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Article by Richard Z. Zhuang & Roberta Lock
Gene Editing Prevents Heart Cell Therapy-Associated Arrhythmias
Source Publication:
Gene editing to prevent ventricular arrhythmias associated with cardiomyocyte cell therapy, Cell Stem Cell 2023
Silvia Marchiano et al., Charles E. Murry Lab
The human heart loses its regenerative capacity soon after birth. The lack of regenerative ability makes heart attacks especially dangerous. The discovery of stem cells has opened the doors to replacing these damaged areas with healthy stem-cell derived cardiomyocytes grown in the lab. However, the past 10 years of research has shown that there are many obstacles to overcome before we can inject cells into a damaged heart. Stem cell-derived cardiomyocytes exhibit a property called automaticity, meaning that they spontaneously beat. One major hurdle in heart cell therapy is that implanted heart cells’ innate beating can cause the patient’s heart to beat out of its regular rhythm –an arrhythmia: a life-threatening symptom that can prevent the heart from pumping enough blood and stopping altogether. Using gene editing technology, scientists are able to edit the DNA of the implanted cells to prevent these engraftment-related arrhythmias upon implantation.
What did these researchers do?
In order to develop a heart cell therapy that does not cause arrhythmias, the researchers set out to engineer stem cell-derived cardiomyocytes that lack automaticity but contract when externally stimulated. By leveraging CRISPR-Cas9 technology, the researchers were able to genetically manipulate a combination of four ion channels (which are proteins that govern the contraction process) in human pluripotent stem cells (hPSCs). Cardiomyocytes generated from these edited stem cells, named MEDUSA (Modification of Electrophysiological DNA to Understand and Suppress Arrhythmias), maintained a constant resting state that only contracted upon external stimulation. They then tested these cells by implanting 150 million MEDUSA stem cell-derived cardiomyocytes into a pig’s heart, and monitored their heart activity over two months using EKG. When transplanted, these cells engrafted and coupled electrically and mechanically with the host’s heart without causing sustained arrhythmias.
Why is this important?
The risks associated with a heart attack not only lies within the episode itself. During a myocardial infarction (heart attack), around ~1 billion adult heart muscle cells (cardiomyocytes) can die and are only replaced by non-contractile scar tissue. Even if a patient survives, the heart’s function is often permanently impaired due to the inability for heart muscle to regenerate after a heart attack. While cell therapy promises the potential for replacing damaged heart muscle, engraftment-related arrhythmias is a major complication that prevents this therapy from reaching the clinic.
How did the researchers do this?
It is known that the automaticity of stem cell-derived cardiomyocytes is strongly associated with their immature nature compared to cells found in the adult heart. To better understand the exact ion channels responsible, the researchers studied how ion channel expression changes over time as they mature. By looking at differences in gene expression, the scientists identified important candidate genes that contribute to the automaticity of hPSC-derived cardiomyocytes. After testing how these individual channels affected automaticity using ion channel-suppressing drugs, the researchers set out to use CRISPR-Cas9 to perturb genes of interest. After several rounds of knocking out and knocking in various combinations of ion channel genes and animal studies, the researchers settled on the triple knockout single knockin MEDUSA cell line that showed little-to-no automaticity in the lab but maintained their ability to depolarize and beat upon external stimulation. 100% of pigs implanted with un-edited cells showed arrhythmias, persisting for multiple weeks, and resulted in 67% mortality. Meanwhile, pigs implanted with MEDUSA cardiomyocytes showed brief self-terminating arrhythmias for 24 hours, but most importantly returned to normal rhythm until endpoint at 3 months.
Immunofluorescence showing engrafted cells inside host pig heart
What comes next?
This research marks a major milestone in cardiac cell therapy. However, much more work must be done before it reaches the clinic. Firstly, engraftment arrhythmias can only be studied in large animal models as small animals have vastly different heart physiologies (and do not exhibit these same arrhythmias. Consequently, this study was largely limited by the number of subjects used. It is therefore important for the MEDUSA cells developed here to be tested in larger studies to further validate their safety and efficacy. Secondly, studies here were conducted on healthy subjects with uninjured hearts. To fully understand whether the cells here are safe to use for patients, studies will need to be performed on injured subjects while not only looking at arrhythmic potential but also the efficacy of the treatment to improve heart function after injury. With this in mind, this work published in this study is groundbreaking, as it potentially makes the possibility of using stem-cell derived cardiomyocytes for transplantation into the heart much safer.