Optimized microfluidic formulation and organic excipients for improved lipid nanoparticle mediated genome editing.

Abstract

mRNA-based gene editing platforms have tremendous promise in the treatment of genetic diseases. However, for this potential to be realized <i>in vivo</i>, these nucleic acid cargos must be delivered safely and effectively to cells of interest. Ionizable lipid nanoparticles (LNPs), the most clinically advanced non-viral RNA delivery system, have been well-studied for the delivery of mRNA but have not been systematically optimized for the delivery of mRNA-based CRISPR-Cas9 platforms. In this study, we investigated the effect of microfluidic and lipid excipient parameters on LNP gene editing efficacy. Through <i>in vitro</i> screening in liver cells, we discovered distinct trends in delivery based on phospholipid, cholesterol, and lipid-PEG structure in LNP formulations. Combination of top-performing lipid excipients produced an LNP formulation that resulted in 3-fold greater gene editing <i>in vitro</i> and facilitated 3-fold greater reduction of a therapeutically-relevant protein <i>in vivo</i> relative to the unoptimized LNP formulation. Thus, systematic optimization of LNP formulation parameters revealed a novel LNP formulation that has strong potential for delivery of gene editors to the liver to treat metabolic disease.