基于CRISPR/Cas9基因编辑技术的声控脑靶向相变纳米载体的构建及治疗胶质瘤的研究

The emergence of CRISPR/Cas9, a new kind of genome-editing technique, opened a new chapter on precise gene therapy. But most of the studies still remained at cellular level recently. The high efficiency delivery of CRISPR/Cas9 to central nervous system is the key for targeted gene therapy of CNS disease. Our group had constructed several targeted gene or drug loaded systems which could across blood brain barrier under ultrasound combined with microbubbles. This project proposed the design of a new vector of sgRNA/Cas9 for nanoscale, multifunctional genomics editing, and achieved a precise treatment for glioma through cascade targeting strategy. The liposome in this system is modified with aptamer, and the sgRNA/Cas9 complex is designed to knock out the MGMT gene, and the acoustic phase change PLGA-liquid fluorocarbon nanoparticle is the core of the system. Ultrasound mediated phase change could open BBB, and the specific binding between aptamer and glioma could promote internalization of sgRNA/Cas9-loaded liposome, leading to down regulation of MGMT expression and enhancement of the sensitivity of glioma on chemotherapy. This nano system will be verified by physical characterization, loading rate, toxicity, targeting efficiency in vitro and in vivo, for achieving accurate delivery and gene editing of CRISPR / Cas9 in CNS. To provide a new way to explore the precise gene therapy for CNS diseases.

新型基因编辑技术CRISPR/Cas9的出现，为精准化基因治疗开启了新的篇章，但目前仍停留在细胞水平。如何将CRISPR/Cas9向CNS有效投递是靶向基因治疗的关键。基于超声介导微泡靶向开放BBB，课题组成功构建多种跨BBB和靶向性载药或载基因体系。本项目提出设计一载sgRNA/Cas9的纳米级、多功能基因组学编辑新型载体，通过级联靶向策略，实现针对胶质瘤的精准治疗。该系统以靶向核酸适配体修饰脂质体，包载靶向敲除MGMT基因的sgRNA/Cas9复合物，以声致相变型PLGA液态氟碳纳米粒为系统核心。在超声介导下相变开放BBB，借助核酸适配体与胶质瘤特异性结合，促进内化，通过下调MGMT表达，增强胶质瘤对化疗的敏感性。所建纳米系统经物理表征、包载率、毒性、离体和载体动物靶向性等试验验证，实现CRISPR/Cas9在CNS的精准投递和基因编辑。为探索CNS疾病的精准化基因治疗提供新的途径。

胶质瘤的治疗一直是临床上的难点和热点，不仅是因为血脑屏障（BBB）的存在，也因其耐药的特性降低了化疗药物的治疗效果。构建一种即能透过血脑屏障，又能降低肿瘤耐药性的载药系统对胶质瘤的治疗具有重要意义。因此，我们设计了一种基于CRISPR/Cas9技术、联合超声-微泡的脑靶向纳米药物递送系统。首先通过纳米粒沉淀法制备了以cRGD为靶向配体、装载靶向耐药基因MGMT的CRISPR/Cas9质粒的脂质-聚合物纳米粒（LPHNs-cRGD），该纳米粒具有典型的壳-核结构，能保护基因免受核酸酶的影响，在体外能够靶向胶质瘤细胞，介导基因转染，并增强胶质瘤细胞对化疗药物替莫唑胺（TMZ）的敏感性。在体内研究中，通过联合聚焦超声辐照，复合物MBs-LPHNs-cRGD能够安全、有效地开放BBB，靶向作用于动物脑内胶质瘤，并增强TMZ对胶质瘤的杀伤效果，延长荷瘤鼠的生存时间，且具有良好的生物安全性。通过本项目的研究能为中枢神经疾病的精准化基因治疗提供实验依据和一种治疗胶质瘤的策略。