title: CFTR gene transfer to human cystic fibrosis pancreatic duct cells using a Sendai virus vector.
abstract: Cystic fibrosis (CF) is a fatal inherited disease caused by the absence or dysfunction of the CF transmembrane conductance regulator (CFTR) Cl- channel. About 70% of CF patients are exocrine pancreatic insufficient due to failure of the pancreatic ducts to secrete a HCO3- -rich fluid. Our aim in this study was to investigate the potential of a recombinant Sendai virus (SeV) vector to introduce normal CFTR into human CF pancreatic duct (CFPAC-1) cells, and to assess the effect of CFTR gene transfer on the key transporters involved in HCO3- transport. Using polarized cultures of homozygous F508del CFPAC-1 cells as a model for the human CF pancreatic ductal epithelium we showed that SeV was an efficient gene transfer agent when applied to the apical membrane. The presence of functional CFTR was confirmed using iodide efflux assay. CFTR expression had no effect on cell growth, monolayer integrity, and mRNA levels for key transporters in the duct cell (pNBC, AE2, NHE2, NHE3, DRA, and PAT-1), but did upregulate the activity of apical Cl-/HCO3- and Na+/H+ exchangers (NHEs). In CFTR-corrected cells, apical Cl-/HCO3- exchange activity was further enhanced by cAMP, a key feature exhibited by normal pancreatic duct cells. The cAMP stimulated Cl-/HCO3- exchange was inhibited by dihydro-4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (H2-DIDS), but not by a specific CFTR inhibitor, CFTR(inh)-172. Our data show that SeV vector is a potential CFTR gene transfer agent for human pancreatic duct cells and that expression of CFTR in CF cells is associated with a restoration of Cl- and HCO3- transport at the apical membrane.
authors.affiliation: Institute for Cell and Molecular Biosciences, University of Newcastle, Newcastle upon Tyne, United Kingdom.
authors.forename: Zoltán
authors.initials: Z
authors.lastname: Rakonczay
authors.forename: Péter
authors.initials: P
authors.lastname: Hegyi
authors.forename: Mamoru
authors.initials: M
authors.lastname: Hasegawa
authors.forename: Makoto
authors.initials: M
authors.lastname: Inoue
authors.forename: Jun
authors.initials: J
authors.lastname: You
authors.forename: Akihiro
authors.initials: A
authors.lastname: Iida
authors.forename: Imre
authors.initials: I
authors.lastname: Ignáth
authors.forename: Eric W F W
authors.initials: EW
authors.lastname: Alton
authors.forename: Uta
authors.initials: U
authors.lastname: Griesenbach
authors.forename: Gabriella
authors.initials: G
authors.lastname: Ovári
authors.forename: János
authors.initials: J
authors.lastname: Vág
authors.forename: Ana C
authors.initials: AC
authors.lastname: Da Paula
authors.forename: Russell M
authors.initials: RM
authors.lastname: Crawford
authors.forename: Gábor
authors.initials: G
authors.lastname: Varga
authors.forename: Margarida D
authors.initials: MD
authors.lastname: Amaral
authors.forename: Anil
authors.initials: A
authors.lastname: Mehta
authors.forename: János
authors.initials: J
authors.lastname: Lonovics
authors.forename: Barry E
authors.initials: BE
authors.lastname: Argent
authors.forename: Michael A
authors.initials: MA
authors.lastname: Gray
mesh_terms: D002460:Cell Line; D003550:Cystic Fibrosis / Q000235:genetics / Q000503:physiopathology; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics*; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006801:Humans; D006863:Hydrogen-Ion Concentration; D010183:Pancreatic Ducts / Q000502:physiology*; D012333:RNA, Messenger / Q000378:metabolism; D029082:Sendai virus / Q000502:physiology*; D001616:beta-Galactosidase / Q000378:metabolism
chemical_list: C505032:CFTR protein, human; D012333:RNA, Messenger; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D001616:beta-Galactosidase