Back to Search
Start Over
Gene transfer of CFTR to airway epithelia: low levels of expression are sufficient to correct Cl- transport and overexpression can generate basolateral CFTR.
- Source :
-
American journal of physiology. Lung cellular and molecular physiology [Am J Physiol Lung Cell Mol Physiol] 2005 Dec; Vol. 289 (6), pp. L1123-30. Date of Electronic Publication: 2005 Aug 05. - Publication Year :
- 2005
-
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.
- Subjects :
- Bronchi cytology
Cell Line
Cystic Fibrosis Transmembrane Conductance Regulator genetics
Cytomegalovirus genetics
Epithelial Cells cytology
Genetic Vectors genetics
Humans
Ion Transport genetics
Keratins genetics
Promoter Regions, Genetic
Trachea cytology
Bronchi enzymology
Chlorides metabolism
Cystic Fibrosis Transmembrane Conductance Regulator metabolism
Epithelial Cells enzymology
Gene Expression
Gene Transfer Techniques
Trachea enzymology
Subjects
Details
- Language :
- English
- ISSN :
- 1040-0605
- Volume :
- 289
- Issue :
- 6
- Database :
- MEDLINE
- Journal :
- American journal of physiology. Lung cellular and molecular physiology
- Publication Type :
- Academic Journal
- Accession number :
- 16085675
- Full Text :
- https://doi.org/10.1152/ajplung.00049.2005