Gene edits quell rhythm problem from heart stem cell grafts


Silvia Machiano conducts  research in the Murry lab in the Institute for Stem Cell and Regenerative Medicine
Murry Lab/ISCRM
Silvia Machiano conducts heart muscle cell research in the Murry lab at the Institute for Stem Cell and Regenerative Medicine.

Regenerative medicine researchers have made progress toward overcoming a seemingly intractable obstacle to stem cell-derived heart cell transplantation for repairing damaged heart muscle. Their approach is a significant advance toward making heart cell transplants a potential reality for patients with cardiac disease.

Their findings were presented today, June 21, at the International Society for Stem Cell Research 2021 Annual Meeting, held virtually this year.

Human heart muscle can be destroyed by a heart attack, persistent high blood pressure, or diseased valves. Cell therapies are a hoped-for option to restore lost function. Many people with heart failure – the progressive weakening of the heart – have trouble carrying out daily activities. Their survival odds five years after diagnosis is greatly diminished.

Unlike skin and many other organs in the body, the heart is a non-regenerative organ. That means it is unable to replace deficiencies in its cell population. However, scientists are learning to harness stem cells to make an unlimited number of heart muscle cells.

Using these stem cell-derived heart muscle cells, or cardiomyocytes, researchers at the UW Medicine Institute for Stem Cell and Regenerative Medicine, led by director Dr. Chuck Murry, have been working to engineer heart transplants. Their goal is to develop safe and effective new treatment options for patients with heart failure.

About nine years ago, the research had a setback: transplanted cardiomyocytes caused engraftment arrhythmias, which are irregular heart rhythms, during the first month following transplantation and healing, in laboratory models. In some cases, the heart rate exceeded 200 beats per minute for more than half the day.

A fast heart rate, if it occurs at rest, can be dangerous in people. Some large mammals, like macaques, can tolerate fast heart rates. For others, such as pigs, they can be fatal. It is not known if humans could tolerate these arrhythmias.

“This engraftment arrhythmia, where the heart races too quickly, has been one of the major hurdles we’ve been trying to overcome en route to clinical trials,” said Murry, who is a professor of laboratory medicine and pathology, of medicine, Division of Cardiology, and of bioengineering at the University of Washington School of Medicine in Seattle.

To get at this issue, the researchers first had to figure out how the transplanted cardiomyocytes might provoke engraftment arrhythmias. Electrophysiological studies, like those done clinically to check heart rhythms, were conducted in the lab on the cells.

“Compelling evidence pointed to an abnormal pace-making activity coming from the grafts,” noted Murry. “The alternate explanation, a zone of slow conduction in the graft that set up a condition called re-entry, had not been ruled out completely. We had to choose a fork in the road and committed to pace-making.”

So, five years ago, his team started sequencing the RNA in the human graft cells as they healed after transplantation.

Chuck Murry lab UW Medicine

Heart muscle regeneration researchers (left to right) Naoto Muraoka, Elaheh Karbassi, and Chuck Murry.

The project team gathered genetic information on the full repertoire of ion channels – pores in the heart cells that generate electrical currents to trigger the heart’s pumping actions. They studied how the RNA data might change as the heart muscle cell grafts matured. Their investigations covered the vulnerable period when engraftment heart rhythm problems emerge.

They named their project after the snake-haired goddess (or multi-tentacled jellyfish) MEDUSA: Modifying Electrophysiological DNA to Understand and Suppress Arrhythmias. The lead on the project was Silvia Marchiano, a postdoctoral researcher in the Murry Lab.

“Being able to work on this project is a privilege because we are making real progress that will benefit health care in the near future,” Marchiano. “I feel that this project was, and still is, like the Aida, the opera by Giuseppe Verde, my favorite.” It was a huge production, she said, and everyone worked in harmony to make it possible.

The researchers then computationally compared the ion channels in the cardiomyocyte grafts with those in adult heart muscle cells. They hypothesized that the impulses propagating the irregular heartbeat were either from ion channels present in the graft but absent in the adult heart, or, conversely, from channels absent in the graft but present in the adult heart.

Because ion channels are protein structures encoded by genes, the researchers decided to try gene editing. They saw this genomic approach as a possible way to banish the rhythm problems perpetrated by the stem cell-derived heart cell implants. They hoped that gene editing could block or turn on the appropriate ion channels.

Their plan was to transplant hundreds of millions of edited stem cell-derived cardiomyocytes into lab models to see if the arrhythmia went away. Their first series of single edits had no effect in preventing arrythmia. A double edit was tried to no avail. Then, they performed a series of triple edits. Yet again the arrythmias persisted.

“This was our darkest period,” Murry said. “After nearly three years of effort at this point, we began to question the validity of our hypothesis that graft pace-making was the cause.”

In a final rally, the team knocked out three ion channel genes and overproduced a fourth channel on the surface of the cells. Unlike the previous series of edits, where rhythm problems persisted, the resulting cultured cells containing the four edits had no spontaneous beating activity.

Critically, the quadruple edits successfully eliminated engraftment arrhythmia in lab models. Those results supported the idea that the problem was a pace-making issue. Although these cells did not beat in isolation, the edited cardiomyocytes could still be excited with electrical pacing. This implies that they should be able to follow the heart’s own pace-making prompts.

“Our findings suggest that the changes we’ve made to these cells to address these safety concerns have not gotten rid of their ability to provide efficacy,” Murry said.

He added, “We think this strategy has potential to markedly improve the safety profile of stem cell-derived heart muscle cells by reducing the risk of engraftment arrhythmias to patients in the first month after transplantation, while preserving the ability of these heart muscle cells to beat in sync with the patient’s heart and improve function.”

From here, the scientists will conduct additional tests to see whether these promising, early results translate into improvement in cardiac function. The hope is that this therapeutic approach might help lead to future clinical trials in patients with heart disease. One of the next steps along that route will be managing immune responses to the cardiomyocyte grafts, which Murry says continues to be a work in progress.

The studies on engraftment arrhythmia were done in conjunction with Sana Biotechnology, a company that is developing and delivering engineered cells as medicines. In addition to his UW Medicine position, Murry is the senior vice president and head of cardiometabolic cell therapy at Sana.

Murry’s presentation at the Presidential Symposium plenary session during the International Society for Stem Cell Research 2021 annual meeting was titled “Genome Editing to Eliminate Engraftment Arrhythmia During Heart Regeneration.”

The work was supported by the National Heart Lung and Blood Institute, National Institutes of Health; NIH 4D Nucleome; UW Heart Regeneration Program; Washington Research Foundation; Mike and Lynn Garvey; and Fondation Leducq.

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