Abstract Body: Background: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a genetic cardiac disorder characterized by severe arrhythmias and heart dysfunction. Mutations in desmosome gene Plakophilin-2 (PKP2) account for 40% of ARVC cases, with current palliative therapies failing to address the genetic cause. Herein, we generated an in vitro ARVC model using human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) harboring a pathogenic PKP2 variant (c.2146G>C), as a platform to test a PKP2-gene replacement approach developed at Tenaya Therapeutics for the treatment of PKP2-associated ARVC patients.
Methods: Three isogenic human iPSC lines (wild-type, heterozygous and homozygous PKP2 mutant) were differentiated to iPSC-CM; and monolayers and engineered heart tissues (EHTs) were generated. Gene and protein expression were assessed by RNA-Seq/RT-qPCR, and immunocytochemistry, respectively. Contractility, electrophysiology and calcium handling were measured. A proprietary adeno-associated virus gene therapy (AAV9:PKP2) was used to transduce human iPSC-CM, and changes in gene and protein expression, and contractile function were evaluated.
Results: A human iPSC-CM beating monolayer was achieved for all three lines. PKP2 expression was depleted in a genotype-dependent manner, and desmosome structure was disrupted in the mutant lines. Functional assessment of human iPSC-CM monolayers showed impaired contractile properties and abnormal electrophysiological and calcium transients in the mutant lines, such as decreased contraction amplitude and prolonged field and action potential duration. Transcriptional analysis revealed changes in desmosome, gap junctions, sarcomere, ion channels, metabolic and apoptosis gene expression. Characterization of EHTs displayed a deficit in contractility, slower action and field potential kinetics and irregular calcium homeostasis in the mutant lines. The administration of AAV9:PKP2 to the mutant lines restored ion channel and desmosome gene and protein expression, and contractile function.
Conclusions: Our PKP2 human iPSC-CM disease model recapitulated the main hallmarks of ARVC phenotype. Administration of AAV9:PKP2 restored desmosome protein expression and contractility. This model lays a foundation for the understanding of the underlying molecular pathophysiological mechanisms of PKP2-ARVC, and the potential for AAV9:PKP2 as a one-time dose to correct the genetic cause of disease in individuals with PKP2-associated ARVC.