRNA- or siRNA- loaded non-targeting exosomes and scRNA- or siRNA-loaded skeletal muscle-targeting exosomes.

[0055] Figure 9 graphically illustrates gene expression of PGC-1 alpha and of downstream targets of PGC-1 alpha (A) and cytochrome c oxidase function (B) in primary mouse muscle cells treated with PGC-1 alpha siRNA or scRNA delivered by naked scRNA or siRNA, scRNA or siRNA transfected with lipofectamine 2000 (LF2000), scRNA- or siRNA-loaded non-targeting exosomes and scRNA- or siRNA-loaded skeletal muscle targeting exosomes.

Detailed Description of the Invention

[0056] A method of treating myopathy in a mammal is provided comprising administering to the mammal exosomes genetically modified to incorporate a muscle protein useful to treat the myopathy or nucleic acid encoding the protein. Thus, exosomes which have been genetically engineered to incorporate a muscle protein, and/or nucleic acid encoding a muscle protein are also provided.

[0057] The term "exosome" refers to cell-derived vesicles having a diameter of between about 20 and 140 nm, for example, a diameter of about 20-120 nm, or a diameter of about 40-100 nm, including exosomes of a diameter of about 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm and/or 120 nm. Exosomes may be isolated from any suitable biological sample from a mammal, including but not limited to, whole blood, serum, plasma, urine, saliva, breast milk, cerebrospinal fluid, amniotic fluid, ascitic fluid, bone marrow and cultured mammalian cells (e.g. immature dendritic cells (wild-type or immortalized), induced and non-induced pluripotent stem cells, fibroblasts, platelets, immune cells, reticulocytes, tumour cells, mesenchymal stem cells, satellite cells, hematopoietic stem cells, pancreatic stem cells, white and beige pre-adipocytes and the like). As one of skill in the art will appreciate, cultured cell samples will be in the cell- appropriate culture media (using exosome-free serum). Exosomes may be identified by specific surface markers not present in other vesicles, including surface markers such as tetraspanins, e.g. CD9, CD37, CD44, CD53, CD63, CD81, CD82 and CD151; targeting or adhesion markers such as integrins, ICAM-1, EpCAM and CD31; membrane fusion markers such as annexins, TSG101, ALIX; and other exosome transmembrane proteins such as Rab5b, HLA-G, HSP70, LAMP2 (lysosome-associated membrane protein) and LIMP (lysosomal integral membrane protein). Exosomes may also be obtained from a non-mammalian biological sample, including cultured non-mammalian cells. As the molecular machinery involved in exosome biogenesis is believed to be evolutionarily conserved, exosomes from non-mammalian sources include surface markers which are isoforms of mammalian surface markers, such as isoforms of CD9 and CD63, which distinguish them from other cellular vesicles. As used herein, the term "mammal" is meant to encompass, without limitation, humans, domestic animals such as dogs, cats, horses, cattle, swine, sheep, goats and the like, as well as non-domesticated animals such as, but not limited to, mice, rats and rabbits. The term "non-mammal" is meant to encompass, for example, exosomes from microorganisms such as bacteria, flies, worms, plants, fruit/vegetables (e.g. corn, pomegranate) and yeast.

[0058] Exosomes may be obtained from the appropriate biological sample using any protocol that yields exosomes useful for therapeutic use, e.g. sufficiently pure, intact exosomes with good stability. For example, one or more of the following isolation techniques may be employed: centrifugation, filtration, ultracentrifugation, ultrafiltration, immunoaffinity capture and solvent or polymer-based methodologies.

[00S9] In one embodiment, the isolation protocol includes the steps of: i) exposing the biological sample to a first centrifugation to remove cellular debris greater than about 7-10 microns in size from the sample and obtaining the supernatant following centrifugation; ii) subjecting the supernatant from step i) to centrifugation to remove microvesicles therefrom; iii) microfiltering the supernatant from step ii) and collecting the microfiltered supernatant; iv) subjecting the microfiltered supernatant from step iii) to at least one round of ultracentrifugation to obtain an exosome pellet; and v) re-suspending the exosome pellet from step iv) in a physiological solution, conducting a second ultracentrifugation In a density gradient and removing the exosome pellet fraction therefrom. [0060] Thus, the process of isolating exosomes from a biological sample includes a first step of removing undesired large cellular debris from the sample, i.e. cells, cell components, apoptotic bodies and the like greater than about 7-10 microns in size. This step is generally conducted by centrifugation, for example, at 1000-4000x g for 10 to 60 minutes at 4°C, preferably at 1500-2SOOx g, e.g. 2000x g, for a selected period of time such as 10-30 minutes, 12-28 minutes, 14-24 minutes, 15-20 minutes or 16, 17, 18 or 19 minutes. Asoneof skill in the art will appreciate, a suitable commercially available laboratory centrifuge, e.g. Thermo-Scientific™ or Cole- Parmer™, is employed to conduct this isolation step. To enhance exosome isolation, the resulting supernatant is subjected to a second optional centrifugation step to further remove cellular debris and apoptotic bodies, such as debris that is at least about 7-10 microns in size, by repeating this first step of the process, i.e. centrifugation at 1000-4000x g for 10 to 60 minutes at 4°C, preferably at 1500-2500x g, e.g. 2000x g, for the selected period of time.

[0061] Following removal of cell debris, the supernatant resulting from the first centrifugation step(s) is separated from the debris-containing pellet (by decanting or pipetting it off) and may then be subjected to an optional additional (second) centrifugation step, including spinning at 12,000-15,000x g for 30-90 minutes at 4°C to remove intermediate-sized debris, e.g. debris that is greater than 6 microns size. In one embodiment, this centrifugation step is conducted at 14,000x g for 1 hour at 4°C . The resulting supernatant is again separated from the debris- containing pellet.

[0062] Alternatively or additionally, the resulting supernatant is collected and subjected to a third centrifugation step, including spinning at between 40,000-60,000x g for 30-90 minutes at 4°C to further remove impurities such as medium to small-sized micro vesicles greater than 0.3 microns in size e.g. in the range of about 0.3-6 microns. In one embodiment, the centrifugation step is conducted at 50,000x g for 1 hour. The resulting supernatant is separated from the pellet for further processing. If the second centrifugation step is conducted, the third centrifugation step is optional.

[0063] The supernatant is then filtered to remove debris, such as bacteria and larger microvesicles, having a size of about 0.22 microns or greater, e.g. using microfiltration. The filtration may be conducted by one or more passes through filters of the same size, for example, a 0.22 micron filter. Alternatively, filtration using 2 or more filters may be conducted, using filters of the same or of decreasing sizes, e.g. one or more passes through a 40-50 micron filter, one or more passes through a 20-30 micron filter, one or more passes through a 10-20 micron filter, one or more passes through a 0.22-10 micron filter, etc. Suitable filters for use in this step include the use of 0.45 and 0.22 micron filters.

[0064] The microfiltered supernatant (filtrate) may then be combined with a suitable physiological solution, preferably sterile, for example, an aqueous solution, a saline solution or a carbohydrate-containing solution, in a 1:1 ratio, e.g. 10 mL of supernatant to lOmL of physiological solution, to prevent clumping of exosomes during the subsequent ultracentrifugation and to maintain the integrity of the exosomes. The exosomal solution is then subjected to ultracentrifugation to pellet exosomes and any remaining contaminating microvesicles (between 100-220 nm). This ultracentrifugation step is conducted at 110,000- 170,000x g for 1-3 hours at 4 °C, for example, 144,000x g for 2 hours or 170,000x g for 3 hours. This ultracentrifugation step may optionally be repeated, e.g. 2 or more times, in order to enhance results. Any commercially available ultracentrifuge, e.g. Thermo-Scientific™ or Beckman™, may be employed to conduct this step. The exosome-containing pellet is removed from the supernatant using established techniques and re-suspended in a suitable physiological solution.

[0065] Following ultracentrifugation, the re-suspended exosome-containing pellet is subjected to density gradient separation to separate contaminating microvesicles from exosomes based on their density. Various density gradients may be used, including, for example, a sucrose gradient, a colloidal silica density gradient, an iodixanol gradient, or any other density gradient sufficient to separate exosomes from contaminating microvesicles (e.g. a density gradient that functions similar to the 1.100-1.200 g/ml sucrose fraction of a sucrose gradient). Thus, examples of density gradients include the use of a 0.25-2.5 M continuous sucrose density gradient separation, e.g. sucrose cushion centrifugation, comprising 20-50% sucrose; a colloidal silica density gradient, e.g. Percoll™ gradient separation (colloidal silica particles of 15-30 nm diameter, e.g. 30%/70% w/w in water (free of KNase and DNase), which have been coated with polyvinylpyrrolidone (PVP)); and an iodixanol gradient, e.g. 6-18% iodixanol. The resuspended exosome solution is added to the selected gradient and subjected to ultracentrifugation at a speed between 110,000- 170,000x g for 1-3 hours. The resulting essentially pure exosome pellet is removed and re- suspended in physiological solution.

