In Vitro Assessment of Variables Affecting the Efficiency and Efficacy of Adenovirus-Mediated Gene Transfer to Cystic Fibrosis Airway Epithelia
- 1 January 1996
- journal article
- research article
- Published by Mary Ann Liebert Inc in Human Gene Therapy
- Vol. 7 (1) , 51-59
- https://doi.org/10.1089/hum.1996.7.1-51
Abstract
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) (103), 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 β-galactosidase (β-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/β-actin (CB) promoters transduced intermediate levels of β-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 ≥6–10% of cells in the epithelial sheet. Ad5-CBCFTR transduction of CF airway epithelial cells (moi = 103) 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. Clinical gene transfer safety and efficacy trials of adenovirus (Ad)-mediated transduction of cystic fibrosis transmembrane conductance regulator (CFTR) to airway epithelia of CF patients have been initiated in the United States. In this study, we quantitatively evaluate the effects of variables derived from these clinical trials on the efficiency and efficacy of Ad-mediated gene transfer to CF airway epithelia using an in vitro model system. The data presented suggest that the variables studied have important effects on gene transfer efficiency and efficacy. These data may also permit comparisons among various clinical trials and better assessment of CFTR gene transfer efficiency and efficacy.Keywords
This publication has 25 references indexed in Scilit:
- Gene Therapy for Cystic Fibrosis Utilizing a Replication Deficient Recombinant Adenovirus Vector to Deliver the Human Cystic Fibrosis Transmembrane Conductance Regulator cDNA to the Airways. A Phase I Study. Children's Hospital Medical Center, Cincinnati, OhioHuman Gene Therapy, 1994
- Gene Therapy for Cystic Fibrosis Using E1-Deleted Adenovirus: A Phase I Trial in the Nasal Cavity. University of North Carolina at Chapel Hill, Chapel Hill, North CarolinaHuman Gene Therapy, 1994
- Gene Therapy of Cystic Fibrosis Lung Disease Using E1 Deleted Adenoviruses: A Phase I Trial. University of Michigan, Ann Arbor, Michigan and University of Pennsylvania, Philadelphia, PennsylvaniaHuman Gene Therapy, 1994
- Development and Analysis of Recombinant Adenoviruses for Gene Therapy of Cystic FibrosisHuman Gene Therapy, 1993
- Direct gene transfer of human CFTR into human bronchial epithelia of xenografts with E1–deleted adenovirusesNature Genetics, 1993
- Widespread long-term gene transfer to mouse skeletal muscles and heart.Journal of Clinical Investigation, 1992
- Correction of the Apical Membrane Chloride Permeability Defect in Polarized Cystic Fibrosis Airway Epithelia Following Retroviral-Mediated Gene TransferHuman Gene Therapy, 1992
- In vivo transfer of the human cystic fibrosis transmembrane conductance regulator gene to the airway epitheliumCell, 1992
- Expression of the cystic fibrosis gene in non-epithelial invertebrate cells produces a regulated anion conductanceCell, 1991
- Expression of cystic fibrosis transmembrane conductance regulator corrects defective chloride channel regulation in cystic fibrosis airway epithelial cellsNature, 1990