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Serum-free lentiviral vector production is compatible with medium-resident nuclease activity arising from adherent HEK293T host cells engineered with a nuclease-encoding transgene

Ali, Sadfer; Rivera, Milena; Ward, John; Keshavarz-Moore, Eli; Mason, Chris; Nesbeth, Darren; (2023) Serum-free lentiviral vector production is compatible with medium-resident nuclease activity arising from adherent HEK293T host cells engineered with a nuclease-encoding transgene. Heliyon , 9 (6) , Article e17067. 10.1016/j.heliyon.2023.e17067. Green open access

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Abstract

At present lentiviral vector production for cell and gene therapy commonly involves transient plasmid transfection of mammalian cells cultivated in serum-containing media and addition of exogenous nuclease to reduce host cell and plasmid DNA impurities. Switching from serum-containing media to chemically-defined, serum free media, and minimising the number of process additions, are both increasingly regarded as necessary steps for simplifying and potentially automating lentiviral vector bioprocessing in future. Here we adapted human embryonic kidney 293T (HEK293T) cells to grow in serum-free media and also modified these cells with transgenes designed to encode a secreted nuclease activity. Stable transfection of HEK293T cells with transgenes encoding the Staphylococcus aureus nuclease B (NucB) open reading frame with either its native secretion signal peptide, the murine Igκ chain leader sequence or a novel viral transport fusion protein, all resulted in qualitatively detectable nuclease activity in serum-free media. Serum-free transient transfection of human embryonic kidney HEK293T cells stably harbouring the transgene for NucB with its native secretion signal produced active lentivirus in the presence of medium-resident nuclease activity. This lentivirus material was able to transduce the AGF-T immortal T cell line with a green fluorescent protein reporter payload at a level of 2.05 × 105 TU/mL (±3.34 × 104 TU/mL). Sufficient nuclease activity was present in 10 μL of this unconcentrated lentivirus material to degrade 1.5 μg DNA within 2 h at 37 °C, without agitation - conditions compatible with lentivirus production. These observations demonstrate that lentiviral vector production, by transient transfection, is compatible with host cells harbouring a nuclease transgene and evidencing nuclease activity in their surrounding growth media. This work provides a solid basis for future investigations, beyond the scope of this present study, in which commercial and academic groups can apply this approach to therapeutic payloads and potentially omit exogenous nuclease bioprocess additions.

Type: Article
Title: Serum-free lentiviral vector production is compatible with medium-resident nuclease activity arising from adherent HEK293T host cells engineered with a nuclease-encoding transgene
Open access status: An open access version is available from UCL Discovery
DOI: 10.1016/j.heliyon.2023.e17067
Publisher version: https://doi.org/10.1016/j.heliyon.2023.e17067
Language: English
Additional information: © 2023 Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Keywords: Lentivirus, Gene therapy, Serum, Medium, Nuclease, Transgene, Transient, Transfection, T-cell
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.ucl.ac.uk/id/eprint/10171358
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