Gene transfer of CFTR to airway epithelia: low levels of expression are sufficient to correct Cl- transport and overexpression can generate basolateral CFTR.
American journal of physiology. Lung cellular and molecular physiology 2005 Dec 1; In press
Farmen S SL, Karp P PH, Ng P P, Palmer D DJ, Koehler D DR, Hu J J, Beaudet A AL, Zabner J J, Welsh M MJ
Howard Hughes Medical Institute, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, 500 EMRB, Iowa City, IA 52242, USA.
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.