AMINOGLYCOSIDE SUPPRESSION OF PREMATURE STOP MUTATIONS IN CFTR

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Many human diseases are caused by premature translation termination mutations. Because of the prevalence of this class of mutation, a mechanism that prevented translation termination at premature stop mutations would have the potential to reduce or eliminate the disease of a subset of individuals afflicted with a wide range of genetic diseases, including cancer, diabetes, Tay Sachs, beta-thalassemia, hypercholesterolemia, and many others. Another example is the disease cystic fibrosis (CF). CF is caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR). While the most common mutation in the CFTR gene is the deletion of a phenylalanine at residue 508 (delta F508), roughly 10 percent of all CF patients carry at least one premature stop mutation. In preliminary studies, we obtained evidence that premature stop mutations found in CF patients can be suppressed by aminoglycoside antibiotics, resulting in the expression of full-length, functional CFTR. These results suggest that a clinical strategy utilizing aminoglycoside treatment may restore CFTR function in CF patients with this class of mutation. However, it is important that we first understand in greater detail the mechanism of translation termination and how aminoglycosides act to subvert this process. To explore the feasibility of using aminoglycoside-based therapy to suppress disease-causing premature stop mutations, we propose the following Specific Aims: SPECIFIC AIM 1: Characterize the aminoglycoside-mediated suppression of premature stop mutations in the CFTR gene. SPECIFIC AIM 2: Investigate the ability of aminoglycosides to suppress a naturally-occurring premature stop mutation in the human CFTR gene expressed in a transgenic mouse model. SPECIFIC AIM 3: Determine how sequence context affects the efficiency of translation termination in human cells. SPECIFIC AIM 4: Characterize the mechanism of translation termination and investigate how aminoglycosides alter this process in mammalian cells.