Next Generation Exon 51 Skipping Antisense Oligonucleotides for Duchenne Muscular Dystrophy

Abstract

In the last two decades, antisense oligonucleotides (AONs) that induce corrective exon skipping have matured as promising therapies aimed at tackling the dystrophin deficiency that underlies the severe and progressive muscle fiber degeneration in Duchenne muscular dystrophy (DMD) patients. Pioneering first generation exon 51 skipping AONs like drisapersen and eteplirsen have more recently been followed up by AONs for exons 53 and 45, with, to date, a total of four exon skipping AON drugs having reached (conditional) regulatory US Food and Drug Administration (FDA) approval for DMD. Nonetheless, considering the limited efficacy of these drugs, there is room for improvement. The aim of this study was to develop more efficient [2′- O -methyl-modified phosphorothioate (2′OMePS) RNA] AONs for DMD exon 51 skipping by implementing precision chemistry as well as identifying a more potent target binding site. More than a hundred AONs were screened in muscle cell cultures, followed by a selective comparison in the hDMD and hDMDdel52/ mdx mouse models. Incorporation of 5-methylcytosine and position-specific locked nucleic acids in AONs targeting the drisapersen/eteplirsen binding site resulted in 15-fold higher exon 51 skipping levels compared to drisapersen in hDMDdel52/ mdx mice. However, with similarly modified AONs targeting an alternative site in exon 51, 65-fold higher skipping levels were obtained, restoring dystrophin up to 30% of healthy control. Targeting both sites in exon 51 with a single AON further increased exon skipping (100-fold over drisapersen) and dystrophin (up to 40%) levels. These dystrophin levels allowed for normalization of creatine kinase (CK) and lactate dehydrogenase (LDH) levels, and improved motor function in hDMDdel52/ mdx mice. As no major safety observation was obtained, the improved therapeutic index of these next generation AONs is encouraging for further (pre)clinical development.

Megabase-Scale Transgene De-Duplication to Generate a Functional Single-Copy Full-Length Human DMD Transgenic Mouse Model

“…Furthermore, derivatives of the hDMDTg line such as the del52 exon deletion disease model are more difficult to engineer and validate. While these double-copy humanised models can be used for the testing of ASOs [15], they are not ideal for evaluating strategies that are reliant on DNA double-stranded breaks such as CRISPR-Cas9 gene editing [16]. This is because large intervening deletions between the target sites of the two transgene copies can complicate the analysis of DNA repair outcomes.…”

Considering the Promise of Vamorolone for Treating Duchenne Muscular Dystrophy

“…The ideal therapy for DMD and BMD is to restore functional dystrophin protein in the muscles with many strategies under investigation. One approach is to replace or correct the defective DMD gene (theoretically applicable to most DMD boys), others target gene transcription to normalise the production of dystrophin protein using specifically engineered molecules to target different mutations (e.g., for exonskipping strategy) and thus is applicable to only sub-groups of DMD boys initially [1,14,15]. While many of these potential therapies produce promising results in pre-clinical studies in dystrophic animals, successful clinical translation remains challenging, and they are not yet available for the wide DMD population [14].…”