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

使用肽-DNA复合物开发简单高效的基因转染

• 批准号: 14598003
• 负责人: AOYAGI Haruhiko
• 金额: $ 2.62万
• 依托单位: Nagasaki University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14598003/
• 关键词: Gene carrier; Dendrimer; Amphiphilic peptide; Transfection; Cultured cells

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.

非病毒基因传递系统的开发是实现治疗基因安全传递的重要关键，而没有病毒载体通常指出的副作用。我们已经表明，第6代（KG 6）的树突状聚（L-赖氨酸）显示出高转染效率到几个培养细胞具有低细胞毒性。在这里，为了研究取代末端阳离子基团对基因递送到细胞中的影响，我们合成了KGR 6和KGH 6，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，KGR 6与质粒DNA的结合能力与KG 6相当，而KGH 6的结合能力较低。KGR 6在几种培养细胞中的转染效率比KG 6高3至12倍。相比之下，KGH 6没有显示转染效率。然而，一旦KGH 6与DNA在酸性条件（pH 5.0）下混合，就形成DNA复合物，并显示出与KG 6介导的转染相当的高转染效率。在酸性条件下形成的KGH_6-DNA复合物为1-2 μm球形，在中性条件下相对稳定。配合物的大小和球形与KG 6相同。为了评价KG 6作为体内非病毒基因载体的潜力，我们研究了KG 6携带质粒DNA在小鼠体内的生物分布。静脉注射后，质粒DNA与KG 6的复合物在血液中循环3小时。KG 6与DNA复合物在血液中的隐形特性将导致肿瘤中增强的渗透性和保留（EPR）效应。KG 6有望成为一个有前途的候选人，使功能性基因在体内输送。

项目成果

T.Okuda et al.: "Time-Dependent Complex Formation of Dendritic Poly(L-lysine) with Plasmid DNA and Correlation with in vitro Transfection Efficiencies."Organic & Biomoleular Chemistry. 1. 1270-1273 (2003)

T.Okuda 等人：“树突状聚（L-赖氨酸）与质粒 DNA 的时间依赖性复合物形成及其与体外转染效率的相关性。”有机

M.Nakayama, M.Kimura, A.Wada, K.Yahiro K.Ogushi, T.Niidome, A.Fujikawa, D.Shirasaka, N.Aoyama, H.Kurazono, M.Noda, J.Moss, T.Hirayama: "Helicobacter pylori VacA activates the p38/ATF-2-mediated signal pathway in AZ521 cells"J.Biol.Chem.. (in press).

M.Nakayama、M.Kimura、A.Wada、K.Yahiro K.Ogushi、T.Niidome、A.Fujikawa、D.Shirasaka、N.Aoyama、H.Kurazono、M.Noda、J.Moss、T.Hirayama

K.Ogushi, A.Wada, T.Niidome, T.Okuda, R.Llanes, M.Nakayama, Y.Nishi, H.Kurazono, K.D.Smith, A.Aderem, J.Moss, T.Hirayama: "Gangliosides act as co-receptors for Salmonella enteritidis FliC and promote FliC-induction of human β-defensin-2 expression in Caco

K.Ogushi、A.Wada、T.Niidome、T.Okuda、R.Llanes、M.Nakayama、Y.Nishi、H.Kurazono、K.D.Smith、A.Aderem、J.Moss、T.Hirayama：“神经节苷脂法作为肠炎沙门氏菌 FliC 的共受体并促进 FliC 诱导 Caco 中的人 β-defensin-2 表达

K.Maruyama et al.: "Novel receptor-mediated gene delivery system comprising plasmid/protamine/sugar-containing polyanion ternary complex"Biomaterials. (印刷中).

K. Maruyama 等人：“包含质粒/鱼精蛋白/含糖聚阴离子三元复合物的新型受体介导的基因传递系统”（正在出版）。

T.Okuda et al.: "Characters of Dendritic Poly(L-lysine) Analogues with the Terminal Lysines Replaced with Arginines and Histidines as Gene Carriers in Vitro"Biomaterials. 25. 537-544 (2004)

T.Okuda 等人：“末端赖氨酸被精氨酸和组氨酸取代的树枝状聚（L-赖氨酸）类似物作为体外基因载体的特征”生物材料。