Pellinen Riikka, Hakkarainen Tanja, Wahlfors Tiina, Tulimäki Kirsi, Ketola Anna, Tenhunen Anni, Salonen Tuula, Wahlfors Jarmo
A.I. Virtanen Institute, Department of Biotechnology and Molecular Medicine, University of Kuopio, Kuopio, Finland.
Int J Oncol. 2004 Dec;25(6):1753-62.
Lentiviruses have been used as gene transfer vectors for almost 10 years and their utility has been demonstrated in a variety of different applications. However, their value in cancer gene therapy has not been studied thoroughly. Here we show that VSV-G pseudotyped HIV-1-based lentiviruses are efficient vectors for human tumor cells in vitro and in vivo. Lentiviral gene transfer efficiency was demonstrated by transducing 42 different cell lines, representing 10 different human tumor types. It was shown that most of the cell lines were good or excellent targets for lentiviral transduction, allowing 50-95% gene transfer efficiency. These results were comparable to those obtained with an E1/E3 deleted, serotype 5 adenovirus vector. Analysis of lentivirus vector structure revealed that virus particles devoid of HIV-1 accessory proteins appeared to be more efficient, but the presence of enhancing elements cPPT and WPRE did not play a major role in transduction efficiency to four different human tumor cell lines. However, their effect on the gene expression level in these cells was apparent. To examine the impact of lentiviral gene expression level on suicide gene therapy approach, human osteosarcoma cells were transduced with lentivirus- or adenovirus vectors carrying the fusion gene HSV-TK-GFP and exposed to ganciclovir. Cell viability analysis after the treatment revealed that both vector types induced similar level of cytotoxicity, suggesting that lentiviral expression of a suicide gene is adequate for tumor cell destruction. Finally, in vivo transduction studies with subcutaneous tumors showed that lentivirus vectors can yield similar gene transfer efficiency than adenovirus vector, despite three orders of magnitude lower titer of the lentiviral preparation. In conclusion, these data show that lentiviruses are efficient gene transfer vehicles for human tumor cells and justify their use in further preclinical cancer gene therapy studies.
慢病毒作为基因转移载体已应用了近10年，其效用已在各种不同的应用中得到证实。然而，它们在癌症基因治疗中的价值尚未得到充分研究。在此我们表明，基于VSV-G假型化HIV-1的慢病毒在体外和体内都是人类肿瘤细胞的有效载体。通过转导代表10种不同人类肿瘤类型的42种不同细胞系，证明了慢病毒基因转移效率。结果表明，大多数细胞系是慢病毒转导的良好或优秀靶标，基因转移效率可达50-95%。这些结果与使用E1/E3缺失的5型腺病毒载体获得的结果相当。对慢病毒载体结构的分析表明，缺乏HIV-1辅助蛋白的病毒颗粒似乎更有效，但增强元件cPPT和WPRE的存在对四种不同人类肿瘤细胞系的转导效率没有主要影响。然而，它们对这些细胞中基因表达水平的影响是明显的。为了研究慢病毒基因表达水平对自杀基因治疗方法的影响，用携带融合基因HSV-TK-GFP的慢病毒或腺病毒载体转导人骨肉瘤细胞，并使其暴露于更昔洛韦。处理后的细胞活力分析表明，两种载体类型诱导的细胞毒性水平相似，这表明自杀基因的慢病毒表达足以破坏肿瘤细胞。最后，皮下肿瘤的体内转导研究表明，尽管慢病毒制剂的滴度比腺病毒载体低三个数量级，但慢病毒载体仍能产生与腺病毒载体相似的基因转移效率。总之，这些数据表明慢病毒是人类肿瘤细胞的有效基因转移载体，证明了它们在进一步的临床前癌症基因治疗研究中的应用价值。
