Structure-Function of AAV - a Viral Gene Therapy Vector

描述（由申请人提供）：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-宿主相互作用和病毒组装的结构和机制基础。这需要对衣壳进行工程修饰，以最大限度地发挥AAV的潜力，将治疗性DNA传递给患有癌症或遗传性疾病的目标细胞。绘制细胞受体结合位点的足迹对于改变组织趋向性至关重要，而绘制中和抗原决定因子的图谱则需要设计病毒，使其能够到达先前暴露于AAV的患者的靶细胞。类似的挑战将面临其他病毒载体，这项工作将是一个范例。

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.