Title: Rapid, simple and versatile manufacturing of recombinant adeno-associated virus vectors at scale
Authors: Lock, Martin
Alvira, Mauricio
Vandenberghe, Luk H
Samanta, Arabinda
Toelen, Jaan
Debyser, Zeger
Wilson, James M # ×
Issue Date: Oct-2010
Publisher: Mary Ann Liebert, Inc.
Series Title: Human Gene Therapy vol:21 issue:10 pages:1259-1271
Abstract: Adeno-associated virus vector manufacturing at scale continues to hinder the application of AAV technology to gene therapy studies. While scalable systems based upon AAV-adenovirus, -herpesvirus and -baculovirus hybrids hold promise for clinical applications, they require time-consuming generation of reagents and are not highly suited to intermediate scale pre-clinical studies in large animals where several combinations of serotype and genome may need to be tested. Recently we observed that during production of many AAV serotypes, large amounts of vector are found in the culture supernatant, a relatively pure source of vector in comparison with cell-derived material. Here we describe a high yielding, recombinant AAV production process based upon PEI-mediated transfection of HEK 293 cells and iodixanol gradient centrifugation of concentrated culture supernatant. The entire process can be completed in one week and the steps involved are universal for a number of different AAV serotypes. Process conditions have been optimized such that final purified yields are routinely greater than 1 x 1014 genome copies per run, with capsid protein purity exceeding 90%. Initial experiments with vectors produced with the new process demonstrate equivalent or better transduction both in vitro and in vivo when compared to small scale, CsCl gradient-purified vectors. In addition, the iodixanol gradient purification process described effectively separates infectious particles from empty capsids, a desirable property for reducing toxicity and unwanted immune responses during pre-clinical studies.
ISSN: 1043-0342
Publication status: published
KU Leuven publication type: IT
Appears in Collections:Molecular Virology and Gene Therapy
× corresponding author
# (joint) last author

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