AAV-Mediated Prime Editing for the Correction of Familial Mutations Associated with Cardiomyopathy

Introduction: The phospholamban (PLN) gene plays a critical role in regulating calcium homeostasis in cardiomyocytes, primarily by inhibiting the sarcoplasmic reticulum Ca²⁺-ATPase (SERCA2a). Certain pathogenic variants in PLN have been associated with severe manifestations of dilated cardiomyopathy (DCM) and arrhythmogenic cardiomyopathy (ACM). Patients often present with low-voltage ECGs, frequent ventricular arrhythmias, and progression to end-stage heart failure, typically emerging by the fourth decade of life, although some carriers remain asymptomatic. These mutations disrupt calcium handling, induce metabolic dysfunction, and promote protein aggregation in cardiomyocytes. Current treatment strategies focus on managing symptoms, preventing sudden cardiac death, and heart transplantation in advanced cases.

Aim: This study aimed to develop an AAV prime editing (PE) approach and evaluate its effectiveness for targeted PLN variant correction in human iPSC-derived cardiomyocytes and in a humanized PLN variant mouse model.

Methods and Results: To develop optimal prime editing (PE) strategies for correcting pathogenic PLN variants, we screened four pegRNAs in combination with various nicking gRNAs and different Cas9 variants in human cell lines carrying the PLN mutation. This comprehensive screening yielded correction efficiencies ranging from 3% to 30%, enabling the identification of the most promising PE components for effective mutation correction. Subsequently, we utilized a dual AAV prime editing system incorporating pegRNA components and additional AAV elements to enhance editing efficiency. This approach was applied to patient iPSC-derived cardiomyocytes, achieving targeted correction efficiencies of 10-13%. To further evaluate the in vivo efficacy of the AAV-PE system, AAV9-PE vectors were administered to humanized PLN variant mice. Five weeks post-treatment, the effectiveness of the intervention was assessed using a stress-induced arrhythmia protocol. Notably, treatment with the AAV9-PE vector in the mouse model led to a reduction in pathogenic PLN expression and a significant decrease in arrhythmic events compared to saline-treated controls.

These findings demonstrate the potential of dual AAV-mediated prime editing as a therapeutic approach for correcting PLN mutations and mitigating associated cardiac dysfunction. The ongoing research focuses on the efficacy of the prime editing approach in correcting PLN mutations in humanized porcine models, aiming to expand the translational potential of this strategy for broader clinical applications.