Adenovirus-mediated generation of cAMP-stimulated Cl- transport in cystic fibrosis airway epithelia in vitro: effect of promoter and administration method.
Cystic fibrosis (CF) is a common autosomal recesses disease in which loss of CFTR-Cl- channel function of defective cAMP-stimulated Cl- transport transfer across airway epithelia. Recombinant adenoviruses have shown progress as vectors with which to transfer CFTR cDNA to CF airway epithelia. Here we investigated variables involved in adenovirus-mediated transfer of CFTR by measuring cAMP- stimulated Cl- transport in CF airway epithelia grown as monolayers on permeable filter supports. When we compared the effects of different promoters, we found that persistent correction of Cl- transport was obtained when the vector contained the E1a promoter, or to a lesser extent the PGK promoter. Vector containing the CMV promoter produced a greater initial cAMP-stimulated Cl- current, but the duration of correction was shorter and the infection procedure itself increased CFTR expression, suggesting that high input doses of virus stimulate expression. We compared the level of expression, measured with a beta-galactosidase reporter of CFTR mRNA, with CFTR-mediated Cl- transport. Even low levels of expression generated significant Cl- current and marked increases in expression produced only modest increments in Cl- current. Correction of the CF Cl- transport defect was also improved when the concentration of adenovirus vector was high and when the duration of contact with the epithelium was prolonged. These findings may help optimize the ability of adenovirus vectors encoding CFTR to correct the CF Cl- transport defect.
Zabner
J
J
Howard Hughes Medical Institute, University of Iowa College of Medicine, Iowa City 52242, USA.
Wadsworth
S C
SC
Smith
A E
AE
Welsh
M J
MJ
D000256:Adenoviridae; D002478:Cells, Cultured; D002712:Chlorides / Q000378:metabolism*; D003550:Cystic Fibrosis / Q000235:genetics* / Q000378:metabolism / Q000628:therapy; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics*; D018076:DNA, Complementary / Q000008:administration & dosage / Q000235:genetics; D004848:Epithelium / Q000378:metabolism; D018014:Gene Transfer Techniques; D015316:Genetic Therapy; D005822:Genetic Vectors; D006801:Humans; D017136:Ion Transport / Q000235:genetics; D014132:Trachea / Q000378:metabolism
C505032:CFTR protein, human; D002712:Chlorides; D018076:DNA, Complementary; D019005:Cystic Fibrosis Transmembrane Conductance Regulator