特异性靶向细胞内环境敏感递药胶束的乙肝基因治疗基础研究

The high gene transfection of the non-viral gene delivery system is importance premise to realize the efficient gene therapy. In this project, we chosen 10-23DNAzyme as model gene, and designed an in vivo stable nano carrier, which display excellent uptake ability by targeting cells and cytoplasm accumulation. Through the hydrophilic polysaccharides was conjugated with octadecylamine by disulfide linkage, a novel intracellular splendid glutathione-responsive glycolipid-like gene delivery vector should be formed. The relationship between the effective distribution in vivo, excellent cellular-uptake and the active targeting modification of the vector will be studied. There are also explored the inherent law of the modification of the histidine and disulfide bonds of shell-sheddable micelles with the genes escaping from lysosome and intracellular release. By the research of gene therapy on HBV model cell lines and HBV model animals, we investigated the relevance between biological targeting and gene transfer efficiency. As considered to be biological stabile and safe, we further expounded the inherent law of both in vitro and in vivo gene therapy. This study will provide new idea, strategy and approach for efficient and safe gene therapy for HBV, and enlarge and develop the HBV gene therapeutical theory.

非病毒载体的高效安全转染，是实现乙肝基因治疗的重要前提。本项目以乙肝病毒基因特异性切割酶10-23DNAzyme作为治疗基因，采用体内稳定、靶细胞高效摄取、具有细胞浆靶向性的纳米载体，通过亲水性多糖与疏水性脂链之间的二硫键化学链接，形成靶细胞内高谷胱甘肽浓度响应性新型糖脂载体基因给药系统。研究基因给药系统主动靶向修饰与体内有效分布、细胞高效摄取之间的相关性，探索基因给药系统的外壳卸载型二硫键修饰、pH响应组氨酸分子修饰，促进基因在细胞内的谷胱甘肽敏感释放及溶酶体逃逸的机理。在基因给药系统生物稳定性、安全性研究的基础上，通过乙肝病毒模型细胞和模型动物的基因治疗研究，探明基因给药系统生物靶向、基因转染效率与基因治疗疗效之间的内在规律性，为实现高效安全的乙肝基因治疗，提供新思路、新策略和新方法，丰富和发展乙肝基因治疗理论体系。

非病毒载体的高效安全转染，是实现乙肝基因治疗的重要前提。本项目以乙肝病毒基因特异性切割酶10-23DNAzyme作为报告基因，采用体内稳定、靶细胞高效摄取、具有细胞浆靶向性的纳米载体，通过亲水性多糖与疏水性脂链之间的二硫键化学链接，形成靶细胞内高谷胱甘肽浓度响应性新型糖脂载体基因给药系统（Chitosan-SS-Octadecylamine，CSSO）。研究基因给药系统主动靶向修饰与体内有效分布、细胞高效摄取之间的相关性，探索基因给药系统的外壳卸载型二硫键修饰、pH响应组氨酸分子修饰，促进基因在细胞内的谷胱甘肽敏感释放及内涵体逃逸的机理。在基因给药系统生物稳定性、安全性研究的基础上，通过乙肝病毒模型细胞和模型动物的基因治疗研究，探明基因给药系统生物靶向、基因介导效率与基因治疗疗效之间的内在规律性，嫁接物载体作用48h后，细胞毒性IC50为1.056mg/ml，CSSO/DrzBS与CSSO/DrzBC基因给药系统转染后72h内对HBsAg和HBeAg最高抑制率分别为76.79±2.18%和83.83±2.34%，DrzBS和DrzBC的脂质体给药系统则分别为22.76±0.67%和23.98±0.81%。相较于脂质体LipofectamineTM2000，CSSO嫁接物胶团在阻断HBV复制上表现出明显的高效低毒的优势。本项目为实现高效安全的乙肝基因治疗，提供了新思路、新策略和新方法，丰富和发展乙肝基因治疗理论体系。