Research ArticleGene Therapy

Titin splicing regulates cardiotoxicity associated with calpain 3 gene therapy for limb-girdle muscular dystrophy type 2A

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Science Translational Medicine  27 Nov 2019:
Vol. 11, Issue 520, eaat6072
DOI: 10.1126/scitranslmed.aat6072

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Safety through splicing

Limb-girdle muscular dystrophy type 2A is characterized by progressive muscle weakness resulting from deficiency in calpain 3. Lostal et al. investigated the therapeutic effect of recombinant adeno-associated viral (AAV) vector expressing CAPN3 and a miR-208a target sequence in mice and nonhuman primates. Mice lacking calpain 3 and dysferlin (a severe model of myopathy) showed reduced dystrophy and restoration of calpain 3 expression after AAV treatment. In contrast to previous murine studies, treatment did not cause cardiotoxicity in nonhuman primates, although transgene was expressed in the heart. The authors identified species-specific differences in calpain 3 binding sites on titin between mice, nonhuman primates, and humans, which could account for differences in cardiotoxicity. Results support further investigation into calpain 3 gene therapy as a treatment for limb-girdle muscular dystrophy.


Limb-girdle muscular dystrophy type 2A (LGMD2A or LGMDR1) is a neuromuscular disorder caused by mutations in the calpain 3 gene (CAPN3). Previous experiments using adeno-associated viral (AAV) vector–mediated calpain 3 gene transfer in mice indicated cardiac toxicity associated with the ectopic expression of the calpain 3 transgene. Here, we performed a preliminary dose study in a severe double-knockout mouse model deficient in calpain 3 and dysferlin. We evaluated safety and biodistribution of AAV9-desmin-hCAPN3 vector administration to nonhuman primates (NHPs) with a dose of 3 × 1013 viral genomes/kg. Vector administration did not lead to observable adverse effects or to detectable toxicity in NHP. Of note, the transgene expression did not produce any abnormal changes in cardiac morphology or function of injected animals while reaching therapeutic expression in skeletal muscle. Additional investigation on the underlying causes of cardiac toxicity observed after gene transfer in mice and the role of titin in this phenomenon suggest species-specific titin splicing. Mice have a reduced capacity for buffering calpain 3 activity compared to NHPs and humans. Our studies highlight a complex interplay between calpain 3 and titin binding sites and demonstrate an effective and safe profile for systemic calpain 3 vector delivery in NHP, providing critical support for the clinical potential of calpain 3 gene therapy in humans.

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