纳米载体递送CRISPR/Cas9基因编辑系统用于离体及在体构建CAR T细胞治疗肿瘤的研究

Chimeric antigen receptor (CAR) T cells therapy is one of the most promising tumor immunotherapies. However, lentivirus and γ-retrovirus are the main vectors for clinical-scale manufacturing of CAR T cells, the random insertion of lentivirus and γ-retrovirus may cause long-term safety risks, such as potential tumorigenicity. CRIPSR/Cas9 system can achieve precise genome editing, which provides a new method for constructing safe and efficient CAR T cells. The applicants previously have successfully disrupted Ntn1 gene of macrophages in vivo by cationic lipid-assisted polymeric nanoparticles (CLAN) encapsulating CRISPR/Cas9 plasmid (ACS Nano, 2018). The pilot experiments also showed that CLAN is capable of delivering CRISPR/Cas9 into T cells. Herein, the applicants plan to further optimize the previous CLAN by modulating the formulation of cationic lipids and polymers to fabricate a library of CLANs with different nano-properties. Then, CLANs of this library will be intravenously injected into mice for screening certain CLAN with the highest efficiency of delivering CRISPR/Cas9 into T cells in vitro and in vivo. The applicants will construct a CRISPR/Cas9 genome editing system that is able to introduce CAR elements of anti-CD19 or anti-EGFRvIII into the genomic region of TCRα chain constant (TRAC). Screened CLAN with highest delivery efficiency will be used to deliver this CRISPR/Cas9 genome editing system into T cells to construct safe and efficient CAR T cells in vitro and in vivo, which can achieve long-term remission of B cell lymphoma and glioma. This project will provide efficient, safe, and broadly applicable CAR T cells-based tumor therapy for clinical use.

嵌合抗原受体（CAR）T细胞是最有前景的肿瘤免疫疗法之一。但是，临床CAR T细胞主要由病毒感染制备，基因随机插入会带来长期的潜在致瘤等安全风险。CRISPR/Cas9系统可实现精确的基因编辑，为构建安全高效的CAR T细胞提供了新方法。申请人前期已通过阳离子脂质辅助的聚合物纳米颗粒(CLAN)递送该系统实现巨噬细胞基因编辑(ACS Nano,2018)，预实验也证明CLAN能够递送该系统到体内外的T细胞。本项目拟在前期发展的CLAN基础上，通过调节阳离子脂质和聚合物组份，制备具有不同纳米特性的颗粒库，并在体内外筛选高效递送该系统到T细胞的纳米颗粒；将构建用于编辑靶向CD19和EGFRvIII的CAR元件到TCRα链恒定区的基因编辑系统，并通过筛选的CLAN递送该系统到T细胞，在体内外构建CAR T细胞，治疗B淋巴瘤和神经胶质瘤，为肿瘤治疗提供潜在高效、安全、易规模化的CAR T细胞疗法。

本项目针对CRISPR-Cas9体内递送效率低和CAR-T细胞临床上主要利用病毒载体在体外制备的难题，致力于发展以T细胞为靶细胞的核酸药物纳米递送载体，通过设计和筛选不同表面特性的纳米载体，为高效递送CRISPR-Cas9到T细胞中以及非病毒载体介导的体内CAR-T细胞构建提供了策略，实现了高效的肿瘤免疫治疗。主要成果包括：（1）发展了调控阳离子脂质辅助纳米载体（CLAN）的表面聚乙二醇密度、电荷和脂质类型的方法，并通过筛选找到了能够高效将CRISPR-Cas9递送到小鼠体内T细胞中的纳米载体。（2）设计了T细胞特异性的纳米操纵子技术，通过该纳米操纵子递送anti-CD19 CAR分子到T细胞膜上，实现了直接在小鼠体内构建anti-CD19 CAR-T细胞，有效抑制了CD19阳性B细胞淋巴瘤的生长。（3）利用CLAN纳米载体递送CRISPR-Cas9等核酸药物，在体内通过增强CAR-T细胞的实体瘤浸润，显著增强了CAR-T细胞的抗肿瘤效果；通过调控树突状细胞的抗原提呈能力等策略，实现了肿瘤等多种疾病的治疗。本项目研究的核酸药物纳米递送载体筛选平台以及CAR-T细胞体内制备新技术，为基因编辑疗法和肿瘤细胞免疫疗法的临床应用提供了新的策略和技术支持。本研究的成果在Biomaterials、ACS Applied Materials & Interfaces、Small和Advanced Drug Delivery Reviews等发表论文7篇，申请发明专利3项，授权专利1项，完成了预期目标。