Neuromuscular Circuits Can Help Evaluate the Functional Outcome of CRISPR in Duchenne Muscular Dystrophy

Mutations in the dystrophin gene (DMD) cause Duchenne muscular dystrophy (DMD), the most common neuromuscular disorder in childhood. DMD patients suffer from progressive skeletal muscle weakness and wasting, resulting in poor quality of life and reduced life expectancy. Unfortunately, the current standard of care can only slow disease progression, and there is still no cure or effective treatment for DMD.

»Progress in finding a cure for neuromuscular disorders has been hampered by the lack of human-relevant pre-clinical models amenable to studying neuromuscular connectivity in healthy and disease conditions and for assessing the efficacy of drug candidates. The aim of our study was to develop such a model by exploiting human pluripotent stem cells, CRISPR-mediated genome editing, bioengineering and light-controllable neuromuscular activity,« says Yung-Yao Lin. He is a geneticist at the Queen Mary University of London and the leader of the team that recently presented such a model in Science Advances.

»To mimic the anatomical arrangement of motor neurons and skeletal muscle, the devices were designed to have a central compartment that accommodates the myofibers and two outer compartments for the aggregates of motor neurons and astrocytes. The outer compartments connect to the central compartment via microchannels so that the motor neurons can project their axons to innervate the myofibers,« says Amaia Paredes-Redondo, a former research scientist in Lin's group and the first author of the paper.

A pulse of light shows the effect of CRISPR gene editing

To allow the model to compare the function of mutated and CRISPR-corrected myofibers, fibroblasts from a DMD patient were reprogrammed to obtain pluripotent stem cells (PSCs). Then, before further differentiating them to myotubes and myofibers, some of the PSCs were subjected to CRISPR correction of the disease-causing mutation. In this case, the DMD c.10141C>T mutation was perfectly corrected using homology-directed repair and piggyBac transposon-based selection. In principle, however, any DMD-causing mutation from other patients could be corrected using different CRISPR strategies. Thus, the main thing was to obtain an isogenic pair of patient-specific cells that only differed in having either a mutated or corrected version of DMD.

»In our micro-physiological model of neuromuscular circuits, the contraction velocity of myofibers reflect the motor function. As we have shown in our paper, inhibition of TGFβ signalling restored motor function in DMD neuromuscular circuits. Thus, our micro-physiological model can be used to de-risk and prioritise potential therapeutics at the pre-clinical stage of the drug development process,« says Yung-Yao Lin.

The model is a drug screening platform for DMD

However, the optogenetic model of DMD can also be used to study the pathophysiology of DMD myofibers and identify potential therapeutic targets. In their paper, Yung-Yao Lin and his co-workers used it to elucidate the role of TGFβ signalling.

»Previous studies have shown that TGFβ signalling regulates skeletal muscle homeostasis and that TGFβ signalling is abnormally upregulated in DMD skeletal muscle. Nevertheless, the role of TGFβ signalling in neuromuscular connectivity remains unclear. Our study found that genes regulating neuromuscular junction assembly and axon guidance were dysregulated in DMD myogenic cultures. However, inhibition of TGFβ signalling normalises the expression of many of these genes leading to a beneficial effect for motor function,« explains Amaia Paredes-Redondo.

This was shown by applying the selective inhibitor of TGFβ signalling, SB-431542, to myofibers in the optogenetic model. The treatment had no effect in CRISPR-corrected myofibers, while SB-431542 treatment in un-corrected, mutated myofibers increased contraction velocity significantly. Furthermore, the research team used the human neuromuscular circuit co-cultures to develop a 96-well high-content imaging-compatible drug screening platform for DMD cellular phenotypes.

CRISPR plays an integral part in DMD research and therapy

»Our study provides a novel human micro-physiological neuromuscular model for studying neuromuscular defects in DMD and evaluating drug candidates or CRISPR-based therapeutics. Furthermore, our findings suggest that enhancing neuromuscular connectivity may be an effective therapeutic strategy,« concludes Yung-Yao Lin.