Systemic AAV-Mediated ƴ- Therapy for Treatment of Muscle Deficits in LGMD2C Mice

Eric R. Pozsgai1,3, Ellyn L. Peterson1,3, Danielle A. Griffin1,3, Jerry R. Mendell1,2, Louise R. Rodino-Klapac1,3 1Center for Gene Therapy, The Research Institute at Nationwide Children’s Hospital, Columbus, Ohio, USA; 2Department of Pediatrics and Neurology, The Ohio State University, Columbus, OH, USA, The Ohio State University, Columbus, OH, USA; 3Sarepta Therapeutics, Inc., Cambridge, MA, USA

2019 ASGCT, April 29-May 2, 2019, Washington, D.C. Disclosures

E.R. Pozsgai, E.L. Peterson, D.A. Griffin, and L.R. Rodino-Klapac are employees of Sarepta. Many LGMDs Are Caused by Defects in the Sarcolemmal That Comprise the -associated Complex

SCHEMATIC OF * prevent muscle damage during contraction1 – in any of the following 4 subunits of the sarcoglycan complex lead to muscular dystrophy1 DYSTROPHIN β-sarcoglycan COMPLEX α-sarcoglycan γ-sarcoglycan δ-sarcoglycan 1 – Sarcoglycan deficiency leads to dystrophin deficiency lamin A/C anoctamin-5 cell nucleus

POMT1 Dysferlin and anoctamin-5 support muscle calpain-3 membrane repair2 FKRP

myotilin – Failed muscle repair leads to chronic muscle degeneration TRIM32

*Image adapted from the MDA website. Causes/Inheritance. www.mda.org/disease/limb-girdle-muscular-dystrophy/causes-inheritance. Accessed December 12, 2018. 1. McNally EM. The Sarcoglycans. Landes Bioscience 2000-2013. www.ncbi.nlm.nih.gov/books/NBK6317/. Accessed December 16, 2018. 2. Liewluck T, Milone M. Muscle Nerve. 2018;58(2):167-177. The Dystrophin-Associated Protein Complex (DAPC)

The relationship between dystrophin and the DAPC is both intricate and dependent The dystrophin complex stabilizes the plasma membrane of striated & cells Loss of function mutations in the encoding dystrophin, or the associated proteins, trigger instability of the plasma membrane and myofiber & cardiomyocyte loss

nNOS, neuronal nitric oxide synthase.

Image adapted from Fairclough RJ, et al. Nat Rev Genet. 2013;14(6):373-378. The Sarcoglycan Complex: Essential Proteins of the DAPC

• LGMD is caused by a broad range of mutations occurring in multiple genes that encode for proteins that play vital roles in muscle function, regulation and repair1 • Sarcoglycanopathies • Sarcoglycans prevent muscle damage during contraction2 • All 4 functional sarcoglycans must be present to form a functional sarcoglycan complex3

Gene/Protein4 Function2 Disease4 Stabilizes DAPC, prevents SGCA muscle damage during LGMD2D α-sarcoglycan contraction Stabilizes DAPC, prevents SGCB muscle damage during LGMD2E β-sarcoglycan contraction Stabilizes DAPC, prevents SGCG muscle damage during LGMD2C γ-sarcoglycan contraction Stabilizes DAPC, prevents SGCD muscle damage during LGMD2F δ-sarcoglycan contraction

1. Mah JK, et al. Can J Neurol Sci. 2016;43(1):163-177. 2. McNally EM. The Sarcoglycans. Landes Bioscience 2000-2013. www.ncbi.nlm.nih.gov/books/NBK6317/. Accessed March 21, 2019. 3. Allen DG, et al. Physiol Rev. 2016;96(1):253- 305. 4. Liewluck T, Milone M. Muscle Nerve. 2018;58(2):167-177. LGMD2C (γ-Sarcoglycanopathy) Overview

• Phenotype can present similar to that of DMD with severe, progressive proximal muscle weakness; however, some patients may exhibit a milder phenotype

• Symptoms may arise early in life, typically before age 10, and presentation may occur up to the second decade of life

• Common examination features include calf hypertrophy, scapular winging, macroglossia and lumbar hyperlordosis

• Diagnosis is typically established by a lack or reduction of γ-sarcoglycan expression on biopsies or genetic testing

• Respiratory failure and cardiomyopathy are common features and should be actively screened for

• Serum CK activity is generally moderate to high, but can range from 1000 U/L to 25,000 U/L

1. Liewluck T, Milone M. Muscle Nerve. 2018;58(2):167-177. 2. Wicklund. 3. Murphy AP, Straub V. Journal of Neuromuscular Diseases.2015;S8 Adeno-associated Virus (AAV)-Mediated Gene Transfer

Adeno-associated viruses (AAV) are small, non- Essential Components of AAV Gene Therapy enveloped viruses that, unlike adenoviruses, have not been associated with human disease.5,6

VECTOR1,2 Several AAV serotypes have been identified, each Must deliver the transgene to target with a different tissue tropism. This allows for cells with minimal immune response specific tissue targeting with AAV-mediated gene PROMOTER1,3 therapies.5 Drives expression in intended tissues

