IN VIVO REPORTER SYSTEM FOR IMAGING GENE TRANSFER

用于成像基因转移的体内报告系统

• 批准号: 6376999
• 负责人: Kurt R Zinn
• 金额: $ 22.3万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-07-06 至 2003-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6376999
• 关键词: athymic mouse; bioimaging /biomedical imaging; fusion gene; gene expression; neoplasm /cancer radiodiagnosis; neoplastic cell; radiography; radiotracer; recombinant proteins; reporter genes; technology /technique development; tissue /cell culture; transfection; transfection /expression vector

The long-term objective is to develop a novel, non-invasive in vivo reporter system to measure gene transfer by gamma camera imaging using a 99mTc-labeled peptide. This will be accomplished by studying a model system that utilizes a recombinant adenoviral vector (Ad5) encoding the human type 2 somatostatin receptor (hSSTr2), together with 99mTc-labeled and 188Re-labeled peptides with high affinity for the receptor. Cancer cells transfected with the Ad5-hSSTr2 will express the receptor which can be measured by binding of the radiolabeled peptides. The proposed reporter system will be important as a part of a larger objective to treat cancer using gene therapy technology. The developed gene therapy vector will include both the reporter hSSTr2 gene and a therapeutic gene (cytosine deaminase, CD), the reporter allowing in vivo imaging of targeted gene delivery. The reporter gene will also be targeted with a 188Re-peptide for therapy, with concurrent imaging. Currently no reliable in vivo method exists to measure gene transfer and expression in cancer cells. Specific aim 1 will use an existing Ad5-hSSTr2 to express high levels of the hSSTr2 protein in transfected cancer cell lines. As part of this aim, chimeric hSSTr2 will be produced with engineered domains to increase specificity and sensitivity of detection. The chimeric hSSTr2 genes will be placed in an Ad5 vector with the CD gene for therapy. Specific aim 2 will evaluate the hSSTr2 protein and newly developed chimeras in vitro. Experiments will evaluate the receptor number, internalization, and recycling kinetics. The hSSTr2 protein will be targeted using the 99mTc-P829 (Diatide, currently in Phase III clinical trials for neuroendocrine cancer) and 188Re-P829 peptides with high affinity for induced hSSTr2 protein. Specific aim 3 will consist of the in vivo testing of the Ad5 vectors encoding the hSSTr2 (and chimeras) using nude mice with tumors. Gamma camera imaging will be conducted in animal tumor models to measure the location and amount of expression of the transferred hSSTr2 gene constructs, by measuring the binding of the 99mTc- P829 peptide to the receptors. Therapy studies will be conducted using 5-flurocytosine, either alone, or in combination with 188Re-P829 targeting the hSSTr2 protein. Preliminary data presented herein indicate the feasibility of the approach. The proposed research is significant for the gene therapy field since the reporter system will eventfully be extended to human trials, and allow for non-invasive imaging of gene transfer, and provide an additional mechanism for therapy. The proposed reporter system will determine if gene therapy vectors are targeted to tumor, and when further gene therapy treatment is necessary.

长期目标是开发一种新型、非侵入性体内报告系统，通过使用 99mTc 标记肽的伽玛相机成像来测量基因转移。 这将通过研究一个模型系统来实现，该模型系统利用编码人 2 型生长抑素受体 (hSSTr2) 的重组腺病毒载体 (Ad5)，以及对该受体具有高亲和力的 99mTc 标记和 188Re 标记肽。 用 Ad5-hSSTr2 转染的癌细胞将表达受体，该受体可以通过放射性标记肽的结合来测量。 拟议的报告系统作为使用基因治疗技术治疗癌症的更大目标的一部分非常重要。 开发的基因治疗载体将包括报告基因 hSSTr2 和治疗基因（胞嘧啶脱氨酶，CD），报告基因允许对靶向基因递送进行体内成像。 报告基因还将以 188Re 肽为目标进行治疗，同时进行成像。 目前还没有可靠的体内方法来测量癌细胞中的基因转移和表达。具体目标 1 将使用现有的 Ad5-hSSTr2 在转染的癌细胞系中表达高水平的 hSSTr2 蛋白。作为该目标的一部分，将生产具有工程化结构域的嵌合 hSSTr2，以提高检测的特异性和灵敏度。 嵌合的 hSSTr2 基因将被置于具有 CD 基因的 Ad5 载体中用于治疗。 具体目标2将在体外评估hSSTr2蛋白和新开发的嵌合体。 实验将评估受体数量、内化和再循环动力学。 hSSTr2 蛋白将使用对诱导 hSSTr2 蛋白具有高亲和力的 99mTc-P829（Diatide，目前处于神经内分泌癌 III 期临床试验）和 188Re-P829 肽来靶向。 具体目标 3 将包括使用带有肿瘤的裸鼠对编码 hSSTr2（和嵌合体）的 Ad5 载体进行体内测试。将在动物肿瘤模型中进行伽玛相机成像，通过测量 99mTc-P829 肽与受体的结合来测量转移的 hSSTr2 基因构建体的表达位置和数量。 治疗研究将单独使用 5-氟胞嘧啶，或与靶向 hSSTr2 蛋白的 188Re-P829 联合使用。 本文提供的初步数据表明该方法的可行性。 拟议的研究对于基因治疗领域具有重要意义，因为报告系统最终将扩展到人体试验，并允许基因转移的非侵入性成像，并提供额外的治疗机制。 拟议的报告系统将确定基因治疗载体是否针对肿瘤，以及何时需要进一步的基因治疗。