Engineered human tmpk/AZT as a novel enzyme/prodrug axis for suicide gene therapy
Engineered human tmpk/AZT as a novel enzyme/prodrug axis for suicide gene therapy
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DOI：
10.1038/mt.sj.6300122
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发表时间：
2007-05-01
影响因子：
12.4
通讯作者：
Medin, Jeffrey A.
中科院分区：
文献类型：
--
作者：
Sato, Takeya;Neschadim, Anton;Medin, Jeffrey A.
通过控制治疗细胞的命运可以提高基因治疗和干细胞移植的安全性。自杀基因疗法使用将前药转化为细胞毒性实体的酶；然而，使用了动力学较差的异源部分。我们描述了一种新型酶/前药组合，用于选择性诱导慢病毒载体转导细胞的细胞凋亡。合理设计的人胸苷酸激酶 (tmpk) 变体可有效磷酸化 3'-叠氮基-3'-脱氧胸苷 (AZT)，并得到有效递送。转导的 Jurkat 细胞系被 AZT 消除。我们证明该方案针对分裂细胞和非分裂细胞，具有一种新颖的杀伤机制，涉及通过破坏线粒体内膜电位和激活 caspase-3 诱导细胞凋亡。原代小鼠和人类 T 细胞也被转导并对 AZT 产生反应。此外，对注射转导的 K562 细胞的非肥胖糖尿病/严重联合免疫缺陷 (NOD/SCID) 小鼠进行低剂量 AZT 给药可抑制肿瘤生长。因此，这种新颖的自杀基因治疗方法可以作为安全开关整合到治疗载体中。
Gene therapy and stem cell transplantation safety could be enhanced by control over the fate of therapeutic cells. Suicide gene therapy uses enzymes that convert prodrugs to cytotoxic entities; however, heterologous moieties with poor kinetics are employed. We describe a novel enzyme/prodrug combination for selectively inducing apoptosis in lentiviral vector - transduced cells. Rationally designed variants of human thymidylate kinase (tmpk) that effectively phosphorylate 3'-azido-3'- deoxythymidine (AZT) were efficiently delivered. Transduced Jurkat cell lines were eliminated by AZT. We demonstrate that this schema targeted both dividing and non-dividing cells, with a novel killing mechanism involving apoptosis induction via disruption of the mitochondrial inner membrane potential and activation of caspase-3. Primary murine and human T cells were also transduced and responded to AZT. Furthermore, low-dose AZT administration to non-obese diabetic/ severe combined immunodeficiency (NOD/SCID) mice injected with transduced K562 cells suppressed tumor growth. This novel suicide gene therapy approach can thus be integrated as a safety switch into therapeutic vectors.