Boosting Intracellular Delivery of Lipid Nanoparticle-Encapsulated mRNA

Intracellular
delivery of mRNA holds great potential for vaccine− and therapeutic discovery and development.
Despite increasing recognition of the utility of lipid-based nanoparticles
(LNPs) for intracellular delivery of mRNA, particle engineering is
hindered by insufficient understanding of endosomal escape, which
is believed to be a main limiter of cytosolic availability and activity
of the nucleic acid inside the cell. Using a series of CRISPR-based
genetic perturbations of the lysosomal pathway, we have identified
that late endosome/lysosome (LE/Ly) formation is essential for functional
delivery of exogenously presented mRNA. Lysosomes provide a spatiotemporal
hub to orchestrate mTOR signaling and are known to control cell proliferation,
nutrient sensing, ribosomal biogenesis, and mRNA translation. Through
modulation of the mTOR pathway we were able to enhance or inhibit
LNP-mediated mRNA delivery. To further boost intracellular delivery
of mRNA, we screened 212 bioactive lipid-like molecules that are either
enriched in vesicular compartments or modulate cell signaling. Surprisingly,
we have discovered that leukotriene-antagonists, clinically approved
for treatment of asthma and other lung diseases, enhance intracellular
mRNA delivery in vitro (over 3-fold, p < 0.005)
and in vivo (over 2-fold, p < 0.005). Understanding
LNP-mediated intracellular delivery will inspire the next generation
of RNA therapeutics that have high potency and limited toxicity.