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Mol Ther Methods Clin Dev. 2018 Nov 22;13:14-26. doi: 10.1016/j.omtm.2018.11.004. eCollection 2019 Jun 14.

Scalable Production of AAV Vectors in Orbitally Shaken HEK293 Cells.

Author information

1
Department of Clinical Neurosciences, Laboratory of Neurotherapies and Neuromodulation (LNTM), Lausanne University Hospital (CHUV), 1011 Lausanne, Switzerland.
2
Neurosciences Research Center (CRN), Laboratory of Neurotherapies and Neuromodulation (LNTM), Lausanne University Hospital, 1011 Lausanne, Switzerland.
3
Brain Mind Institute, Ecole Polytechnique Fédérale de Lausanne (EPFL), 1015 Lausanne, Switzerland.
4
ExcellGene SA, 1870 Monthey, Switzerland.
5
Faculty of Life Science, Ecole Polytechnique Fédérale de Lausanne (EFPL), 1015 Lausanne, Switzerland.

Abstract

Adeno-associated virus (AAV) vectors are currently among the most commonly applied for in vivo gene therapy approaches. The evaluation of vectors during clinical development requires the production of considerable amounts of highly pure and potent vectors. Here, we set up a scalable process for AAV production, using orbitally shaken bioreactors and a fully characterized suspension-adapted cell line, HEKExpress. We conducted a proof-of-concept production of AAV2/8 and AAV2/9 vectors using HEKExpress cells. Furthermore, we compared the production of AAV2/9 vectors using this suspension cell line to classical protocols based on adherent HEK293 cells to demonstrate bioequivalence in vitro and in vivo. Following upstream processing, we purified vectors via gradient centrifugation and immunoaffinity chromatography. The in vitro characterization revealed differences due to the purification method, as well as the transfection protocol and the corresponding HEK293 cell line. The purification method and cell line used also affected in vivo transduction efficiency after bilateral injection of AAV2/9 vectors expressing a GFP reporter fused with a nuclear localization signal (AAV2/9-CBA-nlsGFP) into the striatum of adult mice. These results show that AAV vectors deriving from suspension HEKExpress cells are bioequivalent and may exhibit higher potency than vectors produced with adherent HEK293 cells.

KEYWORDS:

AAV8; AAV9; CNS transduction; adeno-associated virus; immune affinity chromatography; orbitally shaken bioreactors; suspension HEK293 cells; transient transfection

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