Matrilysin-Sensing Gene Delivery Vectors for Colorectal Cancer Therapy

项目总结 一旦结直肠癌转移，它就会成为一种致命的疾病，5年存活率为 大约10%。可以特异性靶向转移性结直肠肿瘤细胞的有效治疗药物是 这是非常需要的。用核酸(如基因或RNAi)来抗击癌症是一种非常有前途的方法 治疗方法；不幸的是，将基因载体定向递送到肿瘤细胞在很大程度上是困难的 来实现。到目前为止，大多数载体靶向方法都依赖于过度表达的细胞表面受体 靶癌细胞的某些亚群。不幸的是，没有唯一的细胞表面生物标记物 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- 使用胞外蛋白水解酶的可激活病毒(PAV)在转移性结直肠癌中的过度表达 微环境作为生物标志物，实现靶向递送。具体地说，母溶素(也称为 基质金属蛋白酶7(MMP7)在结直肠癌中高表达。高水平的 肿瘤微环境中的MMP7将以局部方式激活PAV，并使载体能够 结合广泛表达的细胞受体，包括在结直肠癌细胞上，并介导高效 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)，它 最近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 证明我们已经创造了感知MMP7的PAV，大大提高了它们的基因传递效率 一旦接触到这种蛋白酶。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，我们将综合和 描述一组感应MMP7的PAV的特征。我们的设计过程将同时利用理性和组合 为了加快设计解决方案的实现，需要采取各种方法。在目标2中，我们将测试基因传递 PAV在体外对结直肠癌细胞的作用，并将进行机制研究，以探讨 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。 以确定其在体内的特异性和治疗效果。如果成功，这个项目将 构建有可能成为治疗转移性肿瘤的可行治疗选择的蛋白酶反应型AAV载体 结直肠癌。

PROJECT SUMMARY 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 limitation, 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.