Streamlined Adeno-Associated Virus Production Using Suspension HEK293T Cells.

Recombinant adeno-associated viruses (rAAVs) are valuable viral vectors for in vivo gene transfer, also having significant ex vivo therapeutic potential. Continued efforts have focused on various gene therapy applications, capsid engineering, and scalable manufacturing processes. Adherent cells are commonly used for virus production in most basic science laboratories because of their efficiency and cost. Although suspension cells are easier to handle and scale up compared to adherent cells, their use in virus production is hampered by poor transfection efficiency. In this protocol, we developed a simple scalable AAV production protocol using serum-free-media-adapted HEK293T suspension cells and VirusGEN transfection reagent. The established protocol allows AAV production from transfection to quality analysis of purified AAV within two weeks. Typical vector yields for the described suspension system followed by iodixanol purification range from a total of 1 × 1013 to 1.5 × 1013 vg (vector genome) using 90 mL of cell suspension vs. 1 × 1013 to 2 × 1013 vg using a regular adherent cell protocol (10 × 15 cm dishes). Key features • Adeno-associated virus (AAV) production using serum-free-media-adapted HEK293T suspension cells. • Efficient transfection with VirusGEN. • High AAV yield from small-volume cell culture. Graphical overview.

Characterization and quantification of adeno-associated virus capsid-loading states by multi-wavelength analytical ultracentrifugation with UltraScan.

Aim: We present multi-wavelength (MW) analytical ultracentrifugation (AUC) methods offering superior accuracy for adeno-associated virus characterization and quantification. Methods: Experimental design guidelines are presented for MW sedimentation velocity and analytical buoyant density equilibrium AUC. Results: Our results were compared with dual-wavelength AUC, transmission electron microscopy and mass photometry. In contrast to dual-wavelength AUC, MW-AUC correctly quantifies adeno-associated virus capsid ratios and identifies contaminants. In contrast to transmission electron microscopy, partially filled capsids can also be detected and quantified. In contrast to mass photometry, first-principle results are obtained. Conclusion: Our study demonstrates the improved information provided by MW-AUC, highlighting the utility of several recently integrated UltraScan programs, and reinforces AUC as the gold-standard analysis for viral vectors.