Date of Award:
Master of Science (MS)
D. Keith Roper
D. Keith Roper
Viral vectors are gene carriers that efficiently deliver therapeutic gene constructs to target cells. Viral vectors are frequently produced in suspension-growing cells of mammalian origin, referred to as packaging cells. Lentiviral vectors have become widely used as gene vectors since they were initially developed two and a half decades ago, particularly in research settings. The advantages of lentiviral vectors for treating diseases of genetic origin have driven research into their large-scale manufacture for clinical settings. Currently, three main challenges exist that limit mass production of lentiviral vectors. First, plasmid DNA used in the production process is very expensive. Second, high vector titers have historically only been achievable at low packaging cell densities, limiting the scalability of suspended-culture technologies. And third, vector production frequently suffers from batch-to-batch variability in vector yield. In conjunction, these challenges diminish the economic viability and practical implementation of industrially-scaled processes for lentiviral vector production.
Recent advances in commercial reagents can help mitigate these stated challenges. There is currently a knowledge gap in the utility of cationic lipid-based transfection reagents for producing lentivirus in suspended cultures at high cell density.
In the present work, we investigate the production of lentiviral vectors by transient transfection of high density Human Embryonic Kidney packaging cells with a commercial cationic lipid transfection reagent. Several parameters in the production process were monitored to identify sources of variability in vector titers. We found that as packaging cells were cultured to increasing densities pre-transfection, there was a corresponding increase in lentiviral vector yield. Further, we found that aside from pre-culture density, consistency in culture routine leading up to transfection had the most significant influence on lentiviral vector yields.
Accordino, Jacob G., "Lentiviral Vector Production at High Cell Density by Transient Transfection of Suspended Culture HEK Cells" (2022). All Graduate Theses and Dissertations. 8596.
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