Streamlined Adeno-Associated Virus Production Using Suspension HEK293T Cells

Authors

Aditi A Kulkarni
Austin G Seal
Corinne Sonnet
Kazuhiro Oka

Publication Date

2-5-2024

Journal

Bio-protocol Journal

DOI

10.21769/BioProtoc.4931

PMID

38379831

PMCID

PMC10875358

PubMedCentral® Posted Date

2-5-2024

PubMedCentral® Full Text Version

Post-Print

Published Open-Access

yes

Keywords

AAV, Suspension cells, Serum-free media, Transfection reagent, Iodixanol density gradient

Abstract

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