Extended gene expression for HIV-1 VLPs scale-up and production enhancement using shRNA and chemical additives
June 17-22, 2018
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.
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Laura Cervera Gracia, Javier Fuenmayor, Francesc Gòdia, Irene Gonzalez-Domínguez, and Francesc Gòdia, "Extended gene expression for HIV-1 VLPs scale-up and production enhancement using shRNA and chemical additives" in "Vaccine Technology VII", Amine Kamen, McGill University Tarit Mukhopadhyay, University College London Nathalie Garcon, Bioaster Charles Lutsch, Sanofi Pasteur Eds, ECI Symposium Series, (2018). http://dc.engconfintl.org/vt_vii/85