Structure-Function of AAV - a Viral Gene Therapy Vector.

描述(申请人提供)：腺相关病毒(AAV)是人类基因治疗的主要候选载体。长期目标是了解衣壳宿主相互作用的结构决定因素。这不仅是病毒学的基本兴趣，也是设计改变细胞特异性或增强免疫逃避的重组病毒载体以实现基因治疗的全部潜力所必需的。 随着AAV-2和AAV-6的晶体结构接近完成，AAV-3将被解决为主要人类血清型的完整结构。它们将在目标1中用作了解种群中病毒分离株的结构和序列多样性的基础，以及如何最好地模仿进化来设计抵抗免疫中和的载体。AIM 2包括AAV-2的功能表位图和物理表位图。前者将涉及在体外选择在单抗(MAb)存在下生长的病毒。逃逸突变位点的簇将被映射到原子结构中，以定位主要的构象表位。将通过冷冻电子显微镜(EM)绘制AAV与单抗片段的复合体的足迹图。目标3将在我们描述AAV-2与其主要细胞受体的相互作用之后，描述感染进入所需的二级受体的结合。这将包括通过质谱学分析氢-重氢交换的足迹，以及通过低温电子显微镜成像。目标4将继续描述病毒与之相互作用的细胞成分的特征，现在重点放在AAV通过内体进入细胞核时结合的细胞内伙伴。将使用免疫沉淀和质谱学蛋白质鉴定来确定合作伙伴，这为这些相互作用的未来生物物理表征开辟了前景。 总之，这些研究将在细胞/分子水平上提供对衣壳-宿主相互作用的全面的分子理解，这将是几乎没有先例的，因此在病毒学中具有基本的兴趣。它们的阐明对于理解工程改良基因治疗载体的机会和限制是重要的。如果要实现目前开发具有遗传成分的疾病的治疗方法的希望，这是一个迫切的需要。

DESCRIPTION (provided by applicant): Adeno-Associated Virus (AAV) is a leading candidate vector for human gene therapy. The long-term goal is to understand the structural determinants of capsid host interactions. This is not only of fundamental interest in virology, but is also needed to engineer recombinant viral vectors with changed cellular specificity, or enhanced immune evasion, to realize the full potential of gene therapy. With the crystallographic structure of AAV-2 in-hand and AAV-6 near completion, AAV-3 will be solved to complete structures for the predominant human serotypes. They will be used in Aim 1 as the basis of understanding the structural and sequence diversity of viral isolates in the population, and how best to mimic evolution in designing vectors resistant to immune neutralization. Aim 2 includes both functional and physical epitope mapping for AAV-2. The former will involve in vitro selection of viruses grown in the presence of monoclonal antibodies (MAb). Clusters of escape mutation sites will be mapped to the atomic structure to locate the predominant conformational epitopes. Footprints will be mapped physically through cryo-electron microscopy (EM) of complexes of AAV with MAb fragments. Aim 3 will follow our characterization of the interactions of AAV-2 with its primary cellular receptor, with characterizations of the binding of secondary receptors required for infectious entry. This will include footprinting by hydrogen-deuterium exchange analyzed by mass spectrometry, and imaging by cryo-EM. Aim 4 will continue the characterization of cellular components with which the virus interacts, now focusing on the intracellular partners bound as AAV enters through endosomes and makes its way into the nucleus. Partners will be identified using immuno-precipitation followed by mass spectrometric protein identification, opening prospects of future biophysical characterization of these interactions. Together these studies will provide a comprehensive molecular understanding of capsid-host interactions at the cellular / molecular level that will be with little precedent, and thus of fundamental interest in virology. Their elucidation is important for understanding the opportunities and constraints in engineering improved gene therapy vectors. This is a critical need if current hopes are to be realized of developing therapies for diseases with a genetic component.