D. Frank

Duchenne and Becker Muscular Dystrophies: A Review of Animal Models, Clinical End Points, and Biomarker Quantification:

Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are neuromuscular disorders that primarily affect boys due to an X-linked mutation in the DMD gene, resulting in reduced to near absence of dystrophin or expression of truncated forms of dystrophin. Some newer therapeutic interventions aim to increase sarcolemmal dystrophin expression, and accurate dystrophin quantification is critical for demonstrating pharmacodynamic relationships in preclinical studies and clinical trials. Current challenges with measuring dystrophin include the variation in protein expression within individual muscle fibers and across whole muscle samples, the presence of preexisting dystrophin-positive revertant fibers, and trace amounts of residual dystrophin. Immunofluorescence quantification of dystrophin can overcome many of these challenges, but manual quantification of protein expression may be complicated by variations in the collection of images, reproducible scoring of fluorescent intensity, and bias introduced by manual scoring of typically only a few high-power fields. This review highlights the pathology of DMD and BMD, discusses animal models of DMD and BMD, and describes dystrophin biomarker quantitation in DMD and BMD, with several image analysis approaches, including a new automated method that evaluates protein expression of individual muscle fibers.

Eteplirsen treatment for Duchenne muscular dystrophy: Exon skipping and dystrophin production

Objective To describe the quantification of novel dystrophin production in patients with Duchenne muscular dystrophy (DMD) after long-term treatment with eteplirsen. Methods Clinical 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. Results Reverse 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). Conclusion Taken 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 evidence This 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.

226th ENMC International Workshop: . Towards validated and qualified biomarkers for therapy development for Duchenne muscular dystrophy 20-22 January 2017, Heemskerk, The Netherlands

Twenty-three participants from 6 countries (England; Germany; Italy; Sweden, The Netherlands; USA) attended the 226th ENMC workshop on Duchenne biomarkers “Towards validated and qualified biomarkers for therapy development for Duchenne Muscular Dystrophy.” The meeting was a follow-up of the 204th ENMC workshop on Duchenne muscular dystrophy biomarkers. The workshop was organized with the support of Parent Project Muscular Dystrophy (PPMD) and Marathon Pharmaceuticals, which provided travel support for participants from the US via an unrestricted grant to PPMD in addition to ENMC support. It was attended by representatives of academic institutions, industry working in the Duchenne muscular dystrophy field and patient representatives. 1.1. Background to the workshop 1.1.1. Biomarkers Biomarkers are defined as biological, measurable and quantifiable indicators of underlying biological processes. Different types of biomarkers can be distinguished: diagnostic biomarkers indicate the presence of disease, prognostic biomarkers correlate with predicted disease course, and therapeutic biomarkers are designed to predict or measure response to treatment [1]. Therapeutic biomarkers can indicate whether a therapy is having an effect. This type of biomarker is called a pharmacodynamics biomarker and can be used to e.g. show that a missing protein is restored after a therapy. Safety biomarkers assess likelihood, presence, or extent of toxicity as an adverse effect, e.g. through monitoring blood markers indicative of liver or kidney damage. Sometimes biomarkers can also be used as primary endpoints in clinical trials instead of functional outcome measures, and these are termed “surrogate endpoints”. In Europe [2,3] biomarkers can only be used as surrogate endpoints after going through a rigorous regulatory process to officially qualify them for this purpose. Similar pathways exist in the US, where the Food and Drug Administration (FDA) also supplies a process for qualification of biomarkers for other contexts of use.