COMBINING TRANSLATION READTHROUGH INDUCING DRUGS AND NONSENSE MEDIATED DECAY PATWHAY INHIBITION TO THE CFTR RESCUE IN CYSTIC FIBROSIS CELL MODEL SYSTEM

Abstract

Nonsense mutations affect 10% of patients with cystic fibrosis and produce a premature termination codon in CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) mRNA causing early termination of translation and leading to lack of CFTR function. A potential therapy for nonsense mutations provides the use of small molecules able to overcome the premature stop codon (PTC) by a readthrough mechanism that lead to synthesis a complete CFTR protein. Despite the good results obtained from this approach, TRIDs efficiency is considerably reduced by the poor amount of target transcript, that is the mRNA containing the PTC. The readthrough, indeed, does not occur on the totality of target transcripts because of their degradation due to the nonsense mediated decay pathway (NMD). This pathway provides the degradation of mRNA harboring premature stop codon to prevent the production of altered polypeptides. In contrast, the activity of this pathway interferes with the effectiveness of the readthrough drugs, limiting the mRNA concentration of the target protein. Thus, a promising strategy for nonsense mutation treatment is a combined use of readthrough agents and factors that attenuate the nonsense mRNA decay. By silencing the UPF1 mRNA/protein, the activity of the NMD pathway was reduced, in FRT cells CFTR W1282X. Alternatively, caffeine was used as specific inhibitor of the UPF1 activity, to increase the efficiency of readthrough molecules (NV848 and NV914) in FRT cells CFTRW1282X cells. In both cases, the combined treatment: NV848 or NV914/caffeine and NV848 or NV914/UPF1siRNA caused an increase of CFTRW1282X mRNA level followed by the rescue of the CFTR expression and functionality. However, unexpectedly, despite the higher CFTRW1282X mRNA level in caffeine treated samples, both expression and functionality CFTR rescue resulted slightly lower than the recovery achieved by UPF1 silencing. Our results indicate that modulation of NMD pathway, although still to be optimized, could be a promising approach in order to increase TRIDs effects in presence of stop mutations.