申请者完成2009年NSFC时发现，构建的肿瘤Ki67启动子调控的溶瘤腺病毒Ki67-ZXC2能在肾癌细胞内复制并将其杀伤，但瘤内注射结果不佳。通过预实验发现：（1）缺氧条件下该病毒在肾癌细胞内复制能力下降；（2）病毒瘤体内注射难以播散。此外，病毒感染肾癌细胞无靶向性也限制其临床应用。 为解决上述问题，本研究对Ki67-ZXC2改造提出如下策略：（1）将低氧反应原件（HRE）序列嵌合至Ki67启动子上游，HRE作为增强子能提高Ki67启动子调控病毒在肾癌组织缺氧环境内的复制能力；（2）病毒携带Decorin基因，通过重塑肿瘤细胞外基质，提高病毒在肿瘤组织内的播散能力；（3）利用肿瘤通透肽iRGD偶联的聚乙二醇修饰构建的病毒。iRGD赋予病毒靶向性的同时，其水解产生的小肽能够提高肿瘤组织通透性，使病毒易于播散。我们将在体外及动物实验证实这种病毒的肾癌治疗作用。

We had identified that oncolytic adenovirus Ki67-ZXC2 regulated by tumor specific Ki67 promoter could replicate in and kill renal cancer cells. The therapeutic efficacy of Ki67-ZXC2 was inefficient when with intratumoral injection and the ability to replicate in tumor mass was decreased due to its hypoxia microenvironment. The extracellular matrix (ECM) of tumor cells also inhibited distribution of adenovirus in vivo. Moreover, off-targeting with tumor cells limited the clinical intravenous delivery. Taking both modification of Ki67-ZXC2 and change of tumor microenvironment into account, we proposed following strategies. In order to improve adenoviral replication within hypoxia area of tumor tissue, we incorporate 6 hypoxia response elements (HRE) upstream of Ki67 promoter. A Decorin-expressing cassette is inserted into Ki67-ZXC2 and the spread of adenovirus is increased through remodeling ECM. We construct PEGylated oncolytic adenovirus (iRGD-PEG-6HREs-Ki67-Decorin) conjugated with tumor permeability peptide (iRGD), in which iRGD guarantees viral tumor-targeting . Furthermore, small peptide cleaved by a protease from iRGD could increase the permeability of tumor tissue and enhance the adenoviral spread. We will identify the therapeutic efficiency of iRGD-PEG-6HREs-Ki67-Decorin and the possibility of its intravenous delivery in vitro and in vivo.