Role of Computationally Evaluated Target Specificity in the Hepatotoxicity of Gapmer Antisense Oligonucleotides

Area of Science:

• Oligonucleotide Therapeutics
• Toxicology
• RNA Biology

Background:

• Gapmer antisense oligonucleotides (ASOs) are therapeutic agents that can exhibit off-target RNA cleavage.
• This off-target cleavage is a potential mechanism underlying gapmer-induced hepatotoxicity.
• A hypothesis suggests that increased specificity of gapmers correlates with reduced hepatotoxicity.

Purpose of the Study:

• To investigate the relationship between the specificity of Malat1-targeting gapmers and their associated hepatotoxicity.
• To determine if in silico specificity analysis can predict and mitigate the risk of gapmer-induced liver toxicity.

Main Methods:

• Design and synthesis of various Malat1-targeting gapmer antisense oligonucleotides.
• Computational evaluation of target specificities for each designed gapmer.
• Assessment of hepatotoxicity and transcriptome analysis to identify off-target RNA cleavage events.

Main Results:

• Contrary to expectations, a significant relationship between gapmer specificity and hepatotoxicity was not observed.
• In silico analysis of gapmer sequences did not consistently predict or mitigate hepatotoxicity risk.
• Nontoxic gapmers, despite possessing numerous target-like RNA sequences, did not exhibit off-target RNA cleavage.

Conclusions:

• Gapmer specificity, as predicted by in silico methods, does not reliably correlate with hepatotoxicity.
• The mechanism of gapmer hepatotoxicity may involve off-target RNA cleavage, but specificity alone is not the sole determinant.
• Further research is needed to fully elucidate the complex mechanisms governing gapmer safety and efficacy.