Adenovirus 5–Fiber 35 Chimeric Vector Mediates Efficient Apical Correction of the Cystic Fibrosis Transmembrane Conductance Regulator Defect in Cystic Fibrosis Primary Airway Epithelia

Abstract

In vivo gene transfer to the human respiratory tract by adenovirus serotype 5 (Ad5) vectors has revealed their limitations related to inefficient gene transfer, host antiviral response, and innate adenoviral toxicity. In the present work, we compared the cytotoxicity and efficiency of Ad5 and a chimeric Ad5F35 vector with respect to CFTR gene transfer to cystic fibrosis (CF) and non-CF human airway epithelial cells. We found that high doses of Ad5 vector had an adverse effect on the function of exogenous and endogenous CFTR. Results obtained with Ad5 capsid mutants suggested that the RGD motifs on the penton base capsomers were responsible for the negative effect on CFTR function. This negative interference did not result from a lower level of biosynthesis and/or altered cellular trafficking of the CFTR protein, but rather from an indirect mechanism of functional blockage of CFTR, related to the RGD integrin-mediated endocytic pathway of Ad5. No negative interference with CFTR was observed for Ad5F35, an Ad5-based vector pseudotyped with fibers from Ad35, a serotype that uses another cell entry pathway. In vitro, Ad5F35 vector expressing the GFP-tagged CFTR (Ad5F35-GFP-CFTR) showed a 30-fold higher efficiency of transduction and chloride channel correction in CFTR-deficient cells, compared with Ad5GFP-CFTR. Ex vivo, Ad5F35-GFP-CFTR had the capacity to transduce efficiently reconstituted airway epithelia from patients with CF (CF-HAE) via the apical surface, restored chloride channel function at relatively low vector doses, and showed relatively stable expression of GFP-CFTR for several weeks.

Pseudomonas aeruginosa–Induced Apoptosis in Airway Epithelial Cells Is Mediated by Gap Junctional Communication in a JNK-Dependent Manner

“…Calu-3 cells were maintained in DMEM:F12 (3:1 v/v), supplemented with 10% FCS, 30 U/ml penicillin, and 30 mg/ml streptomycin (Life Technologies). The human nasal epithelial CF 15 cell line, which was derived from a patient homozygous for the F508del mutations of CFTR, was maintained in the same medium supplemented with growth factors, as previously described (13). To obtain a well-polarized cell monolayer, 100,000 Airway cell infection with P. aeruginosa bacterial strains Cells were infected with the well-characterized P. aeruginosa laboratory strain PAO1.…”

“…The chimeric vector HAdV5F35-GFP-CFTR has been previously described (15). HAdV5F35-GFP-CFTR encoded the wild-type allele of the CFTR gene fused to the 39 end of the GFP gene.…”

“…6C). To demonstrate the contribution of CFTR in the regulation of JNK signaling, we measured p-JNK signals in the CF airway CF 15 cell line before and after correction of the CF phenotype by adenoviral-mediated expression of wild-type CFTR (Supplemental Fig. 3E).…”

Cystic Fibrosis - Renewed Hopes Through Research

“…This chimeric vector transduced efficiently well-differentiated human airway epithelium via the apical membrane and showed stable expression of the GFP-CFTR protein. Measurements of transepithelial ion transport showed the correction of the chloride channel function at relatively low vector doses in ∆F508 CF airway epithelial cells (Granio et al, 2010). This is a successful example of a viral vector which was genetically modified to target a receptor on the apical surface of the airway epithelial cells for efficient gene transduction.…”

“…The GFP-CFTR fusion constructs have also been inserted into viral vectors such as Adenovirus (Vais, 2004;Granio et al, 2007;Granio et al, 2010) and Sendai virus (Ban et al, 2007) to facilitate the detection and direct tracking of the protein after gene transfer. When the Ad vectors, Ad5-GFP-CFTR and Ad5-GFP-CFTR∆F508, were used to transduce reconstituted airway epithelium from ∆F508 CF patients, the biologically active GFP-CFTR and the mutant GFP-CFTR∆F508 proteins could be directly tracked in the epithelial cells by confocal fluorescence microscopy due to their GFP-tag (Granio et al, 2007 ;see Figure 2, A and B).…”