基于蛋白多功能输送载体的基因编辑技术及其抗肿瘤治疗

Combination of multi-drug is an effective approach to improve therapeutic effect by regulating multiple networking factors. The multitargeted drugs based on one molecule or nanocarrier play important roles to fulfill the requirements of combined therapy. However, the screening of pharmaceutical molecule targeting multiple factors or genes gene is very laborious and even lead to a negative result. For multitargeted drugs in one nanocarrier, developing multifunctional nanocarrier materials is an essential issue to overcome the drawbacks of conventional combinatory therapy adopting multiple drug formulations, e.g. difficulty in adjusting drug dose and action time, and complicated drug administration. This proposal aims to establish a platform for efficient non-viral genome editing based on multifunctional protein delivery system. Its success will facilitate the widespread clinical use of the CRISPR/Cas9 system to solve health problems that require precise control over tumor cell fate. First, the azide group introduced on the surface of dCas protein in a specific site can be easily used to link with drug loading nanoparticle. Secondly, the small molecule drugs (emoxir) are encapsulated into the micellar core. Then, the modified RNPs (the complexes of Cas and gRNAs) are loaded into nanoparticle by the means of electrostatic interaction or click reaction to inhibit tumor growth. Finally, the negatively charged anionic polymer with cross-link structure via phenyl boronate ester linkages. Therefore, carriers are endowed with properties of long circulation and high intratumoral drug enrichment via carrier size control, zeta potential regulating and cross-linked structures. Additionally, non-viral intracellular delivery of proteins and protein: nucleic acid complexes, as established here, can greatly expand the scope of research and therapeutic applications of proteins and RNA.

联合应用蛋白定点突变、可逆交联捆绑和环境响应性药物释放等策略，发展基于多功能dCas蛋白/gRNA输送载体和基因编辑技术的多靶点药物治疗新策略。首先，在不影响蛋白活性前提下，在dCas蛋白上定点引入叠氮基团；其次，制备带炔基的酸和还原剂双敏感阳离子聚合物，酸敏段负载小分子药物乙莫克舍自组装为正电纳米胶束，载体表面的还原敏感断裂键通过炔基与叠氮反应负载dCas蛋白，正电部分负载gRNA；最后，靶向负电聚合物包覆于外层，形成苯硼酸酯键交联捆绑的负电纳米载体，具有较小粒径、负电长循环、交联捆绑高血液稳定性、多组织靶向输送、肿瘤内酸和还原双敏感释药的特点。通过多功能载体，靶向血液肿瘤细胞及隐匿在脂肪和骨髓组织中的白血病干细胞，联合输送针对多个基因的dCas蛋白/gRNA和小分子抑制剂乙莫克舍，多模式（上调和下调基因表达、竞争抑制）调控，同时促进细胞凋亡和抑制能量代谢剂耐药，协同提高白血病治疗效果。

发展了基于组氨酸标签定位/邻位识别的蛋白修饰技术，并联合应用微环境响应性药物释放和生物正交靶向输送等技术，发展了多种可联合输送Cas9/dCas9蛋白或质粒、小分子药物以及造影剂的纳米载体，通过调控靶向修饰、表面电位、响应性释放等性能，展现出高效的药物传输、基因编辑和协同治疗效果。（1）合成了可定位修饰His标签的反应分子，对Cas9/dCas9蛋白进行定点修饰；合成了末端携带氮川三乙酸（NTA）的邻位修饰分子，选择性地修饰邻位区域的氨基，定点/定位引入叠氮或DBCO等点击反应基团；（2）合成了还原响应的两亲性聚合物NTA-聚乙二醇-聚己内酯（NTA-PEG-PCL），NTA特异性结合携带His标签的Cas9/sgRNA复合物，疏水嵌段PCL负载光敏剂Ce6，通过靶向分子修饰和表面电位调控等策略优化传输性能。光照启动肿瘤内的基因编辑和和光动力治疗，未光照的正常组织中则出现Cas9/sgRNA降解，实现了肿瘤特异性的基因编辑和光动力协同治疗；（3）将叠氮修饰聚合物N3-PEG-PCL、NTA-PEG-PCL、DBCO-PEG-DBCO等聚合物联用，共输送Cas9/sgRNA和紫杉醇，实现生物正交靶向输送、高效溶酶体逃逸、基因编辑和化疗药物的协同治疗；（4）制备了具有还原剂和酸双敏感的聚合物NTA-SS-PEG-聚(二异丙基氨基)甲基丙烯酸乙酯-聚（地西他滨）甲基丙烯酸乙酯，共输送磁共振造影剂、Cas9/sgRNA和表观遗传学药物地西他滨，协同增强肿瘤细胞对PD-1抗体的响应性；（5）发展了具有多重响应性的载体，联合输送Cas9/sgRNA、紫杉醇和PD-L1抗体，响应外源红外光和内源pH刺激逐级释放药物，实现了时空可控的基因编辑和多药联合治疗；（6）合成了具有核定位功能的阳离子聚合物，将编码dCas9/sgRNA的质粒高效导入到肺或成骨细胞中，上调抗病毒基因表达，抑制禽流感病毒在肺中的感染和增殖并表现出低副作用，也可上调成骨基因表达促进成骨。部分研究成果已形成10篇SCI论文，发表于Science Advances、Advanced Science、Small、Materials Today Bio和ACS Applied Materials & Interfaces等高水平期刊，转化为核心期刊教学论文2篇，5项发明专利获得授权，毕业博士生3名和硕士生4名。