628. Transposons Expressing Full-Length Human Dystrophin Enable Genetic Correction of Dystrophic Mesoangioblasts and iPS-Derived Mesoangioblast-Like Cells

Musculo-Skeletal Diseases II entire dystrophin gene, and additionally encompasses the TMEM47 gene. This is reminiscent of the famous B.B. deletion in the DMD patient whose DNA was used for subtractive hybridization in the cloning of the DMD and nearby CGD loci. Further investigation revealed that the homologous canine DNA elements are ferritinlike pseudogenes, and surprisingly that there are several additional copies present on the X-chromosome. Phylogenomic comparison of the physical map positions of the orthologous pseudogenes in proximity to the dystrophin gene shows a pattern of positional synteny, consistent with the expansion of this pseudogene family prior to the mammalian radiation. Unexpectedly, only in the canine lineage have the pseudogene sequences remained virtually identical to one another, suggesting the existence of a heretofore uncharacterized process of concerted molecular evolution spanning large physical distances. The fully characterized deletional null animal model represents an invaluable resource for the evaluation of the immunological consequences of recombinant dystrophin expression via gene therapy, as it provides the only mammalian system in which the complete absence of dystrophin prevents the development of central tolerance, as would be expected in most cases of DMD. Thus the GSHPMD model can be uniquely leveraged to address a major safety concern as the field pursues strategies for systemic gene delivery in DMD. Of note, published studies have revealed evidence for deleterious immunological responses following administration of vectors expressing recombinant HUMAN dystrophin in both DMD and the Golden Retriever canine model (NEJM 363: 1429-1437 and Mol. Ther. 18:1501-1508).

628. Transposons Expressing Full-Length Human Dystrophin Enable Genetic Correction of Dystrophic Mesoangioblasts and iPS-Derived Mesoangioblast-Like Cells

Mariana Loperfido1, Susan Jarmin2, Sumitava Dastidar1, Mario Di Matteo1, Ilaria Perini3, Marc Moore2, Nisha Nair1, Ermira Samara-Kuko1, Takis Athanasopoulos2, Francesco Saverio Tedesco4, George Dickson5, Maurilio Sampaolesi3, Thierry VandenDriessche1, Marinee K. Chuah1 1 Department of Gene Therapy & Regenerative Medicine, Free University of Brussels, Brussels, Belgium, 2Royal Holloway, University of London, London, United Kingdom, 3Translational Cardiomyology Laboratory, University of Leuven, Leuven, Belgium, 4University College London, London, United Kingdom, 5 Royal Holloway, University of London, Brussels, United Kingdom Duchenne muscular dystrophy (DMD) is a genetic neuromuscular disorder caused by the absence of dystrophin. We developed a novel gene therapy approach based on the use of the piggyBac (PB) transposon system to deliver the coding DNA sequence (CDS) of either full-length human dystrophin (DYS: 11.1 kb) or truncated microdystrophins (MD1: 3.6 kb; MD2: 4 kb). PB transposons encoding microdystrophins were transfected in C2C12 myoblasts, yielding 65±2% MD1 and 66±2% MD2 expression in differentiated multinucleated myotubes. A hyperactive PB (hyPB) transposase was then deployed to enable transposition of the large-size PB transposon (17 kb) encoding the full-length DYS and green fluorescence protein (GFP). Stable GFP expression attaining 78±3% could be achieved in the C2C12 myoblasts that had undergone transposition. Western blot analysis demonstrated expression of the full-length human DYS protein in myotubes. Subsequently, dystrophic mesoangioblasts from a Golden Retriever muscular dystrophy dog were transfected with the large-size PB transposon resulting in 50±5% GFP-expressing cells after stable transposition. This was consistent with correction of the differentiated dystrophic mesoangioblasts following expression of full-length human DYS. Alternatively, dystrophic mesoangioblastlike cells were generated from iPS of DMD patients. These iPSMolecular Therapy Volume 24, Supplement 1, May 2016 Copyright © The American Society of Gene & Cell Therapy

derived mesoangioblasts, constitute an essentially unlimited supply of stem/progenitor cells that could be genetically corrected using PB transposons expressing dystrophin. These results pave the way toward a novel non-viral gene therapy approach for DMD using PB transposons underscoring their potential to deliver large therapeutic genes.

629. Intravenous Delivery of a MTMR2-Encoding AAV9 Vector Extends Lifespan and Improves Muscle Function in Mice with X-Linked Myotubular Myopathy Nathalie Danièle, Laura Julien, Christelle Moal, Thibaut Jamet, Alban Vignaud, Ana Buj Bello Genethon, Evry, France

X-linked myotubular myopathy is a rare genetic disease affecting the skeletal musculature. Male patients present profound neonatal muscle hypotonia and weakness, respiratory insufficiency, and in most cases have a very severe reduction in lifespan. At the pathological level, muscle fibers are hypotrophic and contain central nuclei with disorganized mitochondria and triads. The disease is caused by mutations in the MTM1 gene, which encodes myotubularin, the founder member of a family of 15 homologous proteins in mammals (including MTMR1 to 14). We recently demonstrated the therapeutic efficacy of intravenous delivery of rAAV vectors expressing MTM1 in murine and canine models of myotubular myopathy. In the present study, we tested whether Mtmr1 and Mtmr2 overexpression, Mtm1 closest homologs, could also rescue the XLMTM phenotype. Recombinant serotype-9 AAV vectors encoding either MTM1, MTMR1 or MTMR2 under the control of the desmin promoter were compared by injection into the tibialis anterior muscle of twoweek-old Mtm1 deficient mice. Two weeks after vector delivery, a therapeutic effect was observed with Mtm1 and Mtmr2, but not Mtmr1, with Mtm1 being the most efficacious transgene. We further explored a systemic route of administration, intravenous injection of a single dose of rAAV9-Mtmr2 in XLMTM mice ameliorated muscle histology and strength, and extended lifespan throughout the 3-month period of the study. Even though Mtmr2-treated mutant mice remained smaller than their wild-type counterparts, with partial increase in body weight and myofiber size, most importantly, the contractile force of myotubularin-deficient muscles improved strongly. Altogether, these results establish the proof-of-concept that overexpression of MTMR2 in skeletal muscle represents a novel therapeutic approach for myotubular myopathy.

630. AAV Transduction of a Truncated Dysferlin Improves Dysferlinopathy Telmo Llanga1, Bryan Sutton2, Nadia Nagy1, Matthew Hirsch1 University of North Carolina, Chapel Hill, NC, 2Texas Tech University, Lubbock, TX

1

Dysferlinopathy is a progressive muscular dystrophy caused by the absence of functional Dysferlin. Dysferlin cDNA is over the packaging capacity of adeno-associated viral vectors (AAV), complicating potentially therapeutic gene addition strategies. The use of dual or fragmented AAV vectors, while genetically intriguing, is undesired as these formats demonstrate decreased transduction efficiencies. Therefore, an alternative approach using a panel of smaller dysferlin-like molecules was executed in vitro and in vivo in an AAV vector context. Three of the four “hybrid” dysferlin reading frames produced protein which behaved similar to full length dysferlin in localization studies. Upon AAV vector production, only 1 variant (341) demonstrated intact genome packaging despite final cassettes sizes of approximately 5kb. Intramuscular administration of vectors encoding 341 in dysferlin deficient mice resulted in increased muscle S249