Nonclinical Exon Skipping Studies with 2′-O-Methyl Phosphorothioate Antisense Oligonucleotides in mdx and mdx-utrn−/− Mice Inspired by Clinical Trial Results

Abstract

Duchenne muscular dystrophy is a severe, progressive muscle-wasting disease that is caused by mutations that abolish the production of functional dystrophin protein. The exon skipping approach aims to restore the disrupted dystrophin reading frame, to allow the production of partially functional dystrophins, such as found in the less severe Becker muscular dystrophy. Exon skipping is achieved by antisense oligonucleotides (AONs). Several chemical modifications have been tested in nonclinical and clinical trials. The morpholino phosphorodiamidate oligomer eteplirsen has been approved by the Food and Drug Administration, whereas clinical development with the 2′- O -methyl phosphorothioate (2OMePS) AON drisapersen was recently stopped. In this study, we aimed to study various aspects of 2OMePS AONs in nonclinical animal studies. We show that while efficiency of exon skipping restoration is comparable in young and older C57BL/10ScSn- Dmd mdx /J ( mdx /BL10) mice, functional improvement was only observed for younger treated mice. Muscle quality did not affect exon skipping efficiency as exon skip and dystrophin levels were similar between mdx /BL10 and more severely affected, age-matched D2- mdx mice. We further report that treadmill running increases AON uptake and dystrophin levels in mdx /BL10 mice. Finally, we show that even low levels of exon skipping and dystrophin restoration are sufficient to significantly increase the survival of mdx-utrn −/− mice from 70 to 97 days.

Using a State-of-the-Art Toolbox to Evaluate Molecular and Functional Readouts of Antisense Oligonucleotide-Induced Exon Skipping in mdx Mice

“…Previous studies in mdx indicated that twice weekly SC administration of 100 mg/kg M23D gave rise to optimal tissue levels and exon skipping [24,25]. Therefore, this dosing regimen was the starting point of this study, including both an 10).…”

“…Some of the exon skip studies in mdx mice, including recent publications, have reported on AON-induced exon 23 skipping data assessed using nonquantitative, sometimes even nested, PCR with gel-based or densitometric assessment of exon skip levels [42][43][44][45]25]. A disadvantage of this approach is that it has the propensity to overestimate exon skip levels due to the PCR bias, favoring amplification of the smaller exon skip product over the exon 23-containing nonskipped amplicon, in particular, when using a large number of PCR cycles or nested PCR [46,17].…”

“…Five -week-old male mdx mice were treated twice weekly subcutaneously with 100 mg/kg M23D (n = 15) or M23D-scr (n = 15). This SC dose of M23D and even higher doses have been previously used in other mdx studies [24,25]. In addition, a group of mdx mice was treated intravenously with M23D in the tail vein (n = 15).…”

A Decade of Progress in Gene Targeted Therapeutic Strategies in Duchenne Muscular Dystrophy: A Systematic Review

“…One clear advantage of AOs in exon skipping provides the basis for modeling, significantly enhancing its ability to be tailored with, thus improving delivery efficiency and exon skipping efficacy. In this review, 15 out of 23 studies have employed various constructs of different backbones comprising of either phosphorodiamidate morpholino oligomers (PMOs) ( Akpulat et al, 2018 ; Lee et al, 2018 ), 2′-O-methyl phosphorothioate (2OMePS) AO ( Van Putten et al, 2019 ), 2′-deoxy-2′-fluoro phosphorothioate (2FPS) AO ( Jirka et al, 2015 ), or 2′-O-methoxyethyl oligonucleotide (MOE) ( Yang et al, 2013 ). The results were encouraging, supporting the use of these constructs as possible alternatives.…”

Developments in reading frame restoring therapy approaches for Duchenne muscular dystrophy

“…However, one has to bear in mind that in most of these mouse studies the exon skipping and dystrophin restoration levels are much higher than observed in humans. Indeed, when restoring lower dystrophin levels in mdx models, functional effects are more modest [112][113][114]. Another consideration is that most mdx models regenerate extremely well.…”