Dottorato in MORFOGENESI E INGEGNERIA TISSUTALE

Titolo della tesi: Aptamer-conjugated gold nanoparticles for selective delivery of microRNA in dystrophic muscles

Abstract Duchenne Muscular Dystrophy is the most common and severe form of genetic dystrophy affecting around 1 in every 5000 new-born boys. It is caused by the lack of dystrophin protein, that leads to a progressive loss of muscle mass that is then substituted by fibrotic and adipose tissue. Current strategies aimed at restoring functional dystrophin in DMD boys are facing several limitations, including the vast extension of the target tissue and the length of the dystrophin gene. Therefore, strategies aimed at improving skeletal muscle through pharmacological interventions are being actively investigated. Within this context, we aimed at developing a novel approach, based on gold nanoparticles (AuNPs), for targeted delivery of oligonucleotides and drugs into dystrophic muscles. To obtain specific delivery, we have functionalized the therapeutic AuNPs with an aptamer against the alpha7/beta1 integrin dimer, a protein dimer highly enriched in the surface of skeletal muscle, including satellite cells. Functionalized AuNPs were first tested in an in vitro cellular model of murine myoblasts (C2C12 cells) to assess the specific targeting and intra-cellular oligonucleotide delivery. The results showed that over 95% of C2C12 cells incorporated cy5-labeled AuNPs. In addition, to show their efficiency as a therapeutic delivery system we conjugated the AuNPs to microRNA-206 (miR-206), a potent regulator of satellite cells function and also a paracrine molecule in regenerating muscle. We first demonstrated that functionalized AuNPs release a functional copy of the microRNA in C2C12 cells, by using a luciferase reporter containing the miR-206 seed sequencing in the 3’ UTR. We then assessed if AuNPs containing miR-206 modulate satellite cells function ex vivo. To this end, we first isolated satellite cells from dystrophic mice by fluorescence-activated cell sorting (FACS) and treated them with control or miRNA-206-containing AuNPs. We demonstrated that miR-206-containing AuNPs strongly stimulated the differentiation of satellite cells, leading to the formation of larger multinucleated myotubes, compared to controls. In addition, AuNPs containing miR-206 induced satellite cells differentiation also in isolated single myofibers. Finally, experiments in DBA-2 mdx dystrophic mice showed that systemic delivery of AuNPs containing miR-206 efficiently target satellite cells leading to improved skeletal muscle regeneration in vivo. Taken together our results indicate that aptamer-conjugated AuNPs represent a good platform for the systemic delivery of oligonucleotides into dystrophic muscles. Furthermore, the AuNPs’ low toxicity, together with their high capacity of functionalization and their ability to target also satellite cells allows to overcome several of the limitation faced by currently delivery strategies based on adeno-associated virus. This makes of the developed nanosystem a potent tool able to revolutionize the regenerative medicine field for skeletal muscle disorders.