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

• Published in: American journal of physiology. Lung cellular and molecular physiology 2005-12, Vol.33 (6), p.L1123-L1130
• Main Authors: FARMEN, Sara L, KARP, Philip H, NG, Philip, PALMER, Donna J, KOEHLER, David R, HU, Jim, BEAUDET, Arthur L, ZABNER, Joseph, WELSH, Michael J
• Format: Article
• Language: English
• Subjects: Biological and medical sciences; Cystic fibrosis; Errors of metabolism; Fundamental and applied biological sciences. Psychology; Gene expression; Gene therapy; Genes; Medical sciences; Metabolic diseases; Miscellaneous hereditary metabolic disorders; Respiratory system; Vertebrates: respiratory system
• ISSN: 1040-0605; 1522-1504

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 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. [PUBLICATION ABSTRACT]