Transplantation of stem cell-derived cardiomyocytes with a chemogenetic off-on switch to improve cell engraftment and inhibit engraftment induced arrhythmias

缺血性心脏病是世界范围内的主要死亡原因。心肌梗死会导致心肌收缩力永久丧失，并经常导致心力衰竭。多能干细胞衍生的心肌细胞的移植代表了具有治疗心力衰竭患者的巨大潜力的再生治疗概念。它已成功应用于临床前研究，目前正在进行首次临床试验。本项目旨在解决有关这一战略的两个基本问题：1。移植效率低2.移植诱导的心律失常我们假设，移植基因工程干细胞衍生的心肌细胞与一个关闭-打开开关将导致更大的移植物和抑制移植诱导的心律失常。这一假设是基于体外实验与心肌细胞的开关。为此，将由甘氨酸受体的氯离子选择性离子孔结构域和烟碱乙酰胆碱受体的突变配体结合结构域组成的所谓的药理学选择性致动器模块（PSAM）敲入人诱导多能干细胞的AAVS 1基因座。用一种小分子物质（PSEM 89 S）可以可逆地阻断该细胞系的心肌细胞。从这些心肌细胞产生的工程化心脏组织（EHT），用PSAM 89 S停止，有规律地发育，在PSAM 89 S洗出后开始连贯地跳动，并产生与对照EHT类似的力，表明即使在没有主动收缩的情况下也会发生成熟。然而，细胞周期活性在沉默的心肌细胞中更高。干细胞来源的心肌细胞移植导致大型动物模型的室性心律失常。这是由细胞的不成熟和自动性造成的。心律失常发生在最初几天内，三周后，当细胞进一步成熟时，心律失常消退。我们假设，通过在术后关键时期电灭活心肌细胞，有可能抑制移植诱导的心律失常，同时心肌细胞有时间成熟。也有报道称，移植不能搏动的心肌细胞会导致更大的移植物。这一发现导致了第二个假设：心肌细胞失活将导致更大的移植物。为了解决这两个假设，已经产生了具有针对体内应用优化的关-开开关的新干细胞系，并将其用于小型和大型动物模型。

Ischemic heart disease is the main cause of death worldwide. Myocardial infarction results in a permanent loss of contractile myocardium and regularly leads to the development of heart failure. Transplantation of pluripotent stem cell-derived cardiomyocytes represents a regenerative therapeutic concept with great potential for the treatment of heart failure patients. It was successfully applied in preclinical studies and is currently evaluated in first clinical trials. This project aims to address two fundamental problems regarding this strategy: 1. The low transplantation efficiency 2. The transplantation induced arrhythmias We hypothesize that the transplantation of genetically engineered stem cell derived cardiomyocytes with an off-on switch will result in larger grafts and suppress the transplantation induced arrhythmias. This hypothesis is based on in vitro experiments with cardiomyocytes with an off-on switch. For this a so called Pharmacologically Selective Actuator Modul (PSAM), consisting of the chloride-selective ion pore domain of the glycine receptor and a mutated ligand-binding domain of the nicotinic acetylcholine receptor was knocked-into the AAVS1 locus of human induced pluripotent stem cells. Cardiomyocytes derived from this line could be stopped reversibly with a small molecular substance (PSEM89S). Engineered Heart Tissue (EHTs) generated from these cardiomyocytes, that were stopped with PSAM89S developed regularly, started to beat coherently after PSEM89S washed out and developed similar forces as control EHTs, indicating that maturation occurs even in the absence of active contraction. Yet, cell cycle activity was higher in the silenced cardiomyocytes. Stem cell derived cardiomyocyte transplantation resulted in ventricular arrhythmias in large animal models. This was caused by the cell`s immaturity and their automatism. Arrhythmias occurred within the first days and subsided after three weeks when the cells were further matured. We hypothesize that by electrically inactivating cardiomyocytes over the critical postoperative period it is possible to suppress the transplantation induced arrhythmias while the cardiomyocytes have time to mature. It also has been reported that the transplantation of cardiomyocytes that are not able to beat resulted in larger grafts. This finding led to the second hypothesis: cardiomyocyte inactivation will result in larger grafts. To address these two hypotheses a new stem cell line with an off-on switch optimized for an in vivo application has been generated and will be used in a small and large animal model.