[0066] Depending on the density gradient used, the re-suspended exosome pellet resulting from the density gradient separation may be ready for use. For example, if the density gradient used is a sucrose gradient, the exosome pellet is removed from the appropriate sucrose gradient fraction, and is ready for use, or may preferably be subjected to an ultracentrifugation wash step at a speed of 110,000- 170,000x g for 1 -3 hours at 4°C. If the density gradient used is, for example, a colloidal silica or a iodixanol density gradient, then the resuspended exosome pellet may be subjected to additional wash steps, e.g. subjected to one to three ultracentrifugation steps at a speed of 110,000- 170,000x g for 1-3 hours each at 4°C, to yield an essentially pure exosome-containing pellet. As one of skill in the art will appreciate, the exosome pellet from any of the centrifugation or ultracentrifugation steps may be washed between centrifugation steps using an appropriate physiological solution, e.g. saline. The final pellet is removed from the supernatant and may be re- suspended in a physiologically acceptable solution for use. Alternatively, the exosome pellet may be stored for later use, for example, in cold storage at 4°C, in frozen form or in lyophilized form, prepared using well-established protocols. The exosome pellet may be stored in any physiological acceptable carrier, optionally including cryogenic stability and/or vitrification agents (e.g. DMSO, glycerol, trehalose, polyhydroxylated alcohols (e.g. methoxylated glycerol, propylene glycol), M22 and the like).

[0067] In another embodiment, an immunoaffinity method of isolating exosomes from a biological sample may be utilized. The method comprises the steps of: i) optionally exposing the biological sample to a method of pre-enrichment to remove cellular and other debris; and ii) subjecting the sample to immunoaffinity capture with an antibody cocktail comprising at least three different antibodies to different exosome surface proteins, wherein the exosomes bind to the antibodies which bind or are bound to a solid support to yield an exosome-solid support complex from which the exosome are removed to yield isolated exosomes.

[0068] The optional pre-enrichment may include one or more centrifugation steps, microfiltration, and ultracentrifugation with or without a density gradient, as previously described to remove cellular debris, microvesicles and/or bacteria. Biological samples with a high protein concentration (such as serum) may also be immuno-depleted using methods well-established in the art to remove high abundance proteins that may interfere with exosome capture.

[0069] Following pre-enrichment, immunoaffinity exosome capture is conducted using antibodies having specificity for an exosomal marker. Examples of exosomal markers that may be used include the following: CD9 molecule (CD9), programmed cell death 6 interacting protein/ Alix (PDCD6IP), heat shock protein family A (Hsp70) member 8 (HSPA8), glyceraldehyde-3- phosphate dehydrogenase (GAPDH), actin, beta (ACTB), annexin A2 (ANXA2), CD63 molecule (CD63), syndecan binding protein (SDCBP), enolase 1 , (alpha) (ENOl), heat shock protein 90kDa alpha family class A member 1 (HSP90AA1), tumor susceptibility 101 (TSG101), pyruvate kinase, muscle (PKM), lactate dehydrogenase A (LDH A), eukaryotic translation elongation factor 1 alpha 1 (EEF1A1), tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta (YWHAZ), phosphoglycerate kinase 1 (PGK1), eukaryotic translation elongation factor 2 (EEF2), aldolase, fructose-bisphosphate A (ALDOA), heat shock protein 90kDa alpha family class B member 1 (HSP90AB1), annexin A5 (ANXA5), fatty acid synthase (FASN), tyrosine 3- monooxygenase/tryptophan 5-monooxygenase activation protein, epsilon (YWHAE), clathrin, heavy chain (He) (CLTC), CD81 molecule (CD81), albumin (ALB), valosin containing protein (VCP), triosephosphate isomerase 1 (TPI1), peptidylprolyl isomerase A (cyclophilin A) (PPIA), moesin (MSN), cofilin 1 (CFL1), peroxiredoxin 1 (PRDX1), profilin 1 (PFN1), RAP IB, member of RAS oncogene family (RAP IB), integrin subunit beta 1 (ITGB1), heat shock protein family A (Hsp70) member 5 (HSPA5), solute carrier family 3 (amino acid transporter heavy chain), member 2 (SLC3A2), histone cluster 1, H4a (HIST1H4A), guanine nucleotide binding protein (G protein), beta polypeptide 2 (GNB2), ATPase, Na+/K+ transporting, alpha 1 polypeptide (ATP1A1), tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, theta (YWHAQ), flotillin 1 (FLOT1), filamin A, alpha (FLNA), chloride intracellular channel 1 (CLIC1), chaperonin containing TCP1, subunit 2 (CCT2), cell division cycle 42 (CDC42), tyrosine 3- monooxygenase/tryptophan 5-monooxygenase activation protein, gamma (YWHAG), alpha-2- macroglobulin (A2M), tubulin alpha lb (TUBA IB), ras-related C3 botulinum toxin substrate 1 (rho family, small GTP binding protein Racl) (RAC1), lectin, galactoside-binding, soluble, 3 binding protein (LGALS3BP), heat shock protein family A (Hsp70) member 1A (HSPA1A), guanine nucleotide binding protein (G protein), alpha inhibiting activity polypeptide 2 (GNAI2), annexin Al (ANXA1), ras homolog family member A (RHOA), milk fat globule-EGF factor 8 protein (MFGE8), peroxiredoxin 2 (PRDX2), GDP dissociation inhibitor 2 (GDI2), EH domain containing 4 (EHD4), actinin, alpha 4 (ACTN4), tyrosine 3-monooxygenase/tryptophan 5- monooxygenase activation protein, beta (YWHAB), RAB7A, member RAS oncogene family (RAB7A), lactate dehydrogenase B (LDHB), GNAS complex locus (GNAS), RAB5C, member RAS oncogene family (RAB5C), ADP ribosylation factor 1 (ARFl), annexin A6 (ANXA6), annexin Al 1 (ANXA11), actin gamma 1 (ACTG1), karyopherin (importin) beta 1 (KPNB1), ezrin (EZR), annexin A4 (ANXA4), ATP citrate lyase (ACLY), tubulin alpha lc (TUBA1C), transferrin receptor (TFRC), RAB14, member RAS oncogene family (RAB14), histone cluster 2, H4a (HIST2H4A), guanine nucleotide binding protein (G protein), beta polypeptide 1 (GNB1), thrombospondin 1 (THBS1), RAN, member RAS oncogene family (RAN), RAB5A, member RAS oncogene family (RAB5 A), prostaglandin F2 receptor inhibitor (PTGFRN), chaperonin containing TCP1, subunit 5 (epsilon) (CCT5), chaperonin containing TCP1, subunit 3 (CCT3), adenosylhomocysteinase (AHCY), ubiquitin-like modifier activating enzyme 1 (UBA1), RAB5B, member RAS oncogene family (RABSB), RAB1A, member RAS oncogene family (RAB1A), lysosomal-associated membrane protein 2 (LAMP2), integrin subunit alpha 6 (ITGA6), histone cluster 1, H4b (HIST1H4B), basigin (Ok blood group) (BSG), tyrosine 3- monooxygenase/tryptophan S-monooxygenase activation protein, eta (YWHAH), tubulin alpha la (TUBA1A), transketolase (TKT), t-complex 1 (TCP1), stomatin (STOM), solute carrier family 16 (monocarboxylate transporter), member 1 (SLC16A1), RAB8A, member RAS oncogene family (RAB8A), myosin, heavy chain 9, non-muscle (MYH9) and major vault protein (MVP).

[0070] In one embodiment, the antibody cocktail comprises 3 or more antibodies each having a specificity for a different exosome marker selected from the following proteins: Alix, Flotillin 1, CD9, CD63, CD81, TSG101 and LAMP2. For example, the antibody cocktail may comprise an antibody with specificity to each of Alix, Flotillin 1, CD9 and CD63, or an antibody to each of Flotillin 1, CD9, CD63, CD81 and TSG101, or an antibody to each of Flotillin 1, CD9, CD63, CD81, TSG101 and ALIX, or an antibody with specificity to each of ALIX, TSG101 and CD9, or an antibody with specificity to each of CD81, CD63 and CD9.

[0071] Examples of immunoaffmity capture techniques that may be used to capture exosomes using a selected antibody cocktail include, but are not limited to, immunoprecipitation, column affinity chromatography, magnetic-activated cell sorting, fluorescence-activated cell sorting, adhesion-based sorting and microfluidic-based sorting. The antibodies in the antibody cocktail may be utilized together, in a single solution, or may be utilized in two or more solutions that are administered simultaneously or consecutively.

[0072] In a further embodiment, exosome separation methods may be utilized in conjunction with one or more of centrifugation, microfiltration and ultracentrifugation steps, to isolate exosomes from a biological sample. For example, protein organic solvent precipitation (PROSPR) may be used. Briefly, PROSPR, as described in Gallart-Palau et al., Scientific Reports, 2016, Sept. 30,5: 14664 (the contents of which are incorporated herein by reference) is rapid three- step protocol to remove soluble proteins from a sample via solvent-based precipitation (e.g. acetone), leaving lipid-based microvesicles, including exosomes, in suspension within the sample, for further separation as above-described. Similarly, polymer-based separation may be used in which suitable polymers such as polyethylene-glycol and variants thereof are mixed with an exosome-containing sample to separate exosomes from the sample into the polymer layer.

