Costello, Alan (2019) RNA manipulations to improve recombinant protein production in Chinese hamster ovary cells. PhD thesis, Dublin City University.
Abstract
To date the major advances in biopharmaceutical production from Chinese Hamster Ovary (CHO) cells has come from culture vessel development and refining growth media formulation. However, it is believed that there is still room for improvement in the advent of media and process optimisation reaching a plateau. Publication of the first CHO-K1 genome in 2011 has resulted in a shift to focus efforts towards engineering of the CHO cell itself. The use of microRNA (miRNA) manipulation is one approach which has been fruitful in boosting cell specific productivity but at a cost of cellular growth rate, ultimately diminishing volumetric titre. Stable transgenic lines were created in a model IgG producing CHO cell line for six prioritized miRNAs robustly associated with growth rate. Depletion of miR-378-3p via miRNA-sponge decoy, was found to significantly enhance cell growth by ~40% but reduced specific productivity. To overcome the inherent antagonistic trade-off of cell energy driven towards growth or recombinant protein production, a tetracycline inducible (TET-ON) system was employed to manipulate the endogenous levels of different miRNAs at defined stages of culture. Absolute quantification of target miRNA and miRNA-sponge mRNA revealed a limitation of current synthetic sponge-decoy designs in the control of endogenous miRNA species. In efforts to re-engineer synthetic sponge-decoys we encountered circular RNAs (circRNA). circRNA is a closed loop structure and naturally occurring circRNAs demonstrate greater stability over their linear mRNA counterparts. The potential of circular transgene open reading frames (ORF) as a means of improving translational output from an RNA molecule was investigated using a model recombinant glycoprotein, Erythropoietin (EPO). Artificial circularization of gene ORF was achieved by splice signals, made proximal via complementary intronic flanking sequence. Rolling circle translation was explored by the removal of the stop codon from the EPO ORF. To ensure homogenous protein product, the addition of a self-cleavage 2A peptide to the infinite ORF was also assessed. These two constructs exhibited a significant increase in cell specific and volumetric productivity. Additional transgene mRNA engineering was investigated by encoding putative methylation sites in the 5’ un-translated region (UTR) of a model protein therapeutic.
Metadata
Item Type: | Thesis (PhD) |
---|---|
Date of Award: | 8 January 2019 |
Refereed: | No |
Supervisor(s): | Clynes, Martin, Barron, Niall and Doolan, Padraig |
Subjects: | Biological Sciences > Biotechnology Biological Sciences > Cell biology Biological Sciences > Molecular biology |
DCU Faculties and Centres: | DCU Faculties and Schools > Faculty of Science and Health > School of Biotechnology Research Initiatives and Centres > National Institute for Cellular Biotechnology (NICB) |
Use License: | This item is licensed under a Creative Commons Attribution-NonCommercial-No Derivative Works 3.0 License. View License |
Funders: | Science Foundation Ireland (13/IA/1841) |
ID Code: | 22906 |
Deposited On: | 04 Apr 2019 09:34 by Padraig Doolan . Last Modified 08 Jan 2023 04:30 |
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