Mechanistic Basis Underlying Protein Repair of CFTR Nonsense Mutations

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DESCRIPTION (provided by applicant): Suppression of premature termination codons (PTCs) with aminoglycosides and other agents has the potential to treat an underlying cause of cystic fibrosis (CF) and other genetic diseases including Duchenne's muscular dystrophy, Hurler's syndrome, and spinal muscular atrophy. While initial studies with gentamicin, amikacin, and the novel small molecule PTC124 indicate significant promise to the approach, successfully employing the strategy in CF and other diseases will require overcoming important challenges identified by our laboratory, including the need for a better understanding of basic mechanisms of action, defining patient populations most responsive to treatment (in particular, the molecular basis underlying increased response observed for the W1282X cystic fibrosis transmembrane conductance regulator (CFTR) mutation), and identifying pathways to augment rescue of CFTR nonsense codons. Approaches to repair CFTR mutations that confer dysfuction at the plasma membrane (as opposed to those caused by PTCs) have also received increasing attention in multicenter clinical trials. Interim results testing VX-770, a novel small molecule and CFTR modulator that potentiates gating of mutant CFTR localized to the cell surface, indicated improved CFTR activity and lung function in CF patients harboring G551D CFTR (a mutation resident at the cell surface but inactive due to a channel gating defect). These results indicate that correction of basic CF defects by protein repair therapy is feasible, and can result in clinically beneficial effects, even in a short (2 week) time frame. Recent evidence from our laboratory establishes that CFTR potentiators (such as VX-770) also potently activate W1282X CFTR, a relatively common premature stop mutation. Moreover, we observed significantly enhanced Cl- transport when a CFTR potentiator was used in combination with an aminoglycoside to induce translational readthrough of CFTR premature termination codons. These results provide preliminary data for an innovative therapeutic approach to treat CF patients harboring premature termination codons in CFTR. This proposal will test the hypothesis that certain CFTR nonsense mutants such as W1282X exhibit residual activity, even in the truncated state, and may respond to small molecule potentiators that enhance channel gating and restore CFTR activity. The proposed studies will capitalize on data emerging from an international clinical trial testing the stop codon suppressor PTC124 in CF patients (led by Dr. Rowe), and will also examine the combination of stop codon suppression and potentiation of CFTR readthrough product in experimental models, including primary human bronchial epithelial cells and G542X CFTR human transgenic mice. Results will lend insight regarding the mechanistic underpinnings of premature stop codon suppression, an emerging treatment strategy applicable to patients with a variety of genetic diseases, and provide preliminary data necessary to justify clinical testing of a novel approach to restore mutant CFTR function and ameliorate the basic defect in the disease. Correction of nonsense mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) represents a novel treatment strategy to correct a fundamental defect underlying cystic fibrosis (CF). However, a better understanding of the mechanistic basis of the approach is needed to overcome important challenges identified in human clinical trials. These experiments will define mechanisms underlying response to stop codon suppression, and examine whether potentiation of CFTR truncation mutants (and readthrough product following suppression of these mutations) represents a viable approach to restore CFTR function and thus ameliorate disease.