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) (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.

Gene transfer of CFTR to airway epithelia: low levels of expression are sufficient to correct Cl−transport and overexpression can generate basolateral CFTR

“…We used an adenovirus in which the very strong CMV promoter drove the CFTR cDNA (51) and delivered it to the basolateral surface of well-differentiated non-CF airway epithelia. Our earlier work as well as that of others showed that this procedure produced apically localized and functional CFTR in CF epithelia (17,42). Three days later, we measured transepithelial Cl Ϫ current under short-circuit conditions (Fig.…”

Adenovirus-based vascular endothelial growth factor gene delivery to human pancreatic islets

“…20 8,33 Variation in vector MOI, vector promoters, concentration of vector-containing medium and the duration of vector incubation time are known to affect the transfection efficiency of adenovrial vectors in several cell types. 34,35 Therefore, as a first attempt, we sought to optimize the transfection conditions of human islets with Adv-GFP-hVEGF. The GFP intensity of islets after transfection with Adv-GFP-hVEGF increased with increase in MOI from 50 to 5000 (Figure 2), suggesting efficient transfection at high MOI.…”

Evidence that Systemic Gentamicin Suppresses Premature Stop Mutations in Patients with Cystic Fibrosis

“…As has been reported in other human studies to improve CFTR function or to transfer cftr to CF-affected tissues, Na ϩ transport as measured by the nasal PD was unaffected by gentamicin treatment (Figures 4 and 5). This may reflect the higher sensitivity of measurable Cl Ϫ secretion in the context of low-level CFTR activity, compared with attenuation of Na ϩ hyperabsorption (15,24,30,(32)(33)(34)(35)(36)(37).…”