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

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 d...

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Autores principales: Švab, Živa, Braga, Luca, Guarnaccia, Corrado, Labik, Ivan, Herzog, Jeremias, Baralle, Marco, Giacca, Mauro, Skoko, Nataša
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8392531/
https://www.ncbi.nlm.nih.gov/pubmed/34439791
http://dx.doi.org/10.3390/biom11081125
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author Švab, Živa
Braga, Luca
Guarnaccia, Corrado
Labik, Ivan
Herzog, Jeremias
Baralle, Marco
Giacca, Mauro
Skoko, Nataša
author_facet Švab, Živa
Braga, Luca
Guarnaccia, Corrado
Labik, Ivan
Herzog, Jeremias
Baralle, Marco
Giacca, Mauro
Skoko, Nataša
author_sort Švab, Živa
collection PubMed
description 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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spelling pubmed-83925312021-08-28 High Throughput miRNA Screening Identifies miR-574-3p Hyperproductive Effect in CHO Cells Švab, Živa Braga, Luca Guarnaccia, Corrado Labik, Ivan Herzog, Jeremias Baralle, Marco Giacca, Mauro Skoko, Nataša Biomolecules Article 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. MDPI 2021-07-30 /pmc/articles/PMC8392531/ /pubmed/34439791 http://dx.doi.org/10.3390/biom11081125 Text en © 2021 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Švab, Živa
Braga, Luca
Guarnaccia, Corrado
Labik, Ivan
Herzog, Jeremias
Baralle, Marco
Giacca, Mauro
Skoko, Nataša
High Throughput miRNA Screening Identifies miR-574-3p Hyperproductive Effect in CHO Cells
title High Throughput miRNA Screening Identifies miR-574-3p Hyperproductive Effect in CHO Cells
title_full High Throughput miRNA Screening Identifies miR-574-3p Hyperproductive Effect in CHO Cells
title_fullStr High Throughput miRNA Screening Identifies miR-574-3p Hyperproductive Effect in CHO Cells
title_full_unstemmed High Throughput miRNA Screening Identifies miR-574-3p Hyperproductive Effect in CHO Cells
title_short High Throughput miRNA Screening Identifies miR-574-3p Hyperproductive Effect in CHO Cells
title_sort high throughput mirna screening identifies mir-574-3p hyperproductive effect in cho cells
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8392531/
https://www.ncbi.nlm.nih.gov/pubmed/34439791
http://dx.doi.org/10.3390/biom11081125
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