Bhagwan, Jamie R.
(2018)
CRISPR-Cas9 gene targeting of human induced pluripotent stem cells to generate a palette of optical reporter cell lines towards investigating hypertrophic cardiomyopathy.
PhD thesis, University of Nottingham.
Abstract
Hypertrophic cardiomyopathy (HCM) is an inherited cardiomyopathy with a variety of causative mutations, largely within sarcomeric proteins. This can result in thickening of the left ventricle of the heart, altered contractility, arrhythmias, and in some cases, sudden cardiac death.
Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) are a valuable tool for disease modelling cardiomyopathies such as HCM, and screening for potential pharmaceutical interventions. The recent emergence of CRISPR Cas9 has opened new opportunities to carry out precise gene editing to correct disease-causing mutations, as well as providing an accessible method of introducing reporter genes into the genome of hiPSCs.
This project sought to utilise CRISPR-Cas9 to generate a palette of genetically encoded calcium indicator (GECI) hiPSC lines in both healthy and diseased backgrounds to investigate HCM in vitro.
Site-specific guide RNAs and targeting vectors were designed to target the AAVS1 locus, an apparent safe harbour, with the GECIs RGECO or GEM-GECO, using CRISPR Cas9. These reporter lines were generated in three different genetic backgrounds, representing two different mutation backgrounds associated with HCM, two ACTC1 G301A isogenic pairs of healthy and heterozygous mutant hiPSCs and an MYH7 C9123T isogenic trio of healthy, heterozygous and homozygous mutant hiPSCs. These lines were validated with PCR, immunostaining and live imaging microscopy.
By utilising confocal line scan microscopy and repurposing software to quantify and measure calcium transients, an arrhythmic phenotype was found in MYH7 C9123T mutant hiPSC-CMs. This arrhythmic phenotype was also seen in P1 ACTC1 G301A mutant hiPSC-CMs, but not P2 ACTC1 G301A mutant hiPSC-CMs, suggesting that this particular mutation is necessary yet not sufficient to cause an arrhythmic phenotype.
Based on the observation that MYH7 mutants were resistant to calcium channel blockers and the arrhythmic phenotype was rescued in low extracellular calcium conditions, a focused drug screen was performed which identified the late sodium current inhibitor ranolazine, and the ryanodine receptor antagonist dantrolene, as viable drug treatments for phenotypic rescue in mutant hiPSC-CMs.
Through this study, new insights into the mechanisms underlying HCM and the variability between mutations and within individuals have been elucidated.
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