Expression and maturation of Sendai virus vector-derived CFTR protein: functional and biochemical evidence using a GFP-CFTR fusion protein.
Sendai virus (SeV) vector has been shown to efficiently transduce airway epithelial cells. As a precursor to the potential use of this vector for cystic fibrosis (CF) gene therapy, the correct maturation of the SeV vector-derived CF transmembrane conductance regulator (CFTR) protein was examined using biochemical and functional analyses. We constructed a recombinant SeV vector, based on the fusion (F) gene-deleted non-transmissible SeV vector, carrying the GFP-CFTR gene in which the N terminus of CFTR was fused to green fluorescence protein (GFP). This vector was recovered and propagated to high titers in the packaging cell line. Western blotting using an anti-GFP antibody detected both the fully glycosylated (mature) and the core-glycosylated (immature) proteins, indicating that SeV vector-derived GFP-CFTR was similar to endogenous CFTR. We also confirmed the functional channel activity of GFP-CFTR in an iodide efflux assay. The efficient expression of GFP-CFTR, and its apical surface localization, were observed in both MDCK cells in vitro, and in the nasal epithelium of mice in vivo. We concluded that recombinant SeV vector, a cytoplasmically maintained RNA vector, is able to direct production of a correctly localized, mature form of CFTR, suggesting the value of this vector for studies of CF gene therapy.
Ban
H
H
DNAVEC Corporation, Tsukuba-shi, Ibaraki, Japan.
Inoue
M
M
Griesenbach
U
U
Munkonge
F
F
Chan
M
M
Iida
A
A
Alton
E W F W
EW
Hasegawa
M
M
D000818:Animals; D002460:Cell Line; D003550:Cystic Fibrosis / Q000378:metabolism / Q000628:therapy*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics*; D015870:Gene Expression; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000008:administration & dosage* / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D018613:Microscopy, Confocal; D009296:Nasal Cavity; D010477:Perfusion; D011993:Recombinant Fusion Proteins / Q000008:administration & dosage / Q000378:metabolism; D020545:Respiratory Mucosa / Q000378:metabolism; D029082:Sendai virus / Q000235:genetics*; D014161:Transduction, Genetic / Q000379:methods*
D011993:Recombinant Fusion Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D049452:Green Fluorescent Proteins