DEFINITION OF THE DYSTROPHIN GENE TRANSCRIPTOME AND MODULATION OF MUTATIONS BY ANTISENS OLIGONUCLEOTIDES-INDUCED TARGETED EXON SKIPPING

Dystrophin gene mutations are responsible for dystrophinopathies, a group of clinical manifestations affecting skeletal and cardiac muscles. Dystrophin gene is finely regulated and the splicing process plays an important role in modulating the clinical phenotype. Furthermore, it is expected that regulatory sequences belonging to the huge dystrophin introns could be implicated in the pathogenesis of dystrophinopathies. Recently, in vitro as well as in vivo studies demonstrated the possibility to restore the dystrophin protein synthesis by modulating splicing with antisense oligoribonucleotides. The present project takes advantage of a large series of dystrophinopatic patients and aims at: 1) finely characterising intronic transcribed sequences of the dystrophin gene (non coding RNAs–ncRNAs) and evaluating their structural/functional characteristics. Potentially functional ncRNAs will be tested for their transcriptional status and for the presence of mutations in selected patients. This approach will increase both the spectrum of characterised dystrophin pathogenic mutations and the number of regulatory sequences to be targeted for therapeutic purposes. 2) modulate the splicing process by antisense oligoribonucleotides in cells derived from patients’ tissues (myoblast and skin fibroblast induced into myogenesis). This approach will be addressed to severe dystrophin mutations that can be prompted towards a more favourable splicing behaviour, associated to a milder cellular phenotype. Furthermore we will explore the efficacy of novel delivery systems to improve oligoribonucleotides uptake into target cells. This novel approach employs non-toxic polymeric nanospheres and have been proposed for administration of vaccines.