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WO 2017/054086 Al 6 April 2017 (06.04.2017) P O P C T

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WO 2017/054086 Al 6 April 2017 (06.04.2017) P O P C T (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date WO 2017/054086 Al 6 April 2017 (06.04.2017) P O P C T (51) International Patent Classification: (74) Agent: TANDAN, Susan; Gowling WLG (Canada) LLP, C12N 5/ 0 (2006.01) C07K 14/47 (2006.01) One Main Street West, Hamilton, Ontario L8P 4Z5 (CA). A61K 31/7088 (2006.01) C07K 14/705 (2006.01) (81) Designated States (unless otherwise indicated, for every A61K 38/46 (2006.01) C07K 19/00 (2006.01) kind of national protection available): AE, AG, AL, AM, A61K 48/00 (2006.01) C12N 15/12 (2006.01) AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, A61K 9/00 (2006.01) C12N 15/62 (2006.01) BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DJ, DK, DM, A61P 21/00 (2006.01) C12N 15/85 (2006.01) DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, A61P 3/00 (2006.01) C12N 15/87 (2006.01) HN, HR, HU, ID, IL, IN, IR, IS, JP, KE, KG, KN, KP, KR, (21) International Application Number: KW, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, PCT/CA201 6/05 1141 MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, (22) International Filing Date: SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, 30 September 2016 (30.09.201 6) TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, (25) Filing Language: English ZW. (26) Publication Language: English 4) Designated States (unless otherwise indicated, for every kind of regional protection available): ARIPO (BW, GH, (30) Priority Data: GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, 62/235,786 1 October 201 5 (01. 10.2015) US TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, 62/303,089 3 March 2016 (03.03.2016) US TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, (71) Applicant: EXERKINE CORPORATION [CA/CA]; c/o DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, McMaster University Medical Centre, Room 2H26, 1200 LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, Main Street West, Hamilton, Ontario L8N 3Z5 (CA). SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, KM, ML, MR, NE, SN, TD, TG). (72) Inventors: TARNOPOLSKY, Mark; c/o McMaster Uni versity Medical Centre, Room 2H26, 1200 Main Street Published: West, Hamilton, Ontario L8N 3Z5 (CA). SAFDAR, Ad- — with international search report (Art. 21(3)) eel; c/o McMaster University Medical Centre, Room 2H26, 1200 Main Street West, Hamilton, Ontario L8N 3Z5 (CA). (54) Title: TREATMENT OF GENETIC MYOPATHIES USING BIOENGINEERED EXOSOMES Fig. 1 ■ control □ DMD o00 o mRNA exo+mRNA (57) Abstract: A method of treating myopathy in a mammal is provided. The method includes administering to the mammal o somes genetically modified to incorporate a muscle protein useful to treat the myopathy or nucleic acid encoding the protein. 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, a d 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 (C ) 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, trifunctiona! protein deficiency, CPT2 deficiency) and the mitochondrial cytopathies. [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 incoiporate 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 incoiporate a functional muscle protein useful to treat the myopathy and/or nucleic acid encoding the muscle protein. [001 1] 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 incoiporate 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] A l . 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).
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