以促内涵体逃逸聚合物PEG-P[Asp(TEP)]-cholesterol为载体构建双级脑靶向基因传递系统沉默BACE1基因的研究

In order to improve the efficiency of siRNA brain delivery and cell ransfection efficiency.A low toxicity copolymer PEG-P[Asp(TEP)]-cholesterol with endosomal escape function was synthesized.A novel polyion complex(PIC)is prepared by self-assemblely process in the presence of PEG-P[Asp(TEP)]-cholesterol and small interfering RNA plasmid specific for beta-secretase. The noval dual-brain targeting gene delivery system is constructed by using RDP combined with Tet1 peptide as the co-modification ligand and connecting to the surface of polyion complex(RDP/Tet1-PIC). We will investigate and optimize the degree of grafting of the polymer, the the density of the modified ligand on the surface of polyion complex, the compound of the N/P ratio of the polyion complex related to the physicochemical properties and biological properties of the gene delivery system. The brain transport property of the RDP/Tet1-PIC was evaluated, and the gene transfection efficiency of nerve cells and silencing efficiency of b-site amyloid precursor protein-cleaving enzyme1(BACE1) gene was investigated. initially the intracellular transport mechanism was investigated. Furthermore, RDP/Tet1-PIC loaded pGPU6/BACE1a improved the Amyloid-β25-35 induced learning and memory impairments in rats were investigated. After the establishment of the rat model of Alzheimer's disease, and the evaluation of RDP/Tet1-PIC loaded siRNA induce the BACE1 gene silencing in rat model. This study will have application prospects in the prevention and treatment of neurodegenerative diseases and provid a novel active targeting, high efficiency, low toxicity non-viral vectors and provide new ideas and the necessary experimental and theoretical basis.

本项目针对siRNA脑部传递中存在的主要问题，合成具有促内涵体逃逸功能、低毒性的载体聚合物-胆甾醇基修饰的聚乙二醇-b-聚（四亚乙基五胺）天冬氨酸提高转染效率，与BACE1干扰质粒孵育自组装构建可沉默β-分泌酶基因的聚离子复合物。而后将RDP肽和Tet1肽作为双重修饰配体，链接于聚离子复合物表面，构建一种双级脑靶向基因传递系统，增加脑部基因药物递送的选择性。分别考察优化聚合物的接枝度，靶向配体的修饰密度、复合物的N/P比等对系统的物化性能和生物学性能的影响。评价该传递系统细胞毒性，跨血脑屏障转运特性，细胞转染和基因沉默效率，初步考察细胞内转运机制和释放机制。并建立大鼠阿尔茨海默病模型，评价载药系统介导siRNA沉默BACE1基因以及对模型大鼠记忆和认知能力的提高。本研究为主动靶向、高效、低毒的非病毒基因载体构建及神经退行性疾病的基因治疗提供新的思路和必要的实验和理论依据。

神经退行性疾病病理可见神经元退行变性、丢失，导致认知记忆功能和运动功能障碍，发病机制复杂，是神经科学面临的严峻挑战。基因治疗具有分子靶点的高特异性特点，如利用siRNA代替传统反义核酸进行转录后引起生物细胞内同源基因的特异性沉默，从而实现基因表达调控等。许多神经退行性病变与基因的异常表达有关，与其他组织相比，脑组织表达更多的特异基因序列，因此，具有复杂结构的神经系统内高效、准确的沉默靶基因治疗有重要的意义。本项目合成了一种低毒、内源性氨基酸为主要结构的阳离子基因载体PEG-P[Asp(TEP)]-cholesterol，并构建了双级脑靶向基因递释系统RVG29/Tet1-Polyplex，粒径约100nm，Zeta电位+16mV，具有促内涵体逃逸功能，同时通过双功能配体靶向于BBB进而靶向神经细胞，实现更加高效的脑内基因递释，以阿尔茨海默病作为疾病模型，考查载siRNA重组质粒的基因递释系统干扰沉默其致病相关的BACE1基因。本研究构建的基因递释系统细胞及组织毒性均显著低于常规转染试剂Lipofectamine2000。细胞内定位示踪结果显示具有明显的促内涵体逃逸作用。相对于对照组被动靶向载体系统及单配体修饰的载体系统，在具有主动靶向跨血脑屏障的同时神经细胞转染效率显著提高。动物体内药动学和药效学结果表明，RVG29/Tet-1-Polyplex载治疗基因具有更强的脑组织分布和转染效率，同时显著提高了转基因阿尔茨海默病模型小鼠的记忆和空间认知能力，抑制了BACE1蛋白表达。以上结果说明本项目构建的双级靶向载基因递释系统具有较好的中枢神经系统基因递释作用。