PREcision MEDicine with induced pluripotent stem cells for Cardiac Arrhythmias Risk Evaluation

Project Information

PREMEDiCARE

Grant agreement ID: 795209

DOI

10.3030/795209

Project closed

EC signature date 14 March 2018

Start date 1 October 2018

End date 3 July 2021

Funded under

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Total cost

€ 168 277,20

EU contribution

€ 168 277,20

Coordinated by

ISTITUTO AUXOLOGICO ITALIANO
Italy

Periodic Reporting for period 1 - PREMEDiCARE (PREcision MEDicine with induced pluripotent stem cells for Cardiac Arrhythmias Risk Evaluation)

Reporting period: 2018-10-01 to 2020-09-30

Summary of the context and overall objectives of the project

The Long QT Syndrome (LQTS) is one the most common inherited cardiac arrhythmias, representing one of the leading causes of sudden cardiac death below age 20. LQTS is clinically visible as a pathological prolongation of the QT interval on the electrocardiogram as a consequence of genetic variants in genes encoding for cardiac ion channels or other key cardiac proteins, or of exogenous factors such as medications.

The in vitro study of LQTS and congenital cardiac arrhythmias has profoundly changed in the last 10 years. Earlier, aspects of LQTS could be partially reproduced in vitro only in heterologous systems (i.e. by introducing the mutated ion channel of choice into cancer cell lines), by pharmacologically simulating the effect of QT-prolonging drugs in animal cardiomyocytes (CMs) or by using costly and complex transgenic animal models. None of these experimental models is capable of fully reproducing the clinical condition of LQTS patients. Better, more refined and more ethical experimental models to investigate the underlying mechanisms of LQTS and congenital cardiac arrhythmias are needed.
Human induced pluripotent stem cells (hiPSCs) have demonstrated to be a very powerful tool to recreate the clinical condition of a patient in vitro, and this has revealed valid also for LQTS and cardiac arrhythmias; hiPSCs guarantee a virtually unlimited source of human CMs which can be used to identify the molecular mechanisms of a disease and to test drugs on cells directly derived from a patient (i.e. sharing the same diseased genetic background).

However, despite the proven capability of CMs derived from hiPSCs (hiPSC-CMs) to reproduce simple clinical hallmarks of LQTS variants in vitro (e.g. QT prolongation), we do not know how they behave in modelling complex features of LQTS; one aspect poorly investigated in vitro was the incomplete penetrance, the fact that individuals carrying a specific disease-causing variant may exhibit life-threatening symptoms while others, still affected by the same variant, are completely asymptomatic. Logically, this complicates the diagnosis, the risk stratification and the therapy for LQTS patients.

The PREMEDiCARE project has two main goals:
1) Demonstrate that hiPSC-CMs can reproduce the clinical severity observed in symptomatic vs asymptomatic carriers of variants associated with LQTS.
2) Identify and functionally characterize factors correlated with an altered risk of drug-induced LQTS in symptomatic vs asymptomatic LQTS patients.

These aims are challenging but important for society and for the whole scientific community as they will contribute to refine human-based experimental models that can represent reliable alternatives to animal testing and help to develop precision medicine approaches for the prevention and/or treatment of congenital and acquired cardiac arrhythmias such as LQTS.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

The PREMEDiCARE project has demonstrated that it is possible to reproduce in vitro the differences observed in patients affected by the same disease-causing mutation who show different clinical phenotypes (i.e. symptoms).

In our study (Lee & Sala & Mura et al. Cardiovascular Research, 2021, doi: 10.1093/cvr/cvaa019) two relatives were affected by the KCNQ1-p.Y111C variant, which causes a severe loss-of-function in a cardiac potassium channel (KV7.1 IKs). Surprisingly, one of the patients had a prolonged QT interval and was severely symptomatic while the other was completely asymptomatic, and we have investigated whether it was possible to recreate this discrepancy in vitro with hiPSC-CMs.

We have characterized the electrophysiology of hiPSC-CMs from these two subjects and compared to that from a control, and we have found that hiPSC-CMs are capable of discriminate the different clinical severity of LQTS despite the presence of an identical disease-causing variant. We have further investigated the mechanism underlying this discrepancy demonstrating that in this family the phenotypic differences were caused by two common single nucleotide polymorphisms (SNPs) in a gene (MTMR4) involved in the ubiquitination-mediated degradation of ion channels. These “protective” SNPs reduced the efficacy by which potassium channels are degraded and prevented the collateral degradation of another key cardiac potassium channel (KV11.1 hERG), compensating for the loss-of-function induced by the LQTS variant.

In a second study (Sala et al., bioRxiv, 2021, doi: 10.1101/2021.06.25.449913) we further verified whether the different disease severity among carriers of LQTS variant was also inducing a differential drug response to proarrhythmic drugs. In other words, we investigated whether hiPSC-CMs from symptomatic patients with LQTS were more prone to develop drug-induced arrhythmias compared to those from asymptomatic patients or from controls. By testing the effect of the QT-prolonging drug hydroxychloroquine, recently repurposed in the attempt to prevent or treat of COVID-19, we have demonstrated that disease-specific hiPSC-CMs do show a differential sensitivity to QT prolonging drugs that agrees with the underlying genotype. This demonstrates that safety pharmacology may indeed benefit from disease-specific cohorts of hiPSC-CMs in addition to drug testing in hiPSC-CMs from commercial sources or healthy donors.

The results of the PREMEDiCARE project were published in peer-reviewed journals (Cardiovascular Research, European Heart Journal, Stem Cell Reports) and presented in virtual meetings, with the most recent findings also uploaded on preprint servers (bioRxiv) to facilitate the dissemination of the results while the manuscript is undergoing peer review. At the time of writing this report, the PREMEDiCARE project has led to 3 peer-reviewed articles, 2 peer-reviewed reviews, 1 editorial, 1 peer-reviewed book chapter, 1 preprint publication (currently under peer-review), 1 podcast (European Society of Cardiology Cardiotalk).

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

The PREMEDiCARE project has progressed beyond the state of the art in two aspects.
1) The project has identified a novel factor (genetic modifier) involved in the potassium channel turnover that protects its carrier from the consequences of type-1 LQTS;
This will contribute to advancements in patient risk stratification which will ideally improve the quality of life of patients affected by LQTS and identify the most suitable treatment or preventive strategies based on the results of genetic testing. The PREMEDiCARE project has further confirmed that a more comprehensive risk stratification should go beyond the main LQTS disease-causing genes and that hiPSC-CMs can be a valuable tool for the experimental validation of these genetic targets. The results will be validated in future studies in larger cohorts of LQTS patients.
2) The project has also contributed to demonstrate the importance of disease-specific hiPSC-CMs in safety pharmacology, offering a new perspective on the way drugs can be repurposed or tested to investigate the wide spectrum of cohort-specific drug responses.

Phenotypic differences of symptomatic vs asymptomatic patients affected by the same LQTS variant

Differential response to QT-prolonging drugs in hiPSC-CMs from asymptomatic or symptomatic carriers.