Study of cellular trafficking and membrane permeability of antisense DNA

反义DNA的细胞运输和膜通透性研究

• 批准号: 06454252
• 负责人: SHIMADA Jingoro
• 金额: $ 4.42万
• 依托单位: St.Marianna University School of Medicine
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1994
• 资助国家: 日本
• 起止时间: 1994 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-06454252/
• 关键词: antisense DNA; phosphorothioate; cationic liposomes; cellular distribution; geraniol; ゲラニオール; カチオン性リポソーム; リポフェクチン; DOTAP; transfectam; VEGF; HUVEC; S-オリゴ; FITC標識S-オリゴ; 拡散チャンバー; 単純ヘルペスウイルスI型; S-oligo; 共焦点レーザー蛍光顕微鏡システム

SummaryWe synthesized antisense DNA toward to immediate early pre-mRNA 4/5 (IE pre-mRNA4/5) of herpes simplex virus type1 (HSV-1) and evaluated anti-herpetic activity both in vitro and in vivo system. We clarified sequence specificity of antisense DNA,membrane delivery effects, and enhancement of biological activity by using drug delivery system (DDS). Since sequence non-specific activity of antisense DNA could not be neglected, anti-HIV activity of antisense DNA containing consecutive G sequence were evaluated.We observed from this study following points ; 1) Phosphorothioate oligonucleotide possessed most potent anti-herpetic activity, which was paralleled with intercellular amount. 2) Cellular distribution of antisense DNA in infected cells were obviously different from that in non-infected cells. From this results, it was suggested that virus infection may enhance the delivery efficiency of antisense DNA.3) We found that antisense DNA targeted limited splicing site revealed potent anti-herpetic activity. 4) Sequence specific activity of antisense DNA can be delineated from calculation based on the thermodynamics and kinetics of base-pairing. 5) 5'end modification with geraniol enhanced anti-herpetic activity of antisense DNA in a sequence specific manncr. Homogeneous cellular distribution of geranil modified antisense DNA may reflect on anti-herpetic activity. 6) Cationic liposomes altered subcellular distribution of antisense DNA.7) Sequence non-specific biological activity was recognized. Sequence containing consecutive G sequence showed potent anti-HIV activity. 8) Inhibition of virus replication by antisense DNA was recognized on rabbit herpes cornea keratitis.

摘要我们合成了针对 1 型单纯疱疹病毒 (HSV-1) 立即早期前 mRNA 4/5 (IE pre-mRNA4/5) 的反义 DNA，并评估了体外和体内系统的抗疱疹活性。我们阐明了反义DNA的序列特异性、膜递送效应以及通过使用药物递送系统(DDS)增强生物活性。由于反义DNA的序列非特异性活性不可忽视，因此对含有连续G序列的反义DNA的抗HIV活性进行了评估。我们从本研究中观察到以下几点： 1)硫代磷酸酯寡核苷酸具有最有效的抗疱疹活性，其与细胞间量平行。 2)感染细胞中反义DNA的细胞分布与非感染细胞明显不同。由此结果表明，病毒感染可能会增强反义DNA的递送效率。3）我们发现针对有限剪接位点的反义DNA显示出有效的抗疱疹活性。 4) 反义DNA的序列比活性可以根据碱基配对的热力学和动力学计算得出。 5)用香叶醇进行的5'端修饰以序列特异性方式增强了反义DNA的抗疱疹活性。香叶修饰的反义 DNA 的均匀细胞分布可能反映其抗疱疹活性。 6) 阳离子脂质体改变了反义DNA的亚细胞分布。7) 识别了序列非特异性生物活性。含有连续G序列的序列显示出有效的抗HIV活性。 8)在兔疱疹性角膜炎中发现反义DNA对病毒复制的抑制作用。

项目成果

岩谷若夫、東海林洋子、田村信也、乗松美貫、嶋田甚五郎、水島裕: "抗HSV活性を有するアンチセンス・オリゴDNAの安定性およびウイルス感染細胞における動態の検討。" Drug Delivery System. 11. 427-434 (1996)

Wakao Iwatani、Yoko Tokaibayashi、Shinya Tamura、Minuki Norimatsu、Jingoro Shimada、Yutaka Mizushima：“病毒感染细胞中具有抗 HSV 活性和动力学的反义寡核苷酸稳定性研究。”11. 427 -434（ 1996）

東海林洋子、嶋田甚五郎 他: "アンチセンスDNAの細胞内分布に及ぼす要因" Drug Delivery System. (印刷中).

Yoko Tokaibayashi、Jingoro Shimada 等人：“影响反义 DNA 细胞内分布的因素”药物输送系统（正在出版）。

N.Tamura, W.Iwatani, Y.Shoji, M.Norimatsu, J.Shimada, Y.Mizushima.: "Antiviral effects of phosphorothioate oligonucleotides complementary to 3' splice region of herpes simplex virus immediate early pre-mRNA5." Nucleic Acids Symposium Series. 31. 73-74 (19

N.Tamura、W.Iwatani、Y.Shoji、M.Norimatsu、J.Shimada、Y.Mizushima.：“与单纯疱疹病毒立即早期前 mRNA5 3 剪接区域互补的硫代磷酸酯寡核苷酸的抗病毒作用。”

東海林洋子、嶋田甚五郎: "オリゴヌクレオチドのDDS." 臨床薬理の進歩. 16. 9-19 (1995)

Yoko Tokaibayashi、Jingoro Shimada：“寡核苷酸的 DDS”。临床药理学进展 16. 9-19 (1995)。

Y.Shoji,Shimade,et al.: "Anti-HIV activity of phosphorothioate oligodeoxynucleotides containing consecutive G sequences" Int.J.Immunotherapy. XII(1/2). 1-9 (1996)

Y.Shoji，Shimade 等人：“含有连续 G 序列的硫代磷酸寡脱氧核苷酸的抗 HIV 活性”Int.J.Immunotherapy。