Development of hybrid DNA carrier for the improvement of gene therapy

一种用于基因治疗的泛嗜性逆转录病毒以水疱性口炎病毒G蛋白（VSV-G）为包膜蛋白。由于VSV-G蛋白的受体是磷脂，因此该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一个优势，也是从生物危害的角度来看的关键因素。基于这一考虑，我们正在尝试开发一种在自然条件下没有传染性，但在转染试剂（如脂肪转染试剂）存在下表现出传染性的新型修饰病毒。VSV-G蛋白含有28个氨基酸的胞内结构域、15个氨基酸的跨膜结构域和298个氨基酸的胞外结构域。胞外结构域含有2个糖附着位点，糖修饰对蛋白质的稳定性至关重要。我们构建了VSV-G伪型病毒的多种缺失突变体。在一个突变体中，细胞外缺失143个氨基酸，但包含2个完整的糖附着位点，而在另一个突变体中，细胞外缺失298个氨基酸，只包含一个糖附着位点。两者均表现出脂质体依赖性感染。但其传染性仅为完整病毒的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cDNA整合活性。克隆了ni -1蛋白和molony小鼠白血病病毒整合酶。到目前为止，已有报道的ni -1与整合酶蛋白之间的物理相互作用，我们的手还没有检测到。

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.