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

我们试图建立一种新型的以星形细胞瘤为靶点的腺病毒基因治疗系统。为此，使用Cre重组酶(Cre)/IoxP系统和星形细胞瘤(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有两个开关单元的Ad。该Ad含有一个编码GFP的填充序列(AxCALGLTK)，在CAG启动子下的两个IoxP位点之间有一个功能性的多聚腺苷化信号，HSV-TK基因被连接到下游。在该系统中，Cre重组酶的共表达开启了填充序列(GFP)或下游基因(TK)的基因表达。AxGFAPNCre和AxCALGLTK联合感染导致TK在06胶质瘤细胞和反应性星形胶质细胞中表达，而AS GFP在注射部位周围的其他类型细胞中表达。同样，AxGFAPNCre的联合感染导致TK在胶质瘤细胞中表达，而GFP在其余细胞中表达。将06脑胶质瘤移植到大鼠纹状体内2周后，将ADS联合注射到肿瘤区域。联合感染后给予更昔洛韦(GCV)可显著抑制肿瘤生长并杀伤肿瘤细胞，而对照组未见如此显著的肿瘤丢失。目前的结果表明，使用Cre/IoxP腺病毒系统的细胞型特异性基因治疗似乎是有效的，至少对星形细胞瘤是有效的。

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.