Abstract 14905: 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 RyR2R4496C/+ mice (1.3 x 1012 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<0.001). Electrophysiological analysis of isolated myocytes showed that delayed after depolarizations (DADs) and triggered activity (TA) induced by isoproterenol were almost abolished in U10 infected cells in comparison to controls (p<0.001 both for DADs and TA). Rescue of arrhythmic phenotype closely mirrors the ability of miRYR2-U10 treatment to significantly suppress expression of R4496C-RYR2 allele, without affecting wild type mRNA (WT:R4496C allele ratio U10 vs SCR p<0.05). Electron microscopy showed that cardiac tissue from mutant CPVT and SCR injected mice manifested abnormal mitochondria while these alterations were attenuated in mutant mice treated with allele specific silencing (p<0.05).
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.
Author Disclosures: R. Bongianino: None. A. Vollero: None. F. Lodola: None. M. Denegri: Consultant/Advisory Board; Modest; Audentes Therapeutics. S. Boncompagni: None. S. Fasciano: None. A. Mazzanti: None. D. Mangione: None. S. Barbaro: None. G. Rizzo: None. A. Auricchio: None. C. Napolitano: Consultant/Advisory Board; Modest; Audentes Therapeutics. F. Protasi: None. S.G. Priori: Honoraria; Modest; Medtronic Inc., Boston Inc., Gilead. Consultant/Advisory Board; Modest; Audentes Therapeutics, GE Healthcare, Chiesi.
- © 2016 by American Heart Association, Inc.