Consequences of Making the Inactive Active Through Changes in Antisense Oligonucleotide Chemistries

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Antisense oligonucleotides (ASOs) are crucial for modulating pre-messenger RNA (pre-mRNA) processing.
• Standard ASOs show variable efficiency in inducing exon skipping for genetic disorders like Duchenne muscular dystrophy (DMD).
• Ineffective ASO sequences necessitate optimization for enhanced therapeutic potential.

Purpose of the Study:

• To re-evaluate ineffective antisense oligonucleotide sequences by incorporating locked nucleic acid (LNA) residues.
• To assess the splice-switching efficiency of LNA-modified ASOs targeting specific dystrophin (DMD) exons.
• To investigate potential off-target effects and alternative splicing events induced by LNA-ASOs.

Main Methods:

• Synthesis of ASOs targeting human DMD exons 16, 23, and 51 using two chemistries: 2'-O-methyl modified bases and LNA/2'-O-methyl mixmers.
• Transfection of synthesized ASOs into primary human myotubes.
• Analysis of DMD transcripts to quantify exon skipping efficiency and identify alternative splice variants.

Main Results:

• Standard 2'-O-methyl ASOs showed no detectable exon skipping for the targeted DMD exons.
• All corresponding LNA/2'-O-methyl mixmer ASOs successfully induced targeted exon excision.
• The LNA-ASO targeting exon 51 resulted in unexpected transcripts with partial exon 51 retention.

Conclusions:

• LNA/2'-O-methyl mixmer ASOs demonstrate superior efficacy in inducing exon skipping compared to standard ASOs.
• The enhanced efficiency of LNA-ASOs may be accompanied by the activation of cryptic splice sites and potential off-target gene expression effects.
• LNA modification represents a promising strategy for improving ASO-based exon skipping therapies, warranting further investigation into safety profiles.