AAV p51EE Rep mediated integration into Chr19 AAVS1 site

描述(申请人提供)：在过去的10年里，人们已经知道人类细小病毒AAV2整合到人类19号染色体上(主要在被称为AAVS1的特定位置)，在人类细胞中产生AAV2感染的潜伏形式。在我们的研究之前，有三个元件被认为对这个事件很重要，病毒蛋白Rep，AAVS1中的靶点，以及整合的底物，重点是作为Rep依赖的复制起点的病毒ITR元件。与基于病毒ITR元件的重组载体整合效率极低(不到1%)。我们已经开始了一系列研究，以表征高效AAV整合的生物学特征。我们有了几个重要的发现，主要是发现了我们已经确定的一个以前未知的整合元件(P51EE)。使用一种相对简单的Rep介导的有效整合方法，我们发现25%到50%的转导细胞经历了成功的位点特异性整合事件。这项提议描述了整合事件的产物，整合系统的底物，我们正在描述通过遗传策略调节这一事件的分子机制。此外，我们正在调整AAV2整合系统，使其能够用于介导任何所需转基因的组织特异性、物种特异性整合。由于到目前为止所有的数据都表明Rep介导的整合事件没有负面影响，在这个建议中开发的系统不仅将描述一个非常有趣的病毒宿主细胞潜伏期系统，它还将为长期稳定的体外和体内基因转移提供一个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.