Abstract 16753: Efficacy & Efficiency of Genome Editing With CRISPR/Cas9 in a Humanized Knock-In Mouse Model of PLN ^R14del

Introduction: A mutation in the Phospholamban (PLN) gene, a calcium regulating protein, the Arg14 deletion (R14del), has been identified in patients with atypical Arrhythmogenic Cardiomyopathy (ACM). Carriers of this mutation are at high risk for malignant ventricular arrhythmias and ventricular dilation. Objective: To evaluate the efficiency and efficacy of genome editing by using CRISPR/Cas9 and a cardiotropic Adeno-Associated Vector (AAV) in a knock-in mouse model expressing the human coding sequence of mutant PLN (hPLN R14del ). Methods: We generated wild-type (WT), heterozygous (Het) and double mutant (Mut) humanized mice. We designed a CRISPR-Cas9/gRNA construct which cut around the site encoding R14Del-PLN gene. To check for CRISPR-Cas9 efficiency we used droplet digital (ddPCR) Genome Edit Detection assay that consist of primer pair to amplify the sequence that includes the mutation, and a FAM-labeled probe designed to bind the mutant allele. Heterozygous mice were injected with 2x10 12 vg of AAV9.CRISPR/Cas9/gRNA for inactivation of the R14del allele. Cardiac function and heart morphology were assessed by Magnetic Resonance Imaging (MRI), echocardiography and H&E staining. Ventricular tachycardia (VT) threshold was defined as the pacing frequency (f Pacing ) required for induction of sustained VT in ex vivo set-up. Results: Echocardiography and histology showed no significant changes in cardiac function and structure in the R14del Het & Mut mice. Eight weeks after AAV injection the hearts were harvested and analyzed for indel frequencies at on-target site in the genome. ddPCR analysis showed an efficiency of ~13% inactivation of the R14del mutation in AAV9/CRISPR-Cas9 injected mice compared to controls. Seventy-five percent of Het hearts exhibited adrenergic mediated VT (f Pacing = 19.6±4.7Hz) vs. only 30% of their WT counterparts (25.8±1.4Hz, p<0.05). VT susceptibility in Het hearts was significantly suppressed by AAV-mediated CRISPR/Cas9 delivery targeting the mutant allele. Conclusions: Our results demonstrate that mouse model harboring the human R14del mutation does not result in gross cardiac morphological changes. Silencing this mutation by CRISPR/Cas9 AAV based gene therapy reversed the VT susceptibility in the Het mice.