Increased Cytotoxicity of Herpes Simplex Virus Thymidine Kinase Expression in Human Induced Pluripotent Stem Cells
Increased Cytotoxicity of Herpes Simplex Virus Thymidine Kinase Expression in Human Induced Pluripotent Stem Cells
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DOI：
10.3390/ijms20040810
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发表时间：
2019-02-02
影响因子：
5.6
通讯作者：
Miyoshi, Hiroyuki
中科院分区：
文献类型：
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作者：
Iwasawa, Chizuru;Tamura, Ryota;Miyoshi, Hiroyuki
人类诱导多能干细胞（iPSC）为再生医学带来了巨大的希望。主要的安全问题是来自iPSC的移植细胞的致瘤性。一个潜在的解决方案是将自杀基因引入iPSC作为安全开关。单纯疱疹病毒1型胸苷激酶（HSV-TK）基因与更昔洛韦的联合应用是从基础研究到临床应用最广泛的酶/前药自杀系统。在本研究中，我们试图用慢病毒载体或CRISPR/Cas9介导的基因组编辑建立稳定表达HSV-TK的人iPSC。然而，这一任务难以实现，因为HSV-TK的高水平和/或组成型表达导致诱导细胞死亡或HSV-TK表达沉默。核苷酸代谢分析表明，由HSV-TK表达引起的胸苷三磷酸过度蓄积导致dNTP池失衡。这种不平衡状态导致DNA合成抑制和细胞死亡，其过程类似于胸苷阻断，但更严重。我们还证明了Tet-inducible系统是克服HSV-TK表达的细胞毒性的可行解决方案。我们的研究结果为在人类iPSC中使用HSV-TK基因提供了警告，特别是在临床应用中。
Human induced pluripotent stem cells (iPSCs) hold enormous promise for regenerative medicine. The major safety concern is the tumorigenicity of transplanted cells derived from iPSCs. A potential solution would be to introduce a suicide gene into iPSCs as a safety switch. The herpes simplex virus type 1 thymidine kinase (HSV-TK) gene, in combination with ganciclovir, is the most widely used enzyme/prodrug suicide system from basic research to clinical applications. In the present study, we attempted to establish human iPSCs that stably expressed HSV-TK with either lentiviral vectors or CRISPR/Cas9-mediated genome editing. However, this task was difficult to achieve, because high-level and/or constitutive expression of HSV-TK resulted in the induction of cell death or silencing of HSV-TK expression. A nucleotide metabolism analysis suggested that excessive accumulation of thymidine triphosphate, caused by HSV-TK expression, resulted in an imbalance in the dNTP pools. This unbalanced state led to DNA synthesis inhibition and cell death in a process similar to a thymidine block, but more severe. We also demonstrated that the Tet-inducible system was a feasible solution for overcoming the cytotoxicity of HSV-TK expression. Our results provided a warning against using the HSV-TK gene in human iPSCs, particularly in clinical applications.