Enhanced protein production by microRNA-30 family in CHO cells is mediated by the modulation of the ubiquitin pathway

Fischer, Simon; Mathias, Sven; Schaz, Simone; Emmerling, Verena Vanessa; Buck, Theresa; Kleemann, Michael; Hackl, Matthias; Grillari, Johannes; Aschrafi, Armaz; Handrick, René; Otte, Kerstin

doi:10.1016/j.jbiotec.2015.08.002

Cited by 30 publications

(25 citation statements)

References 50 publications

(4 reference statements)

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“…Recent miRNA studies commonly enhanced q p of recombinant CHO cells by stable overexpression of miRNAs (cgr‐miR‐17, cgr‐miR‐19b, cgr‐miR‐20a, cgr‐miR‐92a, cgr‐miR‐2861, mmu‐miR‐30 family members, hsa‐miR‐7, hsa‐miR1287, hsa‐miR‐557) . In this context miR‐7 and miR‐30 had a negative impact on cell growth . Kelly et al depleted miR‐23 in recombinant CHO cells, thus increasing mitochondrial activity and q p .…”

Section: Discussionmentioning

confidence: 99%

Hyperosmotic stimulus study discloses benefits in ATP supply and reveals miRNA/mRNA targets to improve recombinant protein production of CHO cells

Pfizenmaier

Junghans

Teleki

2016

Biotechnology Journal

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Biopharmaceuticals are predominantly produced by Chinese hamster ovary (CHO) cells cultivated in fed-batch mode. Hyperosmotic culture conditions (≥ 350 mOsmol kg(∑1) ) resulting from feeding of nutrients may enhance specific product formation rates (qp ). As an improved ATP supply was anticipated to enhance qp this study focused on the identification of suitable miRNA/mRNA targets to increase ATP levels. Therefor next generation sequencing and a compartment specific metabolomics approach were applied to analyze the response of an antibody (mAB) producing CHO cell line upon osmotic shift (280 → 430 mOsmol kg(-1) ). Hyperosmotic culture conditions caused a ∼2.6-fold increase of specific ATP formation rates together with a ∼1.7-fold rise in cytosolic and mitochondrial ATP-pools, thus showing increased ATP supply. mRNA expression analysis identified several genes encoding glycosylated proteins with strictly tissue related function. In addition, hyperosmotic culture conditions induced an upregulation of miR-132-3p, miR-132-5p, miR-182, miR-183, miR-194, miR-215-3p, miR-215-5p which have all been related to cell cycle arrest/proliferation in cancer studies. In relation to a previous independent CHO study miR-183 may be the most promising target to enhance qp by stable overexpression. Furthermore, deletion of genes with presumably dispensable function in suspension growing CHO cells may enhance mAB formation by increased ATP levels.

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Section: Discussionmentioning

confidence: 99%

Hyperosmotic stimulus study discloses benefits in ATP supply and reveals miRNA/mRNA targets to improve recombinant protein production of CHO cells

Pfizenmaier

Junghans

Teleki

2016

Biotechnology Journal

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“…Based on their broad regulatory role within the cell, miRNAs have gained particular attention for host cell engineering to optimize bioprocess relevant cellular features. In this context, transient or stable overexpression of distinct miRNAs was demonstrated to boost the production of recombinant protein in CHO cells, as reported for Cricetulus griseus ( cgr) ‐miR‐17, cgr ‐miR‐19b, miR‐557/miR‐1287, miR‐2861, miR‐483 as well as for the entire miR‐30 family . The vast number of different miRNAs expressed by a particular cell type has prompted researchers to search for functional target miRNAs in CHO cells .…”

Section: Introductionmentioning

confidence: 94%

miR‐143 targets MAPK7 in CHO cells and induces a hyperproductive phenotype to enhance production of difficult‐to‐express proteins

