title: Expression of the cystic fibrosis transmembrane conductance regulator from a novel adeno-associated virus promoter.
abstract: Adeno-associated virus type 2 (AAV) vectors have been used for gene expression in respiratory epithelial cells and may be useful in gene therapy for diseases like cystic fibrosis (CF) which affect the airways. The AAV p5 promoter together with the AAV inverted terminal repeat (ITR) forms a 263-base pair cassette which mediated efficient expression in a CF bronchial epithelial cell line. We report here that the ITR itself can mediate gene expression. In stable transfection assays, AAV-CF vectors expressing the full-length cystic fibrosis transmembrane conductance regulator (CFTR) cDNA from either the p5 promoter or the ITR restored cAMP regulation of the chloride efflux characteristic of CFTR function. An AAV-ITR-CF vector deleted for the amino terminus of CFTR was also functional. This vector was packaged into AAV particles and used to transduce cells without selection. Transduced cells also exhibited cAMP-regulated Cl- efflux. The complemented cell lines showed increased levels of CFTR protein immunofluorescence, and the presence of intact AAV-CF vector sequence was confirmed by Southern blot analysis of rescued vector sequences. These studies provide novel insights into AAV gene expression, and this newly described promoter allows for the production of AAV vectors expressing CFTR in those differentiated cells affected in CF.
authors.affiliation: Laboratory of Molecular and Cellular Biology, National Institute for Diabetes, Digestive, and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892.
authors.forename: T R
authors.initials: TR
authors.lastname: Flotte
authors.forename: S A
authors.initials: SA
authors.lastname: Afione
authors.forename: R
authors.initials: R
authors.lastname: Solow
authors.forename: M L
authors.initials: ML
authors.lastname: Drumm
authors.forename: D
authors.initials: D
authors.lastname: Markakis
authors.forename: W B
authors.initials: WB
authors.lastname: Guggino
authors.forename: P L
authors.initials: PL
authors.lastname: Zeitlin
authors.forename: B J
authors.initials: BJ
authors.lastname: Carter
mesh_terms: D001483:Base Sequence; D002460:Cell Line; D002712:Chlorides / Q000378:metabolism; D003550:Cystic Fibrosis / Q000235:genetics; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D000229:Dependovirus / Q000235:genetics*; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D007700:Kinetics; D008565:Membrane Proteins / Q000032:analysis / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods; D011401:Promoter Regions, Genetic; D015398:Signal Transduction; D014162:Transfection
chemical_list: C505032:CFTR protein, human; D002712:Chlorides; D008565:Membrane Proteins; D009838:Oligodeoxyribonucleotides; D019005:Cystic Fibrosis Transmembrane Conductance Regulator