Tsuyoshi Yamamoto

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′-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 20u2009mg/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.

Amido-Bridged Nucleic Acids (AmNAs): Synthesis, Duplex Stability, Nuclease Resistance, and in Vitro Antisense Potency

Since their discovery, 2’,4’-bridged nucleic acids (2’,4’-BNAs) [or locked nucleic acids (LNAs)] have been considered promising candidates for antisense technology because of their unprecedented high binding affinities toward complementary strands, 2] sequence selectivities, and potential for in vivo applications. 5] Our group and others are continuing efforts to develop other bridged nucleic acids with improved properties based on the LNA structural concept. Recently, our group has focused on developing new bridged nucleic acids with carbonyl functionality in the bridge. The introduction of a carbonyl group into the bridge would be expected, as a consequence of its trigonal planarity, to restrict the flexibility of the sugar moiety. We have so far reported two analogues of bridged nucleic acids with carbonyl functionality in their bridges; both exhibited interesting properties. In one report we described a seven-membered bridged nucleic acid with a cyclic urea structure, which showed high nuclease resistance along with high RNA selectivity. We recently introduced another interesting class of LNA analogues: hydroxamate-bridged nucleic acids (HxNAs), which possessed unique nuclease resistance. Among the challenges to the development of a potent antisense oligonucleotide, in vivo enzymatic digestion of the oligonucleotide remains an important factor. Previous studies have shown that the nuclease resistance of a bridged nucleic acid can be enhanced by increasing the size of the bridge, but this reduces binding affinity. 7] Optimization of the bridged nucleic acids to improve nuclease resistance without losing binding affinity is therefore needed. To accomplish this, the relationship between the size of the bridge and the properties of the bridged nucleic acid were investigated. Here we report the synthesis and properties of a new set of analogues of LNAs with cyclic amide structures, named amido-bridged nucleic acid (AmNAs). This is the first report of an amide linkage in a bridged nucleic acid, although amide functionality has found other uses in nucleic acid chemistry, for example, in modification of the phosphate linkage 13] and in peptide nucleic acids (PNAs). Structurally, an AmNA has a five-membered bridged structure similar to those of an LNA or an 2’-amino-LNA. Previous reports on 2’-amino-LNAs demonstrated that their binding affinities toward complementary strands are comparable to those of LNAs. Introduction of a carbonyl group into the LNA structure, as in an AmNA, should enhance the rigidity and polarity of the structure. These structural characteristics of an AmNA would be expected to impart properties appropriate for antisense oligonucleotides. Compound 1 (Scheme 1), synthesized by known procedures, was used as the starting material for the synthesis of AmNAs. Triflylation of 1 followed by an SN2 reaction with NaN3 yielded 3 in good yield. The silyl protecting group of 3 was removed (tributylammonium fluoride, TBAF) to produce 4 with a free hydroxy moiety that was oxidized with pyridinium dichromate (PDC) in DMF to afford the carboxylic acid 5. By means of a Staudinger reaction (i.e. , treatment of 5 with PBu3 in THF), the azido group at the 2’-position was converted into an amino group to yield 6. The carboxylic acid function at the 4’-position and the amino function at the 2’-position of 6 were linked together by a condensation reaction, activated by N’-(3dimethylaminopropyl)-N-ethylcarbodiimide (EDCI) to afford the desired cyclic product 7. Debenzylation of 7 by catalytic hydrogenolysis yielded monomer 8 in excellent yield. The desired monomer containing a carbonyl function in a five-membered bridge had thus been successfully synthesized. The functionalizable nitrogen at the 2’-position allowed the synthesis of various N-substituted derivatives of the monomer. The first attempt to methylate this functionalizable nitrogen also caused N-methylation at the thymine moiety, so the thymine moiety was first protected with a benzyloxymethyl (BOM) protecting group, followed by N-methylation of the bridge nitrogen and then by debenzylation by catalytic hydrogenolysis to afford the desired N-methylated monomer 10. To incorporate the synthesized monomers 8 and 10 into oligonucleotides, the primary hydroxy groups of 8 and 10 were selectively tritylated with 4,4’-dimethoxytrityl chloride (DMTrCl), followed by phosphitylation of secondary hydroxy groups with 2-cyanoethyl-N,N,N’,N’-tetraisopropylphosphorodiamidite to afford the desired thymine phosphoramidites 9 and 12. For interconversion of the nucleobase (i.e. , thymine into cytosine), the secondary hydroxy group of tritylated nucleoside 11 was protected with a triethylsilyl (TES) group, and subsequent treatment with 2,4,6-triisopropylbenzenesulfonyl chloride (iPr3ArSO2Cl) in the presence of 4-dimethylaminopyridine (DMAP) and triethylamine (TEA) sulfonated the 4-oxo group of the thymine moiety. The sulfonylated nucleoside was subjected to substitution with ammonia to afford a 5-methylcytidine derivative. The exocyclic amino moiety of the 5-methylcytidine was protected with an acetyl group, followed by desilylation and phosphitylation of the free secondary hydroxy group by 2cyanoethyl-N,N,N’,N’-tetraisopropylphosphordiamidite to afford the desired N-acetyl-protected 5-methylcytidine phosphordiamidite 13 (Scheme 1). [a] A. Yahara, Dr. A. R. Shrestha, Dr. T. Yamamoto, Dr. Y. Hari, T. Osawa, M. Yamaguchi, Dr. M. Nishida, Dr. T. Kodama, Prof. Dr. S. Obika Graduate School of Pharmaceutical Sciences, Osaka University 1-6 Yamadaoka, Suita, Osaka 565-0871 (Japan) E-mail : obika@phs.osaka-u.ac.jp Supporting information for this article is available on the WWW under http ://dx.doi.org/10.1002/cbic.201200506.

