利用細胞週期及微核糖核酸為基礎的邏輯匣專一性殺死上皮癌細胞

Abstract

Anti-mitotic chemotherapy is a cancer therapy that kills cells with high division rate. Since abnormal proliferation is one of the most prominent characteristics of can-cer cells, antimitotic drugs are expected to have high selectivity and sensitivity. How-ever, non-specific targeting of hematopoietic stem cells (HSCs) that also divide rapidly is one of the most severe side effects. To overcome this problem, we constructed a synthetic AND genetic logic, utilizing two sensors, cyclin B1 promoter and miR-142-5p binding sites, in mammalian cells. miR-142-5p was chosen as it is present exclusively in hematopoietic lineages. When cells enter G2 phase of cell cycle, cyclin B1 promoter will be active to express Tet-On 3G transactivator protein. In the presence of doxycycline, the transactivator will then activate a toxic gene, here hBax-β or trun-cated caspase-3/7, tagged with miR-142-5p binding sites. In this setting, HSCs, pro-grammed with miR-142 production, toxic gene will be repressed thus cells are pro-tected, whereas in non-hematopoietic cancer cells, where miR-142 level is significant-ly lower, apoptosis will be triggered. We have successfully cloned and characterized all individual parts in the circuit and have observed the circuit’s capability to render death in HeLa cell. Moreover, we are currently optimizing the circuit by co-expressing an-other apoptotic gene and built the stable cell lines for increasing the transfection effi-cacy. However, when we construct a vector containing the two different types of pro-moter, i.e. constitutive and doxycycline-inducible, there appears to be interference between promoters. In resolving this, we tested an insulator core element and found that two tandem copies of this sequence can effectively suppress the interference. We envision our circuit will serve as a useful alterative tool for targeted cancer therapy in the near future.