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Synthetic Biology Approaches to Lentiviral Packaging Cell Engineering

Ali, Sadfer; (2019) Synthetic Biology Approaches to Lentiviral Packaging Cell Engineering. Doctoral thesis (Eng.D), UCL (University College London).

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In cell and gene therapy lentiviral vectors have become one of the preferred methods for transgene delivery. Scale-up of lentivirus manufacture is highly costly, with key factors including: 1) the requirement for high concentrations of three or more plasmids for transient transfection of cells to produce virus particles, 2) the fact lentivirus ‘packaging’ cells are typically adherent, limiting them to scale-out, as opposed to scale-up, approaches to increase cultivation capacity and 3) transient transfection typically results in significant levels of plasmid DNA impurity persisting in the product stream, necessitating addition of exogenous, clinical-grade nuclease followed by demonstration of nuclease and nucleic acid removal. To obviate the need for transient transfection, a strategy was devised and implemented to generate a cell line that constitutively produces lentiviral particles. The ‘serial insertion of genes for high titer (SIGHT)’ approach involved introducing the genetic components of a packaging cell line alongside cell surface expression markers so cells with high levels of expression of the required transgenes could be ligand-captured. SIGHT and the Sleeping Beauty recombinase system were used to generate the new cell line, 293LV, for high gene dosage of a lentiviral host genome. Measurement of the performance of transient transfection in 3D culture for lentivirus production was attempted using a HF MicroBrx™ device (Cell Culture Company, LLC) and completed with a novel, low-cost ‘tilted vessel extra-capillary space screening (TVECSS)’ method. Finally, the 293LV cell line was further engineered to have a measurable nuclease secretion phenotype in the new, Virase-1 cell line, within a process that was adapted to serum-free conditions for maximum industrial compatibility. Serum-free lentivirus production performance, with T cell transduction at 0.5-1x106 infectious particles per mL un-concentrated growth media, was maintained by the nuclease-secreting Virase-1 cells.

Type: Thesis (Doctoral)
Qualification: Eng.D
Title: Synthetic Biology Approaches to Lentiviral Packaging Cell Engineering
Event: UCL (University College London)
Language: English
Additional information: Copyright © The Author 2019. Original content in this thesis is licensed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0) Licence (https://creativecommons.org/licenses/by-nc/4.0/). Any third-party copyright material present remains the property of its respective owner(s) and is licensed under its existing terms. Access may initially be restricted at the author’s request.
UCL classification: UCL > Provost and Vice Provost Offices
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/10071961
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