Catecholaminergic polymorphic ventriculartachycardia (CPVT) is a lethal human arrhythmia provoked by exercise or emotional stress. It is mediated by abnormal calcium release from the sarcoplasmic reticulum through "leaky" ryanodine channels. Beta-adrenergic blockers are the therapy of choice for human CPVT but they fail to achieve complete arrhythmia control in some of the cases. Gene therapy is a potential treatment for genetic and other diseases associated with inadequate expression of a key protein.We established a new gene-delivery system to target CASQ2 knock-out mice suffering from CPVT2. CASQ2 gene was cloned into pAAV-IRES-hrGFP plasmid. AAV9 recombinant vectors were generated by co-transfecting the expression plasmid into the AAV-293 cells with pHelper (carrying adenovirus-derived genes) and pAAV-RC (carrying AAV-9 replication and capsid genes), which express the transacting factors required for AAV replication and packaging in the AAV-293 cells. Viral particles were purified from crude cell lysates, concentrated and injected into the left ventricle of 12-14-week-old mice. Mice underwent provocation testing for arrhythima 7 weeks post viral infection. Cryosections staining demonstrated that cardiac muscle and lung tissues were CASQ2 and GFP positive while liver and spleen were GFP negative in the infected mice. Calsequestrin protein expression was elevated in the hearts of infected CASQ2 knock-out mice (n=10) compared to controls. A dose-dependent reduction in abnormal ventricular beats was observed in mice that expressed AAV9-delivered myocardial CASQ2 protein (R2=0.723, p<0.005).Viral gene-delivery eliminated sustained ventricular tachycardia in all infected mice (p=0.012) while more than 33% of the normal protein level was required to prevent non-sustained VT (n=5, p=0.003).
In conclusion, we assembled a vector useful for cardiac gene delivery, thus creating a platform for gene therapy studies in inherited heart diseases. In vivo viral delivery of CASQ2 cDNA attenuated ventricular arrythmia in mice with CPVT2.