Improvement of Gene Therapy by Use of Aritificial Virus

为了提高人工活DNA在宿主染色体中的整合效率，我们以Molony鼠白血病病毒前整合复合体(PIC)为模型系统进行了研究。我们发现，整合酶是PIC的主要成分之一，它与转录因子YY1物理上相互作用。我们还发现，Bmi-1是多梳基团抑制复合体的一个亚单位，它与整合酶相互作用蛋白-1(INI-1)相互作用。INI-1与整合酶结合，也是PIC的主要成分。为了阐明整合酶与YY-1的相互作用，我们首先通过GST-Down实验分析了整合酶与YY-1的相互作用。我们发现YY-1的N-末端区域与整合酶相互作用。由于YY-1的N-末端是一个激活区域，而C-末端是DNA相互作用的区域，我们推测整合酶和YY-1与病毒的整合反应可能会影响整合反应。HIV-1整合酶通过GST-Pull-down与YY-1相互作用。为了提高病毒载体的产量，我们将一种瞬时病毒生产系统--所谓的Q-载体系统应用到Molony白血病病毒载体系统中。到目前为止，已经用包装细胞系的方法制备了高滴度的病毒载体。但包装细胞系的建立费时费力。另一方面，Q载体系统可以在2-3天内产生病毒载体。我们发现，Q载体产生的病毒滴度在很大程度上依赖于载体的大小，但当转基因大小在2kb左右时，其滴度与原始的包装细胞法相当。当转基因对动物细胞有细胞毒作用时，Q载体系统比包装细胞法有更高的滴度。

In order to improve the integration of artificial vital DNA into host chromosome, we have studied the components of preintegration complex (PIC) of molony murine leukemia virus as a model system. We found that integrase which is one of the main components of the PIC, physically interacted with a transcription factor YY1. We also found that Bmi-1 which is a subunit of polycomb group repressive complex, interacted with INI-1(integrase interacter-1). INI-1 associates with integrase and is also a main component of PIC. To clarify the interaction between integrase and YY-1, first, we analyzed the interaction by GST-pull down assay. We found that the N-terminal region of YY-1 interacted with the integrase. Because N-terminal region of YY-1 is known as an activation domain and the C-terminal region is a domain for DNA interaction, we assumed that binary binding trough integrase and YY-1 to virus cDNA may affect the integration reaction. Integrase from HIV-1 also interacted with YY-1 by the GST-pull down assay.In order to improve the viral vector production, we applied a transient viral production system, so-calle d Q-vector system, to molony leukemia virus based vector system. So far now, high titer viral vector preparation has been prepared by so-called packaging cell line method. But the establishment of packaging cell line is laborious and it take longer time. On the other hand, virus vector can be produced within 2-3 days by the Q-vector system. We found that the virus titer produced by the Q-vector was largely dependent on vector size but the titer was comparable to the original packaging cell method if the transgene size was around 2 kb. When the transgene has a cytotoxic effect on animal cells, the Q-vector system gave a higher titer comparing to the packaging cell method.