CPVT is an autosomal dominant inherited cardiac arrhythmia. In this disease, the ryanodine receptor type 2 (RyR2) is leaky, leading to aberrant calcium release from the sarcoplasmic reticulum (SR) of cardiomyocytes during diastole, which manifests in delayed afterdepolarizations, potentially culminating in arrhythmia.

We are pursuing two potential gene therapies:

1. Concurrent knockout and transactivation (CONNACT) of functionally equivalent genes.
Here, we focus on knockout of dysfunctional Ryr2 gene and transactivation of a functionally equivalent counterpart i.e., Ryr1 or Ryr3. To this end we used, intraperitoneal injection of dual mRNA trans-splicing AAV9 vectors containing catalytically active Cas9-VPR and sgRNAs with different spacer lengths into WT mice. In qRT-PCR and Western blot experiments of cardiac tissue we show efficient Ryr2 knockdown and an increase in Ryr1 expression. RyR1 immunolabeling revealed the typical striated pattern of SR. However, surface ECG data displayed a reduction in QRS complex amplitude and widened P-waves in AAV-treated mice compared to control, indicating that RyR1 cannot rescue the depletion of RyR2. In further studies, we will test whether RyR3 can functionally replace RyR2 in cardiomyocytes.

2. Transactivation of Calsequestrin 2 (CASQ2).
Several in vitro studies have shown that an increase in CASQ2, the main Ca2+-buffering protein in the SR, promotes RyR2 closure. CASQ2 transactivation is therefore a promising approach to improve RyR2 function in CPVT. Using a catalytically inactive dCas9-VPR and 1 sgRNA resulted in approximately 4-fold overexpression of Casq2 at the transcript level in WT mice, with no negative effects on surface ECG data.