Trial research for practical use of the reagent for gene expression control by Decoy oligo nucleotide.

我们考察了“转录因子诱饵抑制疾病相关基因表达”和“日本血凝病毒(HVJ)-脂质体基因转移”相结合的新型基因治疗方法的有效性、重复性和稳定性。Sp1诱骗基因可同时抑制肿瘤细胞表达组织因子(Tf)，而Sp1诱骗基因不能抑制肿瘤细胞表达组织因子(Tf)。此外，两种细胞的体外侵袭力、尿激酶型纤溶酶原激活物的信使核糖核酸合成及细胞增殖均受到抑制，仅Sp1诱骗基因可抑制细胞增殖达40%。这些结果表明，Sp1诱骗策略将通过同时减少癌细胞(A)血管生成生长因子的表达、(B)增殖和(C)侵袭性而有效地调控肿瘤的生长。在兔球囊损伤颈动脉内膜增厚模型中，核因子-kB诱骗抑制损伤后血管收缩程度约为50%。利用脂质体将核因子-kB诱骗物转移到血管壁上的效率比单纯使用裸露核因子-kB诱骗物高。另外，AP-1对低氧刺激也有效。提示利用脂质体诱骗转移的方法进行基因治疗是非常有效的，与现有的其他方法相比具有很大的优势。然而，脂质体的稳定活性取决于用于脂质体制备的脂类的质量(氧化度)和脂膜的条件。在-20℃氮气条件下，黑暗条件下，基因转移效率较高的保证期为10天左右。因此，我们认为，为了实现HVJ-脂质体的商业化供应，有必要对脂类和产品的保存形式进行改进。

We inspected availability, reproducibility and stability of novel gene therapy using the combination of "suppression of disease-related gene expression by a decoy for transcription factor" and "gene transfer by hemagglutinating virus of Japan (HVJ)- liposome".Using HVJ-liposome method, we transferred Sp1 decoy into cultured cancer cells (A549 and U251 cells). The TNF-alpha-mediated expression of both VEGF and TGF betal and tissue factor (TF) by the cancer cells could be simultaneously suppressed to less than 30% by transfection of Sp1 decoy but not by mutated-Sp1 decoy. In addition, in vitro invasiveness, synthesis of mRNA for urokinase-type plasminogen activator, andcell proliferation of both cell lines were also inhibited to 40% by the transfection of only Sp1 decoy. These results suggested that the Sp1 decoy strategy would be effective for regulating tumor growth by simultaneously reducing cancer cell (a) angiogenic growth factor expression, (b) proliferation, and (c) invasiveness. In rabbit balloon injury arteria carotis intima thickening model, NF-kB decoy suppressed the degree of vasoconstriction after injury to about 50%. By using HVJ- liposome, efficiency of NF-kB decoy transfer to vascular wall becomes higher than that of naked NF-kB decoy only. Otherwise, AP-1 was effective for hypoxia stimuli. These are suggested that gene therapy by decoy transfer by HVJ-liposome was extremely effective and has a great advantage for other current ones.However, stable activity of HVJ- liposome is dependent on quality of the lipids used for liposome preparation (oxidation degrees) and conditions of lipid film. Guarantee period for relatively high efficiency of gene transfer is about 10 days under N2 gas at -20℃ in dark. Therefore, we concluded that the improvements of lipids and conservation form of product are necessary, in order to commercial supply of HVJ-liposome.