666P MyoScreen™ infantile and late-onset Pompe human skeletal muscle disease models for drug discovery and development of next-generation gene therapies.

Pompe Disease (PD) is an inherited metabolic disorder that causes glycogen to accumulate within lysosomes ultimately leading to severe muscle weakness. According to age of onset, it is classified into early and late-onset (EOPD and LOPD). In vitro skeletal muscle 2D models have been reported for EOPD using pluripotent stem cells but concerns exist about their phenotypic and genotypic representation of the disease. To our knowledge there are no reports of a human LOPD skeletal muscle model. We therefore leveraged the MyoScreen platform and our expertise in quantitative image analysis to generate and compare EOPD and LOPD pathological hallmarks. Robust myotube differentiation was obtained from myoblasts from 2 LOPD patients when cultured on micropatterned 96-well plates, while EOPD patient cells differentiate into smaller myotubes and had a lower fusion index (LOPD 40% versus EOPD <20%). Myotubes from both forms of Pompe showed an increase in baseline levels of autophagy compared to Healthy myotubes, as monitored by LC3b intensity (2.3-fold), spot number (1.99-fold) and percent coverage area (2-fold). CQ treatment exacerbated the autophagic build-up further. Only EOPD myotubes presented an accumulation of lysosomes at basal levels (1.7-fold LAMP1 mean intensity versus Healthy). Structurally, the EOPD donor showed large vacuoles filled with LAMP1 positive vesicles that displaced the myofibrillar architecture. Levels of glycogen were quantified biochemically: compared to Healthy myotubes, EOPD and LOPD showed 4 and 1.4-fold increase respectively, and a genotype-phenotype correlation of reduced GAA enzyme activity. Enzyme replacement therapy and liver-directed AAV delivery of the corrective enzyme α-glucosidase have had limited success in improving muscle function due to inefficient internalization. The MyoScreen PD platform provides an unprecedented opportunity for new drug screening, target identification and the assessment of novel emerging RNA and gene therapies. [ABSTRACT FROM AUTHOR]