Duchenne muscular dystrophy

Adenine base editing (ABE) is a technically plausible approach for the DMD c.2609T>G nonsense variant. The T>G transversion is not directly addressable by current ABE or CBE windows, but cytosine base editing of the complementary strand (G>A on the sense strand reading C>T on the antisense strand) could restore the Leu codon. Careful PAM identification and window positioning are required; the alternative is a prime editing strategy targeting the precise G>T reversion. Both modalities warrant feasibility modeling before moving to cell-based validation.

DMD c.2609T>G introduces a premature stop at Leu870 within the rod domain, disrupting dystrophin mechanical function. ClinVar classifies this as Pathogenic (single submitter, criteria provided). Nonsense variants in DMD exons 10�C40 have been addressed experimentally by exon skipping and, more recently, by base editors and prime editors in patient-derived myotubes and mdx mouse models (Chemello et al. 2021, PMID:33861946; Xu et al. 2021, PMID:34380046). ABE8e and PE3 platforms have demonstrated >20% correction efficiency in cardiomyocytes derived from DMD iPSCs, supporting translational feasibility for point variants in this gene. PAM availability across exon 19�C20 junction region (where c.2609 resides) is reasonable given SpCas9-NG and SaCas9 variants with relaxed PAM requirements.

The precise T>G transversion at c.2609 does not fall within the canonical editing window of ABE7.10 or CBE3, so prime editing or a next-generation base editor with shifted windows (e.g., ABE8e with extended window) must be confirmed by in silico modeling. Muscle delivery via AAV remains the primary bottleneck: dual-AAV split-intein strategies for large cargo (PE3 ~6.3 kb) are still under optimization, and transduction efficiency in cardiac muscle lags behind skeletal muscle. Off-target analysis by orthogonal sequencing (CIRCLE-seq, GUIDE-seq) has not been reported for this specific variant site. Long-term dystrophin restoration data beyond 6 months in animal models are lacking. No patient-specific iPSC or organoid work has been published for this allele.

For DMD c.1950T>A (p.Cys650Ter), a nonsense variant in exon 15 of the dystrophin gene, CRISPR-mediated strategies offer two complementary approaches: (1) permanent exon skipping via disruption of the exon 15 splice acceptor or donor to exclude the nonsense-containing exon from mature mRNA, producing an internally truncated but partially functional Becker-like dystrophin, or (2) direct base editing to revert the stop codon if the target adenine falls within an ABE editing window. Both approaches aim to restore dystrophin expression in skeletal and cardiac muscle. CRISPR-based permanent exon skipping has the advantage over ASO-based exon skipping (e.g., eteplirsen-class drugs) of requiring only a single treatment to achieve lifelong reading frame restoration, versus chronic weekly/monthly ASO injections.

CRISPR-mediated exon skipping has been demonstrated in DMD patient-derived iPSC-cardiomyocytes and in deltaE50-mdx mice, where Cas9-mediated disruption of exon splice sites restores dystrophin expression and improves muscle function (Amoasii et al., Science 2018; PMID:29439201). In vivo AAV-CRISPR delivery to skeletal and cardiac muscle has shown sustained dystrophin restoration over 12+ months in canine DMD models (Amoasii et al., Nat Med 2018; PMID:30250058). For p.Cys650Ter in exon 15, exon skipping of exon 15 (or exons 14-15 to maintain reading frame depending on phase) would remove 81-162 amino acids from the spectrin-repeat rod domain — a region tolerant of internal deletions based on Becker muscular dystrophy genotype-phenotype correlations. ClinVar classifies this variant as Pathogenic. Alternatively, ABE could potentially convert the TAA stop codon (from the c.1950T>A change creating a TGA→TAA stop on the sense strand; the antisense target A could be edited to G) to restore a sense codon. Vertex/CRISPR Therapeutics and Editas Medicine are advancing in vivo CRISPR-DMD programs, though none have reached Phase 3.

