Precisely identifying and killing tumor cells are diligent pursuits in oncotherapy. Synthesized gene circuits have emerged as an intelligent weapon to solve these problems. Gene circuits based on post-transcriptional regulation enable a faster response than systems based on transcriptional regulation, which requires transcription and translation, showing superior safety. In this study, synthetic-promoter-free gene circuits possessing two control layers were constructed to improve the specific recognition of tumor cells. Using split-TEV, we designed and verified the basic control layer of protein-protein interaction (PPI) sensing. Another orthogonal control layer was built to sense specific proteins. Two layers were integrated to generate gene circuits sensing both PPI and specific proteins, forming 10 logic gates. To demonstrate the utility of this system, the circuit was engineered to sense alpha-fetoprotein (AFP) expression and the PPI between YAP and 14-3-3σ, the matching profile of hepatocellular carcinoma (HCC). Gene-circuit-loaded cells distinguished HCC from other cells and released therapeutic antibodies, exhibiting in vitro and in vivo therapeutic effects.

精确鉴定和杀死肿瘤细胞是肿瘤疗法中的努力追求。合成基因电路已成为解决这些问题的智能武器。与基于转录调控的系统相比，基于转录后调控的基因电路能够实现更快的响应，后者需要转录和翻译，显示出卓越的安全性。在这项研究中，构建了具有两个控制层的无合成启动子基因电路，以改善对肿瘤细胞的特异性识别。使用split-TEV，我们设计并验证了蛋白质-蛋白质相互作用（PPI）感应的基本控制层。建立了另一个正交控制层以检测特定的蛋白质。整合了两层以生成同时检测PPI和特定蛋白质的基因电路，形成10个逻辑门。为了演示该系统的实用性，对电路进行了工程设计，以检测甲胎蛋白（AFP）的表达以及YAP与14-3-3σ之间的PPI，即肝细胞癌（HCC）的匹配情况。加载基因电路的细胞将HCC与其他细胞区分开，并释放出治疗性抗体，表现出体外和体内治疗作用。