發展一種具備長期表現能力之新式重組桿狀病毒載體

羅文鑫(Wen-Hsin Lo)

指導教授 ： 胡育誠

國立清華大學/工學院/化學工程學系/博士(2010年)

摘要

由於桿狀病毒具備安全性而逐漸被視為一種有潛力的基因釋放載體。然而，基於哺乳動物細胞非其天然宿主細胞，病毒轉導細胞後不會再行複製致使基因表現期較為短暫，因而限制了其應用範圍。為了能延長基因表現，本研究發展一套雙桿狀病毒系統，一株病毒表現FLP重組酶；另一株病毒攜帶基因包含EB病毒(Epstein-Barr virus)之oriP/EBNA1複製系統和轉殖基因表現匣，其兩側加入Frt序列做為切接辨識位置。在共轉導細胞後，藉由FLP重組酶的表現及辨識桿狀病毒基因體上的Frt序列，經切除/接合之重組作用組成一環狀的游離態質體，此游離態質體則藉著oriPEBNA1系統而能夠自行複製。此切除/接合之重組效率可藉由添加丁酸鈉的方式顯著的提升，其游離態質體生成效率在HEK2936約75%、在BHK約85%、在兔子之初代軟骨細胞約77%和在人類的間葉幹細胞(MSCs)約為48%。此外，游離態質體能夠在病毒轉導後兩天內迅速形成。在延長基因表現效期方面，經共轉導後之細胞在無selection pressure環境下，其轉殖基因表現期可達48天；而在selection pressure（50 g/ml G418）環境下，表現期可延長至少63天。由結果顯示，本系統可藉著非崁入式的游離態複製子之形成，來有效改善桿狀病毒表現的時效性，且我們亦發現在游離態質體形成後，桿狀病毒基因體會迅速被分解。此外，本系統亦能在MSCs中有效地表現具骨誘導能力之生長因子(BMP-2)，其除了能延長生長因子表現期，也加速誘導MSC分化進入骨生成。再著，我們建構一baculovirus reprogramming system，其利用self-cleaving 2A sequences將多個再程序化因子基因(再程序化因子可誘導人類體細胞再程序化成誘導式多能性幹細胞(iPSCs))連結成為一多基因表現匣。經轉導人類體細胞，此系統證實具備了可同時表現多個再程序化因子之能力。綜合本研究之數據顯示，新開發之長效型桿狀病毒載體應具有在基因治療上的應用潛力（特別是療程期長的基因治療）。

關鍵字

重組桿狀病毒 ； 延長表現 ； oriP/EBNA1 系統 ； FLP重組酶 ； 2A序列 ； 誘導型多能幹細胞

並列摘要

Baculovirus has been regarded safe and has captured growing attention as an promising gene delivery vector. However, the transient expression nature due to its inability to replicate in mammalian cells limits its widespread application. To prolong the expression, we developed a binary baculovirus system whereby one baculovirus expressed FLP recombinase while the other harbored an Frt-flanking cassette encompassing Epstein-Barr virus oriP/EBN-1, which are essential for the episomal maintenance of the EBV genome in latently infected cells, and the transgene expression cassette. 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 HEK293 cells, 85% in BHK cells, 77% in primary chondrocytes and 48% in mesenchymal stem cells (MSCs), and the episomally-maintained plasmids were rapidly generated at 2 days post-trasnduction. The hybrid baculovirus substantially prolonged the transgene expression to 48 days without selection and >63 days with selection (50 g/ml G418). This results indicated that the system is capable of sustained transgene expression without integration into the host genome, 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 (bone morphogenetic protein-2) was efficiently delivered into MSCs using this system, which not only prolonged the growth factor expression but also potentiated the osteogenesis of MSCs. On the other hand, we constructed a baculovirus reprogramming system in which multiple reprogramming factor genes, which induce the reprogramming of somatic cells into induced pluripotent stem cells (iPSCs), are fused in-frame into a single open reading frame (ORF) via self-cleaving 2A sequences. This system was capable of efficiently expressed multiple reprogramming factors in somatic cells, suggested this system may provide a valuable tool for generation of iPSCs cells. These data collectively implicate the potential of this hybrid baculovirus system in gene therapy applications necessitating sustained transgene expression.

並列關鍵字

baculovirus ； sustained expression ； oriP/EBNA1 system ； FLP recombinase ； 2A sequences ； iPSCs

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