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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...

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Autores principales: van Asbeck, Alexander H., Dieker, Jürgen, Oude Egberink, Rik, van den Berg, Lennard, van der Vlag, Johan, Brock, Roland
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2021
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.
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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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