VEXOSOMES as stealth natural nanosystems to treat monogenic diseases

Duchenne Muscular Dystrophy (DMD) is a severe monogenic disorder characterized by mutations in the DMD gene, which encodes the dystrophin protein responsible for muscle strength. Despite recent advances in gene therapy, nowadays there are no effective treatments to cure the disease. Among tested approaches, Adeno-Associated Viruses (AAVs) revealed to be promising vectors to deliver therapeutic genes directly into target cells. These are designed as gene replacement therapies. However, this strategy presents some limitations, such as the immune recognition of the vectors, that leads to a premature elimination. Thus, the need for innovative strategies to enhance the safety and efficacy of AAV-based therapies is an urgent need. Extracellular vesicles (EVs) have emerged as a natural nanoscale delivery system, advantageous because they are inherently biocompatible and can evade immune detection. The idea at the base of this project was to test a new formulation called "vexosomes" that consisted in the encapsulation of AAVs within extracellular vesicles derived from different cell types. The research began with the isolation of EVs from two cell types: C2C12 myoblast (target cells) and BEAS-2B, non-target epithelial cells. These cell types were chosen to explore whether the origin of EVs affects the properties, functionality and target capacity of the resulting vexosomes. Once isolated, physicochemical and molecular properties of EVs were characterized using techniques such as nanoparticle tracking analysis, western blotting and mass spectrometry to assess particle size, concentration, and protein composition. The next step involved loading AAVs into EVs using two established techniques: extrusion and freeze-thaw cycles. In this way the therapeutic capabilities of AAVs can be coupled with the protective and stealth properties of EVs to overcome existing barriers to the treatment of DMD. Finally, the transduction efficiency was then evaluated in vitro using non-target cells and both C2C12 myoblasts and differentiated myotubes. Results showed that when cells were transduced with vexosomes originating from the same cell line, a slight increase in AAV transduction efficiency was observed, suggesting some stealth properties. However, encapsulation efficiency remained low, indicating that further optimization is needed. Another objective of this study was to examine whether the differentiation of C2C12 cell lines leads to changes in intracellular properties affecting the transduction efficiency of AAVs. To investigate this, myoblasts were first differentiated and then transduced with both vexosome formulations. In this case, an increase in transduction efficiency was observed with EVs isolated from C2C12 cells, while a decrease was noted with EVs from BEAS-2B cells, suggesting two opposing behaviors. While further research is required to refine the synthesis and performance of vexosomes, this study provides a strong foundation for the growing understanding of EV-mediated transduction, encapsulation methods, and skeletal muscle cell differentiation. With continued development, vexosomes could help overcome current limitations in gene therapy and improve treatment options for DMD and similar disorders.