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

Degradation of specific glycosaminoglycans improves transfection efficiency and vector production in transient lentiviral vector manufacturing processes

“…It is recognised that the addition of enzymes, such as Benzonase ® , is an expensive requirement in large-scale viral vector manufacturing processes and alternative approaches are being investigated. One group has engineered an inducible HEK293T cell line with the ability to secrete a Staphylococcus aureus nuclease during LVV production to reduce DNA impurity levels with the ultimate goal of omitting costly Benzonase ® additions ( Ali et al, 2023 ; Howe et al, 2024 ). A similar approach could be taken to digest chondroitin sulphate-based GAGs prior to transfection through the generation of a cell line engineered to secrete chondroitinase ABC, avoiding costly additions of enzyme to bioreactors in the context of larger scale LVV production.…”

Engineering an Autonucleolytic Mammalian Suspension Host Cell Line to Reduce DNA Impurity Levels in Serum-Free Lentiviral Process Streams

“…Stable Transfection of HEK293T cells with pETIP-ThorNucB. Stable HEK293T transfection was performed using 10 μg of pETIP-ThorNucB plasmid 21 and Fugene6 transfection reagent (Promega, UK) as per manufacturer's instructions, followed by selection using 3 μg/mL puromycin dihydrochloride (Thermo Fisher Scientific, UK) 4 days posttransfection.…”

“…Standard molecular biology techniques were used for all plasmid propagation, isolation, and analytical procedures. The plasmid pETIP-ThorNucB 21 encodes the S. aureus nuclease (NucB) with its native translocation signal replaced with an influenza hemagglutinin translocation signal peptide fused directly to an influenza hemagglutinin eptitope tag (HAss-HAtag) followed by a 62-residue region of the amino terminal domain of the Semliki Forest Virus capsid protein (Cp-p62). The following third-generation lentivirus plasmids were used: pLJM1-eGFP (Addgene plasmid no.…”

“…We previously demonstrated the feasibility of engineering adherent human embryonic kidney 293T (HEK293T) cells with transgenes encoding a secreted nuclease activity in a manner compatible with their use as hosts for lentivirus production . The logic for this approach in terms of the topology of the host cell interior and lentiviral budding is set out in Figure .…”

“…We previously demonstrated the feasibility of engineering adherent human embryonic kidney 293T (HEK293T) cells with transgenes encoding a secreted nuclease activity in a manner compatible with their use as hosts for lentivirus production. 21 The logic for this approach in terms of the topology of the host cell interior and lentiviral budding is set out in Figure 1 . Here we sought to re-engineer HEK293T with the ThorNucB transgene encoding Staphylococcus aureus NucB (UniProt: P00644 ) with the Semliki Forest virus capsid translocation signal for rapid egress 22 and further improve the resulting cell line by adapting it to grow in suspension mode and in serum-free media.…”