Boosting Ligand-Mediated Hepatic mRNA Delivery and Gene Editing Through Coordinated Pairing of Polyethylene Glycol-Modified Lipids.

Active targeting can improve the efficacy of mRNA therapeutics, but ligand-mediated targeting with lipid nanoparticles (LNPs) remains challenging. Here, we studied the impact of polyethylene glycol (PEG)-lipid composition and triantennary N-acetylgalactosamine (TriGalNAc)-modification on the hepatic delivery efficiency of mRNA-LNPs. Incorporating TriGalNAc-lipid into LNPs containing dimyristoyl glycerol (DMG)-PEG2000 or distearoylphosphatidylethanolamine (DSPE)-PEG2000, excipients used in FDA-approved RNA-LNPs or liposomes, did not further improve hepatic mRNA delivery. We discovered that incorporating TriGalNAc-lipid into LNPs containing DSPE-PEG350 increased hepatic luciferase expression 4-6-fold over TriGalNAc-conjugated or unconjugated DMG-PEG2000 LNPs, and 60-fold over TriGalNAc-conjugated or unconjugated DSPE-PEG2000 LNPs in vivo. Despite its relatively low molecular weight PEG, unconjugated DSPE-PEG350 LNP showed moderately extended blood circulation but only moderately reduced cellular uptake in vitro, potentially providing a balanced platform for adding TriGalNAc ligands and improving the overall hepatic delivery efficiency in vivo. The pairing of TriGalNAc-lipid and DSPE-PEG350 in LNPs containing adenine base editor RNAs against PCSK9 lowered plasma cholesterol in wild-type mice, while conventional DMG-PEG2000 LNP was ineffective at the same dose. This study highlights the importance of redesigning the LNP surface to potentiate ligand-mediated active targeting to the liver.