title: Development of Endogenous and Synthetic CHO Promoter Expression Systems for Recombinant Protein Production
Creators: name: Binge, Alexandra
name: FUJIFILM Diosynth Biotechnologies
Publisher: name: University of Kent
Year: 2018

description: Chinese hamster ovary (CHO) cells are the most commonly used mammalian cell expression system for the industrial production of biotherapeutic recombinant proteins. Mammalian expression systems are a popular choice for the production of complex biopharmaceuticals due to their ability to correctly fold and assemble recombinant proteins and to carry out necessary human-like post-translational modifications. Traditionally, viral promoters are used to drive transgene expression in mammalian cells. This study reports on the investigation of endogenous CHO promoters as potential alternatives to the commonly used strong viral promoters such as SV40 and CMV. Although these promoters provide high recombinant gene expression, this is not always favourable as constitutive transgene over expression can ultimately lead to cellular stress. Furthermore, viral promoters have been reported to undergo silencing mechanisms over long culture periods, compromising product titers. Endogenous and synthetic CHO putative promoter sequences were investigated for their abilities to drive reporter and recombinant gene expression both in stable and transient systems. Initially a panel of ten putative promoters were identified from the literature, these being identified for this study as the associated gene or protein had been reported to have high transcript or protein amounts in CHO cells. The sequence 400 bp upstream of the transcript or translation start site of these genes was cloned into a promoterless eGFP reporter vector and their ability to drive transient gene expression examined. The ability of these putative promoters to drive eGFP expression was generally inferior to that of the viral SV40 promoter and always many-fold lower than that observed from the viral CMV with enhancer promoter. Regions 2 kb upstream from the transcript or translation site for each gene were then cloned into the promoterless eGFP reporter system to assess for promoter activity further upstream. For one of the 2 kb sequences, eGFP expression was enhanced above that observed from the equivalent 400 bp sequence and was greater than that from the SV40 promoter. A further nine target putative promoters were then identified from published RNAseq studies and sequences 500 bp upstream of the transcriptional start site of identified genes were cloned. Of the nine 500 bp sequences, three displayed promoter activity equivalent to or above that of SV40 and so regions 2 kb upstream of the transcriptional start site of these genes were investigated for further promoter activity. The use of the 2 kb segments resulted in an increase in stable but not transient eGFP expression from that observed from the 500 bp sequences alone. When placed directly downstream of the CMV enhancer two of the 2 kb sequences showed higher eGFP expression than when the CMV enhancer was not present. The CMV enhancer had no impact when upstream of the two other 2 kb sequences investigated showing this to be a sequence dependent effect. The 2 kb sequence which exhibited the strongest promoter activity of targets when driving transient eGFP expression, in addition to the two sequences with the CMV enhancer which showed enhanced transient eGFP expression, were investigated for their ability to drive IgG heavy and light chain, and hence IgG, stable expression in CHO cells compared to the CMV promoter with enhancer. Upon generation of pools and mini pools stably producing IgG under the control of the various promoters, the CHO endogenous 2 kb promoter outperformed the synthetic candidates, the CMV with enhancer and an industrially relevant control promoter. Indeed, the endogenous CHO promoter sequence was shown to achieve product titers of up to 3-fold greater than those from the commonly used CMV promoter with enhancer in CHO cells, demonstrating the potential for endogenous promoters to replace typically used viral promoters for recombinant gene expression. Further, colonies emerged faster after transfection and selection when using this promoter compared to the others investigated and a larger range of higher-expressing pools were available for investigation. In summary, this study has identified an endogenous CHO promoter sequence able to drive IgG expression beyond that of current widely used viral promoters and has generated additional promoters exhibiting a range of abilities to drive recombinant gene expression to varying amounts that could be applied to cell engineering approaches in the future.