Gene transfer of CFTR to airway epithelia: low levels of expression are sufficient to correct Cl- transport and overexpression can generate basolateral CFTR

Sara L Farmen; Philip H Karp; Philip Ng; Donna J Palmer; David R Koehler; Jim Hu; Arthur L Beaudet; Joseph Zabner; Michael J Welsh

doi:10.1152/ajplung.00049.2005

Back

Gene transfer of CFTR to airway epithelia: low levels of expression are sufficient to correct Cl- transport and overexpression can generate basolateral CFTR

Journal article

Open access

Peer reviewed

Gene transfer of CFTR to airway epithelia: low levels of expression are sufficient to correct Cl- transport and overexpression can generate basolateral CFTR

Sara L Farmen, Philip H Karp, Philip Ng, Donna J Palmer, David R Koehler, Jim Hu, Arthur L Beaudet, Joseph Zabner and Michael J Welsh

American journal of physiology. Lung cellular and molecular physiology, Vol.289(6), pp.L1123-1130

12/2005

DOI: 10.1152/ajplung.00049.2005

PMID: 16085675

Files and links (1)

url

https://doi.org/10.1152/AJPLUNG.00049.2005View

Published (Version of record) Open Access

Abstract

Gene transfer of CFTR cDNA to airway epithelia is a promising approach to treat cystic fibrosis (CF). Most gene transfer vectors use strong viral promoters even though the endogenous CFTR promoter is very weak. To learn whether expressing CFTR at a low level in a fraction of cells would correct Cl(-) transport, we mixed freshly isolated wild-type and CF airway epithelial cells in varying proportions and generated differentiated epithelia. Epithelia with approximately 20% wild-type cells generated approximately 70% the transepithelial Cl(-) current of epithelia containing 100% wild-type cells. These data were nearly identical to those previously obtained with CFTR expressed under control of a strong promoter in a CF epithelial cell line. We also tested high level CFTR expression using the very strong cytomegalovirus (CMV) promoter as well as the cytokeratin-18 (K18) promoter. In differentiated airway epithelia, the CMV promoter generated 50-fold more transgene expression than the K18 promoter, but the K18 promoter generated more transepithelial Cl(-) current at high vector doses. Using functional studies, we found that with marked overexpression, some CFTR channels were present in the basolateral membrane where they shunted Cl(-) flow, thereby reducing net transepithelial Cl(-) transport. These results suggest that very little CFTR is required in a fraction of CF epithelial cells to complement Cl(-) transport because transepithelial Cl(-) flow is limited at the basolateral membrane. Thus they suggest a broad leeway in promoter strength for correcting the CF gene transfer, although at very high expression levels CFTR may be mislocalized to the basolateral membrane.

Gene Expression

Cell Line

Cytomegalovirus - genetics

Gene Transfer Techniques

Promoter Regions, Genetic

Bronchi - enzymology

Trachea - cytology

Humans

Cystic Fibrosis Transmembrane Conductance Regulator - metabolism

Genetic Vectors - genetics

Chlorides - metabolism

Epithelial Cells - enzymology

Cystic Fibrosis Transmembrane Conductance Regulator - genetics

Epithelial Cells - cytology

Keratins - genetics

Bronchi - cytology

Trachea - enzymology

Ion Transport - genetics

Details

Title: Subtitle: Gene transfer of CFTR to airway epithelia: low levels of expression are sufficient to correct Cl- transport and overexpression can generate basolateral CFTR
Creators: Sara L Farmen - Howard Hughes Medical Institute, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, 500 EMRB, Iowa City, IA 52242, USA
Philip H Karp
Philip Ng
Donna J Palmer
David R Koehler
Jim Hu
Arthur L Beaudet
Joseph Zabner
Michael J Welsh
Resource Type: Journal article
Publication Details: American journal of physiology. Lung cellular and molecular physiology, Vol.289(6), pp.L1123-1130
DOI: 10.1152/ajplung.00049.2005
PMID: 16085675
NLM abbreviation: Am J Physiol Lung Cell Mol Physiol
ISSN: 1040-0605
eISSN: 1522-1504
Publisher: American Physiological Society; United States
Grant note: DK 54759 / NIDDK NIH HHS HL 51670 / NHLBI NIH HHS T32 GM007337 / NIGMS NIH HHS HL 59314 / NHLBI NIH HHS
Language: English
Date published: 12/2005
Academic Unit: Neurology; Molecular Physiology and Biophysics; Pulmonary, Critical Care, and Occupational Medicine; Neurosurgery; Internal Medicine
Record Identifier: 9984020764802771

Metrics

19 Record Views

113 Times Cited - Web of Science