Dual Ionizable Lipids (ALC0315 and SM102) in a Novel Lipid Nanoparticle Formulation Enhance In Vivo Delivery and Time‐Dependent Expression of Messenger RNA and Self‐Amplifying RNA

This study introduces a novel five‐component LNP formulation incorporating ionizable lipid combination ALC‐0315 and SM‐102 with cholesterol, DSPC, and DMG‐PEG‐2000. LNP‐5 was designed for efficient delivery of mRNA and saRNA. We employed a Design of Experiments approach and microfluidics to optimize the formulation parameters, achieving high encapsulation efficiencies (>80%) for both RNA types. Physicochemical characterization by dynamic light scattering confirmed a controlled size (80–120 nm) and low polydispersity (<0.2). The morphology of investigated LNP‐5 was studied by STEM in comparison with LNP‐4 (with SM‐102 or ALC‐0315). Notably, LNP‐5 demonstrated superior in vivo mRNA delivery efficiency and enhanced target protein expression compared to conventional LNP‐4. The study compares the in vivo performance of mRNA and saRNA platforms formulated in novel LNP‐5 lipid nanoparticles containing dual ionizable lipids. While LNP‐mRNA delivery provides rapid and intense but transient reporter expression, LNP‐saRNA mediates a gradual increase in protein production, peaking at day 7 post‐injection. Quantitative RT‐PCR analysis reveals superior saRNA persistence and a 4.3‐fold higher copy number relative to mRNA by day 7, confirming active intracellular replication of the saRNA platform. These findings demonstrate that LNP‐5 efficiently delivers both platforms, with saRNA offering a more sustained therapeutic window due to its self‐amplifying properties.