Title: Development of Novel Therapeutics for Timothy Syndrome Using Human Induced Pluripotent Stem Cells
Description: Cardiac disease is the leading cause of death in the United States, despite the continuing efforts contributed to scientific research and disease management in the past few decades. However many advances have been made in cardiovascular research in recent decades and one of the advances is the development of human induced pluripotent stem cell(iPSC)-based disease models. The human iPSC-based disease models are derived from the somatic cells of patients with cardiac diseases and capture the genotypes of the original patients, which make them more ideal for mimicking human diseases compared with conventional rodent models. So far, the iPSC-based disease models have been used to model several types of cardiac diseases, one of which is the focus of this work-Timothy syndrome. Timothy syndrome is caused by the missense mutations in the CACNA1C gene encoding the voltage-gated calcium channel CaV1.2, which plays an essential role in cardiac function. The disease is a multisystem disorder that is featured by long QT syndrome and syndactyly.

Timothy syndrome patients are treated clinically with beta-adrenergic blockers, calcium channel blockers, and sodium channel blockers. However, these regimens are insufficient to prevent lethal arrhythmias in Timothy syndrome patients, especially infants with Timothy syndrome. Therefore, new therapeutics to prevent the lethal arrhythmias in Timothy syndrome patients are needed until the age when an implantable defibrillator is available. The iPSC-based model of Timothy syndrome was first reported in 2011. The previous report showed that the Timothy syndrome iPSC-derived cardiomyocytes demonstrated several cellular phenotypes including abnormal contractions, abnormal electrophysiological properties and abnormal calcium handling, which were consistent with the clinical features of the patients that the iPSCs were derived from. In addition, the authors demonstrated that Roscovitine, a cyclin-dependent kinase (CDK) inhibitor, could rescue the cellular phenotypes in Timothy syndrome cardiomyocytes.

However, the mechanisms underlying the beneficial effects of Roscovitine on Timothy syndrome cardiomyocytes were not fully elucidated. This work will employ the iPSC-based model of Timothy syndrome to investigate the mechanisms underlying the beneficial effects of Roscovitine on Timothy syndrome cardiomyocytes and search for additional therapeutic compounds and targets for Timothy syndrome. In chapter 1 of this work, we presented new methods to generate iPSCs from human skin fibroblasts or hair keratinocytes, and to differentiate iPSCs into cardiomyocytes in a monolayer format. The major advantage of the two new methods is that they are technically simple and generally applicable for samples from healthy control donors and patients with cardiac diseases. The new methods enabled us to generate a sufficient amount of Timothy syndrome cardiomyocytes from iPSCs derived from the skin fibroblasts of Timothy syndrome patients, which became the foundation for the subsequent mechanistic study. Chapter 2 presents the identification of CDK5 as a new therapeutic target for Timothy syndrome.

As introduced above, the previous report demonstrated that Roscovitine, a CDK inhibitor, could rescue the cellular phenotypes in Timothy syndrome cardiomyocytes. However, the mechanisms underlying the beneficial effects of Roscovitine on Timothy syndrome cardiomyocytes were not fully elucidated. To identify additional therapeutic compounds for Timothy syndrome and investigate the mechanisms underlying the therapeutic effects of Roscovitine on Timothy syndrome cardiomyocytes, we conducted a phenotypic screen using Timothy syndrome cardiomyocytes to screen through twenty Roscovitine analogs and four CDK inhibitors with different specificities for different CDKs. Four positive compounds were identified from the screen. When we summarized the CDK targets of the four positive compounds and the lead compound Roscovitine, it was found that four out of the five positive compounds shared a common CDK target, which is CDK5, indicating that CDK5 could be involved in the pathogenesis of Timothy syndrome as a therapeutic target.

