IN VIVO REPORTER SYSTEM FOR IMAGING GENE TRANSFER

长期目标是开发一种新的、非侵入性的体内报告系统，通过使用99mTC标记的多肽进行伽马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类2型生长抑素受体(HSSTr2)的重组腺病毒载体(Ad5)，以及与该受体具有高亲和力的99mTc标记和188Re标记的多肽。转染Ad5-hSSTr2的癌细胞将表达该受体，该受体可以通过放射性标记的多肽结合来检测。作为使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hSSTr2基因和治疗性基因(胞嘧啶脱氨酶，CD)，该报告允许靶向基因传递的体内成像。报告基因还将以188Re多肽为靶点进行治疗，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。特定目的1将使用现有的Ad5-hSSTr2在转基因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将与工程化结构域一起产生，以提高检测的特异性和敏感性。嵌合的hSSTr2基因将与CD基因一起放入Ad5载体中进行治疗。特异性目标2将在体外对hSSTr2蛋白和新开发的嵌合体进行评估。实验将评估受体数量、内化和循环动力学。HSSTr2蛋白将被99mTC-P829(Diatide，目前处于神经内分泌癌第三阶段临床试验)和与诱导的hSSTr2蛋白具有高亲和力的188Re-P829肽作为靶点。具体目标3将包括编码hSSTr2(和嵌合体)的Ad5载体在裸鼠体内的测试。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基因结构的位置和表达量。治疗研究将使用5-氟胞嘧啶，要么单独使用，要么与靶向hSSTr2蛋白的188Re-P829联合使用。本文提供的初步数据表明，该方法是可行的。这项拟议的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，并允许对基因转移进行非侵入性成像，并提供一种额外的治疗机制。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。

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.