Herpes simplex virus-1 thymidine kinase mutants created by semi-random sequence mutagenesis improve prodrug-mediated tumor cell killing.
Herpes simplex virus-1 thymidine kinase mutants created by semi-random sequence mutagenesis improve prodrug-mediated tumor cell killing.
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发表时间：
2001-04
期刊：
影响因子：
11.2
通讯作者：
M. Black;M. Kokoris;Peter Sabo
中科院分区：
文献类型：
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作者：
M. Black;M. Kokoris;Peter Sabo
癌症自杀基因治疗提供了使用最佳基因的前景，因为治疗基因的来源通常是不相关的。目前，有许多临床前和临床试验来开发使用病毒、酵母或细菌基因的肿瘤消融疗法。一种这样的基因，单纯疱疹病毒1型（HSV-1）胸苷激酶（TK）被广泛用作与更昔洛韦组合的自杀基因。在本文报道的研究中，将一组有限的随机序列（半随机）引入HSV-1 TK的活性位点，并根据其增加更昔洛韦或阿昔洛韦对大肠杆菌敏感性的能力选择所得变体。序列分析表明，功能突变体含有3至5个氨基酸取代，是独特的和新的组合。在酶活性测定结果的基础上，鉴定了三种突变体用于体外进一步分析。这三种突变体与表达野生型TK的大鼠C6胶质瘤细胞的IC 50相比，对更昔洛韦和阿昔洛韦的敏感性显著增加。在裸鼠的异种移植肿瘤模型中进一步评价了一种突变体SR 39。在不影响表达野生型HSV-1 TK的肿瘤的前药剂量下，肿瘤中SR 39的表达显示出阻止肿瘤生长。任何这些突变体作为自杀基因的使用应该提供一个更有效和更安全的替代野生型TK，因为更低，更少的免疫抑制剂量的更昔洛韦将是必要的肿瘤消融，和阿昔洛韦的使用现在可能。
Cancer suicide gene therapy affords the prospect of using the most optimal genes available because the source of the therapeutic gene is often irrelevant. Currently, there are numerous preclinical and clinical trials to develop tumor ablative therapies that use viral, yeast, or bacterial genes. One such gene, the herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) is widely used as a suicide gene in combination with ganciclovir. In the study reported here, a restricted set of random sequences (semi-random) was introduced into the active site of HSV-1 TK, and the resulting variants were selected on the basis of their ability to confer increased ganciclovir or acyclovir sensitivity to Escherichia coli. Sequence analysis demonstrated that functional mutants contained three to five amino acid substitutions that are unique and novel combinations. On the basis of enzyme assay results, three mutants were identified for further analysis in vitro. These three mutants conferred substantial increased sensitivity to both ganciclovir and acyclovir when compared with IC50s of wild-type TK expressing rat C6 glioma cells. One mutant, SR39, was further evaluated in a xenograft tumor model in nude mice. Expression of SR39 in tumors was shown to prevent tumor growth at prodrug dosages that did not affect wild-type HSV-1 TK-expressing tumors. The use of any of these mutants as a suicide gene should provide a more effective and safer alternative to wild-type TK, because lower, less immunosuppressive doses of ganciclovir will be necessary for tumor ablation, and the use of acyclovir may now be possible.