Mathis J Michael, Williams B Jill, Sibley Don A, Carroll Jennifer L, Li Jie, Odaka Yoshinobu, Barlow Shayne, Nathan Cherie-Ann O, Li Benjamin D L, DeBenedetti Arrigo
Department of Cellular Biology and Anatomy, Louisiana State University Health Sciences Center, 1501 Kings Highway, Shreveport, LA 71130, USA.
J Gene Med. 2006 Sep;8(9):1105-20. doi: 10.1002/jgm.935.
Two technical hurdles, gene delivery and target specificity, have hindered the development of effective cancer gene therapies. In order to circumvent the problem of tumor specificity, the suicide gene, HSV-1 thymidine kinase (HSV-Tk), was modified with a complex 5' upstream-untranslated region (5'-UTR) that limits efficient translation to cells expressing high levels of the translation initiation factor, eIF4E. Since previous studies have shown that most tumor cells express elevated levels of eIF4E, tumor-specific gene delivery was optimized by incorporation of the 5'-UTR-modified suicide gene (HSV-UTk) into an adenovirus vector (Ad-CMV-UTk). The efficacy of this novel approach of targeting suicide gene expression and limiting cytotoxicity by means of translational restriction was tested in vitro with the use of the human breast cancer cell lines (MCF-7, MDA-MB435, and ZR-75-1). As controls, normal MCF10A, HMEC, and HMSC cell lines that express relatively low levels of eIF4E were used. Real-time reverse-transcription polymerase chain reaction (RT-PCR) was used to quantify HSV-Tk mRNA for cells infected with Ad-CMV-UTk as well as with Ad-CMV-Tk (a control adenovirus in which HSV-Tk is not regulated at the level of translation). Translation of HSV-Tk in the Ad-infected cells was measured by Western blot analysis. In addition, cytotoxicity was determined following treatment with the pro-drug ganciclovir (GCV) using an MTT viability assay. Finally, microPET imaging was used to assess cancer cell-specific expression of HSV-Tk and expression in normal tissues in vivo after intraperitoneal injection of Ad-CMV-Tk or Ad-CMV-UTk. These data collectively showed enhanced cancer cell-specific gene expression and reduced normal tissue gene expression for the Ad-HSV-UTk compared to the Ad-CMV-Tk, leading to increased cancer cell-enhanced GCV cytotoxicity. These results indicate that translational targeting of suicide gene expression in tumor cells in vitro and in vivo is effective and may provide a platform for enhanced cancer gene therapy specificity.
基因传递和靶向特异性这两个技术障碍阻碍了有效的癌症基因治疗的发展。为了规避肿瘤特异性问题，自杀基因单纯疱疹病毒1型胸苷激酶（HSV-Tk）用一个复杂的5'上游非翻译区（5'-UTR）进行了修饰，该区域将有效翻译限制在表达高水平翻译起始因子eIF4E的细胞中。由于先前的研究表明大多数肿瘤细胞中eIF4E表达水平升高，通过将5'-UTR修饰的自杀基因（HSV-UTk）整合到腺病毒载体（Ad-CMV-UTk）中优化了肿瘤特异性基因传递。利用人乳腺癌细胞系（MCF-7、MDA-MB435和ZR-75-1）在体外测试了这种通过翻译限制靶向自杀基因表达并限制细胞毒性的新方法的疗效。作为对照，使用了表达相对较低水平eIF4E的正常MCF10A、HMEC和HMSC细胞系。实时逆转录聚合酶链反应（RT-PCR）用于定量感染Ad-CMV-UTk以及Ad-CMV-Tk（一种对照腺病毒，其中HSV-Tk在翻译水平不受调控）的细胞中的HSV-Tk mRNA。通过蛋白质印迹分析测量Ad感染细胞中HSV-Tk的翻译。此外，使用MTT活力测定法在用前药更昔洛韦（GCV）处理后测定细胞毒性。最后，在腹腔注射Ad-CMV-Tk或Ad-CMV-UTk后，使用微型正电子发射断层扫描（microPET）成像评估体内HSV-Tk在癌细胞中的特异性表达以及在正常组织中的表达。这些数据共同表明，与Ad-CMV-Tk相比，Ad-HSV-UTk增强了癌细胞特异性基因表达并降低了正常组织基因表达，导致癌细胞增强的GCV细胞毒性增加。这些结果表明，在体外和体内对肿瘤细胞中自杀基因表达进行翻译靶向是有效的，并且可能为增强癌症基因治疗特异性提供一个平台。
