When healthy, the human heart is a tireless workhorse, sending oxygen and nutrients throughout the body with every life-sustaining beat. However, the heart is also one of the least regenerative organs in the body. When it is injured in a major event like a heart attack, the inability to repair itself can have dire consequences.
Nearly 18 million people die each year from heart disease, making it the leading cause of death in the world. In the United States alone, the annual economic impact of heart disease exceeds $200 billion, a figure that is expected to rise dramatically.
Patients with heart disease experience progressive, significant declines in quality of life marked by reduced activity and higher hospitalization rates. Despite all this, progress toward new treatments has been stagnant. Just three new drugs have been approved in the last 20 years; and even those do not address the root cause of the problem – the loss of cardiac muscle tissue required for normal heart functioning.
At the UW Medicine Institute for Stem Cell and Regenerative Medicine (ISCRM), researchers in multiple labs are using stem cell technology to pioneer novel approaches to treating heart disease that can potentially cure rather than manage this chronic disease. In 2018, a study led by ISCRM Director Dr. Charles Murry demonstrated that stem cell-derived cardiomyocytes have the potential to regenerate heart tissue in large non-human primates, a major step toward human clinical trials.
These findings represented a major step forward for Murry and his team, a landmark moment after years of working to clear difficult hurdles, including learning how to grow the cardiomyocytes from stem cells, how to ensure the survival of the cells, and how to avoid immunological complications – an undertaking that Murry says remains a work in progress.
The team also faced another challenge.
“Through the course of our research, we discovered that transplanting stem cell-derived cardiomyocytes into the hearts of large animals caused irregular heartbeats,” says Dr. Murry. “The engraftment arrhythmia we saw became one of the biggest impediments as we worked to make heart regeneration ready for the clinic.”
Steady electrical signaling is vital for normal heart functioning. When that signaling is disrupted, the heart can beat too quickly, too slowly, or erratically. Such a side-effect in any treatment could potentially expose a patient to an unacceptable level of risk. Murry and his team have spent years attempting to understand and fix the problem. Electrophysiological studies eventually pointed to a potential culprit.
In a cardiovascular coup d’état known as automaticity, cells that were supposed to be followers appeared to become leaders. The transplanted cardiomyocytes were usurping pace-making duties from the heart itself, preventing it from setting its own rhythm. The researchers dubbed this undesired side effect engraftment arrhythmia and established that the irregularity was originating in the engrafted cells.
Throughout the years, researchers have proposed multiple mechanisms to explain the origin of arrhythmia but whether any of these explanations applied to the problem hampering heart regeneration was unclear.
To answer that question, the team conducted RNA sequencing of the grafted cells in the host that led to a dual hypothesis. The first part of the hypothesis was that the arrhythmic currents could result from the presence of ion channels where they should not be, (and conversely, the absence of channels where they should be); the second part of the hypothesis was that manipulating those same currents with gene editing might address the underlying issue and restore full sovereignty to the heart.
The gene-editing effort, which began in 2018, occurred in six-month cycles. First, combinations of genes that regulate electrical signaling in the heart were altered using CRISPR gene editing technology. Second, the cardiomyocytes with the edited genes were tested in dishes, then injected into the hearts of large animals. After three years of experiments, a quadruple edit produced the elusive results – heart muscle cells that can beat when stimulated but without causing any sign of arrhythmia. In June 2021, Murry presented preliminary data from this study in a plenary session of the International Society for Stem Cell Research (ISSCR) annual meeting.
The discovery that the quadruple gene edit reduced engraftment arrhythmia in large animal models was an exciting breakthrough. Since the early data were revealed, Murry and his team have been busy validating the safety and efficacy of transplanting gene edited cells into hearts. Now the results of more rigorous experiments have been published in the journal Cell Stem Cell.
Murry shares lead investigator of the new study with Alessandro Bertero, PhD, who was a Research Assistant Professor in the Murry Lab when the work was conducted. Silvia Marchiano, PhD, a Postdoctoral Fellow in the Murry Lab, is the paper’s first author. ISCRM faculty members Nate Sniadecki, PhD and Robb MacLellan, MD are also authors, along with key contributors Xiulan Yang, PhD, Hans Reinecke, PhD, Kenta Nakamura, MD, Lauren Neidig, DVM. While a majority of the research reported in the paper was conducted at the University of Washington, critical support and expertise was provided by Sana Biotechnology.
Among the findings detailed in the study are three crucial advances for the ongoing heart regeneration effort. First, the researchers have demonstrated for the first time that engraftment arrhythmias resulted from the pacemaking activity of the cells. Second, the data show that the multiple gene edits were necessary to reprogram these cells to ensure safer transplantation. Finally, the gene-edited cells could be manufactured at a scale necessary for therapeutic purposes.
The next step was to demonstrate the same degree of safety with a volume of cells that would be enough to regrow heart muscle after injury. “We knew if we could suppress arrhythmia at a dose that was clinically relevant, we’d prove that the cells were safe enough to be used down the road in clinical trials,” says Murry.
Marchiano, who performed much of the hands-on work in the study, explains the process and the encouraging outcome. “When we gave the increased dose, we saw arrhythmia when we expected to, but in a different way. With non-edited cells, at this same dose, the engraftment arrhythmia appears gradually, and it could last for weeks. With our gene-edited cells, this arrhythmia happened instantaneously, lasted for 24 hours and went away completely, without any sort of intervention from our side. Overall, we saw a 95% reduction in arrhythmia.”
The next question was whether the engrafted gene-edited cells would cooperate with the native heart cells. Would the graft and the host become coupled electrically? This experiment, conducted by Filippo Perbellini, PhD, used electrical stimulation and a calcium sensitive dye to trace and map the electrical signaling. When a jolt was applied to a representative slice of heart tissue, the researchers watched for a flash that would tell them the graft and host cells were integrated.
“We had to confirm that the engrafted cells were following the heart’s natural pacemaker,” says Murry. “We saw that the cells were integrated and beating in synch, which is ultimately the hallmark of heart regeneration.”
Advancing toward human clinical trials also requires the means to manufacture the massive quantities of cells needed for ongoing studies and, eventually, clinical treatment. With guidance from scientists at Sana, the Murry Lab learned to grow and preserve billions of cells suspended in a bioreactor that stirs the 3D culture and delivers oxygen and nutrients to the cells.
The next step is to demonstrate that the gene-edited cells are not only safe, but also capable of remuscularizing the walls of infarcted hearts. While those experiments are underway, Murry speaks to the significance of the recently published study. “We knew we could repair the heart. But we had a problem with arrhythmia. We’ve figured out the mechanism of that arrhythmia and we think we’ve found a way to fix it. It’s a milestone in understanding that moves us closer to an urgently needed treatment for the world’s leading cause of death.”
These studies were supported by the UW Medicine Heart Regeneration Program, the Washington Research Foundation, a gift from Mike and Lynn Garvey, and a sponsored research agreement from Sana Biotechnology. This work also was supported in part by NIH grants R01HL128368, R01HL146868, and R01HL148081, a grant from the Fondation Leducq Transatlantic Network of Excellence, and the Bruce-Laughlin Research Fellowship.