An autonucleolytic suspension HEK293F host cell line for high-titer serum-free AAV5 and AAV9 production with reduced levels of DNA impurity
File(s)
Published version
Author(s)
Type
Journal Article
Abstract
We sought to engineer mammalian cells to secrete nuclease activity as a step toward removing the need to purchase commercial nucleases as process additions in bioprocessing of AAV5 and AAV9 as gene therapy vectors. Engineering HeLa cells with a serratial nuclease transgene did not bring about nuclease activity in surrounding media whereas engineering serum-free, suspension-adapted HEK293F cells with a staphylococcal nuclease transgene did result in detectable nuclease activity in surrounding media of the resultant stable transfectant cell line, “NuPro-1S.” When cultivated in serum-free media, NuPro-1S cells yielded 3.06 × 1010 AAV5 viral genomes (vg)/mL via transient transfection, compared with 3.85 × 109 vg/mL from the parental HEK293F cell line. AAV9 production, followed by purification by ultracentrifugation, yielded 1.8 × 1013 vg/mL from NuPro-1S cells compared with 7.35 × 1012 vg/mL from HEK293F cells. AAV9 from both HEK293F and NuPro-1S showed almost identical ability to transduce cells embedded in a scaffold tissue mimic or cells of mouse neonate brain tissue in vivo. Comparison of agarose gel data indicated that the DNA content of AAV5 and AAV9 process streams from NuPro-1S cells was reduced by approximately 60% compared with HEK293F cells. A similar reduction in HEK293F cells was only achievable with a 50 U/mL Benzonase treatment.
Date Issued
2024-09-12
Date Acceptance
2024-08-07
Citation
Molecular Therapy: Methods & Clinical Development, 2024, 32 (3)
ISSN
2329-0501
Publisher
Elsevier
Journal / Book Title
Molecular Therapy: Methods & Clinical Development
Volume
32
Issue
3
Copyright Statement
© 2024 The Authors. Published by Elsevier Inc. on behalf of The American Society of Gene and Cell Therapy.
License URL
Publication Status
Published
Article Number
101317
Date Publish Online
2024-08-12