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Cellular Localization and Activity of Ad-Delivered GFP-CFTR in Airway Epithelial and Tracheal Cells

¹ Laboratoire de Virologie et Pathologie Humaine, Université de Lyon 1 and CNRS FRE 3011, Faculté de Médecine Laennec and IFR Lyon-Est, Lyon, France; ² Institut de Physiologie et Biologie Cellulaires, CNRS UMR 6187, Université de Poitiers, Poitiers, France; ³ Department of Internal Medicine and ⁴ Howard Hughes Medical Institute, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa; ⁵ Transgene SA, Strasbourg, France; and ⁶ Laboratoire de Virologie Médicale, Centre de Biologie et Pathologie Est, Hospices Civils de Lyon, Bron, France

Correspondence and requests for reprints should be addressed to Saw-See Hong, Ph.D., Laboratoire de Virologie et Pathologie Humaine, CNRS FRE 3011, Faculté de Médecine RTH Laennec, 7 rue Guillaume Paradin, 69372 Lyon, France. E-mail: sawsee.hong{at}sante.univ-lyon1.fr

Abstract

Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, and the cellular trafficking of the CFTR protein is an essential factor that determines its function in cells. The aim of our study was to develop an Ad vector expressing a biologically active green fluorescent protein (GFP)-CFTR chimera that can be tracked by both its localization and chloride channel function. No study thus far has demonstrated a GFP-CFTR construct that displayed both of these functions in the airway epithelia. Tracheal glandular cells, MM39 (CFTRwt) and CF-KM4 (CFTRF508), as well as human airway epithelial cells from a patient with cystic fibrosis (CF-HAE) and from a healthy donor (HAE) were used for the functional analysis of our Ad vectors, Ad5/GFP-CFTRwt and Ad5/GFP-CFTRF508. The GFP-CFTRwt protein expressed was efficiently addressed to the plasma membrane of tracheal cells and to the apical surface of polarized CF-HAE cells, while GFP-CFTRF508 mutant was sequestered intracellularly. The functionality of the GFP-CFTRwt protein was demonstrated by its capacity to correct the chloride channel activity both in CF-KM4 and CF-HAE cells after Ad transduction. A correlation between the proportion of Ad5-transduced CF-KM4 cells and correction of CFTR function showed that 55 to 70% transduction resulted in 70% correction of the Cl^– channel function. In reconstituted CF-HAE, GFP-CFTRwt appeared as active as the nontagged CFTRwt protein in correcting the transepithelial Cl^– transport. We show for the first time a GFP-CFTR chimera that localized to the apical surface of human airway epithelia and restored epithelial chloride transport to similar levels as nontagged CFTR.

Key Words: cystic fibrosis • GFP-CFTR • Cl^– channel • adenovirus • CFTR-deficient airway epithelia

CLINICAL RELEVANCE We show a green fluorescent protein–cystic fibrosis transmembrane conductance regulator chimera that localizes to the apical surface of the airway epithelia and restored the Cl– transport. The cystic fibrosis transmembrane conductance regulator protein can now be tracked in human epithelia which will allow rapid translation of therapeutic interventions for cystic fibrosis.