AAV p51EE Rep mediated integration into Chr19 AAVS1 site

AAV p51EE Rep 介导整合到 Chr19 AAVS1 位点

• 批准号: 6927945
• 负责人: ERIK S FALCK-PEDERSEN
• 金额: $ 29.66万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-08-01 至 2007-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6927945
• 关键词: DNA; Parvoviridae; binding sites; cell line; chemical stability; chromosomes; gene expression; genetic regulatory element; genome; molecular genetics; molecular site; nucleic acid sequence; protein structure function; site directed mutagenesis; transcription factor; transfection; virus genetics; virus integration; virus protein

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.

描述（由申请人提供）：过去十年来，人类细小性病毒AAV2整合到人类19号染色体（主要是在特定位置称为AAVS1）中以产生人类细胞中的潜在AAV2感染形式。在进行研究之前，认为三个要素对这一事件很重要，病毒蛋白REP，AAVS1中的目标位点以及集成的底物，该集合集中于病毒ITR元素，这些元素是复制的依赖性起源的病毒ITR元素。基于病毒元素元素的重组载体的整合非常低效率（小于1％）。我们启动了一系列研究，以表征有效的AAV整合的生物学。我们已经做出了一些重要的发现，以发现以前未知的集成元素（P51EE）的发现。使用相对简单的测定进行有效的REP介导的整合，我们发现25％至50％的转导细胞经历了成功的位点特定集成事件。该提案正在表征整合事件的产物，集成系统的底物，我们正在表征通过遗传策略介导该事件的分子机制。此外，我们正在以一种将其用于介导组织特异性物种特异性整合的方式来适应AAV2集成系统。由于迄今为止的所有数据都表明REP介导的集成事件没有负面副作用，因此该提案中开发的系统不仅会表征非常有趣的病毒宿主细胞潜伏期系统，还将为长期稳定的体外和体内基因转移提供非常强大的系统。