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Development of highly functional medicines targeting genomic DNA/RNA

开发针对基因组 DNA/RNA 的高功能药物

基本信息

批准号：
09557201
负责人：
IMANISHI Takeshi
金额：
$ 6.85万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1999
项目状态：
已结题

项目摘要

In order to develop novel and highly functional medicines which can inhibit a target gene expression selectively, we designed and synthesized nucleoside analogues as a key synthon for an antisense or an antigene molecule. We accomplished the effective synthesis of the novel nucleoside analogues in which the sugar puckering was restricted in N-conformation. These nucleoside analogues were introduced into oligonucleotides by using a DNA synthesizer, giving conformationally locked oligonucleotides. The properties of these modified oligonucleotides as an antisense or an antigene molecule were studied by Tm measurements, gel retardation experiments, DNase I footprinting experiments. From these experiments, unprecedented thermal stabilities of duplex formation towards complementary DNA and RNA were observed. Furthermore, good triplex forming abilities of the modified oligonucleotides were also confirmed. In addition, the selective inhibition of the target gene expression in living cells was accomplished by addition of these modified oligonucleotides into cell culture medium. These results clearly indicate that the conformationally locked oligonucelotide was a promising candidate for an antisense and an antigene molecule.On the other hands, development of an ideal gene delivery system is of great importance for antisense and antigene methodology and other gene therapies. Therefore, we designed and synthesized novel symmetrical cationic lipids which have biodegradable ester linkages. The cationic liposomes prepared from the cationic lipids and natural lipid DOPE show an efficient gene delivery into living cells and low cytotoxicity. It means that this cationic liposome is an effective gene delivery reagent.

为了开发新型的、高功能的药物，选择性地抑制靶基因的表达，我们设计并合成了核苷类似物作为反义或抗基因分子的关键合成子。我们完成了新型核苷类似物的有效合成，其中糖折叠被限制在N-构象。通过使用DNA合成仪将这些核苷类似物引入寡核苷酸中，得到构象锁定的寡核苷酸。通过Tm测量、凝胶阻滞实验、DNA酶I足迹实验研究了这些修饰的寡核苷酸作为反义或反基因分子的性质。从这些实验中，观察到对互补DNA和RNA的双链体形成的前所未有的热稳定性。此外，还证实了修饰的寡核苷酸的良好的三链体形成能力。此外，通过将这些修饰的寡核苷酸添加到细胞培养基中来实现活细胞中靶基因表达的选择性抑制。这些结果清楚地表明，构象锁定的寡核苷酸是一种有希望的反义和反基因分子候选物。另一方面，开发理想的基因递送系统对于反义和反基因方法学以及其他基因治疗具有重要意义。因此，我们设计并合成了具有生物可降解酯键的新型对称阳离子脂质。由阳离子脂质和天然脂质DOPE制备的阳离子脂质体显示出有效的基因递送到活细胞中并且具有低细胞毒性。说明该阳离子脂质体是一种有效的基因载体。