Utilizzo di Nanoparticelle per il trasporto di Molecole Antisenso nel modello murino di Distrofia Muscolare di Duchenne

Duchenne Muscular Dystrophy (DMD) is an X-linked severe neuromuscular disease, mainly caused by mutations that disrupt the mRNA reading frame resulting in the absence of dystrophin protein in skeletal and cardiac muscles. Currently the most promising therapeutic approach, defined as exon-skipping, is based on the use of antisense oligonucleotides (AONs) to recognize specific RNA sequences that induce the exclusion of target exon from the mRNA and the restoration of the frame, allowing the synthesis of a functional protein. One of the main difficulties of this therapeutic approach is to obtain protection from degradation and transport of the AONs to the target tissues. Therefore, to obtain consistent results, high doses of AONs are needed, which are, however, a potential source of adverse reactions to life-long therapies as in DMD. Our research group is testing nanoparticles as a new delivery system for AONs with phosphorothioate backbone (2'-O-methyl-phosphorothioate 2'OMePs) in the mouse model of muscular dystrophy (mdx mice). These nanoparticles consist of an inert biocompatible material (polymethylmethacrylate) with covalently linked cationic groups, which allow the electrostatic bond with the antisense molecules. In an initial pilot experiment, we used nanoparticles of about 500 nm of diameter and we have proved that they are able to bind and release the AONs 2'OMePs, leading to the restoration of dystrophin expression in mdx mouse muscles. Despite the positive results obtained, these nanoparticles have a low capacity to adsorb on the surface AONs, and their diameter does not allow for intravenous administration. In subsequent experiments we therefore used nanoparticles of smaller size (about 130 nm of diameter) but with a 5 times higher adsorption capacity. We treated systemically mdx mice with low doses (52,5 mg AON / kg) of nanoparticle-AON complexes, and we analyzed the mice 1 and 12 weeks after the end of the treatment. At 1 week after the end of the treatment analysis of mRNA (Nested RT-PCR and Real Time-RT-PCR) has allowed us to identify in skeletal muscles and heart the transcript missing the target exon. Protein analysis (Western blot and immunofluorescence) also showed the correct molecular weight of restored dystrophin and proper localization in the sarcolemma of skeletal muscle fibers, cardiac, and arrector pili smooth muscle. At a distance of 12 weeks after treatment the correct transcript was still present in the tissues analyzed, although in lesser quantities, and the protein also detectable by immunofluorescence and Western blot. These results demonstrate that these nanoparticles represent a promising system for AONs delivery, as they are able to: i) ensure the protection of RNA molecules from nucleases, allowing lower doses to produce a measurable effect, ii) spread in target tissues (muscles) where release the adsorbed molecules in a slow and controlled manner with a longer treatment effect