Delivery of exogenous mRNA using lipid nanoparticles (LNPs) is a promising strategy for therapeutics. However, a bottleneck remains in the poor understanding of the parameters that correlate with endosomal escape versus cytotoxicity. To address this problem, we compared the endosomal distribution of six LNP-mRNA formulations of diverse chemical composition and efficacy, similar to those used in mRNA-based vaccines, in primary human adipocytes, fibroblasts, and HeLa cells. Surprisingly, we found that total uptake is not a sufficient predictor of delivery, and different LNPs vary considerably in endosomal distributions. Prolonged uptake impaired endosomal acidification, a sign of cytotoxicity, and caused mRNA to accumulate in compartments defective in cargo transport and unproductive for delivery. In contrast, early endocytic/recycling compartments have the highest probability for mRNA escape. By using super-resolution microscopy, we could resolve a single LNP-mRNA within subendosomal compartments and capture events of mRNA escape from endosomal recycling tubules. Our results change the view of the mechanisms of endosomal escape and define quantitative parameters to guide the development of mRNA formulations toward higher efficacy and lower cytotoxicity.
Lipid nanoparticles (LNPs) are the most clinically advanced delivery system for RNA-based drugs but have predominantly been investigated for intravenous and intramuscular administration. Subcutaneous administration opens the possibility of patient self-administration and hence long-term chronic treatment that could enable messenger RNA (mRNA) to be used as a novel modality for protein replacement or regenerative therapies. In this study, we show that subcutaneous administration of mRNA formulated within LNPs can result in measurable plasma exposure of a secreted protein. However, subcutaneous administration of mRNA formulated within LNPs was observed to be associated with dose-limiting inflammatory responses. To overcome this limitation, we investigated the concept of incorporating aliphatic ester prodrugs of anti-inflammatory steroids within LNPs, i.e., functionalized LNPs to suppress the inflammatory response. We show that the effectiveness of this approach depends on the alkyl chain length of the ester prodrug, which determines its retention at the site of administration. An unexpected additional benefit to this approach is the prolongation observed in the duration of protein expression. Our results demonstrate that subcutaneous administration of mRNA formulated in functionalized LNPs is a viable approach to achieving systemic levels of therapeutic proteins, which has the added benefits of being amenable to self-administration when chronic treatment is required.
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