Structural and chemical changes between empty and full AAV capsids

空 AAV 衣壳和完整 AAV 衣壳之间的结构和化学变化

• 批准号: 10646613
• 负责人: Caryn Heldt
• 金额: $ 22.8万
• 依托单位: MICHIGAN TECHNOLOGICAL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-05-01 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10646613
• 关键词: Address; Affect; Amides; Amino Acids; Atomic Force Microscopy; Binding; Biological Models; Capsid; Capsid Proteins; Cells; Charge; Chemicals; Chemistry; DNA; DNA Packaging; DNA delivery; Data; Deuterium; Dose; Drug Delivery Systems; Encapsulated; Future; Gene Transduction Agent; Genes; Genome; Genomic DNA; Hydrogen; Hydrophobic Surfaces; Hydrophobicity; Life; Life Cycle Stages; Liver Dysfunction; Maintenance; Maps; Marketing; Mass Spectrum Analysis; Measurement; Measures; Mechanics; Mendelian disorder; Methods; Minor; Molecular; Molecular Conformation; Nucleic Acids; Patients; Process; Production; Property; Proteins; Quality Control; Reaction; Recombinant adeno-associated virus (rAAV); Research; Safety; Steroids; Structure; Surface; Techniques; Toxic effect; Viral; Viral Packaging; Virus; Work; adeno-associated viral vector; cost; delivery vehicle; immunogenicity; improved; innovation; insight; interest; large scale production; manufacture; nanoindentation; novel; particle; pressure; protein protein interaction; scale up; side effect; structural biology; success; tissue tropism; trafficking; Address; Affect; Amides; Amino Acids; Atomic Force Microscopy; Binding; Biological Models; Capsid; Capsid Proteins; Cells; Charge; Chemicals; Chemistry; DNA; DNA Packaging; DNA delivery; Data; Deuterium; Dose; Drug Delivery Systems; Encapsulated; Future; Gene Transduction Agent; Genes; Genome; Genomic DNA; Hydrogen; Hydrophobic Surfaces; Hydrophobicity; Life; Life Cycle Stages; Liver Dysfunction; Maintenance; Maps; Marketing; Mass Spectrum Analysis; Measurement; Measures; Mechanics; Mendelian disorder; Methods; Minor; Molecular; Molecular Conformation; Nucleic Acids; Patients; Process; Production; Property; Proteins; Quality Control; Reaction; Recombinant adeno-associated virus (rAAV); Research; Safety; Steroids; Structure; Surface; Techniques; Toxic effect; Viral; Viral Packaging; Virus; Work; adeno-associated viral vector; cost; delivery vehicle; immunogenicity; improved; innovation; insight; interest; large scale production; manufacture; nanoindentation; novel; particle; pressure; protein protein interaction; scale up; side effect; structural biology; success; tissue tropism; trafficking

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.

项目摘要 AAV是一种流行的基因疗法载体，因为它具有较大的组织对流和低毒性。但是，AAV是 在包装基因组方面效率低下 在制造业中删除。空的衣壳可以增加AAV的免疫原性和毒性，尤其是 当需要高剂量时。该项目将采用结构生物学方法来了解 AAV中的基因组的存在会影响衣壳季节结构，并对帽子产生影响 表面化学和结构完整性。这将提供更好的方法来删除空的衣壳或减少 空的衣壳的生产，从而为患者提供更安全的AAV产品。分子变化引起的 通过AAV基因组包装将与病毒构象动力学的变化确定并整合 和理化特性。这种新颖的单个颗粒力测量的集成使用原子 力显微镜（AFM），具有来自酰胺氢交换的表面残留电荷分布 （HDXM）和天然质谱法（MS）将导致人们对与AAV衣壳的基因组相互作用的见解和 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 查看病毒衣壳的结构如何对不同的制造条件和细胞运输反应 AAV生产过程中发生的条件将通过完成以下目的来开发： AIM 1：确定AAV衣壳的电荷和疏水性的差异。 AAV2和AAV8将使用 作为具有三个不同基因组大小的模型系统。化学力显微镜（CFM）专门的AFM 技术，将衡量相关的AAV CAPSID的电荷变化和疏水性的变化 制造和蜂窝运输条件。 AIM 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，部分完整和完整的AAV将揭示DNA对固有的贡献 使用HDXM和本机MS的动力学。此外，这些测量还将映射 AAV CAPSID带有完整和部分完整AAV的封装DNA。 AIM 3：确定AAV衣壳之间的身体刚度和脆性差异。纳米凹痕，an AFM技术将用于确定DNA对AAV CAPSID强度的影响。 完成这项工作后，数据驱动的假设是关于AAV如何与其基因组相互作用以及如何相互作用的假设 将开发带有不同基因组大小的衣壳结构变化。解决方案条件的影响 在病毒生命周期和制造周期中，这将被阐明。 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，并带来更多的救生AAV疗法 上市。