Identification of novel miRNA targets in CHO cell lines and characterization of their impact on protein N-glycosylation

Singh, Ankita; Fan, Yuzhou; Cakal, Selgin D.; Amann, Thomas; Hansen, Anders Højgaard; Borth, Nicole; Lee, Gyun Min; Kildegaard, Helene Faustrup; Andersen, Mikael Rørdam

doi:10.22541/au.164167102.22702519/v1

Cited by 3 publications

(1 citation statement)

References 42 publications

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“…Therefore, the use of miRNAs as engineering tools can be challenging, but offers the opportunity to control entire cellular pathways without any energy‐consuming translational burden [ 15 ]. Therefore, miRNA‐associated engineering approaches were established as efficient tools in CHO cell line engineering for the improvement of productivity, fine‐tuning of product quality, the enhancement of cell growth and survival, and the modulation of metabolic pathways [ 3 , 6 , 8 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 ]. However, quality‐relevant characteristics of therapeutic proteins like antibody fucosylation have so far been engineered using standard techniques like knock‐out of genes [ 5 , 31 , 32 , 33 , 34 ], siRNA mediated transient gene knock‐down [ 27 , 35 , 36 ] or miRNA‐mediated transient gene knock‐down [ 37 ] only.…”

Section: Introductionmentioning

confidence: 99%

Stable overexpression of native and artificial miRNAs for the production of differentially fucosylated antibodies in CHO cells

Schlossbauer,

Naumann,

Klingler

et al. 2024

Engineering in Life Sciences

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Cell engineering strategies typically rely on energy‐consuming overexpression of genes or radical gene‐knock out. Both strategies are not particularly convenient for the generation of slightly modulated phenotypes, as needed in biosimilar development of for example differentially fucosylated monoclonal antibodies (mAbs). Recently, transiently transfected small noncoding microRNAs (miRNAs), known to be regulators of entire gene networks, have emerged as potent fucosylation modulators in Chinese hamster ovary (CHO) production cells. Here, we demonstrate the applicability of stable miRNA overexpression in CHO production cells to adjust the fucosylation pattern of mAbs as a model phenotype. For this purpose, we applied a miRNA chaining strategy to achieve adjustability of fucosylation in stable cell pools. In addition, we were able to implement recently developed artificial miRNAs (amiRNAs) based on native miRNA sequences into a stable CHO expression system to even further fine‐tune fucosylation regulation. Our results demonstrate the potential of miRNAs as a versatile tool to control mAb fucosylation in CHO production cells without adverse side effects on important process parameters.

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Section: Introductionmentioning

confidence: 99%

Stable overexpression of native and artificial miRNAs for the production of differentially fucosylated antibodies in CHO cells

Schlossbauer,

Naumann,

Klingler

et al. 2024

Engineering in Life Sciences

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Overexpression of miR-32 in Chinese hamster ovary cells increases production of Fc-fusion protein

et al. 2023

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The demand for industrial genetically modified host cells were increased with the growth of the biopharmaceutical market. Numerous studies on improving host cell productivity have shown that altering host cell growth and viability through genetic engineering can increase recombinant protein production. During the last decades, it was demonstrated that overexpression or downregulation of some microRNAs in Chinese Hamster Ovary (CHO) cells as the host cell in biopharmaceutical manufacturing, can improve their productivity. The selection of microRNA targets has been based on their previously identified role in human cancers. MicroRNA-32 (miR-32), which is conserved between humans and hamsters (Crisetulus griseus), was shown to play a role in the regulation of cell proliferation and apoptosis in some human cancers. In this study, we investigated the effect of miR-32 overexpression on the productivity of CHO-VEGF-trap cells. Our results indicated that stable overexpression of miR-32 could dramatically increase the productivity of CHO cells by 1.8-fold. It also significantly increases cell viability, batch culture longevity, and cell growth. To achieve these results, following the construction of a single clone producing an Fc-fusion protein, we transfected cells with a pLexJRed-miR-32 plasmid to stably produce the microRNA and evaluate the impact of mir-32 overexpression on cell productivity, growth and viability in compare with scrambled control. Our findings highlight the application of miRNAs as engineering tools and indicated that miR-32 could be a target for engineering CHO cells to increase cell productivity.

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Overexpression of MiR-32 in Chinese Hamster Ovary Cells Increases Production of Fc-fusion Protein

Bazaz

Abdolzadeh

Azizi

et al. 2023

Preprint

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The demand for industrial genetically modified host cells has been increased with the growth of the biopharmaceutical market. Numerous studies on improving host cell productivity have shown that altering host cell growth and viability through genetic engineering can increase recombinant protein production. During the last decades, it has been demonstrated that overexpression or downregulation of some microRNAs in Chinese Hamster Ovary (CHO) cells as the most often employed host cell in biopharmaceutical manufacturing, can improve their productivity. MicroRNAs are small non-coding RNAs that play a key role in controlling cellular mechanisms through their binding to different mRNA targets and negatively regulating gene expression. In efforts to increase the host cell's productivity through microRNA engineering of the cells, some microRNA targets have been selected based on their previously identified role in human cancers. MicroRNA-32 (miR-32), which is conserved between humans and hamsters (Crisetulus griseus), has been shown to play a role in the regulation of cell proliferation and apoptosis in some human cancers. In this study, we investigated the effect of miR-32 overexpression on the productivity of CHO-VEGF-trap cells. Our results indicated that stable overexpression of miR-32 could dramatically increase the productivity of CHO cells by 1.8-fold. It also significantly increases cell viability, batch culture longevity, and cell growth. To achieve these results, following the construction of a single clone producing an Fc-fusion protein, we transfected cells with a pLexJRed-miR-32 plasmid to stably produce the microRNA and evaluate the impact of mir-32 overexcretion on cell productivity, growth and viability in compare with scrambled control. Our findings highlight the application of miRNAs as CHO cell engineering tools and indicated that miR-32 could be a target for engineering CHO cells to increase cell productivity.

show abstract

Identification of novel miRNA targets in CHO cell lines and characterization of their impact on protein N-glycosylation

Cited by 3 publications

References 42 publications

Stable overexpression of native and artificial miRNAs for the production of differentially fucosylated antibodies in CHO cells

Stable overexpression of native and artificial miRNAs for the production of differentially fucosylated antibodies in CHO cells

Overexpression of miR-32 in Chinese hamster ovary cells increases production of Fc-fusion protein

Overexpression of MiR-32 in Chinese Hamster Ovary Cells Increases Production of Fc-fusion Protein

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