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A therapeutic antisense oligonucleotide encompassing 2′-O-methoxyethyl modification triggers unique perturbation of the transcriptome

ABSTRACT: Antisense oligonucleotides (ASOs) are short nucleic acids that act by annealing to complementary target RNA or DNA sequences. It is currently unknown how the transcriptome-wide off-target effects of different ASO chemistries targeting the same sequence differ. Nusinersen, the first FDA-approved treatment for spinal muscular atrophy (SMA), is an ASO that restores Survival motor neuron 2 (SMN2) exon 7 splicing by binding to the intronic splicing silencer N1 (ISS-N1) element in intron 7. We tested the transcriptome-wide off-target effects in SMA patient fibroblasts of three ASOs targeting ISS-N1: F18OMe, a 2'-O-methyl (OMe) ASO incorporating a phosphorothioate backbone, F18MOE, a 2'-O-methoxyethyl (MOE) ASO incorporating a phosphorothioate backbone, and F20PMO, a phosphorodiamidate morpholino (PMO) ASO. Using minigenes, we showed that skipping of POLR2H exon 2 and seven other exons is triggered by direct annealing of F18MOE to target exonic sequences, and the chemistry-specific effects of F18MOE on exon skipping are primarily due to enhanced mismatch tolerance of MOE. We confirmed that reduction of ASO length and mixed MOE/OMe chemistry are both effective strategies for mitigation of off-target effects. Our findings will be instructive for future design of ASO therapies for SMA as well as other diseases treatable by ASOs.

ORGANISM(S): Homo sapiens

PROVIDER: GSE307431 | GEO | 2026/01/06

REPOSITORIES: GEO

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