title: Aminoglycoside suppression of a premature stop mutation in a Cftr-/- mouse carrying a human CFTR-G542X transgene.
abstract: Cystic fibrosis (CF) is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Since approximately 5% of all mutant CF alleles are stop mutations, it can be calculated that approximately 10% of CF patients carry a premature stop mutation in at least one copy of the CFTR gene. Certain ethnic groups, such as the Ashkenazi Jewish population, carry a much higher percentage of CF stop mutations. Consequently, a therapeutic strategy aimed at suppressing this class of mutation would be highly desirable for the treatment of this common genetic disease. We have shown previously that aminoglycoside antibiotics can suppress premature stop mutations in the CFTR gene in a bronchial epithelial cell line [Nat Med (1997) 3:1280]. To address whether aminoglycosides can suppress a CFTR premature stop mutation in an animal model, we constructed a transgenic mouse with a null mutation in the endogenous CFTR locus (Cftr-/-) that also expressed a human CFTR-G542X cDNA under control of the intestinal fatty acid binding protein promoter. We then investigated whether the daily administration of the aminoglycoside antibiotics gentamicin or tobramycin could restore the expression of a detectable level of CFTR protein. Immunofluorescence staining of intestinal tissues from Cftr-/- hCFTR-G542X mice revealed that gentamicin treatment resulted in the appearance of hCFTR protein at the apical surface of the glands of treated mice. Weaker staining was also observed in the intestinal glands following tobramycin treatment. Short-circuit current measurements made on intestinal tissues from these mice demonstrated that a significant number of positive cAMP-stimulated transepithelial chloride current measurements could be observed following gentamicin treatment (P=0.008) and a near significant number following tobramycin treatment (P=0.052). When taken together, these results indicate that gentamicin, and to a lesser extent tobramycin, can restore the synthesis of functional hCFTR protein by suppressing the hCFTR-G542X premature stop mutation in vivo.
authors.affiliation: Department of Microbiology, The University of Alabama at Birmingham, Birmingham, AL 35294-2170, USA.
authors.forename: Ming
authors.initials: M
authors.lastname: Du
authors.forename: Julie R
authors.initials: JR
authors.lastname: Jones
authors.forename: Jessica
authors.initials: J
authors.lastname: Lanier
authors.forename: Kim M
authors.initials: KM
authors.lastname: Keeling
authors.forename: J Russell
authors.initials: JR
authors.lastname: Lindsey
authors.forename: Albert
authors.initials: A
authors.lastname: Tousson
authors.forename: Zsuzsa
authors.initials: Z
authors.lastname: Bebök
authors.forename: Jeffrey A
authors.initials: JA
authors.lastname: Whitsett
authors.forename: Chitta R
authors.initials: CR
authors.lastname: Dey
authors.forename: William H
authors.initials: WH
authors.lastname: Colledge
authors.forename: Martin J
authors.initials: MJ
authors.lastname: Evans
authors.forename: Eric J
authors.initials: EJ
authors.lastname: Sorscher
authors.forename: David M
authors.initials: DM
authors.lastname: Bedwell
mesh_terms: D000200:Action Potentials / Q000187:drug effects; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D005576:Colforsin / Q000494:pharmacology; D003550:Cystic Fibrosis / Q000235:genetics* / Q000473:pathology; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics*; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism; D015870:Gene Expression; D005839:Gentamicins / Q000008:administration & dosage / Q000097:blood / Q000494:pharmacology; D006579:Heterozygote; D006801:Humans; D007413:Intestinal Mucosa / Q000187:drug effects / Q000473:pathology; D051379:Mice; D018345:Mice, Knockout; D008822:Mice, Transgenic; D009154:Mutation; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D013489:Suppression, Genetic; D014031:Tobramycin / Q000008:administration & dosage / Q000494:pharmacology; D019076:Transgenes
chemical_list: D000900:Anti-Bacterial Agents; C505032:CFTR protein, human; D005839:Gentamicins; D012333:RNA, Messenger; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D005576:Colforsin; D014031:Tobramycin