Strategies for In Vivo Screening and Mitigation of Hepatotoxicity Associated with Antisense Drugs

Abstract

Antisense oligonucleotide (ASO) gapmers downregulate gene expression by inducing enzyme-dependent degradation of targeted RNA and represent a promising therapeutic platform for addressing previously undruggable genes. Unfortunately, their therapeutic application, particularly that of the more potent chemistries (e.g., locked-nucleic-acid-containing gapmers), has been hampered by their frequent hepatoxicity, which could be driven by hybridization-mediated interactions. An early de-risking of this liability is a crucial component of developing safe, ASO-based drugs. To rank ASOs based on their effect on the liver, we have developed an acute screen in the mouse that can be applied early in the drug development cycle. A single-dose (3-day) screen with streamlined endpoints (i.e., plasma transaminase levels and liver weights) was observed to be predictive of ASO hepatotoxicity ranking established based on a repeat-dose (15 day) study. Furthermore, to study the underlying mechanisms of liver toxicity, we applied transcriptome profiling and pathway analyses and show that adverse in vivo liver phenotypes correlate with the number of potent, hybridization-mediated off-target effects (OTEs). We propose that a combination of in silico OTE predictions, streamlined in vivo hepatotoxicity screening, and a transcriptome-wide selectivity screen is a valid approach to identifying and progressing safer compounds.

Hepatocyte Deletion of Triglyceride‐Synthesis Enzyme Acyl CoA: Diacylglycerol Acyltransferase 2 Reduces Steatosis Without Increasing Inflammation or Fibrosis in Mice

“…(18) Also, DGAT2 was inhibited by using ASO treatment, which may have off-target effects, such as decreasing DGAT2 expression in the adipose (16) or causing hepatotoxicity independent of the intended target. (19) In our study, the findings of similar degrees of inflammation and fibrosis between genotypes with different hepatic TG content suggests that the amount of hepatic TG is not solely responsible for disease progression. A limitation of interpreting the NASH endpoints in our study is that we found only modest development of fibrosis over several weeks.…”

“…However, the latter study utilized a nutrient‐deficient diet to induce hepatic inflammation and fibrosis, which does not mimic other features of MetS, and, in fact, mice on the MCD diet lose weight . Also, DGAT2 was inhibited by using ASO treatment, which may have off‐target effects, such as decreasing DGAT2 expression in the adipose or causing hepatotoxicity independent of the intended target . In our study, the findings of similar degrees of inflammation and fibrosis between genotypes with different hepatic TG content suggests that the amount of hepatic TG is not solely responsible for disease progression.…”

“…The hepatic inflammation and fibrosis observed in MCD‐diet‐fed mice were exacerbated in DGAT2 ASO‐treated animals, provoking concern for DGAT2 inhibition as a therapy . However, ASO treatments themselves may be associated with toxicity . Because of the uncertainty of DGAT2 deficiency in NAFLD and because de novo lipogenesis appears to be an important contributor to NAFLD in humans, contributing to as much as 26% of liver TG fatty acids, inhibition of DGAT2 warrants further investigation.…”

A Sensitive In Vitro Approach to Assess the Hybridization-Dependent Toxic Potential of High Affinity Gapmer Oligonucleotides

“… 1 , 2 Despite these advantages, ASOs with high-affinity modifications have been associated with a higher risk of inducing liver toxicity. 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 Recent in vivo data suggest that the observed hepatotoxicity is hybridization dependent and requires RNase H1 activity. 10 , 12 Furthermore, the affinity of an ASO is considered to be associated with its hepatotoxic potential, likely because high-affinity ASOs can bind to and mediate cleavage of more unintended target RNAs than lower affinity ASOs.…”

Likelihood of Nonspecific Activity of Gapmer Antisense Oligonucleotides Is Associated with Relative Hybridization Free Energy

“…One proposed mechanism of hepatotoxicity for some high-affinity ASOs is reduction of a high number of unintended transcripts due to RNase H1-dependent RNA degradation [8,12,60]. The 96 ASOs in this study resulted from extensive screening of ASOs and showed no hepatotoxicity or immune stimulation in standard rodent toxicology studies.…”