Homologous Recombination Mediates Functional Recovery of Dysferlin Deficiency following AAV5 Gene Transfer

Department of Pediatrics, The Ohio State University, Columbus, Ohio, United States of America.
PLoS ONE (Impact Factor: 3.53). 06/2012; 7(6):e39233. DOI: 10.1371/journal.pone.0039233
Source: PubMed

ABSTRACT The dysferlinopathies comprise a group of untreatable muscle disorders including limb girdle muscular dystrophy type 2B, Miyoshi myopathy, distal anterior compartment syndrome, and rigid spine syndrome. As with other forms of muscular dystrophy, adeno-associated virus (AAV) gene transfer is a particularly auspicious treatment strategy, however the size of the DYSF cDNA (6.5 kb) negates packaging into traditional AAV serotypes known to express well in muscle (i.e. rAAV1, 2, 6, 8, 9). Potential advantages of a full cDNA versus a mini-gene include: maintaining structural-functional protein domains, evading protein misfolding, and avoiding novel epitopes that could be immunogenic. AAV5 has demonstrated unique plasticity with regards to packaging capacity and recombination of virions containing homologous regions of cDNA inserts has been implicated in the generation of full-length transcripts. Herein we show for the first time in vivo that homologous recombination following AAV5.DYSF gene transfer leads to the production of full length transcript and protein. Moreover, gene transfer of full-length dysferlin protein in dysferlin deficient mice resulted in expression levels sufficient to correct functional deficits in the diaphragm and importantly in skeletal muscle membrane repair. Intravascular regional gene transfer through the femoral artery produced high levels of transduction and enabled targeting of specific muscle groups affected by the dysferlinopathies setting the stage for potential translation to clinical trials. We provide proof of principle that AAV5 mediated delivery of dysferlin is a highly promising strategy for treatment of dysferlinopathies and has far-reaching implications for the therapeutic delivery of other large genes.

  • Gene and Cell Therapy: Therapeutic Mechanisms and Strategies, Fourth edited by Templeton, 01/2015: chapter 41; Taylor & Francis/CRC Press., ISBN: 978-1-4665-7199-0
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    ABSTRACT: Objective Dysferlinopathies are a family of untreatable muscle disorders caused by mutations in the dysferlin gene. Lack of dysferlin protein results in progressive dystrophy with chronic muscle fiber loss, inflammation, fat replacement, and fibrosis; leading to deteriorating muscle weakness. The objective of this work is to demonstrate efficient and safe restoration of dysferlin expression following gene therapy treatment.Methods Traditional gene therapy is restricted by the packaging capacity limit of adeno-associated virus (AAV), however, use of a dual vector strategy allows for delivery of over-sized genes, including dysferlin. The two vector system (AAV.DYSF.DV) packages the dysferlin cDNA utilizing AAV serotype rh.74 through the use of two discrete vectors defined by a 1 kb region of homology. Delivery of AAV.DYSF.DV via intramuscular and vascular delivery routes in dysferlin deficient mice and nonhuman primates was compared for efficiency and safety.ResultsTreated muscles were tested for dysferlin expression, overall muscle histology, and ability to repair following injury. High levels of dysferlin overexpression was shown for all muscle groups treated as well as restoration of functional outcome measures (membrane repair ability and diaphragm specific force) to wild-type levels. In primates, strong dysferlin expression was demonstrated with no safety concerns.InterpretationTreated muscles showed high levels of dysferlin expression with functional restoration with no evidence of toxicity or immune response providing proof of principle for translation to dysferlinopathy patients.
    03/2015; 2(3). DOI:10.1002/acn3.172
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    ABSTRACT: No treatment is available for patients affected by the recessively inherited, progressive muscular dystrophies caused by a deficiency in the muscle membrane repair protein dysferlin. A marked reduction in dysferlin in patients harboring missense mutations in at least one of the two pathogenic DYSF alleles encoding dysferlin implies that dysferlin is degraded by the cell's quality control machinery. In vitro evidence suggests that missense mutated dysferlin might be functional if salvaged from degradation by the proteasome. We treated three patients with muscular dystrophy due to a homozygous Arg555Trp mutation in dysferlin with the proteasome inhibitor bortezomib and monitored dysferlin expression in monocytes and in skeletal muscle by repeated percutaneous muscle biopsy. Expression of missense mutated dysferlin in the skeletal muscle and monocytes of the three patients increased markedly, and dysferlin was correctly localized to the sarcolemma of muscle fibers on histological sections. Salvaged missense mutated dysferlin was functional in a membrane resealing assay in patient-derived muscle cells treated with three different proteasome inhibitors. We conclude that interference with the proteasomal system increases expression of missense mutated dysferlin, suggesting that this therapeutic strategy may benefit patients with dysferlinopathies and possibly other genetic diseases.
    Science translational medicine 08/2014; 6(250):250ra112. DOI:10.1126/scitranslmed.3009612 · 14.41 Impact Factor

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