Targeted gene therapy for brain tumor

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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.