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.