Targeted gene therapy toward astrocytoma using a Cre/loxP-based adenovirus system
Targeted gene therapy toward astrocytoma using a Cre/loxP-based adenovirus system
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DOI：
10.1016/j.brainres.2006.01.105
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发表时间：
2006-04
期刊：
影响因子：
2.9
通讯作者：
M. Maeda;K. Namikawa;Ikue Kobayashi;N. Ohba;Yuki Takahara;C. Kadono;A. Tanaka;H. Kiyama
中科院分区：
文献类型：
--
作者：
M. Maeda;K. Namikawa;Ikue Kobayashi;N. Ohba;Yuki Takahara;C. Kadono;A. Tanaka;H. Kiyama
本研究旨在建立一种新型的以腺病毒为基础的星形细胞瘤基因治疗系统。为此，使用Cre重组酶(Cre)/loxP系统和星形细胞瘤特异性GFAP启动子。我们构建了一个在GFAP启动子(AxGFAPNCre)控制下表达Cre的腺病毒(Ad)载体，以及另一个含有开关单位的Ad载体。后者含有一个编码GFP的填充序列(AxCALGLTK)，在两个loxP位点之间有一个功能性的多聚腺苷化信号，紧随其后的是CAG启动子控制的单纯疱疹病毒胸苷激酶(HSV-TK)基因。在这个系统中，填充序列(GFP)或下游基因(HSV-TK)的基因表达都是通过Cre重组酶的共表达来开启的。Western印迹分析显示，AxGFAPNCre和AxCALGLTK共感染C6胶质瘤细胞后，TK蛋白表达水平明显升高。在体内，AxGFAPNCre/AxCALGLTK裸鼠皮下注射C6胶质瘤后，再经腹腔注射更昔洛韦(GCV)可显著抑制肿瘤的生长。AxGFAPNCre和AxCALNLacZ联合感染导致C6胶质瘤细胞和部分反应性星形胶质细胞表达LacZ，而GFP在注射部位周围的其他类型细胞中表达。联合应用AxGFAPNCre/AxCALGLTK和腹腔注射GCV可显著消退大鼠纹状体C6脑胶质瘤，延长存活时间。目前的结果表明，这种使用Cre/loxP腺病毒系统的细胞类型特异性基因治疗是可行和有效的，至少对星形细胞瘤是有效的。
The aim of this study was to establish a novel adenovirus-based gene therapy system targeting astrocytoma. For this purpose, the Cre recombinase (Cre)/loxP system together with the astrocytoma-specific promoter for GFAP were used. We constructed an adenovirus (Ad) vector that expressed Cre under the control of the GFAP promoter (AxGFAPNCre), as well as another Ad vector containing a switching unit. The latter vector contained a stuffer sequence encoding GFP (AxCALGLTK) with a functional polyadenylation signal between two loxP sites, followed by the herpes simplex virus thymidine kinase (HSV-TK) gene under the control of the CAG promoter. In this system, gene expression of either the stuffer sequence (GFP) or the downstream gene (HSV-TK) was switched on by co-expression of Cre recombinase. Western blot analysis demonstrated specific expression of high levels of TK protein in C6 glioma cells after co-infection of AxGFAPNCre and AxCALGLTK. In vivo, AxGFAPNCre/AxCALGLTK injection into C6 gliomas in the subcutaneous tissue of nude mice followed by intraperitoneal ganciclovir (GCV) treatment significantly suppressed tumor growth compared with control mice. Co-infection of AxGFAPNCre and AxCALNLLacZ resulted in LacZ expression in C6 glioma cells and some reactive astrocytes, whereas GFP was expressed in other cell types surrounding the injected site. Furthermore, a combination of AxGFAPNCre/AxCALGLTK and intraperitoneal GCV injection significantly regressed intracranial C6 gliomas in the rat striatum and prolonged the survival time compared with control rats. The present results indicate that this cell-type-specific gene therapy using a Cre/loxP adenovirus system is both operational and effective, at least against astrocytoma.