Metabolic engineering of HEK293 cells to improve transient transfection and cell budding of HIV‐1 virus‐like particles

Abstract

HIV‐1 Gag virus‐like particles (VLPs) are promising candidates for the development of future vaccines. Recent viral outbreaks have manifested the need of robust vaccine production platforms able to adapt to new challenges while achieving mass production capacity. For the rapid production of VLPs, the method of transient gene expression (TGE) have proved highly efficient. Based on a previous characterization of the HEK293 cell line upon transient transfection using multiplexed quantitative proteomics, molecular production bottlenecks and metabolic pathways likely to be optimized were identified. In this study, these molecular components and metabolic pathways have been explored and modulated via transient metabolic engineering using approaches like design of experiments to fully exploit and optimize VLP production, transfection and budding efficiency. Upon overexpression of endosomal sorting complex required for transport accessory proteins like NEDD4L and CIT, VLP production increased 3.3 and 2.9‐fold, respectively. Overexpression of glycosphingolipid precursor enzyme UGCG improved transfection efficiency by 17% and knocking‐down the Gag‐binding protein CNP improved 2.5‐fold VLP specific productivity. Combining CNP inhibition and UGCG overexpression further improved budding efficiency by 37.3%. Modulating VLP production and accessory pathways like intracellular budding, demonstrated the potential of metabolic engineering to optimize and intensify the development of robust production platforms for future vaccines.

Optimization, Production, Purification and Characterization of HIV-1 GAG-Based Virus-like Particles Functionalized with SARS-CoV-2

“…By staining the lipid membrane with CellMask, the strong co-localization of the S protein (red) and cell membrane (grey) was observed (Figure 1B), as well as the co-localization of the S protein (red) and Gag::eGFP (green) in membrane (Figures 1A,C). These results suggest that the expressed S protein could be dragged and incorporated at the surface of the produced S-VLPs, as they bud from the plasmatic membrane [29], where we observe that spike is present.…”

“…As can be seen in Figure 1A, the green fluorescence channel shows Gag::eGFP along the cytoplasm to the vicinity of the plasmatic membrane. This corresponds to what was already known about Gag polyprotein maturation, which occurs at the cytoplasm until it reaches the plasmatic membrane surroundings, where budding occurs in order to generate the Gag-based VLPs [29]. To determine spike localization, cells were immunostained using an anti-S primary antibody and a fluorocrome-conjugated secondary antibody.…”

Affecting HEK293 Cell Growth and Production Performance by Modifying the Expression of Specific Genes

“…In a follow-up study carried out by the same group, overexpression of members of the endosomal sorting complex, ( NEDD8 , NEDD4L and CIT ), and the glycosphingolipid precursor enzyme UDP-Glucose Ceramide Glucosyltransferase ( UGCG ) in HEK293 cells resulted in respective 1.5-, 3.3-, 2.9- and 2.4-fold improvements in VLP production, accompanied by a 51-61% increase in budding efficiency, and a 17% increase in transfection efficacy. In addition, shRNA Knockdown of the Gag-binding protein 2’,3’-Cyclic Nucleotide 3’ Phosphodiesterase gene, CNP , which inhibits viral replication, resulted in a 2.7-fold increase in VLP production and a 36% increase in budding efficiency [ 121 ].…”

Characterization of HIV‐1 virus‐like particles and determination of Gag stoichiometry for different production platforms

“…Data were analyzed with NanoSight ® NTA 3.1 software. NanoSight ® settings are described elsewhere (Lavado-García, Lavado-García et al, 2021).…”

“…How to cite this article: Lavado-García, J., Jorge, I., Boix-Besora, A., Vázquez, J., Gòdia, F., & Cervera, L. (2021).…”