Restriction of Calpain3 Expression to the Skeletal Muscle Prevents Cardiac Toxicity and Corrects Pathology in a Murine Model of Limb-Girdle Muscular Dystrophy

Abstract

All vectors were constructed by classical molecular biology techniques (see online-only Data Supplement Methods). Plasmid DNA was prepared by using the Maxiprep 500 EF kit (Macherey Nagel), Background-Genetic defects in calpain3 (CAPN3) lead to limb-girdle muscular dystrophy type 2A, a disease of the skeletal muscle that affects predominantly the proximal limb muscles. We previously demonstrated the potential of adeno-associated virus-mediated transfer of the CAPN3 gene to correct the pathological signs in a murine model for limb-girdle muscular dystrophy type 2A after intramuscular and locoregional administrations. Methods and Results-Here, we showed that intravenous injection of calpain3-expressing vector in mice can induce mortality in a dose-dependent manner. An anatomopathological investigation revealed large areas of fibrosis in the heart that we related to unregulated proteolytic activity of calpain3. To circumvent this toxicity, we developed new adeno-associated virus vectors with skeletal muscle-restricted expression by using new muscle-specific promoters that include the CAPN3 promoter itself and by introducing a target sequence of the cardiac-specific microRNA-208a in the cassette. Our results show that CAPN3 transgene expression can be successfully suppressed in the cardiac tissue, preventing the cardiac toxicity, whereas expression of the transgene in skeletal muscle reverts the pathological signs of calpain3 deficiency. Conclusions-The molecular strategies used in this study may be useful for any gene transfer strategy with potential toxicity in the heart.

The potential of adeno-associated viral vectors for gene delivery to muscle tissue

“…Briefly, LGMD2A is a skeletal muscle disease due to loss of calpain-3, an intracellular protease encoded by the CAPN3 gene that is predominantly expressed in skeletal muscle. The intravenous injection of a rAAV9 vector containing CAPN3 driven by a pan-muscle-specific promoter in mice caused severe fibrosis in heart and mortality, which was related to the transduction in heart and unregulated proteolytic activity of calpain-3 therein [76]. To prevent the detrimental effects related to both off-target and prolonged transgene expression, a number of strategies have been developed to regulate gene expression at both transcriptional and post-transcriptional levels.…”

“…Tissue-specific promoter and miRNA-based de-targeting design can be combined to further restrict transgene expression in muscle. In the LGMD2A study described at the beginning of this section, the authors developed new rAAV vectors that carry skeletal muscle-specific promoters and cardiac-specific miRNA-208a target sequence [76]. This vector design successfully suppressed calpain-3 expression in heart, thus preventing cardiac toxicity, and resulted in the correction of LGMD2A disease phenotype in skeletal muscle.…”

The Limb–Girdle Muscular Dystrophies: Is Treatment on the Horizon?

“…This study by Roudaut et al shows that cardiac toxicity from CAPN3 transgene expression can be successfully suppressed. Moreover, its expression in the skeletal muscles was sufficient to revert the myopathological signs of calpain 3 deficiency [16].…”

“…Many challenges lie ahead in translating promising therapeutic strategies into effective and accessible treatments but clinicians are optimistic that this is on the horizon. [16] evaluated the safety and efficacy of adeno-associated virus (AAV)-mediated calpain 3 gene transfer in a mouse model of LGMD2A using rAAV2/1 pseudotyped vectors and muscle-specific promoters to avoid calpain 3 cell toxicity more than a decade ago. They reported efficient and stable transgene expression in muscle with restoration of the proteolytic activity without evident toxicity, plus calpain 3 was correctly targeted to the sarcomere.…”

Meeting Report: New Directions in Biology and Disease of Skeletal Muscle 2014

“…Further study implicated both increased protein aggregation and myofibrillar disintegration; the later mediated through an interaction with BAG3. Isabelle Richard (Généthon), organizer of the session, showed that adenoviral mediated calpain-3 gene therapy rescued skeletal muscle defect in calpain-3 deficient mice, but resulted in cardiotoxicity [75, 76]. Promoter and miR driven selective skeletal muscle expression eliminated the cardiac defects.…”