Oncolytic adenovirus modified to increase its infectivity for gastrointestinal cancer

腺病毒（Ad）具有很强的细胞毒活性，其早期转录产物之一E1A蛋白在病毒感染细胞内的复制中起着关键作用。因此，用假定的肿瘤启动子修饰以控制E1A基因表达的Ad可具有溶瘤作用。我们用荧光素酶检测了肿瘤中主要表达基因的调控区域，发现survivin和midkine基因的上游区域分别为500 bp和600 bp，负责转录控制。Ad 5型的细胞受体是CAR（柯萨奇病毒和腺病毒受体），在胃肠道肿瘤中表达常下调。相反，作为Ad 35型受体的CD46在肿瘤中表达上调，因此，携带Ad的CD46结合位点可以增加对肿瘤的感染性。因此，我们构建了一个载体系统，该载体DNA可以激活带有外源调控区域的E1A和E1B基因，另一个载体包含整个Ad序列的其余部分，其中纤维旋结区域被替换为Ad 35型衍生的区域。载体的体外结扎能够在E3区域构建具有35型纤维旋钮结构的Ad。具有35型纤维旋钮的溶瘤性Ad与传统的需要体内重组的方法相比容易制备。用我们的载体系统制备的嵌合溶瘤性Ad对低car的人肿瘤比传统的5型溶瘤性Ad具有更高的细胞毒性，对高car的肿瘤具有与5型Ad相同的细胞毒性。

Adenovirus (Ad) has a strong cytotoxic activity and the E1A protein, one of the early transcriptional products, has a pivotal role in viral replication in the infected cells. Ad modified to control the E1A gene expression with a putative tumor promoter can thereby be oncolytic. We examined the regulatory regions of the genes which were predominantly expressed in tumors with the luciferase assay and found that a 500 bp and a 600 bp upstream regions of the survivin and midkine genes, respectively, were responsible for the transcriptional control. The cellular receptors of the Ad type 5 is CAR (Coxsackievirus and Adenovirus Receptor) and the expression is often downregulated in gastrointestinal tumors. In contrast, the expression of CD46, which is a receptor of Ad type 35, is rather upregulated in the tumors and consequently, the Ad bearing CD46 binding sites can increase the infectivity to the tumors. We thereby constructed a vector system consisting of vector DNA that could activate the E1A and E1B genes with an exogenous regulatory region and another vector that contained the rest of the whole Ad sequences, in which the fiber-knob region was replaced with the Ad type 35-derived one. In vitro ligation of the vectors enables the construction of the Ad bearing the type 35 fiber-knob structure in the E3 region. The oncolytic Ad with the type 35 fiber-knob can be produced with easy compared with conventional methods that required in vivo recombination. The chimeric oncolytic Ad prepared with our vector system was more cytotoxic to CAR-low human tumors than conventional type 5 oncolytic Ad and was as cytotoxic as the type 5 Ad to CAR-high tumors.