Engineered pluripotent stem cell-derived cardiomyocytes prevent serious engraftment-related arrhythmia in nonhuman primates

Abstract Background Pluripotent stem cell-derived cardiomyocyte (PSC-CM) transplantation is a novel therapy for ischemic cardiomyopathy, but engraftment-related arrhythmia is a major, potentially lethal safety concern (1, 2). We developed a genetically edited human PSC-CM line that is "quiescent-yet-excitable" and prevents fatal arrhythmias in pig (3). In this study, we test the engraftability and degree of arrhythmia suppression conferred by edited cardiomyocytes in a nonhuman primate model of myocardial infarction. Methods Combinatorial CRISPR/Cas9 genome editing to screen for electrically quiescent human PSC-CM yielded a line deficient in the depolarization-associated genes HCN4, CACNA1H, and SLC8A1, combined with overexpression of the hyperpolarization-associated gene KCNJ2. We transplanted 750 million gene-edited (n=3) and wildtype (WT, n=1) human PSC-CMs into infarcted Macaca nemestrina primates two weeks after ischemia/reperfusion injury of the left anterior descending coronary artery. Sham transplant (n=1) was also performed, and all animals were treated with standard immunosuppression (cyclosporine/abatacept/methylprednisolone) and anti-arrhythmic (metoprolol/amiodarone) therapy to prevent xenograft rejection and arrhythmic death, respectively. All animals were monitored with continuous telemetric electrocardiography for up to five months. Results All human PSC-CM grafts were structurally coupled with host myocardium at three months post-transplant except for one (edited #3) where graft was not observed at extended endpoint at five months (Figure 1A/B). No significant arrythmia was observed in nonhuman primates dosed with 750 million edited PSC-CMs compared with WT. Arrhythmia burden (% time in arrhythmia) was reduced by 97% (p=0.003) in edited PSC-CM recipients compared to WT recipient (Figure 1D). The WT recipient experienced incessant ventricular tachycardia characteristic of engraftment arrythmia starting one week after transplantation and continued to increase in burden to 50% until three months post-transplant. In two of three animals transplanted with edited cells, no arrhythmia was observed. In the third recipient (edited #3), isolated premature ventricular beats averaging 15% burden were observed. Given this ectopy was low burden, occurred at the time of transplantation and resolved by two weeks post-transplant, it was not considered serious and likely related to surgery and cardiomyocyte delivery rather than cellular engraftment. Conclusion Genetically-edited PSC-CMs without pacemaker-like activity engraft yet significantly reduce arrythmia burden and prevent serious engraftment-related arrhythmia in infarcted nonhuman primates. This study supports automaticity of donor cardiomyocytes as a central mechanism of engraftment arrythmia. Transplantation of "quiescent-yet-excitable" cardiomyocytes to prevent or reduce engraftment arrythmia warrants further investigation, specifically to ensure that efficacy is not adversely affected.