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

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.