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Successful reprogramming of cellular protein production through mRNA delivered by functionalized lipid nanoparticles

Journal article
Authors Marianna Yanez Arteta
Tomas Kjellman
Stefano Bartesaghi
Simonetta Wallin
Xiaoqiu Wu
Alexander J. Kvist
Aleksandra Dabkowska
Noémi Székely
Aurel Radulescu
Johan Bergenholtz
Lennart Lindfors
Published in Proceedings of the National Academy of Sciences of the United States of America
Volume 115
Issue 15
Pages E3351-E3360
ISSN 00278424
Publication year 2018
Published at Department of Chemistry and Molecular Biology
Pages E3351-E3360
Language en
Links https://doi.org/10.1073/pnas.172054...
www.pnas.org/content/pnas/115/15/E3...
Keywords Adipocytes, Gene therapy, Hepatocytes, HEPO mRNA, Small-angle scattering
Subject categories Physical Chemistry, Surface and colloid chemistry, Pharmaceutical Sciences, Biophysical chemistry

Abstract

© 2018 National Academy of Sciences. All Rights Reserved. The development of safe and efficacious gene vectors has limited greatly the potential for therapeutic treatments based on messenger RNA (mRNA). Lipid nanoparticles (LNPs) formed by an ionizable cationic lipid (here DLin-MC3-DMA), helper lipids (distearoylphos-phatidylcholine, DSPC, and cholesterol), and a poly(ethylene glycol) (PEG) lipid have been identified as very promising delivery ve ctors of short interfering RNA (siRNA) in different clinical phases; however, delivery of high-molecular weight RNA has been proven much more demanding. Herein we elucidate the structure of hEPO modified mRNA-containing LNPs of different sizes and show how structural differences affect transfection of human adipocytes and hepatocytes, two clinically relevant cell types. Employing small-angle scattering, we demonstrate that LNPs have a disordered inverse hexagonal internal structure with a characteristic distance around 6 nm in presence of mRNA, whereas LNPs containing no mRNA do not display this structure. Furthermore, using contrast variation small-angle neutron scattering, we show that one of the lipid components, DSPC, is localized mainly at the surface of mRNA-containing LNPs. By varying LNP size and surface composition we demonstrate that both size and structure have significant influence on intracellular protein production. As an example, in both human adipocytes and hepatocytes, protein expression levels for 130 nm LNPs can differ as much as 50-fold depending on their surface characteristics, likely due to a difference in the ability of LNP fusion with the early endosome membrane. We consider these discoveries to be fundamental and opening up new possibilities for rational design of synthetic nanoscopic vehicles for mRNA delivery.

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