Doxycycline-regulated lentiviral vector system with a novel reverse transactivator rtTA2S-M2 shows a tight control of gene expression in vitro and in vivo
Doxycycline-regulated lentiviral vector system with a novel reverse transactivator rtTA2S-M2 shows a tight control of gene expression in vitro and in vivo
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DOI：
10.1038/sj.gt.3301889
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发表时间：
2003-03-01
期刊：
影响因子：
5.1
通讯作者：
Ylä-Herttuala, S
中科院分区：
文献类型：
--
作者：
Koponen, JK;Kankkonen, H;Ylä-Herttuala, S
治疗基因的调节表达是许多疾病的长期基因治疗应用所必需的。在这里，我们描述了一个多西环素（dox）调节的慢病毒载体系统，由两个基于HIV-1的自失活病毒组成。其中一个载体组成型表达四环素反向反式激活因子rtTA 2（s）-M2的新改进形式，另一个载体具有驱动β-半乳糖苷酶基因（lacZ）表达的rtTA响应启动子。rtTA 2（s）-M2在特异性、稳定性和诱导性方面具有高度改善的特性。在体外共转导中国仓鼠卵巢和人内皮细胞杂交瘤（EAhy 926）细胞后，证实了dox系统的功能。该系统的调节显示了对基因表达的严格控制。在临床实践中通常使用的剂量可以在体内获得的浓度下观察到dox的剂量依赖性。LacZ表达可以在共转导细胞的长期（3个月）培养期间开启/关闭。在共转导到大鼠脑中并研究在dox处理的大鼠和对照大鼠中lacZ基因的表达后，接下来在体内测试该系统。巢式RT-PCR证实，在体内实现了基因表达的严格控制。此外，X-gal染色显示阳性细胞在dox处理的大鼠中，而不是在对照组中，共转导后10天与dox处理前4天。结论：我们的强力霉素调控的载体系统显示出长期基因治疗的显着潜力。
Regulated expression of therapeutic genes is required for long-term gene therapy applications for many disorders. Here we describe a doxycycline (dox)-regulated lentiviral vectorsystem consistingof two HIV-1-based self-inactivating viruses. One of the vectors is constitutively expressing a novel improved version of the tetracycline reverse transactivator rtTA2(s)-M2 and the other has a rtTA responsive promoter driving the expression of beta-galactosidase gene (lacZ). The rtTA2(s)-M2 has highly improved properties with respect to specificity, stability and inducibility. Functionality of the system by dox was confirmed after in vitro cotransduction of Chinese hamster ovary and human endothelial hybridoma (EAhy926) cells. Regulation of the system showed tight control of the gene expression. Dose dependence for dox was seen with concentrations that can be obtained in vivo with doses normally used in clinical practice. LacZ expression could be switched on/off during long-term (3 months) culturing of cotransduced cells. The system was next tested in vivo after cotransduction into rat brain and studying expression of the lacZ gene in dox-treated and control rats. Nested RT-PCR confirmed that the tight control of the gene expression was achieved in vivo. Also, X-gal staining showed positive cells in the dox-treated rats, but not in the controls 10 days after cotransduction with 4 days preceding treatment with dox. It is concluded that our doxycycline-regulated vector system shows significant potential for long-term gene therapy treatments.