Ribonuclease H1-dependent Hepatotoxicity Caused by Locked Nucleic Acid-modified Gapmer Antisense Oligonucleotides

Overview

Journal Sci Rep

Specialty Science

Date 2016 Jul 28

PMID 27461380

Citations 48

Authors

Takeshi Kasuya

Shin-Ichiro Hori

Ayahisa Watanabe

Mado Nakajima

Yoshinari Gahara

Masatomo Rokushima

Toru Yanagimoto

Akira Kugimiya

Affiliations

Soon will be listed here.

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.

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References

Reijns M, Bubeck D, Gibson L, Graham S, Baillie G, Jones E . The structure of the human RNase H2 complex defines key interaction interfaces relevant to enzyme function and human disease. J Biol Chem. 2010; 286(12):10530-9. PMC: 3060506. DOI: 10.1074/jbc.M110.177394. View

Senn J, Burel S, Henry S . Non-CpG-containing antisense 2'-methoxyethyl oligonucleotides activate a proinflammatory response independent of Toll-like receptor 9 or myeloid differentiation factor 88. J Pharmacol Exp Ther. 2005; 314(3):972-9. DOI: 10.1124/jpet.105.084004. View

Lundin K, Hojland T, Hansen B, Persson R, Bramsen J, Kjems J . Biological activity and biotechnological aspects of locked nucleic acids. Adv Genet. 2013; 82:47-107. DOI: 10.1016/B978-0-12-407676-1.00002-0. View

Lazzaro F, Novarina D, Amara F, Watt D, Stone J, Costanzo V . RNase H and postreplication repair protect cells from ribonucleotides incorporated in DNA. Mol Cell. 2012; 45(1):99-110. PMC: 3262129. DOI: 10.1016/j.molcel.2011.12.019. View

Lima W, Nichols J, Wu H, Prakash T, Migawa M, Wyrzykiewicz T . Structural requirements at the catalytic site of the heteroduplex substrate for human RNase H1 catalysis. J Biol Chem. 2004; 279(35):36317-26. DOI: 10.1074/jbc.M405035200. View

Suzuki Y, Holmes J, Cerritelli S, Sakhuja K, Minczuk M, Holt I . An upstream open reading frame and the context of the two AUG codons affect the abundance of mitochondrial and nuclear RNase H1. Mol Cell Biol. 2010; 30(21):5123-34. PMC: 2953059. DOI: 10.1128/MCB.00619-10. View

Hiller B, Achleitner M, Glage S, Naumann R, Behrendt R, Roers A . Mammalian RNase H2 removes ribonucleotides from DNA to maintain genome integrity. J Exp Med. 2012; 209(8):1419-26. PMC: 3409502. DOI: 10.1084/jem.20120876. View

Nowotny M, Gaidamakov S, Ghirlando R, Cerritelli S, Crouch R, Yang W . Structure of human RNase H1 complexed with an RNA/DNA hybrid: insight into HIV reverse transcription. Mol Cell. 2007; 28(2):264-76. DOI: 10.1016/j.molcel.2007.08.015. View

van Poelgeest E, Swart R, Betjes M, Moerland M, Weening J, Tessier Y . Acute kidney injury during therapy with an antisense oligonucleotide directed against PCSK9. Am J Kidney Dis. 2013; 62(4):796-800. DOI: 10.1053/j.ajkd.2013.02.359. View

10.

Kawai T, Akira S . Toll-like receptors and their crosstalk with other innate receptors in infection and immunity. Immunity. 2011; 34(5):637-50. DOI: 10.1016/j.immuni.2011.05.006. View

11.

Burel S, Han S, Lee H, Norris D, Lee B, Machemer T . Preclinical evaluation of the toxicological effects of a novel constrained ethyl modified antisense compound targeting signal transducer and activator of transcription 3 in mice and cynomolgus monkeys. Nucleic Acid Ther. 2013; 23(3):213-27. DOI: 10.1089/nat.2013.0422. View

12.

Watanabe A, Nakajima M, Kasuya T, Onishi R, Kitade N, Mayumi K . Comparative Characterization of Hepatic Distribution and mRNA Reduction of Antisense Oligonucleotides Conjugated with Triantennary N-Acetyl Galactosamine and Lipophilic Ligands Targeting Apolipoprotein B. J Pharmacol Exp Ther. 2016; 357(2):320-30. DOI: 10.1124/jpet.115.230300. View

13.

Wu H, Lima W, Zhang H, Fan A, Sun H, Crooke S . Determination of the role of the human RNase H1 in the pharmacology of DNA-like antisense drugs. J Biol Chem. 2004; 279(17):17181-9. DOI: 10.1074/jbc.M311683200. View

14.

Liang Y, Osborne M, Monia B, Bhanot S, Watts L, She P . Antisense oligonucleotides targeted against glucocorticoid receptor reduce hepatic glucose production and ameliorate hyperglycemia in diabetic mice. Metabolism. 2005; 54(7):848-55. DOI: 10.1016/j.metabol.2005.01.030. View

15.

Burel S, Machemer T, Ragone F, Kato H, Cauntay P, Greenlee S . Unique O-methoxyethyl ribose-DNA chimeric oligonucleotide induces an atypical melanoma differentiation-associated gene 5-dependent induction of type I interferon response. J Pharmacol Exp Ther. 2012; 342(1):150-62. DOI: 10.1124/jpet.112.193789. View

16.

Kawabata T, Kinoshita M, Inatsu A, Habu Y, Nakashima H, Shinomiya N . Functional alterations of liver innate immunity of mice with aging in response to CpG-oligodeoxynucleotide. Hepatology. 2008; 48(5):1586-97. DOI: 10.1002/hep.22489. View

17.

Reijns M, Rabe B, Rigby R, Mill P, Astell K, Lettice L . Enzymatic removal of ribonucleotides from DNA is essential for mammalian genome integrity and development. Cell. 2012; 149(5):1008-22. PMC: 3383994. DOI: 10.1016/j.cell.2012.04.011. View

18.

Swayze E, Siwkowski A, Wancewicz E, Migawa M, Wyrzykiewicz T, Hung G . Antisense oligonucleotides containing locked nucleic acid improve potency but cause significant hepatotoxicity in animals. Nucleic Acids Res. 2006; 35(2):687-700. PMC: 1802611. DOI: 10.1093/nar/gkl1071. View

19.

Cerritelli S, Frolova E, Feng C, Grinberg A, Love P, Crouch R . Failure to produce mitochondrial DNA results in embryonic lethality in Rnaseh1 null mice. Mol Cell. 2003; 11(3):807-15. DOI: 10.1016/s1097-2765(03)00088-1. View

20.

Kakiuchi-Kiyota S, Koza-Taylor P, Mantena S, Nelms L, Enayetallah A, Hollingshead B . Comparison of hepatic transcription profiles of locked ribonucleic acid antisense oligonucleotides: evidence of distinct pathways contributing to non-target mediated toxicity in mice. Toxicol Sci. 2013; 138(1):234-48. DOI: 10.1093/toxsci/kft278. View