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

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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.