Abstract
Gag polyprotein from HIV-1 is able to generate virus-like particles (VLPs) when recombinantly expressed in animal cell platforms. HIV-1 VLP production in HEK293 cells can be improved by the use of 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 is the 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. Here, the combination of the EGE protocol with additive supplementation to maximize VLP production is first tested. As an alternative to the direct additive supplementation, the replacement of these chemical additives by iRNA for obtaining the same inhibition action is also tested. 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, representing an overall 18-fold improvement over conventional batch cultivation. shRNAs encoded in the expression vector were tested to substitute valproic acid and caffeine. This novel strategy enhanced VLP production by 2.3 fold without any detrimental effect on cell viability (91.7%) compared with the batch cultivation (92.0%). Finally, the combination of shRNA with EGE resulted in more than 15.6-fold improvement compared with the batch standard protocol traditionally used. The methodology developed enables the production of high titers of HIV-1 VLPs avoiding the toxic effects of additives.
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Acknowledgements
The authors wish to thank Dr. Amine Kamen (McGill University, Montreal, Canada) for providing theHEK293SF-3F6 used in this work. We would also like to thank Dr. Julià Blanco at IRSI Caixa (Badalona, Spain) for providing the plasmid construct for Gag-GFP. The contribution of Manuela Costa (Institut de Biotecnologia i Biomedicina, UAB) to the FACS analysis is deeply appreciated. The support of Dr. Salvador Bartolomé (Department de Bioquímica i de Biologia Molecular, UAB) in fluorimetry analysis is recognized.
Funding
This study is supported by a grant of SEIDI – Ministerio de Economía y Competitividad of SPAIN (BIO2012-31251).
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This article does not contain any studies with human participants or animals performed by any of the authors.
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Fuenmayor, J., Cervera, L., Rigau, C. et al. Enhancement of HIV-1 VLP production using gene inhibition strategies. Appl Microbiol Biotechnol 102, 4477–4487 (2018). https://doi.org/10.1007/s00253-018-8930-8
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DOI: https://doi.org/10.1007/s00253-018-8930-8