Block or degrade? Balancing on- and off-target effects of antisense strategies against transcripts with expanded triplet repeats in DM1

• Published in: Molecular therapy. Nucleic acids 2023-06, Vol.32, p.622-636
• Main Authors: El Boujnouni, Najoua, van der Bent, M. Leontien, Willemse, Marieke, ’t Hoen, Peter A.C., Brock, Roland, Wansink, Derick G.
• Format: Article
• Language: English
• Subjects: antisense oligonucleotide; DMPK; gapmer; MBNL1; MT: Oligonucleotides: Therapies and Applications; myotonic dystrophy type 1; off-target; Original; steric hindrance; triplet repeat
• ISSN: 2162-2531; 2162-2531
• DOI: 10.1016/j.omtn.2023.04.010

Antisense oligonucleotide (ASO) therapies for myotonic dystrophy type 1 (DM1) are based on elimination of transcripts containing an expanded repeat or inhibition of sequestration of RNA-binding proteins. This activity is achievable by both degradation of expanded transcripts and steric hindrance, although it is unknown which approach is superior. We compared blocking ASOs with RNase H-recruiting gapmers of equivalent chemistries. Two DMPK target sequences were selected: the triplet repeat and a unique sequence upstream thereof. We assessed ASO effects on transcript levels, ribonucleoprotein foci and disease-associated missplicing, and performed RNA sequencing to investigate on- and off-target effects. Both gapmers and the repeat blocker led to significant DMPK knockdown and a reduction in (CUG)exp foci. However, the repeat blocker was more effective in MBNL1 protein displacement and had superior efficiency in splicing correction at the tested dose of 100 nM. By comparison, on a transcriptome level, the blocking ASO had the fewest off-target effects. In particular, the off-target profile of the repeat gapmer asks for cautious consideration in further therapeutic development. Altogether, our study demonstrates the importance of evaluating both on-target and downstream effects of ASOs in a DM1 context, and provides guiding principles for safe and effective targeting of toxic transcripts. [Display omitted] Wansink and colleagues compared mechanistically different ASOs as therapeutics for myotonic dystrophy type 1, which revealed differences in efficacies that—combined with the distinct off-target profiles of the triplet repeat blocker and gapmer oligos used—highlight the need for multiple biological readouts for preclinical ASO selection.