Lipid nanoparticle mRNA systems containing high levels of sphingomyelin engender higher protein expression in hepatic and extra-hepatic tissues

Lipid nanoparticles (LNPs) for delivery of mRNA usually contain ionizable lipid/helper lipid/cholesterol/PEG-lipid in molar ratios of 50:10:38.5:1.5, respectively. These LNPs are rapidly cleared from the circulation following intravenous (i.v.) administration, limiting uptake into other tissues. Here, we investigate the properties of LNP mRNA systems prepared with high levels of helper lipids such as 1,2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC) or N-(hexadecanoyl)-sphing-4-enine-1-phosphocholine (egg sphingomyelin [ESM]). We show that LNP mRNAs containing 40 mol % DSPC or ESM have a unique morphology with a small interior solid core situated in an aqueous compartment that is bounded by a lipid bilayer. The encapsulated mRNA exhibits enhanced stability in the presence of serum. LNP mRNA systems containing 40 mol % DSPC or ESM exhibit significantly improved transfection properties in vitro compared with systems containing 10 mol % DSPC or ESM. When injected i.v., LNP mRNAs containing 40 mol % ESM exhibit extended circulation lifetimes compared with LNP mRNA systems containing 10 mol % DSPC, resulting in improved accumulation in extrahepatic tissues. Systems containing 40 mol % ESM result in significantly improved gene expression in spleen and bone marrow as well as liver post i.v. injection compared with 10 mol % DSPC LNP mRNAs. We conclude that LNP mRNAs containing high levels of helper lipid provide a new approach for transfecting hepatic and extrahepatic tissues.

Automated summary

By studying LNP mRNA systems with high levels of helper lipids, it was found that LNP mRNA systems containing 40 mol% of DSPC or ESM exhibited significantly improved transfection properties in vitro and extended circulation lifetimes and better tissue accumulation in vivo, especially in terms of gene expression in spleen, bone marrow, and liver.