Cryogenic electron microscopy-based understanding of viral vector heterogeneity to aide process development

病毒载体可作为溶瘤病毒、基因疫苗和基因治疗载体。然而，正如最近在ChAdOx1 nCoV-19疫苗和基因治疗试验中观察到的不良反应所强调的那样，我们对这些产品的了解仍然存在差距。这目前限制了我们设计病毒的趋向性、免疫原性和可制造性的能力。我们力求解决病毒颗粒合成的质量问题，并提高产品中成熟病毒颗粒的比例。这将通过利用多尺度分辨率的计算能力来实现，以门脉蛋白的作用为中心，捕捉对病毒衣壳生物组装和拆卸途径的新见解，从而实现病毒传染性的“计算显微镜”。使用腺病毒（Ad5）作为我们的模型系统，我们已经证明低温电镜分析可以识别颗粒表面形态，例如纤维分子，并且是一种可以应用于识别一系列病毒载体样生物治疗药物的关键质量标记的技术。通过该项目收集更大的低温电子显微镜数据集将使我们能够探测对病毒传染性至关重要的关键结构特征，包括衣壳和刺突蛋白中的门户结构。关键是我们能够与劳斯实验室（第二主管）合作实现原子水平的模拟，并创建样品，其中处理条件的变化可用于创建已知病毒异质性变化的样品。一旦优化，这些新方法将扩展到分析慢病毒（LV）和腺相关病毒（AAV）载体系统，以充分补充应用于基因传递的主要平台。

Viral vectors can be used as oncolytic viruses, gene-based vaccines, and gene therapy vectors. However, as highlighted by the adverse reactions observed with the recent ChAdOx1 nCoV-19 vaccine and gene therapy trials, gaps remain in our understanding of these products. This currently limits our ability to engineer the virus's tropism, immunogenicity and manufacturability. We seek to address the quality of virus particle synthesis and improve the proportion of mature virus particles in the product. This will be achieved by harnessing the power of computation at multiscale resolution to capture new insights into the biological assembly and disassembly pathways of the viral capsid, centering on the role of the portal protein, towards 'computational microscopy' of viral infectivity.Using adenovirus (Ad5) as our model system we have demonstrated that cryo-EM analysis can identify particle surface morphologies, e.g. fiber molecules and is a technique that could be applied to identify key quality markers on a range of viral vector-like biotherapeutics. Gathering larger cryo-electron microscopy datasets via this project will allow us to probe key structural features critical to viral infectivity, including the portal structure in the capsid and the spike proteins. Key to this is our ability to implement atomic level simulations in collaboration with the Rouse lab (the secondary supervisor) and create samples where changes to the processing conditions can be used to create samples with known changes to viral heterogeneity. Once optimised, these novel methods will be expanded to analyse lentiviral (LV) and adeno-associated virus (AAV) vector systems to fully complement the predominant platforms being applied to gene delivery.