ROS响应CRISPRi系统和表观遗传抑制剂共递送纳米粒用于肿瘤PD-L1调控及肿瘤免疫治疗增强

肿瘤可借助其表面表达的程序性死亡-配体1（PD-L1）与免疫细胞表面的受体PD-1相结合，传导抑制性信号而导致免疫逃逸。PD-L1的免疫检查点抑制剂在临床治疗中已取得了显著疗效，但仍存在副作用大及具有个体选择性等问题。为此，申请人在前期研究基础上，拟采用CRISPR干扰（CRISPRi）技术下调肿瘤PD-L1表达，并结合表观遗传抑制剂阿扎胞苷（AZA）以协同增强抗肿瘤免疫效应。体系从活性氧(ROS)响应聚（β-氨基酯）（PBAE）-S-AZA阳离子聚合物前药出发，经静电力将质粒压缩形成纳米粒。经肿瘤细胞摄取后，质粒在PBAE作用下从溶酶体逃逸，释放质粒，下调PD-L1表达，解除免疫检查点抑制；同时，由硫醚键连接的AZA在胞内高ROS环境下快速释放，协同增强抗肿瘤免疫效应。本项目构建的纳米粒实现了基因编辑、免疫调节和表观遗传治疗的有机结合，为肿瘤免疫治疗的发展提供新的治疗策略。

Engagement of Programmed death-ligand 1（PD-L1）on tumor cells with its receptor PD-1 on immune cells can transmit an inhibitory signal to induce immune evasion. Although the immune checkpoint inhibitor PD-L1 antibody has got significant antitumor efficiency in clinical treatment, its wide clinic application still faces with several side effects and individual selectivity. Based on our previous research, we intend to use CRISPR interference (CRISPRi) system to suppress PD-L1 expression in tumor cells and combine with epigenetic inhibitor azacytidine (AZA) for enhanced cancer immunotherapy. We first fabricate reactive oxygen species (ROS)-responsive poly(β-amino ester) (PBAE)-S-AZA cationic polymer prodrug. CRISPRi plasmid and PBAE-S-AZA prodrug are complexed to form the final nanoparticles via electrostatic interaction. The nanoparticles are able to uptake in tumor cells with high efficiency, followed with the endosome/lysosome escape of plasmid with the cooperation of PBAE. The release of CRISPRi plasmid can lead to suppression of PD-L1 in tumor cells and relief of immune checkpoint blockade. At the mean time, the epigenetic inhibitor AZA is also released from the nanoparticles due to the high intracellular ROS level to enhance the efficacy of immunotherapy synergically. The nanoparticles help to realize the combination of genome editing, immunotherapy and epigenetic regulation effectively. It provides a novel platform for promoting antitumor treatment and precision medicine.