跨血脑屏障CRISPR/Cas13a递送系统的构建及其对狂犬病的治疗研究

RABV as a highly fatal central nervous system disease causes symptoms of the central nerve system diseases with nearly 100% mortality rate, because the blood-brain barrier (BBB) restricts the drug delivery to brain. However, biomimetic mineralization can confer new characteristics to biomacromolecule by the controllable assembling of inorganic functional materials and organic matrix. In our project, we chose CSV-B2c as the model, the BABV nucleoprotein gene as the target, the blood-brain barrier (BBB) penetrating CRISPR/Cas13a delivery system are designed based on the strategy combined with biomimetic mineralization and polypeptide modification. We then evaluate the therapeutic potential of the complex against rabies virus (RABV) infection. Therefore, the therapeutic CRISPR/Cas13a complex are supposed to target to the brain area that are abundant in nucleoprotein gene of CSV-B2c due to virus infection. Using the imaging techniques, the ability of CRISPR/Cas13a complex to cross BBB, target to infection area, and edit the functional genes in the central nervous system are evaluated both in vitro and in vivo. Furthermore, construction the animal model of the recombinant rabies virus infection which steadily expresses the green fluorescent protein (GFP) and investigation the therapeutic effect of CRISPR/Cas13a complex based on the small animal imaging (In-Vivo) system. Strategies and mechanisms of CRISPR/Cas13a complex to target to the brain and clear the virus infection in the brain are also investigated. Our project will provide a new strategies for anti-rabies therapy, and indicate great promise of such strategy in gene therapy of other brain diseases.

狂犬病是由狂犬病病毒（RABV）感染引起的一种高度致死性中枢神经系统疾病，一旦出现临床症状其致死率几乎100%，其主要原因是血脑屏障阻碍了药物到达脑部发挥作用。而仿生矿化通过无机功能材料与有机基质的可控组装赋予了生物大分子新的功能。因此，本项目以RABV固定毒株CVS-B2c的核蛋白基因为靶点，拟采用仿生矿化与多肽修饰等手段，构建新型跨血脑屏障CRISPR/Cas13a递送系统，将具有基因编辑能力的CRISPR/Cas13a靶向递送到脑内的病毒感染区域，用于狂犬病治疗研究。利用成像技术，从细胞和动物水平评价其跨血脑屏障、中枢神经系统靶向递送及基因编辑效率。构建绿色荧光蛋白稳定表达的重组RABV感染动物模型，通过活体成像系统评价CRISPR/Cas13a对小鼠病毒感染后的治疗效果，探讨脑内靶向清除病毒的策略和机制。为狂犬病暴露后治疗提供新的手段及工具，同时也为其它脑内疾病的基因治疗提供参考。

感染性疾病对人类健康和全球公共卫生安全构成重大威胁，亟需开发新的治疗策略加以应对，而纳米药物的出现为感染性疾病的精准防控提供了新的曙光。狂犬病是由狂犬病病毒（RABV）感染引起的一种高度致死性中枢神经系统疾病，一旦出现临床症状其致死率几乎100%，其主要原因是血脑屏障阻碍了药物到达脑部发挥作用。因此，设计和开发一种透血脑屏障、靶向识别和清除脑内RABV的策略，对于RABV暴露后的治疗具有重要的理论价值和现实意义。针对上述问题，本项目采用纳米策略，从病原微生物感染的诊断与治疗两个方面开展相关研究工作：（1）构建纳米传感器实现了对狂犬病病毒核蛋白的双模式灵敏检测；（2）病原微生物感染治疗方面：基于血脑屏障的特殊结构和性质，设计和构建突破血脑屏障的纳米递送系统；利用生物矿化策略，构建CRISPR纳米递送系统并将其用于细胞内基因编辑；针对耐药性病原微生物感染，设计响应性纳米载药系统以及非抗生素的纳米药物，实现了对耐药性病原微生物感染的精准治疗。本项目可以为感染性疾病的精准治疗提供新的策略。在课题执行过程中，项目负责人以并列通讯、并列一作发表论文12篇，申请专利2项，已授权1项，参与相关学术会议4次，分会场报告3次。