Targeted genome editing in human induced pluripotent stem cells from arrhythmogenic cardiomyopathy using CRISPR-Cas9

Introduction: Application of genome editing for cardiomyopathies has been recently suggested as a potential therapeutic intervention. We examined the ability of CRISPR/Cas9 to serve as a therapeutic intervention for arrhythmogenic cardiomyopathy (AC).

Methods: Patient-specific-hiPSCs were generated from AC patient carrying a pathogenic mutation in PKP2. Patient’s hiPSC mutation was genetically corrected using CRISPR/Cas9-based mutation correction and homologous recombination. The corrected cells served as isogenic-controls for the AC-hiPSCs. Molecular, structural and functional assessment was conducted to assess the phenotype of the diseased and corrected cells. Optical mapping was used to characterize signal propagation using engineered hiPSC-derived cardiac tissues. Finally, we established a model for stratifying the risk for arrhythmogenesis using an electrophysiological study in a dish (EPS-in-a-dish) approach.

Results: CRISPR/Cas9 corrected-hiPSC expressed normal mRNA and protein levels of PKP2 based on real-time PCR and western-blots /immunofluorescence, respectively. Lipid droplet accumulation was significantly reduced in the corrected-hiPSCs when compared to AC-hiPSCs and was at similar levels to that of healthy-control-hiPSCs. Optical mapping revealed a significant increase in conduction velocity in the corrected-hiPSCs 61±2cm/s compared with AC-hiPSCs 33±0.6cm/s (p<0.001) and was similar to that of control cells. The improved conduction velocity was associated with improved expression of Cx-43 and improved kinetics of I_Na in patch-clamp studies. Finally, the corrected-hiPSCs demonstrated decreased vulnerability for developing arrhythmia based on the EPS-in-a-dish study when compared to AC-hiPSCs.

Conclusion: CRISPR/Cas9 based therapeutic genome editing results in the restoration of functional and structural abnormalities associated with AC and may serve as a potential therapeutic intervention for genetic cardiomyopathies.