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Identification of novel miRNA targets in CHO cell lines and characterization of their impact on protein N-glycosylation
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  • Ankita Singh,
  • Yuzhou Fan,
  • Selgin Cakal,
  • Thomas Amann,
  • Anders Hansen,
  • Nicole Borth,
  • Gyun Min Lee,
  • Helene Faustrup Kildegaard,
  • Mikael Andersen
Ankita Singh
Technical University of Denmark

Corresponding Author:[email protected]

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Yuzhou Fan
Technical University of Denmark
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Selgin Cakal
Technical University of Denmark
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Thomas Amann
Technical University of Denmark
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Anders Hansen
Technical University of Denmark
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Nicole Borth
BOKU
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Gyun Min Lee
KAIST
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Helene Faustrup Kildegaard
Novo Nordisk AS Gobal Research
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Mikael Andersen
Technical University of Denmark
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Abstract

CHO cell lines are a workhorse for the production of pharmaceutical proteins, but show some limitations in the variability and stability of N-glycosylation profiles. One promising approach to addressing this at the required systems-level is miRNA, which can regulate a large number of genes and have predictable targets. Herein, we first identified de novo 656 potential miRNAs in the CHO genome based on a combination of literature, database searching, and miRNA sequencing. We further sequenced mRNA from the same cultures, and used a combination of mRNA-miRNA correlation analysis, target prediction and literature searches to find miRNAs potentially targeting N-glycosylation. Our ten best miRNA candidates were subjected to miRNA overexpression, knockdown, or knock-out in CHO cell lines. Out of the ten candidates, four (miR-128, miR-34c, miR-30b, and miR-449a) showed positive effects on N-glycosylation and could be applied directly for CHO cell engineering. The fact that 40% of the screened targets had a desired effect, and the prediction of 656 miRNAs illustrates the massive potential of miRNA engineering in CHO.