Mild Innate Immune Activation Overrides Efficient Nanoparticle-Mediated RNA Delivery

Area of Science:

• Biomedical Engineering
• Immunology
• Molecular Biology

Background:

• Clinical mRNA delivery using lipid nanoparticles (LNPs) faces challenges due to underexplored physiological influences.
• Inflammation's impact on LNP-mediated mRNA delivery, particularly for treating inflammatory diseases, remains understudied.

Purpose of the Study:

• To investigate how toll-like receptor 4 (TLR4) activation, a key inflammatory pathway, affects LNP-mediated mRNA delivery.
• To quantify LNP uptake, endosomal escape, and mRNA translation under conditions of TLR4 activation.

Main Methods:

• Utilized in vivo DNA barcoding to identify novel LNPs targeting Kupffer cells at clinical doses.
• Quantified LNP uptake, endosomal escape, and mRNA translation with and without TLR4 activation.
• Assessed the impact of inhibiting TLR4 or its downstream effector, protein kinase R, on mRNA delivery.

Main Results:

• TLR4 activation significantly blocked mRNA translation in all tested cell types without reducing LNP uptake.
• A novel LNP was identified that delivers mRNA to Kupffer cells, distinct from hepatocyte-targeting LNPs.
• Inhibition of TLR4 or protein kinase R restored mRNA translation, improving LNP-mediated delivery.

Conclusions:

• TLR4 activation can override LNP targeting by blocking mRNA translation post-uptake, impacting therapeutic efficacy.
• Understanding inflammatory signaling is crucial for optimizing mRNA delivery strategies in clinical trials for inflammatory diseases.
• The efficacy of an LNP for one inflammatory condition may not translate to another due to differing inflammatory signaling contexts.