Biology of adeno-associated viral vectors

腺相关病毒载体的生物学

• 批准号: 7669754
• 负责人: NICHOLAS MUZYCZKA
• 金额: $ 19.39万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-04-01 至 2014-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7669754
• 关键词: Automobile Driving; Biology; CDC45L gene; Capsid; Complex; Cryoelectron Microscopy; Cystic Fibrosis; DNA biosynthesis; Dependovirus; Development; Gene Transfer; Genes; In Vitro; Lead; Maps; Methods; Motor; Mutagenesis; Positioning Attribute; Production; Proteins; Recombinant adeno-associated virus (rAAV); Role; Signal Transduction; Site; Surface; Viral Packaging; Work; X-Ray Crystallography; adeno-associated viral vector; gene therapy; helicase; improved; next generation; tomography; vector; viral DNA

Our work on Adeno-associated virus (AAV)vectors has focused on the basic biology of AAV DNA replication and capsid assembly. This has lead to important information that has allowed the development of new vector production strategies and the promise of targeted vectors. In this application, we propose to continue this work by focusing on three problems related to viral DNA replication and packaging: 1) Identification of essential components of AAV DNA replication. This aim will determine which of the proteins associated with the MCM complex (MCM2-7) are required for in vitro AAV DNA replication. It has previously been established that MCM 4, 6 and 7 form a minimal complex that contains DNA helicase activity. We wish to establish if this minimal complex alone is capable of AAV leading strand DNA synthesis, or if other components, e.g. MCM2,3,5 and 10, CDC45, cdtl, GINS and Cdc6 are also required for AAV DNA replication. Additionally no studies have shown directly that MCM helicase activity is involved in leading strand AAV DNA synthesis. In this aim we plan to determine by mutagenesis if the helicase activity of MCM is essential for AAV DNA replication. 2) The identification of protein interacting partners in DNA replication and packaging and the mechanism of strand elongation. We will use defined substrates and the essential purified components required for AAV DNA replication to ask basic questions about the mechanism of AAV DNA replication. These include whether MCM loading requires Rep protein, whether the pol 6 complex maintains contact with Rep and MCM during strand elongation, and what the role of the Rep-capsid complex in DNA replication and packaging. Finally, we intend to identify the specific interacting partners in DNA replication using electron microscopy and cryo-EM. 3) Mapping the position of the AAV Rep proteins on the surface of the AAV capsid. It is now well established that Rep is the packaging signal and provides the helicase motor for driving AAV DNA into the capsid. We will use cryo-EM, cryo-tomography and X-ray crystallography to determine the interaction site between Rep and the capsid that is believed to initiate packaging. RELEVANCE (Seeinstructions): Recombinant Adeno-associated virus vectors (rAAV) are efficient and safer gene transfer vehicles that show enormous promise for gene therapy. This proposal focuses on the basic biology of AAV DNA replication and viral packaging. Our hope is that the information obtained will enable us to improve production methods, find ways of creating improved vectors that will become the next generation of gene therapy transfer vehicles.

我们对腺相关病毒(AAV)载体的研究主要集中在AAV DNA复制的基础生物学上 和衣壳组装。这导致了重要的信息，使新的开发 载体生产策略和目标载体的承诺。在此应用程序中，我们建议继续 这项工作的重点是与病毒DNA复制和包装有关的三个问题： 1)AAVDNA复制必需成分的鉴定。这一目标将决定哪一个 与MCM复合体(MCM2-7)相关的蛋白质是AAVDNA体外复制所必需的。它有 先前已确定MCM 4、6和7形成包含DNA解旋酶的最小复合体 活动。我们希望确定这个最小的复合体是否能够单独合成AAV前导链DNA， 或者如果AAV DNA还需要其他成分，如MCM2、3、5和10、CDC45、cdt1、gins和cdc6 复制。此外，没有研究直接表明MCM解旋酶活性参与了 AAV链DNA合成。为此，我们计划通过突变来确定MCM的解旋酶活性是否 是AAV DNA复制所必需的。 2)DNA复制包装中蛋白质相互作用伙伴的确定及其作用机制 股线伸长。我们将使用AAV所需的特定底物和必要的纯化成分 DNA复制：询问有关AAV DNA复制机制的基本问题。这些措施包括 MCM负载是否需要Rep蛋白，Pol6复合体是否与Rep和MCM保持接触 在链延长过程中，以及Rep-衣壳复合体在DNA复制和包装中的作用。 最后，我们打算使用电子显微镜确定dna复制中特定的相互作用伙伴。 和冷冻-EM。 3)定位AAV Rep蛋白在AAV衣壳表面的位置。现在已经很好了 确定Rep是包装信号，并提供解旋酶马达，将AAV DNA驱动到 凯西德。我们将使用低温电子显微镜、冷冻断层扫描和X射线结晶学来确定相互作用的位置 在Rep和衣壳之间，这被认为是启动包装的。 相关性(请参阅说明)： 重组腺相关病毒载体是一种高效、安全的基因转移载体。 基因治疗的巨大希望。这项建议侧重于AAV DNA复制的基础生物学和 病毒包装。我们希望所获得的信息将使我们能够改进生产方法，找到 创造将成为下一代基因治疗转移载体的改进载体的方法。