| Budget Amount *help |
¥9,700,000 (Direct Cost: ¥9,700,000)
Fiscal Year 1996: ¥3,000,000 (Direct Cost: ¥3,000,000)
Fiscal Year 1995: ¥6,700,000 (Direct Cost: ¥6,700,000)
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| Research Abstract |
We developed a novel gene delivery vector system called HVJ-liposome. In this delivery system, DNA was entrapped into negative-charge liposome and the liposome was fused with UV-inactivated HVJ,Sendai virus, to form HVJ-liposome. HVJ-liposome is now widely used as an efficient non-viral vector for introducing DNA,oligonucleotides and proteins into various animal organs. Using this vector system, we succeeded in prevention of restenosis of balloon injured areteries of rats, suppression of glomerulosclerosis, protection of transplanted organs, and inhibition of disseminated brain tumors. However, the system should be improved for more efficient delivery system both in vitro and in vivo toward future gene therapy.
When HVJ-cationic liposome having cationic DC-cholesterol (DC-Chol) was prepared by vortexing and extrusion, the delivery of FITC-labeled antisense oligonucleotides or ribozymes was greatly enhanced, and luciferase gene expression was also increased about 70 times more than conve
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ntional HVJ-liposome containing phosphatidylserine (PS). The HVJ-DC-Chol-liposome was very useful for suicide gene therapy of disseminated glioblastoma in mouse brain. We further optimized lipid components of liposome and developed a novel HVJ-liposome consisting of sphingomyelin (Sph), dioleoylphosphatidylethanolamine (DOPE), phosphatidylcholine (PC), cholesterol (Chol) and DC-Chol (1.7 : 1.7 : 1.7 : 4.0 : 1.0 in molar ratio) as the most efficient vector for gene expression in culture cells (100-800 times higher than conventional HVJ-liposome). However, the use of cationic lipids rather reduced gene expression in animal organs, compared with conventional HVJ-liposome. Then, HVJ-liposome containing Sph, DOPE,PC,PS and Chol (1.3 : 1.3 : 1.0 : 5.0 in molar ratio) mimicking HIV envelope (called HVJ-AVE liposome) raised gene expression in either liver or muscle 5-10 times more than conventional HVJ-liposome. Thus, cationic lipids appeared to have reciprocal effects in gene expression in vitro and in vivo, and our finding indicates the alternative usage of liposomes for in vitro and in vivo gene transfer. Less
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