Nonsense mutations suppression in Cystic fibrosis model systems by nonsense readthrough-inducing drugs (TRIDs)

Background: 10% of cystic fibrosis (CF) patients are affected by a nonsense mutation in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene (1). The consequence is the formation of a premature termination codon (PTC) in the mRNA and the production of a truncated protein, rapidly degraded (2). Among different therapeutic approaches, suppression therapy, by Translational Readthrough Inducing Drugs (TRIDs), is the most promising strategy against this alteration. It is aimed to skip the presence of the PTC, by forcing the insertion of a nearcognate tRNA (nc-tRNA), allowing to the production of a full-length and possibly functional protein (3). In the last decade, three new TRIDs, named NV848, NV914 and NV930 [Italian Patent N.102017000134511, European Patent N.3713934, US Patent N.11,203,578], have been optimized and tested in different nonsense cell models, showing high readthrough activity on the PTCs. The molecules were also validated in vitro and in vivo for their safety (4, 5). Objectives and methods: This study aims to examine different aspects of NV compounds from molecular and physiological points of view. The first purpose was to investigate the biodistribution of one of the three compounds, NV848, in target organs interested in CF (lungs, intestines, pancreas, plasma, kidney, brain). The analyses were performed at different time-points by HPLC, after oral administration of 60 mg/Kg of NV848 molecule. This experiment was necessary and preventive to the following step, that was to evaluate CFTR expression in CFTRG542X/G542X murine model after oral administration of 60 mg/Kg of NV848 molecule. Finally, to explore the possible biological target and mechanism of action (MoA) of the three NV compounds, the last objective was to study the possible interaction between the NV molecules and the FTSJ1 methyltransferase, responsible for tryptophane tRNA modifications (6). Results and conclusions: Biodistribution results showed that the NV848 molecule has reached the organs of interest and in particular lungs, pancreas, and intestines. Moreover, the administration of the NV848 molecule to CFTRG542X/G542X mice has evidenced a sensible increase of the CFTR mRNA and the rescue of CFTR protein expression in the lung of the treated mice. In addition, the experiments performed to study the biological target of the NV molecules have suggested that the NV914 molecule could interact with the FTSJ1 methyltransferase during the readthrough process. In conclusion, our data are promising and pave the way to the development of the future steps in order to optimize the suppression therapy by our NV TRIDs.