title: Functional characterization of a recombinant adeno-associated virus 5-pseudotyped cystic fibrosis transmembrane conductance regulator vector.
abstract: Despite extensive experience with recombinant adeno-associated virus (rAAV) 2 vectors in the lung, gene expression has been low in the context of cystic fibrosis (CF) gene therapy, where the large size of the cystic fibrosis transmembrane conductance regulator (CFTR) coding sequence has prompted the use of compact endogenous promoter elements. We evaluated the possibility that gene expression from recombinant adeno-associated virus (rAAV) could be improved by using alternate AAV capsid serotypes that target different cell-surface receptors (i.e., rAAV5) and/or using stronger promoters. The relative activities of the cytomegalovirus (CMV) Rous sarcoma virus (RSV) promoter, the CMV enhancer/beta-actin (CB) promoter combination, and the CMV enhancer/RSV promoter hybrid were assessed in vitro in a CF bronchial cell line. The CB promoter was the most efficient. AAV capsid serotypes, rAAV2 and rAAV5, were also compared, and rAAV5 was found to be significantly more efficient. Based on these studies a rAAV5-CB-promoter-driven CFTR minigene vector was then used to correct the CF chloride transport defect in vitro, as well as the hyperinflammatory lung phenotype in Pseudomonas-agarose bead challenged CF mouse lungs in vivo. These studies provide functional characterization of a new version of rAAV-CFTR vectors.
authors.affiliation: Powell Gene Therapy Center, UF Genetics Institute, Department of Pediatrics and Molecular Genetics and Microbiology, University of Florida College of Medicine, Gainesville, FL 32619-0296, USA.
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Sirninger
authors.forename: Christian
authors.initials: C
authors.lastname: Muller
authors.forename: Sofia
authors.initials: S
authors.lastname: Braag
authors.forename: Qiushi
authors.initials: Q
authors.lastname: Tang
authors.forename: Hungwen
authors.initials: H
authors.lastname: Yue
authors.forename: Carol
authors.initials: C
authors.lastname: Detrisac
authors.forename: Thomas
authors.initials: T
authors.lastname: Ferkol
authors.forename: William B
authors.initials: WB
authors.lastname: Guggino
authors.forename: Terence R
authors.initials: TR
authors.lastname: Flotte
mesh_terms: D000199:Actins / Q000235:genetics; D000818:Animals; D018118:Chloride Channels / Q000187:drug effects; D003001:Cloning, Molecular; D003550:Cystic Fibrosis / Q000235:genetics / Q000628:therapy*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics*; D004274:DNA, Recombinant / Q000627:therapeutic use; D000229:Dependovirus / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D015316:Genetic Therapy; D005822:Genetic Vectors; D008156:Luciferases / Q000032:analysis / Q000235:genetics; D051379:Mice; D011014:Pneumonia / Q000382:microbiology / Q000473:pathology; D011401:Promoter Regions, Genetic; D011550:Pseudomonas aeruginosa / Q000472:pathogenicity; D015431:Weight Loss
chemical_list: D000199:Actins; D018118:Chloride Channels; D004274:DNA, Recombinant; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D008156:Luciferases