具有高度仿病毒形态和功能的基因递送系统的构建与性能研究

In recent years, artificial virus gene delivery system has been the research hotspots in the field of gene therapy. However, despite the existing artificial virus gene delivery system have gained some progress in virus size, morphology, envelope and capsid simulation, respectively, they rarely have both virus morphology and function. The project aims to construct an artificial virus gene delivery system with high similarity in virus morphology and function, which is composed of an artificial virus envelope and capsid. The multiple functionalized (PEGylated, biotinylated, 2,3-dimethylmaleic anhydride modified) poly-L-lysine dendrimer with a protein-like globular structure was served as an artificial virus envelope to fulfill the long circulation, active targeting and programmed detachment of the envelope outside the cells. The disulfide bond cross-linked fluorinated poly-L-lysine dendrimer was served as an artificial virus capsid to fulfill the rapid endocytosis, endosome escape, as well as polyplex disassembly and DNA release. Owing to its high similarity in virus morphology and function, this system is expected to have an efficient and safe gene delivery performance both in vitro and in vivo. The implementation of this project will put forward the design principle of artificial virus gene delivery system with both virus morphology and function, reveal its in vitro and in vivo gene delivery mechanisms and influence regularity, obtain an efficient and safe gene delivery system, lay the foundation for the development of gene delivery systems with high clinical values.

仿病毒基因递送系统是近年来基因治疗领域的研究热点。已有的仿病毒基因递送系统虽然分别在模拟病毒尺寸、形貌、包膜、衣壳等方面取得了一定进展，但同时具有病毒形态和功能的仿病毒基因递送系统还较少。本项目拟构建一类由仿病毒包膜和衣壳组成的具有高度仿病毒形态和功能的基因递送系统。该系统以具有类蛋白一样球状结构且尺度为纳米级，并经PEG、生物素、2,3-二甲基马来酸酐修饰的肽类树状大分子为仿病毒包膜用于实现长循环、靶向、细胞外程序化脱包膜。以二硫键交联的氟化肽类树状大分子为衣壳用于实现快速入胞、溶酶体逃逸、胞内复合物解组装及DNA释放。由于在形态、功能等方面都具有高度仿病毒特性，该系统预期会有高效安全的体内外基因递送性能。通过项目的实施，提出具有病毒形态和功能的仿病毒基因递送系统的设计原理，揭示其体内外基因递送机制与影响规律，获得一类高效安全的基因递送系统，为研发具有临床应用价值的基因递送系统奠定基础。

本课题以构建具有高转染效率且生物相容性优异的仿病毒基因递送系统为出发点，首先成功合成了氟化修饰的赖氨酸树状多肽，并建立了利用还原敏感性双硫键交联低代数树状多肽以构建树状多肽纳米凝胶的新方法。我们研究发现：该树状多肽纳米凝胶在负载了DNA或siRNA后可形成类似于单纯疱疹病毒拓扑结构的DNA或siRNA复合物，即具有仿病毒结构的仿病毒基因递送系统。更重要的是，该递送系统可携载DNA或siRNA高效突破多重生理屏障，并显著提高其入胞、溶酶体逃逸、胞质内转运、耐血清能力，以及体内外基因转染效率。且进一步携载siVEGF进行抗肿瘤研究发现：siVEGF复合粒子能高效快速地被细胞摄取，并实现溶酶体逃逸；双硫键在胞内还原敏感性断裂可促使复合粒子快速解组装并释放siVEGF；高效的VEGF沉默效果及动物体内抗肿瘤作用。本课题的开展对仿病毒基因递送系统在克服多重细胞外和细胞内屏障方面提供了重要参考意义，且为高效安全的仿病毒基因递送系统的研发奠定了一定理论基础。