Effect of CRE-AAV excision of the rescue cassette in a pig model of PLN

C. Cabrera-Gomez (München)¹, T. Bozoglu (München)¹, P. Hoppmann (München)¹, M. Cambra (München)¹, E. Wolf (Oberschleissheim)², N. Klymiuk (München)¹, A. Bähr (München)³, C. Kupatt (München)^4
1Klinikum rechts der Isar der Technischen Universität München Klinik und Poliklinik für Innere Medizin I München, Deutschland; ²LMU München Lehrstuhl für molekulare Tierzucht und Biotechnologie Oberschleissheim, Deutschland; ³Klinikum rechts der Isar der Technischen Universität München Klinik und Poliklinik für Innere Medizin I Heidelberg, Deutschland; ⁴TUM Klinikum Rechts der Isar Klinik und Poliklinik für Innere Medizin I München, Deutschland

Phospholamban (PLN) protein has a prominent role in the regulation of cardiac activity, regulating the calcium influx as a primary inhibitor of the sarcoplasmic Ca2+-ATPase (SERCA). Mutations in the PLN gene have been associated with dilated and/or arrhythmogenic cardiomyopathies, the Arg(R)14del being one of the most widespread forms. For the study of this pathology, a porcine model was created by humanizing the second exon of the PLN and introducing the R14del mutation. However, due to the dominant character of the disease, conditional expression was ensured by placing a healthy human exon 2 sequence flanked by loxP sites in front of the mutated allele. This rescue cassette was flanked by two loxP sites, making it possible to artificially induce the cassette excision with a Cre recombinase nuclease.
In this porcine PLN model, we administered adeno-associated virus (AAV) containing Cre mRNA (Cre-AAV) with specific cardiac tropism in a postnatal injection. Two rescue cassettes carrier pigs were intravenously injected with 10¹⁴ gc/kg body weight of Cre-AAV at the age of 6 weeks while two control animals (1 carrier and 1 wild type) remained un-injected. After six months’ follow-up, all animals were evaluated by cardiac electrophysiology to study the phenotypic effect of the cassette excision. Heart samples were collected and processed in order to study the excision efficiency at the genomic and transcriptomic level in the heart tissue.
Evaluation on the level of the cardiomyocyte population, by analysing the expression of the PLN transcript for the mutated form, indicated editing efficiency to 15-25% in cardiomyocytes. Functional electrophysiological examination of the left and right ventricles revealed voltage map alterations in both injected animals that correspond to the human pathology, indicating a potential biological effect of AAV induced cardiomyocyte editing.
The results obtained suggest that postnatal administration of Cre-AAV with excision of the rescue cassette in a fraction of the cardiomyocyte population is capable of generating an arrhythmogenic phenotype in PLNR14del pigs. This animal model will serve for further characterization of development of the ARVC and its prevention/treatment by gene therapy and gene editing.