设计、构建白藜芦醇调控的基因开关系统及其在肿瘤治疗中的应用研究

Resveratrol (RES) is a famous natural product which has been considered to play physiological roles such as anticancer, anti-inflammatory and anti-aging effects. So, it is of great significance to design RES-inducible transgene expression device for tumor therapy. The adoptive transfer of synthetic Chimeric Antigen Receptor (CAR) T cells has shown tremendous promise for eradicating leukemia in clinical trials. However, it is hard to be widely applied as such engineered T cells can cause severe toxicities because of their excessive activities and poor control. In this project, we use a synthetic biology strategy for controlling CAR-T cells by designing and creating RES-controlled devices. In addition, we want to confirm the potential benefits of this system for mitigating toxicity and curing leukemia. We first design and validate the RESON and RESOFF orthogonal gene switches and then we explore transgene expression kinetics of these two gene switches in mammalian cells and mice. Finally, we design two types of RES-controlled CAR-T cells based on these two gene switches and we preliminary test the therapeutic activity to kill leukemic cells of the controllable CAR-T cells induced by varying concentration of resveratrol in vitro. This approach will provide a new therapeutic strategy for CAR-T cellular immunotherapy which is more precise, controllable and safer.

白藜芦醇（RES）是具有抗肿瘤、抗炎症、抗衰老等多种生物活性的天然药物。因此，设计合成用于肿瘤治疗的RES调控系统具有极大意义。目前CAR-T细胞治疗白血病在临床上取得巨大成功。但CAR-T的不可控性导致严重的毒副作用，阻碍了CAR-T治疗的广泛应用。因此，本研究旨在探索利用合成生物学方法设计的RES调控系统调控CAR-T细胞的可行性，评估其用于规避传统方式毒副作用及治疗白血病的潜力。首先利用合成生物学的理念，设计合成RESON和RESOFF 两种基因开关；其次，对RES调控的两种基因开关分别在细胞水平和动物水平研究其调控基因表达的动力学特征；最后，利用该基因开关设计两种可控的CAR-T细胞并初步在细胞水平研究RES调控CAR-T杀伤白血病细胞的能力。该研究可为下一代精准可控、更安全有效的CAR-T细胞治疗提供新方法和新策略。

白藜芦醇（RES）是一种具有抗肿瘤、抗炎症、抗衰老等多种生物活性的天然药物，具有很大的应用前景。CAR-T疗法作为新兴的肿瘤免疫治疗方法已被批准上市，但在临床使用中仍存在细胞因子风暴等副作用。因此，本项目设计、构建了一个由白藜芦醇调控的转基因调控平台，并将其应用于缓解CAR-T免疫疗法在临床治疗过程中出现的副作用。我们成功构建了灵敏度高、激活或抑制效果好的RES-ON和RES-OFF 两种基因开关，分别在30μM浓度下可激活144倍或者抑制47倍基因表达。并且该调控系统在细胞水平具有很好的可逆性和长期性。其次，在野生型小鼠体内实验中，该两种调控系统在体内转基因表达具有很好的调控效果，移植小鼠后在250 mg/kg的给药浓度下RES-ON系统可激活30倍，RES-OFF系统可抑制4.3倍。最后将这两种基因开关系统导入T细胞中，在T细胞中可以很好地调控CAR的表达，在加入阳性肿瘤细胞刺激后，可激活IL2、CD69等T细胞激活蛋白的表达，证明该调控CAR-T系统的可行性，同时该系统本底极低，灵敏度也有所提高，为以后的CAR-T动物体内肿瘤免疫治疗奠定了基础。