Endocytosis-dependent and spleen-selective mRNA expression enabled by ionizable lipidoid linkers enhances therapeutic...

mRNA therapy offers precise control, scalability, and a favorable safety profile, making it a promising modality for disease treatment. However, its clinical success critically depends on effective mRNA delivery systems. Currently approved lipid nanoparticles (LNP) predominantly accumulate in the liver, leading to hepatotoxicity and restricting their application to liver-related diseases. Here, we synthesized a series of ionizable lipidoids with distinct linker structures-alkylated, hydroxylated, and esterified-and combined with DOPE and cholesterol to construct three-component lipid nanoparticles (tLNP). We demonstrate that the linker structure of ionizable lipidoids is a key determinant of both the mRNA delivery efficiency and organ selectivity of tLNP. Compared with alkylated or esterified counterparts, tLNP formulated with hydroxylated-linker ionizable lipidoids exhibit enhanced cellular uptake, superior mRNA delivery, and pronounced spleen-selective mRNA expression. Notably, these tLNP achieve a spleen-to-liver mRNA expression ratio of up to 85.88, effectively reducing liver toxicity and enabling extrahepatic mRNA therapies. Leveraging this platform, we developed a therapeutic mRNA vaccine (mLMP2A@BO10-tLNP) for EBV-associated cancers. The immunological role of the spleen and PEG-free tLNP design enhance vaccine safety and efficacy. This work provides design principles for efficient mRNA delivery and introduces strategies for treating EBV-associated cancers. STATEMENT OF SIGNIFICANCE: mRNA-based therapies have demonstrated transformative potential, with multiple applications advancing toward clinical translation. A critical factor in this process is the optimization of lipid nanoparticle (LNP) delivery systems. While current research has largely focused on the design of ionizable lipidoids, the regulatory role of their linker structures remains underexplored. This study reveals that linker structures critically govern LNP-mediated mRNA delivery efficiency and organ-selective expression, providing design principles for next-generation LNPs. Furthermore, by leveraging virus-derived antigens, this work presents a therapeutic mRNA tumor vaccine strategy that significantly reduces off-target cytotoxicity, addressing a key limitation of tumor antigen specificity. Together, these findings offer important insights for improving the safety and translational potential of therapeutic mRNA cancer vaccines.