[0073] The described exosome isolation protocols advantageously provide a means to obtain exosomes, such as mammalian exosomes, which are at least about 90% pure, and preferably at least about 95% or greater pure, i.e. referred to herein as "essentially free" from cellular debris, apoptotic bodies and microvesicles having a diameter less than 20 or greater than 140 nm, for example, free from particles having a diameter of less than 40 or greater than 120 nm (as measured, for example, by dynamic light scattering), and which are biologically intact, e.g. not clumped or in aggregate form, and not sheared, leaky or otherwise damaged. Exosomes isolated according to the methods described herein exhibit a high degree of stability, evidenced by the zeta potential of a mixture/solution of such exosomes, for example, a zeta potential of at least a magnitude of 30 mV, e.g. < -30 or > +30, and preferably, a magnitude of at least 40 mV, 50 mV, 60 mV, 70 mV, 80 mV, or greater. The term "zeta potential" refers to the electrokinetic potential of a colloidal dispersion, and the magnitude of the zeta potential indicates the degree of electrostatic repulsion between adjacent, similarly charged particles (exosomes) in a dispersion. For exosomes, a zeta potential of magnitude 30 mV or greater indicates moderate stability, i.e. the solution or dispersion will resist aggregation, while a zeta potential of magnitude 40-60 mV indicates good stability, and a magnitude of greater than 60 mV indicates excellent stability. [0074] Moreover, high quantities of exosomes are achievable by the present isolation method, e.g. exosomes in an amount of about 100-2000 μg total protein can be obtained from 1-4 mL of mammalian serum or plasma, or from 15-20 mL of cell culture spent media (from at least about 2 x 10 6 cells). Thus, solutions comprising exosomes at a concentration of at least about S μg/μL, and preferably at least about 10-25 μg/μL, may readily be prepared due to the high exosome yields obtained by the present method. The term "about" as used herein with respect to any given value refers to a deviation from that value of up to 10%, either up to 10% greater, or up to 10% less.

[0075] Exosomes isolated in accordance with the methods herein described, which beneficially retain integrity, and exhibit a high degree of purity (being "essentially free" from entities having a diameter less than 20 nm and greater than 140 ran and/or greater than 120 nm), stability and biological activity both in vitro and in vivo, have not previously been achieved. Thus, tile present exosomes are uniquely useful, for example, diagnostically and/or therapeutically, e.g. for the in vivo delivery of protein and/or nucleic acid. They have also been determined to be non- allergenic/non-immunogenic, and thus, safe for autologous, allogenic, xenogenic and cross- kingdom use.

[0076] Isolated exosomes may then be genetically engineered to incorporate an exogenous protein, e.g. an exogenous protein such as a muscle protein, or exogenous nucleic acid encoding a selected muscle protein, or both. The term "muscle protein" is used herein to refer to any protein that is expressed in, or active in, muscle, including skeletal, smooth and cardiac muscle. The term "loaded exosome" is used herein to refer to exosomes which have been genetically engineered to incorporate an exogenous protein, nucleic acid encoding a protein or other biological substances. The term "empty exosome" is used herein to refer to exosomes which have not been genetically engineered to incorporate an exogenous protein, nucleic acid encoding a protein or other biological substances. The term "exogenous" is used herein to refer to protein or nucleic acid originating from a source external to the exosomes. Nucleic acid encoding the protein may be produced using known synthetic techniques, incorporated into a suitable expression vector using well established methods to form a protein-encoding expression vector which is introduced into isolated exosomes using known techniques, e.g. electroporation, transfection using cationic lipid-based transfection reagents, and the like. Similarly, the selected protein may be produced using recombinant techniques, or may be otherwise obtained, and then may be introduced directly into isolated exosomes by electroporation or transfection. More particularly, electroporation applying voltages in the range of about 20-1000 V/cm may be used to introduce nucleic acid or protein into exosomes. Transfection using cationic lipid-based transfection reagents such as, but not limited to, Lipofectamine® MessengerMAX™ Transfection Reagent, Lipofectamine® RNAiMAX Transfection Reagent, Lipofectamine® 3000 Transfection Reagent, or Lipofectamine® LTX Reagent with PLUS™ Reagent, may also be used. The amount of transfection reagent used may vary with the reagent, the sample and the cargo to be introduced. For example, using Lipofectamine® MessengerMAX™ Transfection Reagent, an amount in the range of about 0.15 uL to 10 uL may be used to load 100 ng to 2500 ng mRNA or protein into exosomes. Other methods may also be used to load protein into exosomes including, for example, the use of cell- penetrating peptides.

[0077] Exosomes isolated in accordance with the methods herein described, which beneficially retain integrity, and exhibit a high degree of purity and stability, readily permit loading of exogenous protein and/or nucleic acid in an amount of at least about 1 ng nucleic acid (e.g. mRNA) per 10 ug of exosomal protein or 30 ug protein per 10 ug of exosomal protein.

[0078] In another embodiment, a protein-encoding expression vector as above described, may be introduced directly into exosome-producing cells, e.g. autologous, allogenic, or xenogenic cells, such as immature dendritic cells (wild-type or immortalized), induced and non-induced pluripotent stem cells, fibroblasts, platelets, immune cells, reticulocytes, tumour cells, mesenchymal stem cells, satellite cells, hematopoietic stem cells, pancreatic stem cells, white and beige pre-adipocytes and the like, by electroporation or transfection as described above. Following a sufficient period of time, e.g. 3-7 days to achieve stable expression of the protein, exosomes incorporating the expressed protein may be isolated from the exosome-producing cells as described herein.

[0079] Alternatively, prior to incorporation into exosomes of a selected muscle protein, and/or nucleic acid encoding the protein, exosomes may be modified to express or incorporate a target-specific fusion product. The term "targeting exosome" is used herein to refer to exosomes which have been modified to express or incorporate a target-specific fusion product. The term "non-targeting exosome" is used herein to refer to exosomes which have not been modified to express or incorporate any target-specific fusion product. For the delivery of muscle proteins, the target-specific fusion product comprises a muscle targeting sequence, linked to an exosomal membrane marker. The exosomal membrane marker of the fusion product will localize the fusion product within the membrane of the exosome to enable the targeting sequence to direct the exosome to the intended target. Examples of exosome membrane markers include, but are not limited to: tetraspanins such as CD9, CD37, CD53, CD63, CD81, CD82 and CD151; targeting or adhesion markers such as integrins, ICAM-1 and CDD31; membrane fusion markers such as annexins, TSO101, ALIX; and other exosome transmembrane proteins such as LAMP (lysosome- associated membrane protein), e.g. LAMP 1 or 2, and LIMP (lysosomal integral membrane protein). All or a fragment of an exosome membrane marker may be utilized in the fusion product provided that any fragment includes a sufficient portion of the membrane marker to enable it to localize within the exosome membrane, i.e. the fragment comprises at least one intact transmembrane domain to permit localization of the portion of the membrane marker into the exosomal membrane.

[0080] The target-specific fusion product also includes a muscle targeting sequence, i.e. a protein or peptide sequence which facilitates the targeted uptake of the exosome into muscle. Examples of suitable skeletal muscle targeting sequences include, but are not limited to, muscle- binding peptides such as SERCA2, acetylcholine receptor epsilon, SCN4A, muscle specific creatine kinase (CK-MM) and fragments thereof, including the peptide sequence, TARGEHKEEELI (SEQ ID NO: 1). Cardiac muscle targeting sequences include sequences derived from cardiac troponin.

[0081] Exosomes incorporating a muscle-specific fusion product may be produced using recombinant technology. In this regard, an expression vector encoding the muscle-specific fusion product is introduced by electroporation or transfection into exosome-producing cells isolated from an appropriate biological sample. As one of skill in the art will appreciate, it is also possible to produce the fusion product using recombinant techniques, and then introduce the fusion product directly into exosome-producing cells using similar techniques, e.g. electroporation, transfection using cationic lipid-based transfection reagents, and the like. Following a sufficient period of time, exosomes generated by the exosome-producing cells, and including the fusion product, may be isolated as described.

[0082] The desired muscle protein, nucleic acid encoding the protein or both may be introduced into isolated exosomes incorporating a muscle-targeting fusion product (muscle- targeting exosomes) as previously described, using electroporation or transfection methods. Addition to the exosome of both the desired muscle protein and nucleic acid encoding the same muscle protein may increase delivery efficiency of the protein.

[0083] Exosomes genetically engineered to incorporate a muscle protein, and/or nucleic acid encoding the protein, may be used to deliver the protein and/or nucleic acid to a mammal in vivo in the treatment of a pathological myopathy in which the protein is defective or absent (due to a genetic mutation in the gene encoding the protein) to upregulate the activity of the protein and thereby treat the myopathy. Exosomes either incorporating a muscle-targeting fusion product or not including such a targeting fusion product may be used to deliver the muscle protein (and/or nucleic acid encoding the protein) to muscle tissue in vivo in the treatment of a myopathy. Examples of myopathies that may be treated using the present engineered exosomes include, genetic myopathies including muscular dystrophy, congenital myopathy and metabolic myopathies. At the genetic level, myopathies include disease inherited in an autosomal recessive (AR), autosomal dominant (AD) or X-Hnked recessive (XLR) manner. Examples of muscular dystrophy that are caused by genetic mutations and that may be treated using the present engineered exosomes are set out in Table 1 below. Table 1 identifies the disease, the affected protein and affected or mutated gene involved in each disease, the type of mutation, the mRNA transcript sequence information (via NCBI (National Centre for Biotechnology Information) GenBank accession numbers) for the functional gene (which could be incorporated into the exosomes to treat a disease), and the corresponding protein sequence information for the proteins useful to treat each disease.

[0084] Examples of congenital and other myopathies that are caused by genetic mutations and that may be treated using the present engineered exosomes are set out in Table 2 below. Table 2 identifies the disease, the affected protein and affected or mutated gene involved in each disease, the type of mutation (Mut), the mRNA transcript sequence information (via NCBI GenBank accession numbers) for the functional gene (which could be incorporated into the exosomes to treat a disease), and the corresponding protein sequence information for the proteins useful to treat each disease. [0085] Examples of metabolic myopathies that are caused by genetic mutations and that may be treated using the present engineered exosomes are set out in Table 3 below. Table 3 identifies the disease, the affected protein and affected or mutated gene involved in each disease, the type of mutation, the mRNA transcript sequence information (via the NCBI GenBank accession numbers) for the functional gene (which could be incorporated into the exosomes to treat a disease), and the corresponding protein sequence information for the proteins useful to treat each disease.

