Tijani, Maha; (2018) Vesiculovirus G protein-based stable cell lines for continuous lentiviral vector production. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Lentiviral vectors (LV) are often pseudotyped with the envelope G protein from vesicular stomatitis virus Indiana strain (VSVind.G). However, VSVind.G based continuous LV producer cell lines have not been reported; it has been assumed that VSVind.G is fusogenic and cytotoxic. To find alternative G proteins for LV production, we investigated other vesiculovirus G proteins (VesG) from VSV New Jersey strain (VSVnj), Cocal virus (COCV), Piry virus (PIRYV), VSV Alagoas virus (VSVala), and Maraba virus (MARAV). All these VesG envelopes were used in transient transfection to produce infectious particles that were robust during concentration and freeze-thawing. We then found, surprisingly, that VSVind.G and all the other VesG proteins could be constitutively expressed in 293T cells, and showed no cytotoxicity when compared to a retroviral Env protein. These VesG expressing cells could support LV production when other components were transiently supplied. However, we showed that VesG expressing cells do not show receptor interference hence LV can superinfect their producer cells, resulting in vector genome accumulation and possible toxicity. We attempted to knock-out the low- density lipoprotein receptor (LDLR) gene on producer cells, which was reported to be the primary cell entry receptor for VSVind.G. However, only a slight reduction in LV transduction was observed in LDLR-KO cells. Hence, other methods such as using anti-retroviral drugs to block superinfection may be necessary to allow construction of stable producer cell lines.

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