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Improvement of Gene Therapy by Use of Aritificial Virus

利用人工病毒改进基因治疗

基本信息

批准号：
16360410
负责人：
IIJIMA Shinji
金额：
$ 9.66万
依托单位：
Nagoya University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2005
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-16360410/
关键词：
Integration Artificial virus Integrase YY1 Q-vector RNAガンウイルス遺伝子治療核移行

项目摘要

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.

为了提高人工活DNA在宿主染色体上的整合效率，我们以小鼠白血病病毒为模型系统，研究了小鼠白血病病毒整合前复合物（PIC）的组成。我们发现，整合酶是PIC的主要成分之一，与转录因子YY 1物理相互作用。我们还发现多梳族阻遏复合物的亚基Bmi-1与整合酶相互作用因子INI-1相互作用。INI-1与整合酶结合，也是PIC的主要成分。为了阐明整合酶和YY-1之间的相互作用，首先，我们通过GST-下拉分析来分析相互作用。我们发现YY-1的N端区域与整合酶相互作用。由于YY-1的N-末端区域被认为是激活结构域，而C-末端区域是DNA相互作用的结构域，因此我们假设通过整合酶和YY-1与病毒cDNA的二元结合可能影响整合反应。为了提高病毒载体的产量，我们将一种瞬时病毒生产系统（Q-vector system）应用到以白血病病毒为载体的系统中。迄今为止，高滴度病毒载体制剂已通过所谓的包装细胞系方法制备。但包装细胞系的建立费时费力。另一方面，通过Q-载体系统可以在2-3天内产生病毒载体。我们发现，由Q-载体产生的病毒滴度在很大程度上取决于载体大小，但如果转基因大小为约2kb，则滴度与原始包装细胞方法相当。当转基因对动物细胞具有细胞毒性作用时，与包装细胞方法相比，Q-载体系统给出了更高的滴度。