Innovative Strategies to Enable Prime Editing of PLN R14del-induced Cardiomyopathy

Abstract

This thesis addresses the genetic basis and therapeutic opportunities for PLN R14del-induced cardiomyopathy, a severe inherited cardiac disease characterized by impaired calcium handling, protein aggregation, and arrhythmogenic remodeling. Current management strategies alleviate symptoms but fail to correct the underlying mutation. The work presented here investigates prime editing (PE) as a precise genome-editing tool to repair the causative mutation in patient-derived induced pluripotent stem cell–derived cardiomyocytes (iPSC-CMs) and preclinical models. Part I establishes the conceptual and methodological foundation. It reviews recent advances in CRISPR-based technologies, with an emphasis on prime editing, and evaluates techniques for measuring editing efficiency, including reporter systems and molecular assays. Using a tailored reporter cell line, the thesis demonstrates how pegRNA design and secondary structure strongly influence PE efficiency, providing insights for rational pegRNA optimization. Part II explores delivery strategies for clinical translation. Adenoviral vectors were engineered to co-deliver SpGPEmax and optimized pegRNAs into iPSC-CMs and humanized PLN R14del mice, achieving efficient correction. Complementary non-viral systems were developed, including cell-penetrating peptide–mediated PE-RNP delivery (LAH5) and photoporation-based approaches, both of which enable transient, efficient, and scalable delivery. These methods reduced off-target effects and immunogenicity while preserving cell viability. Together, the findings provide proof-of-principle that prime editing can precisely repair PLN R14del mutations across multiple delivery platforms. This work highlights key technical considerations—pegRNA design, editing evaluation, and delivery optimization—and establishes a translational framework for correcting inherited cardiomyopathies. Beyond PLN R14del, the approaches developed here pave the way for targeted genome editing of diverse genetic cardiac diseases, advancing precision cardiovascular medicine.