Development of hybrid DNA carrier for the improvement of gene therapy

用于基因治疗的泛嗜性逆转录病毒以水泡性口炎病毒G蛋白(VSV-G)为囊膜蛋白。由于VSV-G蛋白的受体是磷脂，病毒基本上可以感染所有类型的细胞。这种广泛的宿主范围是该病毒的优势，也是从生物危害的角度来看的关键因素。基于这一考虑，我们试图开发一种在自然条件下不具有感染性，但在脂质体等转染剂存在下具有感染性的新型修饰病毒。VSV-G蛋白包括28个氨基酸的胞内区、15个氨基酸的跨膜区和298个氨基酸的胞外区。胞外区含有2个糖结合位点，糖的修饰对蛋白质的稳定性具有重要意义。我们构建了VSV-G伪型病毒的各种缺失突变体。在一个突变体中，胞外143个氨基酸缺失，但含有2个完整的糖结合位点；在另一个突变体中，胞外298个氨基酸缺失，仅含有一个糖结合位点。两者均表现出脂联素依赖的感染性。但其感染力仅为完整病毒的1/1000。另一方面，病毒颗粒的形成效率是完整病毒的1/10，为了研究INI-1蛋白是否影响逆转录病毒的cDNA整合活性。克隆了INI-1蛋白和Molony小鼠白血病病毒整合酶。到目前为止，INI-1与整合酶蛋白之间的物理相互作用尚未被我们亲手检测到。

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.