Introduction:
The phospholamban (PLN) gene is a key regulator of calcium homeostasis in cardiomyocytes through its inhibitory effect on the sarcoplasmic reticulum Ca²⁺-ATPase (SERCA2a). Pathogenic variants in PLN are associated with severe forms of dilated (DCM) and arrhythmogenic cardiomyopathy (ACM). Affected individuals often display low-voltage ECGs, frequent ventricular arrhythmias, and progressive heart failure, typically manifesting by the fourth decade of life, although some carriers remain asymptomatic. These mutations disrupt calcium cycling, induce metabolic stress, and promote protein aggregation in cardiomyocytes. Current therapies remain symptomatic, focusing on arrhythmia prevention and heart transplantation in end-stage disease.
Aim:
This study aimed to establish an adeno-associated virus (AAV)–based prime editing (PE) platform to correct pathogenic PLN variants and evaluate its therapeutic potential in human iPSC-derived cardiomyocytes and a humanized PLN mouse model.
Methods and Results:
To establish a screening platform for prime editing optimization, we generated a human cell line carrying the PLN mutation in its endogenous locus. Four pegRNAs combined with five nicking guide RNAs and two Streptococcus pyogenes Cas9 variants were screened, yielding correction efficiencies between 3% and 18% as determined by targeted sequencing. The most efficient configuration was packaged into an intein-split dual AAV system for in vivo delivery. In patient-derived iPSC cardiomyocytes carrying the R14del mutation, AAV-mediated PE achieved up to 11% precise correction. To confirm in vivo applicability, we performed proof-of-concept editing at the Dnmt1 locus in neonatal mice, resulting in up to 20% correction in heart tissue and up to 43% in the liver. Subsequently, dual AAV9-PE vectors targeting the humanized PLN R14del allele were systemically administered to neonatal mice. Eight weeks post-injection, we observed up to 19% allele frequency with the precise correction at the transcript level, indicating robust editing efficiencies in mouse cardiomyocytes. Functionally, stress-induced arrhythmia testing demonstrated a marked reduction in ventricular arrhythmias compared with saline-treated controls.
Conclusion:
Dual AAV–mediated prime editing enables efficient and therapeutically relevant correction of the PLN R14del mutation in both in vitro and in vivo models. These results establish prime editing as a promising therapeutic strategy for inherited cardiomyopathies. Ongoing studies in humanized porcine models will further evaluate its translational potential toward clinical application.
