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The asialoglycoprotein receptor (ASGPr) and N -acetylgalactosamine (GalNAc) is one of the most reliable receptor-ligand combinations for delivering antisense oligonucleotides (ASOs) to the liver. Here, we show that a modular GalNAc conjugation strategy allows us to reinforce the activity of the parent, naked 2',4'-BNA/LNA gapmer targeting apolipoprotein B. The conjugation partly reduced a possible hepatotoxicity of the parent ASO. The structure-activity study revealed the significance of the metabolic susceptibility of the GalNAc moiety to nucleolytic cleavage that results in exposure of the parent gapmer. The broad usefulness of our delivery strategy of ASOs to the liver has been demonstrated.

Recently, some studies have reported nephrotoxicity associated with a certain class of antisense oligonucleotides (ASOs) in humans. One possibility for reducing the potential nephrotoxicity of ASOs is to alter their pharmacokinetics. In this study, we investigated the effect of a ligand conjugation strategy on the renal accumulation of ASOs. We selected two ligands, cholesterol and N-acetylgalactosamine (GalNAc), with the purpose of reducing renal distribution and liver targeting, and then designed a series of cholesterol-GalNAc dual conjugated ASOs. The gene-silencing activity of the cholesterol-GalNAc dual conjugated ASO in the liver was slightly lower than that of a GalNAc-conjugated ASO. On the other hand, the renal distribution of the cholesterol-GalNAc dual conjugated ASO was considerably decreased compared with the GalNAc-conjugated ASO, as we expected. As dual conjugation was successful in reducing the renal distribution of ASO, it should be an effective strategy for reducing the nephrotoxic potential of ASOs.

We synthesized thymidine derivatives of 2'-C,4'-C-ethyleneoxy-bridged 2'-deoxyribonucleic acids with an 8'-methyl group ((R)-Me-EoDNA and (S)-Me-EoDNA) and without any substituent (EoDNA). Oligonucleotides including these EoDNAs showed high hybridization abilities with complementary RNA and excellent enzymatic stabilities compared with natural DNA. Moreover, the in vitro antisense potency of oligonucleotides with these EoDNAs and our recently reported methylene-EoDNAs was investigated and compared with that of LNA, which is a practical chemical modification for oligonucleotide-therapeutic agents. The results showed that EoDNAs and methylene-EoDNAs could be promising candidates for antisense technology.

Antisense oligonucleotides are attractive therapeutic agents for several types of disease. One of the most promising modifications of antisense oligonucleotides is the introduction of bridged nucleic acids. As we report here, we designed novel bridged nucleic acids, triazole-bridged nucleic acid (TrNA), and tetrazole-bridged nucleic acid (TeNA), whose sugar conformations are restricted to N-type by heteroaromatic ring-bridged structures. We then successfully synthesized TrNA and TeNA and introduced these monomers into oligonucleotides. In UV-melting experiments, TrNA-modified oligonucleotides exhibited increased binding affinity toward complementary RNA and decreased binding affinity toward complementary DNA, although TeNA-modified oligonucleotides were decomposed under the annealing conditions. Enzymatic degradation experiments demonstrated that introduction of TrNA at the 3'-terminus rendered oligonucleotides resistant to nuclease digestion. Furthermore, we tested the silencing potencies of TrNA-modified antisense oligonucleotides using in vitro and in vivo assays. These experiments revealed that TrNA-modified antisense oligonucleotides induced potent downregulation of gene expression in liver. In addition, TrNA-modified antisense oligonucleotides showed a tendency for increased liver biodistribution. Taken together, our findings indicate that TrNA is a good candidate for practical application in antisense methodology.

Abnormal elevation of low-density lipoprotein (LDL) and triglyceride-rich lipoproteins in plasma as well as dysfunction of anti-atherogenic high-density lipoprotein (HDL) have both been recognized as essential components of the pathogenesis of atherosclerosis and are classified as dyslipidemia. This review describes the arc of development of antisense oligonucleotides for the treatment of dyslipidemia. Chemically-armed antisense candidates can act on various kinds of transcripts, including mRNA and miRNA, via several different endogenous antisense mechanisms, and have exhibited potent systemic anti-dyslipidemic effects. Here, we present specific cutting-edge technologies have recently been brought into antisense strategies, and describe how they have improved the potency of antisense drugs in regard to pharmacokinetics and pharmacodynamics. In addition, we discuss perspectives for the use of armed antisense oligonucleotides as new clinical options for dyslipidemia, in the light of outcomes of recent clinical trials and safety concerns indicated by several clinical and preclinical studies.

