In vitro assessment of variables affecting the efficiency and efficacy of adenovirus-mediated gene transfer to cystic fibrosis airway epithelia.
Primary cultures of airway epithelia were used to evaluate variables pertinent to adenovirus (Ad)-mediated gene transfer efficiency and efficacy including: (i) Ad-vectors with different promoters, (ii) the duration of vector incubation with cells, (iii) the concentration and depth of vector-containing medium at constant multiplicity of infection (moi) (10(3)), and (iv) the relative sensitivity of reverse transcription polymerase chain reaction (RT-PCR) versus functional analysis for the detection of transduced cystic fibrosis transmembrane conductance regulator (CFTR). An Ad5-lacZ vector with a cytomegalovirus (CMV) enhancer/promoter transduced the greatest amount of beta-galactosidase (beta-Gal) activity, while an Ad2-lacZ vector with an E1a enhancer/promoter transduced the least. Ad5-lacZ vectors with the Rous sarcoma virus (RSV), E1a/RSV, or CMV enhancer/beta-actin (CB) promoters transduced intermediate levels of beta-Gal. Optimal gene transfer efficiency was detected with a 4-8 hr incubation of Ad5-CMVlacZ with cells, although optimal CFTR Cl-transport function was detectable after only a 30 min incubation of Ad5-CBCFTR with cells, consistent with correction of > or = 6-10% of cells in the epithelial sheet. Ad5-CBCFTR transduction of CF airway epithelial cells (moi = 10(3)) was optimal when higher concentrations, lower volumes, or smaller depths of vector-containing medium were utilized. RT-PCR was at least 100-fold more sensitive for the detection of transduced CFTR than functional analysis, and could detect as few as 0.001% Ad5-CBCFTR-infected CF cells admixed with uninfected CF cells. In summary, the variables studied clearly affect the efficiency of Ad-mediated gene transfer in vitro and potentially in vivo. They also suggest that RT-PCR is a poor marker of gene transfer efficiency and efficacy.
Johnson
L G
LG
CF/Pulmonary Research and Treatment Center, University of North Carolina at Chapel Hill 27599, USA.
Pickles
R J
RJ
Boyles
S E
SE
Morris
J C
JC
Ye
H
H
Zhou
Z
Z
Olsen
J C
JC
Boucher
R C
RC
D000260:Adenoviruses, Human / Q000235:genetics*; D001483:Base Sequence; D002460:Cell Line; D002478:Cells, Cultured; D002712:Chlorides / Q000378:metabolism; D003550:Cystic Fibrosis / Q000378:metabolism* / Q000473:pathology; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics*; D018076:DNA, Complementary; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D017136:Ion Transport; D007763:Lac Operon; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic
C505032:CFTR protein, human; D002712:Chlorides; D018076:DNA, Complementary; D019005:Cystic Fibrosis Transmembrane Conductance Regulator