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

描述(由申请人提供)：将转录图谱、癌细胞靶向和分子遗传成像集成到一个平台中将有许多生物学应用，并有可能改善医学实践。我们设计并验证了一种由腺相关病毒(AAV)和噬菌体(称为AAVP)的基因元件组成的新的杂交病毒系统。这些配基导向的颗粒使转基因报告的靶向系统传递和成像成为可能。实验非侵入性监测报告的反式激活，可遵循体外和体内。靶向分子成像将代表前列腺癌治疗方面的重大进步。这个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结合起来，作为一种基于选定标记物的转录活性开发改进的肿瘤监测工具的方式。这些启动子将通过对癌细胞和肿瘤血管转录组的大规模评估，使用公共数据库的广泛搜索，以及通过构建SAGE文库和使用现代低成本高通量焦磷酸测序方法进行大规模测序来确定。识别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，我们希望有一个全面的肿瘤标记物小组，覆盖在不同患者肿瘤样本中看到的大多数基因表达变异性。建立一个由多个标记物组成的小组的概念与被广泛宣传的个性化医学时代是一致的，应该首次推出一个在体内进行时间和空间成像的系统，即肿瘤内的转录图谱。在肿瘤特异性启动子的控制下，携带自杀/报告基因(HSVtk)的载体与展示肿瘤靶向多肽的载体相结合，应该能够以非常特定的方式进行转录成像和抑制肿瘤生长。这种载体的成像和治疗可能性加强了药物诊断共同发展的想法，如果在此之前对基因表达进行单独评估(例如，在前列腺癌患者的尿液中)，可能会导致基于单个患者上调的标志物的个性化诊断/治疗。这种与配套药物相结合的个性化成像测试，与美国食品和药物管理局最近提出的药物诊断共同开发概念相结合。根据这一设想，特定肿瘤基因提供的转录成像也将触发肿瘤细胞凋亡，从而允许成像以及治疗和监测疾病进展。我们的具体目标是：(I)使用大规模转录组分析来识别和验证前列腺癌中上调的转录本。(Ii)结合配体定向的AAVP靶向和转录靶向，以及(Iii)通过将转录组定向的启动子克隆到RGD/GRP78靶向的AAVP构建体中，评价体内成像前列腺癌特异性转录本的效率。建立一个由多个标记物组成的小组的概念与被广泛宣传的个性化医学时代是一致的，应该首次推出一个在体内进行时间和空间成像的系统，即肿瘤内的转录图谱。

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.