Identification and Molecular Characterization of Process Relevant MicroRNAs in Chinese Hamster Ovary (CHO) Cells: CHO Optimization for the Production of Recombinant Proteins of Pharmaceutical Interest

Švab, Živa (2021). Identification and Molecular Characterization of Process Relevant MicroRNAs in Chinese Hamster Ovary (CHO) Cells: CHO Optimization for the Production of Recombinant Proteins of Pharmaceutical Interest. PhD thesis The Open University.



Biologics are highly effective therapies that have revolutionized the treatment of cancers, autoimmune disorders and other non-communicable diseases, however they have a high cost and are often used for chronic conditions requiring long treatment. An increasing need to improve patients access to these therapies is addressed by development of high-quality but lower cost biotherapeutics. Animal cell cultures, in particular Chinese hamster ovary (CHO) cells, are the cell line of choice for the manufacturing of many complex biotherapeutics. The constant upgrading of cell productivity is needed to meet the growing demand for these life-saving drugs. CHO cell culture technology has advanced significantly over the last few decades and is now generally considered a reliable, robust and mature technology. CHO cell factories have been improved with a plethora of genetic engineering approaches targeting different cell pathways, such as glycosylation, apoptosis, autophagy, cell cycle, cellular protein biosynthesis machinery, metabolism and secretory pathways. Manipulation with small non-coding RNAs, miRNAs, is a good alternative to a single gene knockdown approach due to their post-transcriptional regulation of entire cellular pathways without posing translational burden to the production cell. The aim of the present study was to identify and characterize miRNAs that can increase production of the recombinant proteins of pharmaceutical interest in CHO cells. Therefore, a functional screen using a library of 2042 synthetic human microRNA mimics transiently transfected into the CHO recombinant cell line producing erythropoietin has been performed. Viable cell density was increased by 14 miRNAs while 1015 miRNAs enhanced cell-specific erythropoietin productivity and 370 miRNAs resulted in higher volumetric productivity. 124 best miRNA candidates were further tested in the etanercept producing CHO cells. We selected seven miRNAs (miR-143-3p, miR-18b-3p, miR-132-5p, miR-521-1-3p, miR-574-3p, miR-3667-5p and miR-3939-3p), which significantly improved both erythropoietin and etanercept production in CHO cells, for further studies. Moreover, we compared the effect of Chinese hamster and human flanking sequences on the biogenesis of selected miRNAs in CHO cells. Stable CHO cell overexpressing miRNAs from endogenous CHO pri-miRNA sequences outperformed the cells with human pri-miRNA sequences. This work offers a novel strategy for the stable expression of any miRNA in CHO cells through site-directed mutagenesis of hamster-specific flanking sequences of pri-miR-143. It also highlights the importance of flanking genomic sequences, and their secondary structure features on pri-miRNA processing, offering a cost-effective and fast strategy as a valuable tool for efficient miRNAs engineering in CHO cells. Further on, we selected two process relevant microRNAs 574-3p and 3667-5p for a scale-up studies, their molecular characterization and target identification. In this study we identified a number of putative new targets of miR-574 using SILAC-based proteomic analysis. In addition, we validated several targets of miR-574-3p and identified p300 as a main target of miR-574-3p in CHO cells. Indeed, siRNA-mediated knockdown of p300 was able to induce pro-productive effects. In summary, our data suggest that miR-574-3p and p300 might represent novel genetic elements to enhance production in CHO cells, which may be mediated by down-regulation of p53.

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