Enhanced RNAi Potency

Background

Despite the clinical success of RNA interference (RNAi), existing therapeutic siRNAs face significant structural bottlenecks that limit their efficacy beyond liver-targeted delivery. A primary challenge lies in the thermodynamic and enzymatic selection of the “guide” strand; if the antisense strand is not efficiently loaded into the RNA-induced silencing complex (RISC), the resulting gene knockdown is weak, requiring higher, potentially toxic doses. Furthermore, while chemical modifications (such as 2’-O-methyl) have improved nuclease resistance, they often interfere with the Argonaute 2 (Ago2) binding pocket, reducing the catalytic turnover of the silencing machinery.

Current state-of-the-art siRNAs rely on 3’ overhangs or internal modifications that do not fully optimize the 5’ terminus—the most critical region for RISC recognition and strand selection. There remains an urgent need for structural innovations that improve the “intrinsic potency” of the siRNA molecule itself, enabling robust gene silencing at lower concentrations to treat aggressive, extrahepatic targets like metastatic cancer.

Technology Overview

This technology provides a novel platform for enhancing RNAi potency through specific DNA-based modifications at the 5’ end of the antisense strand. By incorporating a nucleotide overhang of two or more deoxythymidines (dT) specifically at the 5’ terminus, the researchers have engineered a “sticky” antisense lead that significantly optimizes the loading and catalytic efficiency of the RISC complex.

In head-to-head studies targeting KRAS and MYC oncogenes, these 5’ modified dsRNAs showed remarkably superior inhibition across multiple cell lines (including A-431 and MIA-PaCa2) compared to standard commercial modifications. For example, the 2dT and 3dT antisense modifications achieved significantly lower ED50 values (effective dose for 50% inhibition) than standard “Hi2OMe” modified siRNAs. This platform is versatile and can be applied to any gene target, including mutant-specific sequences such as G12V KRAS, providing a highly potent tool for precision oncology.

Benefits

• Enhanced Potency: Significant reduction in ED50/GI50 compared to conventional chemically modified siRNAs, allowing for higher efficacy at lower doses.
• Optimized RISC Loading: DNA-based 5’ modifications facilitate superior antisense strand selection and Argonaute-mediated activity.
• Mutation Specificity: Demonstrated effectiveness in silencing specific oncogenic mutations (e.g., G12V KRAS) without affecting wild-type variants.
• Versatile Platform: Seamlessly integrates with existing delivery vehicles (LNPs, ligands) and various internal chemical modification patterns.
• Reduced Toxicity: Enhanced intrinsic activity minimizes the required dose, reducing the risk of off-target effects and systemic toxicity.

Applications

• Precision Oncology: Targeting traditionally “undruggable” transcription factors and oncogenes (MYC, KRAS, etc.).
• Therapeutic Development: A platform for improving the efficacy of any siRNA-based drug candidate across diverse therapeutic areas.
• Research Tools: High-potency gene silencing for in vitro and in vivo functional genomics.