Exon skipping is a leading therapeutic approach for Duchenne muscular dystrophy (DMD), aprogressive muscle wasting disorder caused by pathogenic variants in the DMD gene that typically disrupt the translation reading frame. This approach aims to modulate DMD pre-mRNA splicing to reframe the transcript and generate an internally deleted but partially functional quasi-dystrophin protein. >> Learn More: What is exon skipping?
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FDA-approved exon skipping therapies clinical efficacy is very limited
The study mentioned four FDA-approved exon skipping therapies and clearly stated that their clinical efficacy is quite limited. To investigate how treatment timing affects exon skipping outcomes, this scientific study involved injecting dystrophin-deficient mdx mice with peptide-phosphorodiamidate morpholino oligonucleotide (PPMO) exon skipping conjugates, starting from adult (12 weeks) or aged (75 weeks) stages, and collecting tibialis anterior muscle tissue for biochemical and transcriptomic analyses.
In individuals affected by DMD, initiating exon skipping therapies early increases their effectiveness
Mean Dmd exon 23 skipping was 79% in adults and 44% in aged PPMO-treated mdx mice, whereas dystrophin protein restoration was 35% and 8%, respectively. Histopathological improvements were only evident in the adult-treated mice. PPMO-treatment in adult mdx mice induced a broad transcriptomic shift towards a wild-type signature, whereas treatment in aged mice resulted in negligible gene expression changes, indicating that late intervention is ineffective at reversing diseaseassociated pathologies despite low-level dystrophin restoration. Dystrophin transcript imbalance was corrected only in adult-treated mdx mice. Increased expression of the dystrophin-repressing microRNA miR-31-5p, which was more strongly upregulated in aged mdx muscle, provides a potential mechanistic explanation. In conclusion, PPMO-mediated exon skipping is substantially more effective when initiated in adult rather than aged dystrophic muscle, supporting early therapeutic intervention in DMD-affected individuals. >> Learn More: Potential Duchenne Population Amenable for Exon Skipping
FDA-approved DMD therapies have limited effectiveness and are very expensive
Recent years have seen substantial progress in the field of experimental therapeutics for DMD. Various strategies have been employed to restore dystrophin. For example, antisense oligonucleotide (ASO)-mediated exon skipping can be utilised to modulate alternative splicing such that the dystrophin translation reading frame is restored. Four such antisense oligonucleotide drugs received accelerated approval from the US FDA for the treatment of DMD boys (Eteplirsen, Viltolarsen, Golodiresen, and Casimersen). While a major achievement for the DMD research and patient communities, the dystrophin restoration achieved by these compounds is minimal, and of debatable clinical benefit.
Similarly, the FDA approved the first gene therapy for DMD (Elevidys) in 2023, despite failure to reach its clinical endpoint in a Phase 3 trial. Furthermore, the safety of Elevidys (and high dose adeno-associated virus-based gene therapies in general) has been called into question following multiple patient deaths. >> Discover More: Why Elevidys Was Not Approved by the European Medicines Agency (EMA)?
What is the difference between PMOs and PPMOs?
The current generation of exon skipping ASOs are all phosphorodiamidate morpholino oligonucleotides (PMOs). This chemistry does not occur in nature, and is widely regarded as being safe. However, PMO delivery to skeletal (and especially cardiac) muscle is very limited, which has motivated a search for delivery-assisting moieties that can enhance their activity. The scientists has extensively explored the use of peptide-conjugated PMOs (PPMOs) for such purposes. PPMOs are much more potent than unconjugated PMOs, and exhibit exon skipping activity in cardiac tissue. PPMOs are currently being commercially developed by PepGen Ltd and Entrada Therapeutics. Similarly, Avidity Biosciences and Dyne Therapeutics are developing PMOantibody and PMO-FAb fragment therapeutics for DMD. >> Read More: Next Generation Exon Skipping Therapies
What is an mdx mouse, and what are its characteristics?
The mdx mouse is the most commonly-used murine model of DMD, which carries a premature termination codon in Dmd exon 23, skipping of which results in an X-linked muscular dystrophy that exhibits some aspects of DMD pathology. In this study scientists have previously performed a variety of omics analyses in this mouse with a focus on the effects of dystrophin restoration therapies, including gene expression/microRNA microarray, small RNA-Seq, and high resolution mass spectrometry-based proteomics.
Efficacy of dystrophin restoration therapies
The total amount of dystrophin restored.
It is widely accepted that the more dystrophin that can be restored the better. Various lines of evidence suggest that ~10% of healthy dystrophin levels is a sensible threshold for therapeutic benefit.
The quality of dystrophin restored.
Exon skipping splice correction and microdystrophin gene therapies both rely on the generation of internally-deleted quasi-dystrophin proteins that resemble those found in some Becker muscular dystrophy patients, and which are associated with mild pathology. However, such quasi-dystrophins are unlikely to fully compensate for the loss of the full-length dystrophin protein. There is a rowing appreciation that fulllength dystrophins are desirable, which presents a challenge given that the size of the dystrophin ORF (~11 kb) exceeds the packaging capacity of AAV vectors (~4.6 kb). Restoration of full-length dystrophin has been demonstrated in human clinical trial participants treated with an exon skipping therapy to treat DMD exon2 duplication. Furthermore, there are multiple other efforts to develop full-length dystrophin delivery strategies using split-vector approaches, or by using vectors with higher packaging capacities.
Correct localization of dystrophin at the sarcolemma.
This study has shown that uniform distribution of dystrophin protein along the sarcolemma within a myofiber is an important determinant of therapeutic success. Importantly, exon skipping treatment with PPMO conjugates induced a uniform pattern of sarcolemmal dystrophin coverage (even at low doses), while CRISPR-Cas9-mediated exon deletion resulted in a patchy pattern of dystrophin distribution. This is an important observation, as incomplete sarcolemmal dystrophin coverage is likely to be insufficient to prevent myofiber turnover and correct disease pathology.
Optimal timing for initiation of treatment.
Early initiation of dystrophin restoration therapies may be beneficial to prevent or delay pathological degeneration of muscle. However, for ‘one-and-done’ therapies like microdystrophin gene therapy or CRISPR-Cas9-mediate gene correction, the number of corrected nuclei will be progressively diluted as non-corrected myonuclei are added to myofibers as a consequence of growth and regeneration.
In this study, scientists assessed the efficacy of exon skipping using PPMO antisense oligonucleotide conjugates when the treatment was initiated at the adult and aged stages. These data support the notion that early treatment may be beneficial in the treatment of DMD.
Exon skipping therapies may also be implemented as part of a combination therapy
Exon skipping therapy could be initiated in young DMD boys with the aim of maintaining their muscle function until such an age that treatment with a gene therapy may be safe and appropriate. Indeed, preclinical evidence is supportive of a synergistic effect of combined PMO-mediated exon skipping with either AAV-U7-mediated exon skipping or AAV-microdystrophin gene therapy. This study adds to the discourse concerning the appropriate time to start exon skipping therapy in DMD patients. Evidence from the preclinical data presented herein supports the notion of initiating exon skipping therapy at an early age, before the establishment of advanced tissue pathology.
Keep Reading: Timing matters: exon skipping therapy is most effective when initiated early in a mouse model of Duchenne muscular dystrophy
