Shorter Phosphorodiamidate Morpholino Splice-Switching Oligonucleotides May Increase Exon-Skipping Efficacy in DMD

Abstract

Duchenne muscular dystrophy is a fatal muscle disease, caused by mutations in DMD, leading to loss of dystrophin expression. Phosphorodiamidate morpholino splice-switching oligonucleotides (PMO-SSOs) have been used to elicit the restoration of a partially functional truncated dystrophin by excluding disruptive exons from the DMD messenger. The 30-mer PMO eteplirsen (EXONDYS51) developed for exon 51 skipping is the first dystrophin-restoring, conditionally FDA-approved drug in history. Clinical trials had shown a dose-dependent variable and patchy dystrophin restoration. The main obstacle for efficient dystrophin restoration is the inadequate uptake of PMOs into skeletal muscle fibers at low doses. The excessive cost of longer PMOs has limited the utilization of higher dosing. We designed shorter 25-mer PMOs directed to the same eteplirsen-targeted region of exon 51 and compared their efficacies in vitro and in vivo in the mdx52 murine model. Our results showed that skipped-dystrophin induction was comparable between the 30-mer PMO sequence of eteplirsen and one of the shorter PMOs, while the other 25-mer PMOs showed lower exon-skipping efficacies. Shorter PMOs would make higher doses economically feasible, and high dosing would result in better drug uptake into muscle, induce higher levels of dystrophin restoration in DMD muscle, and, ultimately, increase the clinical efficacy.

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

“…Eteplirsen, which is an AO that was granted accelerated approval by the FDA in 2016, induces exon 51 skipping that could benefit around 14% of DMD patients ( Syed, 2016 ; Łoboda and Dulak, 2020 ). However, the efficacy of Eteplirsen is rather controversial as claims of Eteplirsen demonstrating significant dystrophin restoration in phase III of the PROMOVI trial (NCT02255552) were only evidenced by 12 evaluable patients ( Syed, 2016 ; Akpulat et al, 2018 ). The relatively short half-life of AOs and its rapid clearance from the circulation were two challenges that were continuously addressed; the solution would be to increase AO dosage, but this, too, is faltered by excessive cost.…”

“…The relatively short half-life of AOs and its rapid clearance from the circulation were two challenges that were continuously addressed; the solution would be to increase AO dosage, but this, too, is faltered by excessive cost. To remedy this issue, structural modification such that was done by Akpulat et al (2018) in shortening the commercial 30-mer Eteplirsen into 25-mer AOs could allow cost-efficient administration of higher doses.…”

“…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.…”

Restoration of Normal NF1 Function with Antisense Morpholino Treatment of Recurrent Pathogenic Patient-Specific Variant c.1466A>G; p.Y489C

“…PMOs are built on a backbone of mopholine rings connected by phosphorodiamidate linkages and are resistant to a variety of enzymes. Antisense oligos are currently being used to treat diseases like B-thalassemia [9], Duchenne muscular dystrophy [10], and several other genetic conditions. Such oligos have also been evaluated for deep intronic NF1 variants and were shown to restore normal splicing in primary fibroblast and lymphoblast cell lines bearing three different deep intronic variants (c.288+2025T>G; c.5749+332A>G; and c.7908−321C>G) [11].…”

Detailed genetic and functional analysis of the hDMDdel52/mdx mouse model

“…AONs are chemically modified short synthetic RNA-like molecules that have the ability to interfere with the splicing process of pre-mRNAs in a highly sequence-specific manner. By inducing the skipping of an exon adjacent to a deletion of one or more exons, the disrupted reading frame can be restored [ 12 ]. This allows production of truncated but partly functional dystrophin proteins such as produced in Becker muscular dystrophy (BMD), a milder form of dystrophinopathy [ 13 , 14 ].…”