Single Alkyl Phosphonate Modification of the siRNA Backbone in the Seed Region Enhances Specificity and Therapeutic Profile

We evaluated the effect of alkyl phosphonate linkages in enhancing the specificity and therapeutic profile of siRNA when incorporated into the seed region. Modified siRNAs with a single alkyl phosphonate linkage demonstrated improved specificity, particularly when positioned at the internucleotide linkage between positions 6 and 7 from the 5'-end of the guide strand. These modifications maintained robust on-target activity both in vitro and in vivo while significantly reducing off-target effects, as shown by differential gene expression analysis. Additionally, we observed enhanced safety in mice, as evidenced by reduced ALT/AST elevation and the absence of histopathological changes. To validate the target specificity of siRNAs with a single alkylphosphonate modification in the seed region, we employed differential gene expression (DGE) analysis. This approach was applied to data from both cultured cells and in vivo models, offering a detailed assessment of the modification's impact on gene expression.