No in vivo CRISPR gene editing therapy for DMD has been approved or completed Phase 3 trials as of early 2026. AAV-mediated delivery of CRISPR components to sufficient skeletal muscle mass (30-40% of body weight) requires high systemic AAV doses with associated hepatotoxicity and immune response risks. Pre-existing anti-Cas9 immunity (present in ~65% of humans for SpCas9) may limit editing efficiency. Cardiac muscle transduction is critical for long-term survival but may require separate dosing optimization. For p.Cys650Ter specifically, the exon skipping strategy depends on exon 15 removal maintaining the reading frame — this requires confirmation that the exon 14-exon 16 junction produces an in-frame transcript. Off-target editing in the large dystrophin gene (2.4 Mb) and elsewhere in the genome requires thorough safety characterization. The Becker-like truncated dystrophin produced by exon skipping provides partial function but is not equivalent to full-length dystrophin — the degree of clinical benefit depends on which spectrin repeats are deleted and whether the nNOS-binding domain is preserved. Long-term durability of editing in post-mitotic myonuclei is expected to be stable, but editing in proliferating satellite cells is needed for regeneration-dependent muscle maintenance.

Exon skipping using antisense oligonucleotides (ASOs) targeting the exon containing the c.2916T>G nonsense mutation is a plausible therapeutic strategy for this DMD variant. The premature stop codon at p.Tyr972Ter truncates dystrophin in the central rod domain, but skipping the affected exon (exon 22) could restore the reading frame and produce a partially functional, internally deleted dystrophin—analogous to the milder Becker muscular dystrophy phenotype. Multiple exon-skipping ASOs have received FDA approval for other DMD exons (eteplirsen for exon 51, golodirsen for exon 53, viltolarsen for exon 53, casimersen for exon 45), establishing clinical precedent for this modality.

1. The c.2916T>G variant introduces a premature stop codon (p.Tyr972Ter) in exon 22 of the DMD gene, classified as Pathogenic in ClinVar. The mutation falls in the spectrin-like repeat region of the dystrophin rod domain. 2. Exon-skipping ASOs have demonstrated partial dystrophin restoration in DMD patients with out-of-frame deletions: eteplirsen (exon 51), golodirsen and viltolarsen (exon 53), and casimersen (exon 45) have FDA approval, though with modest functional benefit. 3. For nonsense mutations, skipping the exon containing the stop codon can restore the reading frame if the flanking exons maintain frame compatibility. Exon 22 skipping in DMD has been explored in preclinical models. 4. Translational read-through compounds (e.g., ataluren) represent an alternative RNA-level approach for nonsense mutations, though clinical efficacy data for ataluren remain limited and its EU approval was not renewed in 2024.

1. Exon-skipping efficiency for exon 22 specifically has limited clinical data compared to the more commonly targeted exons (51, 53, 45). ASO design, delivery, and tissue penetration in cardiac muscle remain significant challenges. 2. The resulting internally deleted dystrophin from exon 22 skipping may have variable functionality depending on the structural role of the skipped region in the rod domain. 3. Systemic delivery to both skeletal and cardiac muscle is a persistent bottleneck—current ASOs show preferential skeletal muscle uptake with limited cardiac penetration. 4. Long-term efficacy and durability data for exon-skipping therapies remain limited, with modest dystrophin restoration levels (typically <5-10% of normal) in approved products. 5. This analysis is based on general exon-skipping principles applied to this specific variant; dedicated preclinical validation for exon 22 skipping with this particular nonsense mutation would be needed.

Prime Editing (PE5max/PEmax) via Dual-AAV delivery is a rationale-driven therapeutic strategy for Duchenne muscular dystrophy targeting the DMD c.1A>T (p.Met1Leu) variant (Pathogenic, missense variant, initiator_codon_variant, intron variant). The editing system (PEmax with engineered pegRNA) search-and-replace editing that directly rewrites the pathogenic transversion back to wild-type without requiring DSBs. Target tissue: Muscle. Therapeutic goal: Correct or bypass pathogenic DMD mutations in skeletal and cardiac muscle to restore sufficient dystrophin expression and stabilize muscle function. Risk profile: off-target Low (prime editing has inherently low off-target rate), delivery complexity Medium, immunogenicity High.

