Development of hybrid DNA carrier for the improvement of gene therapy

Research Project

Project/Area Number	13450342
Research Category	Grant-in-Aid for Scientific Research (B)
Allocation Type	Single-year Grants
Section	一般
Research Field	生物・生体工学
Research Institution	Nagoya University
Principal Investigator	IIJIMA Shinji Nagoya Univ., Dept.Biotechnol., Professor, 工学研究科, 教授 (00168056)
Co-Investigator(Kenkyū-buntansha)	NISHIJIMA Ken-ichi Nagoya Univ., Dept.Biotechnol., Assistant Professor, 工学研究科, 助手 (10262891)
Project Period (FY)	2001 – 2002
Project Status	Completed (Fiscal Year 2002)
Budget Amount *help	¥14,900,000 (Direct Cost: ¥14,900,000) Fiscal Year 2002: ¥5,600,000 (Direct Cost: ¥5,600,000) Fiscal Year 2001: ¥9,300,000 (Direct Cost: ¥9,300,000)
Keywords	Retrovirus / Liposome / Gene trabsfection / Gene Therapy / Integrase / Gag
Research Abstract	A pantropic retrovirus used for gene therapy contained Vesicular Stomatitis Virus G protein (VSV-G) as the envelope protein. Since the receptor of VSV-G protein is phospholipid, the virus can basically infect to all kind of cell types. This wide host range is an advantage of this virus, as well as critical factor from the viewpoint of biohazard. From this consideration, we are trying to develop a new class of modified virus which has no infectivity under natural condition hut shows infectivity in the presence of transfection reagent such as lipofection reagents. VSV-G protein contains intra-cellular domain of 28 amino acids, transmembrane domain of 15 amino acids and extra-cellular domain of 298 amino acids. Extra-cellular domain contains 2 sugar attachment sites and the sugar modification is important for the protein stability. We constructed various deletion mutant of the VSV-G pseudo-typed virus. In a mutant, extra-cellular 143 amino acids were deleted, but contained 2 sugar attachment sites intact, and in another mutant, extra-cellular 298 amino acids were deleted and contained only one sugar attachment site. Both of them showed lipofectin dependent infectivity. However, the infectivity was 1/1000 of that obtained with intact virus. On the other hand, the efficiency of virus particle formation was 1/10 of that obtained with intact virus. In order to study whether INI-1 protein affects cDNA integration activity of retrovirus. INI-1 protein and molony murine leukemia virus integrase were cloned. Up to now, physical interaction between INI-1 and integrase proteins so far reported, has not been detected by our hand.