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Molecular Fluorescent Imaging of Metastatic Tumors with Conditionally Replication-Selective Adenovirus and GFP Gene

使用条件复制选择性腺病毒和 GFP 基因对转移性肿瘤进行分子荧光成像

基本信息

批准号：
15591475
负责人：
FUJIWARA Toshiyoshi
金额：
$ 2.24万
依托单位：
OKAYAMA UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15591475/
关键词：
Telomerase Adenovirus hTERT Virotherapy Gene Therapy Diagnosis

项目摘要

Currently available methods for detection of tumors in vivo such as X-ray, computed tomography, and ultrasonography are noninvasive and well studied ; the images, however, are not specific for tumors. Direct optical imaging of tumor cells in vivo that can clearly distinguish them from surrounding normal tissues may be clinically useful. Here, we describe a new approach to visualizing tumors whose fluorescence can be detected using tumor-specific replication-competent adenovirus (OBP-301, "Telomelysin") in combination with replication-deficient adenovirus expressing GFP (Ad-GFP). Human telomerase reverse transcriptase (hTERT) is the catalytic subunit of telomerase, which is highly active in cancer cells, but is quiescent in most normal somatic cells. We constructed an adenovirus 5 vector, in which the hTERT promoter element drives expression of E1A and E1B genes linked with an IRES, and showed that OBP-301 replicated efficiently exclusively in human cancer cells, but not in normal cells … More such as human fibroblasts. When the human lung and colon cancer cell lines H1299 and SW620 were infected with Ad-GFP at low multiplicity of infection(MOI), GFP expression could not be detected under a fluorescence microscope ; in the presence of OBP-301, however, Ad-GFP replicated in these tumor cells and showed strong green signals. In contrast, co-infection of OBP-301 and Ad-GFP did not show any signals in normal cells such as WI-38 fibroblasts. We also found that established subcutaneous tumors could be visualized following intratumoral injection of OBP-301 and Ad-GFP. A549 human lung tumors and SW620 human colon tumors transplanted into BALB/c nu/nu mice were intratumorally injected with 8 x 10^5 plaque forming units(PFU) of Ad-GFP in combination with 8 x 10^6 PFU of OBP-301. Within 3 days of treatment, the fluorescence of the expressed GFP became visible by 3CCD camera in these tumors, whereas intratumoral injection of Ad-GFP alone could not induce GFP fluorescence. Moreover, intrathoracic administration of Ad-GFP and OBP-301 could visualize disseminated A549 tumor nodules in mice following intrathoracic implantation. Our results indicate that intratumoral or intrathoracic injection of Ad-GFP in combination with OBP-301 might be a useful diagnostic method that provides a foundation for future clinical application. Less

目前可用于检测体内肿瘤的方法，如X射线，计算机断层扫描和超声检查是非侵入性的，并进行了充分的研究;然而，图像并不特异于肿瘤。直接光学成像的肿瘤细胞在体内，可以清楚地区分他们从周围的正常组织可能是临床上有用的。在这里，我们描述了一种新的方法来可视化肿瘤，其荧光可以检测使用肿瘤特异性复制能力腺病毒（OBP-301，“端粒溶素”）与表达GFP的复制缺陷型腺病毒（Ad-GFP）的组合。人端粒酶逆转录酶（Human telomerase reverse transcriptase，hTERT）是端粒酶的催化亚单位，在癌细胞中高度活跃，而在正常体细胞中处于静止状态。我们构建了腺病毒5载体，其中hTERT启动子元件驱动与IRES连接的E1 A和E1 B基因的表达，并显示OBP-301仅在人癌细胞中有效复制，而在正常细胞中不复制 ...更多信息如人成纤维细胞。当用Ad-GFP以低感染复数（MOI）感染人肺癌和结肠癌细胞系H1299和SW 620时，在荧光显微镜下不能检测到GFP表达;然而，在存在OBP-301的情况下，Ad-GFP在这些肿瘤细胞中复制并显示出强的绿色信号。相反，OBP-301和Ad-GFP的共感染在正常细胞如WI-38成纤维细胞中没有显示任何信号。我们还发现，在瘤内注射OBP-301和Ad-GFP后，可以观察到已建立的皮下肿瘤。向移植到BALB/c nu/nu小鼠中的A549人肺肿瘤和SW 620人结肠肿瘤瘤内注射8 × 10^5空斑形成单位（PFU）的Ad-GFP和8 × 10^6 PFU的OBP-301。在治疗的3天内，表达的GFP的荧光在这些肿瘤中通过3CCD照相机变得可见，而单独的Ad-GFP瘤内注射不能诱导GFP荧光。此外，胸腔内施用Ad-GFP和OBP-301可以使胸腔内植入后小鼠中的播散性A549肿瘤结节可视化。我们的研究结果表明，Ad-GFP联合OBP-301瘤内或胸腔内注射可能是一种有用的诊断方法，为未来的临床应用提供了基础。少