Scientists Use CRISPR To Fight Duchenne Muscular Dystrophy
By Rajaneesh K. Gopinath, Ph.D.
Duchenne Muscular Dystrophy (DMD) is an X-linked recessive disorder predominantly affecting male children. This debilitating, muscle-weakening disease is the most common and severe form of muscular dystrophy. It is caused due to defective frameshift mutations in the largest known human gene, DMD that encodes for dystrophin. The protein is crucial for muscle integrity as it helps connect the muscle fiber’s actin cytoskeleton to the cell membrane. Patients without dystrophin suffer from loss of muscle as early as age five and most couldn’t walk by the age of 12. The symptoms worsen progressively and result in a fatality due to terminal muscle and heart failure.
Besides inheritance, the disease-causing mutations could also be acquired spontaneously by children with no previous history of DMD in the family. That, along with the belated sighting of symptoms, makes diagnosis a major challenge. DMD could be effectively screened in newborns by identifying the elevated levels of creatine kinase (CK) biomarker in the patient serum. In December 2019, PerkinElmer’s GSP Neonatal Creatine Kinase-MM kit became the first assay to be authorized by the FDA for this purpose. However, in older children, CK levels fluctuate and are not very reliable for diagnosis.
DMD has no cure but corticosteroids such as prednisone and deflazacort were the standard of care for over two decades. Emflaza (deflazacort) became the first FDA approved corticosteroid for DMD only a few years ago. Apart from that, Sarepta’s Exondys 51 (eteplirsen) and Vyondys 53 (golodirsen) are two FDA approved antisense oligonucleotides available for treatment today. The drugs bind to the dystrophin pre mRNA at exon 51 and 53 respectively to produce a truncated yet functional protein through exon skipping.
A collaborative study involving researchers from the Technical University of Munich (TUM), Ludwig Maximilian University of Munich (LMU) and the German Research Center for Environmental Health has successfully used CRISPR-Cas9 to correct the defective dystrophin gene in a pig model of DMD that lacked exon 52 (DMDΔ52). “These gene scissors are highly efficient and specifically corrected the dystrophin gene,” said Prof. Wolfgang Wurst, developmental geneticist at TUM and the German Research Center for Environmental Health. By using AAV vector-mediated delivery of Cas9 and gRNAs targeting the flanking regions of Exon 51 (AAV9-Cas9-gE51), they were able to achieve excision of exon51 (DMDΔ51–52). This restored the DMD reading frame resulting in the production of a truncated yet partially functional dystrophin in vivo. The injected animals lived longer as a result of improved muscle function and lower susceptibility to cardiac arrhythmia.
“Muscle and heart cells are long-lived cell structures. One-half of all myocardial cells remain functional from birth throughout the entire lifecycle of a human being,” says Prof. Christian Kupatt, cardiologist at university hospital TUM Klinikum rechts der Isar. “The genome of a cell is used for protein biosynthesis as long as the cell is alive, and once a cell has been affected by the therapy, it remains corrected. So if we change the genome of a myocardial cell, the correction is a long-term success, in contrast to the results of previous methods.”
The Relevance of the Study
This is not the first time CRISPR is used to correct the dystrophin protein. Previous successful attempts have been made in mice and dog models. However, the authors claim this study is performed in a disease model much closer to humans. “Our results are very promising, since, for the first time, we have now been able to demonstrate therapeutic success in a clinically relevant large animal model,” says Prof. Maggie Walter, neurologist at the LMU university hospital.
“Since the disease proceeds faster in our pig model, we were able to verify the efficacy of the therapeutic approaches within a manageable period of time,” says Prof. Eckhard Wolf, LMU specialist in veterinary medicine.
The researchers also demonstrated equivalent results in vitro using human models of DMD. Experiments were performed with induced pluripotent stem cell-derived myoblasts and cardiomyocytes of a DMDΔ52 patient. The excision of exon 51 resulted in functional dystrophin that ameliorated skeletal myotube formation as well as abnormal cardiomyocyte Ca2+ handling and arrhythmogenic susceptibility. In summary, the study suggests that we are one step closer to achieving a cure for DMD in humans. The results are published in the journal Nature Medicine.
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