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Protein Expression Correlates Linearly with mRNA Dose over Up to Five Orders of Magnitude In Vitro and In Vivo
Messenger RNA is rapidly gaining significance as a therapeutic modality. Here, we address the dependence of dose–response functions on the type of delivery vehicle, cell line, and incubation time. Knowledge of these characteristics is crucial for the application of mRNA. As delivery vehicles, a lipi...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8148180/ https://www.ncbi.nlm.nih.gov/pubmed/34063094 http://dx.doi.org/10.3390/biomedicines9050511 |
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author | van Asbeck, Alexander H. Dieker, Jürgen Oude Egberink, Rik van den Berg, Lennard van der Vlag, Johan Brock, Roland |
author_facet | van Asbeck, Alexander H. Dieker, Jürgen Oude Egberink, Rik van den Berg, Lennard van der Vlag, Johan Brock, Roland |
author_sort | van Asbeck, Alexander H. |
collection | PubMed |
description | Messenger RNA is rapidly gaining significance as a therapeutic modality. Here, we address the dependence of dose–response functions on the type of delivery vehicle, cell line, and incubation time. Knowledge of these characteristics is crucial for the application of mRNA. As delivery vehicles, a lipid-based formulation and the cell-penetrating peptide Pepfect14 (PF14) were employed. As cell lines, we included a glomerular endothelial cell line (mGEnC) as a model for differentiated cells, HeLa cells, and SKOV-3 ovarian carcinoma cells. Uptake and expression were detected by flow cytometry, using a Cy5-labelled mRNA coding for enhanced green fluorescent protein (EGFP). There was a linear correlation of dose, uptake, and expression, and this correlation was maintained for over up to 72 h. Through application of a multistep kinetic model, we show that differences in expression levels can already be explained by the number of mRNAs packaged per delivery vehicle. Using luciferase as a reporter protein, linearity of expression was observed over 5 orders of magnitude in vitro and 3 orders of magnitude in vivo. Overall, the results demonstrate that mRNA provides excellent quantitative control over protein expression, also over extended periods of time. |
format | Online Article Text |
id | pubmed-8148180 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-81481802021-05-26 Protein Expression Correlates Linearly with mRNA Dose over Up to Five Orders of Magnitude In Vitro and In Vivo van Asbeck, Alexander H. Dieker, Jürgen Oude Egberink, Rik van den Berg, Lennard van der Vlag, Johan Brock, Roland Biomedicines Article Messenger RNA is rapidly gaining significance as a therapeutic modality. Here, we address the dependence of dose–response functions on the type of delivery vehicle, cell line, and incubation time. Knowledge of these characteristics is crucial for the application of mRNA. As delivery vehicles, a lipid-based formulation and the cell-penetrating peptide Pepfect14 (PF14) were employed. As cell lines, we included a glomerular endothelial cell line (mGEnC) as a model for differentiated cells, HeLa cells, and SKOV-3 ovarian carcinoma cells. Uptake and expression were detected by flow cytometry, using a Cy5-labelled mRNA coding for enhanced green fluorescent protein (EGFP). There was a linear correlation of dose, uptake, and expression, and this correlation was maintained for over up to 72 h. Through application of a multistep kinetic model, we show that differences in expression levels can already be explained by the number of mRNAs packaged per delivery vehicle. Using luciferase as a reporter protein, linearity of expression was observed over 5 orders of magnitude in vitro and 3 orders of magnitude in vivo. Overall, the results demonstrate that mRNA provides excellent quantitative control over protein expression, also over extended periods of time. MDPI 2021-05-05 /pmc/articles/PMC8148180/ /pubmed/34063094 http://dx.doi.org/10.3390/biomedicines9050511 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 van Asbeck, Alexander H. Dieker, Jürgen Oude Egberink, Rik van den Berg, Lennard van der Vlag, Johan Brock, Roland Protein Expression Correlates Linearly with mRNA Dose over Up to Five Orders of Magnitude In Vitro and In Vivo |
title | Protein Expression Correlates Linearly with mRNA Dose over Up to Five Orders of Magnitude In Vitro and In Vivo |
title_full | Protein Expression Correlates Linearly with mRNA Dose over Up to Five Orders of Magnitude In Vitro and In Vivo |
title_fullStr | Protein Expression Correlates Linearly with mRNA Dose over Up to Five Orders of Magnitude In Vitro and In Vivo |
title_full_unstemmed | Protein Expression Correlates Linearly with mRNA Dose over Up to Five Orders of Magnitude In Vitro and In Vivo |
title_short | Protein Expression Correlates Linearly with mRNA Dose over Up to Five Orders of Magnitude In Vitro and In Vivo |
title_sort | protein expression correlates linearly with mrna dose over up to five orders of magnitude in vitro and in vivo |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8148180/ https://www.ncbi.nlm.nih.gov/pubmed/34063094 http://dx.doi.org/10.3390/biomedicines9050511 |
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