Development of easy and high efficient gene transfection using peptide-DNA complex

开发一种非病毒基因导入细胞系统是实现治疗性基因安全传递而不产生病毒载体常被指出的副作用的重要关键。结果表明，第六代L赖氨酸树枝状多聚物(KG6)具有较高的细胞转染率和较低的细胞毒性。在这里，为了研究末端阳离子基团对基因进入细胞的影响，我们合成了KGR6和KGH6，其中末端氨基酸分别被精氨酸和组氨酸取代。DNA结合分析表明，KGR6与DNA的结合能力与KG6相当，而KGH6的结合能力较KG6减弱。KGR6对几种培养细胞的转染率是KG6的3-12倍。相反，KGH6没有显示出转染率。然而，一旦KGH6与DNA在酸性条件下(pH 5.0)混合，就会形成DNA-复合体，并且显示出与Kg6介导的转染率相当的高转染率。KGH6在酸性条件下形成的DNA络合物为1-2μm，呈球形，在中性条件下相对稳定。络合物的大小和球形与KG6相同。KGH6的独特特性将为我们在体外和体内构建有功能的基因转染系统提供基础和有价值的工具之一。为了评价KG6作为非病毒基因载体在体内发挥作用的潜力，我们研究了KG6携带的质粒DNA在小鼠体内的生物分布。Kg6与质粒DNA的复合体在静脉注射后在血液中循环3h。血液中Kg6与DNA络合物的隐形特性将导致肿瘤的渗透性和滞留(EPR)效应增强。KG6有望成为体内实现功能性基因传递的候选基因。

The development of a non-viral gene delivery system into cells is an important key to realize the safe delivery of therapeutic genes without the side effects often pointed out for viral vectors. We have shown that dendritic poly (L-lysine) of the 6th generation (KG6) shows high transfection efficiency into several cultivated cells with low cytotoxicity. Here, to investigate the effect of substituting terminal cationic groups on the gene delivery into cells, we synthesized KGR6 and KGH6, in which terminal amino acids were replaced by arginines and histidines, respectively. DNA-binding analysis showed that KGR6 could bind to the plasmid DNA as strongly as KG6, whereas KGH6 showed decreased binding ability. KGR6 showed 3-to 12-fold higher transfection efficiency into several cultivated cells than KG6. In contrast, KGH6 showed no transfection efficiency. However, once KGH6 was mixed with the DNA under acidic conditions (pH 5.0), DNA-complexes were formed and showed high transfection efficiency comparable to KG6-mediated transfection. DNA-complexes of KGH6 formed under acidic conditions were 1-2 μm and spherical, and relatively stable under neutral conditions. The size and spherical shape of the complexes were the same as those of KG6. The unique character of KGH6 will be one of the basic and valuable tools which will enable us to construct a functional gene transfection system in vitro and in vivo.To evaluate the potential of KG6 as a non-viral gene carrier that works in vivo, we investigated the biodistribution of plasmid DNA delivered with KG6 in mice after intravenous administration. The plasmid DNA complexes with KG6 circulated in the blood for 3 h after intravenous injection. The stealth character of DNA complexes with KG6 in the blood would cause an enhanced permeability and retention (EPR) effect in the tumor. KG6 is expected to be a promising candidate that enables functional gene delivery in vivo.