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

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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.