利用E. coli融合表达体系及群体感应系统构建抗肿瘤工程菌株

Synthetic biology has the potential to engineer bacteria that specifically kill tumors and deliver cytokines that have antitumour effects, which allowing the engineering of ‘perfect’ cancer therapies. However, it is still remained challenges before bacteria can be used in the clinic, such as the body resistant of intravenous injection of bacteria which exprssion cytokines. Oral route could avoid this risk, but the cytokines almostbe degraded by the digestive enzymes. In the previous research, we developed a recombinant expression system which could extracellular expression, improve the solubility and stability of the target proteins. In this project, Escherichia coli Nissle 1917 is chosen as a basic strain. We plan to use the density sensing module to engineer invasive E. coli strain which could kill the tumor cells by the endogenous cytosine deaminase (CDase/5-FC). And further develop an oral cytokines delivery pathway based on Cel-CD recombinant expression system to stimulate the immune response. In this project, the Cel-CD recombinant expression system is applied to the construction of biological therapy engineering bacteria, which protect the extracellular expressed cytokines from digestive enzymes. It is provides a new application example for tumor therapy and has good clinical application prospect.

随着合成生物学的发展，构建能特异性杀伤肿瘤细胞、同时表达细胞因子激发机体抗肿瘤免疫应答的工程菌，是肿瘤生物治疗的全新发展方向。由于静脉注射的安全性及机体耐受性尚缺乏保证，口服是生物工程菌肿瘤治疗的理想给药方式，但细胞因子易被消化酶降解影响药物利用率。申请人前期研究中构建了大肠杆菌融合表达平台，载体蛋白Cel-CD不仅克服了大肠杆菌分泌限制，还能增加细胞因子在消化道中的稳定性。在本项目中，我们选取益生菌E.coli Nissle 1917作为平台菌株，利用群体感应系统构建侵袭性工程菌，借助大肠杆菌内源性胞嘧啶脱氨酶 (CDase/5-FC)杀伤肿瘤细胞。在此基础上通过Cel-CD分泌表达白介素-18，激发机体自身免疫反应进一步抑制肿瘤生长。本项目将Cel-CD融合表达平台应用于生物治疗工程菌的构建，提高了细胞因子的药物利用率，为肿瘤微生物治疗提供了全新的应用实例，具有良好的临床应用前景。

大肠杆菌E. coli nissle 1917（EcN）是临床治疗新生儿腹泻的益生菌，不具有致病性并且不会在人体内长期定植。在借助合成生物学手段编辑携带抗肿瘤药物蛋白或相关酶类后，可保护药物蛋白不受消化道酶的降解, 到达目标区域后直接释放药物蛋白到效应部位, 减轻全身副作用，在应用于消化道疾病治疗时具有良好的前景。.本项目借助合成生物学技术改造EcN，尝试了多种基因组编辑技术，最后采用Tn-7转座子成功编辑获得基因组整合有荧光素基因簇luxCDABE的工程菌菌株。在此基础上，1）利用前期构建的分泌表达体系成功融合分泌了一系列药物蛋白，最后选取了分泌效果较好的AGR2蛋白进行了肠炎的干预治疗，进行了小鼠DSS肠炎模型验证。2）针对目前工程菌株的应用限制，进一步构建了侵袭性工程菌以及裂解表达系统。本项目所构建的大肠杆菌工程菌一方面通过通过脂多糖激发机体天然免疫， 起到治疗佐剂作用，一方面进入到目标组织释放药物蛋白，实现了“一菌多用”， 具有一定的理论创新性。