建立一快速且便利的方式以增強轉殖基因在特定細胞中的表現

Abstract

基因治療對癌症病患提供了前所未有的治療策略及希望。不幸的是，無論是基因傳遞或啟動子系統迄今仍未達到專一性之療效，除此之外，任一系統的最佳化都極端困難。在本研究中我們希望介紹一個簡單的概念：亦即部分專一的基因傳遞系統以及部分專一的啟動子相結合，將可對標靶細胞達到更加專一性的表現。在第一部分，我們首先檢測與腫瘤相關的轉錄因子在腫瘤或快速生長細胞中的活性。接著利用表現量較高的轉錄因子(NF-kB，CREB以及HIF-1)之反應片段，取三倍體構築一轉錄因子相關之合成啟動子(Transcription factor-based synthetic promoter，TSP) 。實驗結果證實TSP在特定細胞中具有活性並有部分專一性。此外相對於NF-□B 或HIF-1迷你啟動子，TSP在抑制劑存在之下表現較佳的抵抗性。 在第二部分，多功能胜□RGD-4C-HA可專一性結合至B16-F10細胞表面之integrin alphavbeta3並且吸附至聚乙烯亞胺(Polyethyleneimine，PEI)。實驗結果顯示RGD-4C-HA能與聚乙烯亞胺形成複合物並且在in vitro實驗中引導專一性的指向。最後，聚乙烯亞胺及胜□複合物與TSP的結合能夠使轉殖基因專一性的表現在B16-F10細胞中。這種策略在in vitro實驗中已經證實為可行，並且在in vivo的專一性基因治療可能也具有潛力。

Gene therapy provides a novel strategy and a new hope for the patients with cancer. Unfortunately, the specificity of the delivery systems or the promoters did not achieve the specific efficacy so far and the perfection of either system will be extremely difficult. In this study we had introduce a simple concept that the combination of partial specific delivery and partial specific promoter activity may achieve more specific effect for specific expression in target cells. In the first part, the tumor related transcription factors were assayed in tumor or rapid-proliferating cells to determine their activities. The activities of NF-□B, CREB, and HIF-1 were higher and three copies of each response elements were used to construct a transcription factor-based synthetic promoter (TSP). The results showed that the expression of TSP was truly active and partial specific to cell types. In addition, it was more resistant than NF-kB or HIF-1 mini-promoters at the presence of inhibitors. In the second part, the multi-functional peptide RGD-4C-HA was designed to specifically target integrin alphavbeta3 on B16-F10 cells and absorbed to polyethyleneimine (PEI) molecules. The results showed that RGD-4C-HA could associate with PEI to form complex and mediate specific targeting in vitro. Finally, the combination of PEI-peptide complex and TSP could enhance the specifically transgenic expression in B16-F10 cells. This strategy had been proven to work in vitro and might be also potential in specific gene therapy in vivo.