Characterization of Storage-Induced mRNA Modifications in ( <i>4S</i> )-KEL12 LNP: Adduct Formation Kinetics, mRNA Decay, and Translational Competence

Lipid-mRNA adducts form during storage for several types of lipid nanoparticles (LNPs) and impair therapeutic efficacy, yet their structural drivers and functional consequences remain incompletely characterized, especially for novel lipids with distinct structures. Here, we investigated adduct formation between mRNA and several impurities derived from an immunotropic ionizable lipid (<i>4S</i>)-KEL12, which has been used to develop therapeutic mRNA cancer vaccines approved for human clinical studies. Elevated storage temperatures promoted both adduct accumulation and the loss of mRNA integrity with divergent kinetics at 25 °C, suggesting their independence. Mechanistically, degradation impurities of (<i>4S</i>)-KEL12, particularly its aldehyde derivative (Z4) and N-oxide derivative (Z1) dominated adduct generation, with Z4 exhibiting ∼4-fold higher activity than Z1. Moreover, mRNA adduction with Z4 did not reduce mRNA integrity by capillary electrophoresis, further supporting independent pathways. Mass spectrometry characterization unambiguously identified cytidines as the primary target on mRNA for Z4 adduction. Functionally, while adducted mRNAs exhibited poor capacity for protein expression in cultured human 293T cells, they did not stimulate significant gene expression involved in innate immunity for RNA sensing and downstream type I interferon pathway activation in human THP1 cells. These findings not only clarify important functional consequences of adducted mRNAs, but also establish impurity control and thermal management as actionable strategies for advancing mRNA therapeutics.