滚轮微针电极阵列电穿孔递送CpG和抗PD-L1 siRNA介导肿瘤免疫治疗

The key scientific issue for small nucleic acid (e.g., siRNA) drug development is how to achieve safe and efficient delivery in targeted tissue or cell. Electroporation is a representative physical delivery technology. How to achieve efficient and low-damage siRNA delivery at low voltage is a pivotal issue for electroporation. Immunotherapy based on immune checkpoints (e.g., PD-1/PD-L1 signal pathway) is a promising strategy for cancer treatment. This project plans to develop a roller microneedle electrode array (RMEA) for in vivo delivery of immunoadjuvant CpG and PD-L1 siRNA for cancer immunotherapy. . Firstly, I will design and screen anti-PD-L1 siRNA. By chemically modify this siRNA, a lead sequence with high potency and stability will be selected. Then, roller microneedle array will be designed and manufactured with micromachining technology. The in vivo and intratumoral delivery conditions for siRNA and CpG will be determined by optimizing the voltage, pulse width and wavelength. Finally, I will perform comprehensive investigations with CT26 or B16F10 tumor-bearing mice, to evaluate its delivery efficiency, anti-tumor effect and in vivo toxicity. This proposal aims to establish a state-of-the-art nucleic acid delivery platform, to provide new theories, new technologies and new solutions for broader nucleic acid drug development and cancer treatment.

小核酸（如siRNA）是继小分子化合物和单克隆抗体后最具战略前景的生物制药技术。电穿孔是一种代表性的对细胞和局部组织行之有效的物理递送技术，如何在低电压下实现高效、低/无损伤的核酸电穿孔是该领域的核心科学问题。本项目拟设计滚轮微针电极阵列，围绕免疫检查点（PD-1/PD-L1）研究和发展基于核酸的肿瘤免疫治疗策略。.首先设计筛选抗PD-L1 siRNA，精细优化其化学修饰方案，获得高活性、高稳定性siRNA；设计新型微针滚轮器件，该器件可按需滚动、支持连续或大面积的组织电穿孔，通过微加工技术制备该器件，摸索优化输出电压、脉宽和波长等条件，确定对siRNA及免疫佐剂CpG最优的电穿孔参数；并在CT26和B16F10小鼠肿瘤模型上，深入研究该器件递送核酸的效率、核酸药物治疗肿瘤的效果及安全性，阐明该策略的理论机理和可行性，以建立优良的核酸递送平台，为更广泛的疾病治疗提供新理论、新技术、新方案。

核酸药物的局部给药与静脉注射相比，代表了一种具有变革性的创新药物开发方法。为了在体内实现高效广泛的siRNA药物治疗，本项目利用边缘有微针阵列的平行圆形叶片作为电极，设计了一种滚动微针电极阵列(RoMEA)。RoMEA集成了紧密间隔的微针电极和滚动结构，允许低损伤和大面积的siRNA转染。通过RoMEA在活体皮肤生成微孔，实现了高效的siRNA传递和基因沉默。此外，siRNA靶向程序性死亡配体1 (PD-L1)单独或联合抗程序性死亡-1 (PD-1)抗体或CpG2395免疫佐剂在两个肿瘤异种移植小鼠模型中的应用表明，所提出的策略恢复了有效的T细胞免疫反应，具有显著的肿瘤生长抑制作用，且具有良好的安全性。因此，RoMEA是一种前所未有的、巧妙的用于癌症免疫治疗的大面积局部核酸递送临床解决方案。.通过本项目的开展，针对局部病灶或手术部位进行按需、大面积、安全高效的核酸药物递送有了创新性的方法技术突破。有助于通过物理方法进行局部操作来输运调控免疫系统的核酸药物，实现新型的肿瘤高效治疗。