Establishment of a producer cell line for adeno-associated virus vector using a novel system regulating nuclear protein

我们探索了一种新的方法来调节核蛋白的功能；改变它们的亚细胞定位。为了将含有SV40核定位信号的核蛋白β-半乳糖苷酶(n-β-GAL)与粒细胞集落刺激因子受体(G-CSFR)的跨膜区融合到胞浆内。为了从融合蛋白中释放出n-β-Gal部分，我们使用了来自三叶草黄脉病毒的一种蛋白水解酶NIA，它的识别序列插入在G-β-Gal和n-Csfr-Gal之间。Western分析表明，在293细胞中，该融合蛋白在蛋白酶存在的情况下被切割，而没有识别序列的融合蛋白保持完整。该嵌合蛋白仅定位在细胞质中，通过X-Gal染色和使用抗P-Gal抗体的免疫荧光染色显示。当与NIA一起表达时，β-GAL主要在细胞核中检测到。此外，我们还分离到了293个细胞克隆，表明该酶不具有细胞毒性。在此基础上，我们构建了在脑心肌炎病毒内部核糖体进入位点的翻译控制下，融合了由P5启动子驱动的G-CSFR和杀菌素S抗性基因的Rep蛋白的双顺反子表达载体。将该重组表达载体导入293细胞，经杀菌素S筛选后，筛选出小Rep/Cap、腺病毒辅助表达载体和Nia重组表达载体。与NIA共转染后，rAAV的产量提高了10倍(约105个颗粒/10-cmDISH)。然而，随着传代的进行，这些细胞株产生的rAAV滴度逐渐下降。这些结果表明，即使在没有NIA蛋白酶的情况下，嵌合蛋白也存在泄漏的核运输。

We explored a novel approach to the functional regulation of nuclear proteins; altering their subcellular localization. To anchor a nuclear protein, β-galactosidase with the nuclear localization signal of SV40 (nβ-gal), within the cytoplasm, it was fused to the transmembrane domain of granulocyte colony-stimulating factor receptor(G-CSFR). To liberate the nβ-gal portion from the fusion protein, we used a protease derived from clover yellow vein virus, Nia protease, whose recognition sequence was inserted between the G-CSFR and nβ-gal. Western analysis showed that the chimeric protein was cleaved in the presence of the protease in 293 cells and that the fusion protein without the recognition sequence remained intact. This chimeric protein was localized exclusively in the cytoplasm as visualized by X-gal staining and immunofluorescent staining using an anti-P-gal antibody. When expressed together with the Nia, β-gal was predominantly detected in the nuclei. Moreover, we isolated 293-cell clones constitutively expressing the Nia, indicating this protease is not cytotoxic. Based on these results, we constructed a dicistronic plasmid expressing an Rep protein fused with G-CSFR driven by the p5 promoter and the blasticidin S resistance gene under the translational control of the encephalomyocarditis virus internal ribosome entry site (IRES). Following transfection of 293 cells with this plasmid and the selection with blasticidin S, we isolated cell clones that could produce recombinant AAV after transfection with small Rep/Cap plasmid, Adenovirus helper plasmid, and Nia plasmid. The yield of rAAV was increased 10-fold when the Nia plasmid was cotransfected (about 105 particles/10-cm dish). However, the titer of rAAV produced by these cell lines gradually decreased as they were passaged. These results indicated the presence of a leaky nuclear transport of the chimeric protein even in the absence of the Nia protease.