Structure-Function of AAV - a Viral Gene Therapy Vector

描述(申请人提供)：腺相关病毒(AAV)是人类基因治疗的首选候选载体。长期目标是了解衣壳-宿主相互作用和病毒组装的结构和机制基础。这是对衣壳进行工程改造所必需的，以最大限度地发挥AAV向患有癌症或遗传性疾病的靶细胞运送治疗性DNA的潜力。绘制细胞受体结合位点的足迹图是改变组织嗜性的关键，而需要绘制中和抗原决定簇的图来设计能够到达先前接触AAV患者的靶细胞的病毒。其他病毒载体也将面临类似的挑战，这项工作将成为一个范例。 这项研究将建立在我们最近的AAV血清2型(AAV-2)的3埃分辨率晶体结构的基础上，并对其进行持续的完善和分析。与细胞受体硫酸乙酰肝素蛋白多糖的相互作用将通过对AAV-2与小肝素片段复合的结晶学分析、通过与肯·泰勒合作的大片段复合体的冷冻电子显微镜(EM)以及通过结合位点的定点突变来表征。 AAV与抗体的相互作用将使用Barrie Carter和Jurgen Kleinschmidt准备的一组单抗(MAb)进行研究。功能表位将通过对突变体的测序来绘制，这些突变体将在抗体存在的情况下通过病毒繁殖选择。具有代表性的单抗的物理表位将通过低温电磁成像进行映射，并使用已知的AAV-2结构以分子分辨率进行解释。 将启动对其他4种血清型中至少一种的结构研究。比较分析将显示受体结合区的保守程度，并揭示最受免疫监视的区域的可变性程度。所有拟议的结构研究都将补充和加速其他地方开发基于AAV的疗法的广泛努力，为目前通过开明的试错法进行的修改提供结构基础和一组限制限制。

DESCRIPTION (provided by applicant): Adeno-associated virus (AAV) is a prime candidate vector for human gene therapy. The long term objective is an understanding of the structural and mechanistic bases of capsid-host interactions and viral assembly. This is needed to engineer modifications of the capsid to maximize AAV's potential to deliver therapeutic DNA to targeted cells afflicted with cancer or an inherited disorder. Mapping the footprint of the cellular receptor binding site is critical to modifying tissue tropism, while mapping of the neutralizing antigenic determinants is needed to engineer viruses that can reach target cells in patients previously exposed to AAV. Similar challenges will be faced with other viral vectors, for which this work will be a paradigm. The research will build upon our recent 3 Angstrom resolution crystallographic structure of AAV serotype 2 (AAV-2), and its continuing refinement and analysis. Interactions with the cellular receptor, heparan sulfate proteoglycan, will be characterized through crystallographic analysis of AAV-2 complexed with small heparan fragments, through cryo-electron microscopy (EM) of complexes with larger fragments (in collaboration with Ken Taylor), and through site-directed mutagenesis of the binding site. AAV-antibody interactions will be studied using panels of monoclonal antibodies (MAb) prepared by Barrie Carter and Jurgen Kleinschmidt. Functional epitopes will be mapped through the sequencing of mutants to be selected by viral propagation in the presence of antibodies. Physical epitopes for representative MAb will be mapped by cryo-EM-imaging, interpreted at molecular resolution using the known AAV-2 structure. Structural studies of at least one of the other 4 serotypes will be initiated. Comparative analysis will show the extent of conservation of receptor-binding regions, and reveal the extent of variability of regions most subject to immune surveillance. All of the proposed structural studies will complement and accelerate extensive efforts elsewhere to develop AAV-based therapies, by providing a structural rationale and set of limiting constraints for modifications that currently are being made by enlightened trial-and-error.