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Correlation of mRNA delivery timing and protein expression in lipid-based transfection

A. Reiser, D. Woschée, N. Mehrotra, R. Krzysztoń, View ORCID ProfileH. H. Strey, J. O. Rädler
doi: https://doi.org/10.1101/607986
A. Reiser
1Faculty of Physics and Center for NanoScience, Ludwig-Maximilians-University, Geschwister-Scholl-Platz 1, 80539 Munich, Germany
2Graduate School of Quantitative Biosciences (QBM), Ludwig-Maximilians-University, Fedor-Lynen-Straße 25, 81377 Munich, Germany
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D. Woschée
1Faculty of Physics and Center for NanoScience, Ludwig-Maximilians-University, Geschwister-Scholl-Platz 1, 80539 Munich, Germany
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N. Mehrotra
1Faculty of Physics and Center for NanoScience, Ludwig-Maximilians-University, Geschwister-Scholl-Platz 1, 80539 Munich, Germany
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R. Krzysztoń
1Faculty of Physics and Center for NanoScience, Ludwig-Maximilians-University, Geschwister-Scholl-Platz 1, 80539 Munich, Germany
2Graduate School of Quantitative Biosciences (QBM), Ludwig-Maximilians-University, Fedor-Lynen-Straße 25, 81377 Munich, Germany
3Department of Biomedical Engineering and Laufer Center for Quantitative Biology, Stony Brook University, Stony Brook, NY 11794, USA
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H. H. Strey
3Department of Biomedical Engineering and Laufer Center for Quantitative Biology, Stony Brook University, Stony Brook, NY 11794, USA
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  • ORCID record for H. H. Strey
J. O. Rädler
1Faculty of Physics and Center for NanoScience, Ludwig-Maximilians-University, Geschwister-Scholl-Platz 1, 80539 Munich, Germany
2Graduate School of Quantitative Biosciences (QBM), Ludwig-Maximilians-University, Fedor-Lynen-Straße 25, 81377 Munich, Germany
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  • For correspondence: raedler@lmu.de
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Abstract

Non-viral gene delivery is constrained by the dwell time that most synthetic nucleic acid nanocarriers spend inside endosomal compartments. In order to overcome this endosomal-release bottleneck, methods are required that measure nanocarrier uptake kinetics and transfection efficiency simultaneously. Here, we employ live-cell imaging on single-cell arrays (LISCA) to study the delivery-time distribution of lipid-based mRNA complexes under varied serum conditions. By fitting a translation-maturation model to hundreds of individual eGFP reporter fluorescence time courses, the protein expression onset times and the expression rates after transfection are determined. Using this approach, we find that delivery timing and protein expression rates are not intrinsically correlated at the single-cell level, even though population-averaged values of both parameters conjointly change as a function of increasing external serum protein fraction. Lipofectamine mediated delivery showed decreased transfection efficiency and longer delivery times with increasing serum protein concentration. This is in contrast to ionizable lipid nanoparticles (LNPs) mediated transfer, which showed increased efficiency and faster uptake in the presence of serum. In conclusion, the interdependences of single-cell expression rates and onset timing provide additional clues on uptake and release mechanisms, which are useful for improving nucleic acid delivery.

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Posted April 13, 2019.
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Correlation of mRNA delivery timing and protein expression in lipid-based transfection
A. Reiser, D. Woschée, N. Mehrotra, R. Krzysztoń, H. H. Strey, J. O. Rädler
bioRxiv 607986; doi: https://doi.org/10.1101/607986
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Correlation of mRNA delivery timing and protein expression in lipid-based transfection
A. Reiser, D. Woschée, N. Mehrotra, R. Krzysztoń, H. H. Strey, J. O. Rädler
bioRxiv 607986; doi: https://doi.org/10.1101/607986

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