Antisense oligonucleotides (AONs) have emerged as a promising therapy for Duchenne Muscular Dystrophy (DMD), a severe neuromuscular disease caused by dystrophin deficiency. Early AON drugs like drisapersen and eteplirsen achieved limited success, leading researchers to develop more efficient next-generation AONs. This study focused on refining AONs for exon 51 skipping through advanced chemical modifications and identifying optimal target sites to enhance efficacy and safety.

Methods and Target Optimization

Researchers screened over 100 AONs in muscle cell cultures and humanized mouse models, using novel chemical modifications such as 5-methylcytosine and locked nucleic acids (LNAs). AONs targeting both established and alternative exon 51 sites were evaluated. One AON targeting a newly identified site achieved a 65-fold higher exon skipping and restored dystrophin levels to 30-40% of normal. Synergistic effects were observed when combining two target sites, improving dystrophin expression and reducing biomarkers like creatine kinase and lactate dehydrogenase, indicators of muscle damage.

Preclinical Findings

Preclinical studies in hDMDdel52/mdx mouse models revealed that the new AONs not only improved dystrophin expression but also enhanced motor functions and normalized serum biomarkers. The best-performing AONs demonstrated significant efficiency and a favorable safety profile, with minimal histopathological effects.

Comparison to Becker Muscular Dystrophy

Importantly, these AONs restored muscle integrity to levels comparable to those seen in Becker Muscular Dystrophy (BMD), a milder form of dystrophinopathy. Using techniques like droplet digital PCR (ddPCR) and capillary Western immunoassays, researchers quantitatively confirmed the enhanced efficacy of these next-generation AONs.

Conclusion and Future Directions

This study emphasizes the benefits of targeting alternative sites within exon 51 and optimizing chemical structures. Such advancements allow for lower dosing while maintaining therapeutic efficacy, a crucial step toward safer, more effective treatments for DMD patients.

Future work will involve further preclinical safety studies and potentially clinical trials. These findings represent a significant leap in the development of exon-skipping therapies, offering hope for improved outcomes for DMD patients.