Gene expression dynamics limit efficacy of ASOs in kinetic simulations

Poster presented on Oct. 20, 2025 by Aleksandra Khatova at 21st Annual Meeting of the Oligonucleotide Therapeutics Society, Budapest, Hungary.

Abstract: Antisense oligonucleotides (ASOs) are usually optimized under the simplifying assumption that their target mRNA is at steady state. However, gene expression is a highly dynamic process, and the abundance of transcripts of most human genes fluctuates widely [1, 2]. In this work, we extend the kinetic model of RNase-H-mediated ASO action of Pedersen et al. [3] to study the performance of ASOs under the temporal regulation of the target mRNA transcription. Stochastic simulations were performed for the systems where the production rates of target mRNA were undergoing periodic or stochastic pulses of upregulation modeling gene expression dynamics in the cell. The simulations showed that ASOs may not be able to efficiently handle the increase of mRNA levels during the periods of unregulated transcription, especially if the half-life of target mRNA is short. Increasing ASO dose or the ASO:mRNA duplex association rate dampened, but did not eliminate, the peaks in mRNA levels. Altering the dissociation rate of ASO:mRNA duplex had little effect on the amplitude of the peaks. Additionally, we have augmented the Pedersen system with the subset of reactions describing interactions with a close off-target. Stochastic simulations of this system in the presence of transcriptional pulses showed that when ASO is designed to have the best efficacy during the peaks of target upregulation, it may have increased knockdown of the off-target mRNA in periods when no upregulation is happening. These results demonstrate that gene expression dynamics is another criterion to consider when selecting targets and ASOs.

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