Gene therapy utilizing of Cre/IoxP system selectively suppress brain tumor

我们试图建立一种新的腺病毒基因治疗系统，在星形细胞瘤的目标。为此目的，使用Cre重组酶（Cre）/IoxP系统连同星形细胞瘤（06胶质瘤细胞）特异性启动子GEAP启动子。我们构建了在GFAP启动子（AxGFAPNCre）控制下表达Cre的腺病毒载体（Ad）。构建了另一个在载体中具有两个开关单元的Ad。该Ad含有编码GFP（AxCALGLTK）的填充序列，其在CAG启动子下的两个IoxP位点之间具有功能性聚腺苷酸化信号，并且单纯疱疹病毒胸苷激酶（HSV-TK）基因连接在下游。在该系统中，填充序列（GFP）或下游基因（TK）的基因表达通过Cre重组酶的共表达来开启。AxGFAPNCre和AxCALGLTK的共感染导致TK在06胶质瘤细胞和反应性星形胶质细胞中表达，而GFP在注射部位周围的其他类型的细胞中表达。同样地，AxGFAPNCre的共感染导致TK在神经胶质瘤细胞中表达，而GFP在其余细胞物种中表达。将06胶质瘤移植到大鼠纹状体中后两周，将Ads的组合物注射到肿瘤区域中。合并感染后给予更昔洛韦（GCV）可显著抑制肿瘤生长并杀死肿瘤细胞，而对照组未见明显的肿瘤消失。目前的研究结果表明，细胞类型特异性基因治疗使用的Cre/IoxP腺病毒系统出现工作和有效的至少对星形细胞瘤。

We attempted to establish a novel adenovirus-based gene therapy system, in which astrocytoma is targeted. Forthis purpose the Cre recombinase (Cre)/IoxP system together with astrocytoma (06 glioma cell) specific promoter, GEAP promoter, was used. We constructed adenovirus vector (Ad), which expresses Cre under the control of the GFAP promoter (AxGFAPNCre). Another Ad having two switching unit in the vector was constructed. This Ad contains a stuffer sequence encoding GFP (AxCALGLTK) with a functional polyadenylation signal between two IoxP sites under the CAG promoter, and the herpes simplex virus thymidine kinase (HSV-TK) gene is attached downstream. In this system, gene expression of either stuffer sequence (GFP) or downstream gene (TK) is switched on by the co-expression of Cre recombinase. The co-infection of AxGFAPNCre and AxCALGLTK resulted in expression of TK in 06 glioma cells and reactive astrocytes, where as GFP was expressed in other types of cells around the injected site. Likewise the co-infection of AxGFAPNCre resulted in expression of TK in the glioma cells, whereas GFP was expressed in the remaining cell species. Two weeks after the transplantation of 06 glioma into the rat striatum the combination of Ads was injected into the tumor region. The ganciclovir (GCV) administration after the co-infections significantly suppressed the tumor growth and killed the tumor cells, while such significant loss of tumor was not seen in control. The present results suggest that cell-type specific gene therapy using the Cre/IoxP adenovirus system appears working and effective at least against astrocytoma.