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

Abstract

SignificancemRNA treatments represent an exciting approach to cure diseases that cannot be tackled with current therapeutics. However, the delivery of mRNA to target cells remains a challenge, but among the existing alternatives, lipid nanoparticles (LNPs) offer a promising answer to this. Here we determine the structure of LNPs encapsulating mRNA, consisting of a lipid mixture already evaluated in clinical trials. We show that the lipids are not homogeneously distributed across the LNP, and one of the lipids is localized mainly at its surface. The structural information enabled us to design LNPs that successfully modify intracellular protein production in two clinically relevant cell types. Our findings and approach provide a framework for understanding and optimizing vehicles for mRNA delivery.

Deconvoluting Lipid Nanoparticle Structure for Messenger RNA Delivery

“…Interestingly, Sito and Stig LNPs formed faceted structures, suggesting the formation of two-dimensional crystals. 8 Camp LNPs, however, show a predominantly smooth curvature (much like the previously proposed "onion" structure). 16 Chol LNPs have a small fraction of particles with internal defects (8%) but hardly any multilamellarity.…”

“…However, the question remains whether or not the lessons learned from the improvements of the LNP structure for siRNA delivery apply to the mRNA due to its larger size (10 3 nucleotides). 1 Evidence suggests that the incorporation of mRNA can rearrange the LNP organization, causing the formation of inverted-hexagonal nanostructures, 8 unlike siRNA that is confined in lamellar nanostructures by ionizable lipids. 9 This is further complicated by the multidimensional nature of both LNP lipid formulations and the RNA nucleotide composition.…”

“…Upon further inspection of Sito LNPs with a cryo-transmission electron microscope (cryo-TEM), we found that Sito LNPs have a faceted surface, as opposed to spherical Chol LNPs with smooth curvature, likely due to phase separation of comprising lipids, 11 and potentially, the formation of two-dimensional lipid crystals in the LNP membrane. 8 In this study, we investigated the structure of LNPs containing naturally occurring phytosterols (namely, βsitosterol (Sito), fucosterol (Fuco), campesterol (Camp), and This article is made available via the ACS COVID-19 subset for unrestricted RESEARCH re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic.…”

ObLiGaRe doxycycline Inducible (ODIn) Cas9 system driving pre-clinical drug discovery, from design to cancer treatment

“…LNPs were manufactured using microfluidic mixing as described previously by Cullis [46, 47]. In brief, stocks of lipids were dissolved in ethanol to obtain a lipid concentration of 1-2 mM.…”

Using macropinocytosis for intracellular delivery of therapeutic nucleic acids to tumour cells

“…dilinoleylmethyl-4-dimethyl- [42]) helper lipids (1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesterol) and a PEG-based lipid (DMG-PEG). This system was originally developed for intravenous siRNA delivery to hepatocytes but has since been applied to deliver multiple types of nucleic acids (DNA [43], mRNA [44]) using multiple administration routes [45]. Dlin-X-DMA-based LNPs are now in late-stage clinical development and approaching commercialization [46].…”