Production of High-Yield Adeno Associated Vector Batches Using HEK293 Suspension Cells

Kimberly L. Pietersz, Paul J.H. Nijhuis, Matthijs H.M. Klunder, Joëlle van den Herik, Barbara Hobo, Fred de Winter, Joost Verhaagen*

*Corresponding author for this work

Research output: Contribution to JournalArticleAcademicpeer-review

Abstract

Adeno-associated viral vectors (AAVs) are a remarkable tool for investigating the central nervous system (CNS). Innovative capsids, such as AAV.PHP.eB, demonstrate extensive transduction of the CNS by intravenous injection in mice. To achieve comparable transduction, a 100-fold higher titer (minimally 1 x 1011 genome copies/mouse) is needed compared to direct injection in the CNS parenchyma. In our group, AAV production, including AAV.PHP.eB relies on adherent HEK293T cells and the triple transfection method. Achieving high yields of AAV with adherent cells entails a labor-and material-intensive process. This constraint prompted the development of a protocol for suspension-based cell culture in conical tubes. AAVs generated in adherent cells were compared to the suspension production method. Culture in suspension using transfection reagents Polyethylenimine or TransIt were compared. AAV vectors were purified by iodixanol gradient ultracentrifugation followed by buffer exchange and concentration using a centrifugal filter. With the adherent method, we achieved an average of 2.6 x 1012 genome copies (GC) total, whereas the suspension method and Polyethylenimine yielded 7.7 x 1012 GC in total, and TransIt yielded 2.4 x 1013 GC in total. There is no difference in in vivo transduction efficiency between vectors produced with adherent compared to the suspension cell system. In summary, a suspension HEK293 cell based AAV production protocol is introduced, resulting in a reduced amount of time and labor needed for vector production while achieving 3 to 9 times higher yields using components available from commercial vendors for research purposes.

Original languageEnglish
Article numbere66532
JournalJournal of Visualized Experiments
Volume2024
Issue number206
Early online date26 Apr 2024
DOIs
Publication statusPublished - Apr 2024

Bibliographical note

Publisher Copyright:
© 2024 JoVE Journal of Visualized Experiments.

Funding

This work was supported by a grant from the Royal Netherlands Academy of Arts and Sciences (KNAW) research fund and a grant from Start2Cure (0-TI-01). We thank Leisha Kopp for her input and advice in the setup of the protocol. Figures were created using Biorender.

FundersFunder number
Koninklijke Nederlandse Akademie van Wetenschappen0-TI-01
Koninklijke Nederlandse Akademie van Wetenschappen

    Fingerprint

    Dive into the research topics of 'Production of High-Yield Adeno Associated Vector Batches Using HEK293 Suspension Cells'. Together they form a unique fingerprint.

    Cite this