Synthetic biology approach to nucleic acid clearance in lentiviral vector production
File(s)
Published version
Author(s)
Type
Conference Paper
Abstract
To improve lentiviral vectors (LVV) purity for cell and gene therapy applications manufacturers’ currently remove residual nucleic acids using a DNase treatment step with endonucleases such as Benzonase. 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 adapted adherent HEK293T cells to serum-free culture and then engineered them to secrete nuclease enzymes to remove nucleic acid 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 novel virus-encoded transport protein. The Tet repressor system was selected to regulate expression of nucB to reduce unwanted cell cytotoxicity and any negative effects on lentivirus production from potential intracellular nuclease expression and constitutive nuclease secretion. 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 base pairs to 10 kilobase pairs), 2-hour incubations at 37ºC 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 at each step of the LVV production process and had no measurable effect on infectious titre of the clarified supernatant when used to transduce both HEK293T and AGF-T cells under serum-free conditions. This provides a holistic approach to remove residual nucleic acids 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 nucleic acids.
Date Issued
2023-05-01
Date Acceptance
2023-01-23
Citation
Molecular Therapy, 2023, 31 (4, Supplement 1)
ISSN
1525-0016
Publisher
Cell Press
Journal / Book Title
Molecular Therapy
Volume
31
Issue
4, Supplement 1
Copyright Statement
© 2023. Creative Commons Attribution – NonCommercial – NoDerivs (CC BY-NC-ND 4.0)
Source
American Society of Cell and Gene Therapy
Subjects
Bioprocessing
Cell line engineering
Cell Therapy
Gene Therapy
Lentiviral Vector
Nuclease
Publication Status
Published
Start Date
2022-05-02
Coverage Spatial
Washington DC, USA