Elucidate the structural role of helper lipids in modulating hepatic expression following repeated intravenous administration of mRNA-LNPs

Lipid nanoparticles (LNPs) are the most advanced non-viral platform for hepatic mRNA delivery, yet the functional role of helper phospholipids, particularly under repeated dosing regimens required for chronic protein replacement therapies, remains poorly understood. Here, we systematically investigated six helper phospholipids varying in alkyl chain length, headgroup, and unsaturation, evaluating LNP properties and in vivo efficacy after single and repeated intravenous administration (weekly, biweekly, or triweekly). DSPE-based LNPs consistently achieved the highest mRNA expression in vitro and in vivo. Mechanistically, DSPE's superior performance involves a two-step cooperative process: enhanced ApoE adsorption promoting cellular uptake and optimal pKa (6.6) for pH-responsive endosomal protonation. Notably, DSPC induced the highest IL-6 levels under the weekly dosing regimen, suggesting that vaccine-approved lipids may be suboptimal for low-inflammation therapies requiring frequent administration. Importantly, substituting DSPC with DSPE consistently enhanced expression across multiple ionizable lipid platforms (ALC-0315, MC3, and Lipid 5), and significantly improved PCSK9 gene editing efficiency as well as exogenous OTC protein expression. These findings redefine helper phospholipids as active regulators of repeated-dose mRNA delivery and identify DSPE as a promising candidate for chronic hepatic protein replacement.