PERSONALIZED MEDICINE FIGHTING NONSENSE DISEASES: EXPLORATION OF NV MOLECULES MECHANISM OF ACTION IN PURE-LITE SYSTEM AND THEIR READTHROUGH ACTIVITY IN CHOROIDEREMIA NONSENSE MODEL

Precision medicine represents a new approach in genetic medicine for treating a patient's mutation profile. Nonsense mutations cause 11% of inherited genetic diseases, including Choroideremia (CHM), Cystic fibrosis (CF), Duchenne Muscular Dystrophy (DMD), and some Cancers. Choroideremia is an X-linked disease associated with many retinal disorders. This retinal dystrophy causes the progressive degeneration of the choriocapillaris, photoreceptors, and retinal pigment epithelium. The CHM gene codifies for a 95 kDa, known as rab escort protein 1 (Rep1), involved in the intracellular trafficking and prenylation of polypeptides, a post-translational modification fundamental for the correct functionality of specific proteins. Nonsense mutations represent about 34% of mutations in the CHM gene. These mutations prematurely introduce a premature termination codon (PTC: TGA, TAG, and TAA) in the mRNA frame, causing a truncated and non-functional protein that will be eliminated from intracellular surveillance pathways. Nowadays there is no cure for diseases caused by nonsense mutations, but in the last decades, promising results have come from a pharmacological approach, called suppression therapy. This approach uses specific drugs having readthrough activity, known as TRIDs (Translational Readthrough Inducing Drugs), which permit the insertion of a near-cognate-tRNA in correspondence with a PTC during protein translation, allowing the correct development of a functional full-length protein. Our study is focused on investigating three molecules, NV848, NV914 and NV930, patented by Pibiri-Lentini group, that have shown readthrough activity in different nonsense model diseases. Specifically, elucidating their Mechanism of Action in the PURE-LITE system and exploring their readthrough activity in a choroideremia nonsense model system have been the goals of our efforts. PURE-LITE system is a highly purified, eukaryotic cell-free protein synthesis system, that allows the investigation of both the termination of protein synthesis in the P-site catalyzed by the RF complex (eRF1/eRF3/ribosome) and the readthrough via mispairing at the termination codon by a near-cognate tRNA when a PTC enter into the 40S A-site ribosome subunit. Simultaneously, chronic treatment and protein expression analyses was performed on Choroideremia nonsense cell model system to study their readthrough activity. Our results have shown both that the mechanism of action of these molecules is different by their precursor Ataluren, and in addition their readthrough activity with the rescue of Rep1 protein.