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Abstract：
Human pluripotent stem cells, including induced pluripotent stem cells (iPSCs) and embryonic stem cells, hold great promise for cell-based therapies, but safety concerns that complicate consideration for routine clinical use remain. Installing a \"safety switch\" based on the inducible caspase-9 (iCASP9) suicide gene system should offer added control over undesirable cell replication or activity. Previous studies utilized lentiviral vectors to integrate the iCASP9 system into T cells and iPSCs. This method results in random genomic insertion of the suicide switch and inefficient killing of the cells after the switch is \"turned on\" with a small molecule (eg, AP1903). To improve the safety and efficiency of the iCASP9 system for use in iPSC-based therapy, we precisely installed the system into a genomic safe harbor, the AAVS1 locus in the PPP1R12C gene. We then evaluated the efficiencies of different promoters to drive iCASP9 expression in human iPSCs. We report that the commonly used EF1α promoter is silenced in iPSCs, and that the endogenous promoter of the PPP1R12C gene is not strong enough to drive high levels of iCASP9 expression. However, the CAG promoter induces strong and stable iCASP9 expression in iPSCs, and activation of this system with AP1903 leads to rapid killing and complete elimination of iPSCs and their derivatives, including MSCs and chondrocytes, in vitro. Furthermore, iPSC-derived teratomas shrank dramatically or were completely eliminated after administration of AP1903 in mice. Our data suggest significant improvements on existing iCASP9 suicide switch technologies and may serve as a guide to other groups seeking to improve the safety of stem cell-based therapies.
摘要：
人类多能干细胞，包括诱导多能干细胞（iPSCs）和胚胎干细胞，对细胞疗法大有希望，但在常规临床使用时考虑复杂的安全性问题仍然存在.安装基于诱导型caspase-9（iCASP9）自杀基因系统的“安全开关”应提供对不良细胞复制或活性的额外控制。先前的研究利用慢病毒载体将iCASP9系统整合到T细胞和iPSC中。这种方法导致自杀开关的随机基因组插入和小分子开关“打开”后细胞的低效杀伤（例如，AP1903).为了提高iCASP9系统在基于iPSC的治疗中使用的安全性和效率，我们精确地将系统安装到基因组安全港，PPP1R12C基因中的AAVS1基因座。然后我们评估了不同启动子在人iPSC中驱动iCASP9表达的效率。我们报告说，常用的EF1α启动子在iPSCs中被沉默，PPP1R12C基因的内源启动子不足以驱动高水平的iCASP9表达。然而,CAG启动子诱导iPSCs中强而稳定的iCASP9表达，和激活该系统与AP1903导致快速杀死和完全消除的iPSC及其衍生物，包括MSCs和软骨细胞，在体外。此外，在小鼠中施用AP1903后,iPSC衍生的畸胎瘤显著缩小或完全消除。我们的数据表明，对现有的iCASP9自杀开关技术进行了重大改进，并可作为其他寻求提高干细胞疗法安全性的团体的指南。
