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

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.