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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. [ABSTRACT FROM AUTHOR]

[Display omitted] Synthetic siRNA molecules without chemical modifications are easily degraded in the body, and 2'- O -modifications are frequently introduced to enhance stability. However, such chemical modifications tend to impact the gene knockdown potency of siRNA negatively. To circumvent this problem, we previously developed a prodrug-type siRNA bearing 2'- O -methyldithiomethyl (MDTM) groups, which can be converted into unmodified siRNA under the reductive environment in cells. In this study, we developed a nuclease-resistant prodrug-type 2'- O -MDTM siRNA for deployment in future animal experiments. To rationally design siRNA modified with a minimal number of 2'- O -MDTM nucleotide residues, we identified the sites susceptible to nuclease digestion and tolerant to 2'- O -methyl (2'-OMe) modification in the antisense strand of apolipoprotein B-targeted siRNA. Subsequently, we optimized the positions where the 2'-OMe and 2'- O -MDTM groups should be incorporated. siRNA bearing the 2'- O -MDTM and 2'-OMe groups at their respective optimized positions exhibited efficient knockdown potency in vitro and enhanced stability in serum. [ABSTRACT FROM AUTHOR]

Small interfering RNAs (siRNAs) are an active agent to induce gene silencing and they have been studied for becoming a biological and therapeutic tool. Various 2′- O -modified RNAs have been extensively studied to improve the nuclease resistance. However, the 2′- O -modified siRNA activities were often decreased by modification, since the bulky 2′- O -modifications inhibit to form a RNA-induced silencing complex (RISC). We developed novel prodrug-type 2′- O -methyldithiomethyl (MDTM) siRNA, which is converted into natural siRNA in an intracellular reducing environment. Prodrug-type 2′- O -MDTM siRNAs modified at the 5′-end side including 5′-end nucleotide and the seed region of the antisense strand exhibited much stronger gene silencing effect than non-prodrug-type 2′- O -methyl (2′- O -Me) siRNAs. Furthermore, the resistances for nuclease digestion of siRNAs were actually enhanced by 2′- O -MDTM modifications. Our results indicate that 2′- O -MDTM modifications improve the stability of siRNA in serum and they are able to be introduced at any positions of siRNA. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]