Lentivirus Production and Transduction of T-Cells Using Packaging Cells Engineered to Secrete Active Nuclease into Serum-Free Media

To improve the purity of lentiviral vectors (LVV) for cell and gene therapy applications manufacturers’ currently remove residual DNA using a DNase treatment step. This is a prohibitively expensive but necessary step in the manufacturing process as regulators require concentrations of less than 10 ng/dose at sizes less than 200 base pairs in the final product. As an alternative we have engineered serum-free adapted HEK293T cells to express nuclease enzymes to remove DNA impurities from LVV supernatant. Three nuclease expression cassettes were designed by fusing the N-terminus of the Staphylococcus aureus nuclease B (nucB) open reading frame to: the native S. aureus nuclease signal peptide, the mammalian murine Igκ chain leader sequence, and a virus-encoded transport protein, under the Tet repressor system. All three nuclease expression cassettes demonstrated levels of activity equivalent to or better than Benzonase at 250 units in growth medium harvested from engineered cell lines 24 hours post tetracycline induction. When treating 1.5 µg 1 kb DNA ladder (500 bp to 10 Kbp), incubations at 37ºC for 2-hours with 10 µL of growth media reduced DNA ladder to non-visible sizes on 1% agarose gel_. To verify that a nuclease secreting cell line can support LVV production, a LVV encoding for green fluorescent protein was prepared by transient transfection. Nuclease activity was demonstrated in fractions collected during LVV production and had no measurable effect on infectious titer of the clarified supernatant when transducing both HEK293T and AGF-T cells under serum-free conditions. This provides a holistic approach to remove residual DNA early in the process stream and improve the purity of the final product, whilst avoiding the addition of exogenous nuclease and its associated costs at scale. This may also potentially reduce any deficiencies of downstream processing attributed to the viscosity of residual DNA.