Optimizing Peptide Ionizable Lipids Enables Efficient and Low-Toxicity mRNA Delivery for In Vivo Prime Editing and Protein Replacement Therapy

Highly efficient mRNA lipid nanoparticle (LNP) often presents potential safety risks. Here, we establish a structure-activity relationship framework for peptide ionizable lipids (PILs) to facilitate the rational design of safe and effective mRNA-LNPs. The PIL structure comprises three modular components: building block, side-chain length, and hydrophobic tail. Through systematic optimization, a lead compound (Dab4) with four building blocks and a moderate side chain length was identified, demonstrating minimized hepatotoxicity while maintaining superior delivery performance. Leveraging this framework, a series of Dab4-derived PILs with three tail types, including alkyl (a-tail), ester (aat-tail), and hydroxyl (e-tail), were synthesized. This tail chemistry determined organ tropism, with B12-a13Dab4 (a-tail) showing optimal performance in the liver. The B12-a13Dab4 LNP exhibited significantly higher hepatic delivery efficiency and markedly improved biosafety compared with the FDA-approved SM-102 formulation. Moreover, B12-a13Dab4 LNP efficiently triggers in vivo prime editing by co-delivering PE7 mRNA and epegRNA, and achieves significant therapeutic effects in a Hereditary Tyrosinemia Type 1 (HT-1) model through repeated delivery fumarylacetoacetate hydrolase (FAH) mRNA. This study establishes rational design principles for PILs that strike a balance between efficacy and safety, offering a versatile mRNA-LNP platform for the advancement of gene editing and protein replacement therapies.

gene editing; lipid nanoparticle; mRNA delivery; peptide ionizable lipids; protein replacement therapy.