Gentamicin-Induced Correction of CFTR Function in Patients with Cystic Fibrosis and<i>CFTR</i>Stop Mutations

Wilschanski, Michael; Yahav, Yaacov; Yaacov, Yasmin; Blau, Hannah; Bentur, Lea; Rivlin, Joseph; Aviram, Micha; Bdolah‐Abram, Tali; Bebök, Zsuzsa; Shushi, Liat; Kerem, Batsheva; Kerem, Eitan

doi:10.1056/nejmoa022170

Cited by 463 publications

(344 citation statements)

References 24 publications

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“…This is being critically evaluated for cystic fibrosis (13)(14)(15)(16) and muscular dystrophy (17)(18)(19)(20)(21)(22). After animal studies, gentamicin-induced CFTR protein was recently reported to improve transmembrane conductance across the nasal mucosa in a group of 10 patients with homozygous PTC mutations in the CTRF gene; patients with the more common del508F mutation were used as controls and showed no improvement of conductance (16). Studies treating mdx mice with gentamicin were reported to restore dystrophin function in skeletal muscle (17).…”

Section: Discussionmentioning

confidence: 99%

Correction of ATM gene function by aminoglycoside-induced read-through of premature termination codons

Lai

Chun

Nahas

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

146

102

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Cells were irradiated with 10 Gy and were incubated for 45 min at 37°C before fixation on coverslips. ATM pS1981 IRIFs were not observed in untreated cells; maximum intensity of staining was seen in the nuclei of cells exposed to 75 g͞ml. Nonirradiated cells, untreated or exposed to comparable concentrations of geneticin, showed only occasional foci. A taxia-telangiectasia (A-T) is an autosomal recessive neurodegenerative disorder with onset in early childhood, resulting from mutations in the A-T mutated (ATM) gene (1). The ATM protein is a hierarchical serine-threonine kinase, phosphorylating many substrates involved in repair of doublestranded DNA breaks, control of cell cycle checkpoints, and responses to oxidative stress, as well as in radiosensitivity, cancer susceptibility, immune function, and neurological development (2). ATM protein levels are undetectable in the cells of most A-T patients by conventional testing. However a few patients, often with a milder phenotype, have some detectable ATM protein (3,4). This finding encouraged us to seek compounds, such as aminoglycosides, that have the potential to read through premature termination codons and restore ATM protein function (5, 6).To test these effects, we selected 13 lymphoblastoid cell lines (LCLs) with primary premature termination codon (PTC) mutations from a repository of Ͼ400 lines derived from A-T patients. Herein, we show representative data from 5 of the 13 LCLs. Using protein truncation testing (PTT) driven by plasmid templates containing PTC mutations in the ATM gene (7), we observed in vitro read-through effects of various magnitudes with three of four aminoglycosides tested. Full-length ATM protein was also documented ex vivo by immunoprecipitation. Correction of radioresistant DNA synthesis and radiosensitivity, as well as autophosphorylation of ATM, suggested that this readthrough produces functional ATM protein. Experimental ProceduresCell Lines. Lymphoblastoid cell lines were maintained in RPMI medium 1640 (Invitrogen) with 15% FBS (HyClone) and 1% penicillin͞streptomycin (Invitrogen) at 37°C and 5% CO 2 . In ex vivo experiments, aminoglycosides were added daily into culture media at the indicated doses and times. Because streptomycin is also an aminoglycoside that can perturb proofreading by binding to another ribosomal site, ex vivo experiments were performed with or without streptomycin; no differences were noted.Mutations. We selected only PTC mutations that resulted directly from the mutation, i.e., primary PTC mutations, and not those caused indirectly by upstream mutations or aberrant splicing, because the latter would have no potential therapeutic benefits. The LCLs carried the following mutations (www.benaroyaresearch.org͞bri investigators͞atm.htm): AT185LA, homozygous 3673C 3 T (a UAA G stop codon in PTT fragment 4); TAT51, homozygous 5623C 3 T (a UGA C stop codon in PTT fragment 5); AT187LA, homozygous 5908C 3 T (a UAA G stop codon in PTT fragment 6); AT153LA, homozygous 8977C 3 T (a UGA A stop codon in fragment 8); and AT...

