All ETDs from UAB

Advisory Committee Chair

David Bedwell

Advisory Committee Members

Zsuzsanna Bebok

Robert A Kesterson

Kevin L Kirk

Steven M Rowe

Document Type

Dissertation

Date of Award

2015

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

A nonsense mutation introduces a premature terminations codon (PTC) into an open reading frame (ORF) upstream of a normal stop codon and results in truncated protein production. In frame PTCs account for ~11% of all mutations that cause human genetic diseases. To treat PTC-associated disorders, one approach is to use drugs that facilitate the insertion of amino acids carried by near-cognate aminoacyl-tRNAs at PTCs. Aminoglycosides were shown to suppress nonsense mutations and restore full-length protein production. This process is also termed as readthrough. However, their low efficiency and their potential to induce ototoxicity and nephrotoxicity limits their long-term application for PTC suppression. Therefore, large-scale efforts have been directed towards the discovery of less toxic and more efficient nonsense suppression compounds. Cystic Fibrosis (CF) is an autosomal recessive disease caused by mutations in Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. Previous studies from our laboratory discovered that CFTR PTCs can be treated with agents that suppress PTCs. In our study, we tested a broad range of new synthetic aminoglycoside derivatives in vitro and in vivo using a CFTR-G542X mouse model of CF. NB124 showed the highest suppression activity in our initial screening. It restored CFTR function in Fischer Rat Thyroid (FRT) cells (G542X), primary human bronchial epithelial (HBE) CF cells (G542X/delF508) as well as a CF mouse model expressing a human CFTR-G542X transgene better than the known suppression drug gentamicin. NB124 was also less cytotoxic than gentamicin in a tissue-based model for ototoxicity. These results provide evidence that NB124 is a promising drug for a wide array of CFTR nonsense mutations. Little is know about the identity and the functional consequences of the amino acids inserted during PTC suppression in mammalian cells. To identify the inserted amino acids and to understand the consequences of PTC suppression on CFTR biogenesis and function, we designed a system that allowed us to purify full-length tagged reporter proteins induced by PTC suppression. We then identified the amino acid(s) inserted using tandem mass spectrometry (MS-MS). Using constructs expressing each of the amino acid identified upon PTC suppression of either the G542X or W1282X nonsense mutations, we found that many CFTR variants produced by suppression showed reduced cAMP-stimulated chloride channel activity, which correlated with reduced CFTR processing and/or stability. We further found the CFTR modulators improved CFTR variant function. The CFTR corrector lumacaftor (VX-809) improved the processing and the CFTR potentiator ivacaftor (VX-770) enhanced the activity of CFTR in some of these variants. In addition, the combination of nonsense suppression drug G418, lumacaftor and ivacaftor showed synergistic increases in CFTR function for both CFTR nonsense alleles G542X and W1282X. Our results suggest that nonsense suppression frequently results in the restoration of partial CFTR function that can be further augmented by corrector-potentiator therapy. We conclude that nonsense suppression in combination with CFTR modulators may be beneficial for the treatment of CF patients with nonsense mutations.

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