Aminoglycoside antibiotics restore CFTR function by overcoming premature stop mutations

Abstract

Cystic fibrosis (CF) is caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR). A single recessive mutation, the deletion of phenylalanine 508 (deltaF508), causes severe CF and resides on 70% of mutant chromosomes. Severe CF is also caused by premature stop mutations, which are found on 5% of CF chromosomes. Here we report that two common, disease-associated stop mutations can be suppressed by treating cells with low doses of the aminoglycoside antibiotic G-418. Aminoglycoside treatment resulted in the expression of full-length CFTR and restored its cyclic AMP-activated chloride channel activity. Another aminoglycoside, gentamicin, also promoted the expression of full-length CFTR. These results suggest that treatment with aminoglycosides may provide a means of restoring CFTR function in patients with this class of mutation.

Premature stop codons involved in muscular dystrophies show a broad spectrum of readthrough efficiencies in response to gentamicin treatment

“…First, a precise quantification of readthrough was obtained using reporter systems, either by transfection of cultured cells or through expression of synthetic RNAs in rabbit reticulocyte lysates. 5,15 Secondly, the re-expression of a mutated gene after gentamicin treatment has been assessed in animal models, such as Mdx mdx or CFTR mutant mice. 6,8 However, the correlation between readthrough levels obtained in vitro or ex vivo and the re-expression observed in vivo remained uncertain, since several processes might modulate the primary molecular effect of the antibiotic on readthrough, in particular mRNA stabilization and protein accumulation.…”

“…Bedwell and co-workers 4,5 have also shown that G-418 and gentamicin can restore the expression of the cystic fibrosis transmembrane conductance regulator (CFTR) in a bronchial cell line with a nonsense mutation in the CFTR gene. Similarly, a Hurler syndrome fibroblast cell line heterozygous for a stop mutation showed a significant increase in alpha-L-iduronidase when cultured in the presence of gentamicin.…”

“…Similarly, a Hurler syndrome fibroblast cell line heterozygous for a stop mutation showed a significant increase in alpha-L-iduronidase when cultured in the presence of gentamicin. 4,5 The strength of this approach was first demonstrated in vivo by the work of Barton-Davis and co-workers, who demonstrated that gentamicin could partially restore expression of fulllength dystrophin in mdx mice with the X-linked muscular dystrophy mutation (Mdm mdx ), a C-T transition at position 3185, resulting in a termination codon replacing a glutamine codon in the dystrophin gene. 6 Negamycin, a dipeptide antibiotic with similar stop codon-misreading activity, has also been shown to restore dystrophin synthesis in the mdx mouse skeletal muscle.…”

The Molecular Bases of Phenylketonuria (PKU) in New South Wales, Australia: Mutation Profile and Correlation with Tetrahydrobiopterin (BH4) Responsiveness

“…Dietary supplementation of sapropterin, a synthetic form of BH 4 , the cofactor of PAH, is efficacious in a subset of PAH mutations in lowering blood Phe levels (Kure et al 1999;Bernegger and Blau 2002). More recently, a novel set of compounds have been developed specifically targeting nonsense mutations and in vitro studies have shown that these compounds may be of therapeutic benefit in PKU patients carrying nonsense mutations (Howard et al 1996;Barton-Davis et al 1999;Welch et al 2007;Du et al 2009;Nudelman et al 2010;Ho et al 2013). Mutation screening would identify those patients for whom these approaches, including other mutation-specific therapies such as anti-aggregation compounds for p.G46S (Leandro et al 2011), may be applicable.…”

Novel Animal Models of Pediatric Epilepsy

“…In vitro and in vivo studies have suggested that this strategy may be relevant for human diseases. Suppression of a nonsense mutation in the cystic fibrosis transmembrane conductance regulator gene by growth in gentamicin increased cystic fibrosis transmembrane conductance regulator expression by 10 to 20 % compared to control cells and restored cAMP-activated chloride transport [149,150]. In a mouse model of Duchenne muscular dystrophy, gentamicin was used to disrupt translational fidelity to phenotypically correct a nonsense mutation [151].…”