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1 Departments of Physiology and Biophysics, University of Iowa Roy J and Lucille A Carver College of Medicine, Iowa City, IA, USA
2 Department of Internal Medicine, University of Iowa Roy J and Lucille A Carver College of Medicine, Iowa City, IA, USA; Howard Hughes Medical Institute, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, IA, USA
3 Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA
4 Lung Biology Research Program, Hospital for Sick Children, Toronto, ON, Canada
5 Department of Internal Medicine, University of Iowa Roy J and Lucille A Carver College of Medicine, Iowa City, IA, USA
6 Departments of Physiology and Biophysics, University of Iowa Roy J and Lucille A Carver College of Medicine, Iowa City, IA, USA; Department of Internal Medicine, University of Iowa Roy J and Lucille A Carver College of Medicine, Iowa City, IA, USA; Howard Hughes Medical Institute, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, IA, USA
* To whom correspondence should be addressed. E-mail: michael-welsh{at}uiowa.edu.
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 ~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.
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