Peng K W, Morling F J, Cosset F L, Murphy G, Russell S J
Cambridge Centre for Protein Engineering, Medical Research Council Centre, UK.
Hum Gene Ther. 1997 Apr 10;8(6):729-38. doi: 10.1089/hum.1997.8.6-729.
We are developing protease-activatable gene delivery vehicles for selective gene delivery to protease-expressing cells. Angiogenesis, inflammation, and cancer invasion are linked to the overexpression of matrix metalloproteinases (MMPs), which destroy the extracellular matrix. Therefore, the MMPs are promising targets for therapy. We have displayed epidermal growth factor (EGF) on retroviral vector particles as an MMP-cleavable amino-terminal extension of the 4070A murine leukemia virus (MLV) envelope glycoprotein. This was achieved by engineering an MMP-cleavage signal (PLGLWA) into the linker between the EGF domain and the 4070A SU. The chimeric envelope was expressed and incorporated into viral particles, and the EGF domain could be cleaved from the surface of the viral particles by gelatinase A (MMP-2). The MMP-sensitive vector and control MMP-insensitive vectors could bind, via their displayed EGF domains, to EGF receptors on A431 cells but were unable to infect them because the EGF receptor (EGFR) does not support postbinding steps required for retroviral entry. In the presence of exogenous MMPs, the infectivity of the MMP-sensitive vector, but not of the MMP-insensitive vectors, was restored on A431 cells, and this cleavage activation could be partially blocked by MMP inhibitors. Endogenous MMPs produced by EGFR-positive HT 1080 cells could selectively activate the MMP-sensitive vector giving rise to a titer that was 1,000-fold higher on HT 1080 cells than on MMP-negative A431 cells. Inhibitor studies and gelatin zymograms indicated that the membrane-associated MT-MMP expressed on the HT 1080 cells played an important role in cleavage activation of the vector. When presented simultaneously with both EGFR-positive cell lines A431 and HT 1080, the vector could efficiently discriminate between the two different cell types, infecting the MMP-positive HT 1080 cells in preference over the A431 cells.
我们正在开发蛋白酶可激活的基因递送载体，用于将基因选择性递送至表达蛋白酶的细胞。血管生成、炎症和癌症侵袭与基质金属蛋白酶（MMPs）的过表达有关，MMPs会破坏细胞外基质。因此，MMPs是很有前景的治疗靶点。我们已将表皮生长因子（EGF）展示在逆转录病毒载体颗粒上，作为4070A小鼠白血病病毒（MLV）包膜糖蛋白可被MMP切割的氨基末端延伸。这是通过在EGF结构域与4070A SU之间的连接子中设计一个MMP切割信号（PLGLWA）来实现的。嵌合包膜得以表达并整合到病毒颗粒中，并且EGF结构域可被明胶酶A（MMP - 2）从病毒颗粒表面切割下来。MMP敏感型载体和对照MMP不敏感型载体可通过其展示的EGF结构域与A-431细胞上的EGF受体结合，但无法感染它们，因为EGF受体（EGFR）不支持逆转录病毒进入所需的结合后步骤。在存在外源性MMPs的情况下，MMP敏感型载体而非MMP不敏感型载体在A-431细胞上的感染性得以恢复，并且这种切割激活可被MMP抑制剂部分阻断。EGFR阳性的HT 1080细胞产生的内源性MMPs可选择性激活MMP敏感型载体，使得在HT 1080细胞上的滴度比在MMP阴性的A-431细胞上高1000倍。抑制剂研究和明胶酶谱分析表明，HT 1080细胞上表达的膜相关MT - MMP在载体的切割激活中起重要作用。当同时与EGFR阳性细胞系A-431和HT 1080呈现时，该载体可有效区分这两种不同的细胞类型，优先感染MMP阳性的HT 1080细胞而非A-431细胞。