Design and evaluation of locked nucleic acid-based splice-switching oligonucleotides in vitro

Antisense-mediated modulation of pre-mRNA splicing is an attractive therapeutic strategy for genetic diseases. Currently, there are few examples of modulation of pre-mRNA splicing using locked nucleic acid (LNA) antisense oligonucleotides, and, in particular, no systematic study has addressed the optimal design of LNA-based splice-switching oligonucleotides (LNA SSOs). Here, we designed a series of LNA SSOs complementary to the human dystrophin exon 58 sequence and evaluated their ability to induce exon skipping in vitro using reverse transcription-polymerase chain reaction. We demonstrated that the number of LNAs in the SSO sequence and the melting temperature of the SSOs play important roles in inducing exon skipping and seem to be key factors for designing efficient LNA SSOs. LNA SSO length was an important determinant of activity: a 13-mer with six LNA modifications had the highest efficacy, and a 7-mer was the minimal length required to induce exon skipping. Evaluation of exon skipping activity using mismatched LNA/DNA mixmers revealed that 9-mer LNA SSO allowed a better mismatch discrimination. LNA SSOs also induced exon skipping of endogenous human dystrophin in primary human skeletal muscle cells. Taken together, our findings indicate that LNA SSOs are powerful tools for modulating pre-mRNA splicing.

MicroRNAs Induce Epigenetic Reprogramming and Suppress Malignant Phenotypes of Human Colon Cancer Cells

Although cancer is a genetic disease, epigenetic alterations are involved in its initiation and progression. Previous studies have shown that reprogramming of colon cancer cells using Oct3/4, Sox2, Klf4, and cMyc reduces cancer malignancy. Therefore, cancer reprogramming may be a useful treatment for chemo- or radiotherapy-resistant cancer cells. It was also reported that the introduction of endogenous small-sized, non-coding ribonucleotides such as microRNA (miR) 302s and miR-369-3p or -5p resulted in the induction of cellular reprogramming. miRs are smaller than the genes of transcription factors, making them possibly suitable for use in clinical strategies. Therefore, we reprogrammed colon cancer cells using miR-302s and miR-369-3p or -5p. This resulted in inhibition of cell proliferation and invasion and the stimulation of the mesenchymal-to-epithelial transition phenotype in colon cancer cells. Importantly, the introduction of the ribonucleotides resulted in epigenetic reprogramming of DNA demethylation and histone modification events. Furthermore, in vivo administration of the ribonucleotides in mice elicited the induction of cancer cell apoptosis, which involves the mitochondrial Bcl2 protein family. The present study shows that the introduction of miR-302s and miR-369s could induce cellular reprogramming and modulate malignant phenotypes of human colorectal cancer, suggesting that the appropriate delivery of functional small-sized ribonucleotides may open a new avenue for therapy against human malignant tumors.

