Optimized Chemical Modifications Enhance Lipid Nanoparticle-Mediated siRNA Silencing of YAP1 and WWTR1

Lipid nanoparticles (LNPs) are clinically validated carriers for the delivery of small interfering RNA (siRNA). Their efficient tissue accessibility and cellular uptake complement conjugated siRNA approaches. While chemical modifications are critical for stabilizing siRNAs and enhancing their potency (particularly in conjugated formats), their contribution in LNP delivery is not fully understood. Here, we systematically evaluated advanced sequence-modification patterns in siRNAs targeting YAP1 and WWTR1 to define their impact on LNP-siRNAs. We found that siRNAs fully incorporating 2'-O-methyl (2'-OMe), 2'-fluoro nucleotides achieved superior knockdown efficacy in vivo compared with partially modified LNP-siRNAs typically used in clinical drugs. Further increasing the proportion of 2'-OMe to 86% did not impair RNA interference activity, and DNA substitutions provide additional opportunities for structural optimization to collectively enhance knockdown efficacy in mouse liver. In contrast, 5'-(E)-vinylphosphonate modification of the guide strand provides no benefit to knockdown efficacy in vitro or in vivo, likely owing to differences in endosomal trafficking between LNP and conjugated siRNAs. These findings provide a framework for chemical optimization of LNP-based siRNAs. The generation of siRNAs with greater efficacy and longer durability facilitates lower doses with less frequent administration, which mitigates LNP-associated toxicity and improves the therapeutic potential of LNP delivery systems.