Triazole- and Tetrazole-Bridged Nucleic Acids: Synthesis, Duplex Stability, Nuclease Resistance, and in Vitro and in Vivo Antisense Potency.

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.

In vitro and in vivo biophysical properties of oligonucleotides containing 5'-thio nucleosides.

Phosphorothioate modification is one of the most widely investigated and promising chemical modifications in oligonucleotide (ON) based therapeutics. Structurally similar 5'-thio or phosphorothiolate-modified nucleotides, in which a 5'-bridging oxygen is replaced with a sulfur atom, are gaining importance for ON-based research. Several reports have been published describing the synthesis of 5'-thio-modified ONs but no detailed in vitro and in vivo data are available. Here, we report the synthesis of 5'-thio-modified 2'-deoxy-5-methylcytidine. 5'-Thio-modified thymidine and 2'-deoxy-5-methylcytidine were incorporated into target ONs, then we evaluated their binding affinity, nuclease stability, RNase H mediated scission, stability in blood serum, and in vitro and in vivo activity. This is the first report showing the influence of 5'-thio-modified antisense ONs in in vitro and in vivo experiments.

Effect of an N-substituent in sulfonamide-bridged nucleic acid (SuNA) on hybridization ability and duplex structure.

A sulfonamide-bridged nucleic acid without an N-substituent (SuNA[NH]) was successfully synthesized. A comparison of the SuNA[NMe]- and SuNA[NH]-modified oligonucleotides revealed that the duplex-forming abilities of the SuNA[NMe]-modified oligonucleotides with complementary DNA and RNA were higher than those of the SuNA[NH]-modified oligonucleotides. The crystal structures of DNA duplexes containing a SuNA[NR] revealed that the helical structures of the two duplexes and hydration patterns around the bridge moiety were different. These results provide insights into hydration patterns and rationale for the high RNA affinity of SuNA-modified oligonucleotides.

Evaluation of the effects of chemically different linkers on hepatic accumulations, cell tropism and gene silencing ability of cholesterol-conjugated antisense oligonucleotides.

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.

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 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.

Amido-bridged nucleic acids with small hydrophobic residues enhance hepatic tropism of antisense oligonucleotides in vivo.

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.

Sulfonamide-bridged nucleic acid: synthesis, high RNA selective hybridization, and high nuclease resistance.

2'-N,4'-C-(N-methylamino)sulfonylmethylene-bridged thymidine (SuNA), which has a six-membered linkage including a sulfonamide moiety, was synthesized and introduced into oligonucleotides. The oligonucleotides containing SuNA exhibited excellent nuclease resistance, a high affinity toward single-stranded RNA, and a low affinity toward single-stranded DNA compared to the natural oligonucleotide.

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.

Development of a system to sensitively and specifically visualize c-fos mRNA in living cells using bispyrene-modified RNA probes.

In situ visualization of c-fos mRNA was shown both in fixed cells and in living cells using bispyrene-modified 2'-O-methyl RNA probes (OMUpy2) or phosphorothioate modified OMUpy2 (OMUpy2-S), which was RNA-specific with high sensitivity, and a system for time-lapse imaging of c-fos mRNA was successfully developed.

Real-time monitoring of mRNAs with fluorescence-modified RNA probes in living cells.

In our previous study, we reported that bispyrene-modified RNA probes (OMUpy2) were useful for RNA detection in homogeneous physiological media(1-2). The aim of this study is to establish in situ monitoring system to detect mRNAs in living cells by OMUpy2. We chose c-fos mRNA as a target RNA which is known as one of the immediate-early genes. The real-time moni- toring of mRNAs was carried out in living C4II cells. After transfection of OMUpy2 into the cells which had been incubated with serum-free medium, cells were stimulated by media with serum and the resulting expression of mRNA was monitored by a fluorescence microscope. In the case of OMUpy2-CF3S, which was complementary to c-fos mRNA, fluorescence emission around 480 nm was observed obviously. These results suggest that mRNA expressed by the serum stimulation was successfully visualized in a real-time manner.