In Vitro Assessment of Variables Affecting the Efficiency and Efficacy of Adenovirus-Mediated Gene Transfer to Cystic Fibrosis Airway Epithelia

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.

Overview summary

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.