Design of bifunctional RNA-binding compounds that can modulate siRNA hydrophobicity for deep penetration and gene...

Small interfering RNA (siRNA) could be a promising new modality to block the neovascularization in retinal diseases. For ocular siRNA delivery, hydrophobic modification is essential to increase the siRNA membrane compatibility, self-deliverability, and retinal penetration. Current covalent approach such as cholesterol or fatty acid conjugation can only tether one hydrophobic ligand to siRNA molecule, which shows limitations in the ocular delivery. Here, we identified a siRNA-binding drug, quinacrine, and conjugated it with cholesterol to obtain a bifunctional RNA-binding compound that can act as a tag to tether multiple cholesterol molecules on siRNA non-covalently. Despite a moderate affinity to siRNA (K_D = 932 ± 26 nM), the tag can convert siRNA into a fully hydrophobic (logP = 1.86), membrane-permeable and self-deliverable (IC₅₀ = 166 ± 59 nM) molecule. Tagged with a miniature and chemically modified anti-VEGF siRNA, a single intravitreal administration enables siRNA penetration of all retinal cell layers and induces effective gene silencing over 1 month in the mouse model. The data suggest that the bifunctional RNA-binding compound could be a useful small-molecule carrier to promote siRNA delivery in the retina.