[0086] As one of skill in the art will appreciate, a muscle protein or nucleic acid encoding the muscle protein for incorporation into exosomes according to the invention may be a functional native mammalian protein or nucleic acid, including for example, a protein or nucleic acid from human and non-human mammals, or a functionally equivalent protein or nucleic acid. The term "functionally equivalent" is used herein to refer to a protein which exhibits the same or similar function to the native protein (retains at least about 30% of the activity of native protein), and includes all isoforms, variants, recombinant produced forms, and naturally-occurring or artificially modified forms, i.e. including modifications that do not adversely affect activity and which may increase cell uptake, stability, activity and/or therapeutic efficacy. The term "functionally equivalent" also refers to nucleic acid, e.g. mRNA, DNA or cDNA, encoding a muscle protein, and is meant to include any nucleic acid sequence which encodes a functional muscle protein, including all transcript variants, variants that encode protein isoforms, variants due to degeneracy of the genetic code, artificially modified variants, and the like. Protein modifications may include, but are not limited to, one or more amino acid substitutions (for example, with a similarly charged amino acid, e.g. substitution of one amino acid with another each having non-polar side chains such as valine, leucine, alanine, isoleucine, glycine, methionine, phenylalanine, tryptophan, proline; substitution of one amino acid with another each having basic side chains such as histidine, lysine, arginine; substitution of one amino acid with another each having acidic side chains such as aspartic acid and glutamic acid; and substitution of one amino acid with another each having polar side chains such as cysteine, serine, threonine, tyrosine, asparagine, glutamine), additions or deletions; modifications to amino acid side chains, addition of a protecting group at the N- or C- terminal ends of the protein, fusion products (e.g. with Fc peptide), and the like. Suitable modifications will generally maintain at least about 70% sequence similarity with the active site and other conserved domains of a native muscle protein, and preferably at least about 80%, 90%, 95% or greater sequence similarity. Nucleic acid modifications may include one or more base substitutions or alterations, addition of 5' or 3' protecting groups, and the like, preferably maintaining significant sequence similarity, e.g. at least about 70%, and preferably, 80%, 90%, 95% or greater.

[0087] Targeting and non-targeting exosomes including a selected protein, such as a muscle protein, or nucleic acid encoding the protein in accordance with the invention, may be formulated for therapeutic use by combination with a pharmaceutically or physiologically acceptable carrier. The expressions "pharmaceutically acceptable" or "physiologically acceptable" mean acceptable for use in the pharmaceutical and veterinary arts, i.e. not being unacceptably toxic or otherwise unsuitable for physiological use. As one of skill in the art will appreciate, the selected carrier will vary with the intended utility of the exosome formulation. In one embodiment, exosomes are formulated for administration by infusion or injection, e.g. subcutaneously, intraperitoneally, intramuscularly or intravenously, and thus, are formulated as a suspension in a medical-grade, physiologically acceptable carrier, such as an aqueous solution in sterile and pyrogen-free form, optionally, buffered or made isotonic. The carrier may be distilled water (DNase- and RNase-free), a sterile carbohydrate-containing solution (e.g. sucrose or dextrose) or a sterile saline solution comprising sodium chloride and optionally buffered. Suitable sterile saline solutions may include varying concentrations of sodium chloride, for example, normal saline (0.9%), half-normal saline (0.45%), quarter-normal saline (0.22%), and solutions comprising greater amounts of sodium chloride (e.g. 3%-7%, or greater). Saline solutions may optionally include additional components, e.g. carbohydrates such as dextrose and the like. Examples of saline solutions including additional components, include Ringer's solution, e.g. lactated or acetated Ringer's solution, phosphate buffered saline (PBS), TRIS (hydroxymethyl) aminomethane hydroxymethyl) aminomethane)-buffered saline (TBS), Hank's balanced salt solution (HBSS), Earle's balanced solution (EBSS), standard saline citrate (SSC), HEPES- buffered saline (HBS) and Gey's balanced salt solution (GBSS).

[0088] In other embodiments, the present exosomes are formulated for administration by routes including, but not limited to, oral, intranasal, enteral, topical, sublingual, intra-arterial, intramedullary, intrauterine, intrathecal, inhalation, ocular, transdermal, vaginal or rectal routes, and will include appropriate carriers in each case. For oral administration, exosomes may be formulated in normal saline, complexed with food, in a capsule or in a liquid formulation with an emulsifying agent (honey, egg yolk, soy lecithin, and the like). Oral compositions may additionally comprise adjuvants including sugars, such as lactose, trehalose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and derivatives thereof, such as sodium carboxymethylcellulose, ethylcellulose and cellulose acetates; powdered tragancanth; malt; gelatin; talc; stearic acids; magnesium stearate; calcium sulfate; vegetable oils, such as peanut oils, cotton seed oil, sesame oil, olive oil and corn oil; polyols such as propylene glycol, glycerine, sorbital, mannitol and polyethylene glycol; agar; alginic acids; water; isotonic saline and phosphate buffer solutions. Wetting agents, lubricants such as sodium lauryl sulfate, stabilizers, colouring agents and flavouring agents may also be present Exosome compositions for topical application may be prepared including appropriate carriers. Creams, lotions and ointments may be prepared for topical application using an appropriate base such as a triglyceride base. Such creams, lotions and ointments may also contain a surface active agent. Aerosol formulations may also be prepared in which suitable propellent adjuvants are used. Other adjuvants may also be added to the composition regardless of how it is to be administered, for example, anti-microbial agents, antioxidants and other preservatives may be added to the composition to prevent microbial growth and/or degradation over prolonged storage periods.

[0089] Targeting and non-targeting exosomes according to the present invention are useful in a method to treat myopathies involving a defective/missing muscle protein, or a condition involving lack of expression of a muscle protein. The terms 'treat", "treating" or 't reatment" are used herein to refer to methods that favorably alter a myopathy, including those that moderate, reverse, reduce the severity of, or protect against, the progression of the myopathy. Thus, for use to treat a pathological myopathy, a therapeutically effective amount of exosomes (targeting or non- targeting) loaded with a selected muscle protein, and/or nucleic acid encoding a muscle protein, are administered to a mammal. The term "therapeutically effective amount" is an amount of exosome required to treat the myopathy, while not exceeding an amount which may cause significant adverse effects. Exosome dosages that are therapeutically effective will vary on many factors including the nature of the condition to be treated as well as the particular individual being treated. Appropriate exosome dosages for use include dosages sufficient to result in an increase in activity or amount of the target muscle protein in the patient by at least about 10%, and preferably an increase in activity of the protein or amount of the target muscle protein of greater than 10%, for example, at least 20%, 30%, 40%, 50% or greater. In one embodiment, the dosage may be a dosage of exosome that delivers from about 0.1 mg/kg to about 100 mg/kg, such as 0.1- 50 mg/kg, or 0.1-10 mg/kg, of the desired protein, or an exosome dosage that delivers a sufficient amount of nucleic acid to yield about 0.1 mg/kg to about 100 mg/kg, such as 0.1 -50 mg/kg, or 0.1 - 10 mg/kg, of the desired protein. For example, the dosage of mRNA encoding a particular protein may be in the range of about 1 ug/kg to 1 mg/kg to treat a given myopathy. In another embodiment, for the delivery of a gene-editing system, the dosage may be a dosage of exosomes sufficient to deliver about lng/kg to about lOOmg/kg of gene-editing protein or nucleic acid. The term "about" is used herein to mean an amount that may differ somewhat from the given value, by an amount that would not be expected to significantly affect activity or outcome as appreciated by one of skill in the art, for example, a variance of from 1-10% from the given value. [0090] As will be appreciated by one of skill in the art, exosomes comprising a muscle protein, and/or nucleic acid encoding the protein, for example, to treat a myopathy, may be used in conjunction with (at different times or simultaneously, either in combination or separately) one or more additional therapies to facilitate treatment, including but not limited to, exercise, physiotherapy, stretching, high protein and/or carbohydrate diet, creatine monohydrate supplementation and corticosteroid treatment.

[0091] In another embodiment, the present method of treating myopathy in a mammal may include administration to the mammal of exosomes (for example, isolated as described above), genetically modified to incorporate gene-silencing systems (e.g., siRNA) to reduce the expression of a mutated gene followed by administration to the mammal of exosomes genetically modified to incorporate a protein useful to treat the myopathy and/or nucleic acid encoding the protein. In one embodiment, siRNA suitable to target a mutated gene that expresses a product that causes or amplifies the myopathy. Examples of mutations that may be targeted with siRNA include, but are not limited to, mutations that result in myotonic muscular dystrophy type 1 and 2, occulopharyngeal muscular dystrophy, malignant hyperthermia (RYR1 and CACNA1S mutations), and other dominant-negative myopathies due to mutations in TPM3, COL6A1, COL6A2, COL6A3, αB-crystalin, and filamin.

