Howe, Geoffrey;
Wasmuth, matthew;
Emanuelle, Pamela;
Massaro, Giulia;
Rahim, Ahad;
Ali, sadfer;
Rivera, Milena;
... Nesbeth, Darren; + view all
(2024)
Engineering an autonucleolytic mammalian suspension host cell line to reduce DNA impurity levels in serum-free lentiviral process streams.
ACS Synthetic Biology
, 13
(2)
pp. 408-692.
10.1021/acssynbio.3c00682.
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Abstract
We engineered HEK293T cells with a transgene encoding tetracycline-inducible expression of a Staphylococcus aureus nuclease incorporating a translocation signal. We adapted the unmodified and nuclease-engineered cell lines to grow in suspension in serum-free media, generating the HEK293TS and NuPro-2S cell lines, respectively. Transient transfection yielded 1.19 × 106 lentiviral transducing units per milliliter (TU/mL) from NuPro-2S cells and 1.45 × 106 TU/mL from HEK293TS cells. DNA ladder disappearance revealed medium-resident nuclease activity arising from NuPro-2S cells in a tetracycline-inducible manner. DNA impurity levels in lentiviral material arising from NuPro-2S and HEK293TS cells were undetectable by SYBR Safe agarose gel staining. Direct measurement by PicoGreen reagent revealed DNA to be present at 636 ng/mL in lentiviral material from HEK293TS cells, an impurity level reduced by 89% to 70 ng/mL in lentiviral material from NuPro-2S cells. This reduction was comparable to the 23 ng/mL achieved by treating HEK293TS-derived lentiviral material with 50 units/mL Benzonase.
| Type: | Article |
|---|---|
| Title: | Engineering an autonucleolytic mammalian suspension host cell line to reduce DNA impurity levels in serum-free lentiviral process streams |
| Open access status: | An open access version is available from UCL Discovery |
| DOI: | 10.1021/acssynbio.3c00682 |
| Publisher version: | https://doi.org/10.1021/acssynbio.3c00682 |
| Language: | English |
| Additional information: | This version is the author accepted manuscript. For information on re-use, please refer to the publisher's terms and conditions. |
| Keywords: | lentivirus, mammalian cells, bioprocess, gene therapy, nuclease |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices > UCL BEAMS UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Engineering Science UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Engineering Science > Dept of Biochemical Engineering |
| URI: | https://discovery-pp.ucl.ac.uk/id/eprint/10185876 |
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