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Title: Production of self-inactivating lentiviral vectors by constitutive packaging cell lines for gene therapy clinical applications
Author: Sanber, K. S. R.
ISNI:       0000 0004 7229 4458
Awarding Body: UCL (University College London)
Current Institution: University College London (University of London)
Date of Award: 2015
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Lentiviral vectors (LVs) are useful experimental tools for stable gene delivery and have been used to treat human inherited genetic disorders and hematologic malignancies with promising results. Because some of the LV components are cytotoxic, transient plasmid transfection has been used to produce the large batches needed for clinical trials. However, this method is costly, poorly reproducible and hard to scale up. Generation of stable packaging cell lines (PCLs) that continuously produce LVs can potentially overcome these limitations. The WinPac-RDpro cell line was developed between Collins and Takeuchi laboratories in Division of Infection and Immunity, UCL by inserting a codon-optimized HIV-1 Gag-Pol expression cassette in a continuously expressed locus in 293FT cells using Cre recombinase-mediated cassette exchange (RMCE). Subsequently HIV-1 Rev and RDpro envelope expression cassettes were serially transfected. In this thesis, WinPac-RDpro cells were used to generate model producer cells by stably transfecting a plasmid expressing a SIN GFP-encoding LV. Vector titers in excess of 106 293T transducing units (TU)/ml could be repeatedly harvested from the final producer clones in a volume of >0.5 L even under reduced serum conditions. Titers could be increased to around 1 x 10^8 293T TU/ml by concentration using scalable tangential flow filtration (TFF). Additionally, these LVs efficiently transduced human T cells and CD34+ cells at low multiplicities of infection (MOI). Titers in excess of 10^6 TU/ml were achieved using an RMCE-based strategy that was aimed at introducing a SIN LV expression cassette at a pre-selected locus. Similar titers were also achieved by using a promoterless selectable marker cloned in cis to the vector genome expression cassette. Furthermore, the Cocal Virus G protein (COCV-G) was stably expressed in WinPac cells to generate WinPac-CVG cells. These packaging cells were able to support the production of COCV-G pseudotyped SIN LVs at high titers (up to 106 TU/ml) following transient supplementation of a SIN LV expression plasmid. The efficient and stable expression of SIN LV genomes in these cells is expected to facilitate high-titer production of vectors with favorable characteristics. In conclusion, the work presented here provides significant improvements to available LV production methods. This will be of use to all basic and clinical investigators who wish to produce large batches of LVs, and addresses an important issue that has hindered large-scale LV clinical testing and application.
Supervisor: Collins, M. K. ; Takeuchi, Y. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available