Comparison of Different Suicide-Gene Strategies for the Safety Improvement of Genetically Manipulated T Cells
Comparison of Different Suicide-Gene Strategies for the Safety Improvement of Genetically Manipulated T Cells
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DOI：
10.1089/hgtb.2012.050
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发表时间：
2012-12-01
影响因子：
--
通讯作者：
Pule, Martin
中科院分区：
文献类型：
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作者：
Marin, Virna;Cribioli, Elisabetta;Pule, Martin
过继T细胞治疗(ACT)的使用正在增加；然而，T细胞治疗会导致严重的毒性反应。因此，已经描述了几种自杀基因策略，这些策略允许选择性地破坏注入的T细胞。我们在体外比较了四种策略在EB病毒(EBV)-细胞毒性T淋巴细胞(CTL)中的有效性。将单纯疱疹病毒胸苷激酶(HSV-TK)、人诱导型半胱氨酸天冬氨酸氨基转移酶9(ICaspase 9)、突变型人胸腺苷酸激酶(MTMPK)和人CD20密码子优化基因克隆到逆转录病毒载体中。密码子优化显著提高了CD20的表达。EBV-CTL可以在所有载体中高效转导，转基因表达与单独含有dCD34的对照载体相似。在长时间的培养中保持了表达。自杀基因的表达与免疫表型、增殖或CTL功能的改变无关。HSV-TK、iCasp9和CD20的激活最终导致转导的T细胞同样有效的破坏。然而，虽然iCasp9和CD20可以立即诱导细胞死亡，但表达HSV-TK的T细胞需要暴露在更昔洛韦3天才能完全发挥作用。转导mTMPK的细胞在各个时间点均表现出较低的T细胞杀伤率。我们的结果表明，iCasp9较快的活性可能有利于治疗某些类型的急性危及生命的毒性。密码子优化的CD20有可能成为自杀基因。
Use of adoptive T-cell therapy (ACT) is increasing; however, T-cell therapy can result in severe toxicity. Consequently, several suicide-gene strategies that allow selective destruction of the infused T cells have been described. We compared effectiveness of four such strategies in vitro in Epstein Barr virus (EBV)-cytotoxic T lymphocytes (CTLs). Herpes simplex virus thymidine kinase (HSV-TK), human inducible caspase 9 (iCasp9), mutant human thymidylate kinase (mTMPK), and human CD20 codon optimized genes were cloned in frame with 2A-truncated codon optimized CD34 (dCD34) in a retroviral vector. Codon-optimization considerably improved CD20 expression. EBV-CTLs could be efficiently transduced in all constructs, with transgene expression similar to the control vector containing dCD34 alone. Expression was maintained for prolonged cultures. Expression of the suicide genes was not associated with alterations in immunophenotype, proliferation, or function of CTLs. Activation of HSV-TK, iCasp9, and CD20 ultimately resulted in equally effective destruction of transduced T cells. However, while iCasp9 and CD20 effected immediate cell-death induction, HSV-TK-expressing T cells required 3 days of exposure to ganciclovir to reach full effect. mTMPK-transduced cells showed lower T-cell killing all time points. Our results suggest that the faster activity of iCasp9 might be advantageous in treating certain types of acutely life-threatening toxicity. Codon-optimized CD20 has potential as a suicide gene.