title: Ribonuclease H1-dependent hepatotoxicity caused by locked nucleic acid-modified gapmer antisense oligonucleotides.
abstract: Gapmer antisense oligonucleotides cleave target RNA effectively in vivo, and is considered as promising therapeutics. Especially, gapmers modified with locked nucleic acid (LNA) shows potent knockdown activity; however, they also cause hepatotoxic side effects. For developing safe and effective gapmer drugs, a deeper understanding of the mechanisms of hepatotoxicity is required. Here, we investigated the cause of hepatotoxicity derived from LNA-modified gapmers. Chemical modification of gapmer's gap region completely suppressed both knockdown activity and hepatotoxicity, indicating that the root cause of hepatotoxicity is related to intracellular gapmer activity. Gene silencing of hepatic ribonuclease H1 (RNaseH1), which catalyses gapmer-mediated RNA knockdown, strongly supressed hepatotoxic effects. Small interfering RNA (siRNA)-mediated knockdown of a target mRNA did not result in any hepatotoxic effects, while the gapmer targeting the same position on mRNA as does the siRNA showed acute toxicity. Microarray analysis revealed that several pre-mRNAs containing a sequence similar to the gapmer target were also knocked down. These results suggest that hepatotoxicity of LNA gapmer is caused by RNAseH1 activity, presumably because of off-target cleavage of RNAs inside nuclei.
authors.affiliation: Discovery Research Laboratories for Innovative Frontier Medicines, Shionogi &Co., LTd., Osaka, 561-0825, JAPAN.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Kasuya
authors.affiliation: Discovery Research Laboratories for Innovative Frontier Medicines, Shionogi &Co., LTd., Osaka, 561-0825, JAPAN.
authors.forename: Shin-Ichiro
authors.initials: S
authors.lastname: Hori
authors.affiliation: Research Laboratory for Development, Shionogi &Co., LTd., Osaka, 561-0825, JAPAN.
authors.forename: Ayahisa
authors.initials: A
authors.lastname: Watanabe
authors.affiliation: Discovery Research Laboratories for Innovative Frontier Medicines, Shionogi &Co., LTd., Osaka, 561-0825, JAPAN.
authors.forename: Mado
authors.initials: M
authors.lastname: Nakajima
authors.affiliation: Discovery Research Laboratories for Innovative Frontier Medicines, Shionogi &Co., LTd., Osaka, 561-0825, JAPAN.
authors.forename: Yoshinari
authors.initials: Y
authors.lastname: Gahara
authors.affiliation: Discovery Research Laboratories for Innovative Frontier Medicines, Shionogi &Co., LTd., Osaka, 561-0825, JAPAN.
authors.forename: Masatomo
authors.initials: M
authors.lastname: Rokushima
authors.affiliation: Discovery Research Laboratories for Innovative Frontier Medicines, Shionogi &Co., LTd., Osaka, 561-0825, JAPAN.
authors.forename: Toru
authors.initials: T
authors.lastname: Yanagimoto
authors.affiliation: Discovery Research Laboratories for Innovative Frontier Medicines, Shionogi &Co., LTd., Osaka, 561-0825, JAPAN.
authors.forename: Akira
authors.initials: A
authors.lastname: Kugimiya
mesh_terms: D000818:Animals; D020868:Gene Silencing; D008099:Liver / Q000378:metabolism*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D009841:Oligonucleotides / Q000235:genetics / Q000633:toxicity*; D016376:Oligonucleotides, Antisense / Q000235:genetics / Q000633:toxicity*; D016914:Ribonuclease H / Q000235:genetics* / Q000378:metabolism
chemical_list: D009841:Oligonucleotides; D016376:Oligonucleotides, Antisense; C477371:locked nucleic acid; D016914:Ribonuclease H; C078856:ribonuclease HI