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.

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.许多研究表明，辅助位点特异性集成仅需要三个参与者：a）AAV REP78或REP68非结构性蛋白质，b）含有Rep结合位点（RBS）和C）细胞AAVS AAVS1整合位点的AAV DNA元件。在决定性的事件中，这些参与者聚集在一起形成蛋白质-DNA复合物，该复合物是集成过程的起点，该过程被认为与AAV复制启动过程平行。这种预一体化复合物取决于REP68/78蛋白结合整合位点AAVS1，促进其融化并催化链特异性的刻痕反应的能力，该反应使Rep68/78蛋白质共同结合到整合位点。尽管通过研究AAV DNA复制，已经实现了这种机制的一般观点，但关于Rep-Aavs1复合物的结构以及导致其形成的步骤，知之甚少。该提案的长期目标是建立一个结构，生物物理和生化框架，以了解AAV介导的网站特异性集成的机制。我们的方法是使用X射线晶体学，冷冻EM和小角度X射线散射（SAXS）进行结构研究，并具有生物物理，动力学和功能研究，以完全了解导致特定于位点集成的事件。我们预计，在本研究建议中获得的结果将是生成准确模型以了解这种独特机制的起点，并且还将为我们提供具有新型特异性设计潜在设计REP蛋白质的知识和工具，这些特异性可用于在基因治疗领域开发新技术。 公共卫生相关性：这项研究将有助于理解AAV介导的特定地点整合的机制。此外，它将有助于我们对原点DNA复制启动的理解。从该提案中获得的知识将是设计新型基因治疗载体的基础，这些基因治疗载体可以针对染色体中的特定区域。