利用CRISPR/Cas9基因编辑实现肿瘤免疫治疗的靶向纳米递送系统的优化与效果研究

Traditional treatments, such as chemotherapy and radiotherapy, have poor therapeutic effects on malignant tumor. Cancer immunotherapies are treatments that restore or enhance the immune system's ability to fight cancer. The development of cancer immunotherapies progresses at a growing pace with a variety of technological approaches. A successful immunotherapy treatment must induce active immune responses against cancer cell in the human body, including “effector cell therapy”, classified as passive immunotherapy, and “inhibition of immunosuppression”, which intends to break immunological tolerance to autoantigens or immunosuppressive environments. Cancer immune evasion is a major stumbling block in designing effective anticancer therapeutic strategies. The therapeutic efficacy will be significantly improved when a combination treatment against various immune escape mechanisms are used. Genome-editing technologies have brought revolutionary progresses in cancer immunotherapy. Among the well-known engineered nucleases systems, CRISPR/Cas9 is becoming a most popular genome editing tool due to its simplicity, versatility and high efficiency, which takes cancer immunotherapy to the next level. Nano drug carriers can enrich gene drugs in cells, while free gene drugs alone cannot cross the cell membrane because the anionic property and stiff structure. Nanoparticles used as drug delivery system has a vast application prospect. Cationic solid lipid nanoparticles (cSLN) as gene drug delivery vehicles have attracted spotlight for they significantly enhanced the activity of gene drugs in cells. In this project, we will develop and optimize the biodegradable cSLN for targeted delivery CRISPR/Cas9 system (Cas9 mRNA and sgRNA) into colorectal cancer cells formed by poly (lactide-co-glycolide) (PLGA) and lipids, based on the results of our previous studies. The cSLN has several advantages, such as the structure and surface properties are controllable, and it’s easily functionalized and modified. In this project, we will investigate the genome editing effects of optimized cSLN delivered Cas9 mRNA and sgRNA both in vitro and in vivo, and to understand the underlying mechanisms. This project is also expected to provide methodological and theoretical supports for the development of nano drug carriers for cancer immunotherapy.

传统治疗手段对恶性肿瘤的疗效不佳。利用免疫系统识别并清除肿瘤细胞的免疫疗法有望成为一类安全、有效的抗肿瘤疗法。消除肿瘤细胞的免疫逃逸是实现肿瘤免疫疗法的关键。同时针对肿瘤细胞的多种免疫逃逸机制设计的治疗策略将显著提高疗效。借助CRISPR/Cas9基因编辑技术同时敲除肿瘤细胞多种基因可阻断其多种免疫逃逸机制，提高免疫系统抗肿瘤效果。纳米药物载体能有效克服CRISPR/Cas9基因编辑元件体内应用的障碍，实现其在肿瘤免疫疗法中的应用。阳离子固体脂质纳米载体作为基因药物输送载体显著增强药物活性而备受关注。本项目将在已有预研结果基础上，发展基于聚乳酸-羟基乙酸共聚物和脂质分子的阳离子固体脂质纳米载体，优化其体内药物递送性能，实现高效靶向肿瘤细胞输送Cas9 mRNA和sgRNA，并对体内外生物学效应和作用机制进行研究，为发展可用于肿瘤免疫治疗的纳米载体提供方法学和理论上的依据及支持。

肿瘤的免疫疗法有望成为继放疗、化疗之后一类安全、有效的抗肿瘤疗法。消除肿瘤细胞的免疫逃逸是实现肿瘤免疫疗法的关键。CRISPR/Cas9 基因编辑技术提高了细胞基因组中基因表达的效率，为抑制肿瘤细胞免疫逃逸提供了有效途径。然而，核酸药物在生理环境中的稳定性和细胞膜穿透能力较差，成为CRISPR/Cas9 基因编辑技术用于体内肿瘤治疗的主要障碍。纳米药物递送系统在改善和提高核酸药物的体内靶向输运效果方面具有较好的优势。其中，阳离子纳米药物载体用于核酸药物递送具有高效携载、保护降解、可控释放、易于靶向修饰等优势，是CRISPR/Cas9 基因编辑元件体内靶向递送的理想载体。.针对同时敲除肿瘤细胞中CCL2和PD-L1 表达是恢复并增强机体免疫系统抗肿瘤作用的这一关键问题，本项目拟制备具有结构和性能可控的阳离子脂质纳米药物载体，研究并优化其对Cas9mRNA 和sgRNA 的负载、释放和输送性能，同时借助靶向分子的官能化修饰构建具有靶向细胞输送针对CCL2 和PD-L1 的CRISPR/Cas9 基因编辑元件能力的生物可降解纳米载体，并研究CRISPR/Cas9 基因编辑元件输送系统的体内、外高效克服肿瘤免疫逃逸的能力和肿瘤免疫治疗效果，探索机制。.本项目在纳米药物载体的基础和应用研究中取得创新性进展，发展出新型具有应用潜力的基于生物可降解高分子的阳离子脂质纳米载体用于CRISPR/Cas9基因编辑元件的体内输送，实现体内靶细胞的高效基因编辑；进一步揭示了该纳米载体消除肿瘤细胞免疫逃逸、激活抗肿瘤免疫反应的作用机制。我们所制备的纳米药物在小鼠模型中实现了显著抑制肿瘤生长的目的，具有较好的临床转化前景,也为发展用于体内递送CRISPR/Cas9基因编辑元件的纳米药物载体提供方法学和理论上的依据及支持。本项目研究成果主要以论文形式发表，发表高水平SCI论文17篇，培养博士研究生5名，硕士研究生6名。