In recent years, several synthetic nucleic acid sequences that modulate gene expression known as antisense oligonucleotides (ASOs) have been approved to treat various neurological and neuromuscular disorders, including spinal muscular atrophy, Batten disease and Duchenne muscular dystrophy. These therapies primarily work by sterically blocking complementary sequences on RNA molecules, thereby selectively altering gene expression. Decades of research have been dedicated to chemically modifying ASOs to enhance their stability, specificity, and affinity while reducing toxicity from hybridisation-dependent and independent off-target effects.

While there is a wealth of knowledge on the off-target effects of nucleic acids that recruit RNase H for their activities, few reports discuss the off-target effects of steric-blocking ASOs. This study investigated sequence-independent off-target splicing defects caused by ASOs with a combination of ribose and backbone modifications designed as steric blockers. Various ASO chemistries were delivered into cultured cells, and RT-PCR, Sanger sequencing, RNA sequencing, and immunolabeling were used to observe off-target effects. The results indicated chemistry-dependent impacts on gene expression, including cryptic splicing, exon skipping, up- and down-regulation, and isoform-switching. Notably, these off-target defects were absent when the same sequences were synthesised as phosphorodiamidate morpholino oligomers. This highlights that chemistry matters when developing therapeutic ASOs.

As research into tailored splice-modulating therapies continues to expand, our findings provide valuable data that will aid in identifying safe and effective therapeutic candidates.