Efficient In Vivo Delivery of siRNA to the Liver by Conjugation of α-Tocopherol

TLDR

RNA interference enables target‑specific gene knockdown, but safe and efficient in vivo delivery to organs remains unestablished. This study investigates α‑tocopherol as a carrier molecule for delivering siRNA in vivo. α‑Tocopherol was covalently attached to the 5′‑end of the antisense strand of a 27/29‑mer siRNA, designed to be cleaved by Dicer into a mature 21‑mer while the C6 hydroxyl antioxidant group was removed. Intravenous injection of 2 mg kg⁻¹ Toc‑siRNA targeting apolipoprotein B reduced hepatic apoB mRNA, evidenced by lipid droplet accumulation, without inducing interferons or other side effects, demonstrating effective and safe in vivo gene silencing.

Abstract

RNA interference is a powerful tool for target-specific knockdown of gene expression. However, efficient and safe in vivo delivery of short interfering RNA (siRNA) to the target organ, which is essential for therapeutic applications, has not been established. In this study we used α-tocopherol (vitamin E), which has its own physiological transport pathway to most of the organs, as a carrier molecule of siRNA in vivo. The α-tocopherol was covalently bound to the antisense strand of 27/29-mer siRNA at the 5'-end (Toc-siRNA). The 27/29-mer Toc-siRNA was designed to be cleaved by Dicer, producing a mature form of 21/21-mer siRNA after releasing α-tocopherol. The C6 hydroxyl group of α-tocopherol, associated with antioxidant activity, was abolished. Using this new vector, intravenous injection of 2 mg/kg of Toc-siRNA, targeting apolipoprotein B (apoB), achieved efficient reduction of endogenous apoB messenger RNA (mRNA) in the liver. The downregulation of apoB mRNA was confirmed by the accumulation of lipid droplets in the liver as a phenotype. Neither induction of interferons (IFNs) nor other overt side effects were revealed by biochemical and pathological analyses. These findings indicate that Toc-siRNA is effective and safe for RNA interference-mediated gene silencing in vivo.