Cardiac in vivo correction of the PLN R14del cardiomyopathy mutation using prime editing

Background Phospholamban (PLN) R14del is a founder mutation associated with progressive dilated and arrhythmogenic cardiomyopathy characterised by impaired sarcoplasmic reticulum calcium cycling, ventricular arrhythmias and high rates of heart transplantation. No disease-modifying therapy targeting the underlying genetic cause is available. Prime editing (PE) enables precise sequence modification without inducing double-strand DNA breaks and, combined with dual adeno-associated virus (AAV) delivery, represents a programmable strategy for cardiac genome editing. Purpose To develop and validate a dual AAV PE platform for in vivo correction of PLN R14del in patient-derived induced pluripotent stem cell (iPSC) cardiomyocytes, murine models and a translational large-animal setting. Methods A human PLN R14del cell line was generated by PE for guide optimisation. Four pegRNAs were screened with multiple nicking guide RNAs across two Streptococcus pyogenes Cas9 variants. Editing efficiencies were quantified by deep sequencing. In vivo cardiac delivery was benchmarked using AAV9 vectors targeting the Dnmt1 locus, comparing neonatal and adult administration. The optimised SpCas9 configuration was delivered via intein-split dual AAV9 to patient-derived PLN R14del iPSC cardiomyocytes and to humanised PLN R14del mice. Arrhythmia burden was assessed using caffeine–epinephrine electrocardiogram challenge. Translational delivery feasibility was evaluated in wild-type porcine hearts following systemic and intracardiac AAV administration. Results In a PE-generated human PLN R14del cell line, SpCas9-based PE achieved 15.8% precise correction, whereas SpG-Cas9 variants yielded 1–12%. In patient-derived PLN R14del iPSC cardiomyocytes, dual AAV9 delivery achieved ~10% precise correction. In vivo, proof-of-concept PE at the Dnmt1 locus resulted in 15–20% cardiac editing following neonatal delivery compared with 4–5% after adult delivery, indicating age-dependent differences in efficiency. In humanised PLN R14del mice, neonatal systemic delivery achieved ~20% cardiac correction versus 2–3% in adults. Neonatally treated mice demonstrated reduced stress-induced ventricular arrhythmias compared with controls (10–15% vs 45–60%; p<0.01), whereas adult-treated mice showed no functional improvement. In wild-type porcine hearts, systemic and intracardiac AAV delivery resulted in myocardial expression of dual AAV-delivered PE components across ventricular regions, confirming large-animal cardiac transduction feasibility. Conclusion Dual AAV-mediated PE enables functionally relevant correction of PLN R14del in vivo and reveals age-dependent differences in cardiac editing efficiency. Large-animal studies demonstrate scalable myocardial delivery, supporting translational development of cardiac genome editing strategies.