Ban H, Inoue M, Griesenbach U, Munkonge F, Chan M, Iida A, Alton E W F W, Hasegawa M
DNAVEC Corporation, Tsukuba-shi, Ibaraki, Japan.
Gene Ther. 2007 Dec;14(24):1688-94. doi: 10.1038/sj.gt.3303032. Epub 2007 Sep 27.
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.
仙台病毒（SeV）载体已被证明能有效地转导气道上皮细胞。作为将该载体潜在用于囊性纤维化（CF）基因治疗的前期研究，我们使用生化和功能分析方法检测了SeV载体衍生的囊性纤维化跨膜电导调节因子（CFTR）蛋白的正确成熟情况。我们构建了一种基于缺失融合（F）基因的非传染性SeV载体的重组SeV载体，其携带GFP-CFTR基因，其中CFTR的N端与绿色荧光蛋白（GFP）融合。该载体在包装细胞系中回收并增殖至高滴度。使用抗GFP抗体进行的蛋白质印迹检测到了完全糖基化（成熟）和核心糖基化（未成熟）的蛋白质，这表明SeV载体衍生的GFP-CFTR与内源性CFTR相似。我们还在碘外流试验中证实了GFP-CFTR的功能性通道活性。在体外的MDCK细胞和体内的小鼠鼻上皮中均观察到了GFP-CFTR的高效表达及其顶端表面定位。我们得出结论，重组SeV载体作为一种在细胞质中维持的RNA载体，能够指导产生正确定位的成熟形式的CFTR，这表明该载体在CF基因治疗研究中的价值。