[0092] In another aspect of the invention, a method of treating myopathy in a mammal may include administering to the mammal exosomes genetically modified to incorporate genome- editing systems to correct the inherent primary mutation leading to the myopathy. Genome editing may include gene insertions, deletions, modifications (e.g. nucleotide transitions, transversions, insertions or deletions of one or more nucleotides or duplications of any nucleotide sequence), gene activation and gene silencing. As will be appreciated by one of skill in the art, genome editing may be for the purpose of correcting an undesirable gene mutation, introducing a gene mutation (e.g. including gene mutations to yield a transgenic animal model), altering a gene sequence (e.g. to improve, enhance or inhibit gene function), inserting a gene sequence (e.g. to activate or inhibit gene expression), and the like. Examples of nuclease genome editing systems include, but are not limited to, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) nuclease system, e.g. including a targeting gRNA and a CRISPR-associated (Cas) gene, such as CRISPR-Cas9, Transcription Activator-Like Effector Nucleases (TALEN) and mito-TALEN, Zinc-Finger Nucleases (ZFN) and aptamer-guided delivery of therapeutic nucleic acids, e.g. small interfering RNA, micro RNA, anti-microKNA, antagonist RNA, small hairpin RNA and other RNA, DNA or peptide (including affuners) based aptamers.

[0093] In one embodiment, the exosome is genetically modified to express or incorporate a CRISPR nuclease system, such as a CRISPR/Cas9 Type II genome editing system, including a Cas 9 nuclease, and a guide RNA (gRNA), which comprises a fusion of crRNA (CRISPR RNA) and a trans-activating RNA (tracrRNA). CRISPR RNA includes a targeting RNA sequence and a distinctive array of non-coding direct RNA repeats. The crRNA and tracrRNA are related to a selected Cas nuclease. As one of skill in the art will appreciate, the crRNA and tracrRNA (components of the gRNA) and the Cas nuclease are indicated to be "related" which means that the crRNA and tracrRNA are specific for and recognized by one or more particular Cas nucleases.

[0094] [0087] The targeting sequence of the guide RNA (gRNA) is a strand of RNA that is homologous to a region on a target gene, i.e. a gene to be edited or silenced, associated with a myopathy. Target genes may be genes associated with genetic disease, including hereditary disease such as autosomal dominant, autosomal recessive and X-linked myopathies. The targeting RNA may comprise from 10-30 nucleotides, e.g. from 15-25 nucleotides, such as 20 nucleotides, and may comprise a OC content of about 40-80%. The CRISPR system may be utilized to disrupt expression of a gene by insertion or deletion of nucleotides to disrupt the Open Reading Frame (ORF) of a target gene, or to introduce a premature stop codon therein. Non-Homologous End Joining (NHEJ) DNA repair may be used in this instance. The CRISPR system may also be used to edit (e.g. to correct a gene mutation) by utilizing homology directed repair, in which an editing region (also known as a repair template) is included in the CRISPR system. The editing region or repair template incorporates an edit (e.g. a healthy or wild-type DNA sequence to replace an undesirable DNA mutation in a target gene or any other edit as above), flanked by a region of homology (homologous arms) on either side thereof. The size of the editing region or repair template is not particularly restricted, and may include a single nucleotide edit, or edits of up to 100 nucleotides or more. The targeting sequence of the gRNA is selected such that it targets a site within the target gene that is proximal (e.g. within about 2-5 nucleotides or more) to a protospacer adjacent motif (PAM) located within the target gene. The PAM is recognized by the Cas nuclease and permits Cas nuclease binding. The homologous arms will generally increase in size with the size of the editing region or repair template, for example, for edits of less than about 50 nucleotides, the homologous arms may be in the range of about 100- 150 nucleotides in length, while larger editing regions may incorporate homologous arms of about 200-800 nucleotides, or more. Edits may also be introduced using CRISPR which facilitate expression of a target gene, e.g. edits which introduce a transcription factor that promote gene expression. The gRNA additionally incorporates related crRNA and a tracrRNA sequences, which interact with and function to direct the Cas nuclease to the target gene to catalyze cleavage of the target gene by the Cas nuclease. As will be understood by one of skill in the art, while each of the crRNA, tracrRNA, and Cas nuclease sequences are related, these sequences may be native or mutated sequences, provided that any mutations thereof do not have an adverse impact on function. Selection of suitable gRNA sequences can be achieved using methods known in the art, such as using programs for this purpose (e.g. CRISPR-MIT (http://crispr.mit.edu/), CHOPCHOP (https://chopchop.rc.fas.harvard.edu/index.php), or E-CRISP (http://www.e-crisp.org/E- CRISP/index.html).

[009S] The Cas nuclease may, for example, be a Cas 9 -based nuclease. Examples of a Cas 9 nuclease include wild-type Cas 9 (a double nickase) from Streptococcus pyogenes (SP), Staphylococcus aureus (SA), Neisseria meningitidis (NM), Streptococcus thermophilus (ST), and Treponema denticola (TD), as well as mutated recombinant Cas 9, e.g. mutated to function as a single nickase such as Cas9 D10A and Cas9 H840A, which may be used with 2 or more gRNAs to achieve a genome edit with increasing targeting efficiency that prevents non-specific genomic editing. Wild-type and single nickase Cas 9 may be used to edit genes, for example, that result in autosomal recessive, X-linked recessive or autosomal dominant myopathies, in order to correct the mutation. The mutated Cas 9 may also be a nuclease-deficient Cas (for example, incorporating both D10A and H840A to inactivate nuclease function) which binds but does not cleave and thereby silences a gene. Nuclease-deficient Cas 9 may be used to treat an autosomal recessive myopathy, to prevent or minimize expression of a dysfunctional mutated protein, which may interfere with the activity of the desired functional protein.

[0096] The targeting RNA is an RNA strand complementary to a site on the target gene which is 3-4 nucleotides upstream of a PAM sequence recognized by the Cas nuclease. The targeting RNA does not itself include a PAM sequence. PAM sequences differ for various Cas nucleases. For example, for Streptococcus pyogenes (SP), the PAM sequence is NGG; for S. aureus, the PAM sequence is NNORRT or NNGRR(N); for Neisseria meningitides, the PAM sequence is NNNNGATT; for Streptococcus (hemophilus, the PAM sequence is NNAGAAW; for Treponema denticola (TD), the PAM sequence is NAAAAC. "N" represents any nucleotide, W = weak (A or T) and R = A or G.

[0097] For introduction into exosomes, nucleic acid encoding a nuclease genome editing system, such as a selected CRISPR nuclease system including gRNA, DNA repair template and a Cas nuclease, may be produced using known synthetic techniques and then incorporated into the same or different expression vectors under the control of an appropriate promoter. Suitable vectors for such expression are known in the art. Alternatively, expression vectors incorporating the selected nuclease genome editing system may be obtained commercially. Expression vectors incorporating the nuclease genome editing system may be introduced into exosomes using electroporation or transfection using cationic lipid-based transfection reagents. Alternatively, the components of the nuclease editing system may be introduced directly into exosomes as single- stranded (ss) DNA using similar introduction techniques. For example, the DNA repair template may be introduced into exosomes as ssDNA, the gRNA and Cas nuclease may be introduced into exosomes as oligonucleotides/mRNA, or the Cas nuclease may be introduced into exosomes as a protein (produced using recombinant techniques, or otherwise obtained). Thus, as would be recognized by one of skill in the art, the different components of the nuclease editing system may be introduced into exosomes in various forms and/or combinations of nucleic acid, protein and expression vector incorporating DNA.

[0098] In another embodiment, Class 2 CRISPR technology (such as CRISPR-spCAS9- HF) can be incorporated into exosomes and used as a gene editing system.

[0099] The exosomes of the present invention provide many advantages over current treatment methods. At the outset, exosomes represent a physiological treatment method since they are an entity that naturally exists within a mammal. In addition, the present exosomes provide stability and protection from degradation and denaturation to the proteins/enzymes and/or nucleic acid that they deliver. This in turn facilitates the delivery of higher levels of protein or nucleic acid than is achieved by many other modes of delivery. Further, the use of exosomes to deliver protein/nucleic acid results in a minimal immune reaction to the protein or nucleic acid being delivered because exosomes may be obtained from cells that do not induce any significant immunogenic response or which are not toxic (e.g. exosomes from immature dendritic cells). Moreover, the present exosomes may be tailored to incorporate a targeting sequence that results in enhanced recognition and fusion with the target tissue, e.g. muscle, to result in increased specificity with respect to targeted protein/nucleic acid delivery.

[00100] Embodiments of the invention are described in the following specific examples which are not to be construed as limiting.

Example 1 - Dystrophin rescue in fibroblasts from Duchenne Muscular Dystrophy patients

[00101] To determine if dystrophin could be delivered to cells using genetically modified exosomes, the following experiment was conducted.

[00102] Exosome isolation - Dendritic cells (DC) were isolated from mouse bone marrow progenitor cells and from human peripheral blood mononuclear cells (collected using Ficoll gradient separation of human blood). Briefly, femur and tibia were carefully harvested from mice and were flushed with HBSS media to collect bone marrow progenitor cells. The bone marrow progenitor cells were cultured in OlutaMAX-DMEM media (Life Technologies) containing 10% FBS, ImM sodium pyruvate, 0.5% penicillin-streptomycin, and mouse recombinant granulocyte/macrophage colony-stimulating factor (R&D Systems). For human dendritic cell isolation, blood was collected in EDTA-lavender tubes followed by dilution of blood with 4x PBS buffer (pH 7.2 and 2 mM EDTA). 40 mL of diluted cell suspension was carefully layered over 20 mL of Ficoll gradient. The gradient was centrifuged at 400x g for 60 minutes followed by collection of the interphase layer containing the mononuclear cells. The mononuclear cells were cultured in IMDM media (BD Biosciences) containing 10% FBS, 1% glutamine, 0.5% penicillin- streptomycin, and human recombinant granulocyte/macrophage colony-stimulating factor (R&D Systems). Both human and mouse dendritic cells were further purified using EasySep™ Mouse and Human Pan-DC Enrichment Kit (Stem Cell Technologies). Dendritic cells were then cultured with the aforementioned media (GlutaMAX-DMEM media for mouse DC and IMDB media for human DC). Media was pre-spun at 170,000x g for 2 hours at 37 °C for 4 days to ensure that the subsequent exosome pellet would not be contaminated with bovine microvesicles and/or exogenous exosomes.

