High Throughput miRNA Screening Identifies miR-574-3p Hyperproductive Effect in CHO Cells

Abstract

CHO is 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. Manipulation of 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. In this study, we performed a high-throughput screening of 2042-human miRNAs and identified several candidates able to increase cell-specific and overall production of Erythropoietin and Etanercept in CHO cells. Some of these human miRNAs have not been found in Chinese hamster cells and yet were still effective in them. We identified miR-574-3p as being able, when overexpressed in CHO cells, to improve overall productivity of Erythropoietin and Etanercept titers from 1.3 to up to 2-fold. In addition, we validated several targets of miR-574-3p and identified p300 as a main target of miR-574-3p in CHO cells. Furthermore, we demonstrated that stable CHO cell overexpressing miRNAs from endogenous CHO pri-miRNA sequences outperform the cells with human pri-miRNA sequences. Our findings highlight the importance of flanking genomic sequences, and their secondary structure features, on pri-miRNA processing offering a novel, cost-effective and fast strategy as a valuable tool for efficient miRNAs engineering in CHO cells.

Multi‐lipase gene knockdown in Chinese hamster ovary cells using artificial microRNAs to reduce host cell protein mediated polysorbate degradation

“…Since the KO of every conceivable PS degrading HCP in the host cell line might not be practical, amiRNAs represent a flexible alternative. It has been shown several times that effects of transient miRNA transfections can be translated to a stable expression system (Fischer, Marquart, et al, 2017; Jadhav et al, 2014; Strotbek et al, 2013; Švab et al, 2021; Xu et al, 2019). Nevertheless, it is hypothesized that stable expression of amiRNAs could have even more pronounced effects on PS degradation, since transient transfection may not effectively reduce expression of HCPs with a long half‐life and amiRNAs will be degraded relatively quickly over time.…”

The Effect of microRNA on the Production of Recombinant Protein in CHO Cells and its Mechanism

“…miR-31* improves the unit productivity of CHO cells by promoting oxidative phosphorylation in mitochondria. miR-23 can increase the activity of mitochondria without affecting growth ( Švab et al, 2021 ). Inhibition of endogenous miR-378-3p in CHO-DP12 cells can increase the abundance of ubiquitin carboxyl-terminal hydrolase 14 (USP14) and increase the peak density of viable cells by 59%, but the specific cell productivity has not been improved ( Costello et al, 2018 ).…”

“…In addition, in the CEVEC’s amniocyte production cell line, the antibody concentrations of overexpressing miR-136, miR-3074, and miR-219 were 1.13, 1.06, and 1.15 times that of the negative control, respectively ( Weis et al, 2018 ). When miR-574-3p is overexpressed in CHO cells, it can increase the titer of erythropoietin and etanercept from 1.3 to 2 times ( Švab et al, 2021 ). Plasma cells (PCs), as part of the adaptive immune system, are naturally specialized in their structure, function, and cell signaling pathways for stable and high-level antibody production.…”

“…The cell cycle analysis of CHO-ETN cells stably expressing miR-574-3p by flow cytometry showed that the number of cells in the G0/G1 phase was less than the control cells after four days of culture. miR-574-3p shows anti-apoptotic effect ( Švab et al, 2021 ). Up-regulation of miR-7 by targeting the key regulators’ ubiquitin E3 ligase S-phase kinase-associated protein 2 (Skp2) and Proteasome activator complex subunit 3(Psme3) of the transition from G1 to S phase of the cell cycle induces G1 phase halt but does not promote cell apoptosis ( Sanchez et al, 2013 ).…”

“…It acetylates p53 to stabilize p53, which inhibits CMV promoter activity and interferes with basic transcription. When miR-574-3p is overexpressed, down-regulation of mRNA levels and protein levels of p300, which reduces the acetylation, destabilization, and degradation of p53 eliminates the inhibition of the CMV promoter and increases the expression of recombinant protein ( Švab et al, 2021 ).…”