Uncovering Molecular Targets for Arrhythmogenic Cardiomyopathy Therapeutics

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Abstract Arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C) is an incurable genetic based cardiac disease that causes sudden death in young adults and athletes. ARVD/C is termed a “disease of the desmosome” as 40-50% of mutations in ARVD/C patients are found in desmosomal (junctional anchor) genes, with plakophilin-2 (PKP2) being the most frequently mutated desmosomal gene. Evidence suggests that altered RNA splicing may be a critical mechanism through which PKP2 patient genetics drive ARVD/C. However, no models and limited mechanistic insights exist into how human desmosomal mutations in RNA splicing impact ARVD/C and what form of therapeutics would be impactful in these settings. Through CRISPR-Cas9 we generated a novel mouse model globally harboring a human PKP2 mutation (IVS10-1 G>C) that impacts RNA splicing. PKP2 homozygous mutant (PKP2 Hom) mice selectively display all adult hallmarks of ARVD/C including sudden death. RNA and sequencing analyses revealed low levels of a larger PKP2 transcript that retains an intronic sequence. Protein analyses of PKP2 Hom hearts revealed low levels of a higher molecular weight PKP2 mutant protein that was expressed in the absence of endogenous PKP2. Strategies to increase wild type PKP2 and mutant PKP2 protein in PKP2 mutant neonatal cardiomyocytes suggested that splicing effects on PKP2 haploinsufficiency mechanistically drive cell junction deficits in early ARVD/C. Targeted restoration of PKP2 protein dose in neonatal PKP2 Hom mice had therapeutic potential in late ARVD/C as it restored cardiac mechanical junction complex and prolonged life in adult PKP2 Hom mice. PKP2 Hom mice provide an ideal test platform to assess the impact and mechanism of PKP2 restoration in circumventing ARVD/C in classic patient- centric models during early and late stages of disease. Prime editing (search-and-replace) strategies have come to age as novel methods to correct single base mutations and address the “root cause” of ARVD/C, though limited studies have applied this technology towards therapeutic use in disease settings. We hypothesize the PKP2 RNA splicing mutation is sufficient to drive ARVD/C through a mechanism impacting splicing consequences on PKP2 protein dose. PKP2 targeted strategies (gene therapy and prime base editor-directed correction) can be exploited to therapeutically alter ARVD/C. We aim to determine: (i) the pathogenic mechanism by which PKP2 RNA splicing mutations drive ARVD/C, (ii) the impact and mechanism of early and late PKP2 restoration in our novel PKP2 mutant mouse and human ARVD/C models, and (iii) a base editing strategy to correct the PKP2 (IVS10-1 G>C) mutation and assess its impact in our novel PKP2 mutant ARVD/C model.