[00103] The dendritic cells were then grown to about 80% confluency in alpha minimum essential medium supplemented with ribonucleosides, deoxyribonucleosides, 4 mM L-glutamine, 1 mM sodium pyruvate, 5 ng/mL murine GM-CSF, and 20% fetal bovine serum. For conditioned media collection, cells were washed twice with sterile PBS (pH 7.4, Life Technologies) and the aforementioned media (with exosome-depleted fetal bovine serum) was added. Conditioned media from human and mouse immature dendritic cell culture was collected after 48 hours. The media (10 mL) was spun at 2,000x g for 15 min at 4°C to remove any cellular debris. This was followed by an optional 2000x g spin for 60 min at 4°C to further remove any contaminating nonadherent cells. The supernatant was then spun at 14,000x g for 60 min at 4°C. The resulting supernatant was optionally spun at S0,000x g for 60 min at 4°C. The supernatant was then filtered through a 40 μm filter, followed by filtration through a 0.22 um syringe filter (twice). The supernatant was then carefully transferred into ultracentrifuge tubes and diluted with an equal amount of sterile PBS (pH 7.4, Life Technologies). This mixture was then subjected to ultracentrifugation at 100,000x-170,000x g for 2 hours at 4°C using a fixed-angle rotor. The resulting pellet was re-suspended in PBS and re-centrifuged at 100,000x-170,000x g for 2 hours at 4°C. The pellet was resuspended carefully with 25 mL of sterile PBS (pH 7.4, Life Technologies) and then added gently on top of 4 mL of 30%/70% Percoll™ gradient cushion (made with 0.22 μm filter sterilized water) in an ultracentrifuge tube. This mixture was spun at 100,000x-170,000x g for 90 minutes at 4°C. With a syringe, the exosomal pellet-containing fraction at the gradient interface was isolated carefully, diluted in 50 mL of sterile PBS (pH 7.4, Life Technologies), followed by a final spin for 90 minutes at 100,000x-170,000x g at 4°C to obtain purified exosomes. The resulting exosomal pellet was resuspended in sterile PBS or sterile 0.9% saline for downstream use. Exosomal fraction purity was confirmed by sizing using a Beckman DelsaMax dynamic light scattering analyzer showing minimal contamination outside of the 40-120 nm size range, and by immuno-gold labelling/Western blotting using the exosome membrane markers, CD9, CD63, TSG101 and ALIX.

[00104] Exosomes were loaded with dystrophin mRNA using a cationic lipid-based transfection reagent (Lipofectamine® MessengerMAX™ Transfection Reagent, Life Technologies). After transfection, exosomes were spun for 2 hours at 170,000x g at 4°C followed by re-suspension in 5% (wt/vol) glucose in 0.9% sterile saline solution.

[00105] Treatment with Dmd mRNA-loaded exosomes - Primary dermal fibroblasts isolated from four DMD patients, and two age/gender-matched controls, were treated with human Dmd mRNA (100 ng), empty exosomes (10 ug of total exosomal protein in 0.9% sterile saline), and with human Dmd mRNA-loaded exosomes (100 ng of mRNA packaged in 10 ug of total exosomal protein in 0.9% sterile saline) for 48 hours. Cells were then seeded on glass-bottom chamber slides, washed with PBS, fixed with 4% paraformaldehyde, and incubated with anti- dystrophin (Abeam) antibody. Subsequent to a secondary incubation with an Alexa-fluor 568 conjugated anti-Rabbit antibody, cells were washed with phosphate buffered saline (PBS) and mounted with DNA-specific fluorescent dye, 4'-6-diamidino-2-phenylindole (DAPI)-containing hardset Vecta shield mounting fluid (Vector Laboratories, Inc.). Representative 3D z-stack images were taken using an Olympus laser scanning confocal microscope, which were then rendered into 2D images using the NIH Image J, Version 1.37, analysis software (Scion Image, NIH). The images confirm that bioengineered exosomes loaded with dystrophin {Dmd) mRNA rescue dystrophin deficiency in primary dermal fibroblasts isolated from Duchenne Muscular Dystrophy (DMD) patients. Quantification of confocal microscopy images to determine dystrophin protein content in primary dermal fibroblasts from control and DMD patients after treatment with exosomes loaded with dystrophin (Dmd) mRNA was then conducted. Total fluorescence per image was analyzed using the NIH Image J, Version 1.37, analysis software (Scion Image, NIH), and the data are represented as a bar graph. N=2 Controls (Con) and N=4 DMD patients. Results show almost complete rescue of dystrophin protein in fibroblasts treated with exosomes loaded with dystrophin (Dmd) mRNA as compared to those treated with either dystrophin (Dmd) mRNA or exosomes not loaded with dystrophin mRNA (Figure 1).

[00106] Immunoblot analysis of dystrophin protein expression in primary dermal fibroblasts was then conducted. Cells were washed with cold PBS, scraped, solubilized in radioimmuno-precipitation assay (RIP A) buffer and were resolved on 4-15% Criterion™ gradient gels (Biorad). The gels were transferred onto PVDF membranes using Trans-Blot® Turbo™ Transfer Starter System (Biorad), followed by blocking with 3% milk in TBS-Tween at room temperature. Immunoblotting was carried out using anti-dystrophin (Abeam) overnight at 4°C . Membranes were then incubated with anti-mouse horseradish peroxidase-linked secondary antibody (OE Healthcare) and were visualized by enhanced chemiluminescence detection reagent (Amersham Biosciences). Relative intensities of the protein bands were digitally quantified by using NIH Image J, Version 1.37, analysis software (Scion Image, NIH). Data were analyzed using an unpaired /-test and are presented as mean ± SEM, *P<0.05, N=3 Control (Con), N=4 dystrophin (DMD). Results show complete rescue of dystrophin protein in fibroblasts treated with exosomes loaded with dystrophin (Dmd) mRNA as compared to those treated with either dystrophin (Dmd) mRNA or exosomes not loaded with dystrophin mRNA (Figure 2).

Example 2 - Treatment of a Genetic Myopathy In Vivo with Dystrophin mRNA loaded Exosomes

[00107] Genetically engineered exosomes comprising dystrophin mRNA as described in Example 1 were used for the in vivo treatment of a myopathy resulting from genetic mutation, namely, one of the most common and representative muscular dystrophy disorders: Duchenne Muscular Dystrophy (DMD).

In vivo experiments

[00108] Hemizygous male CS7BL/10ScSn-Dmdmdx (mdx+Iy or mdx) mice (lacking functional dystrophin gene) were obtained from Jackson Laboratories (Strain 001801), and housed at McMaster University's Central Animal Facility. Mice were bred in accordance with rules set by McMaster University's Animal Research and Ethics board, following guidelines set forth by the Canadian Council of Animal Care. Twelve male mdx mice, ~100 days old, were randomly divided into various treatment groups as follows: VEH (vehicle, 150 uL of sterile 0.9% saline injection per animal per week, n=4), EXO (empty exosomes, 10 μg of total exosomal protein in 150 μL of sterile 0.9% saline per mouse per week, n=4), EXO-mRNA (exosomes loaded with mRNA, 100 ng of human dystrophin mRNA packaged in 10 ug of total exosomal protein in 150 μL of sterile 0.9% saline per animal per week, n=4) and prednisolone, which is the gold-standard therapy for Duchenne muscular dystrophy (PRED; administered at a dosage of 2 mg/kg body weight/day, delivered as two bolus i.p. injections per week, n=4). All animals were treated for six weeks via intravenous tail vein injections. WT mouse values were used as controls and indicated with a dashed line. Data is represented as mean ± SEM. *P < 0.0S. Data were analyzed using an unpaired t-test. Exosomes used were isolated and loaded with Dmd mRNA as described in Example 1. [00109] Maximum grip strength was measured by placing mice on a level wire mesh connected to a force transducer. The mouse was gently pulled away from the mesh by the base of the tail until release, while the transducer recorded the peak force (N). Trials were performed in triplicate, and mice were allowed time between trials to rest. The maximum grip strength was recorded, and corrected for muscle mass.

[00110] Mice were sacrificed two days after the last injection by cervical dislocation. Blood was collected immediately after sacrifice in EDTA-coated microfuges, followed by centrifugation at 1500 g for 15 mins at 4°C, supernatant (plasma) collected and frozen at -80 °C Tissues (EDL, SOL, TA and diaphragm) were extracted, snap frozen, weighed and stored at -80 °C until further analysis.

Measurement of dystrophin content

[00111] Whole tissue R1PA lysates were prepared from the TA, EDL, SOL and diaphragm muscle groups using established methods. Protein concentrations of whole tissue lysates were determined using a commercial assay (BCA Protein Assay; Pierce). Muscle extracts were separated on a 4-15% gradient SDS-PAGE, and subsequently transferred onto PVDF membranes (Biorad). Membranes were then blocked with 3% skim milk in lx TBS-Tween20 solution (Tris- buffered saline-Tween-20: 25 mM Tris-HCl, pH 7.5, 1 mM NaCl and 0.1% Tween-20) at room temperature, followed by overnight incubation in blocking solution with an antibody directed towards dystrophin (Abeam). Membranes were then incubated with the appropriate anti-rabbit HRP-linked secondary antibody (1 : 10,000) and visualized with an enhanced chemiluminescence detection reagent (GE Healthcare, Cleveland, OH) and the ChemiDoc MP system (Biorad). Ponceau staining was used to ensure equal loading across all gels (not shown).

