Development of a Hybrid Baculoviral Vector for Sustained Transgene Expression
Development of a Hybrid Baculoviral Vector for Sustained Transgene Expression
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DOI：
10.1038/mt.2009.13
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发表时间：
2009-04-01
影响因子：
12.4
通讯作者：
Hu, Yu-Chen
中科院分区：
文献类型：
--
作者：
Lo, Wen-Hsin;Hwang, Shiaw-Min;Hu, Yu-Chen
杆状病毒是一种很有前途的基因传递载体，但由于其只能介导转基因在哺乳动物细胞中的瞬时表达，因此其广泛应用受到阻碍。为了延长表达，我们开发了一种双杆状病毒系统，其中一种杆状病毒表达FLP重组酶，而另一种杆状病毒含有包含转基因和来源于Epstein-Barr病毒的oriP/EBNA 1的Frt侧翼盒。共转导细胞后，表达的FLP将Frt侧翼盒从杆状病毒基因组上切割下来并催化环状附加体形成，然后盒内的oriP/EBNA 1使附加体能够自我复制。丁酸钠显著增强切除/重组效率，在人胚肾-293（HEK 293）细胞中达到75%，在幼仓鼠肾（BHK）细胞中达到85%，在原代软骨细胞中达到77%，在间充质干细胞（MSC）中达到48%。由于复制子和转基因转录的维持，杂合杆状病毒将转基因表达显著延长至约48天（无选择）和>63天（有选择）。与复制的附加体相反，杆状病毒基因组被迅速降解。此外，骨诱导生长因子基因被有效地传递到MSC，这不仅延长了生长因子的表达，而且还增强了MSC的成骨能力。这些数据共同暗示了这种杂交杆状病毒系统在需要持续转基因表达的基因治疗应用中的潜力。
Baculovirus is a promising gene delivery vector but its widespread application is impeded as it only mediates transient transgene expression in mammalian cells. To prolong the expression, we developed a dual baculovirus system whereby one baculovirus expressed FLP recombinase while the other harbored an Frt-flanking cassette encompassing the transgene and oriP/EBNA1 derived from Epstein-Barr virus. After cotransduction of cells, the expressed FLP cleaved the Frt-flanking cassette off the baculovirus genome and catalyzed circular episome formation, then oriP/EBNA1 within the cassette enabled the self-replication of episomes. The excision/recombination efficiency was remarkably enhanced by sodium butyrate, reaching 75% in human embryonic kidney-293 (HEK293) cells, 85% in baby-hamster kidney (BHK) cells, 77% in primary chondrocytes, and 48% in mesenchymal stem cells (MSCs). The hybrid baculovirus substantially prolonged the transgene expression to approximate to 48 days without selection and >63 days with selection, thanks to the maintenance of replicons and transgene transcription. In contrast to the replicating episomes, the baculovirus genome was rapidly degraded. Furthermore, an osteoinductive growth factor gene was efficiently delivered into MSCs using this system, which not only prolonged the growth factor expression but also potentiated the osteogenesis of MSCs. These data collectively implicate the potential of this hybrid baculovirus system in gene therapy applications necessitating sustained transgene expression.