Abstract 18712: Adeno-associated Viral Gene Delivery of Calsequestrin 2 Protects Adult Calsequestrin 2-R33Q Knock-in Mice From Developing Ventricular Tachycardias

Abstract

Background. Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT) is a highly lethal recessive arrhythmogenic disease associated with mutations in the calsequestrin 2 ( CASQ2 ) gene. We previously demonstrated that CASQ2 gene delivery was able to rescue the arrhythmic phenotype in CASQ2 knock-out (KO) mice 5 months after the viral infection. The aim of the present study is to investigate the long-term effects of viral gene delivery in CASQ2-R33Q missense mutation mouse model. Methods. Newborn CPVT mice were infected with adeno-associated viral vector 9 (AAV9) containing the coding sequence of the wild type CASQ2 co-expressed with green fluorescent protein (GFP) gene. Furthermore, we evaluated the effect at 7 and 12 months after viral infection by in vivo ECG analysis, in vitro electrophysiological and molecular assays. Results. Telemeter recording of ECG demonstrated in vivo ventricular tachycardias (VT) after epinephrine (2 mg/Kg) administration in only 17% (2 out of 12) of the infected mice, while 87% (7/8) of the control CASQ2-R33Q homozygous mice presented a clear arrhythmic phenotype (p<0.005). Additionally, electrophysiological analysis showed that delayed after depolarization (DADs) and triggered activity (TA) were almost abolished in GFP-positive cardiomyocytes (7 months: DADs: 0%, TA 0%, n=18; 12 months: DADs: 8%, TA: 8%, n=12) in comparison with their internal control (GFP-negative cells, 7 months: DADs:100%, TA: 83%, n=6; 12 months: DADs: 100%, TA: 71%, n=7; p<0.001). Finally, western blot revealed increased CASQ2 expression in the infected mice and immunofluorescence assay indicated its correct localization along the z-lines. Conclusions. In the present work we show that the viral expression of CASQ2 is effective for a long period of time and is able to revert the functional abnormalities of the mutant endogenous protein and to prevent life-threatening arrhythmias in the knock-in CASQ2-R33Q mice. These data suggest that despite R33Q mice, at variance with KO mice, express abnormal CASQ2, the gene replacement therapy is still able to prevent arrhythmogenic mechanisms in vitro and VTs in vivo thus suggesting that CASQ2-gene transfer may become a novel therapy for recessive CPVT.