Dystrophin mRNA-loaded exosome therapy restores dystrophin content and improves functional outcomes in mdx mice

[00112] Treatment with dystrophin mRNA-loaded exosomes restored dystrophin protein content in the Extensor digitorum longus (EDL) muscle (A), soleus (SOL) muscle (B), heart (C) (Figure 3) and diaphragm of mdx mice to levels comparable to those seen in WT mice, an effect which was not present in any other treatment groups. Body weight was unchanged in all groups treated (Figure 4A). The fast and slow twitch skeletal muscles of mdx mice treated with dystrophin mRNA-loaded exosomes demonstrated a significant reduction in size, suggesting that pseudohypertrophy (an early sign of DMD) was reduced and thus, an improvement in the quality of muscle present when compared to the other treatment groups (Figure 4B-D). Corresponding with an improvement in muscle quality, only the mdx mice treated with dystrophin mRNA-loaded exosomes experienced a significant increase in muscle strength, indicating improved muscle functionality (Figure 5). Lastly serum CK (a hallmark characteristic of the active muscle damage from DMD) was reduced to WT levels only in mdx mice treated with dystrophin mRNA-loaded exosomes, further demonstrating improved muscle health in this group (Figure 6).

[00113] These findings show that the administration of dystrophin mRNA-loaded exosomes was able to largely protect mdx mice from the development of a muscular dystrophy phenotype, a finding not observed with prednisolone therapy. Thus, exosomes loaded with mRNA encoding a functional protein were confirmed herein to be delivered to muscle and subsequently to restore the amount of the dystrophin protein to wildtype levels, indicating the efficacy of the disclosed method for treating genetic myopathies.

Example 3 - Treatment of acid alpha glucosidase deficiency with protein loaded exosomes

[00114] To determine the efficacy of exosomes to efficiently deliver cargo to muscle, exosomes were engineered to incorporate alpha acid glucosidase (OAA) and deliver the protein to the muscle of OAA deficient mice.

Production of Mouse GAA Protein

[00115] Mouse alpha acid glucosidase (OAA) (NCBI Reference Sequence: NM_008064.3) cDNA from skeletal muscle was sub-cloned into a mammalian vector (pOEX GST-fusion vector; OE Healthcare Life Sciences). The vector was maintained using the competent E. coli DHSalpha line (Life Technologies). The pGEX mammalian vector was then transfected into Chinese Hamster Ovary Cells (CHO; ATCC Cat. CCL-661) for mass production of active GAA enzyme. To isolate recombinant GAA in its processed active form, CHO cells transfected with G AA-pOEX vector were lysed and CHO cell lysate was cleared using ultra-performance resins for OST-tagged fusion protein purification (GE Healthcare Life Sciences). Over 80% of the recombinant protein was eluted after 3 washes. Elution #1 and Elution #2 were combined to obtain a high yield of protein. GST tag was removed from active GAA using PreScission Protease (GE Healthcare Life Sciences).

Introduction of GAA protein into exosomes

[00116] Exosomes were produced as described in Example 1. Exosomes were loaded with GAA protein using cationic lipid-based transfection reagents (Lipofectamine® MessengerMAX™ Transfection Reagent, Life Technologies). After transfection, exosomes were spun for 2 hours at 170,000x g at 4°C followed by re-suspension in 5% (wt/vol) glucose in 0.9% sterile saline solution.

Breeding of GAA mouse and littermate wildtype mice

[00117] Four GAA heterozygous breeding pairs (GAA-/+) were obtained from Jackson Laboratories (Maine, USA) to generate homozygous GAA knock-outs (GAA-/-) and wild-type littermates (GAA+/+). Mice were genotyped at 1 month of age using a standard genotyping kit (REDExtract-N-Amp Tissue PCR Kit; Sigma Aldrich). The birth rate of homozygous mutants was 18%, and significant deficits in front-limb muscle strength were detected as early as 1.5 months of age. During breeding, all animals were housed three to five per cage in a 12-h light/dark cycle and were fed ad libitum (Harlan-Teklad 8640 22/5 rodent diet) after weaning. The study was approved by the McMaster University Animal Research and Ethics Board under the global Animal Utilization Protocol # 12-03-09, and the experimental protocol strictly followed guidelines put forth by Canadian Council of Animal Care.

[00118] Primary mononuclear cells were isolated from skeletal muscle of GAA+/+ (wild- type mice with normal GAA activity) and GAA-/- mice (GAA knock-out mice, a model of Pompe Disease) (n = 3 per group). Cells were pre-plated to obtain a pure population of myoblasts. Cells were differentiated for S days into myotubes, followed by treatment with naked murine GAA recombinant protein (40 mg/kg recombinant GAA in PBS), empty exosomes (10 ug of total exosomal protein in PBS) or GAA protein-loaded exosomes (40 mg/kg of recombinant GAA in 10 ug of total exosomal protein in PBS) for 48 hours in pre-spun growth media devoid of bovine microvesicles and exosomes, Cells were harvested after 48 hours and GAA activity was measured. GAA enzyme activity

[00119] A standard fluorometric enzyme assay, originally described by Reuser et al. (Am J Hum Genet 30(2), 132-43, 1978), was used to determine GAA (EC 3.2.1.20) activity. In brief, cellular lysates were prepared by homogenizing cells (fibroblasts and myotubes) in 200 μL mannitol buffer (70 mM sucrose, 220 mM mannitol, 10 mM HEPES, 1 mM EGTA, protease inhibitor mixture (Complete Tablets, Roche), pH 7.4). Following BCA assay (Pierce) for colorimetric determination of protein concentration, 10 uL of each sample was loaded in triplicates and mixed with 20 μL. of the artificial substrate (4-methyl-umbelliferyl a-d-gluco-pyranoside in 0.2 M sodium acetate [NaAc] buffer, pH 3.9, heated to 65 °C) in a 96 well black plate. Standards were prepared from a S mM 4-methylumbelHferone/50% ethanol stock by serial dilution in 0.2 M NaAc buffer (pH 3.9), loaded in 10 uL triplicates, and mixed with 20 uL 0.2 M NaAc buffer (pH 3.9). The samples were then incubated in the dark for 1 h at 37.S °C and the reaction was terminated by adding 200 uL of 0.S M sodium carbonate (pH 10.7). The release of the product 4- methylumbelliferone from the artificial substrate 4-methyl-umbelliferyl a-d-gluco-pyranoside is proportionate to acid a-glucosidase activity (nmol/mg protein/hr), and the resulting fluorescence was read at 360 nm excitation/460 nm emission with a monochromator-based microplate detection system (Tecan Safire 2). RESULTS

[00120] Primary myotubes from GAA+/+ (wild-type mice with normal GAA activity) and GAA-/- (GAA knock-out mice, a model of Pompe Disease) were treated with naked murine GAA recombinant protein, empty exosomes (not loaded with GAA recombinant protein) or GAA protein-loaded exosomes, for 48 hours. Primary myotubes show a greater rescue of GAA activity when treated with GAA protein-loaded exosomes in comparison with naked GAA (currently utilized enzyme replacement therapy for Pompe disease) (Figure 7). Data is represented as mean ± SD (experiment independently repeated three times). *P < 0.05. Data were analyzed using an unpaired t-test.

[00121] Thus, exosomes modified to incorporate functional protein were shown to effectively deliver protein to muscle cells. Example 4 - Biogengineering Tangerine Exosomes Enhances Exosome Delivery to Muscle

[00122] To prepare non-targeting exosomes (not tagged with a membrane fusion product), DC cells were grown to about 80% confluency before exosome collection as described above. To prepare targeting exosomes (tagged with a membrane fusion product), on the third day of growth, DC were transfected with mammalian expression Lamp 1 -skeletal muscle targeting sequence (MTS) fusion plasmid 0.1-1 ug (depending on cell density) using Lipofectamine 2000 reagent (Life Technologies). The Lampl -MTS fusion plasmid was made using Gateway® technology and vectors (Life Technologies) with amplified mouse skeletal muscle cDNA that corresponds to the skeletal muscle targeting sequence (TARGEHKEEELI; SEQ ID NO: 1). The mouse LAMP1 cDNA exosome marker (NM_001317353.1) was amplified from mouse dendritic cell cDNA. The skeletal muscle targeting sequence and the exosome marker were linked via PCR, and then were incorporated into a mammalian expression vector. On the fifth day, the dendritic cells were washed and combined with fresh growth media (GlutaMAX-DMEM, pre-spun at 170,000x g for 2 hours to generate media virtually free of contamination from bovine exosomes or microvesicles).

[00123] Primary mouse muscle (differentiated into myotubes) were isolated from C57B1/6 mice using standard protocols. The cells were differentiated for 2-5 days prior to treatment. To test the efficiency of in vitro delivery of potential therapeutic agents (siRNA, proteins, mRNA, DNA, or small molecules), siRNA targeting a house-keeping gene GAPDH (abundantly expressed in almost all cell types) was used as proof of principle. Primary mouse myotubes were treated with 100 ng of GAPDH siRNA or scRNA (scrambled RNA obtained from Sigma; control for siRNA) as follows: l OOng of naked scRNA or siRNA, scRNA or siRNA transfected with Lipofectamine 2000 (LF2000), scRNA- or siRNA-loaded non-targeting exosomes, and scRNA- or siRNA-loaded skeletal muscle targeting exosomes (100 ng of scRNA or siRNA packaged in 10 ug of total exosomal protein in 0.9% sterile saline). scRNA and GAPDH siRNA were purchased from Sigma.

