Induction of a cAMP–Stimulated Chloride Secretion in Regenerating Poorly Differentiated Airway Epithelial Cells by Adenovirus-Mediated CFTR Gene Transfer

Abstract

In cystic fibrosis (CF), the airway epithelium is in the process of injury and regeneration. In the context of the CF gene therapy, we previously reported that regenerating poorly differentiated (PD) cells of human airway epithelium represent preferential cell targets for recombinant adenoviral gene vectors. To define whether PD non-CF and CF epithelial cells possess a functional cystic fibrosis transmembrane conductance regulator protein (CFTR) chloride channel, we analyzed the CFTR expression and the regulation of chloride secretion under cyclic (c)AMP stimulation in these regenerating PD epithelial cells of non-CF and CF airway tissue. Moreover, we studied the effects of CFTR gene transfer mediated by a replication-defective adenovirus containing the wild-type CFTR gene (AdCFTR) on CFTR expression and on cAMP-stimulated chloride secretion. Distribution of the CFTR protein was evaluated in regenerating PD airway cells by light fluorescence microscopy and scanning laser confocal microscopy. The cAMP-mediated regulation of cell membrane chloride secretion was investigated using the whole-cell patch clamp and SPQ (6-methoxy-N-[3-sulfopropyl]quinolinium) techniques. Compared with the absence of CFTR expression and cAMP-regulated chloride secretion in nontransduced regenerating PD cells of either non-CF or CF origin, transduction with AdCFTR induces a CFTR expression and a cAMP-regulated stimulation of the cell membrane chloride secretion in the regenerating PD cells. These results suggest that, out of the context of CF, remodeled and poorly differentiated airway epithelium may present abnormalities in ion transport. Moreover, our data suggest that, in the context of CF gene therapy, adenoviral vectors can be efficient in correcting, at least partially, the chloride secretion defect in the remodeled CF airway epithelium.

Overview summary

Gene therapy for cystic fibrosis (CF) requires delivery of the normal cystic fibrosis transmembrane conductance regulator (CFTR) gene to the CF airway epithelium. Therefore, gene transfer in human airways by adenoviral vectors has been shown to be inefficient in differentiated epithelial cells, and efficient in poorly differentiated cells. Because poorly differentiated airway epithelial cells are not a priori the cells required for CFTR gene transfer, we addressed the question whether these cells express CFTR and whether their preferential transduction in CF airways using an adenoviral vector may be relevant for the functional correction of the ion transport defect in CF. Here, we present in vitro experiments showing no CFTR expression in poorly differentiated epithelial cells of either non-CF or CF airways. We also demonstrate that CFTR gene expression in poorly differentiated airway epithelial cells of CF or non-CF origin, mimicking cells involved in airway regeneration, induces a cAMP-stimulated chloride secretion.