IN VIVO REPORTER SYSTEM FOR IMAGING GENE TRANSFER

长期的目标是开发一种新的，非侵入性的体内报告系统来测量基因转移的γ照相机成像使用99 mTc标记的肽。 这将通过研究利用编码人2型生长抑素受体（hSSTr 2）的重组腺病毒载体（Ad 5）以及对受体具有高亲和力的99 mTc标记和188 Re标记肽的模型系统来实现。 用Ad 5-hSSTr 2转染的癌细胞将表达受体，其可以通过放射性标记的肽的结合来测量。 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。 所开发的基因治疗载体将包括报告基因hSSTr 2基因和治疗基因（胞嘧啶脱氨酶，CD），报告基因允许靶向基因递送的体内成像。 报告基因也将与188 Re肽靶向治疗，同时成像。 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Ad 5-hSSTr 2在转染的癌细胞系中表达高水平的hSSTr 2蛋白。作为这一目标的一部分，嵌合hSSTr 2将与工程结构域一起产生，以增加检测的特异性和灵敏度。 嵌合hSSTr 2基因将与CD基因一起置于Ad 5载体中用于治疗。 具体目标2将在体外评价hSSTr 2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。 将使用对诱导的hSSTr 2蛋白具有高亲和力的99 mTc-P829（Diploid，目前处于神经内分泌癌的III期临床试验中）和188 Re-P829肽靶向hSSTr 2蛋白。 具体目标3将包括使用具有肿瘤的裸鼠体内测试编码hSSTr 2（和嵌合体）的Ad 5载体。将在动物肿瘤模型中进行γ照相机成像，以通过测量99 mTc-P829肽与受体的结合来测量转移的hSSTr 2基因构建体的表达的位置和量。 将使用5-氟胞嘧啶单独或与靶向hSSTr 2蛋白的188 Re-P829组合进行治疗研究。 本文提供的初步数据表明该方法的可行性。 这项研究对基因治疗领域具有重要意义，因为报告系统将被扩展到人体试验，并允许基因转移的非侵入性成像，并提供额外的治疗机制。 所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。

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.