RNA-based gene therapy of Duchenne Muscular Dystrophy: role of miRNA deregulation in the pathogenesis of DMD and their possible use for improving the exon skipping strategy

Among primary myopathies, the Duchenne Muscular Dystrophy (DMD) is certainly the most relevant because of diffusion and severity. The defect resides in mutations in the X-linked dystrophin gene: in the absence of such protein the muscles gradually begin to deteriorate. Since this disease is a monogenic disorder, it was considered from the very beginning for a gene therapy approach, although technically very challenging due to the large size of the protein whose coding sequences are difficult to accommodate in most viral vectors. In the last years we have pioneered a strategy, different from gene replacement, consisting in the modification of the dystrophin mRNA: by preventing the inclusion of specific mutant exons in the mature mRNA (exon skipping) it is possible to restore a shorter but functional dystrophin. In previous work we proved that the delivery of antisense molecules through AAV recombinant viruses in the mdx mouse produced a body wide distribution in muscle districts of therapeutic sequences, including the heart and diaphragm, and restored both dystrophin synthesis and muscular functionality for the entire life of the animal. Therefore, this approach seemed to be very promising in view of a possible gene therapy of DMD. The aim of this project is now to elucidate several new aspects linked to the DMD physiopathology: on one side we want to improve the translation of dystrophin in exon skipping treatments through the inhibition of a repressor microRNA (miR-31); on the other side we want to elucidate the molecular mechanisms by which dystrophin localizes and activates nNOS thus leading to a cascade of effects impinging on gene expression. Finally, since we identified muscle miRNAs leakage into the blood as a consequence of muscle damage, we want to test whether such miRNAs can be utilized as biomarkers not only for the diagnosis of DMD onset and progression but also for measuring and monitoring the outcomes of therapeutic interventions on humans.