title: Gene transfer of CFTR to airway epithelia: low levels of expression are sufficient to correct Cl- transport and overexpression can generate basolateral CFTR.
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.
authors.affiliation: Howard Hughes Medical Institute, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, 500 EMRB, Iowa City, IA 52242, USA.
authors.forename: Sara L
authors.initials: SL
authors.lastname: Farmen
authors.forename: Philip H
authors.initials: PH
authors.lastname: Karp
authors.forename: Philip
authors.initials: P
authors.lastname: Ng
authors.forename: Donna J
authors.initials: DJ
authors.lastname: Palmer
authors.forename: David R
authors.initials: DR
authors.lastname: Koehler
authors.forename: Jim
authors.initials: J
authors.lastname: Hu
authors.forename: Arthur L
authors.initials: AL
authors.lastname: Beaudet
authors.forename: Joseph
authors.initials: J
authors.lastname: Zabner
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Welsh
mesh_terms: D001980:Bronchi / Q000166:cytology / Q000201:enzymology*; D002460:Cell Line; D002712:Chlorides / Q000378:metabolism*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics / Q000378:metabolism*; D003587:Cytomegalovirus / Q000235:genetics; D004847:Epithelial Cells / Q000166:cytology / Q000201:enzymology*; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D017136:Ion Transport / Q000235:genetics; D007633:Keratins / Q000235:genetics; D011401:Promoter Regions, Genetic; D014132:Trachea / Q000166:cytology / Q000201:enzymology*
chemical_list: C505032:CFTR protein, human; D002712:Chlorides; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D007633:Keratins