Mild Innate Immune Activation Overrides Efficient Nanoparticle‐Mediated RNA Delivery

Clinical mRNA delivery remains challenging, in large part because how physiology alters delivery in vivo remains underexplored. For example, mRNA delivered by lipid nanoparticles (LNPs) is being considered to treat inflammation, but whether inflammation itself changes delivery remains understudied. Relationships between immunity, endocytosis, and mRNA translation lead to hypothesize that toll‐like receptor 4 (TLR4) activation reduced LNP‐mediated mRNA delivery. Therefore, LNP uptake, endosomal escape, and mRNA translation with and without TLR4 activation are quantified. In vivo DNA barcoding is used to discover a novel LNP that delivers mRNA to Kupffer cells at clinical doses; unlike most LNPs, this LNP does not preferentially target hepatocytes. TLR4 activation blocks mRNA translation in all tested cell types, without reducing LNP uptake; inhibiting TLR4 or its downstream effector protein kinase R improved delivery. The discrepant effects of TLR4 on i) LNP uptake and ii) translation suggests TLR4 activation can “override” LNP targeting, even after mRNA is delivered into target cells. Given near‐future clinical trials using mRNA to modulate inflammation, this highlights the need to understand inflammatory signaling in on‐ and off‐target cells. More generally, this suggests an LNP which delivers mRNA to one inflammatory disease may not deliver mRNA to another. Lipid nanoparticle (LNP)‐mediated mRNA delivery is being considered to treat inflammation, but whether inflammation itself changes delivery remains unclear. Relationships between immunity and endocytosis lead to the hypothesis that toll‐like receptor 4 (TLR4) affects LNP delivery. It is found that low doses of a TLR4 agonist reduce mRNA delivery, illustrating the need to understand how dysregulated cell signaling affects nanoparticle delivery.