Effect of modular conjugation strategy for N-acetylgalactosamine-targeted antisense oligonucleotides.

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.

Cholesterol-GalNAc Dual Conjugation Strategy for Reducing Renal Distribution of Antisense Oligonucleotides.

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.

Synthesis, duplex-forming ability, enzymatic stability, and in vitro antisense potency of oligonucleotides including 2'-C,4'-C-ethyleneoxy-bridged thymidine derivatives.

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.

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.

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.

Serial incorporation of a monovalent GalNAc phosphoramidite unit into hepatocyte-targeting antisense oligonucleotides.

The targeting of abundant hepatic asialoglycoprotein receptors (ASGPR) with trivalent N-acetylgalactosamine (GalNAc) is a reliable strategy for efficiently delivering antisense oligonucleotides (ASOs) to the liver. We here experimentally demonstrate the high systemic potential of the synthetically-accessible, phosphodiester-linked monovalent GalNAc unit when tethered to the 5'-terminus of well-characterised 2',4'-bridged nucleic acid (also known as locked nucleic acid)-modified apolipoprotein B-targeting ASO via a bio-labile linker. Quantitative analysis of the hepatic disposition of the ASOs revealed that phosphodiester is preferable to phosphorothioate as an interunit linkage in terms of ASGPR binding of the GalNAc moiety, as well as the subcellular behavior of the ASO. The flexibility of this monomeric unit was demonstrated by attaching up to 5 GalNAc units in a serial manner and showing that knockdown activity improves as the number of GalNAc units increases. Our study suggests the structural requirements for efficient hepatocellular targeting using monovalent GalNAc and could contribute to a new molecular design for suitably modifying ASO.