Delivers transgenes via nonintegrating, stable, TRANSGENE1,4 extrachromosomal episomes to the nucleus, Produces a functioning version of the protein thereby limiting the risk of insertional mutagenesis of interest seen with other viral vectors.5

1. Naso MF, et al. BioDrugs. 2017;31(4):317-334. 2. US National Library of Medicine, Lister Hill National Center for Biomedical Communications. Genetics Home Reference. Help me Understand Genetics: Gene Therapy. Bethesda, Maryland: 2013. https://ghr.nlm.nih.gov/primer/therapy/genetherapy. Accessed August 29, 2018. 3. Zheng C, Baum J. Methods Mol Biol. 2008;434:205-219. 4. Chamberlain K, et al. Hum Gene Ther Methods. 2016;27(1):1-12. 5. Balakrishnan B, Jayandharan GR, Curr Gene Ther. 2014; 14:1-15. 6. Atchison RW et al. Science 1965; 149(3685): 754-6. scAAVrh74.MHCK7.hSGCG for the Treatment of LGMD2C (γ- sarcoglycanopathy)

AAVrh74.MHCK7.hSGCC The scAAVrh74.MHCK7.hSGCG construct was designed to restore functional full-length γ- VECTOR sarcoglycan to muscle cells AAVrh74 provides AAVrh74 was chosen based on superior systemic transduction in preclinical testing1-3 delivery with high muscle tropism PROMOTER • Because muscle cells do not divide, AAVrh74-transduced muscle MHCK7 allows for fibers may theoretically be protected indefinitely robust and • AAVrh74 provides systemic delivery, including to widespread cardiac muscle skeletal and TRANSGENE cardiac muscle • Preclinical data support single-administration hypothesis Full-length transgene γ-sarcoglycan expression transgene construct for full-length protein The MHCK7 promoter is optimized for the desired production expression in skeletal and cardiac muscle expression regulated by α-MHC to enhance cardiac expression2,3

ƴ-sarcoglycan transgene is a Full-Length gene construct scAAVrh.74.MHCK7.hSGCG for ƴ-sarcoglycan4 p MHCK7 INTRON hSGCG cDNA PROMOTER A ITR ITR

ITR, inverted terminal repeat; hSGCB, human β-sarcoglycan; pA, poly-A tail 1. Chicoine LG et al. Mol Ther 2013;22:338-47. 2. Pozsgai ER. Mol Ther 2017;25:855-69. 3. Salva MZ, et a. Mol Ther. 2007;15(2):320-329. l 4. Colella P, et al. Mol Ther Methods Clin Dev 2017;8:87-104. Systemic scAAVrh.74.MHCK7.hSGCG Restores γ-Sarcoglycan Expression in GSG KO Mice

• hSGCG Expression in Three Dosing Cohorts 12 Weeks Post IV Gene Delivery • Widespread Expression in Skeletal and Cardiac Muscle • hSGCG Correctly Localizes to Sarcolemma

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• Re-assembly of dystrophin associated protein complex (DAPC) at sarcolemma • GSG KO mice show absent or reduced sarcolemma expression of α-sarcoglycan, β- sarcoglycan, and dystrophin. • Treatment with AAV.MHC7.hSGCG • Increased α-sarcoglycan and β- sarcoglycan subunit expression at the sarcolemma in GSG KO mice • Increased expression of dystrophin at the sarcolemma in GSG KO mice Improved Muscle Morphology Following γ-Sarcoglycan Gene Transfer

55% average reduction in central nucleation across skeletal of GSG KO mice after ƴ-sarcoglycan gene transfer

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DIA Systemic γ-Sarcoglycan Gene Delivery to Diseased GSG KO Mice Reduced Muscle Fibrosis

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Distribution of Fiber Size is Normalized After hSGCG Gene Delivery Functional Improvement Resulting from Systemic γ-Sarcoglycan Gene Transfer

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*NF – No histopathological findings in independent veterinary review Conclusions from γ-Sarcoglycan Pre-Clinical Studies in GSG KO Mice

• GSG KO mice are complete KO of SGCG, recapitulate LGMD2C disease phenotype

• Widespread high level expression of hSGCG transgene

• Upregulation and re-assembly of DAPC

• Improved muscle morphology following γ-sarcoglycan gene transfer

• Restored muscle strength and increased ambulation in treated GSG KO mice

• No Observed Adverse Effect Level (NOAEL) in treated GSG KO mice AAV γ-Sarcoglycan Gene Therapy Program Synopsis

scAAVrh.74.MHCK7.hSGCG

MHCK7 PROMOTER INTRON hSGCG cDNA pA ITR ITR

Design Consideration Driven By Output Tissue targeting AAVrh74 Transduction and expression in (skeletal and cardiac) Promoter muscles and heart AAVrh74 No Safety Events Full-Length No Toxic Histopathology Transgene Correct localization Promoter Restoration of DAPC (hSGCG and DAPC proteins) Transgene Improvements in specific force, resistance to Function Transgene injury, ambulation Thank you