Multiple approaches for Duchenne muscular dystrophy therapy

Exon Skipping has been demonstrated to be a successful strategy for the gene therapy of Duchenne Muscular Dystrophy (DMD): the rational is to convert severe Duchenne forms into milder Becker ones. Here we show the selection of U1 snRNA-antisense constructs able to confer effective rescue of dystrophin synthesis in a Δ44 Duchenne genetic background, through skipping of exon 45; moreover, we demonstrate that the resulting dystrophin is able to recover the correct timing of myogenic marker expression, to re-localize nNOS and to rescue expression of miRNAs previously shown to be sensitive to the Dystrophin-nNOS-HDAC2 pathway. Becker mutations display different phenotypes, likely depending on whether the shorter protein is able to reconstitute the wide range of wild type functions. Among them, efficient assembly of the dystrophin associated protein complex (DAPC) and Nitric Oxide Synthase (nNOS) localization are important. Comparing different Becker deletions we demonstrate the correlation between the ability of the mutant dystrophin to re-localize nNOS and the expression levels of two miRNAs, miR-1 and miR29c, known to be involved in muscle homeostasis and to be controlled by the Dys-nNOS-HDAC2 pathway. Since the gene responsible for the disease has been identified, several aberrant pathways have been characterized and many therapeutic approaches have been proposed to face all the symptoms associated to the pathology. What is now quite clear is that the best way to cure the disease is to apply different strategies in parallel, to enhance the beneficial effect that could be obtained from a single treatment. With this concept in mind we identified a microRNA, miR-31, that is deregulated in DMD conditions if compared to a healthy control. This miRNA represses dystrophin expression by targeting its 3′UTR region. In human DMD myoblasts treated with exon skipping, we demonstrate that miR-31 inhibition increases dystrophin rescue. These results indicate that interfering with miR-31 activity can provide an ameliorating strategy for those DMD therapies that are aimed at efficiently recovering dystrophin synthesis.