We next validated CDK5 as a new therapeutic target for Timothy syndrome using two independent approaches. The two approaches are expressing a dominant negative mutant of CDK5 and expressing short hairpin RNAs targeting CDK5 in Timothy syndrome cardiomyocytes using lentiviruses. Both approaches led to CDK5 inhibition in Timothy syndrome cardiomyocytes and we examined the changes in the cellular phenotypes in Timothy syndrome cardiomyocytes with CDK5 inhibition. The results indicated that CDK5 inhibition alleviated all the previously-reported phenotypes in Timothy syndrome cardiomyocytes. To investigate the mechanisms underlying the beneficial effects of CDK5 inhibition on Timothy syndrome cardiomyocytes, we examined the expression of CDK5 activator p35 and the activity of CDK5 in Timothy syndrome cardiomyocytes. We found that Timothy syndrome cardiomyocytes showed a higher expression of CDK5 activator p35 and a higher activity of CDK5 compared with control cardiomyocytes. When we over-expressed CDK5 in control cardiomyocytes, we found that CDK5 over-expression caused a change in the function of CaV1.2 channels in control cardiomyocytes that resembled the phenotype in Timothy syndrome cardiomyocytes.

In summary of the results, we propose that in Timothy syndrome cardiomyocytes, the increased expression of CDK5 activator p35 causes CDK5 hyper-activation, which enhances the abnormal function of the mutant CaV1.2 channels, leading to more severe phenotypes. Thus, CDK5 inhibition alleviates the phenotypes in Timothy syndrome cardiomyocytes. The results in this chapter reveal that CDK5 is a new therapeutic target for Timothy syndrome and CDK5-specific inhibitors can potentially be developed into new therapeutics for Timothy syndrome. However, we found that the currently-available chemical inhibitors for CDK5 are not highly-selective and have several significant side effects that make them not ideal candidates to be developed into new therapeutics for cardiac diseases. Therefore new therapeutic compounds and targets are still needed for Timothy syndrome. Chapter 3 presents the identification of the sigma-1 receptor as a new therapeutic target for Timothy syndrome. Due to the side effects associated with the currently-available chemical inhibitors for CDK5, we made an effort to search for an additional therapeutic target and therapeutic compounds for Timothy syndrome.

We reasoned that instead of directly inhibiting CDK5, we could potentially alleviate the phenotypes in Timothy syndrome cardiomyocytes by affecting the CDK5 activator p35 and this idea led us to the sigma-1 receptor. After we looked into the sigma-1 receptor, we found that in addition to being reported to modulate p35 protein level, the sigma-1 receptor had also been reported to modulate calcium homeostasis, which is another favorable effect for Timothy syndrome cardiomyocytes. Therefore we hypothesized that the activation of the sigma-1 receptor could be beneficial for Timothy syndrome cardiomyocytes, which feature an increased expression of p35 and a dysregulation of calcium homeostasis. To test this hypothesis, we examined the effects of two sigma-1 receptor agonists, one of which is a FDA-approved drug, on the phenotypes in Timothy syndrome cardiomyocytes. The results demonstrated that the treatment of the two sigma-1 receptor agonists alleviated the previously-reported phenotypes in Timothy syndrome cardiomyocytes.

We also examined the effects of the two sigma-1 receptor agonists on the functions of control cardiomyocytes and found that the treatment of the two sigma-1 receptor agonists did not have significant side effects on the regular contractions and normal calcium transients in control cardiomyocytes. Overall, the results reveal that the sigma-1 receptor is a new therapeutic target for Timothy syndrome. The results also demonstrate that the two sigma-1 receptor agonists that we tested are promising lead compounds that can developed into novel therapeutics for Timothy syndrome in the future. Since one of the sigma-1 receptor agonists that we tested is a FDA-approved drug, this drug could potentially be used directly in Timothy syndrome patients for treating the cardiac arrhythmias in the near future. In summary, this work is a proof of concept that the iPSC-based models of cardiac diseases can be used to generate novel insights into disease pathogenesis, and to identify new therapeutic targets and compounds for cardiac diseases, and in particular for Timothy syndrome.

The therapeutic targets and compounds that we have identified in this work would be helpful for the development of novel therapeutics for treating the lethal arrhythmias in Timothy syndrome patients in the future.