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Title: Synthetic biology approaches to lentiviral packaging cell engineering
Author: Ali, Sadfer
Awarding Body: UCL (University College London)
Current Institution: University College London (University of London)
Date of Award: 2019
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Full text unavailable from EThOS. Thesis embargoed until 01 May 2024
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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.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available