Prime editing corrects the dilated cardiomyopathy causing RBM20-P633L-mutation in human cardiomyocytes.

Area of Science:

• Molecular Biology
• Genetics
• Cardiology

Background:

• Prime editing (PE) is a novel gene editing technology with therapeutic potential for post-mitotic organs like the heart.
• The efficacy of PE in non-proliferating cells, such as human cardiomyocytes, remains largely uncharacterized.

Purpose of the Study:

• To evaluate the applicability and efficiency of prime editing in human cardiomyocytes derived from induced pluripotent stem cells (hi-CMs).
• To investigate the potential of PE to correct mutations causing inherited cardiac diseases, specifically dilated cardiomyopathy (DCM).

Main Methods:

• Developed a screening platform using HEK293T cells with a target array (TA-HEK) to identify efficient prime editing guide RNAs (pegRNAs).
• Applied the PE4 system to correct the RBM20P633L mutation in hi-CMs carrying patient-derived DCM mutations.
• Assessed editing efficiency, off-target effects, RBM20 protein localization, and downstream splicing changes.

Main Results:

• Identified efficient pegRNAs for targeting specific mutations, including LMNA K117fs, RBM20 P633L, and RBM20 R634Q.
• Achieved an average of 34.8% T-to-C editing efficiency for the RBM20P633L mutation in homozygous hi-CMs with minimal off-target edits.
• Restored RBM20 nuclear localization and normalized CAMK2D splicing, demonstrating functional rescue.

Conclusions:

• Prime editing can be effectively applied to correct disease-causing mutations in human cardiomyocytes, representing a significant advancement.
• This study provides the first demonstration of PE-mediated phenotypic rescue in a human post-mitotic model of DCM.
• The developed strategy offers a promising pathway for developing in vivo therapies for RBM20P633L-mediated DCM and other inherited cardiac conditions.