Schoellhorn

Fischer

Wagner

et al. 2017

Biotechnology Progress

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In recent years, the number of complex but clinically effective biologicals such as multi-specific antibody formats and fusion proteins has increased dramatically. However, compared to classical monoclonal antibodies (mAbs), these rather artificially designed therapeutic proteins have never undergone millions of years of evolution and thus often turn out to be difficult-to-express using mammalian expression systems such as Chinese hamster ovary (CHO) cells. To provide access to these sophisticated but effective drugs, host cell engineering of CHO production cell lines represents a promising approach to overcome low production yields. MicroRNAs (miRNAs) have recently gained much attention as next-generation cell engineering tools. However, only very little is known about the capability of miRNAs to specifically increase production of difficult-to-express proteins. In a previous study we identified miR-143 amongst others to improve protein production in CHO cells. Thus, the aim of the present study was to examine if miR-143 might be suitable to improve production of low yield protein candidates. Both transient and stable overexpression of miR-143 significantly improved protein production without negatively affecting cell growth and viability of different recombinant CHO cells. In addition, mitogen-activated protein kinase 7 (MAPK7) was identified as a putative target gene of miR-143-3p in CHO cells. Finally, siRNA-mediated knock-down of MAPK7 could be demonstrated to phenocopy pro-productive effects of miR-143. In summary, our data suggest that miR-143 might represent a novel genetic element to enhance production of difficult-to-express proteins in CHO cells which may be partly mediated by down-regulation of MAPK7. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1046-1058, 2017.

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“…Furthermore, individual miRNAs are capable of regulating entire cellular pathways (Fischer et al, ; Hackl et al, ), and thus might have a substantial impact on CHO cell phenotypes. Currently available literature on miRNA research in CHO cells mostly focusses on the identification of miRNAs to improve recombinant protein production (Emmerling et al, ; Fischer et al, , ; Jadhav et al, ; Kelly et al, ; Loh et al, ), or cell growth (Barron et al, ; Druz et al, ; Sanchez et al, ), while other studies were dedicated to basic principles of miRNA biogenesis and function (Diendorfer et al, ; Fischer et al, ,; Hackl et al, , ; Hernandez Bort et al, ). We have previously identified miR‐557 to increase productivity of a recombinant easy‐to‐express mAb in CHO cells (Strotbek et al, ).…”

Section: Introductionmentioning

confidence: 99%

miRNA engineering of CHO cells facilitates production of difficult‐to‐express proteins and increases success in cell line development

Fischer

Marquart

Pieper

et al. 2017

Biotech & Bioengineering

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In recent years, coherent with growing biologics portfolios also the number of complex and thus difficult‐to‐express (DTE) therapeutic proteins has increased considerably. DTE proteins challenge bioprocess development and can include various therapeutic protein formats such as monoclonal antibodies (mAbs), multi‐specific affinity scaffolds (e.g., bispecific antibodies), cytokines, or fusion proteins. Hence, the availability of robust and versatile Chinese hamster ovary (CHO) host cell factories is fundamental for high‐yielding bioprocesses. MicroRNAs (miRNAs) have emerged as potent cell engineering tools to improve process performance of CHO manufacturing cell lines. However, there has not been any report demonstrating the impact of beneficial miRNAs on industrial cell line development (CLD) yet. To address this question, we established novel CHO host cells constitutively expressing a pro‐productive miRNA: miR‐557. Novel host cells were tested in two independent CLD campaigns using two different mAb candidates including a normal as well as a DTE antibody. Presence of miR‐557 significantly enhanced each process step during CLD in a product independent manner. Stable expression of miR‐557 increased the probability to identify high‐producing cell clones. Furthermore, production cell lines derived from miR‐557 expressing host cells exhibited significantly increased final product yields in fed‐batch cultivation processes without compromising product quality. Strikingly, cells co‐expressing miR‐557 and a DTE antibody achieved a twofold increase in product titer compared to clones co‐expressing a negative control miRNA. Thus, host cell engineering using miRNAs represents a promising tool to overcome limitations in industrial CLD especially with regard to DTE proteins. Biotechnol. Bioeng. 2017;114: 1495–1510. © 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.

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Enhanced protein production by microRNA-30 family in CHO cells is mediated by the modulation of the ubiquitin pathway

Cited by 30 publications

References 50 publications

Hyperosmotic stimulus study discloses benefits in ATP supply and reveals miRNA/mRNA targets to improve recombinant protein production of CHO cells

Hyperosmotic stimulus study discloses benefits in ATP supply and reveals miRNA/mRNA targets to improve recombinant protein production of CHO cells

miR‐143 targets MAPK7 in CHO cells and induces a hyperproductive phenotype to enhance production of difficult‐to‐express proteins

miRNA engineering of CHO cells facilitates production of difficult‐to‐express proteins and increases success in cell line development

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