Matrilysin-Sensing Gene Delivery Vectors for Colorectal Cancer Therapy

描述(申请人提供)：一旦结直肠癌转移，它就成为一种致命的疾病，5年存活率约为10%。目前迫切需要能够针对转移性大肠肿瘤细胞的有效治疗方法。用核酸(如基因或RNAi)来对抗癌症是一种非常有前途的治疗方法；不幸的是，将基因载体定向运送到肿瘤细胞在很大程度上是很难实现的。到目前为止，大多数载体靶向方法都依赖于在某些亚群的靶癌细胞上过度表达的细胞表面受体。不幸的是，没有唯一的细胞表面生物标记物来专门识别肿瘤中的所有细胞。为了克服这一限制，我们建议开发蛋白酶激活病毒(PAV)，它使用在转移性结直肠癌微环境中过度表达的胞外蛋白酶作为生物标志物，以实现靶向递送。具体地说，基质溶素(也称为基质金属蛋白酶7，MMP7)在结直肠癌中过表达。肿瘤微环境中高水平的MMP7将以局部的方式激活PAV，使载体能够结合广泛表达的细胞受体，包括在结直肠癌细胞上，并介导有效的基因传递。我们的PAV技术是基于临床上有前景的腺相关病毒(AAV)，该病毒最近已被批准为欧洲第一个人类基因治疗产品。我们有关键的试点数据表明，我们已经创造了MMP7感应PAV，一旦接触到蛋白酶，它们的基因传递效率就会大大提高。此外，在原位癌症模型中，PAV原型能够显著增加肿瘤中的转基因输送和表达。在目标1中，我们将合成和表征一组MMP7敏感的PAV。我们的设计过程将利用理性和组合方法，以加快设计解决方案的实现。在目标2中，我们将测试PAV在体外对结直肠癌细胞的基因传递性能，并将进行机制研究，以探索PAV与细胞的相互作用。最后，我们将在转移性结直肠癌的原位模型中测试PAV，以确定它们的体内特异性和治疗效果。如果成功，该项目将产生蛋白酶反应性AAV载体，可能成为转移性结直肠癌的可行治疗选择。

DESCRIPTION (provided by applicant): Once colorectal cancer metastasizes, it becomes a lethal disease with a 5-year survival rate of approximately 10%. Effective therapeutics that can specifically target metastatic colorectal tumor cells are sorely needed. Delivery of nucleic-acids (e.g. genes or RNAi) to combat cancer is a highly promising therapeutic approach; unfortunately, targeted delivery of gene vectors to tumor cells has been largely difficult to achieve. Most vector targeting approaches to date have relied on cell surface receptors overexpressed on some subpopulation of target cancer cells. Unfortunately, there is no unique cell surface biomarker that specifically identifies all cells in a tumor. To overcome this limitatin, we propose to develop protease-activatable viruses (PAVs) that use extracellular proteases overexpressed in metastatic colorectal tumor microenvironments as the biomarkers to achieve targeted delivery. Specifically, matrilysin (also known as matrix metalloproteinase 7, MMP7) has been shown to be overexpressed in colorectal cancer. High levels of MMP7 in the tumor microenvironment will activate the PAVs in a localized manner and enable the vectors to bind cellular receptors that are broadly expressed, including on colorectal cancer cells, and mediate efficient gene delivery. Our PAV technology is based on the clinically promising adeno-associated virus (AAV), which has recently been approved as the first human gene therapy product in Europe. We have key pilot data demonstrating we have created MMP7-sensing PAVs that dramatically increase their gene delivery efficiency once exposed to the protease. Moreover, in an orthotopic cancer model, a PAV prototype is able to significantly increase transgene delivery and expression in tumors. In aim 1, we will synthesize and characterize a panel of MMP7-sensing PAVs. Our design process will harness both rational and combinatorial approaches in order to expedite achievement of the design solution. In aim 2, we will test the gene delivery performance of PAVs in vitro on colorectal cancer cells, and mechanistic studies will be done to probe the interaction of PAVs with the cells. Finally, we will test the PAVs in an orthotopic model of metastatic colorectal cancer in order to determine their in vivo specificity and therapeutic efficacy. If successful, this project will generate protease-responsive AAV vectors that may become viable therapeutic options for metastatic colorectal cancer.