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Therapeutic genome editing using CRISPR/Cas9 for arrhythmogenic cardiomyopathy induced pluripotent stem cells derived cardiac tissues

Session HFA Discoveries - ePublications

Speaker Oren Caspi

Event : HFA Discoveries 2020

  • Topic : basic science
  • Sub-topic : Basic Science - Cardiac Diseases: Cardiomyopathies
  • Session type : ePublication

Authors : O Caspi (Haifa,IL), S Chrona (Haifa,IL), A Gepstein (Haifa,IL), A Shiti (Haifa,IL), I Huber (Haifa,IL), G Arbel (Haifa,IL), L Gepstein (Haifa,IL)

O Caspi1 , S Chrona2 , A Gepstein2 , A Shiti2 , I Huber1 , G Arbel2 , L Gepstein1 , 1Rambam Health Care Campus, Cardiology - Haifa - Israel , 2Technion - Israel Institute of Technology - Haifa - Israel ,

Basic Science - Cardiac Diseases: Cardiomyopathies

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 for 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 INa 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 INa 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.

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