Transfection of miR-31* boosts oxidative phosphorylation metabolism in the mitochondria and enhances recombinant protein production in Chinese hamster ovary cells

Martínez-López, Jesús; Coleman, Orla; Clynes, Martin

doi:10.1016/j.jbiotec.2021.04.012

Cited by 5 publications

(3 citation statements)

References 76 publications

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“…After identifying ATF6β as a target gene of microRNA miR-1287, knockdown of ATF6β by nucleofection increased fed-batch VCD and increased titer of a DG44-IgG 1 CHO cell line by 1.36-fold 20 . Despite WFS1 being identified in multiple CHO cell ER stress-related studies, there is no validation of its role in CHO cell UPR regulation 33 , 66 , 80 , 81 . The proteins WFS1 and ATF6β are both considered negative regulators of the ATF6 arm of the UPR with one pancreatic β cell study reporting WFS1 as a target mRNA of ATF6β 20 – 22 , 29 – 32 .…”

Section: Discussionmentioning

confidence: 99%

RNASeq highlights ATF6 pathway regulators for CHO cell engineering with different impacts of ATF6β and WFS1 knockdown on fed-batch production of IgG1

Rives,

Peak,

Blenner

2024

Sci Rep

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Secretion levels required of industrial Chinese hamster ovary (CHO) cell lines can challenge endoplasmic reticulum (ER) homeostasis, and ER stress caused by accumulation of misfolded proteins can be a bottleneck in biomanufacturing. The unfolded protein response (UPR) is initiated to restore homeostasis in response to ER stress, and optimization of the UPR can improve CHO cell production of therapeutic proteins. We compared the fed-batch growth, production characteristics, and transcriptomic response of an immunoglobulin G1 (IgG1) producer to its parental, non-producing host cell line. We conducted differential gene expression analysis using high throughput RNA sequencing (RNASeq) and quantitative polymerase chain reaction (qPCR) to study the ER stress response of each cell line during fed-batch culture. The UPR was activated in the IgG1 producer compared to the host cell line and our analysis of differential expression profiles indicated transient upregulation of ATF6α target mRNAs in the IgG1 producer, suggesting two upstream regulators of the ATF6 arm of the UPR, ATF6β and WFS1, are rational engineering targets. Although both ATF6β and WFS1 have been reported to negatively regulate ATF6α, this study shows knockdown of either target elicits different effects in an IgG1-producing CHO cell line. Stable knockdown of ATF6β decreased cell growth without decreasing titer; however, knockdown of WFS1 decreased titer without affecting growth. Relative expression measured by qPCR indicated no direct relationship between ATF6β and WFS1 expression, but upregulation of WFS1 in one pool was correlated with decreased growth and upregulation of ER chaperone mRNAs. While knockdown of WFS1 had negative impacts on UPR activation and product mRNA expression, knockdown of ATF6β improved the UPR specifically later in fed-batch leading to increased overall productivity.

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Section: Discussionmentioning

confidence: 99%

RNASeq highlights ATF6 pathway regulators for CHO cell engineering with different impacts of ATF6β and WFS1 knockdown on fed-batch production of IgG1

Rives,

Peak,

Blenner

2024

Sci Rep

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“…The introduction of miR-483 mimics will also significantly increase the production of antibodies in CHO cells ( Emmerling et al, 2016 ). Overexpression of miR-31* can increase IgG production and cell-specific productivity ( Martinez-Lopez et al, 2021 ). Overexpression of miR-219-1-3p and miR-3074-5p can increase the production of antibodies in high-yielding CHO cell lines.…”

Section: Effect Of Mirna On the Production Of Recombinant Proteinmentioning

confidence: 99%

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

Liu

Dong

Lin³

et al. 2022

Front. Bioeng. Biotechnol.

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Recombinant protein production by mammalian cells is the initial step in the manufacture of many therapeutic proteins. Chinese hamster ovary (CHO) cells are the most common host system to produce recombinant therapeutic proteins (RTPs). However, it is still challenging to maintain high productivity ensuring the good quality of RTPs produced by CHO cells. MicroRNAs(miRNAs) are short regulatory non-coding RNAs that can regulate cellular behavior and complex phenotypes. It has been found that miRNAs can enhance the expression level of recombinant proteins in CHO cells by promoting proliferation, resisting apoptosis, and regulating metabolism. miRNAs also can affect the quality of RTPs. In this review, we will discuss the effect and mechanism of miRNA on the expression level and quality of recombinant proteins in CHO cells.

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“…So far, just a few miRNAs, when overexpressed, were able to enhance recombinant protein production in CHO cells. These include miR-466h [21,22], miR-17, -19b and 92a [23], miR-557 and miR-1287 [24], miR-17 [25], miR-483 [26], miR-30 family [27], miR-2861 [28], miR-143 [29], miR-31 [30] and miR-200a [31]. On the other hand, downregulation or knock-out of miRNAs, such as miR-23b [32] or miR-744-3p [33], increased recombinant protein expression as well.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2021

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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.

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Transfection of miR-31* boosts oxidative phosphorylation metabolism in the mitochondria and enhances recombinant protein production in Chinese hamster ovary cells

Cited by 5 publications

References 76 publications

RNASeq highlights ATF6 pathway regulators for CHO cell engineering with different impacts of ATF6β and WFS1 knockdown on fed-batch production of IgG1

RNASeq highlights ATF6 pathway regulators for CHO cell engineering with different impacts of ATF6β and WFS1 knockdown on fed-batch production of IgG1

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

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

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