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。

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.