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 × 10
13
to 1.5 × 10
13
vg
(vector genome) using 90 mL of cell suspension vs. 1 × 10
13
to 2 ×
10
13
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