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Updated: Jan 2, 2026
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Evaluation of Exon Inclusion Induced by Splice Switching Antisense Oligonucleotides in SMA Patient Fibroblasts
Published on: May 11, 2018
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Hybridization-mediated off-target effects of splice-switching antisense oligonucleotides
Juergen Scharner1, Wai Kit Ma1, Qian Zhang1
1Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA.
Nucleic Acids Research
|December 6, 2019
Summary
Splice-switching antisense oligonucleotides (ASOs) can cause unintended mis-splicing. Strategies like mixed chemistry and strategic mismatches reduce these off-target effects, improving ASO safety for genetic disease therapies.
Area of Science:
- Molecular Biology
- Genetics
- RNA Therapeutics
Background:
- Splice-switching antisense oligonucleotides (ASOs) are a promising therapeutic approach for genetic diseases by modulating pre-mRNA splicing.
- ASOs are known for their specificity but potential for off-target effects has not been systematically studied.
Purpose of the Study:
- To investigate whether splice-modulating ASOs induce hybridization-dependent mis-splicing of unintended RNA targets.
- To identify factors influencing off-target splicing events and explore strategies to mitigate them.
Main Methods:
- In vitro testing of splice-modulating ASOs against 108 predicted off-target sequences.
- Analysis of mis-splicing events induced by different ASO chemistries, lengths, and delivery methods.
- Evaluation of ASOs with strategically placed mismatches.
Main Results:
- 17 mis-splicing events were identified for one ASO targeting unintended sequences.
- Off-target effects were found to be difficult to predict and influenced by ASO chemistry.
- Mixed-chemistry ASOs, shorter ASOs, combined ASOs, and free uptake delivery reduced off-target activity.
Conclusions:
- ASO off-target effects are a concern that requires careful consideration during therapeutic design.
- ASO chemistry, sequence design, and delivery methods significantly impact off-target splicing.
- Strategies such as mixed chemistry and strategic mismatches can effectively reduce unwanted off-target splicing events, enhancing ASO safety and efficacy.
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