Sara Aguti, Shuzhi Cheng, Pierpaolo Ala, Sean Briggs, Francesco Muntoni, Haiyan Zhou (Molecular Therapy Nucleic Acids)
June 4, 2024
Gapmer antisense oligonucleotides (ASOs) hold therapeutic promise for allele-specific silencing, but face challenges in distinguishing between mutant and wild-type transcripts. This study explores new design strategies to enhance ASO specificity, focusing on a common dominant mutation in COL6A3 gene associated with Ullrich congenital muscular dystrophy. Initial gapmer ASO design exhibited high efficiency but poor specificity for the mutant allele. We then adopted a mixmer design, incorporating additional RNA bases based on computational predictions of secondary structures for both mutant and wild-type alleles, aiming to enhance ASO accessibility to mutant transcripts. The mixmer ASO design demonstrated up to a 3-fold increase in specificity compared with the classical gapmer design. Further refinement involved introducing a nucleotide mismatch as a structural modification, resulting in a 10-fold enhancement in specificity compared with the gapmer design and a 3-fold over the mixmer design. Additionally, we identified for the first time a potential role of the RNA-induced silencing complex (RISC), alongside RNase H1, in gapmer-mediated silencing, in contrast with what was observed with mixmer ASOs, where only RNase H1 was involved. In conclusion, this study presents a novel design concept for allele-specific ASOs leveraging mRNA secondary structures and nucleotide mismatching and suggests a potential involvement of RISC in gapmer-mediated silencing.
Aguti, S., Ala, P., Briggs, S., Muntoni, F., & Zhou, H. (2024, June 4). Strategies to improve the design of gapmer antisense oligonucleotide on allele-specific silencing. https://www.cell.com/molecular-therapy-family/nucleic-acids/fulltext/S2162-2531(24)00124-0#articleInformation
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