Sizing up viruses: application of emerging structural techniques for the characterisation of recombinant adeno-associated viruses (rAAVs)

背景：重组腺相关病毒（raav）和其他形式的核酸包封技术（旨在将遗传物质传递到细胞中）的最新应用进展表明，迫切需要一套全面的分析工具来描述这些颗粒的结构特征及其目标相互作用。完全组装的颗粒的特征以及与衣壳组装、制造和储存相关的详细结构变化带来了挑战。除了衣壳结构，我们对这些生物制药制剂中封装的单链DNA的高阶结构及其决定因素的理解也存在明显的缺陷，部分原因是缺乏适当有效的结构和分析工具。这一点尤其重要，因为该领域试图包装超过自然容纳的4.7 kbp的核酸，然后试图将这些大型治疗性基因货物运送到癌细胞中，并对抗恶性肿瘤。实验方法：在利兹大学的博士研究生项目中，该学生将与阿斯利康合作，部署并开发新的结构生物学技术，重点是结构质谱分析方法。这些能够为rAAV衣壳结构、ssDNA包装、它们之间的关系以及在生物制药相关条件下发生的变化提供有价值的见解。补充工作：该学生将接受正交结构成像方法的培训，用于完整组装的raav及其单个部件的高阶结构和功能。此外，还将接触到额外的质谱技术，如自上而下变性和天然完整质谱、交联质谱（XL-MS）和离子迁移质谱（IM-MS），用于寡核苷酸、ssDNA、病毒蛋白、包衣病毒颗粒和非共价配体的研究。最后，将有可能在其他实验室复制内部建立的方法，用于仪器间/实验室可重复性研究。培训地点：利兹大学和阿斯利康剑桥大学。

Background:Recent developments in the application of recombinant adeno-associated viruses (rAAVs) and other forms of nucleic acid encapsulation which aim to deliver genetic material to cells, have highlighted the urgent need for a comprehensive package of analytical tools for the structural characterisation of these particles and their target interactions. Challenges arise around the characterisation of the fully assembled particles and the detailed structural changes associated with capsid assembly, manufacture and storage.In addition to capsid structure, there are clear deficits in our understanding of the higher-order structure of the single stranded (ss)DNA encapsidated in these biopharmaceutical formulations and what determines it, in part due to a lack of suitably efficient structural and analytical tools. This is particularly important as the field attempts to package nucleic acid exceeding the 4.7 kbp that is naturally accommodated, in an attempt to then deliver these large therapeutic genetic cargoes to cancer cells, and fight malignancy.Experimental Approach:In this CASE PhD studentship at the University of Leeds, in collaboration with AstraZeneca, the student will deploy established and develop novel structural biology techniques, with an emphasis on structural mass spectrometry methods. These are capable of providing valuable insights into rAAV capsid structure, ssDNA packaging, and the relationship between these, and changes that occur under biopharmaceutically relevant conditions.Supplementary Work:The student will be trained in orthogonal structural imaging methods for the higher-order structure and function of both fully assembled rAAVs and their individual parts. Furthermore,there will be exposure to additional MS techniques such as top-down denaturing and native intact mass MS, cross-linking mass spectrometry (XL-MS) and Ion-mobility mass spectrometry (IM-MS) for oligonucleotides, ssDNA, viral proteins, encapsidated viral particles and non-covalent ligand studies. Finally, there will be the possibility to reproduce in-house established methods at other laboratories for inter-instrument/laboratory reproducibility studies.Training Locations:University of Leeds and AstraZeneca - Cambridge.