[00124] GAPDH qPCR was carried out to assess down-regulation of GAPDH mRNA. Treatment of myotubes with GAPDH siRNA-loaded skeletal muscle targeting exosomes exhibited greater down-regulation of GAPDH compared to GAPDH siRNA loaded non-targeting exosomes and conventional GAPDH siRNA transfected with LF2000 (Figure 8). Example 5 - Muscle targetins exosomes efficiently deliver PGC-1 alpha siRNA to muscle cells

[00125] Skeletal muscle-targeting exosomes prepared as described in Example 4 were transfected with PGC-1 alpha siRNA or scRNA. Primary mouse myotubes, obtained as described in Example 4, were treated with 100 ng of naked PGC-1 alpha siRNA or scRNA, scRNA or PGC- 1 alpha siRNA transfected with LF2000, 100 ng of scRNA- or PGC-1 alpha siRNA-loaded non- targeting exosomes and scRNA- or PGC-1 alpha siRNA-loaded skeletal muscle targeting exosomes (100 ng of scRNA or siRNA packaged in 10 ug of total exosomal protein in 0.9% sterile saline). PGC-1 alpha siRNA as purchased from Sigma.

[00126] PGC-1 alpha siRNA-loaded skeletal muscle targeting exosomes and PGC-1 alpha siRNA loaded non-targeting exosomes were determined to down-regulate PGC-1 alpha expression, which resulted in the subsequent down-regulation of mitochondrial genes that are known to be regulated by PGC-1 alpha including: NRF-1, Tfam, COX-IV, COX-VIIa, and Cycs (Figure 9A). This reduction in PGC-1 alpha mRNA and downstream mitochondrial genes also resulted in a reduction in mitochondrial electron transport chain complex IV (cytochrome c oxidase; COX) activity (Figure 9B). Importantly, the PGC-1 alpha siRNA had no effect on those genes which are not believed to be under the influence of PGC-1 alpha (being PGC-1 beta and PRC), demonstrating both the desired specificity and functionality of the siRNA administered via exosomes. Furthermore, the down-regulation in gene expression and reduction in mitochondrial biofunctional capacity was more effective in cells treated with PGC-1 alpha siRNA-loaded skeletal muscle targeting exosomes in comparison to cells treated with PGC-1 alpha siRNA-loaded non- targeting exosomes or PGC-1 alpha siRNA transfected with LF2000 (Figure 9A-B). Thus, exosomes can be bioengineered to deliver cargo to skeletal muscle for the purpose of modulating cellular activity.

[00127] The foregoing demonstrates for the first time that the present genetically modified exosomes can be used to treat genetic myopathies (such as muscular dystrophy, congenital myopathy, and metabolic myopathies), and that muscle targeting exosomes may also be used to deliver cargo into muscle. Example 6 - Exosome Isolation from human biological samples

[00128] The following is another method that may be used to isolate exosomes.

[00129] For serum isolation, blood was allowed to clot for 1 hour at room temperature followed by spinning at 2,000x g for 15 min at 4°C. Human immature dendritic cells (IDC) were grown to 65-70% confluency in alpha minimum essential medium supplemented with ribonucleosides, deoxyribonucleosides, 4 mM L-glutamine, 1 mM sodium pyruvate, 5 ng/mL murine OM-CSF, and 20% fetal bovine serum. For conditioned media collection, cells were washed twice with sterile phosphate buffered saline (PBS) (pH 7.4, Life Technologies) and the aforementioned media (with exosome-depleted fetal bovine serum) was added. Conditioned media from human immature dendritic cell culture was collected after 48 hours. From this point onwards, all samples (serum- lmL and IDC media- 10mL) were treated exactly the same. Debris was removed from the biological sample by centrifugation of the supernatant at 2000x g for 24 min at 4°C. The supernatant was then spun at 2000x g for 60 min at 4°C and the subsequent supernatant was spun at 14,000x g for 60 min at 4°C. The resulting supernatant was then filtered through a 0.22 μm syringe filter (twice). This resultant filtrate is ready for purification via immunoaffinity.

[00130] Magnetic beads were prepared by vortexing magnetic bead-protein A solution (30 mg Dynabeads® suspension/mL in PBS, pH 7.4, with 0.01% Tween®-20 and 0.09% sodium azide) (Dynabeads®-Protein A; Cat. no. 10610D, Life Technologies) for 15-30 seconds to resuspend the beads. This sequence was repeated with the magnetic bead-protein O solution (30 mg Dynabeads® suspension/mL in PBS, pH 7.4, with 0.01% Tween®-20 and 0.09% sodium azide) (Dynabeads®-Protein O; Cat. no. 10612D, Life Technologies). 500 each μL of magnetic bead-protein A and magnetic bead-protein G (herein referred to as magnetic bead-protein A/G) were transferred into a microfuge tube. The tube was placed on a magnetic rack for 1 min at room temperature (RT) to separate the magnetic bead-protein A/O from the solution, and the supernatant was carefully removed without disturbing the magnetic beads. The tube was removed from the magnet. A selected antibody was then bound to the magnetic bead-protein A/O complex. This involved the following: 100 ug each of antibodies (ALIX, TSG101 and CD9 (referred to as 3Ab- a method); or CD9, CD81 or CD63 (referred to as 3Ab-b method); and CD9 (ab92726, Abeam), CD63 (abl93349, Abeam), CD81 (sc-9158, SantaCruz), ALIX (abl l7600, Abeam), TSG101 (ab83, Abeam) and flotillin 1 (ab41927, Abeam) for the antibody cocktail (6Ab) method) was diluted in a protease inhibitor solution (1 mL of sterile PBS (pH 7.4) including 0.1% Tween®-20 and 1/10 th of a Roche complete mini protease inhibitor cocktail tablet). The antibody solution was added to the magnetic bead-protein A/O. The resultant solution was men incubated at 4°C overnight using an end-over-end mixer. The tube was placed on the magnet for 1 min at RT and then the supernatant was removed carefully without disturbing the magnetic bead-protein A/G- antibody complex. The tube was removed from the magnet and the magnetic bead-protein A/G- antibody complex was resuspended in 0.1 mL of protease inhibitor solution. The magnetic bead- protein A/G-antibody complex was washed for 5 minutes with 1 mL of fresh protease inhibitor solution, using an end-over-end mixer. This was repeated three more times.

[00131] The antibody was then crosslinked to the protein A/O affinity ligand. This involved the following. The magnetic bead-protein A/O-Antibody complex was resuspended in 500 μL of 5 mM Bis(sulfosuccinimidyl)suberate in antibody conjugation buffer (20 mM sodium phosphate, 0.1SM NaCl (pH 7.4,prepared fresh on day of use). The resultant solution was incubated at room temperature for 40 min with gentle mixing using an end-over-end mixer. 25 μL of quenching buffer (1M Tris HC1 (pH 7.4,prepared fresh on day of use) was then added and the solution was incubated at room temperature for 24 min with gentle mixing using an end-over-end mixer. The tube was placed on the magnet for 1 min and the supernatant carefully removed without disturbing the magnetic bead-protein A/G-antibody complex. The crosslinked magnetic bead-protein A/G- antibody complex was washed for 5 minutes with 1 mL of fresh protease inhibitor solution, using an end-over-end mixer. This was repeated three more times.

[00132] The exosomes were then isolated using the following steps. 1 mL of the exosome filtrate was resuspended in 400 uL of magnetic bead-protein A/G-antibody complex and incubated overnight at 4°C with gentle mixing using an end-over-end mixer. The magnetic bead-protein A/G-antibody-exosome complex was then washed for 5 minutes with 1 mL of fresh PBS (pH 7.4) with 0.1% Tween®-20 and protease inhibitor solution, using an end-over-end mixer. This wash was repeated three more times. The exosomes were eluted by resuspending the magnetic bead- protein A/G-antibody-exosome complex in elution buffer (20 mMTris HC1 (pH 8), 137 mM NaCl, 1% Nonidet P-40, 2 mM EDTA). [00133] A BCA assay (Pierce™) was used to determine the yield of exosomes in each sample. Total protein yield obtained from human serum for the three-antibody cocktail was 5.2 μg/μL for 3Ab-a and 11.9 μg/μL for 3Ab-b, and for the six-antibody cocktail was 17.0 μg/μL for 6Ab. The total protein yield obtained from human IDC cells was 4.0 μg/μL for 3Ab-a and 5.7 μg/μL, for 3Ab-b, and 8.7 μg/μL for 6Ab. Since it is assumed that all exosomes bear certain standard exosomal markers (such as CD9, CD63, ALIX, etc.), it would be expected that immunoprecipitation using any antibody having specificity for these markers in isolation should be equally effective at immunocapturing all exosomes.

[00134] The size distribution and stability of the exosomes was also determined using a Beckman DelsaMax dynamic light scattering analyzer. Exosomes isolated from human serum using the three antibody cocktail (3Ab) and six antibody cocktail (6Ab) were each within the established exosomal size range of 20-100nm, indicating a high degree of purity. Correspondingly, the zeta potential of exosomes isolated from human serum using each antibody cocktail (3Ab and 6Ab) was determined to be between 102 to 164mV, indicating excellent stability.

[00135] Relevant portions of references referred to herein are incorporated by reference.