Intracellular Routing and Recognition of Lipid-Based mRNA Nanoparticles

Abstract

Messenger RNA (mRNA) is being extensively used in gene therapy and vaccination due to its safety over DNA, in the following ways: its lack of integration risk, cytoplasmic expression, and transient expression compatible with fine regulations. However, clinical applications of mRNA are limited by its fast degradation by nucleases, and the activation of detrimental immune responses. Advances in mRNA applications, with the recent approval of COVID-19 vaccines, were fueled by optimization of the mRNA sequence and the development of mRNA delivery systems. Although delivery systems and mRNA sequence optimization have been abundantly reviewed, understanding of the intracellular processing of mRNA is mandatory to improve its applications. We will focus on lipid nanoparticles (LNPs) as they are the most advanced nanocarriers for the delivery of mRNA. Here, we will review how mRNA therapeutic potency can be affected by its interactions with cellular proteins and intracellular distribution.

Keywords: intracellular routing; lipid-based nanoparticles; mRNA delivery.

Figure 1

Critical parameter to consider regarding LNPs formulation and cellular interactions. To perform efficient transfection, LNPs should protect mRNA with ionizable lipid until its final destination and should be able to release it efficiently. Surface functionalization through incorporation of targeted lipid will help to reach specific organ/cells while PEGylated lipid will help the circulation of particles in vivo. Finally, incorporation of helper lipids will help either the formation of LNPs or its interaction with biological membrane. Altogether, those lipids need to trigger an efficient cellular uptake of the particle, which leads to the cytosol and avoids the lysosome, without triggering deleterious cell sensors, so that mRNA can lead to an efficient protein production in the cell.

Figure 2

Principal modes of endocytosis, reproduced from [39], MDPI, 2020, under creative common license.

Figure 3

Flow cytometry study of mRNA NP uptake and localization. (A) Schematic representation of uptake study process. Cell-associated fluorescence intensity (MFI: mean fluorescence intensity) was measured in PBS (total florescence) after TB treatment (intra-cellular fluorescence) and after monensin treatment (fluorescence recovery in acidic compartments). (B) Histograms showing the effects of different treatments on recorded MFI. (C–E) corresponding to the cell surface-associated fluorescence, the intracellular fluorescence and the recovery of quenched fluorescence expressed and monensin increase. Shown are unpublished results corresponding to murine dendritic (DC2.4) cells incubated for 4 h with cationic mRNA–lipopolyplexes (LPR C) or cationic mRNA–lipopolyplexes bearing a trimannose ligand (LPR C-TM), without serum.

Figure 4

Improvement in mRNA expression in vivo by tuning the intracellular degradation. (A) Structures of the original lipid (ssPalmO), the lipid with a degradable phenyl ester (ssPalmO-Phe), and the lipid with a non-degradable benzyl ester (ssPalmO-Ben); (B) LNPs prepared with EPO mRNA were intravenously injected in mice (0.05 mg kg⁻¹) before determination of blood EPO concentration by ELISA 24 h after injection (** p < 0.01). Reproduced from [73], Wiley, 2020, under creative common license.

Figure 5

Late endosomes are essential for mRNA transfection wild-type (WT) and HAP1 cells with deleted Rab7 (Rab7-KO) were transfected with luciferase mRNA lipoplexes. A clear decrease in luciferase expression in Rab7-KO cells was evidenced. Adapted with permission from [88]. Copyright (2021) American Chemical Society. (* 0.05 ≥ p > 0.01, ** 0.01 ≥ p > 0.005, *** 0.005 ≥ p).

Figure 6

β-sitosterol enhanced mRNA-based gene transfection. (A) The structure of cholesterol and β-sitosterol. (B) Particles made of cholesterol or β-sitosterol were screened for size (nm), mRNA encapsulation (percent) and transfection efficiency (200 ng of mRNA). (C) Endosomal escape was visualized using smFISH. Representative fluorescent images showing mRNA, LNPs, and image analysis after delivery with LNPs (control) or eLNP (containing C-24 alkyl phytosterols) in HeLa cells. Adapted from Patel et al. [77], Nature Portfolio, 2020, under creative common license.

Figure 7

Parameter estimation of single-cell expression time courses using the translation-maturation model. Protein adsorption on the nanocarrier’s surface has the opposite effect on ionizable lipid nanoparticles i-LNPs compared to lipoplexes. Scatterplots of expression onset time t0 vs. expression rate m0kTL of lipoplexes (left) and i-LNPs (right) with (orange) and without serum (blue). The arrows indicate the opposite effect on transfection efficiency and timing induced by addition of FBS. Each data point corresponds to a single cell. The median value is indicated as full dot. Adapted from Reiser et al., [113], Oxford University Press, 2019, under creative common license.

Figure 8

Co-transfection of osteoprogenitor cells with BMP2 mRNA and NS1 mRNA resulted in decreasing type I interferon response together with enhanced therapeutic BMP2 mRNA expression. Adapted from [132], Elsevier, 2021, with permission. ** represents p < 0.01.