Cholesterol analogs modulate lipid nanoparticle performance for mRNA delivery after lyophilization and enable ocular disease therapy.

Lipid nanoparticles (LNPs) are essential nucleic acid delivery carriers, with cholesterol being crucial for their structural integrity and intracellular transport. Although C24-alkyl phytosterols (cholesterol analogs) promote gene transfection in HeLa cells, their broader impact on mRNA-LNP performance across organs and organ-derived cell lines remains unknown. We investigated the effects of C24-alkyl phytosterols on mRNA-LNP morphology, stability, transfection efficiency, and in vivo biodistribution before and after lyophilization. Freshly prepared β-sitosterol-containing mRNA-LNPs exhibited the highest transfection efficiency in most cell lines. Stigmasterol-containing mRNA-LNPs demonstrated superior performance after lyophilization. In vivo, compared with cholesterol-based controls, β-sitosterol-formulated ALC-0315-based mRNA-LNPs and fucosterol-formulated MC3-based LNPs yielded increased bioluminescence in target organs. β-sitosterol-mRNA-LNPs delivering (MERTK)mRNA robustly restored visual function in a retinal degeneration rat model. These findings highlight how subtle structural modifications of cholesterol analogs can influence mRNA-LNP therapeutic efficacy, providing ideas for the rational design of next-generation gene delivery systems. We demonstrated the therapeutic effects of mRNA-LNPs in an animal model of eye disease (RCS rats), paving the way for application in ocular gene therapy.