Biology of adeno-associated viral vectors

我们在腺相关病毒（AAV）载体方面的工作主要集中在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，使新的发展， 载体生产策略和靶向载体的前景。在本申请中，我们建议继续 这项工作通过关注与病毒DNA复制和包装相关的三个问题： 1)鉴定AAV DNA复制的必要组分。这一目标将决定 与MCM复合物相关的蛋白质（MCM 2 -7）是体外AAV DNA复制所需的。它有 先前已经确定MCM 4、6和7形成含有DNA解旋酶的最小复合物 活动我们希望确定这种最小复合物是否能够单独进行AAV前导链DNA合成， 或者如果AAV DNA也需要其它组分，例如MCM 2、3、5和10、CDC 45、cdt 1、GINS和Cdc 6 复制的此外，没有研究直接表明MCM解旋酶活性参与导致 链AAV DNA合成。在这个目标中，我们计划通过诱变来确定MCM的解旋酶活性， 是AAV DNA复制所必需的。 2)DNA复制包装中蛋白质相互作用伴侣的鉴定及其作用机制 股线伸长我们将使用确定的底物和AAV所需的基本纯化组分， DNA复制提出有关AAV DNA复制机制的基本问题。这些包括 MCM加载是否需要Rep蛋白，pol 6复合物是否与Rep和MCM保持接触 在链延伸过程中，以及Rep-capsid复合物在DNA复制和包装中的作用。 最后，我们打算用电子显微镜来鉴定DNA复制中的特定相互作用伙伴 和冷冻电镜 3)绘制AAV Rep蛋白在AAV衣壳表面上的位置。现在好了 确定Rep是包装信号，并提供了驱动AAV DNA进入包装的解旋酶马达。 衣壳。我们将使用冷冻电镜，冷冻断层扫描和X射线晶体学来确定相互作用的网站 Rep和据信启动包装的衣壳之间的相互作用。 相关性（参见说明）： 重组腺相关病毒载体（rAAV）是一种高效、安全的基因转移载体， 基因治疗的巨大前景。该提案侧重于AAV DNA复制的基础生物学， 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， 创造改良载体的方法，这些载体将成为下一代基因治疗转移载体。

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.