Abstract
Recombinant adeno-associated virus virus-derived vectors (rAAVs) are among the most used viral delivery system for in vivo gene therapies with a good safety profile. However, rAAV production methods often lead to a heterogeneous vector population, in particular with the presence of undesired empty particles. Analytical ultracentrifugation sedimentation velocity (AUC-SV) is considered as the gold analytical technique allowing the measurement of relative amounts of each vector subpopulation and components like particle aggregates, based on their sedimentation coefficients. This letter presents the principle and practice of AUC experiments for rAAVs characterization. We discuss our results in the framework of previously published works. In addition to classical detection at 260 nm, using interference optics in the ultracentrifuge can provide an independent estimate of weight percentages of the different populations of capsids, and of the genome size incorporated in rAAV particles.
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Acknowledgements
This work used the platforms of the Grenoble Instruct-ERIC center (ISBG; UAR 3518 CNRS-CEA-UGA-EMBL) within the Grenoble Partnership for Structural Biology (PSB), supported by FRISBI (ANR-10-INBS-0005-02) and GRAL, financed within the University Grenoble Alpes graduate school (Ecoles Universitaires de Recherche) CBH-EUR-GS (ANR-17-EURE-0003). IBS acknowledges integration into the Interdisciplinary Research Institute of Grenoble (IRIG, CEA). We acknowledge the translational vector core “Centre de Production des Vecteurs” (CPV) in Nantes University (INSERM UMR 1089, TaRGeT laboratory) which is labelled “industrial integrator within the frame of “Le grand défi national de bioproduction” (ANR-20-F2II-006) and “La stratégie d’accélération de Biothérapies” funded by France 2023 (ANR-22_IBB_00XX.1). This work was performed under the financial support of Pre-industrial Gene Therapy Consortium (PGT-ANR-10-DPBS-01-01) and the national GIS-IBiSA and regional Biogenouest (Region Pays de la Loire) core networks.
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Special Issue: Analytical Ultracentrifugation 2022.
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Saleun, S., Mas, C., Le Roy, A. et al. Analytical ultracentrifugation sedimentation velocity for the characterization of recombinant adeno-associated virus vectors sub-populations. Eur Biophys J 52, 367–377 (2023). https://doi.org/10.1007/s00249-023-01650-3
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DOI: https://doi.org/10.1007/s00249-023-01650-3