Locked nucleic acid antisense inhibitor targeting apolipoprotein C-III efficiently and preferentially removes triglyceride from large very low-density lipoprotein particles in murine plasma.

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.

Cholesterol-lowering Action of BNA-based Antisense Oligonucleotides Targeting PCSK9 in Atherogenic Diet-induced Hypercholesterolemic Mice.

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'-BNA(NC) 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'-BNA(NC)-AON showed an earlier LDL-C-lowering effect and was more tolerable in mice. Our results validate the optimization of 2',4'-BNA(NC)-based anti-PCSK9 antisense molecules to produce a promising therapeutic agent for the treatment of hypercholesterolemia.

Development of a 2',4'-BNA/LNA-based siRNA for Dyslipidemia and Assessment of the Effects of Its Chemical Modifications In Vivo.

Recent advances in RNA interference (RNAi)-based drug development have partially allowed systemic administration of these agents in vivo with promising therapeutic effects. However, before chemically modified small-interfering RNAs (siRNAs) can be applied clinically, their in vivo effects should be thoroughly assessed. And while many studies have assessed the effects of chemically modified siRNAs in vitro, there has been no comprehensive assessment of their effects in vivo. Here, we aimed to elucidate the effects of administering chemically modified siRNAs in vivo and to propose a 2',4'-bridged nucleic acid (BNA)/locked nucleic acid (LNA)-based siRNA candidate for dyslipidemia. A potentially therapeutic siRNA, siL2PT-1M, was modified with phosphorothioate (PS) and 2',4'-BNA/LNA in its sense strand and with 2'-methoxy (2'-OMe) nucleotides in its immunostimulatory motif; administration of siL2PT-1M resulted in sustained reductions in serum total cholesterol (TC) (24 days) and a concomitant apolipoprotein B (apoB) mRNA reduction in liver without adverse effects. The 2',4'-BNA/LNA modification in the sense strand was greatly augmented the duration of the RNAi effect, whereas cholesterol conjugation shortened the duration. Cholesterol-conjugated immunostimulatory siRNA (isRNA) induced higher serum interferon-α (IFN-α) levels than did nonmodified isRNA, indicating that the immune reaction was facilitated by cholesterol conjugation. Our results indicated that modification of the adenosine residues complementary to the immunostimulatory motif and of central 5'-UG-3' in the sense strand would ameliorate the negative immune response.Molecular Therapy - Nucleic Acids (2012) 1, e45; doi:10.1038/mtna.2012.32; published online 18 September 2012.

Antisense drug discovery and development.

Numerous chemically modified oligonucleotides have been developed so far and show their own unique chemical properties and pharmacodynamic/pharmacokinetic characteristics. Among all non-natural nucleotides, to the best of our knowledge, only five chemistries are currently being tested in clinical trials: phosphorothioate, 2´-O-methyl RNA, 2´-O-methoxyethyl RNA, 2´,4´-bridged nucleic acid/locked nucleic acid and the phosphorodiamidate morpholino oligomer. Since phosphorothioate modification can improve the pharmacokinetics of oligonucleotides, this modification is currently used in combination with all other modifications except phosphorodiamidate morpholino oligomer. For the treatment of metabolic, cardiovascular, cancer and other systemic diseases, the phosphorothioate class of drugs is obviously helpful, while superior efficacies can be observed in phosphorodiamidate morpholino oligomer compared to other classes of oligonucleotides for the treatment of Duchenne muscular dystrophy. Which properties of antisense molecules are actually essential for clinical applications? In this article, we provide an overview of the medicinal chemistry of existing non-natural antisense molecules, as well as their clinical applications, to discuss which properties of antisense oligonuculeotides affect therapeutic potency.