Structural and chemical changes between empty and full AAV capsids

项目摘要 腺相关病毒（AAV）具有大组织嗜性和低毒性，是一种流行的基因治疗载体.然而，AV是 在包装其基因组方面效率低下，这使得许多空的或部分完整的衣壳需要 在生产过程中去除。空衣壳可以增加AAV的免疫原性和毒性，特别是 当需要高剂量时。这个项目将采取结构生物学的方法来了解如何 AAV中基因组的存在影响衣壳四级结构和对衣壳的最终影响 表面化学和结构完整性。这将提供更好的方法来去除空衣壳或减少 生产空衣壳，从而为患者提供更安全的AAV产品。诱导的分子变化 通过AAV基因组包装将确定并整合病毒构象动力学的变化 和物理化学性质。这种新的集成的单粒子力测量使用原子 力显微镜（AFM）与表面残基电荷分布从酰胺氢-氘交换 （HDXMS）和天然质谱（MS）将导致深入了解基因组与AAV衣壳的相互作用， 基因组的存在如何改变衣壳结构、化学和完整性。前所未有的 观察病毒衣壳的结构如何对不同的生产条件和细胞运输做出反应 将通过完成以下目标来开发AAV生产期间发生的条件： 目的1：确定腺相关病毒衣壳的电荷和疏水性差异。将使用AAV 2和AAV 8 作为具有三种不同基因组大小的模型系统。化学力显微镜（CFM）是一种专门的原子力显微镜 技术，将测量在相关条件下AAV衣壳的电荷和疏水性的变化。 制造和细胞贩运条件。 目的2：研究衣壳蛋白-DNA和蛋白-蛋白相互作用对AAV病毒颗粒的影响 动力学空的、部分满的和满的AAV的比较将揭示DNA对内源性AAV的贡献。 此外，这些测量还将映射 在完全和部分完全的AAV中具有包封的DNA的AAV衣壳。 目的3：确定AAV衣壳之间的物理刚性和脆性差异。纳米压痕 AFM技术将用于确定DNA对AAV衣壳强度的影响。 在完成这项工作后，关于AAV如何与其基因组相互作用以及AAV如何与其基因组相互作用的数据驱动的假设被提出。 将开发出不同基因组大小的衣壳结构变化。溶液条件的影响， 在病毒生命周期和生产周期中变化很大。DNA的描述 将完成AAV中的包装和由于DNA包装而发生的结构变化。这 这些信息将改善AAV的生产、质量控制和安全性，并带来更多挽救生命的AAV疗法。 到市场。

Project Summary AAV is a popular gene therapy vector because it has large tissue tropism and low toxicity. However, AAV is inefficient at packaging its genome, and this leaves many empty or partially full capsids that need to be removed in manufacturing. Empty capsids can increase the immunogenicity and toxicity of the AAV, especially when high doses are required. This project will take a structural biology approach to understand how the presence of the genome in AAV affects the capsid quaternary structure and the resulting effects on capsid surface chemistry and structural integrity. This will provide better methods to remove empty capsids or reduce the production of empty capsids, thus providing a safer AAV product for patients. Molecular changes induced by AAV genome packaging will be determined and integrated with changes in viral conformational dynamics and physicochemical properties. This novel integration of single particle force measurements using an atomic force microscopy (AFM) with surface residue charge distributions from amide hydrogen-deuterium exchange (HDXMS) and native mass spectrometry (MS) will lead to insight into genome interaction with AAV capsids and how the presence of the genome changes the capsid structure, chemistry, and integrity. An unprecedented view into how the structure of a viral capsid reacts to different manufacturing conditions and cellular trafficking conditions that occur during AAV production will be developed by completing the following aims: Aim 1: Ascertain the difference in the charge and hydrophobicity of AAV capsids. AAV2 and AAV8 will be used as model systems with three different genome sizes. Chemical force microscopy (CFM) a specialized AFM technique, will measure the changes in charge and hydrophobicity of AAV capsids under relevant manufacturing and cellular trafficking conditions. Aim 2: Identify the contributions of capsid protein-DNA and protein-protein interactions on AAV viral particle dynamics. Comparison of empty, partially full, and full AAV will reveal contributions of DNA on intrinsic dynamics using HDXMS and native MS. Further, these measurements will also map interaction interfaces of AAV capsid with encapsulated DNA in full and partially full AAV. Aim 3: Determine the physical rigidity and brittleness difference between AAV capsids. Nanoindentation, an AFM technique, will be used to determine the effects of DNA on AAV capsid strength. Upon completion of this work, a data driven hypothesis on how AAV interacts with its genome and how the capsid structure changes with different genome sizes will be developed. The effect of solution conditions, which vary greatly during the virus life cycle and manufacturing cycle, will be elucidated. Descriptions of DNA packaging in AAV and the structural changes that occur due to DNA packaging will be completed. This information will improve production, quality control, and safety of AAV and bring more lifesaving AAV therapies to market.