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: 2899971
• 负责人: Kurt R Zinn
• 金额: $ 17.93万
• 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/2899971
• 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的、无创的体内报告系统，通过伽马相机成像使用99mtc标记的肽来测量基因转移。这将通过研究一个模型系统来完成，该模型系统利用重组腺病毒载体（Ad5）编码人类2型生长抑素受体（hSSTr2），以及99mtc标记和188re标记的高亲和力受体肽。用Ad5-hSSTr2转染的癌细胞会表达这种受体，这种受体可以通过结合放射性标记肽来测量。作为利用基因治疗技术治疗癌症的更大目标的一部分，拟议的报告系统将是重要的。开发的基因治疗载体将包括报告基因hSSTr2基因和治疗基因（胞嘧啶脱氨酶，CD），报告基因允许靶向基因传递的体内成像。报告基因也将被188 - re -肽靶向治疗，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。Specific aim 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。作为这一目标的一部分，嵌合hSSTr2将产生工程结构域，以提高检测的特异性和敏感性。嵌合的hSSTr2基因将与CD基因一起放置在Ad5载体中进行治疗。特异性目的2将在体外评价hSSTr2蛋白和新形成的嵌合体。实验将评估受体数量、内化和循环动力学。hSSTr2蛋白将使用99mTc-P829 （Diatide，目前处于神经内分泌癌的III期临床试验）和188Re-P829肽靶向，这些肽对诱导的hSSTr2蛋白具有高亲和力。特异性目标3将包括使用带肿瘤的裸鼠对编码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马相机成像，通过测量99mTc- P829肽与受体的结合，来测量转移的hSSTr2基因构建物的位置和表达量。治疗研究将使用5-氟胞嘧啶单独或与靶向hSSTr2蛋白的188Re-P829联合进行。本文提供的初步数据表明了该方法的可行性。该研究对基因治疗领域具有重要意义，因为报告系统将最终扩展到人体试验，并允许基因转移的非侵入性成像，并为治疗提供额外的机制。提出的报告系统将确定基因治疗载体是否靶向肿瘤，以及何时需要进一步的基因治疗。