Quiescent-yet-excitable pluripotent stem cell-derived cardiomyocytes engraft without fatal arrhythmias in minipig and primates
Authors: Nakamura K , Marchiano S M , Neidig L E , Ryan J K , Blakely L P , Reinecke H , Klaiman J M , Kattman S , Yang X , Knollmann B C , Thies R S , Sniadecki N J , Maclellan W R , Bertero A , Murry C E 
Published: November 2023

Abstract

Background
Pluripotent stem cell-derived cardiomyocyte (PSC-CM) transplantation is a promising therapy for subacute myocardial infarction (MI), but transient engraftment-related arrhythmia (EA) is major barrier to clinical development (1, 2). We hypothesize that PSC-CM genetically edited to remove automaticity but otherwise functionally intact will engraft in vivo with greater arrhythmic safety.

Methods
Combinatorial CRISPR/Cas9 genome editing to screen for electrically quiescent PSC-CM yielded a line deficient in the depolarization-associated genes HCN4, CACNA1H, and SLC8A1, combined with overexpression of the hyperpolarization-associated gene KCNJ2. Initially, we transplanted 150 million (n=3) and 500 million gene-edited (n=2) and 150 million wildtype (n=7) PSC-CMs into a minipig model of EA, and we monitored the animals for up to seven weeks on continuous ECG for arrythmia without anti-arrhythmic therapy. In a second study, we transplanted 750 million gene-edited (n=2) and wildtype (n=1) PSC-CMs into sub-acutely infarcted Macaca nemestrina primates. Sham transplant (n=1) was also performed, and all animals were observed for up to three months with immunosuppression and beta-blocker/amiodarone therapy.

Results
Gene-edited PSC-CM successfully engrafted and structurally coupled with the host myocardium of minipigs (Figure 1) and nonhuman primates (Figure 2). No significant EA occurred after transplanting edited PSC-CM at the 150 million dose, whereas brief EA (&lt;24 hours) was observed and well tolerated at the higher 500 million dose. All five minipigs survived to prespecified timepoints without complication. Conversely, wildtype PSC-CM controls demonstrated uncontrolled and fatal ventricular tachycardia (VT). Of the seven WT control minipigs, only four survived (43% mortality); three expired or were euthanized due to unstable VT or ventricular fibrillation. Arrythmia burden for both 150 and 500 million doses were significantly reduced compared to the 150 million wildtype dose in the first 30-days post-transplant (p&lt;0.0001 for both). We then tested edited PSC-CM in a clinically relevant nonhuman primate model of sub-acute myocardial infarction using standard medical therapy including modest antiarrhythmic therapy. No arrythmia was noted in both nonhuman primates dosed with 750 million edited PSC-CM whereas significant arrhythmia was observed with wildtype despite beta blocker and amiodarone therapy. All pig and primate grafts were structurally coupled with host myocardium.

Conclusion
Genetically-edited PSC-CM without pacemaker-like activity significantly reduced arrythmia burden and prevented arrhythmic death in a dose-escalation study in minipig. No arrhythmia was observed in a clinically relevant nonhuman primate model of cardiac remuscularization for sub-acute myocardial infarction. This study supports continued investigation of this novel strategy to improve safety and tolerability of PSC-CM transplantation.Edited PSC-CM transplant in minipigEdited PSC-CM transplant in primates