以桿狀病毒載體表現特異性之microRNA並結合Sleeping Beauty 長效表現系統長期抑制目標基因

Development of Baculovirus Vectors for microRNA Expression and Sustained Gene Suppression

陳秋伶(Chiu-Ling Chen)

指導教授 ： 胡育誠

國立清華大學/工學院/化學工程學系/碩士(2010年)

摘要

microRNA (miRNA)已知為一可參與基因調控之生物過程，在基因治療上是新穎且有效的基因調控工具。本研究以桿狀病毒載體傳遞基因改質後之內源性microRNA155 (engineered pre-miRNA sequences)，藉由基因序列的設計，可表現出特異性之miRNA 以調控目標基因，我們首先建構數組可特定互補於egfp 序列上不同位置之engineered pre-miRNA 而其mature miRNA 片段可特異性抑制EGFP 表現，我們稱此miRNA 表現匣為EGFP-miR，以此基因表現匣轉殖入桿狀病毒載體後所製備出之桿狀病毒為BacEGFP-miR。BacEGFP-miR(病毒劑量MOI 500)與重組桿狀病毒BacCE (Ho et al. 2004)共同轉導人類上皮細胞，egfp 之蛋白與基因表現量分別降為11%及10%。關節炎為一種慢性疾病且影響全球數億人口，而細胞激素持續產生導致長期發炎，本研究亦將engineered miRNA 基因調控系統應用於骨關節炎中， 抑制Fibroblast-like synoviocytes (from osteoarthritis patients)之proinflammatory cytokine (TNF-a)表現以減緩發炎反應。為了探討miRNA 能否有效應用於TNF-a的抑制，我們建構重組桿狀病毒BacTNF-miR (可表現特異性抑制TNF-a之engineered pre-miRNA)轉導滑膜細胞，經病毒轉導後1 天TNF-a之基因及蛋白表現皆有顯著之抑制效果(p<0.05)。後續我們以 BacTNF-miR 與BacTNF-miRscr (負對照組)分別轉導滑膜細胞，分析桿狀病毒轉導後之細胞凋亡程度，兩組細胞凋亡比例分別為4.2%與5.7%而假性轉導組(mock)為1.1%，顯示病毒轉導後無顯著之滑膜細胞凋亡且不影響基因調控過程。我們結合engineered pre-miRNA 表現與Sleeping Beauty 長效系統將BacSB 病毒與BacT2dE-miR 共同轉導改質之H-d2E 細胞 (此細胞穩定表現
destabilized EGFP，d2EGFP，half-life: 2hr)以延長miRNA 表現期，長效表現之miRNA 可持續抑制20-25% 之d2EGFP 表現達24 天。本研究以桿狀病毒載體表現長效RNAi 基因調控系統作為關節炎治療法為一嶄新之治療策略也替RNAi系統開創更廣泛的應用性。

關鍵字

桿狀病毒 ； 關節炎

並列摘要

microRNA (miRNA) can play significant roles in regulating gene expression, which implicates its potential in gene therapy. However, effective delivery of miRNA remains a hurdle, thus entailing a vector system for efficient delivery. Since baculovirus mediates efficient gene transfer to mammalian cells, here we aimed to develop the baculovirus vectors to carry engineered pre-miRNA sequences to knock down selected genes. We first constructed baculoviruses that harbored the miRNA sequences targeting different regions of the egfp gene and demonstrated that these baculoviruses suppressed the EGFP expression in a sequence- and dose-dependent manner. The attenuation of EGFP expression was up to 10% and 11 % in the protein and mRNA levels. Osteoarthritis (OA) is a chronic, progressive joint disease within the cartilage and is accompanied by deregulated activities of cytokine such as TNF-a. To exploit the miRNA therapy for OA treatment, we constructed another baculovirus harboring the pre-miRNA sequences targeting TNF-a (BacTNF-miR) and showed that BacTNF-miR was effectively suppressed the mRNA and protein levels of TNF-a from the fibroblast-like synoviocytes isolated from the OA patients, without induction of appreciable apoptosis. To provide long-term suppression, we exploited the Sleeping Beauty system and constructed Bac-SB that expressed the SB
transposase and BacT2dE-miR that harbored the destabilized EGFP (d2EGFP)-specific miRNA expression cassette flanked by the IR/DR sequences recognizable by the SB. Co-transduction of a cell line stably expressing d2EGFP with BacSB and BacT2dE-miR led to prolonged repression of the d2EGFP
expression for up to 24 days. These data collectively proved that baculovirus is an effective vector for miRNA delivery and downregulation of the target gene in mRNA/protein level. Combination of the baculovirus vector with SB system further enabled sustained expression of miRNA, rendering it a feasible approach to treating
such diseases as OA.

並列關鍵字

microRNA ； baculovirus ； Sleeping Beauty

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