Single alkyl phosphonate modification of the siRNA backbone in the seed region enhances specificity and therapeutic...

We evaluated the effect of alkyl phosphonate linkages in enhancing the specificity and therapeutic profile of siRNA when incorporated into the seed region. siRNAs modified with a single alkyl phosphonate linkage demonstrated enhanced specificity and therapeutic profile compared to the parent siRNA. We found that these modifications are most effective when positioned at the internucleotide linkages 6-7 from the 5'-end of the guide strand. Our findings reveal that siRNAs with this modification maintain robust on-target activity both in vitro and in vivo. Importantly, differential gene expression (DGE) analysis showed a significant reduction in off-target effects across in vitro and in vivo, leading to an improved therapeutic profile. We also demonstrate enhanced safety in mice, as evidenced by reduced ALT/AST elevation and the absence of histopathological changes. This novel chemical approach to siRNA design provides impetus to advancing RNA interference-based treatments for various diseases.