Acute hepatotoxicity of 2' fluoro-modified 5-10-5 gapmer phosphorothioate oligonucleotides in mice correlates with intracellular protein binding and the loss of DBHS proteins

Abstract

We reported previously that a 2' fluoro-modified (2' F) phosphorothioate (PS) antisense oligonucleotides (ASOs) with 5-10-5 gapmer configuration interacted with proteins from Drosophila behavior/human splicing (DBHS) family with higher affinity than PS-ASOs modified with 2'-O-(2-methoxyethyl) (2' MOE) or 2',4'-constrained 2'-O-ethyl (cEt) did. Rapid degradation of these proteins and cytotoxicity were observed in cells treated with 2' F PS-ASO. Here, we report that 2' F gapmer PS-ASOs of different sequences caused reduction in levels of DBHS proteins and hepatotoxicity in mice. 2' F PS-ASOs induced activation of the P53 pathway and downregulation of metabolic pathways. Altered levels of RNA and protein markers for hepatotoxicity, liver necrosis, and apoptosis were observed as early as 24 to 48 hours after a single administration of the 2' F PS-ASO. The observed effects were not likely due to the hybridization-dependent RNase H1 cleavage of on- or potential off-target RNAs, or due to potential toxicity of 2' F nucleoside metabolites. Instead, we found that 2' F PS-ASO associated with more intra-cellular proteins including proteins from DBHS family. Our results suggest that protein-binding correlates positively with the 2' F modification-dependent loss of DBHS proteins and the toxicity of gapmer 2' F PS-ASO in vivo.

Figure 1.

PS-ASOs induce hepatotoxicity in mice in a sugar modification-dependent manner. (A) Schematic representation of the 5–10–5 Pten gapmer ASO. (B) Plasma levels of ALT and AST upon a single administration of Pten PS-ASOs modified with 2′ MOE (ION-116847), cEt (ION-582801) or 2′ F (ION-404130) at 100 or 400 mg/kg for 96 h. N = 3 per group. (C) Plasma levels of ALT and AST upon a single administration of Pten LNA (ION-390896) at 100 or 400 mg/kg for 96 h. N = 3 per group. (D) Plasma levels of ALT and AST upon repeated administration of 2′ F ASO (ION-404130) at 20 mg/kg per dose, three doses per week. S-saline, and F-2′ F PS-ASO (ION-404130). ( E) qRT-PCR of on-target Pten mRNA. Mice were treated as was described in B. The error bars indicate data acquired from three individual animals. (F) qRT-PCR showed dose-dependent reduction of target Pten mRNA in mouse hepatocytes by Pten ASOs modified with 2′ MOE (ION-116847), cEt (ION-582801), or 2′ F (ION-404130). ASOs were delivered by lipid transfection. The error bars represent standard deviation from three separate experiments. (G) Plasma levels of ALT and AST upon a single administration of PS-ASOs modified with either 2′ MOE or 2′ F at 400 mg/kg for 96 h. Seven pairs of 2′ MOE or 2′ F-modified sequences were included. Hepatotoxic potentials of the 2′ F PS-ASOs were rank-ordered and color-coded (red: high hepatotoxic potentials; and green: low hepatotoxic potentials) based on plasma levels of ALT and AST. N = 3 per group. F.D. = found death.

Figure 2.

2′ F PS-ASO (ION-404130) activated multiple stress pathways and induced expression of genes involved in apoptosis. (A) Numbers of genes altered in liver transcriptomes of mice 96 h after the administration of saline or 400 mg/kg 2′ MOE ASO (ION-116847), cEt ASO (ION-582801) or 2′ F ASO (ION-404130), as detected by microarray analyses. (B) Unsupervised hierarchical clustering analysis of 273 genes differentially expressed in ASO-treated mice compared to saline-treated mice. Logarithmic values of treated to untreated expression ratios are shown in the heat map using red and green color codes for up- and down-regulation, respectively. Experiments were done with three animals per group. (C) qRT-PCR of transcripts regulated downstream of P53 pathway, including Cdkn1a/p21 and Gadd45a. The error bars indicate data acquired from three individual animals. (D) Western analysis of liver proteins isolated from animals administrated with saline, 2′ MOE (ION-116847), cEt (ION-582801), or 2′ F (ION-404130) PS-ASOs. ASOs were administered at 400 mg/kg and animals were sacrificed at 96 h after the administration of ASOs. Experiments were done with three animals per individual group. Bip serves as a loading control. PARP_CL-cleaved PARP. ( E) Western analysis of liver proteins isolated from animals administrated with saline, 2′ MOE (ION-141923), or F (ION-804856) PS-ASOs. ASOs were administered at 400 mg/kg and animals were sacrificed at 96 h after the administration of ASOs. Experiments were done with three animals per individual group. HSP90 serves as a loading control. PARP_CL-cleaved PARP.

