Plakophilin‐2 Gene Therapy Prevents Arrhythmogenic Right Ventricular Cardiomyopathy Development In A Novel Mouse Model Harboring Patient Genetics

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a genetic cardiac disease that causes sudden death in young adults and athletes. ARVC is characterized by extensive electrical involvement, though structural defects are equally important as fibro‐fatty replacement of myocardium leads to ventricular dysfunction and failure. ARVC is termed a “disease of the desmosome” as 40% of mutations in ARVC patients are found in the cardiac desmosomal components, with plakophilin‐2 (PKP2) being the most frequently mutated desmosomal gene in ARVC. Studies of PKP2 ARVC populations suggest that a majority of mutations impact PKP2 protein levels via diverse mechanisms (nonsense mutation, insertion/deletion mutation, splice site mutation). There are currently no effective treatments or cures for ARVC, thus, strategies elevating PKP2 protein levels would represent a clinically relevant avenue to address a large portion of ARVC populations. We hypothesize that PKP2 protein dose is a critical driver of ARVC, and that early delivery of PKP2 via adeno‐associated virus (AAV) can prevent disease development. Through CRISPR‐Cas9 we generated a novel knock‐in mouse model harboring a human equivalent PKP2 mutation (IVS10‐1 G>C) that impacts PKP2 RNA splicing, PKP2 protein levels, and is sufficient to recapitulate all classic ARVC disease features. PKP2 homozygous mutant (PKP2 Hom) mice are viable at birth yet display adult hallmarks of ARVC including ventricular arrhythmias, right and left ventricular dysfunction, and fibro‐fatty replacement of myocardium leading to sudden death starting at 4 weeks of age. PKP2 Hom mice display reduced PKP2 protein levels, which results in a disruption of desmosomal and gap junction proteins by the onset of disease features. Using a cardiotropic AAV9, early delivery at postnatal day 2 of PKP2 (AAV9‐PKP2) resulted in restoration of PKP2 protein to wild type levels by 4 weeks of age in PKP2 Hom mice. This caused a significant improvement in cell‐cell junction protein levels. Cardiac function (both left and right ventricles) and electrophysiology were significantly improved at 4 weeks of age as well. Histological analysis further showed improved morphology in PKP2 Hom mice treated with AAV9‐PKP2, as well as significantly less fibrosis throughout ventricular myocardium. Kaplan‐Meier survival curves highlighted a dramatic improvement in survival in PKP2 Hom mice treated with AAV9‐PKP2, suggesting early administration of AAV9‐PKP2 resulted in a beneficial and durable impact on cardiac function. We provide a novel mouse model that incorporates PKP2 patient genetics and serves as an ideal platform to evaluate therapies for ARVC. Early administration of PKP2 via AAV9 represents an effective and clinically relevant approach to prevent ARVC disease development.