Development of targeted gene therapy to incurable sarcoma and malignant mesothelioma

我们在HSV-1 ICP-4蛋白的氨基末端添加了HIFl α ODD序列中57个氨基酸的多肽，包括564位的脯氨酸残基。将CMV-NLS-ODD-ICP 4-IRES-LacZ-polyA片段插入pKpX 2的Stu I位点（J.Virol.62，196 - 205，1988）（由Conevolent大学的Weller博士友情提供），并用Xho I消化使其线性化。将得到的11.3kb靶向重组载体UL 39-CMV-NLS-ODD-ICP 4-IRES-LacZ-polyA-UL 39与突变体HSV-1 DNA，d120共转染到Vero细胞中（2.5 × 105个细胞/孔）。重组HSV-1在低氧条件下（O2 1%）优先复制，并显示出更强的抗肿瘤活性。ICP 4的表达在缺氧条件下也增强。重组HSV-1经瘤内注射对人恶性间皮瘤、平滑肌肉瘤和乳腺癌均有显著的抗肿瘤活性。

We added 57 amino acids polypeptide within the ODD sequence of HIFlalpha, including proline residue at 564, to the amino terminal of HSV-1 ICP4 ptotein. Further, we added nuclear localization signal (NLS) comprising 23 amino acids to the amino terminus of ODD-ICP4.The CMV-NLS-ODD-ICP4-IRES-LacZ-polyA fragment was inserted into the Stu I site of pKpX2 (J. Virol.62,196-205,1988) (kindly provided by Dr. Weller in Coneticut University), and was linealized by digestion with XhoI. The resulting 11.3kb targeted recombination vector, UL39-CMV-NLS-ODD-ICP4-IRES-LacZ-polyA-UL39 was co-transfected with a mutant HSV-1 DNA, d120 into Vero cells (2.5x105cells/well). We isolated a single plaque of the recombinant HSV-1.The recombinant HSV-1 preferentially replicated in the hypoxic condition (O2 1%) and showed more potent anti-tumor activity. Expression of ICP4 was also enhanced in the hypoxic condition. The recombinant HSV-1 showed prominent anti-tumor activity against human malignant mesothelioma, leiomyosarcoma and breast cancer by direct injection into the tumors.