Therapeutic Genome editing using CRISPR/Cas9 for an induced pluripotent stem cell model of arrhythmogenic cardiomyopathy

Introduction: CRISPR/Cas9 provides a novel methodology for selective gene editing. Application of genome editing for cardiomyopathies has been recently suggested as a potential therapeutic intervention. In the current study, we examine the ability of combined CRISPR/Cas9 and homologous recombination 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 by using CRISPR/Cas9-based mutation correction and homologous recombination. The corrected cells served as isogenic-controls for the AC-hiPSCs. Real-time-PCR and Western-Blot analysis were used to assess the molecular signature of the disease. Structural abnormalities were evaluated by immunostainings for desmosomal proteins and lipid-droplet accumulation. Patch-Clamp was used to assess I_Na current-kinetics at the cellular level. Conduction abnormalities and arrhythmogenesis using an electrophysiology study in a dish (EPS-in-a-dish) were evaluated at the tissue level using multilayered confluent hiPSC-derived cardiac cell tissues with optical mapping.

Results: CRISPR/Cas9 corrected hiPSCs demonstrated appropriate stem cell characteristics and differentiated to cardiomyocytes expressing typical molecular markers. The 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±2 cm/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 using 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 arrhythmogenic cardiomyopathy and may serve as a potential therapeutic intervention for genetic cardiomyopathies.