VViral-delivered Gene Therapy for Treatment of CPVT

Efrat Kurtzwald- Josefson ^1,2 Edith Hochauser ² Shiraz Khun ¹ Asher Shainberg ³ Dan Aravot ² Michael Eldar ¹ Michael Arad ¹

¹Heart Institute, Sheba Medical Center, Tel Hashomer
²Cardiac Research Laboratory, Felsenstein Medical Research Center, Sackler School of Medicine, Tel Aviv University, Petah Tikva
³The Mina & Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan

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. AAV9 recombinant plasmids were generated by cloning the CASQ2 gene into the pAAV-IRES-hrGFP vector. The expression plasmid was co-transfected 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. Examination of cryosections demonstrated that cardiac muscle and lung tissues were CASQ2 and GFP positive while liver and spleen were GFP negative in AAV injected mice. Calsequestrin protein expression was elevated in the hearts of transfected 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 transfected 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.