721. Proper Maturation of CFTR Protein Expressed by SeV Vector Was Proved by Using a GFP-CFTR Fusion Protein

Top of pageAbstract Sendai virus (SeV) vector has been shown to transduce the airway epithelial cells very efficiently. This property makes the vector promising for cystic fibrosis (CF) gene therapy. Previously, we generated a series of SeV vectors carrying the cystic fibrosis transmembrane conductance regulator (CFTR) gene and showed that SeV can mediate CFTR gene transfer both in vitro and in vivo. CFTR activity in such experiments was confirmed in C127 cells in vitro using a radioactive iodide efflux assay and a whole cell perforated patch-clamp assay, and in CF knockout mice in vivo. These results indicated that SeV vector was a good candidate vector for CF gene therapy. However, there was not clear correlation between the expression levels of CFTR estimated from the design of a series of the vectors and observed activities of them. Additionally, cytotoxicity was observed in cells transduced by some types of SeV vectors probably or largely because of the overexpression of CFTR. To clarify these matters, biochemical analysis for CFTR protein itself is indispensable because CFTR is known to be highly glycosylated and glycosylation is responsible for mature CFTR function. However, we could not detect the CFTR by Western blotting using anti-CFTR antibody in SeV vector-transduced cells. Thus, we constructed a recombinant SeV vector designated SeV(HNL)-GFPCFTR/ΔF in which the N-terminus of CFTR was fused with an useful tag, GFP gene. The fusion gene was introduced into the junction between the hemagglutinin-neuraminidase (HN) and large protein (L) genes on the F gene-deleted SeV (SeV/ΔF) genome. It has previously been shown that GFP fusion to the N-terminus of CFTR did not affect processing, localization and function of CFTR (Bryan D. et al. 1998, J Biol Chem 273, 21759-68). We characterized the GFP-CFTR in the cells transduced with SeV(HNL)-GFPCFTR/ΔF. Western blotting using an anti-GFP antibody detected two proteins at 190 kDa and 170 kDa. These correspond in size to fully glycosylated mature CFTR and to the core-glycosylated immature protein, respectively. The mature and immature CFTR are known to be distinguishable by their different sensitivity to glycosidases. As expected, the 190 kDa protein was sensitive only to PNGaseF glycosidase and 170 kDa protein was to both PNGaseF and EndoH glycosidase. These results demonstrate that glycosylation of GFP-CFTR fusion protein derived from SeV(HNL)-GFPCFTR/ΔF was quite similar to endogenous CFTR protein. The subcellular localization of GFP-CFTR in MDCK cells infected with SeV(HNL)-GFPCFTR/ΔF was further analyzed by confocal laser microscopy. The GFP-CFTR was found to predominantly localize to the apical membrane. Thus, the proper maturation of CFTR protein in SeV vector-transduced cells was proved, indicating again that SeV is useful for CF gene therapy. Additionally, the generated SeV(HNL)-GFPCFTR/ΔF would be an useful system to analyze dynamics of CFTR protein including the tracking both in vitro and in vivo. The quantitative and functional analyses are now under investigation.