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Published ahead of print on July 19, 2007, doi:10.1165/rcmb.2007-0026TE

Am. J. Respir. Cell Mol. Biol., Volume 37, Number 6, December 2007, 631-639

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Submitted on January 29, 2007
Revised on July 19, 2007

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

Ophelia Granio1, Caroline Norez2, Katherine J.D. Ashbourne Excoffon3, Philip H Karp4, Monika Lusky5, Frederic Becq2, Pierre A Boulanger6, Joseph Zabner3, and Saw-See Hong1*

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

* To whom correspondence should be addressed. E-mail: sawsee.hong{at}sante.univ-lyon1.fr.

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 which determines its function in cells. The aim of this study was to develop an Ad vector expressing a biologically active GFP-CFTR chimera which can be tracked by both its localization and chloride channel function. No study thus far, has demonstrated a GFP-CFTR construct which displayed both of these functions in the airway epithelia. Tracheal glandular cells, MM39 (CFTRwt) and CF-KM4 (CFTR{Delta}F508), as well as human airway epithelial cells from a CF patient (CF-HAE) and a healthy donor (HAE) were used for the functional analysis of our Ad vectors, Ad5/GFP-CFTRwt and Ad5/GFP-CFTR{Delta}F508. 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, whilst GFP-CFTR{Delta}F508 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 following Ad-transduction. A correlation between the proportion of Ad5-transduced CF-KM4 cells and correction of CFTR function showed that 55-70% transduction resulted in 70% correction of the Cl- channel function. In reconstituted CF-HAE, GFP-CFTRwt appeared as active as the non-tagged CFTRwt protein in correcting the transepithelial Cl- transport. We show for the first time a GFP-CFTR chimera which localized to the apical surface of human airway epithelia and restored epithelial chloride transport to similar levels as non-tagged CFTR.




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