Integration of Vascular Genomics and Proteomics for Diagnosis and Therapy of Canc

描述（由申请人提供）：将转录谱分析、癌细胞靶向和分子遗传成像整合到一个单一平台中，将具有许多生物学应用和改善医学实践的潜力。我们设计并验证了一种由腺相关病毒（AAV）和噬菌体（AAVP）遗传元件组成的新型杂交病毒系统。这些配体定向颗粒能够实现转基因报告基因的靶向系统递送和成像。报告基因反式激活的实验性无创监测，可以在体内和体外进行。靶向分子成像将是前列腺癌治疗的一大进步。该项目的总体目标是将AAVP的归巢和肿瘤转导能力与癌症特异性启动子相结合，以此开发基于选定标记物转录活性的肿瘤监测改进工具。这些启动子将通过大规模评估癌细胞和肿瘤血管的转录组来确定，使用公共数据库的广泛搜索，以及通过构建和大规模测序SAGE文库，使用现代低成本高通量焦磷酸测序方法。鉴定的上调基因将在两个独立的样本集中进行验证，确认的上调转录本的启动子将被鉴定、认证并克隆到aavp中。随着一组可靠基因的定义，我们期望有一个全面的肿瘤标志物面板，涵盖在不同患者肿瘤样本中看到的大多数基因表达变异性。建立一个由多种标记物组成的小组的概念与个性化医疗的时代是一致的，并且应该首次启动一个在时间和空间上成像的系统，在体内，肿瘤内的转录谱。结合显示针对肿瘤设计的肽的载体，同时携带自杀/报告基因（HSVtk），在肿瘤特异性启动子的控制下，应该能够以非常特定的方式实现转录成像和肿瘤生长抑制。该载体的成像和治疗可能性加强了药物诊断共同开发的理念，如果在此之前对基因表达进行个体评估（例如，在前列腺癌患者的尿液中），则可能导致基于个体患者上调标记的个性化诊断/治疗。美国食品和药物管理局（fda）最近提出了药物诊断共同开发的概念，这种个性化成像测试与一种伴用药合并在一起。在这一愿景下，特定肿瘤基因提供的转录成像也将触发肿瘤凋亡，从而实现成像以及疾病进展的治疗和监测。我们的具体目标是：(i)通过大规模转录组分析识别和验证前列腺癌中上调的转录本。（ii）将AAVP的配体定向靶向与转录靶向相结合；（iii）利用克隆到RGD/ grp78靶向AAVP构建体中的转录组定向启动子，评估在体内成像前列腺癌特异性转录物表达的效率。建立一个由多种标记物组成的小组的概念与个性化医疗的时代是一致的，并且应该首次启动一个在时间和空间上成像的系统，在体内，肿瘤内的转录谱。

DESCRIPTION (provided by applicant): The integration of transcriptional profiling, cancer cells targeting and molecular-genetic imaging into a single platform would have many biological applications and the potential to improve the practice of medicine. We have designed and validated a new hybrid viral system composed of genetic elements from adeno-associated virus (AAV) and phage (termed AAVP). These ligand-directed particles enable targeted systemic delivery and imaging of transgene reporters. Experimental non-invasive monitoring of reporter trans-activation, may be followed ex-vivo and in vivo. Targeted molecular imaging would represent a major advance in the management of prostate cancer. The overall goal in this project is to combine the homing and tumor transducing capabilities of AAVP with cancer-specific promoters, as a way to develop improved tools for tumor monitoring based on the transcriptional activity of selected markers. These promoters will be identified by the large-scale evaluation of the transcriptome of cancer cells and tumor vasculature, using extensive searches in public databases, as well as by the construction and large-scale sequencing of SAGE libraries, using modern low-cost high throughput pyrosequencing approaches. The identified upregulated genes will be validated in two independent sample sets, and the promoters of confirmed upregulated transcripts will be identified, certified and cloned into AAVPs. With the definition of a reliable set of genes we expect to have comprehensive panel of tumor markers covering most of the gene expression variability seen in distinct patient tumor samples. The concept of establishing a panel of multiple markers is in line with the much heralded era of personalized medicine, and should launch, for the first time, a system for imaging temporally and spatially, in vivo, the transcriptional profile within tumors. The combination of a vector displaying peptides designed to target tumors, which also carries a suicide/reporter transgene (HSVtk) under the control of a tumor-specific promoter should enable transcriptional imaging and tumor growth suppression in a very specific fashion. The imaging and treatment possibilities of this vector reinforce the idea of drug-diagnostic co-development that, if preceded by an individual evaluation of gene expression (in the urine of patients with prostate cancer, for instance), could lead to a personalized diagnosis/treatment based on the up-regulated markers of an individual patient. This personalized imaging test merged with a companion drug comes together with the drug-diagnostic co-development concept recently put forward by the US Food and Drug Administration. Under this vision, the transcriptional imaging provided by a specific tumor gene will also trigger tumor apoptosis, allowing imaging as well as treatment and monitoring of disease progression. Our Specific Aims are: (i) To identify and to validate transcripts upregulated in prostate cancer using large scale transcriptome analysis. (ii) To combine ligand-directed targeting of AAVP to transcriptional targeting, and (iii) To evaluate the efficiency of imaging in vivo the expression of prostate cancer specific transcripts by using transcriptome-directed promoters cloned into RGD/GRP78-targeted AAVP constructs. The concept of establishing a panel of multiple markers is in line with the much heralded era of personalized medicine, and should launch, for the first time, a system for imaging temporally and spatially, in vivo, the transcriptional profile within tumors.