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

Ophélia Granio; Caroline Norez; Katherine J.D Ashbourne Excoffon; Philip H Karp; Monika Lusky; Frédéric Becq; Pierre Boulanger; Joseph Zabner; Saw See Hong

doi:10.1165/rcmb.2007-0026TE

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

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

Ophélia Granio, Caroline Norez, Katherine J.D Ashbourne Excoffon, Philip H Karp, Monika Lusky, Frédéric Becq, Pierre Boulanger, Joseph Zabner and Saw See Hong

American journal of respiratory cell and molecular biology, Vol.37(6), pp.631-639

06/08/2007

DOI: 10.1165/rcmb.2007-0026TE

PMID: 17641299

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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 CFKM4 (CFTRDF508), 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-CFTRDF508. 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-CFTRDF508 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 Cl2 channel function. In reconstituted CF-HAE, GFP-CFTRwt appeared as active as the nontagged CFTRwt protein in correcting the transepithelial Cl2 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.

Life Sciences

Human health and pathology

Molecular biology

Biochemistry, Molecular Biology

Tissues and Organs

Details

Title: Subtitle: Cellular Localization and Activity of Ad-Delivered GFP-CFTR in Airway Epithelial and Tracheal Cells
Creators: Ophélia Granio - Virologie et pathologie humaine
Caroline Norez - Institut de physiologie et biologie cellulaires
Katherine J.D Ashbourne Excoffon - Department of Internat Medicine
Philip H Karp - Department of Internat Medicine
Monika Lusky - TRANSGENE SA
Frédéric Becq - Institut de physiologie et biologie cellulaires
Pierre Boulanger - Laboratoire de Virologie Médicale
Joseph Zabner - Department of Internat Medicine
Saw See Hong - Virologie et pathologie humaine
Resource Type: Journal article
Publication Details: American journal of respiratory cell and molecular biology, Vol.37(6), pp.631-639
DOI: 10.1165/rcmb.2007-0026TE
PMID: 17641299
NLM abbreviation: Am J Respir Cell Mol Biol
ISSN: 1044-1549
eISSN: 1535-4989
Publisher: American Thoracic Society
Language: English
Date published: 06/08/2007
Academic Unit: Pulmonary, Critical Care, and Occupational Medicine; Internal Medicine
Record Identifier: 9984094547902771

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