EpCAM-targeted delivery of nanocomplexed siRNA to tumor cells with designed ankyrin repeat proteins.
EpCAM-targeted delivery of nanocomplexed siRNA to tumor cells with designed ankyrin repeat proteins.
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DOI：
10.1158/1535-7163.mct-09-0402
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发表时间：
2009-09
影响因子：
5.7
通讯作者：
Zangemeister-Wittke U
中科院分区：
文献类型：
--
作者：
Winkler J;Martin-Killias P;Plückthun A;Zangemeister-Wittke U
核酸的特异性递送和细胞的有效摄取仍然是基因靶向癌症治疗的主要挑战。在此，我们报告了使用针对上皮细胞粘附分子 (EpCAM) 的设计锚蛋白重复蛋白 (DARPin) 作为与 bcl-2 mRNA 互补的 siRNA 的载体。为了实现 siRNA 的电荷复合，将 DARPin 与截短的人鱼精蛋白 1 序列融合。为了增加细胞结合亲和力和递送到细胞中的 siRNA 量，生成了 DARPin 二聚体并用作与鱼精蛋白的融合蛋白。所有蛋白质在大肠杆菌中均表达良好，为了去除紧密结合的细菌核酸，在变性条件下通过固定化金属离子亲和层析纯化它们，然后重折叠。融合蛋白能够复合每个鱼精蛋白 4-5 个 siRNA 分子，并完全保留对 EpCAM 的结合特异性，如 MCF-7 乳腺癌细胞上所证明的。与非特异性 lipofectamine 转染相反，使用融合蛋白复合 siRNA 的抗凋亡 bcl-2 下调严格依赖于 EpCAM 结合和内化。抑制 bcl-2 表达可促进肿瘤细胞凋亡，这通过抗癌药物阿霉素的敏感性增加得到证明。
Specific delivery to tumors and efficient cellular uptake of nucleic acids remain major challenges for gene-targeted cancer therapies. Here we report the use of a Designed Ankyrin Repeat Protein (DARPin) specific for the Epithelial Cell Adhesion Molecule (EpCAM) as carrier for siRNA complementary to the bcl-2 mRNA. For charge complexation of the siRNA, the DARPin was fused to a truncated human protamine-1 sequence. To increase the cell binding affinity and the amount of siRNA delivered into cells, DARPin dimers were generated and used as fusion proteins with protamine. All proteins expressed well in E. coli and, to remove tightly bound bacterial nucleic acids, they were purified under denaturing conditions by immobilized metal ion affinity chromatography, followed by refolding. The fusion proteins were capable of complexing 4-5 siRNA molecules per protamine, and fully retained the binding specificity for EpCAM as demonstrated on MCF-7 breast carcinoma cells. In contrast to unspecific lipofectamine transfection, down-regulation of anti-apoptotic bcl-2 using fusion protein complexed siRNA was strictly dependent on EpCAM binding and internalization. Inhibition of bcl-2 expression facilitated tumor cell apoptosis as demonstrated by increased sensitivity to the anti-cancer agent doxorubicin.