Studying how administration route and dose regulates antibody generation against LNPs for mRNA delivery with single-particle resolution

Following the recent approval of both siRNA- and mRNA-based therapeutics, nucleic acid therapies are considered a game changer in medicine. Their envisioned widespread use for many therapeutic applications with an array of cellular target sites means that various administration routes will be employed. Concerns exist regarding adverse reactions against the lipid nanoparticles (LNPs) used for mRNA delivery, as PEG coatings on nanoparticles can induce severe antibody-mediated immune reactions, potentially being boosted by the inherently immunogenic nucleic acid cargo. While exhaustive information is available on how physicochemical features of nanoparticles affects immunogenicity, it remains unexplored how the fundamental choice of administration route regulates anti-particle immunity. Here, we directly compared antibody generation against PEGylated mRNA-carrying LNPs administered by the intravenous, intramuscular, or subcutaneous route, using a novel sophisticated assay capable of measuring antibody binding to authentic LNP surfaces with single-particle resolution. Intramuscular injections in mice were found to generate overall low and dose-independent levels of anti-LNP antibodies, while both intravenous and subcutaneous LNP injections generated substantial and highly dose-dependent levels. These findings demonstrate that before LNP-based mRNA medicines can be safely applied to new therapeutic applications, it will be crucial to carefully consider the choice of administration route.