肿瘤通透肽修饰的HRE调控、携带Decorin基因的溶瘤腺病毒抗肾癌作用研究

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.

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。

本研究针对病毒Ki67-ZXC2存在的问题：肿瘤组织内部低氧环境下复制能力减弱；难以进入肿瘤组织并在其中播散；感染肿瘤细胞没有靶向性等，构建由了嵌合6个拷贝HREs的Ki67启动子调控、携带Decorin基因的溶瘤腺病毒6HREs-Ki67-Decorin；然后用偶联iRGD的PEG对其修饰，形成iRGD-PEG-6HREs-Ki67-Decorin复合物，通过体外及动物实验证实这种新型OAd能有效提高治疗效果，为其临床应用奠定实验基础。.实验结果显示：携带Decorin的病毒能够抑制2种肾癌细胞（786-O、Ketr-3）生长，具有体外抗肿瘤活性；表达Decorin的6HREs-Ki67-Decorin抑制肾癌细胞迁移和侵袭； Decorin的过表达上调细胞内E-cadherin表达、下调VEGF表达；iRGD-PEG自身对细胞的毒性作用很低，生物相容性较好；iRGD-PEG修饰可显著增加病毒在血液中的循环时间，iRGD-PEG修饰的腺病毒在体内产生低水平的中和抗体，有利于腺病毒的静脉给药；iRGD-PEG-6HREs-Ki67-Decorin在肝脏中的分布较低，在肿瘤组织中的分布较高， iRGD-PEG修饰的病毒具有更好的肿瘤靶向性；iRGD-PEG-6HREs-Ki67-Decorin治疗组肿瘤生长抑制作用更明显。结果表明：iRGD-PEG修饰的携带Decorin基因的腺病毒iRGD-PEG-6HREs-Ki67-Decorin可以有效在肿瘤组织中播散、抑制人肾癌裸鼠移植瘤的生长，为肾癌的生物与基因治疗奠定了基础。