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

Najoua El Boujnouni; M Leontien van der Bent; Marieke Willemse; Peter A C 't Hoen; Roland Brock; Derick G Wansink

doi:10.1016/j.omtn.2023.04.010

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

Mol Ther Nucleic Acids. 2023 Apr 20:32:622-636. doi: 10.1016/j.omtn.2023.04.010. eCollection 2023 Jun 13.

Authors

Najoua El Boujnouni¹, M Leontien van der Bent¹, Marieke Willemse¹, Peter A C 't Hoen¹, Roland Brock^{1

2}, Derick G Wansink¹

Affiliations

¹ Department of Medical BioSciences, Research Institute for Medical Innovation, Radboud University Medical Center, 6525 GA Nijmegen, the Netherlands.
² Department of Medical Biochemistry, College of Medicine and Medical Sciences, Arabian Gulf University, Manama 293, Bahrain.

Abstract

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.

Keywords: DMPK; MBNL1; MT: Oligonucleotides: Therapies and Applications; antisense oligonucleotide; gapmer; myotonic dystrophy type 1; off-target; steric hindrance; triplet repeat.