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的，无创的在体内报告基因系统，以使用99mTC标记的肽通过伽马相机成像来测量基因转移。 这将通过研究使用编码人类2型生长抑素受体（HSSTR2）的重组腺病毒载体（AD5）的模型系统来实现，并与99mTC标记和188re标记的肽一起使用，具有高亲和力。 用AD5-HSSTR2转染的癌细胞将表达可以通过放射性标记肽结合来测量的受体。 拟议的记者系统将作为使用基因治疗技术治疗癌症的更大目标的一部分很重要。 开发的基因治疗载体将包括报告基因HSSTR2基因和治疗基因（胞质脱氨酶，CD），这是记者，允许对靶向基因递送的体内成像进行体内成像。 记者基因还将以188Re肽进行治疗，并并发成像。 目前尚无可靠的体内方法来测量癌细胞中基因转移和表达。特定的目标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。作为此目标的一部分，将使用工程域生产嵌合HSSTR2，以提高检测的特异性和敏感性。 嵌合HSSTR2基因将与CD基因进行治疗放置在AD5载体中。 特定的目标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，内在化和回收动力学。 HSSTR2蛋白将使用99MTC-P829（目前正在III期神经内分泌癌临床试验中）和188RE-P829肽对诱导HSSTR2蛋白的高亲和力进行靶向。 具体的目标3将包括使用带有肿瘤的裸小鼠编码HSSTR2（和嵌合体）的AD5载体的体内测试。通过测量99MTC-P829肽与受体的结合，将在动物肿瘤模型中进行伽马摄像机成像，以测量转移的HSSTR2基因构建体的表达和量。 治疗研究将使用5-氟细胞的单独或与针对HSSTR2蛋白的188RE-P829结合进行。 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很重要，因为报告基因系统最终将扩展到人体试验，并允许对基因转移的非侵入性成像，并提供了一种额外的治疗机制。 拟议的报告基因系统将确定基因治疗载体是否针对肿瘤，并且需要进一步的基因治疗治疗。