Stevenson M, Hale A B H, Hale S J, Green N K, Black G, Fisher K D, Ulbrich K, Fabra A, Seymour L W
Department of Clinical Pharmacology, University of Oxford, Oxford, UK.
Cancer Gene Ther. 2007 Apr;14(4):335-45. doi: 10.1038/sj.cgt.7701022. Epub 2007 Jan 19.
Effective gene therapy for disseminated metastatic cancer is currently impossible because of poor delivery of vector to target sites. Modification of viral vectors to target advanced cancer has long been a challenge. In this study, we aimed to redirect adenovirus tropism to infect prostate cancer cells via alpha6beta1 integrins, whose expression is upregulated during prostate cancer progression. To ablate normal mechanisms of infection and provide a framework for attachment of targeting ligands, viruses were non-genetically modified with pHPMA-ONp polymer. Addition of polymer-coated virus to prostate cells showed significantly reduced transgene expression compared with unmodified virus. To restore infectivity, an alpha6-integrin binding peptide (-SIKVAV-) derived from laminin was incorporated onto the surface of the polymer-coated viruses. Photon correlation spectroscopic analysis revealed a small increase in the mean diameter of the particles following retargeting. Addition of -SIKVAV- peptide restored virus infectivity of PC-3 cells in a ligand concentration-dependent manner that was significantly improved following removal of unincorporated polymer and peptide. Competition assays using cells preincubated with Ad5 fiber protein or free -SIKVAV- peptide confirmed that entry of retargeted viruses was mediated via the incorporated ligand. Application of retargeted viruses to a panel of human cell lines revealed varying levels of transduction efficiency. Flow cytometric analysis of cells using anti-alpha6 integrin and anti-beta1 integrin antibodies demonstrated that for prostate cells, greater transduction efficiency correlated with higher levels of expression of both integrin subunits. Furthermore with the exception of LNCaP cells, increased alpha6beta1 integrin expression correlated with advanced disease. Intravenous administration of retargeted viruses to tumor-bearing mice resulted in slower plasma clearance and greatly reduced liver tropism, and hence toxicity compared with unmodified virus, while maintaining reporter gene expression in the tumor. The data suggest that YESIKVAVS-retargeted viruses have potential for systemic delivery for the treatment of metastatic disease.
由于载体难以有效递送至靶位点，目前针对播散性转移性癌症的有效基因治疗尚无法实现。长期以来，改造病毒载体以靶向晚期癌症一直是一项挑战。在本研究中，我们旨在通过α6β1整合素将腺病毒嗜性重定向至感染前列腺癌细胞，该整合素在前列腺癌进展过程中表达上调。为消除正常感染机制并提供靶向配体附着的框架，我们用聚（N-（2-羟丙基）甲基丙烯酰胺）-邻硝基苯甲酰胺（pHPMA-ONp）聚合物对病毒进行了非基因改造。与未修饰的病毒相比，向前列腺细胞中添加聚合物包被的病毒显示转基因表达显著降低。为恢复感染性，将源自层粘连蛋白的α6整合素结合肽（-SIKVAV-）掺入聚合物包被病毒的表面。光子相关光谱分析显示，重靶向后颗粒的平均直径略有增加。添加-SIKVAV-肽以配体浓度依赖性方式恢复了PC-3细胞的病毒感染性，在去除未结合的聚合物和肽后，感染性得到显著改善。使用预先与Ad5纤维蛋白或游离-SIKVAV-肽孵育的细胞进行的竞争试验证实，重靶向病毒的进入是通过掺入的配体介导的。将重靶向病毒应用于一组人类细胞系显示出不同水平的转导效率。使用抗α6整合素和抗β1整合素抗体对细胞进行流式细胞术分析表明，对于前列腺细胞，更高的转导效率与两种整合素亚基的更高表达水平相关。此外，除LNCaP细胞外，α6β1整合素表达增加与疾病进展相关。与未修饰的病毒相比，向荷瘤小鼠静脉注射重靶向病毒导致血浆清除减慢，肝脏嗜性大大降低，因此毒性降低，同时在肿瘤中维持报告基因表达。数据表明，YESIKVAVS重靶向病毒具有全身递送治疗转移性疾病的潜力。
