Dantrolene Enhances Antisense-Mediated Exon Skipping in Human and Mouse Models of Duchenne Muscular Dystrophy

The ryanodine receptor antagonist dantrolene facilitates exon skipping of dystrophin for treatment of Duchenne muscular dystrophy. Superior Skipping with a Little Help from Dantrolene Hopes have been raised for patients with Duchenne muscular dystrophy, a genetic disease in which muscles rapidly degenerate in young boys: An antisense oligonucleotide can restore the missing protein dystrophin. Usually, its transcription is truncated because of a mutation in the dystrophin gene, but the antisense drug targets the defect and allows transcription to bypass the mutation—a process called exon skipping. Two clinical trials have shown promising results, with restoration of 2 to 16% of the normal amount of dystrophin. However, these results were too variable and dystrophin levels too low to realize the full potential of exon skipping to improve muscle function. Therefore, to augment the effects of this promising drug, Kendall et al. screened a large number of small molecules and found one—dantrolene—that increases antisense-induced exon skipping in a mouse model of muscular dystrophy and in patient cells. In testing 300 compounds from a drug library on cells also treated with an exon-skipping antisense directed to a human dystrophin reporter gene, the authors found several that scored positive. Of these, they chose dantrolene for further follow-up because it is already used in the clinic (for malignant hyperthermia) and has been tested in Duchenne muscular dystrophy patients, where it showed few side effects. The investigators found that dantrolene promoted exon 23 skipping in cells from mdx mice (a mouse model of muscular dystrophy) and exon 51 skipping in patient fibroblasts induced to form myotubes. Dantrolene worked in an animal model of DMD as well. When antisense was injected intravenously into mdx mice, dantrolene given systemically augmented exon skipping, resulting in additional dystrophin multiple muscles including the tibialis anterior, gastrocnemius, diaphragm, and quadriceps, but not heart or the triceps. Dantrolene-treated mice were also stronger than those treated with antisense alone and could hang on a wire for 50% longer. Other drugs that, like dantrolene, are antagonists at the ryanodine receptor have the same effect, suggesting that this intracellular calcium channel is the critical target for dantrolene’s effect. Exactly how and in what cells this occurs is not yet clear. This class of ryanodine-targeting drugs may be able to change what has been a promising therapy for patients with muscular dystrophy to a reality, ready for testing. Duchenne muscular dystrophy (DMD) causes profound and progressive muscle weakness and loss, resulting in early death. DMD is usually caused by frameshifting deletions in the gene DMD, which leads to absence of dystrophin protein. Dystrophin binds to F-actin and components of the dystrophin-associated glycoprotein complex and protects the sarcolemma from contraction-induced injury. Antisense oligonucleotide–mediated exon skipping is a promising therapeutic approach aimed at restoring the DMD reading frame and allowing expression of an intact dystrophin glycoprotein complex. To date, low levels of dystrophin protein have been produced in humans by this method. We performed a small-molecule screen to identify existing drugs that enhance antisense-directed exon skipping. We found that dantrolene, currently used to treat malignant hyperthermia, potentiates antisense oligomer–guided exon skipping to increase exon skipping to restore the mRNA reading frame, the sarcolemmal dystrophin protein, and the dystrophin glycoprotein complex in skeletal muscles of mdx mice when delivered intramuscularly or intravenously. Further, dantrolene synergized with multiple weekly injections of antisense to increase muscle strength and reduce serum creatine kinase in mdx mice. Dantrolene similarly promoted antisense-mediated exon skipping in reprogrammed myotubes from DMD patients. Ryanodine and Rycal S107, which, like dantrolene, targets the ryanodine receptor, also promoted antisense-driven exon skipping, implicating the ryanodine receptor as the critical molecular target.