n engl j med 349;15 www.nejm.org october 9, 2003 The new england journal of medicine 1433 original article Gentamicin-Induced Correction of CFTR Function in Patients with Cystic Fibrosis and CFTR Stop Mutations Michael Wilschanski, M.D., Yaacov Yahav, M.D., Yasmin Yaacov, B.Sc.,Hannah Blau, M.D., Lea Bentur, M.D., Joseph Rivlin, M.D., Micha Aviram, M.D., Tali Bdolah-Abram, M.Sc., Zsuzsa Bebok, M.D., Liat Shushi, M.Sc., Batsheva Kerem, Ph.D., and Eitan Kerem, M.D. From the Department of Pediatrics, CysticFibrosis Center, Shaare Zedek MedicalCenter (M.W., Y. Yaacov, E.K.), and Hadas-sah University Hospital, Mount Scopus(E.K.), Hebrew University Medical School,Jerusalem, Israel; the Cystic Fibrosis Cen-ter, Sheba Medical Center, Tel Hashomer,Israel (Y. Yahav); the Graub Cystic FibrosisCenter, Schneider Children’s Medical Cen-ter of Israel, Petah Tikva, Israel (H.B.); theCystic Fibrosis Center, Rambam MedicalCenter, Haifa, Israel (L.B.); the Cystic Fibro-sis Center, Carmel Medical Center, Haifa,Israel (J.R.); the Cystic Fibrosis Center,Soroka Medical Center, Beer Sheva, Israel(M.A.); the Department of Medical Statis-tics (T.B.-A.) and the Department of Genet-ics, Life Sciences Institute (L.S., B.K.), He-brew University, Jerusalem, Israel; and theDepartment of Cell Biology and the Grego-ry Fleming James Cystic Fibrosis ResearchCenter, University of Alabama at Birming-ham, Birmingham (Z.B.).N Engl J Med 2003;349:1433-41. Copyright © 2003 Massachusetts Medical Society. background Mutations in the cystic fibrosis transmembrane conductance regulator ( CFTR ) genecontaining a premature termination signal cause a deficiency or absence of functionalchloride-channel activity. Aminoglycoside antibiotics can suppress premature termi-nation codons, thus permitting translation to continue to the normal end of the tran-script. We assessed whether topical administration of gentamicin to the nasal epithe-lium of patients with cystic fibrosis could result in the expression of functional CFTRchannels. methods In a double-blind, placebo-controlled, crossover trial, patients with stop mutations in CFTR or patients homozygous for the ∆ F508 mutation received two drops containinggentamicin (0.3 percent, or 3 mg per milliliter) or placebo in each nostril three timesdaily for two consecutive periods of 14 days. Nasal potential difference was measuredat base line and after each treatment period. Nasal epithelial cells were obtained be-fore and after gentamicin treatment from patients carrying stop mutations, and theC-terminal of surface CFTR was stained. results Gentamicin treatment caused a significant reduction in basal potential difference inthe 19 patients carrying stop mutations (from ¡45 ± 8 to ¡34 ± 11 mV, P=0.005) and a sig-nificant response to chloride-free isoproterenol solution (from 0 ± 3.6 to ¡5 ± 2.7 mV,P<0.001). This effect of gentamicin on nasal potential difference occurred both in pa-tients who were homozygous for stop mutations and in those who were heterozygous,but not in patients who were homozygous for ∆ F508. After gentamicin treatment, a sig-nificant increase in peripheral and surface staining for CFTR was observed in the nasalepithelial cells of patients carrying stop mutations. conclusions In patients with cystic fibrosis who have premature stop codons, gentamicin can causetranslational “read through,” resulting in the expression of full-length CFTR protein atthe apical cell membrane, and thus can correct the typical electrophysiological abnor-malities caused by CFTR dysfunction.abstractDownloaded from www.nejm.org at Stanford University on March 24, 2004.Copyright © 2003 Massachusetts Medical Society. All rights reserved.n engl j med 349;15 www.nejm.org october 9 , 2003 The new england journal of medicine 1434ystic fibrosis is caused by muta- tions in the cystic fibrosis transmembraneconductance regulator ( CFTR ) gene thatlead to dysfunction of the CFTR protein, which isan apical membrane protein regulating the trans-port of chloride and sodium in secretory epithelialcells. 1 Since the discovery of the CFTR gene, morethan 1000 mutations have been identified, includ-ing missense, deletion or insertion, frame shift,splice site, and nonsense mutations. 2 Nonsenseor stop mutations contain signals that cause a trun-cated or unstable protein, should result in a defi-ciency or absence of CFTR chloride channels, andare associated with a severe cystic fibrosis pheno-type in most cases. 3,4 In addition to their antimicrobial activity, ami-noglycoside antibiotics can suppress premature ter-mination codons by allowing an amino acid to beincorporated in place of the stop codon, thus per-mitting translation to continue to the normal endof the transcript. The mechanism of translation ter-mination is highly conserved among most organ-isms and is almost always signaled by an amber(UAG), ochre (UAA), or opal (UGA) terminationcodon. The nucleotide sequence surrounding thetermination codon has an important role in deter-mining the efficiency of translation termination. 5-7 Aminoglycoside antibiotics can reduce the fidel-ity of translation, 8 predominantly by inhibiting ri-bosomal “proofreading,” a mechanism to excludepoorly matched amino acyl–transfer RNA from be-coming incorporated into the polypeptide chain. 9 In this way aminoglycosides increase the frequencyof erroneous insertions at the nonsense codon andpermit translation to continue to the end of the gene,as has been shown in eukaryotic cells, 10 includinghuman fibroblasts. 11 The susceptibility to suppres-sion by aminoglycosides depends on the stop codonitself and on the sequences surrounding it.Howard et al. demonstrated that two CFTR -asso-ciated stop mutations could be suppressed by treat-ing cells with low doses of an aminoglycoside an-tibiotic. 12 Bedwell et al. demonstrated that afterincubation of bronchial epithelial cell line IB3-1,which carries a W1282X mutation of CFTR, with ami-noglycosides, cyclic AMP (cAMP)–activated chlo-ride conductance and the expression of function-al CFTR were restored to the apical membrane. 13 Recently, Zsembery et al. isolated cholangiocytesfrom the liver of a patient carrying the G542X mu-tation of CFTR and incubated them with gentami-cin, resulting in the expression of cAMP-activatedchloride transport. 14 Thus, in vitro, gentamicin ob-viated the effect of stop-codon mutations on thetranscription and translation of CFTR . This effecthas