In this study we analyzed two ways of retargeting of Ad-vectors to human pancreatic carcinoma with the aim of enhancing the gene transfer efficiency. First, we analyzed the expression of the epidermal growth factor receptor (EGFR) on primary, as well as established pancreatic carcinoma cells by flow cytometry which revealed high expression levels of EGFR on the surface of these cells. We showed that EGFR-retargeted entry pathway using a bispecific fusion protein formed by a recombinant soluble form of truncated Coxsackie and Adenovirus Receptor (sCAR) genetically fused with human EGF (sCAR-EGF) redirects them to the EGFR leading to an enhanced gene transfer efficiency to pancreatic carcinoma cells. Since flow cytometry revealed absence of CAR expression, but the presence of at least one of both alphav integrins on the pancreatic carcinoma cells, a second way of targeting was investigated using a genetically modified Ad vector which has an RGD (Arg-Gly-Asp)-containing peptide inserted into the HI-loop of the fiber knob. This RGD targeted Ad (AdlucRGD) revealed efficient CAR-independent infection by allowing binding to cellular integrins resulting in a dramatic enhancement of gene transfer. These findings have direct relevance for Ad-vector based gene therapy strategies for pancreatic carcinoma.

译文

:在这项研究中,我们分析了将Ad载体重新靶向人胰腺癌的两种方法,目的是提高基因转移效率。首先,我们通过流式细胞术分析了表皮生长因子受体(EGFR)在原发性和已建立的胰腺癌细胞上的表达,揭示了这些细胞表面上EGFR的高表达水平。我们显示,使用由人源EGF(sCAR-EGF)基因融合的截短的柯萨奇和腺病毒受体(sCAR)的重组可溶性形式形成的双特异性融合蛋白,通过双特异性融合蛋白形成的EGFR靶向进入途径将其重定向至EGFR,从而提高了基因转移效率胰腺癌细胞。由于流式细胞仪显示不存在CAR表达,但在胰腺癌细胞上同时存在两种alphav整合素中的至少一种,因此使用具有RGD(Arg-Gly-Asp)的基因修饰的Ad载体研究了第二种靶向方法插入到纤维结的HI-环中的含-肽。该RGD靶向广告(AdlucRGD)通过与细胞整联蛋白结合,从而显着增强了基因转移,从而揭示了与CAR无关的有效感染。这些发现与基于Ad载体的胰腺癌基因治疗策略直接相关。

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