Figure 3.

Time course of mRNA alterations and stress phenotypes induced by 2′ F PS-ASO (ION-404130) in vivo. ( A–F) Mice were treated with a single 400 mg/kg 2′ F PS-ASO (ION-404130) or with saline and indicated parameters were quantified at 24, 48, 72 and 96 h after dosing. N = 3 per group. The error bars indicate data acquired from three individual animals. S-saline, and F-2′ F PS-ASO (ION-404130). (A) qRT-PCR quantification of on-target Pten mRNA levels in liver. (B) Plasma levels of ALT and AST. (C) qRT-PCR of mRNA transcripts Cdkn1a/p21 and Gadd45a. (D) qRT-PCR of liver mRNA levels of anti-apoptosis markers Mcl1 and Bcl-xl. (E) qRT-PCR of liver mRNA levels of necrosis markers that are up-regulated (Map3k6 and Cd68) or down-regulated (Lgr5 and Rhbg) by 2′ F ASO. (F) Representative images of H&E staining and active caspase 3 staining of liver sections.

Figure 4.

2′ F PS-ASO causes a reduction of DBHS proteins in vivo. (A and B) Mice were treated with a single dose of 400 mg/kg 2′ F PS-ASO (ION-404130) or with saline and indicated parameters were quantified at 24, 48, 72 and 96 h after dosing. N = 3 per group. (A) Western analyses of isolated liver proteins. HSP90 serves as a loading control. PARP_CL-cleaved PARP. Levels of p54nrb and PSF proteins were quantitate by ImageJ. N.D.-band not detected. (B) qRT-PCR of mRNA transcripts of P54nrb and PSF. The error bars indicate data acquired from three individual animals. S-saline, and F-2′ F PS-ASO (ION-404130). (C) HeLa cells were treated with control luciferase siRNA (Con), P54nrb siRNA, PSF siRNA or both P54nrb and PSF siRNAs for 96 h. Treated cells were then either mock-transfected or transfected with 200 nM 2′ F ASO (ION-404130) for another 12 h. Western analysis was performed. GAPDH serves as a loading control.

Figure 5.

Adverse effects observed in vivo with 2′ F PS-ASO are not likely due to the hybridization-dependent off-target cleavage events. Potential off-target liver transcripts identified based on sequence homology. Mismatches and indels on these RNAs relative to the Pten ASO sequence are indicated in red. mRNA levels of these transcripts are based on microarray results (N = 3). Transcripts were significantly altered if P-value <0.05. M-2′ MOE PS-ASO (ION-116847), C-cEt PS-ASO (ION-582801), F-2′ F PS-ASO (ION-404130) and S-saline.

Figure 6.

2′ F PS-ASO associates with more intracellular proteins than do 2′ MOE or cEt PS-ASOs. (A) ASO-binding proteins were pulled down from HeLa lysate with a 5′ biotinylated Pten ASO modified with cEt (ION-586183: 5′-biotinylated-582801) and were eluted by completion using 2′ MOE (ION-116847), cEt (ION-582801) or 2′ F (ION-404130). Sliver staining was performed to show the eluted proteins. Western blot was performed for P54nrb, PSF and SSBP1. (B) ASO-binding proteins were pulled down from HeLa lysate with ION-116847, ION-582801 and ION-404130 duplexed with a 5′ biotinylated complementary RNA. Western blot was performed for UPF1, STAU1, KU80 and AGO2.