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. 2016 Jul 27:6:30377.
doi: 10.1038/srep30377.

Ribonuclease H1-dependent hepatotoxicity caused by locked nucleic acid-modified gapmer antisense oligonucleotides

Takeshi Kasuya  1 , Shin-Ichiro Hori  1 , Ayahisa Watanabe  2 , Mado Nakajima  1 , Yoshinari Gahara  1 , Masatomo Rokushima  1 , Toru Yanagimoto  1 , Akira Kugimiya  1
Affiliations

Ribonuclease H1-dependent hepatotoxicity caused by locked nucleic acid-modified gapmer antisense oligonucleotides

Takeshi Kasuya et al. Sci Rep. .

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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Figures

Figure 1
Figure 1. Hepatotoxicity of gapmer and ‘non-gapmer’.
(a) Schematic illustration of a gapmer and non-gapmer. The non-gapmer was designed to reduce gapmer activity by substituting two naïve deoxyribonucleotides for LNA at the centre of the gap. Plasma ALT (b), AST (c) levels in mice received 10 mg/kg of GR ASO were analysed at 4, 7 and 10 days after administration. Whole liver weight (d) and GR mRNA level (e) were measured on day 10. The same experiments were performed with Acsl1 gapmer and non-gapmer (20 mg/kg): plasma ALT (f), AST (g), liver weight (h) Acsl1 mRNA level (i), and mRNA level of IFNγ, TNFα, IL6 (j). (n = 4, mean ± S.D. *p < 0.05, Mann–Whitney U test, vs saline).
Figure 2
Figure 2. Effect of RNaseH1 knockdown for GR gapmer-derived hepatotoxicity.
Mice were administrated an siRNA–invivofectamine complex 2 days prior to s.c. injection of 10 mg/kg gapmer. Plasma was collected 4, 7 and 10 days later, and mouse liver was isolated on day 10. (a) Plasma ALT level; (b) plasma AST level; (c) Expression level of RnaseH1 and (d) GR mRNA in liver (n = 4, mean ± S.D., *p < 0.05, Mann–Whitney U test, vs saline + gapmer).
Figure 3
Figure 3. Effect of RNaseH1 knockdown on Acsl1 gapmer-derived hepatotoxicity.
Acsl1 gapmer (10 mg/kg) was subcutaneously administrated 2 days after injection of siRNA-invivofectamine complex, and the liver and plasma were isolated on day 10 after ASO administration. Plasma ALT level (a); plasma AST level (b); expression level of RnaseH1 mRNA (c); Western blot analysis of RnaseH1 and Acsl1 protein (d). Two homogenates of liver in each group were analysed using β-actin as internal standard. Expression level of RnaseH1 mRNA (e) and RnaseH2a mRNA (f). In situ detection of apoptosis (g). Liver sections of mice that received both siRNA and Acsl1 gapmer was subjected to TUNEL assay and observed under confocal laser microscopy (TM; transmission, Scale bar: 5 μm). (n = 4, mean + S.D., *p < 0.05, Mann–Whitney U test, vs si-N.C. + gapmer).
Figure 4
Figure 4. Effect of RNaseH1 knockdown on other hepatotoxic gapmers.
Mice that had received siRNA–invivofectamine complex were administrated hepatotoxic gapmers targeting ApoB (20 mg/kg), Hprt1 (80 mg/kg), or human Kif11 (80 mg/kg). Upper panels show plasma ALT; lower panels show plasma AST. ApoB (a,d), Hprt1 (b,e), and human Kif11 (c,f). (n = 3, mean ± S.D.) #Mouse was euthanized because of acute weight loss.
Figure 5
Figure 5. Comparison of hepatotoxicity and knockdown activity using a gapmer with GalNAc3-siRNA.
(a) Schematic of GalNAc3-siRNA. The siRNA was designed to target almost same position of Acsl1 mRNA as does the gapmer. To avoid degradation, 2′-fluoro- (2′F) and 2′-O-methyl (2′OMe) nucleotides were utilized. (b) Structure of GalNAc3 probe. (c) Acls1 mRNA level. Administration of GalNAc3-siRNA resulted in almost the same level of knockdown as 5 mg/kg/day of gapmer. (d) Plasma ALT and (e) AST levels. (f) Expression profile of cytokines. (n = 3, mean + S.D., *p < 0.01, Mann–Whitney U test, vs saline).
Figure 6
Figure 6. Analysis of off-target knockdown.
(a) Scatter plot of expression level data from Acsl1 gapmer- and saline-administrated mice liver. (b) Presumptive off-target protein-coding genes of the Acsl1 gapmer which showed greater than two-fold significant change. qRT-PCR analysis of the gene expression in liver of gapmer- or GalNAc3-siRNA-treated mice (c), non-gapmer- or gapmer-treated mice (d), and si-RNaseH + gapmer (e) (n = 3 or 4, mean + S.D.).
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