PEG-associated efficacy loss in lipid nanoparticles persists across formulation modifications for mRNA delivery

Lipid nanoparticles (LNPs) are clinically proven mRNA delivery vehicles, and repeated administration is often required to maintain therapeutic or prophylactic benefit. However, a single dose can provoke adaptive immune responses against polyethylene glycol (PEG), a polymeric excipient on the LNP surface. In this event, resultant anti-PEG antibodies neutralize subsequent LNP doses, causing efficacy loss and limiting therapeutic utility. Here, we investigated whether altering the LNP formulation between initial and subsequent doses could mitigate PEG-associated efficacy loss. Following the administration of an LNP known to elicit anti-PEG antibodies, we re-dosed with LNPs formulated with varied PEG-lipid chemistry, PEG concentration, mRNA cargo, and administration route. None of these adjustments prevented efficacy loss, which consistently correlated with elevated anti-PEG immunoglobulin M (IgM) and, in some cases, immunoglobulin G (IgG). Reduced PEG concentration diminished immunogenicity but also potency, and intraperitoneal dosing elicited the strongest responses upon repeat injection. Further, LNPs that elicit anti-PEG antibodies also impaired the subsequent delivery of an FDA-approved MC3 formulation, suggesting that anti-PEG immune responses can compromise any PEGylated LNP formulation. Together, these findings demonstrate that PEG-associated efficacy loss cannot be resolved through formulation optimization alone, when the ionizable lipid fails to engage the innate immune receptors TLR4 and CD1d. Instead, they suggest that the ionizable lipid's engagement of innate immune receptors is the primary determinant of repeat dosing success.