In situ generation of pseudotyped retroviral progeny by adenovirus-mediated transduction of tumor cells enhances the killing effect of HSV-tk suicide gene therapy in vitro and in vivo
In situ generation of pseudotyped retroviral progeny by adenovirus-mediated transduction of tumor cells enhances the killing effect of HSV-tk suicide gene therapy in vitro and in vivo
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DOI：
10.1002/jgm.490
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发表时间：
2004-03-01
影响因子：
3.5
通讯作者：
Ozawa, K
中科院分区：
文献类型：
--
作者：
Okada, T;Caplen, NJ;Ozawa, K
背景腺病毒-逆转录病毒混合载体系统利用腺病毒的高效转导来指导逆转录病毒子代的原位生产。在这项研究中，我们表明，一个单一的步骤转导胶质瘤细胞与陷阱互补的杂交腺逆转录病毒载体有效地把这些细胞转化为逆转录病毒载体生产细胞，方法采用腺病毒-逆转录病毒混合病毒载体系统，将单纯疱疹病毒胸苷激酶（HSV-tk）基因转染到胶质瘤细胞中，增强更昔洛韦（GCV）对胶质瘤细胞的杀伤作用。基因为了评估原位产生逆转录病毒载体对神经胶质瘤细胞转导效率的影响，用分别表达逆转录病毒基因组（AVC2.GCEGFP或AVC2.GCTK）和逆转录病毒包装蛋白（AxTetGP和AxTetVSVG）的腺病毒-逆转录病毒杂合载体转导9 L细胞。通过分析侧翼逆转录病毒LTR序列，验证了通过腺病毒载体的陷阱互补产生整合的HSV-tk前病毒。用病毒注射在nu/nu小鼠上建立的肿瘤，然后腹膜内注射PBS或GCV。我们还估计了治疗小鼠肿瘤中存在的HSV-tk转基因的拷贝数。结果AVC2.GCEGFP与表达逆转录病毒包装蛋白的载体共转导大鼠9 L胶质瘤细胞，可提高转导效率。与不能产生逆转录病毒的对照细胞相比，用AVC2.GCTK与包装载体一起转导使细胞对前药GCV的体外敏感性增加一个对数。在体内，用AVC2.GCTK和包装载体的组合注射在无胸腺小鼠上建立的皮下9 L肿瘤，随后进行GCV处理，导致在第22天50%的肿瘤完全肿瘤消退，而在对照动物中没有观察到肿瘤消退。在从转导的肿瘤中提取的基因组DNA中检测到诊断体内3' LTR重复的逆转录病毒序列，表明源自腺病毒-逆转录病毒杂合载体的逆转录病毒基因组的前病毒整合。此外，在用腺病毒-逆转录病毒载体治疗的肿瘤中HSV-tk基因的相对拷贝数比对照肿瘤高250倍。原位杂交表明分散的HSV-tk产品在更广泛的区域的肿瘤比在控制肿瘤，这表明原位产生的逆转录病毒的传播。结论虽然该系统的疗效进行了评估，在原位模型，我们的观察表明，这种混合腺病毒逆转录病毒载体系统可以提高自杀基因治疗肿瘤。版权所有（C）2004约翰威利父子有限公司。
Background Hybrid adeno-retroviral vector systems utilize the high efficiency of adenovirus transduction to direct the in situ production of retroviral progeny. In this study, we show that a single-step transduction of glioma cells with traps-complementing hybrid adeno-retroviral vectors effectively turns these cells into retrovirus vector-producing cells, which in turn facilitates the transduction of adjacent cells.Methods We have adapted the adeno-retroviral hybrid viral vector system to enhance the ganciclovir (GCV) killing of glioma cells following transfer of the herpes simplex virus thymidine kinase (HSV-tk) gene. To assess the effect of the in situ production of retroviral vectors on the transduction efficiency of glioma cells, 9L cells were transduced with adeno-retroviral hybrid vectors that separately express a retroviral genome (AVC2.GCEGFP or AVC2.GCTK) and retroviral packaging proteins (AxTetGP and AxTetVSVG). The generation of an integrated HSV-tk provirus by traps-complementation of the adeno-retroviral vectors was verified by analysis of the flanking retroviral LTR sequences. Tumors established on nu/nu mice were injected with the viruses followed by intraperitoneal injections of either PBS or GCV. We also estimated the copy numbers of the HSV-tk transgene present in the tumors of the treated mice. To determine the expression pattern of the HSV-tk transcripts within a tumor, in situ hybridization analysis was performed using an RNA probe specific for HSV-tk.Results The co-transduction of rat 9L glioma cells with AVC2.GCEGFP together with vectors expressing packaging proteins of retroviruses increased the transduction efficiency. Transduction with AVC2.GCTK together with packaging vectors increased the in vitro sensitivity of cells to the pro-drug GCV by one log compared with control cells that were incapable of generating retrovirus. In vivo, the injection of established subcutaneous 9L tumors on athymic mice with a combination of AVC2.GCTK and packaging vectors followed by GCV treatment resulted in complete tumor regression in 50% of tumors at day 22 while no tumor regression was observed in control animals. Retroviral sequences diagnostic of 3' LTR reduplication in vivo were detected in genomic DNA extracted from the transduced tumors, indicating pro-viral integration of the retroviral genome derived from the adeno-retroviral hybrid vector. Furthermore, the relative copy number of the HSV-tk gene in tumors treated with the adeno-retroviral vectors was up to similar to250-fold higher than in control tumors. In situ hybridization suggested dispersion of the HSV-tk product across a wider area of the tumor than in control tumors, which indicates the spread of the in situ generated retroviruses.Conclusions Although the efficacy of this system has to be evaluated in orthotopic models, our observations suggest that this hybrid adeno-retroviral vector system could improve the suicide gene therapy of tumors. Copyright (C) 2004 John Wiley Sons, Ltd.