Hepatotoxicity of high affinity gapmer antisense oligonucleotides is mediated by RNase H1 dependent promiscuous reduction of very long pre-mRNA transcripts

Abstract

High affinity antisense oligonucleotides (ASOs) containing bicylic modifications (BNA) such as locked nucleic acid (LNA) designed to induce target RNA cleavage have been shown to have enhanced potency along with a higher propensity to cause hepatotoxicity. In order to understand the mechanism of this hepatotoxicity, transcriptional profiles were collected from the livers of mice treated with a panel of highly efficacious hepatotoxic or non-hepatotoxic LNA ASOs. We observed highly selective transcript knockdown in mice treated with non-hepatotoxic LNA ASOs, while the levels of many unintended transcripts were reduced in mice treated with hepatotoxic LNA ASOs. This transcriptional signature was concurrent with on-target RNA reduction and preceded transaminitis. Remarkably, the mRNA transcripts commonly reduced by toxic LNA ASOs were generally not strongly associated with any particular biological process, cellular component or functional group. However, they tended to have much longer pre-mRNA transcripts. We also demonstrate that the off-target RNA knockdown and hepatotoxicity is attenuated by RNase H1 knockdown, and that this effect can be generalized to high affinity modifications beyond LNA. This suggests that for a certain set of ASOs containing high affinity modifications such as LNA, hepatotoxicity can occur as a result of unintended off-target RNase H1 dependent RNA degradation.

Figure 1.

(A) Plasma ALT levels from of Balb/c mice treated with increasing doses of LNA ASO were measured at 96 h (single subcutaneous injection). Animals found dead prior to 96 h were arbitrarily assigned an ALT value of 20 000 IU/ml if they had shown increase ALT at 48 or 72 h (open circles). (B) Oneway hierarchical clustering of transcripts significantly modulated by LNA ASOs. A single dose for each LNAASO is shown. The highest tested dose (300 mg/kg) for safe ASOs (ALT < 200 IU/mL at 96 h) is represented, whereas the lowest dose producing an increase in ALT level of approximately 1000 IU/ml is represented for toxic ASO (Union of unpaired Student's t-test assuming equal variance, 2-fold change, FDR ≤ 0.001, 1788 Transcripts). Transcription profiles from 12 saline treated livers and 4 treated livers per ASO/dose group are represented. Down-regulated transcripts are represented in shades of blue proportional to the intensity of change, while up-regulated transcripts are represented in shades of red proportional to the intensity of change. Unchanged transcripts are colored in gray.

Figure 2.

(A) Increased ALT levels in mice is correlated with an increased number of genes with lowered expression. Skew was calculated using standard methods as implemented in the python scipy package. Each dot represents an LNA ASO at a specific dose represented by the color of the dot. (B–D) Cumulative distribution function plot demonstrating that hepatotoxic LNA ASOs (FXI-1, SOD1–2 and PTEN LNA, respectively) are down-regulating a greater proportion of significantly modulated transcripts (P-value ≤ 0.05) and to a greater intensity (absolute fold change) than significantly up-regulated transcripts (P-value ≤ 0.05).

Figure 3.

(A) Upper panel: Mean of plasma ALT (IU/ml) measured 96 h post treatment. Bottom panel: Oneway hierarchical clustering of transcripts significantly modulated in the liver at 24 h post treatment by LNA ASOs in correlation with ALT increase at 96 h. (Prob > 0.000001, r² > 0.54). Thirteen LNA ASOs were tested at 1 or 2 doses (min 11 mg/kg, max 300 mg/kg as detailed in Table 3). Each column represents the average expression for a given ASO/dose. Up-regulated transcripts are represented in shades of red (n = 353) and down-regulated transcripts in shades of blue (n = 240). (B) Top 5 mRNA transcripts whose modulation 24 h post treatment is the most correlated to ALT increase 96 h post treatmentselected from panel A. Normalized liver mRNA transcripts level 24 h post ASO treatment (y-axis) versus Log2 of serum ALT level 96 h post treatment (x-axis) for the R² is shown.

Figure 4.

(A) Median length of mRNA and pre-mRNA transcripts expressed on the microarray as a function of the modulation: ‘Down’ represents the number of transcripts down-regulated 24 h post treatment (240) negatively correlated to ALT increase at 96 h, ‘Up’ represents the number of transcripts up-regulated 24 h post treatment (353) positively correlated to ALT increase at 96 h (Prob > 0.000001, r² > 0.54). Distribution of mRNA (B) and pre-mRNA (C) transcripts expressed on array. The blue horizontal line represents the median length of down-regulated transcripts, in gray unchanged transcripts and in red, up-regulated transcripts. (D) Cumulative probability plot of the basal expression levels of long (pre-mRNA length > 125 000 b) down-regulated transcripts with high (red), medium (green) or minimal correlation to ALT increase, shows transcripts with low level of basal expression tend to exhibit a low level of correlation with ALT increase following treatment with hepatotoxic ASOs. (E–G) Cumulative probability plot of fold change of modulated transcripts binned by pre-mRNA length for three hepatotoxic LNA ASOs (FXI-1, SOD1–2 and FVII-3 LNA at 33 mg/kg, respectively) treatment group are represented. The proportion of transcripts as well as the intensity of transcript down-regulation increases as a function of the length of pre-mRNA transcripts.

Figure 5.

Effect of anti-RNase H1 MOE ASO pretreatment on LNA ASO mediated hepatotoxicity as measure by plasma ALT increase. Mice were pretreated with either ISIS 104838 (black, control MOE ASO) or ISIS 385005 (gray, anti-RNase H1 MOE ASO) prior to treatment with hepatotoxic LNA ASOs at 25 and 75 mg/kg. (A) mRNA transcript levels were measured by RT–PCR in the liver of mice treated for 24 h with the hepatotoxic LNA ASOs at 25 or 75 mg/kg. (B) Plasma ALT levels are measured 72 h after LNA ASO administration.

Figure 6.

Different combination of MOE ASO mixtures to modulate levels of various combination of mRNA target reduction results in synergistic induction of hepatotoxicity in conjunction with targeted mRNA and liver protein reduction. (A) Effect of MOE ASO mixtures on plasma ALT levels (top panel) and five different mRNA target down-regulation (bottom five panels). MOE ASO targets are ordered according to t-test significance (derived from panel B) from bottom to top. ‘0’ and ‘50’ indicates the absence or presence of a given MOE ASO in the mixture. (B) Effect of specific mRNA down-regulation by MOE ASOs on the circulating serum ALT levels regardless of the presence of other MOE ASOs present in the mixture. The level of significance is indicated lower left corner below the target name. (C) Specific protein reduction (Rptor and Ppp3ca) in the liver of mice treated with ASO mixtures measured by western blot.