Abstract
Transient gene expression (TGE) in mammalian cells is a method of rapidly generating recombinant protein material for initial characterisation studies that does not require time-consuming processes associated with stable cell line construction. High TGE yields are heavily dependent on efficient delivery of plasmid DNA across both the plasma and nuclear membranes. Here, we harness the protein nucleoside diphosphate kinase (NDPK-A) that contains a nuclear localisation signal (NLS) to enhance DNA delivery into the nucleus of CHO cells. We show that co-expression of NDPK-A during transient expression results in improved transfection efficiency in CHO cells, presumably due to enhanced transportation of plasmid DNA into the nucleus via the nuclear pore complex. Furthermore, introduction of the Epstein Barr Nuclear Antigen-1 (EBNA-1), a protein that is capable of inducing extrachromosomal maintenance, when coupled with complementary oriP elements on a transient plasmid, was utilised to reduce the effect of plasmid dilution. Whilst there was attenuated growth upon introduction of the EBNA-1 system into CHO cells, when both NDPK-A nuclear import and EBNA-1 mediated technologies were employed together this resulted in enhanced transient recombinant protein yields superior to those generated using either approach independently, including when expressing the complex SARS-CoV-2 spike (S) glycoprotein.
Highlights
Chinese hamster ovary (CHO) cells are the most commonly utilised expression hosts for the production of recombinant biotherapeutic proteins such as monoclonal antibodies as they are able to generate complex, multi-domain, multi-chain proteins with human-like posttranslational modifications (Godfrey et al, 2017)
There was a higher mean fluorescence from the transient population transfected with the nucleoside diphosphate kinase (NDPK-A) nuclear localisation signal (NLS) containing gene alone when compared to the control after 48 h (Figure 1B)
A combination of the NDPKA NLS containing gene construct and binding sequences (BS) together resulted in a small increase in the percentage of cells exceeding the fluorescence threshold compared to the control and a statistically significant increase in the overall geometric mean fluorescence value, but this increase was not as substantial as when either component was included alone
Summary
Chinese hamster ovary (CHO) cells are the most commonly utilised expression hosts for the production of recombinant biotherapeutic proteins such as monoclonal antibodies (mAbs) as they are able to generate complex, multi-domain, multi-chain proteins with human-like posttranslational modifications (Godfrey et al, 2017). Technologies and processes underpinning the generation of recombinant material in CHO cells have been extensively developed enabling manufacturers to achieve product titres greater than 5 g/L of mAb (Marichal-Gallardo and Álvarez, 2012; Budge et al, 2019) Despite these considerable advances, the processes required to stably integrate genes of interest into a CHO host cell chromosome followed by screening, selection and adaption of a high yielding clone which is stable in both product quality and productivity of a desired molecule takes up to 12 months (Wurm, 2004). But further, suggests that a decrease in the rate of nuclear import of plasmid DNA may be linked to the reduced growth observed when the EBNA-1 gene is introduced in the Lonza CHOK1SV or CHOK1SV GS-KO cells either transiently or stably, respectively. The data presented here shows that cells only expressing exogenously added EBNA-1 gene have compromised growth and consistently decreased expression of both eGFP and an IgG4 molecules in the absence of the oriP elements
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