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

开发一种进入细胞的非病毒基因传递系统是实现治疗性基因安全传递的重要关键，而不会出现病毒载体经常指出的副作用。我们已经证明，第6代树突状聚l -赖氨酸（KG6）在几种培养细胞中转染效率高，细胞毒性低。为了研究末端阳离子基团取代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KG6的结合能力相同，而KGH6的结合能力较弱。KGR6对多个培养细胞的转染效率是KG6的3 ~ 12倍。而KGH6则没有转染效率。然而，一旦在酸性条件下（pH 5.0）将KGH6与DNA混合，DNA复合物就会形成，并显示出与kg6介导的转染相当的转染效率。KGH6在酸性条件下形成的dna复合物为1 ~ 2 μm，呈球形，在中性条件下相对稳定。配合物的大小和球形形状与KG6相同。KGH6的独特特性将成为我们构建功能完备的体内外基因转染系统的基础和有价值的工具之一。为了评估KG6在体内作为非病毒基因载体的潜力，我们研究了KG6在静脉给药后质粒DNA在小鼠体内的生物分布。与KG6的质粒DNA复合物在静脉注射后可在血液中循环3小时。含有KG6的DNA复合物在血液中的隐蔽性会增强肿瘤的渗透性和滞留性（EPR）效应。KG6有望成为在体内实现功能性基因传递的有希望的候选药物。

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.