ECTENDED GENE EXPRESSION FOR HIV-1 VLPs AND PRODUCTION ENHANCEMENT USING shRNA AND CHEMICAL ADDITIVES

摘要

Gag polyprotein from HIV-1 can generate Virus-Like Particles (VLPs) when recombinantly expressed in animal cell platforms. HIV-1 VLP production in HEK293 cells can be improved using different strategies for increasing product titers. One of them is the so-called Extended Gene Expression (EGE), based on repeated medium exchanges and retransfections of the cell culture to prolong the production phase. Another approach to improve transient transfection results is media supplementation with gene expression enhancers such as valproic acid and caffeine, despite their detrimental effect on cell viability. Valproic acid is a histone deacetylase inhibitor while caffeine has a phosphodiesterase inhibition effect. The work presented has three main objectives. First, the combination of the EGE protocol with valproic acid and caffeine supplementation to maximize VLP production; second, the replacement of these chemical additives by shRNA for obtaining the same inhibition action and third the bioreactor scale-up of the process. The combination of the EGE protocol with caffeine and valproic acid supplementation resulted in a 1.5-fold improvement in HIV-1 VLP production compared with the EGE protocol alone, which is an 18-fold improvement over a conventional batch cultivation. shRNAs encoded in the expression vector were tested to substitute valproic acid and caffeine. This novel strategy enhanced VLP production by 2.5-fold without any detrimental effect on cell viability, which results in obtaining higher quality VLPs. Finally, the combination of shRNA with EGE resulted in more than 14-fold improvement compared with the batch standard protocol traditionally used. This protocol enables the production of high-quality HIV-1 VLPs avoiding toxic effects of the additives but maintaining high product titers. When EGE process was scaled-up, cell viability was comparable through all processes of the two systems tested; however, the bioreactor allowed for much higher cell densities and specific growth rates. Transfection efficiency was also comparable and successfully achieved in both systems. GagGFP fluorescence quantification revealed similar VLP titres in both shake flasks and bioreactor. A product quality assessment was also carried out to evaluate the two systems. Presence of VLPs in all samples was confirmed by transmission electron microscopy. Nanoparticle tracking analysis showed that the ratio of VLPs/total particles (VLPs and cell vesicles) was higher in the shake flask than in the bioreactor, possibly due to higher cell densities achieved in the bioreactor. Similarly, host cell DNA and host cell protein analyses revealed higher impurity concentrations for the bioreactor compared with shake flasks. Furthermore, the budding process of VLP in the production process was observed using super resolution confocal microscopy. This technique allowed to quantify the percentage of Gag-GFP that colocalize in the cell membrane and the percentage of the membrane that is occupied by VLPs budding from the cell. This increase in resolution also enabled the direct quantification of VLPs in supernatant samples by confocal microscopy that correlated with the quantification obtained by nanoparticle tracking analysis.