Optimization of Lipid Nanoparticle Formulations for mRNA Delivery in Vivo with Fractional Factorial and Definitive Screening Designs

TLDR

Intracellular mRNA delivery can induce therapeutic protein production, yet lipid nanoparticles—successful for siRNA—have only recently been explored for mRNA, and their component ratios critically influence potency. We develop a generalized strategy that employs Design of Experiment methods, specifically Definitive Screening and Fractional Factorial Designs, to optimize lipid nanoparticle formulations for liver mRNA delivery. The optimized formulation, which incorporates DOPE and a higher ionizable lipid:mRNA weight ratio, increased erythropoietin‑mRNA potency 7‑fold compared to siRNA‑based formulations and did not improve siRNA delivery, underscoring distinct optimization spaces for mRNA and siRNA.

Abstract

Intracellular delivery of messenger RNA (mRNA) has the potential to induce protein production for many therapeutic applications. Although lipid nanoparticles have shown considerable promise for the delivery of small interfering RNAs (siRNA), their utility as agents for mRNA delivery has only recently been investigated. The most common siRNA formulations contain four components: an amine-containing lipid or lipid-like material, phospholipid, cholesterol, and lipid-anchored polyethylene glycol, the relative ratios of which can have profound effects on the formulation potency. Here, we develop a generalized strategy to optimize lipid nanoparticle formulations for mRNA delivery to the liver in vivo using Design of Experiment (DOE) methodologies including Definitive Screening and Fractional Factorial Designs. By simultaneously varying lipid ratios and structures, we developed an optimized formulation which increased the potency of erythropoietin-mRNA-loaded C12-200 lipid nanoparticles 7-fold relative to formulations previously used for siRNA delivery. Key features of this optimized formulation were the incorporation of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and increased ionizable lipid:mRNA weight ratios. Interestingly, the optimized lipid nanoparticle formulation did not improve siRNA delivery, indicating differences in optimized formulation parameter design spaces for siRNA and mRNA. We believe the general method described here can accelerate in vivo screening and optimization of nanoparticle formulations with large multidimensional design spaces.