Chimeric Antisense Oligonucleotide Conjugated to α-Tocopherol

Abstract

We developed an efficient system for delivering short interfering RNA (siRNA) to the liver by using α-tocopherol conjugation. The α-tocopherol–conjugated siRNA was effective and safe for RNA interference–mediated gene silencing in vivo. In contrast, when the 13-mer LNA (locked nucleic acid)-DNA gapmer antisense oligonucleotide (ASO) was directly conjugated with α-tocopherol it showed markedly reduced silencing activity in mouse liver. Here, therefore, we tried to extend the 5′-end of the ASO sequence by using 5′-α-tocopherol–conjugated 4- to 7-mers of unlocked nucleic acid (UNA) as a “second wing.” Intravenous injection of mice with this α-tocopherol–conjugated chimeric ASO achieved more potent silencing than ASO alone in the liver, suggesting increased delivery of the ASO to the liver. Within the cells, the UNA wing was cleaved or degraded and α-tocopherol was released from the 13-mer gapmer ASO, resulting in activation of the gapmer. The α-tocopherol–conjugated chimeric ASO showed high efficacy, with hepatic tropism, and was effective and safe for gene silencing in vivo. We have thus identified a new, effective LNA-DNA gapmer structure in which drug delivery system (DDS) molecules are bound to ASO with UNA sequences.

Comparative Characterization of Hepatic Distribution and mRNA Reduction of Antisense Oligonucleotides Conjugated with Triantennary N-Acetyl Galactosamine and Lipophilic Ligands Targeting Apolipoprotein B

“…2), and intact GN3-ASO was not detected (Supplemental Fig. 3) in the whole liver samples at day 1 after administration of 1 mg/kg, in agreement with previous reports on GN3-ASO (Prakash et al, 2014) and Toc-ASO (Nishina et al, 2015b). These results suggested that the phosphodiester bond between the ASO and these ligands is promptly cleaved to liberate unconjugated ASO in the liver cells, and these ligands serve as prodrugs targeting hepatocytes and/or NP cells.…”

“…We observed that the efficacy increased proportionally with the amount of ASO, unlike what was found in a previous study (Nishina et al, 2015b). The phosphodiester bond and the length effect of the linker were required to improve the efficacy of the ASO in the liver.…”

“…Although lipophilic ligand conjugations increased the delivery of ASO to the liver, especially NP cells as found in a previous study (Crooke et al, 1996;Bijsterbosch et al, 2000;Wolfrum et al, 2007;Nishina et al, 2015b), no marked enhancement of efficacy in the whole liver, hepatocytes, and NP cells has been reported. We observed that the efficacy increased proportionally with the amount of ASO, unlike what was found in a previous study (Nishina et al, 2015b).…”

Surface Plasmon Resonance Assay of Binding Properties of Antisense Oligonucleotides to Serum Albumins and Lipoproteins

“…Recently, it was reported that ASOs conjugated with cholesterol, tocopherol (vitamin E), and triantennary N-acetyl galactosamine (GalNAc) were especially available for delivery of ASOs to the liver. [4][5][6][7][8] ASOs conjugated with cholesterol and tocopherol as lipophilic ligands could improve their uptake by the liver via receptor-mediated endocytosis after interaction with albumin and lipoproteins, such as low-density lipoprotein (LDL) and high-density lipoprotein (HDL) in circulation. The liver takes up most of the LDL via LDL receptor-mediated endocytosis.…”

Drug delivery system of therapeutic oligonucleotides

“…Toc-ASO in mouse liver was more than 3.5 fold compared to tocopherol unconjugated ASO with intravenous administration. Alfa-tocopherol also improved the pharmacokinetics of the ASO (31).…”