Antisense oligonucleotides (ASOs) are recognized therapeutic agents for the modulation of specific genes at the post-transcriptional level. Similar to any medical drugs, there are opportunities to improve their efficacy and safety. Here we develop a short DNA/RNA heteroduplex oligonucleotide (HDO) with a structure different from double-stranded RNA used for short interfering RNA and single-stranded DNA used for ASO. A DNA/locked nucleotide acid gapmer duplex with an α-tocopherol-conjugated complementary RNA (Toc-HDO) is significantly more potent at reducing the expression of the targeted mRNA in liver compared with the parent single-stranded gapmer ASO. Toc-HDO also improves the phenotype in disease models more effectively. In addition, the high potency of Toc-HDO results in a reduction of liver dysfunction observed in the parent ASO at a similar silencing effect. HDO technology offers a novel concept of therapeutic oligonucleotides, and the development of this molecular design opens a new therapeutic field.

Antisense oligonucleotide (ASO) has the potential to induce off‐target effects due to complementary binding between the ASO and unintended RNA with a sequence similar to the target RNA. Conventional animal studies cannot be used to assess toxicity induced by off‐target effects because of differences in the genome sequence between humans and other animals. Consequently, the assessment of off‐target effects with in silico analysis using a human RNA database and/or in vitro expression analysis using human cells has been proposed.Our previous study showed that the number of complementary regions of ASOs with mismatches in the human RNA sequences increases dramatically as the number of tolerated mismatches increases. However, to what extent the expression of genes with mismatches is affected by off‐target effects at the cellular level is not clear. In this study, we evaluated off‐target effects of gapmer ASOs, which cleave the target RNA in an RNase H‐dependent manner, by introducing the ASO into human cells and performing microarray analysis. Our data indicate that gapmer ASOs induce off‐target effects depending on the degree of complementarity between the ASO and off‐target candidate genes. Based on our results, we also propose a scheme for the assessment of off‐target effects of gapmer ASOs.

Recent findings in molecular biology implicate the involvement of proprotein convertase subtilisin/kexin type 9 (PCSK9) in low-density lipoprotein receptor (LDLR) protein regulation. The cholesterol-lowering potential of anti-PCSK9 antisense oligonucleotides (AONs) modified with bridged nucleic acids (BNA-AONs) including 2′,4′-BNA (also called as locked nucleic acid (LNA)) and 2′,4′-BNANC chemistries were demonstrated both in vitro and in vivo. An in vitro transfection study revealed that all of the BNA-AONs induce dose-dependent reductions in PCSK9 messenger RNA (mRNA) levels concomitantly with increases in LDLR protein levels. BNA-AONs were administered to atherogenic diet-fed C57BL/6J mice twice weekly for 6 weeks; 2′,4′-BNA-AON that targeted murine PCSK9 induced a dose-dependent reduction in hepatic PCSK9 mRNA and LDL cholesterol (LDL-C); the 43% reduction of serum LDL-C was achieved at a dose of 20 mg/kg/injection with only moderate increases in toxicological indicators. In addition, the serum high-density lipoprotein cholesterol (HDL-C) levels increased. These results support antisense inhibition of PCSK9 as a potential therapeutic approach. When compared with 2′,4′-BNA-AON, 2′,4′-BNANC-AON showed an earlier LDL-C–lowering effect and was more tolerable in mice. Our results validate the optimization of 2′,4′-BNANC-based anti-PCSK9 antisense molecules to produce a promising therapeutic agent for the treatment of hypercholesterolemia.

The multiple-turnover ability of a series of locked nucleic acid (LNA)-based antisense oligonucleotides (AONs) in the RNase H-mediated scission reaction was estimated using a newly developed cell-free reaction system. We determined the initial reaction rates of AONs under multiple-turnover conditions and found that among 24 AONs tested, AONs with melting temperatures (Tm) of 40°C-60°C efficiently elicit multiple rounds of RNA scission. On the other hand, by measuring Tm with two 10-mer RNAs partially complementary to AONs as models of cleaved 5' and 3' fragments of mRNA, we found that AONs require adequate binding affinity for efficient turnover activities. We further demonstrated that the efficacy of a set of 13-mer AONs in mice correlated with their turnover efficiency, indicating that the intracellular situation where AONs function is similar to multiple-turnover conditions. Our methodology and findings may provide an opportunity to shed light on a previously unknown antisense mechanism, leading to further improvement of the activity and safety profiles of AONs.