Gene transfer of CFTR to airway epithelia: low levels of expression are sufficient to correct Cl- transport and overexpression can generate basolateral CFTR.
Am J Physiol Lung Cell Mol Physiol
; 289(6): L1123-30, 2005 Dec.
Article
em En
| MEDLINE
| ID: mdl-16085675
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.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Traqueia
/
Brônquios
/
Expressão Gênica
/
Cloretos
/
Técnicas de Transferência de Genes
/
Regulador de Condutância Transmembrana em Fibrose Cística
/
Células Epiteliais
Limite:
Humans
Idioma:
En
Revista:
Am J Physiol Lung Cell Mol Physiol
Assunto da revista:
BIOLOGIA MOLECULAR
/
FISIOLOGIA
Ano de publicação:
2005
Tipo de documento:
Article
País de afiliação:
Estados Unidos