Abstract
In Chapter 1B and chapter 1C, we provide an overview of the challenges encountered within the cardiac biology research field and how the CRISPR/Cas9 toolbox can facilitate in overcoming these limitations.
Chapter 2 describes a novel heterozygous knock-in mouse model bearing a known pathogenic variant in PKP2. We demonstrate the
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value of matching this model with an equivalent hiPSC-derived cardiomyocyte model bearing the same heterozygous mutation, revealing that degradation of desmosomal proteins is a driving force behind ACM pathogenesis. These findings were further validated in human explanted ACM hearts.
In chapter 3 we identified a novel DSP mutation found in a patient experiencing arrhythmogenic episodes. Using CRISPR/Cas9, we created two isogenic pairs of hiPSC lines to study the clinical relevance of this mutation. In one set we corrected patient-derived cells and in the other set we introduced the mutation in the endogenous locus of commercially bought control hiPSCs. This approach underscored the advantages but also the disadvantages of hiPSC-derived cardiomyocyte models and allowed us to rigidly identify PITX2 as a novel factor involved in ACM pathology.
In chapter 4 we performed spatial transcriptomics on the left ventricular wall of an explanted heart obtained from an ACM patient bearing a nonsense mutation in DSP. The overall goal was to identify disease-driving factors by comparing relatively healthy myocardial regions with areas of active remodeling. We used a KI hiPSC-derived cardiomyocyte model, bearing a known pathogenic nonsense mutation in DSP, to assess the relevance of the identified candidates.
Chapter 5 summarizes the results obtained from two novel heterozygous KI mouse models bearing mutations in DSP, which are equivalent to the human mutations introduced in chapters 3 and 4. This study highlights the challenges and dissimilarities between a rationally designed mouse model and the clinical situation observed in patients.
Chapter 6 is an introduction to CRISPR prime editing. Not only do we show that this novel methodology has the potential to overcome limitations inherent to the basic CRISPR/Cas9 machinery, but also how we want to exploit this system to efficiently restore the PKP2 mutation introduced in chapter 2.
Chapter 7 summarizes all the studies and their contribution towards furthering our understanding of ACM pathology. Finally, a brief outlook is given with respect to CRISPR-based methodologies and ACM pathogenesis.
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