Albert Heck

Exploring AAV subspecies using single molecule mass detection

Eduard H.T.M. Ebberink1, Alisa Ruisinger2, Markus Neubel2, Marco Thomann2, Albert J.R. Heck1

1 Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, University of Utrecht, Padualaan 8, 3584 CH Utrecht, the Netherlands
2 Roche Diagnostics, Penzberg, Germany

Introduction: Adeno-associated viruses (AAV) have proven to be the most promising vehicles for gene therapy. Because of their stability, low immunogenicity and non-pathogenicity, AAVs have ideal attributes as gene vector and delivery system. Unfortunately, disparity in AAV sample preparations (e.g. in capsid protein composition, DNA packaging and impurities) following large-scale production gives rise to AAV heterogeneity, with as of yet an unknown effect on gene delivery. Therefore, accurate analysis of AAV composition is important. Moreover, in a therapeutic context, to have complete control of AAV structure and its genetic payload a robust, quick and low material consuming method would be desirable.

Methods: Here we explore novel, analytical methods to accurately measure AAV molecular weight and unravel capsid structure, DNA packaging and sample purity. Single-particle techniques such as charge-detection mass spectrometry (CDMS) and mass photometry (MP) were performed side-by-side to allow for direct comparison. Two different serotypes (AAV8 and AAV2) were evaluated, either lacking DNA content or with a CMV-GFP transgene. In addition, we were able to compare AAVs from different vendors that utilize different production and purification workflows.

Preliminary data: Subtle differences between the AAV sets could be characterized by MP and CD-MS. We detected different molecular weights of the viral capsids of the same serotype but from different suppliers. Moreover, once a transgene was introduced, next to the expected empty or single genome packed capsids, a third population was measured yielding extra mass (potentially a double packed genome). Strikingly, the CD-MS measurements showed good agreement with the MP data. This work demonstrates that both techniques can be valuable tools in studying AAVs for gene therapy.

Novel aspect: Side-by-side comparison of two single molecule mass analysis techniques to assess structural integrity of adeno-associated virus gene-delivery vehicles.

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