AAV p51EE Rep mediated integration into Chr19 AAVS1 site

描述（由申请人提供）：在过去10年中，已知人细小病毒AAV 2整合到人染色体19中（主要在称为AAVS 1的特定位点），以在人细胞中产生潜伏形式的AAV 2感染。在我们的研究之前，三个元件被认为对该事件是重要的，即病毒蛋白Rep、AAVS 1中的靶位点和用于整合的底物，其集中于作为Rep依赖性复制起点起作用的病毒ITR元件。与基于病毒ITR元件的重组载体的整合是极其低效的（小于1%）。我们已经启动了一系列研究来表征有效AAV整合的生物学。我们已经取得了几个重要的发现，集中在发现一个以前未知的整合元件（p51 EE），我们已经确定。使用相对简单的有效Rep介导整合的测定，我们发现25 - 50%的转导细胞经历成功的位点特异性整合事件。该提案描述了整合事件的产物，整合系统的底物，并且我们描述了通过遗传策略介导该事件的分子机制。此外，我们正在以允许其用于介导任何所需转基因的组织特异性、物种特异性整合的方式调整AAV 2整合系统。因为迄今为止的所有数据表明Rep介导的整合事件没有负面副作用，所以在该提议中开发的系统将不仅表征非常有趣的病毒宿主细胞潜伏系统，它还将提供用于长期稳定的体外和体内基因转移的令人难以置信的强大系统。

DESCRIPTION (provided by applicant): It has been known for past 10 years that the human parvovirus AAV2 integrates into human chromosome 19 (predominantly at a specific site referred to as AAVS1) to generate a latent form of AAV2 infection in human cells. Prior to our studies, three elements were thought to be important to this event, the viral protein Rep, the target site in AAVS1, and the substrate for integration which focused on the viral ITR elements that function as Rep dependent origins of replication. Integration with recombinant vectors based on viral ITR elements is extremely inefficient (less than 1%). We have initiated a series of studies to characterize the biology of efficient AAV integration. We have made several important discoveries, centered on the discovery of a previously unknown integration element (p51EE) that we have identified. Using a relatively simple assay for efficient Rep mediated integration we have found that between 25 and 50% of transduced cells undergo a successful site specific integration event. This proposal is characterizing the products of the integration event, the substrates of the integration system and we are characterizing the molecular mechanisms that mediate this event through genetic strategies. In addition, we are adapting the AAV2 integration system in a manner that will allow it to be used to mediate tissue specific, species specific integration of any desired transgene. Because all data to date indicates the Rep mediated integration event to be free of negative side effects, the system developed in this proposal will not only characterize a very interesting virus host cell latency system, it will also provide an incredibly powerful system for long term stable in vitro and in vivo gene transfer.