Repurposing Dantrolene for Long-Term Combination Therapy to Potentiate Antisense-Mediated DMD Exon Skipping in the mdx Mouse

Abstract

Duchenne muscular dystrophy (DMD) is caused by mutations in DMD, resulting in loss of dystrophin, which is essential to muscle health. DMD “exon skipping” uses anti-sense oligo-nucleotides (AONs) to force specific exon exclusion during mRNA processing to restore reading frame and rescue of partially functional dystrophin protein. Although exon-skipping drugs in humans show promise, levels of rescued dystrophin protein remain suboptimal. We previously identified dantrolene as a skip booster when combined with AON in human DMD cultures and short-term mdx dystrophic mouse studies. Here, we assess the effect of dantrolene/AON combination on DMD exon-23 skipping over long-term mdx treatment under conditions that better approximate potential human dosing. To evaluate the dantrolene/AON combination treatment effect on dystrophin induction, we assayed three AON doses, with and without oral dantrolene, to assess multiple outcomes across different muscles. Meta-analyses of the results of statistical tests from both the quadriceps and diaphragm assessing contributions of dantrolene beyond AON, across all AON treatment groups, provide strong evidence that dantrolene modestly boosts exon skipping and dystrophin rescue while reducing muscle pathology in mdx mice (p < 0.0087). These findings support a trial of combination dantrolene/AON to increase exon-skipping efficacy and highlight the value of combinatorial approaches and Food and Drug Administration (FDA) drug re-purposing for discovery of unsuspected therapeutic application and rapid translation.

Targeting RyR Activity Boosts Antisense Exon 44 and 45 Skipping in Human DMD Skeletal or Cardiac Muscle Culture Models

“…Likewise, dantrolene boosts exon 44 skipping in patient-derived iDRM DMD myotube cultures carrying a mutation amenable to reframing by exon 44 skipping (deletion of exon 45 of DMD ). While the degree of boost is modest, it is in keeping with the levels boosted in mdx mice that were previously shown to positively decrease disease pathology and in keeping with the literature indicating that even low levels of dystrophin can impact muscle function 18 , 21 . Boost activity is seen in both patient iDRM- and iPSC-derived myotube models, adding confidence to our findings.…”

“…Combination therapy also increased dystrophin protein expression as measured by western blotting (Figure 6). These findings demonstrate that observed increases in exon skipping translate into increased dystrophin protein rescue, consistent with our studies in mdx mice in vivo 20 , 21 Figure 5Rycal ARM210 and Dantrolene Boost AO-Mediated Exon44 Skipping in Patient iDRM-Derived Myotubes to Induce Dystrophin Protein ExpressionDystrophin immunostaining was performed using NCL-dys3 (Dys3, N-term), NCL-Dys1 (Dys1, rod domain), and NCL-Dys 2 (Dys 2, C-term) antibodies.…”

“…Dantrolene AO skip-boosting activity has now been validated in mdx mice over both long- and short-term systemic AO treatment. In these studies, exon 23 AO/dantrolene combination therapy demonstrated higher levels of exon 23-skipped mRNA and rescued dystrophin protein, improved muscle function, and diminished pathophysiology relative to those treated with AO or dantrolene alone 20 , 21 …”

“…We previously published that dantrolene boosts DMD AO-mediated exon 51 skipping in human DMD patient-derived fibroblasts, which were directly reprogrammed to myotubes through myogenic differentiation 1 ( Myod1 ) induction (induced directly reprogrammed myotubes, termed iDRM) 20 . Further, systemic codelivery of dantrolene was able to increase AO-mediated exon 23 skipping and dystrophin protein rescue in mdx mice 20 , 21 . However, it remains unclear whether dantrolene is able to increase the amount of dystrophin produced when used in combination with AO targeting other exons and how it functions to promote skipping.…”

Hexose Potentiates Peptide-Conjugated Morpholino Oligomer Efficacy in Cardiac Muscles of Dystrophic Mice in an Age-Dependent Manner

“…Nanoparticles including synthetic and natural biological nanoscale vesicles such as exosomes have been explored as delivery vehicles for AOs, wherein the former showed limited efficacy and the latter is still in early development 6, 8. Recently, small compounds have found favor, particularly the ones used in the clinic for other therapeutic purposes, such as dantrolene and hexose, which showed promise as adjuvants to enhance AOs’ efficacy and can be fast-tracked to the clinic 9, 10, 11, 12…”