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Section: Discussionmentioning

confidence: 99%

Correction of ATM gene function by aminoglycoside-induced read-through of premature termination codons

Lai

Chun

Nahas

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

146

102

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“…The importance of NMD has led to efforts to develop therapeutic agents that modulate this process, especially whereby suppression of a disease-related mutation that normally triggers NMD permits translation read-through and partial restoration of critical protein products (24,25). NMD also plays an important role in the posttranscriptional regulation of numerous RNA binding and processing factors (14,15,18,26).…”

Section: Discussionmentioning

confidence: 99%

Smg1 is required for embryogenesis and regulates diverse genes via alternative splicing coupled to nonsense-mediated mRNA decay

McIlwain

Pan

Reilly

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

162

166

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Smg1 is a PI3K-related kinase (PIKK) associated with multiple cellular functions, including DNA damage responses, telomere maintenance, and nonsense-mediated mRNA decay (NMD). NMD degrades transcripts that harbor premature termination codons (PTCs) as a result of events such as mutation or alternative splicing (AS). Recognition of PTCs during NMD requires the action of the Upstream frameshift protein Upf1, which must first be phosphorylated by Smg1. However, the physiological function of mammalian Smg1 is not known. By using a gene-trap model of Smg1 deficiency, we show that this kinase is essential for mouse embryogenesis such that Smg1 loss is lethal at embryonic day 8.5. High-throughput RNA sequencing (RNA-Seq) of RNA from cells of Smg1-deficient embryos revealed that Smg1 depletion led to pronounced accumulation of PTC-containing splice variant transcripts from approximately 9% of genes predicted to contain AS events capable of eliciting NMD. Among these genes are those involved in splicing itself, as well as genes not previously known to be subject to AS-coupled NMD, including several involved in transcription, intracellular signaling, membrane dynamics, cell death, and metabolism. Our results demonstrate a critical role for Smg1 in early mouse development and link the loss of this NMD factor to major and widespread changes in the mammalian transcriptome.gene trap | Smg-1

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“…In this study, 19 CF patients were either homozygous (n511) or heterozygous (n58) for CFTR stop mutations and a total of five patients were homozygous for the Class II mutation F508del [39]. Gentamicin was shown to significantly reduce the nasal potential difference (a measure of CFTR function) in those patients with stop mutations (from basal potential difference 45¡8 to 34¡11 mV, p50.005), whereas no significant difference was seen in patients homozygous for F508del [39]. Positive effects were also demonstrated by SERMET-GAUDELUS et al [40], although no effect was noted in another study [41].…”

Section: Cftr Modulatorsmentioning

confidence: 99%

Targeting a genetic defect: cystic fibrosis transmembrane conductance regulator modulators in cystic fibrosis

Derichs

2013

Eur Respir Rev

105

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Cystic fibrosis (CF) is caused by genetic mutations that affect the cystic fibrosis transmembrane conductance regulator (CFTR) protein. These mutations can impact the synthesis and transfer of the CFTR protein to the apical membrane of epithelial cells, as well as influencing the gating or conductance of chloride and bicarbonate ions through the channel. CFTR dysfunction results in ionic imbalance of epithelial secretions in several organ systems, such as the pancreas, gastrointestinal tract, liver and the respiratory system. Since discovery of the CFTR gene in 1989, research has focussed on targeting the underlying genetic defect to identify a disease-modifying treatment for CF. Investigated management strategies have included gene therapy and the development of small molecules that target CFTR mutations, known as CFTR modulators. CFTR modulators are typically identified by high-throughput screening assays, followed by preclinical validation using cell culture systems. Recently, one such modulator, the CFTR potentiator ivacaftor, was approved as an oral therapy for CF patients with the G551D-CFTR mutation. The clinical development of ivacaftor not only represents a breakthrough in CF care but also serves as a noteworthy example of personalised medicine.

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Gentamicin-Induced Correction of CFTR Function in Patients with Cystic Fibrosis andCFTRStop Mutations

Cited by 463 publications

References 24 publications

Correction of ATM gene function by aminoglycoside-induced read-through of premature termination codons

Correction of ATM gene function by aminoglycoside-induced read-through of premature termination codons

Smg1 is required for embryogenesis and regulates diverse genes via alternative splicing coupled to nonsense-mediated mRNA decay

Targeting a genetic defect: cystic fibrosis transmembrane conductance regulator modulators in cystic fibrosis

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