Targeted gene therapy for brain tumor

为了引入仅在胶质瘤和黑色素瘤细胞血管中表达的蛋白多糖NG2的肿瘤特异性，我们在Adv F/40S纤维的c端遗传地掺入了NG2结合肽TAASGVRSMH （TAA）。流式细胞术检测了NG2蛋白多糖在不同肿瘤细胞和正常细胞中的特异性。NG2在人黑色素瘤A375和胶质瘤A172细胞中高表达，而在人结肠癌dpd -1细胞、正常人黑色素细胞和肝细胞原代培养物中不表达。Adv F/40S-TAA对ng2阳性细胞的遗传转导效率显著提高。在表达NG2的A375和A172细胞中，与Adv F/40S或Adv F/wt相比，Adv F/40S- taa的转导量增加了3 - 5倍。相反，在几种不同的ng2阴性细胞中，Adv F/40S或Adv F/40S/TAA的基因转导很低，而Adv F/wt的基因转导很高。此外，Adv F/40S-TAA的基因转导被TAA肽剂量依赖性阻断，但重组Ad5纤维或Ad40S纤维不抑制转导。这些结果表明，Adv F/40S-TAA通过其靶NG2受体特异性感染A375黑色素瘤和A172胶质瘤细胞。为了在体内靶向肿瘤细胞，我们检测了携带ng2阳性A375细胞的小鼠静脉给药后Adv F/40S-TAA在体内的分布。Adv F/40S-TAA基因在肿瘤血管中选择性表达，而非肿瘤细胞，在肺、心、肝、脾、肾和血液中均有表达。相反，Adv F/40S-TAA在受体阴性的dld -1小鼠中不能靶向肿瘤。因此，纤维突变体Adv F/40S-TAA在体外和体内均具有不依赖car、靶向ng2的黑色素瘤细胞基因递送能力。

In order to introduce tumor-specific tropism for the proteoglycan NG2, which is exclusively expressed in the vasculature of glioma and melanoma cells, we genetically incorporated an NG2-binding peptide, TAASGVRSMH (TAA), in the C-terminal end of the fiber of Adv F/40S. The specificity for the NG2 proteoglycan was tested by flow cytometric analysis using various kinds of tumor and normal cells. The expression of NG2 was high in human melanoma A375 and human glioma A172 cells, but NG2 was not expressed in human colon carcinoma DLD-1 cells and primary cultures of normal human melanocytes and hepatocytes. The Adv F/40S-TAA showed a remarkably enhanced efficiency in genetic transduction of NG2-positive cells. In A375 and A172 cells expressing NG2, Adv F/40S-TAA increased transduction threefold to fivefold compared with Adv F/40S or Adv F/wt. In contrast, in the several different NG2-negative cells, the gene transduction of Adv F/40S or Adv F/40S/TAA was very low, while that of Adv F/wt was high. Furthermore, the gene transduction of Adv F/40S-TAA was dose-dependently blocked by the TAA peptide, but the recombinant Ad5 fiber or Ad40S fiber did not inhibit the transduction. These results suggested that Adv F/40S-TAA specifically infected A375 melanoma and A172 glioma cells through its target NG2 receptor. In order to target tumor cells in vivo, we examined the in vivo distribution of the Adv F/40S-TAA after intravenous admini stration to mice carrying NG2-positive A375 cells. The bgal of Adv F/40S-TAA was selectively expressed in the tumor vasculature rather than tumor cells, sparing the lung, heart, liver, spleen, kidney, and blood. In contrast, the Adv F/40S-TAA was incapable of targeting the tumor in receptor-negative DLD-1-bearing mice. Thus the fiber-mutant Adv F/40S-TAA achieved a CAR-independent, NG2-targeted gene delivery capacity into melanoma cells in vitro and in vivo.