Author: van Asbeck, Alexander H.; Dieker, Jürgen; Oude Egberink, Rik; van den Berg, Lennard; van der Vlag, Johan; Brock, Roland
Title: Protein Expression Correlates Linearly with mRNA Dose over Up to Five Orders of Magnitude In Vitro and In Vivo Cord-id: 54c652xr Document date: 2021_5_5
ID: 54c652xr
Snippet: 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 c
Document: 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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