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Stem Cells for Cardiac Arrhythmia Risk Assessment

Periodic Reporting for period 3 - STEMCARDIORISK (Stem Cells for Cardiac Arrhythmia Risk Assessment)

Reporting period: 2018-11-01 to 2020-04-30

Cardiovascular disease is a major cause of morbidity and mortality in Europe. Genetic studies have been pivotal in identifying the genetic basis of many inherited cardiac diseases such as primary electric disorders, where potentially lethal arrhythmias arise from abnormalities in the electrical function of the heart. Acquired cardiac arrhythmias, predominantly caused by an adverse response to medication and a major challenge for clinicians and pharmaceutical companies, also have a significant heritable component. However a complicating factor in predicting patients at risk of these disorders is that they are clinically characterised by a broad spectrum of phenotypic expression, even within families. This variability in severity is related not only to the type and position of the mutation within the gene, but is influenced by variants in regulatory regions or in secondary modifier genes. Understanding these genetic contributions has been hampered by the lack of suitable model systems to perform such studies. Human induced pluripotent stem cells (hiPSCs), have the ability to not only self-renew indefinitely but also to differentiate into any cell type in the human body, including cardiomyocytes. As many cardiac diseases are autonomous to the cardiomyocyte, hiPSCs are promising in vitro paradigms for understanding cardiovascular disease pathophysiology and for identifying pathways to target in ameliorating the conditions. Indeed we have shown that cardiomyocytes generated from hPSC lines derived from patients with arrhythmogenic diseases exhibit the characteristic electrophysiological features of the respective disorders, but it is unclear how well these models reflect the genotype-phenotype relationship. The overall objective of STEMCARDIORISK is to establish whether cardiomyocytes derived from hPSCs can replicate in vitro the variable disease severity and incomplete penetrance commonly observed in patients, and whether we can use these models to pinpoint and assess the pathogenicity of novel variants identified by genetic studies. These models will be of important value in the areas of drug safety and personalised medicine, particularly with regards to improved individual risk stratification and patient-specific pharmacotherapy.
Most studies that have used hiPSCs to model diseases have compared the patient-derived iPSCs to iPSC lines generated from unrelated healthy donors. A problem with this approach is that the genomes of the patient and control iPSCs differ not only at the candidate disease-causing mutation, but also at many other loci including potential genetic modifiers. These additional genetic variations may contribute to phenotypic and drug response differences observed between patient and control cardiomyocytes, or even result in the loss of detectable phenotypes if the effect is subtle.

An elegant solution to help solve this issue is to use genetically-matched cell lines in which the cardiac disease-linked genetic variant is the sole modified variable. To demonstrate the power of this approach we have established a wild-type iPSC line that we introduce these variants into. We have extensively characterized this line both genetically and functionally to establish a clear baseline and then used state of the art gene editing tools (CRISPR/Cas9) to introduce specific mutations into a cardiac ion channel. In patients it was shown that the region of the ion channel containing the mutation influences the risk of an arrhythmic event occurring. In our genetically matched hPSC models it appears that we can also detect such differences in the cardiomyocyte’s electrophysiology. We are currently investigating whether our models respond differently to pharmacological compounds (i.e. exacerbate or protect) depending on the genetic variant.

To further reduce the heterogeneity that we have previously observed in our electrophysiological assays, we have refined our cardiomyocyte differentiation strategy leading to improvements in yield and purity. To evaluate the composition of the resulting cardiomyocytes we have established a panel of cardiac markers that ensures that the resulting populations we compare between the different lines are equivalent. We have also established a protocol for cryopreserving these hPSC-derived cardiomyocytes meaning the same stock of cardiomyocytes can be used for multiple assays over a prolonged period of time. These developments have clearly reduced the level of variability we observe in our measurements thereby also improving the sensitivity of our models to detect subtle functional differences.
While the genetic modification approach that we are currently using has provided us with evidence for the first time that differences in disease severity can be detected in hPSCs, the method is time-consuming. We are therefore developing a novel allele replacement strategy that will enable the rapid generation of panels of isogenic hPSC lines differing exclusively at candidate genetic variants thought to be associated with inherited or acquired cardiac arrhythmias. Testing of this method indicates that we can efficiently target 20kb genetic fragments into the hPSCs and up to 170 kb genomic regions can be integrated. This technology simplifies the procedure for generating panels of lines with a wider range and combination of genetic variants while maintaining control of the allele being modified. We believe that this strategy will assist in determining whether our PSC models can distinguish causal from benign variants in genes encoding ion channels, something that has proved difficult to predict based solely on genotype, and also address STEMCARDIORISK’s other main objective – whether we can use hPSC models to interpret and validate results obtained from genome-wide association studies (GWAS).