MYOTONIC DYSTROPHY: EP.235 MyoScreen, a robust muscle-based assay platform validated for screening pharmacologicals against myotonic dystrophy

Abstract

Development of a predictive in vitro model for deciphering the mechanisms involved in Myotonic Dystrophy type 1 (DM1) and accelerating the identification of new therapies remains a crucial unmet need. Our objective was to provide such an in vitro DM1 model capitalizing on the use of MyoScreen™, a well-established high-throughput screening platform integrating human primary skeletal myotubes that demonstrate mature sarcomeric organization and expected responses to chemical/electrical stimulation and pharmacologically relevant drugs. Primary myoblasts from three DM1 patients (blood CTG repeats from 300-1300) and one healthy donor were obtained, amplified and differentiated into aligned myotubes. Main hallmarks of the disease, such as DMPK mRNA foci colocalizing with MBNL1 protein aggregates, were present in the DM1 myotubes and quantified using high-content analysis. No direct relationship between the CTG expansion size and foci number was noted. The splicing profile of several genes involved in the Excitation-Contraction (EC) coupling machinery are altered in DM1. Thus, calcium flux upon chemical induction of EC coupling was investigated and defects in calcium transients were detected in two DM1 donors. To further characterize DM1 myotubes, Cell Profiling was applied to extract over 300 MBNL1 expression features from the generated images. We found this to be a powerful method to discriminate between DM1 and healthy myotube populations. A corresponding machine learning model was built to measure the efficacy of a (CAG)7 ASO (antisense oligonucleotide) to reverse the DM1-associated characteristics. A dose-dependent rescue by up to 60% was observed in DM1 donors. Overall, the DM1 MyoScreen platform will be useful for predictive preclinical testing to rapidly identify new therapeutic targets and select candidate therapeutics targeting skeletal muscle including ASO conjugates, compounds, siRNAs and AAVs (adenovirus associated vectors) producing antisense RNAs and proteins.