ObjectiveTo describe the quantification of novel dystrophin production in patients with Duchenne muscular dystrophy (DMD) after long-term treatment with eteplirsen.MethodsClinical study 202 was an observational, open-label extension of the randomized, controlled study 201 assessing the safety and efficacy of eteplirsen in patients with DMD with a confirmed mutation in the DMD gene amenable to correction by skipping of exon 51. Patients received once-weekly IV doses of eteplirsen 30 or 50 mg/kg. Upper extremity muscle biopsy samples were collected at combined study week 180, blinded, and assessed for dystrophin-related content by Western blot, Bioquant software measurement of dystrophin-associated immunofluorescence intensity, and percent dystrophin-positive fibers (PDPF). Results were contrasted with matched untreated biopsies from patients with DMD. Reverse transcription PCR followed by Sanger sequencing of newly formed slice junctions was used to confirm the mechanism of action of eteplirsen.ResultsReverse transcription PCR analysis and sequencing of the newly formed splice junction confirmed that 100% of treated patients displayed the expected skipped exon 51 sequence. In treated patients vs untreated controls, Western blot analysis of dystrophin content demonstrated an 11.6-fold increase (p = 0.007), and PDPF analysis demonstrated a 7.4-fold increase (p < 0.001). The PDPF findings were confirmed in a re-examination of the sample (15.5-fold increase, p < 0.001). Dystrophin immunofluorescence intensity was 2.4-fold greater in treated patients than in untreated controls (p < 0.001).ConclusionTaken together, the 4 assays, each based on unique evaluation mechanisms, provided evidence of eteplirsen muscle cell penetration, exon skipping, and induction of novel dystrophin expression.Classification of evidenceThis study provides Class II evidence of the muscle cell penetration, exon skipping, and induction of novel dystrophin expression by eteplirsen, as confirmed by 4 assays.
504. Restoration of Dystrophin Expression by Gene Editing with S. aureus Cas9 in Models of Duchenne Muscular Dystrophy
