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

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 × 10 6 lentiviral transducing units per milliliter (TU/mL) from NuPro-2S cells and 1.45 × 10 6 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.

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.…”