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ONLY A SMALL FRACTION OF CELLS PRODUCE ASSEMBLED CAPSIDS DURING TRANSFECTION-BASED MANUFACTURING OF ADENO-ASSOCIATED VIRUS VECTORS
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  • Shantoshini Dash,
  • David Sharon,
  • Alaka Mullick,
  • Amine Kamen
Shantoshini Dash
McGill University

Corresponding Author:[email protected]

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David Sharon
McGill University Faculty of Engineering
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Alaka Mullick
National Research Council Canada
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Amine Kamen
McGill University
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Abstract

Plasmid transfection of mammalian cells is the dominant platform used to produce adeno-associated virus (AAV) vectors for clinical and research applications. Low yields from this platform currently make it difficult to supply these activities with adequate material. In an effort to better understand the current limitations of transfection-based manufacturing, this study examines what proportion of cells in a model transfection produce appreciable amounts of assembled AAV capsid. Using conformation-specific antibody staining and flow cytometry we report the surprising result that despite obtaining high transfection efficiencies and nominal vector yields in our model system, only 5-10% of cells appear to produce measurable levels of assembled AAV capsids. This finding implies that considerable increases in vector titer could be realized through increasing the proportion of productive cells. Furthermore, we suggest that the flow cytometry assay used here to quantify productive cells may be a useful metric for future optimization of transfection-based AAV vector manufacturing platforms.
07 Nov 2021Submitted to Biotechnology and Bioengineering
08 Nov 2021Submission Checks Completed
08 Nov 2021Assigned to Editor
14 Nov 2021Reviewer(s) Assigned
22 Nov 2021Review(s) Completed, Editorial Evaluation Pending
22 Nov 2021Editorial Decision: Revise Major
14 Jan 20221st Revision Received
17 Jan 2022Submission Checks Completed
17 Jan 2022Assigned to Editor
20 Jan 2022Reviewer(s) Assigned
13 Feb 2022Review(s) Completed, Editorial Evaluation Pending
13 Feb 2022Editorial Decision: Accept
Jun 2022Published in Biotechnology and Bioengineering volume 119 issue 6 on pages 1685-1690. 10.1002/bit.28068