Allele Specific Silencing Prevents Malignant Arrhythmias And Ultrastructural Abnormalities In Ryanodine Receptor Mutant Mice

Introduction: Mutations in the cardiac Ryanodine Receptor gene (RYR2) are causative of dominant Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT), a leading cause of sudden death in apparently healthy individuals when exposed to emotions or physical exercise. Hypothesis: We investigated the efficacy of allele specific silencing by RNA interference as an innovative therapeutic approach to attenuate clinical manifestations of dominant CPVT. Methods: We developed an in vitro mRNA and protein-based assays to screen multiple siRNAs for their ability to selectively silence the mutant RYR2-R4496C mRNA over the corresponding wild-type allele. For the most performant of these siRNAs (siRYR2-U10), we evaluated the specificity and efficacy of an adeno-associated serotype 9 viral vector (AAV9) expressing the homologous miRNA (miRYR2-U10) in correcting RyR2 function following in vivo delivery in pups RyR2 R4496C/+ mice (1.3 x 10 12 viral particles, 1 week after birth). Results: ECG analysis revealed a high incidence of adrenergically mediated ventricular tachycardia (VT) in AAV9-miRNA-scramble injected mice (SCR), but not in those treated with AAV9-miRYR2-U10 (U10; p Conclusions: The evidence that RNA interference of RyR2-R4496C transcripts preventing life-threatening arrhythmias in CPVT mice raises the possibility that mutant allele specific silencing might benefit CPVT patients.