B-PO01-008 MULTIPLEX GENOME EDITING FOR THE TREATMENT OF CATECHOLAMINERGIC POLYMORPHIC VENTRICULAR TACHYCARDIA

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Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited cardiac arrhythmia disorder that can cause sudden cardiac death. Variants in the ryanodine receptor type 2 gene (RYR2) cause >60% of CPVT cases. These variants increase RyR2 calcium (Ca2+) leak from the sarcoplasmic reticulum that can trigger ventricular arrythmias. Current therapeutic options are partially effective. Previously, our lab showed that using the CRISPR/Cas9 genome editing system targeting a silent restriction site could disrupt a disease-causing allele preventing VT in a CPVT mouse model with RyR2-R176Q (RQ). To develop a single CRISPR/Cas9 vector that can be used to correct one of several CPVT variants. We designed single guide RNAs (sgRNA) to target the N-terminal RQ and central domain RyR2-R2474S (RS) mutation sites. We cloned vectors with sgRNA targeting RS or RQ, dual guides targeting RS + RQ, and no gRNA as control. We used an in-vitro luciferase assay to determine on and off target editing efficiencies. We generated AAV9 with CRISPR/Cas9 and injected SC p5 mice. 6 weeks after injection, mice underwent ECG stress testing with isoproterenol, caffeine, and programmed electrical stimulation. Isolated ventricular cardiomyocytes loaded with Fluo4 dye were analyzed with confocal line scanning. We found by targeting the RQ site with sgRNA/Cas9, 0/8 mice had pacing-induced VT compared to 6/8 control (p=0.03). Edited cardiomyocytes showed a significant reduction in Ca2+ spark frequency (p<0.01). When targeting the RS site with sgRNA/Cas9, 0/7 mice had inducible VT compared to 6/7 control (p=0.04). The luciferase assay showed similar editing efficiencies with sgRNA and dual gRNA (Δ <10%) with preserved specificity to mutant alleles (>80%). In ongoing experiments, we expect similar efficacies in CPVT mice treated with dual gRNA/Cas9. CRISPR/Cas9 gene editing can treat CPVT by specifically targeting causative mutation sites in multiple channel domains, reducing mutant allele expression and preventing Ca2+ leak. Through successful targeting of the mutation sites, we have promising preclinical data for gene editing as a permanent cure for CPVT.