Lipid nanoparticles optimized for large RNA cargo and tissue targeting enhance in vivo genome editing.

Although lipid nanoparticles are clinically validated for RNA delivery, their potency often declines as transcript size increases. Here we report a large-cargo-informed lipid discovery strategy that incorporates mRNA size into ionizable lipid screening. A combinatorial library of 384 lipids was screened using a 5.7-kb ABE-NanoLuc reporter mRNA, identifying large-cargo-optimized lipids led by LC-1. Following intravenous, intrathecal and intratracheal administration, LC-1 achieved up to 79% Cas9-mediated knockout in liver and 48% and 27% in brain and lung, up to fourfold higher than LP-01 and ALC-0315, and produced higher ABE-mediated reporter correction across all three routes. LC-1 supported base editing of PCSK9, CFTRR55X and Ube3a-ATS. Mechanistic studies showed that, unlike the benchmarks, LC-1 forms stronger lipid-RNA interactions and retains an ordered, fusogenic inverted-hexagonal structure and pH-responsive membrane disruption as mRNA size increases, rather than becoming disordered. These findings establish cargo size as a key design parameter for ionizable lipid discovery.