Yang Jiong, Ding Shigang
Department of Gastroenterology, Peking University Third Hospital, Beijing 100191, China.
Beijing Key Laboratory for Helicobacter Pylori Infection and Upper Gastrointestinal Diseases, Beijing 100191, China.
Mol Ther Nucleic Acids. 2021 Aug 19;25:683-695. doi: 10.1016/j.omtn.2021.08.012. eCollection 2021 Sep 3.
Cancer cell-specific killing switches are synthetic circuits developed as an intelligent weapon to specifically eliminate malignant cells. RNA-delivered synthetic circuits provide safer means to control oncolytic functions, in which proteolysis-responding capsid-cNOT7 is developed to enable logic computation and modular design. Unfortunately, although circuits containing these capsid-cNOT7s exhibited good performance when introduced as replicons, in modified mRNA (modRNA) delivery, the performance was not quite as good. To improve this situation, alternative modules suitable for modRNA delivery need to be developed. An attractive option is RNA-binding protein (RBP)/riboswitches. In this study, RBPs were engineered by fusing with degron and cleavage sites. The compatibility of these chimeric RBPs with proteolysis-based sensing units were tested. Eight two-input logic gates and four three-input logic gates were implemented. After building this chimeric RBP-based system, we constructed a hepatocellular carcinoma (HCC) cell-specific killing circuit using two proteolysis-based sensing units, a two-input logic OR gate, and a leakproof apoptosis-inducing actuator, which distinguished HCC cells and induced apoptosis in a mixed IMR90-PLC/PRF/5 population.
癌细胞特异性杀伤开关是作为一种智能武器开发的合成电路，用于特异性消除恶性细胞。RNA传递的合成电路提供了更安全的方式来控制溶瘤功能，其中开发了蛋白水解响应衣壳-cNOT7以实现逻辑计算和模块化设计。不幸的是，尽管包含这些衣壳-cNOT7的电路作为复制子引入时表现出良好的性能，但在修饰mRNA（modRNA）递送中，性能却不尽如人意。为改善这种情况，需要开发适合modRNA递送的替代模块。一个有吸引力的选择是RNA结合蛋白（RBP）/核糖开关。在本研究中，通过与降解结构域和切割位点融合对RBP进行了工程改造。测试了这些嵌合RBP与基于蛋白水解的传感单元的兼容性。实现了八个双输入逻辑门和四个三输入逻辑门。构建基于这种嵌合RBP的系统后，我们使用两个基于蛋白水解的传感单元、一个双输入逻辑或门和一个防泄漏凋亡诱导执行器构建了一个肝细胞癌（HCC）细胞特异性杀伤电路，该电路能够区分HCC细胞并在IMR90-PLC/PRF/5混合群体中诱导凋亡。