Superior Silencing by 2′,4′-BNANC-Based Short Antisense Oligonucleotides Compared to 2′,4′-BNA/LNA-Based Apolipoprotein B Antisense Inhibitors

The duplex stability with target mRNA and the gene silencing potential of a novel bridged nucleic acid analogue are described. The analogue, 2′,4′-BNANC antisense oligonucleotides (AONs) ranging from 10- to 20-nt-long, targeted apolipoprotein B. 2′,4′-BNANC was directly compared to its conventional bridged (or locked) nucleic acid (2′,4′-BNA/LNA)-based counterparts. Melting temperatures of duplexes formed between 2′,4′-BNANC-based antisense oligonucleotides and the target mRNA surpassed those of 2′,4′-BNA/LNA-based counterparts at all lengths. An in vitro transfection study revealed that when compared to the identical length 2′,4′-BNA/LNA-based counterpart, the corresponding 2′,4′-BNANC-based antisense oligonucleotide showed significantly stronger inhibitory activity. This inhibitory activity was more pronounced in shorter (13-, 14-, and 16-mer) oligonucleotides. On the other hand, the 2′,4′-BNANC-based 20-mer AON exhibited the highest affinity but the worst IC50 value, indicating that very high affinity may undermine antisense potency. These results suggest that the potency of AONs requires a balance between reward term and penalty term. Balance of these two parameters would depend on affinity, length, and the specific chemistry of the AON, and fine-tuning of this balance could lead to improved potency. We demonstrate that 2′,4′-BNANC may be a better alternative to conventional 2′,4′-BNA/LNA, even for “short” antisense oligonucleotides, which are attractive in terms of drug-likeness and cost-effective bulk production.

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.

Glucocorticoid-induced reduction of poly(ADP-ribose) synthetase in nuclei from chick embryo liver

Abstract The effect of glucocorticoid hormone administration on the nuclear poly(ADP-ribose) synthetase activity of chick embryoliver was investigated. Compared with the values obtained with control nuclei, the enzyme activity was markedly reduced in the nuclei of liver prepared from chick embryo treated with 0.1 mg hydrocortisone for 12 hours or longer. The possible relationship between the reduction of poly(ADP-ribose) synthetase activity and decrease in DNA synthesis is discussed.

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.

Ca2+ enrichment in culture medium potentiates effect of oligonucleotides

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 Ca2+ 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 Ca2+-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.

XRN2 is required for the degradation of target RNAs by RNase H1-dependent antisense oligonucleotides.

Antisense oligonucleotides (ASOs) can suppress the expression of a target gene by cleaving pre-mRNA and/or mature mRNA via RNase H1. Following the initial endonucleolytic cleavage by RNase H1, the target RNAs are degraded by a mechanism that is poorly understood. To better understand this degradation pathway, we depleted the expression of two major 5 to 3 exoribonucleases (XRNs), named XRN1 and XRN2, and analyzed the levels of 3 fragments of the target RNAs in vitro. We found that the 3 fragments of target pre-mRNA generated by ASO were almost completely degraded from their 5 ends by nuclear XRN2 after RNase H1-mediated cleavage, whereas the 3 fragments of mature mRNA were partially degraded by XRN2. In contrast to ASO, small interference RNA (siRNA) could reduce the expression level of only mature mRNA, and the 3 fragment was degraded by cytoplasmic XRN1. Our findings indicate that the RNAs targeted by RNase H1-dependent ASO are rapidly degraded in the nucleus, contrary to the cytoplasmic degradation pathway mediated by siRNA.