1. Molecular basis: DMD NM_004006.3(DMD):c.1A>T (p.Met1Leu) is classified as Pathogenic (ClinVar variation ID 4774215). Molecular consequence: missense variant, initiator_codon_variant, intron variant. Protein change: M1L. 2. Epidemiology: Duchenne muscular dystrophy (DMD) is an X-linked recessive neuromuscular disorder with an estimated birth prevalence of ~1 in 3,500–5,000 live male births, characterized by onset of muscle weakness in early childhood, loss of ambulation around 9–13 years without effective disease-modifying therapy, 3. Standard of care: Current standard care combines long-term glucocorticoid therapy, multidisciplinary cardiac and respiratory management, and orthopedic and rehabilitative support. Mutation-specific drugs such as ataluren for nonsense mutations and exon-skipping ASOs (e.g., eteplirsen, golodirsen) are available for su 4. Pipeline: Multiple AAV micro-dystrophin gene therapies (e.g., ELEVIDYS, fordadistrogene) have completed Phase I/II and Phase III trials with mixed efficacy and important safety signals; one product has FDA approval with confirmatory studies ongoing. Exon-skipping and read-through agents have completed Phase I 5. Prime editing validation: PEmax (Chen et al. 2021, Cell) enables precise insertions, deletions, and all 12 point mutations without DSBs. Prime Medicine is advancing PE programs into clinical development. LNP and dual-AAV delivery of PE have been demonstrated in preclinical liver and CNS models.

1. No published data specifically correcting DMD c.1A>T (p.Met1Leu) with Prime Editing (PE5max/PEmax); strategy is based on general principles and must be validated preclinically. 3. Dual-AAV delivery requires intein-mediated protein reconstitution with lower efficiency than single-AAV. Pre-existing AAV immunity in the patient population may limit eligibility. 4. Delivery to Muscle tissue remains a major translational bottleneck. Current vectors have limited transduction efficiency in these compartments. 4. Long-term durability, off-target genome-wide effects, and immunogenicity in the target patient population require thorough preclinical and clinical evaluation.

For DMD c.8547+2T>C, a likely pathogenic splice donor variant at the intron 59 boundary that disrupts normal pre-mRNA splicing and is predicted to cause out-of-frame exon skipping or intron retention leading to Duchenne phenotype, antisense oligonucleotide-mediated exon skipping represents the most variant-class-appropriate RNA therapy approach. The c.8547+2T>C variant destroys the canonical GT splice donor of exon 59. Therapeutic exon skipping using phosphorodiamidate morpholino oligomers (PMOs) could target exon 59 (or adjacent exons depending on reading frame analysis) to restore an in-frame transcript encoding a Becker-like internally deleted but partially functional dystrophin. Four exon-skipping ASOs are FDA-approved for DMD (eteplirsen for exon 51, golodirsen for exon 53, viltolarsen for exon 53, casimersen for exon 45), validating the platform.

Exon-skipping ASOs for DMD have demonstrated proof-of-concept across multiple exon targets. Eteplirsen (Exondys 51) received accelerated FDA approval in 2016 based on modest dystrophin restoration (~0.9% of normal by Western blot). Viltolarsen (Viltepso, exon 53 skipping) showed ~5.7% dystrophin restoration at 24 weeks in the Phase 2 trial (PMID: 32981936). The reading frame rule (Monaco et al., 1988) predicts that in-frame deletions within the central rod domain produce milder Becker phenotype rather than severe Duchenne. For exon 59, which encodes part of spectrin-like repeat 22-23 near the C-terminal region, skipping feasibility depends on whether removal maintains an open reading frame with the adjacent exons 58 and 60. Next-generation peptide-conjugated PMOs (PPMOs) are in clinical development with substantially improved tissue penetration to cardiac and skeletal muscle compared to unconjugated PMOs.

Current exon-skipping ASOs achieve low levels of dystrophin restoration (typically 1-5% of normal), which may be below the threshold needed for meaningful clinical benefit (~15-20% is thought necessary based on Becker dystrophy genotype-phenotype studies). All approved exon-skipping ASOs required accelerated approval pathways based on the surrogate endpoint of dystrophin protein restoration rather than functional clinical endpoints. Confirmatory trials have shown inconsistent functional benefit. ASO therapy requires lifelong weekly IV infusions (for PMOs) or subcutaneous injections, creating substantial treatment burden and cost. For c.8547+2T>C specifically, the amenability to exon 59 skipping requires detailed reading frame analysis — not all splice-site variants in this region will produce a functional protein with exon 59 removal. PPMOs (peptide-PMOs) under development by Sarepta and others may improve tissue delivery and dystrophin restoration levels, but are not yet approved.