肿瘤细胞膜包被ROS响应纳米粒用于CRISPR/Cas9基因编辑系统的靶向递送及EB病毒感染相关鼻咽癌的治疗

The CRISPR/Cas9 gene editing system has received increasing attention due to its simplicity and ease of use, which makes it an attractive candidate treatment for a range of diseases. However, the size of CRISPR/Cas9 plasmid is relatively large, which becomes an obstacle to reach its effective delivery. Another challenge of CRISPR/Cas9 is the potential off-target effects. Off-target editing may bring potential risk of disease so work needs to be done to improve the specificity and reduce the off-target effects of CRISPR/Cas9 system. We first construct a CRISPR/Cas9 system with the capability of specifically knocking out Epstein-Barr virus (EBV) genome in EBV associated nasopharyngeal carcinoma cells. Then, a biomimetic system, cancer cell membrane-coated nanoparticles was designed and applied to realize the targeting delivery of CRISPR/Cas9 plasmid. The core of the biomimetic nanoparticles is fabricated via electrostatic interaction between CRISPR/Cas9 plasmid and reactive oxygen species (ROS)-responsive cationic polymer (hyperbranched poly(β-amino ester), HPBAE). After being modified by a charge-reversible polymer (CRP), completely cancer cell membrane (CCm)-covered nanoparticle can be prepared, which is able to realize the deep penetration in tumor tissue and specifical uptake in tumor cells. Owing to the fast hydrolysis in endosome/lysosome, pH-dependent protonation of CRP can be achieved and then promotes the endosome/lysosome escape of CRISPR/Cas9 plasmid with the cooperation of HPBAE. At the same time, HPBAE degrades due to the high intracellular ROS level, which triggers the release of plasmid. Finally, CRISPR/Cas9 knockout of EBV genome will induce the apoptosis of nasopharyngeal carcinoma cells and thus realize the efficient and complete gene therapy on nasopharyngeal carcinoma.

CRISPR/Cas9系统是一种高效的基因编辑工具，它简便易行，已被广泛应用于多种疾病的基因治疗。但其也存在质粒较大、递送困难，可能因脱靶而带来潜在的致病风险等问题。为此，申请人拟构建和筛选能特异性切割鼻咽癌EBV致病基因的CRISPR/Cas9质粒，并制备肿瘤细胞膜包被仿生纳米粒子用于质粒的靶向递送。粒子从ROS响应阳离子型超支化聚合物聚（β-氨基酯）（HPBAE）出发，经静电力将质粒压缩为纳米结构，再通过可电荷翻转聚合物（CRP）调节粒子的表面电荷以实现肿瘤细胞膜的完整包覆。所形成的仿生纳米粒可实现肿瘤组织深部渗透并被肿瘤细胞专一性摄取；进入内涵体/溶酶体后，CRP发生电荷翻转并质子化，与HPBAE协同促进质粒的溶酶体逃逸；同时，HPBAE在胞内高浓度ROS环境下降解，释放出质粒；最终特异性地对致病基因进行切割，诱导肿瘤细胞凋亡，实现对EBV感染相关鼻咽癌高效、彻底的基因治疗。

项目针对鼻咽癌EBV致病基因LMP1，以经典的spCas9和最新报导的frCas9基因编辑为基础，设计、评价了13条靶向LMP1基因的sgRNA，优选出3条编辑效率较高的sgRNA用于构建CRISPR/Cas9基因编辑质粒；合成了18种ROS响应PBAE共聚物（超支化4种、线型13种、嵌段1种）和3种非ROS响应PBAE（超支化1种、线型1种），对其结构、转染能力进行研究，在此基础上构建了3类ROS响应PBAE-质粒复合纳米粒用于CRISPR/Cas9基因编辑质粒的递送：（1）肿瘤细胞膜包被ROS响应超支化PBAE（PAM-PBAE）复合纳米粒，（2）ROS响应后交联PBAE复合纳米粒，（3）ROS响应PBAE-ThE复合纳米粒；通过低渗-剪切法制备得到鼻咽癌细胞C666-1膜（C6M）囊泡，经共挤出或共孵育即可在复合纳米粒表面形成完整的膜包覆结构，该仿生纳米粒在同源肿瘤细胞的摄取率显著提高，给药后可富集于肿瘤原位或鼻腔，并实现肿瘤组织深部的渗透；经肿瘤细胞摄取后，纳米粒可促进质粒的溶酶体逃逸并释放出质粒，最终特异性地对致病基因LMP1进行切割，调节相关信号通路，诱导肿瘤细胞凋亡，进而实现了对EBV感染相关鼻咽癌高效的基因治疗；其它两类ROS响应复合纳米粒也观察到了较非ROS响应系统更好的基因编辑效率和病毒感染相关癌症治疗结果。在此基础上，还将PBAE拓展到了HPV感染相关肿瘤、椎间盘退变、猪内源性逆转录病毒等领域的基因治疗，以及多肽、小分子药物的递送中，丰富了PBAE递送系统的应用范围。总之，项目证实了通过CRISPR/Cas9系统设计和仿生智能载体构建可有效编辑致病基因，实现对EBV相关鼻咽癌等疾病的安全、有效的基因治疗。此外，项目创新性开发了一种适用于腔道基因治疗的凝胶剂，研究了其跨黏液屏障基因递送能力，为PBAE-质粒复合纳米粒的临床转化提供了有价值的铺垫。