Allele Specific Silencing of Mutant mRNA Rescues Ultrastructural and Arrhythmic Phenotype in Mice Carriers of the R4496C Mutation in the Ryanodine Receptor Gene (RYR2)
Rationale: Mutations in the cardiac Ryanodine Receptor gene (RYR2) cause dominant Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT), a leading cause of sudden death in apparently healthy individuals exposed to emotions or physical exercise.
Objective: We investigated the efficacy of allele specific silencing by RNA interference to prevent CPVT phenotypical manifestations in our dominant CPVT mice model carriers of the heterozygous mutation R4496C in RYR2.
Methods and Results: We developed an in vitro mRNA and protein-based assays to screen multiple siRNAs for their ability to selectively silence mutant RYR2-R4496C mRNA over the corresponding wild-type (WT) allele. For the most performant of these siRNAs (siRYR2-U10), we evaluated the efficacy of an adeno associated serotype 9 viral vector (AAV9) expressing miRYR2-U10 in correcting RyR2 function following in vivo delivery by intraperitoneal injection in neonatal and adult RyR2R4496C/+ heterozygous CPVT mice. Transcriptional analysis showed that after treatment with miRYR2-U10 the ratio between WT and mutant RYR2 mRNA was doubled (from 1:1 to 2:1) confirming the ability of miRYR2-U10 to selectively inhibit RYR2-R4496C mRNA, while protein quantification showed that total RyR2 was reduced by 15% in the heart of treated mice. Furthermore, AAV9-miRYR2-U10 effectively: 1) reduced isoproterenol-induced delayed after depolarizations and triggered activity in infected cells, 2) reduced adrenergically mediated ventricular tachycardia in treated mice, 3) reverted ultrastructural abnormalities of junctional sarcoplasmic reticulum and T-tubules and 4) attenuated mitochondrial abnormalities.
Conclusions:The study demonstrates that allele-specific silencing with miRYR2-U10 prevents life-threatening arrhythmias in CPVT mice suggesting that reduction of mutant RyR2 may be a novel therapeutic approach for CPVT.
- sudden cardiac death
- gene therapy
- cardiac arrhythmia
- catecholaminergic polymorphic ventricular tachycardia
- ryanodine receptor
- genetics, animal models
- Received February 22, 2017.
- Revision received June 7, 2017.
- Accepted June 14, 2017.