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

Abstract

Systemic delivery of antisense oligonucleotides (AO) for DMD exon skipping has proven effective for reframing DMD mRNA, rescuing dystrophin expression, and slowing disease progression in animal models. In humans with Duchenne muscular dystrophy treated with AOs, low levels of dystrophin have been induced, and modest slowing of disease progression has been observed, highlighting the need for improved efficiency of human skipping drugs. Here, we demonstrate that dantrolene and Rycals S107 and ARM210 potentiate AO-mediated exon skipping of exon 44 or exon 45 in patient-derived myotube cultures with appropriate mutations. Further, dantrolene is shown to boost AO-mediated exon skipping in patient-derived, induced cardiomyocyte cultures. Our findings further validate the ryanodine receptors (RyR) as the likely target responsible for exon skip boosting and demonstrate potential applicability beyond exon 51 skipping. These data provide preclinical support of dantrolene trial as an adjuvant to AO-mediated exon-skipping therapy in humans and identify a novel Rycal, ARM210, for development as a potential exon-skipping booster. Further, they highlight the value of mutation-specific DMD culture models for basic discovery, preclinical drug screening and translation of personalized genetic medicines.

Skeletal Ryanodine Receptors Are Involved in Impaired Myogenic Differentiation in Duchenne Muscular Dystrophy Patients

“…In summary, our results emphasize the key role of RYR1 in human DMD pathophysiology: (i) RYR1-mediated Ca 2+ leakage through SR due to calstabin1 depletion delays skeletal muscle differentiation in DMD, and this can be improved by S107; (ii) the elevated cytosolic Ca 2+ concentration is correlated with endomysial fibrosis, suggesting its involvement in DMD prognosis. As suggested by the promising results of combining RYR stabilizers and exon skipping therapies in DMD iDRMs, iPSC-derived myotubes [ 56 , 57 ] and patient iPSCs-derived cardiomyocytes [ 64 ] and in muscle function in mdx mice [ 38 , 62 ], our study highlights the potential effect of RYR1 stabilization as an adjunctive therapeutic strategy in patients with DMD.…”

“…Moreover, Kendall et al reported that dantrolene, a RYR-targeting compound, which is currently used as a chronic treatment for malignant hyperthermia and muscle spasticity, synergizes with antisense-mediated exon skipping therapies in mdx mice and in inducible directly reprogrammable myotubes (iDRMs) derived from fibroblasts of patients with DMD [ 63 ]. Ryanodine and S107 also enhanced the effect of exon-skipping antisense oligonucleotides in iDRMs cells from patients with DMD [ 64 ]. The same team recently showed that dantrolene, S107 and ARM210 similarly increased antisense oligonucleotide-mediated exon skipping in DMD iDRMs and in inducible pluripotent stem cells (iPSC)-derived myotubes [ 63 ].…”

Power and Promise of Next-Generation Sequencing in Liquid Biopsies and Cancer Control

“…Tumor cells secrete and express new antigens on the surface of cells to escape recognition of T cells. [93][94][95][96] Patient-derived T cells can grow in vitro and can be stimulated with these new antigens to elicit a strong T-cell response. 97 To further enhance the ability of T cells to recognize tumors, CAR-encoded DNA was introduced into T cells (CAR-T cell therapy).…”

Abnormal Calcium Handling in Duchenne Muscular Dystrophy: Mechanisms and Potential Therapies

“…Rycal is suggested to improve binding of calstabin to RyR and to prevent SR Ca 2+ leak, thereby restoring Ca 2+ i homeostasis (Capogrosso et al, 2018). A recent study showed that Rycal treatment bolsters antisense oligonucleotidemediated exon skipping in patient-derived myotubes and induced pluripotent stem cell-derived diseased cardiomyocytes (Barthelemy et al, 2019). Although the therapeutic value of Rycal needs to be further validated, these studies suggest that targeting the RyR will boost treatment efficiency in DMD patients.…”