Innovative therapeutic approaches for the recovery of protein expression in rare genetic diseases characterized by the presence of nonsense variants

Approximately 11% of inherited genetic diseases worldwide are caused by nonsense variants. These single-nucleotide substitutions occur within coding DNA sequences and result in the introduction of a premature termination codon (PTC) into the corresponding mRNA. As a consequence, they lead to the production of truncated, non-functional proteins or, in some cases, to their complete absence.Several genetic disorders are associated with nonsense variants, including Choroideremia (CHM), Cystic Fibrosis (CF), Duchenne Muscular Dystrophy (DMD), and numerous other genetic diseases. Notably, nonsense variants account for approximately 34% of pathogenic mutations in Choroideremia and 15% in Cystic Fibrosis. Currently, no specific cure or therapy exists for these types of genetic defects. However, in recent decades, the field of personalized medicine has made significant progress in developing targeted treatments, particularly through nonsense suppression therapy. This pharmacological strategy employs readthrough agents to restore full-length protein synthesis.The first aim of this Ph.D. thesis project was to evaluate the rescue of the CFTR protein in a Cystic Fibrosis G542X murine model by assessing the readthrough activity of two novel compounds, NV914 and NV930, specifically focusing on the restoration of CFTR in the lung, one of the primary organs affected by the disease.The second objective consisted of two different aspects. The first one, was to generate a Choroideremia cellular model carrying nonsense mutations to investigate the restoration of the REP1 protein following treatment with NV molecules (NV848, NV914, and NV930), thereby evaluating their ability to induce readthrough of premature stop codons in CHM mRNA. The second aspect was focused on assessing REP1 recovery directly in primary cell cultures derived from patients harbouring nonsense variants.Finally, the third objective of the project was to investigate the biological target of the three NV848, NV914, and NV930 molecules using the advanced cell-free experimental PURE-LITE system.