(353c) Lipid Nanoparticle- Mediated Delivery of Chemically Modified mRNA Significantly Enhances Protein Expression in Mice

Therapeutic messenger RNA (mRNA) delivery can treat or prevent many diseases by inducing expression of specific proteins in target cells. The versatility of mRNA-based drugs has made it a leading gene therapy platform, with dozens of studies utilizing it for applications such as such as protein replacement, prophylactic vaccines, cancer immunotherapy, gene editing, and cellular reprogramming. However, the clinical translation of mRNA drugs is hindered by the body’s natural immune response to foreign RNAs- a defense mechanism that was acquired to protect from viral infections. Both immune and non-immune cells have specialized receptors that bind to foreign RNAs, leading to inflammation, RNA degradation, and stalled translation. Fortunately, it has been shown that chemically modifying the bases that make up the mRNA backbone can reduce interactions with immune receptors, diminishing unwanted immune responses and increasing efficacy. These modifications “trick” the immune system into thinking the mRNA is endogenously produced, as base modifications are a naturally occurring post-transcriptional process.

To gain an understanding of how base modifications affected efficacy in animals, mRNAs encoding the reporter protein firefly luciferase were synthesized with five different base modifications: psuedouridine (Ψ), N1-methylpseudouridine (m1Ψ), 5-methylcytidine (m5C), 5-methyluridine (m5U), and 2-thiouridine (s2U). These mRNAs were delivered to mice intravenously using three distinct lipid nanoparticles, each targeting a different organ (liver, spleen, and lungs). It was found that m1Ψ was the most effective modification regardless of delivery vehicle, with up to an 11-fold increase in total luciferase expression observed compared to unmodified mRNA. The modifications Ψ and m5C were also effective for all delivery vehicles, resulting in up to a 5-fold increase in expression. Furthermore, the enhanced luciferase expression was almost entirely in the spleen for all effective modifications, regardless of the delivery vehicle. m1Ψ modified mRNA resulted in up to a 54-fold increase in spleen luciferase expression, while the liver and lungs benefitted only modestly from base modifications. These data highlight the importance of base modifications for enhancing mRNA delivery efficacy in vivo. Additionally, it was shown that regardless of the nanoparticle’s primary organ target, the spleen was the primary beneficiary of base modifications as it is composed mostly of immune cells that normally degrade and clear unmodified mRNA.