Rational design of prodrug-type apoB-targeted siRNA for nuclease resistance improvement without compromising gene silencing potency

Synthetic siRNA molecules without chemical modifications are easily degraded in the body, and 2́-O-modifications are frequently introduced to enhance stability. However, such chemical modifications tend to impact the gene knockdown potency of siRNA negatively. To circumvent this problem, we previously developed a prodrug-type siRNA bearing 2́-O-methyldithiomethyl (MDTM) groups, which can be converted into unmodified siRNA under the reductive environment in cells. In this study, we developed a nuclease-resistant prodrug-type 2́-O-MDTM siRNA for deployment in future animal experiments. To rationally design siRNA modified with a minimal number of 2́-O-MDTM nucleotide residues, we identified the sites susceptible to nuclease digestion and tolerant to 2́-O-methyl (2́-OMe) modification in the antisense strand of apolipoprotein B-targeted siRNA. Subsequently, we optimized the positions where the 2́-OMe and 2́-O-MDTM groups should be incorporated. siRNA bearing the 2́-O-MDTM and 2́-OMe groups at their respective optimized positions exhibited efficient knockdown potency in vitro and enhanced stability in serum.