Novel image analysis to assess molecular functionality of dystrophin and myofibre regeneration in Duchenne Muscular Dystrophy clinical trials

Topic:

Gene targeted therapies

Poster Number: 84

Author(s):

Dominic Scaglioni, Francesco Catapano, Matthew Ellis, Silvia Torelli, Darren Chambers, Lucy Feng, Saleh Husayni, Jyoti Malhotra, Shawn Harriman, Erica Koenig, Ashish Dugar, Deborah Steiner, Jennifer Morgan, Rahul Phadke, Francesco Muntoni, MD

Institutions:

1. Dubowitz Neuromuscular Centre, UCL Great Ormond Street Institute of Child Health, London, UK, 3. Department of Neurodegenerative Diseases, UCL Queen Square Institute of Neurology, London, UK, 4. Dubowitz Neuromuscular Centre, UCL Great Ormond Street Institute of Child Health, London, UK, 5. Dubowitz Neuromuscular Centre, UCL Queen Square Institute of Neurology & Great Ormond Street Hospit, 6. Dubowitz Neuromuscular Centre, UCL Queen Square Institute of Neurology & Great Ormond Street Hospit, 9. Sarepta Therapeutics, Cambridge, MA, United States, 10. Sarepta Therapeutics, 11. Sarepta Therapeutics, 12. Sarepta Therapeutics, 13. Dubowitz Neuromuscular Centre, UCL Great Ormond Street Institute of Child Health, London, UK, 14. Dubowitz Neuromuscular Centre, UCL Queen Square Institute of Neurology & Great Ormond Street Hospit, 15. Dubowitz Neuromuscular Centre, UCL Institute of Child Health, London, UK

Background: The primary molecular endpoint for many DMD clinical trials and the proof of concept for therapeutic approaches is the induction or increase in the production of de novo dystrophin. However, it is important to not only quantify levels of restored dystrophin following treatment but to determine its molecular functionality using unbiased methodologies.
Objectives: We assessed if induced dystrophin following 48 weeks treatment with an exon 53 skipping morpholino (golodirsen), which we previously demonstrated to be capable of inducing dystrophin restoration, also results in enhanced levels of dystrophin-associated proteins (DAPs) and a reduction in regenerating myofibres in post-treatment biopsies. To do this, we developed an automated, high-throughput method for multiplexed immunofluorescent analysis of dystrophin, DAPs and myofibre regeneration in entire transverse sections of baseline and 48-week skeletal muscle biopsies from the biceps brachii of 25 DMD boys with eligible deletions, enrolled in the SKIP-NMD study. Using Definiens Developer XD (Munich), we investigated the functionality of restored dystrophin by fluorescent quantification of DAPs across entire transverse sections and also in discrete dystrophin positive sarcolemmal regions. Baseline and week 48 skeletal muscle biopsies were also quantified for the number of regenerating myofibres and correlated with levels of dystrophin expression.
Results: While analysis of the entire cohort is ongoing, the initial assessment reveals an increase in DAP intensity in regions of dystrophin positive sarcolemma in post-treatment samples compared to dystrophin negative regions. Furthermore, many patients show a reduction in the percentage of regenerating myofibres at 48 weeks compared to baseline, which contrasts with the known increase in regenerating fibres in DMD during a 48 week period. We also demonstrated a correlation between the percentage of regenerating myofibres and the mean fluorescence intensity of dystrophin; increased mean dystrophin intensity associated with a decrease in myofibre regeneration.
Conclusion: We suggest that quantitative dystrophin-DAP colocalisation data and potentially the percentage of regenerating fibres could be used as a surrogate marker of the functionality of restored dystrophin in future DMD clinical trials.