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

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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.