SK Yator(1), PR Vochozkova(1) JM Cambra(1) E Wolf(2) C Kupatt(1) N Klymiuk(1)

1. TUM University Clinics, Clinic for Cardiology, Technical University of Munich; 2. Molecular Animal Breeding and Biotechnology, LMU Munich

Genetically inherited mutations in PKP2 account for approximately half of desmosome-related cases of arrhythmogenic cardiomyopathy (ACM). PKP2 dysfunction is caused by mutations of a single allele, making haploinsufficiency the proposed pathogenic mechanism. To overcome shortage for PKP2 transcripts, we plan test transcriptional upregulation in a novel PKP2 pig model, carrying a patient-relevant mutation in the pig PKP2 gene, combined with a transgene for CRISPR activation (CRISPRa).

Anticipating potential detrimental effects of a PKP2 mutation on pig reproduction, our strategy involves a rescue cassette and a Cre-inducible mutation at the PKP2exon 10. We set up a targeting vector, based on a BAC, covering the pig PKP2 gene, and modified it by recombineering in one go. Combining the modified BAC with CRISPR/Cas to stimulate homologous recombination at the PKP2 locus, we produced single-cell clones that were verified for correct integration of the genetic modification.

In parallel, we evaluated CRISPRa for transcriptional activation by inducing a dCas9-VPR transgene in the pig ROSA26 locus, in vitro. Primary porcine cells were nucleofected with gRNAs targeting promoter regions of PKP2, MEG3, and USH1C candidate genes, along with a CRE-expressing plasmid to trigger CRISPRa expression. Some but not all of the designated gRNA combinations led to significant gene upregulation of the genes, confirming the system's functionality.

Prospectively, we will combine PKP2 with CRISPRa modifications in pigs by breeding, by genetic engineering or a combination of both for in vivo testing. Further, we aim at testing transcriptional upregulation in iPSC-derived cardiomyocytes derived from patients with PKP2 mutations.