Howe, Geoffrey;
Bal, Mehtap;
Wasmuth, Matthew;
Massaro, G;
Rahim, Ahad;
Ali, Sadfer;
Rivera, Milena;
... Nesbeth, Darren; + view all
(2024)
An autonucleolytic suspension HEK293F host cell line for high-titre serum-free AAV5 and AAV9 production with reduced levels of DNA impurity.
Molecular Therapy - Methods and Clinical Development
, Article 101317. 10.1016/j.omtm.2024.101317.
(In press).
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PIIS2329050124001335.pdf - Accepted Version Download (15MB) | Preview |
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 HEK293-F 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 3.06x1010 AAV5 viral genomes (vg) / mL via transient transfection, compared to 3.85x109 vg /mL from the parental HEK293-F cell line. AAV9 production, followed by purification by ultracentrifugation, yielded 1.8x1013 vg /mL from NuPro-1S cells compared to 7.35x1012 vg /mL from HEK293-F cells. AAV9 from both HEK293-F 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 to HEK293-F cells. A similar reduction in HEK293-F cells was only achievable with a 50 U / mL Benzonase® treatment.
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