Matrilysin-Sensing Gene Delivery Vectors for Colorectal Cancer Therapy

描述（申请人提供）：结直肠癌一旦转移，将成为一种致命疾病，5年生存率约为10%。目前迫切需要针对转移性结直肠肿瘤细胞的有效治疗方法。递送核酸（例如基因或RNAi）来对抗癌症是一种非常有前途的治疗方法；不幸的是，靶向传递基因载体到肿瘤细胞在很大程度上是难以实现的。迄今为止，大多数载体靶向方法依赖于靶癌细胞的某些亚群上过度表达的细胞表面受体。不幸的是，没有一种独特的细胞表面生物标志物可以特异性地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），利用转移性结直肠肿瘤微环境中过表达的细胞外蛋白酶作为生物标志物来实现靶向递送。具体来说，基质溶素（也称为基质金属蛋白酶7，MMP7）已被证明在结直肠癌中过度表达。肿瘤微环境中高水平的MMP7将以局部方式激活pav，使载体能够结合广泛表达的细胞受体，包括结直肠癌细胞，并介导有效的基因传递。我们的PAV技术基于具有临床前景的腺相关病毒（AAV），该病毒最近已被批准为欧洲首个人类基因治疗产品。我们有关键的试点数据表明，我们已经创建了mmp7感应pav，一旦暴露于蛋白酶，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原型能够显著增加转基因在肿瘤中的传递和表达。在目标1中，我们将合成并表征一组mmp7传感pav。我们的设计过程将利用合理和组合的方法，以加快实现设计解决方案。在目标2中，我们将在体外测试pas在结直肠癌细胞上的基因传递性能，并进行机制研究以探索pas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，以确定其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的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.