BLOCKING HEPARANASE TRANSCRIPTION TO CONTROL METASTASIS

肝素酶是一种内切糖苷酶，通过侵入细胞，特别是转移的肿瘤细胞和迁移的白细胞，降解细胞外基质中的硫酸乙酰肝素蛋白多糖。乙酰肝素酶基因的转导增强了啮齿动物肿瘤细胞的转移潜能，为乙酰肝素酶在侵袭中的作用提供了直接证据。肝素酶抑制剂(主要基于肝素和类似的多糖)在实验模型中已被证明在某些情况下可以抑制肿瘤的生长或转移、血管生成和血管损伤。在这里，我们建议利用锌指DNA结合蛋白设计的最新进展来创造能够在体内抑制乙酰肝素酶基因表达的新型转录抑制蛋白。嵌合抑制蛋白将被设计成与乙酰肝素酶基因中的相关靶点结合，并抑制乙酰肝素酶的转录。我们将通过瞬时转染法和稳定细胞系的产生来确定所设计的蛋白质在哺乳动物细胞中抑制乙酰肝素酶mRNA和蛋白表达的能力。乙酰肝素酶转录的抑制将通过在没有和存在锌指结合蛋白的情况下测量报告基因的表达水平来确定。这项创新研究将直接导致针对转移性癌症和潜在炎症性疾病的新的基因治疗方法的开发。拟议的商业应用：该项目的成功将是“设计者抑制器”技术原理的关键证明。设计的DNA结合蛋白和嵌合抑制物组成了一类新的治疗药物，有可能用于治疗大量人类疾病。

Heparanase is an endoglycosidase that degrades the heparan sulfate proteoglycan of the extracellular matrix by invading cells, notably metastatic tumor cells and migrating leukocytes. Transfection of the heparanase gene enhances the metastatic potential of rodent tumor cells, providing direct evidence for a role of heparanase in invasion. Heparanase inhibitors (mainly based on heparin and similar polysaccharides) have been shown to inhibit tumor growth or metastasis, angiogenesis and vascular damage in some cases in experimental models. Here, we propose to use recent advances in zinc finger DNA binding protein design to create novel transcription repressor proteins which are capable of repressing heparanase gene expression in vivo. Chimeric repressor proteins will be designed to bind to relevant target sites in the heparanase gene and repress heparanase transcription. We will determine the ability of the designed proteins to repress heparanase mRNA and protein expression in mammalian cells through transient transfection assays and the production of stable cell lines. Repression of heparanase transcription will be determined by measuring the level of reporter gene expression in the absence and presence of zinc finger binding protein. This innovative research will lead directly to the development of novel gene therapy approaches for metastatis cancer and potentially inflammatory diseases. PROPOSED COMMERCIAL APPLICATIONS: The success of this project will be a critical proof of principle for "designer repressor" technology. Designer DNA binding proteins and chimeric repressors comprise a new class of therapeutic drugs that potentially could be used to treat a large number of human diseases.