Cholesterol conjugation of oligonucleotides is an attractive way to deliver the oligonucleotides specifically to the liver. However cholesterol-conjugated antisense oligonucleotides (ASOs) mainly accumulate in non-parenchymal cells (NPCs) such as Kupffer cells. In this study, to increase the hepatic accumulation of cholesterol-conjugated ASOs, we prepared a variety of linkers for cholesterol conjugation to anti-Pcsk9 ASOs and examined their effects on pharmacological parameters. Hepatic accumulation of ASO was dramatically increased with cholesterol conjugation. The increase in hepatic accumulation depended largely on the linker chemistry of each cholesterol-conjugated ASO. In addition to hepatic accumulation, the cell tropism of each cholesterol-conjugated ASO tended to depend on their linker. Although a linker bearing a disulfide bond accumulated mainly in NPCs, hexamethylene succinimide linker accumulated mainly in hepatocytes. To estimate the benefits of releasing ASO from the conjugated cholesterol in hepatocyte, we designed another linker based on hexamethylene succinimide, which has a phosphodiester bond between the linker and the ASO. The cholesterol-conjugated ASO bearing such a phosphodiester bond showed a significantly improved Pcsk9 mRNA inhibitory effect compared to its counterpart, cholesterol-conjugated ASO with a phosphorothioate bond, while the hepatic accumulation of both cholesterol-conjugated ASOs was comparable, indicating the effectiveness of removing the conjugated cholesterol for ASO activity. In toxicity analysis, some of the linkers induced lethal toxicities when they were injected at high concentrations (>600μM). These toxicities were attributed to decreased platelet levels in the blood, suggesting an interaction between cholesterol-conjugated ASO and platelets. Our findings may provide a guideline for the design of molecule-conjugated ASOs., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Antisense and RNAi-related oligonucleotides have gained attention as laboratory tools and therapeutic agents based on their ability to manipulate biological events in vitro and in vivo. We show that Ca(2+) enrichment of medium (CEM) potentiates the in vitro activity of multiple types of oligonucleotides, independent of their net charge and modifications, in various cells. In addition, CEM reflects in vivo silencing activity more consistently than conventional transfection methods. Microscopic analysis reveals that CEM provides a subcellular localization pattern of oligonucleotides resembling that obtained by unassisted transfection, but with quantitative improvement. Highly monodispersed nanoparticles ~100 nm in size are found in Ca(2+)-enriched serum-containing medium regardless of the presence or absence of oligonucleotides. Transmission electron microscopy analysis reveals that the 100-nm particles are in fact an ensemble of much smaller nanoparticles (ϕ ∼ 15 nm). The presence of these nanoparticles is critical for the efficient uptake of various oligonucleotides. In contrast, CEM is ineffective for plasmids, which are readily transfected via the conventional calcium phosphate method. Collectively, CEM enables a more accurate prediction of the systemic activity of therapeutic oligonucleotides, while enhancing the broad usability of oligonucleotides in the laboratory., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

High scalability of a novel bicyclic nucleoside building block, amido-bridged nucleic acid (AmNA), to diversify pharmacokinetic properties of therapeutic antisense oligonucleotides is described. N2'-functionalization of AmNA with a variety of hydrophobic groups is straightforward. Combinations of these modules display similar antisense knockdown effects and improve cellular uptake, relative to sequence-matched conventional 2',4'-bridged nucleic acid (2',4'-BNA) in vivo.

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.

A 20-mer phosphorothioate antisense oligodeoxyribonucleotide having locked nucleic acids (LNA-AON) was used to reduce elevated serum triglyceride levels in mice. We repeatedly administered LNA-AON, which targets murine apolipoprotein C-III mRNA, to high-fat-fed C57Bl/6J male mice for 2 weeks. The LNA-AON showed efficient dose-dependent reductions in hepatic apolipoprotein C-III mRNA and decreased serum apolipoprotein C-III protein concentrations, along with efficient dose-dependent reductions in serum triglyceride concentrations and attenuation of fat accumulation in the liver. Through precise lipoprotein profiling analysis of sera, we found that serum reductions in triglyceride and cholesterol levels were largely a result of decreased serum very low-density lipoprotein (VLDL)-triglycerides and -cholesterol. It is noteworthy that larger VLDL particles were more susceptible to removal from blood than smaller particles, resulting in a shift in particle size distribution to smaller diameters. Histopathologically, fatty changes were markedly reduced in antisense-treated mice, while moderate granular degeneration was frequently seen the highest dose of LNA-AON. The observed granular degeneration of hepatocytes may be associated with moderate elevation in the levels of serum transaminases. In conclusion, we developed an LNA-based selective inhibitor of apolipoprotein C-III. Although it remains necessary to eliminate its potential hepatotoxicity, the present LNA-AON will be helpful for further elucidating the molecular biology of apolipoprotein C-III., (© 2013 Published by Elsevier B.V.)