Structural and chemical changes between empty and full AAV capsids

项目摘要 AAV具有组织趋向性大、毒性低等优点，是一种广受欢迎的基因治疗载体。然而，AAV是 在包装其基因组时效率低下，这使得许多空壳或部分充满的衣壳需要 在制造过程中被移除。空衣壳可增强AAV的免疫原性和毒性，尤其是 当需要高剂量的时候。这个项目将采用结构生物学的方法来理解 甲型肝炎病毒基因组的存在影响衣壳四级结构及其对衣壳的影响 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。引起的分子变化 通过AAV，基因组包装将被确定并与病毒构象动力学的变化相结合 和物理化学性质。这种新的单粒子力测量积分使用原子 用原子力显微镜(AFM)研究酰胺氢-氚交换过程中的表面剩余电荷分布 (HDXMS)和自然质谱学(MS)将深入了解基因组与AAV衣壳和 基因组的存在如何改变衣壳结构、化学成分和完整性。史无前例的 观察病毒衣壳的结构如何对不同的制造条件和细胞贩运做出反应 将通过完成以下目标来开发AAV生产过程中发生的条件： 目的1：确定AAV衣壳的电荷和疏水性的差异。将使用AAV2和AAV8 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AAV衣壳在相关条件下的电荷和疏水性的变化 制造和贩卖手机的条件。 目的2：确定衣壳蛋白-DNA和蛋白质-蛋白质相互作用对AAV病毒颗粒的贡献 动力学。比较空的、部分满的和满的AAV将揭示DNA对固有的贡献 使用HDXMS和本地MS的动力学此外，这些测量还将映射 AAV衣壳带有完全和部分完全AAV包裹的DNA。 目的3：测定不同AAV衣壳的物理硬度和脆性差异。纳米压痕，一种 AFM技术，将用于确定DNA对AAV衣壳强度的影响。 这项工作完成后，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假说 随着基因组大小的不同，衣壳结构将发生变化。溶液条件的影响， 在病毒的生命周期和制造周期中有很大的不同，将被阐明。关于DNA的描述 AAV中的包装以及由于DNA包装而发生的结构变化将完成。这 信息将改善AAV的生产、质量控制和安全性，并带来更多拯救生命的AAV疗法 投放市场。

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.