AAV Rep-DNA Complexes Underlaying Site-specific Integration

AAV Rep-DNA 复合物是位点特异性整合的基础

• 批准号: 8040650
• 负责人: Carlos R Escalante
• 金额: $ 27.8万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-30 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8040650
• 关键词: AAV; Rep; DNA; Complexes; Underlaying

DESCRIPTION (provided by applicant): Adeno-Associated Virus type 2 (AAV-2) is unique among animal viruses in its ability to establish a latent infection by integrating site-specifically into a locus of chromosome 19. Moreover, because of its apparent non- pathogenicity, broad cell and tissue tropism and ability to infect in a cell-cycle independent fashion, AAV has emerged as one of the most promising vectors for gene therapy. A number of studies indicate that AAV- mediated site-specific integration requires only three players: a) the AAV Rep78 or Rep68 non-structural proteins, b) an AAV DNA element containing a Rep Binding Site (RBS) and c) the cellular AAVS1 integration site. During a decisive event, these players come together to form a protein-DNA complex that serves as the starting point for an integration process that is considered to be parallel to the AAV replication initiation process. This pre-integration complex is dependent on the ability of Rep68/78 proteins to bind the integration site AAVS1, promote its melting and catalyze a strand-specific nicking reaction that leaves a Rep68/78 protein covalently bound to the integration site. Although a general view of this mechanism has been achieved through the study of AAV DNA replication, little is known regarding the architecture of the Rep-AAVS1 complex and the steps that lead to its formation. The long-term goal of this proposal is to establish a structural, biophysical and biochemical framework required to understand the mechanism of AAV-mediated site-specific integration. Our approach is to couple structural studies using X-ray crystallography, cryo-EM and small angle X-ray scattering (SAXS) with biophysical, kinetic and functional studies to get a complete understanding of the events that lead to site-specific integration. We expect that the results obtained in this research proposal will serve both as a starting point to generate accurate models to understand this unique mechanism and also will give us the knowledge and tools to potentially design Rep proteins with novel specificities that could be used to develop new technologies in the gene therapy field. PUBLIC HEALTH RELEVANCE: This study will contribute significantly toward understanding the mechanism of AAV mediated site-specific integration. In addition, it will contribute to our understanding of origin DNA replication initiation. Knowledge gained from this proposal will serve as the basis for designing novel gene therapy vectors that could be targeted to a specific region in the chromosome.

描述（由申请方提供）：2型腺相关病毒（AAV-2）在动物病毒中是独特的，其能够通过位点特异性整合到19号染色体的基因座中来建立潜伏感染。此外，由于其明显的非致病性、广泛的细胞和组织向性以及以细胞周期非依赖性方式感染的能力，AAV已经成为用于基因治疗的最有希望的载体之一。许多研究表明，AAV介导的位点特异性整合仅需要三个参与者：a）AAV Rep 78或Rep 68非结构蛋白，B）含有Rep结合位点（RBS）的AAV DNA元件和c）细胞AAV 1整合位点。在一个决定性的事件中，这些参与者聚集在一起形成蛋白质-DNA复合物，作为整合过程的起点，该过程被认为与AAV复制起始过程平行。该整合前复合物依赖于Rep 68/78蛋白结合整合位点AAVS 1、促进其解链并催化链特异性切口反应的能力，所述切口反应使Rep 68/78蛋白共价结合至整合位点。虽然已经通过研究AAV DNA复制实现了对这种机制的一般看法，但关于Rep-AAVS 1复合物的结构和导致其形成的步骤知之甚少。该提案的长期目标是建立理解AAV介导的位点特异性整合机制所需的结构、生物物理和生物化学框架。我们的方法是将使用X射线晶体学，cryo-EM和小角X射线散射（SAXS）的结构研究与生物物理，动力学和功能研究相结合，以全面了解导致位点特异性整合的事件。我们希望在这项研究计划中获得的结果将作为一个起点，以生成准确的模型来理解这种独特的机制，也将为我们提供知识和工具，以潜在地设计具有新特异性的Rep蛋白，这些蛋白可用于开发基因治疗领域的新技术。 公共卫生相关性：这项研究将有助于了解AAV介导的位点特异性整合的机制。此外，它将有助于我们理解的起源DNA复制启动。从这个提议中获得的知识将作为设计新的基因治疗载体的基础，这些载体可以靶向染色体中的特定区域。