Mechanism of nonsense mutation suppression therapy

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DESCRIPTION (provided by applicant): Nonsense mutations inactivate gene function by promoting synthesis of truncated polypeptides and accelerating mRNA decay. These mutations are a significant cause of genetic disorders, giving rise to 15- 30% of all disease-causing alleles. Recent studies have shown that the antibiotic, gentamicin, can suppress translation termination at premature nonsense codons and produce limited but functionally significant quantities of essential proteins in mouse models of cystic fibrosis and muscular dystrophy. While this has led to initial clinical evaluations of gentamicin therapy in human inherited disorders, long-term application of this approach is compromised by the considerable adverse effects of this drug. PTC Therapeutics, Inc. (PTC) has pursued the concept of a pharmaceutical approach to treating diseases caused by nonsense mutations and has identified a novel, low molecular weight compound (PTC124) that is considerably more potent than gentamicin in both cell and animal models. PTC124 has the potential for near-term broad application in numerous genetic disorders, but such widespread utility requires mechanism of action analyses which demonstrate that it: i) promotes insertion of near-cognate tRNAs at premature termination codons; ii) principally targets premature translational termination, not normal translational termination; iii) has limited host-transcriptional effects on non-targeted mRNAs; iv) does not elicit toxic side effects in whole animals; tnd v) promotes nonsense suppression in additional animal models of disease states. This fast-track STTR proposal describes molecular biological approaches to all of these issues. In Phase I, we will use cultured =ells to address items i-iii and to establish the technological approaches required for addressing the remaining issues in animal models in Phase II. More specifically, we will treat HeLa cells with PTC124 and: I) define the events promoting nonsense suppression and II) determine whether PTC124-mediated nonsense suppression has global consequences for cellular mRNA decay and translation.