Enhancement of anti-cancer effect with genetically modified adenovirus in gene therapy

根据研究计划，利用基于5-型腺病毒和5/3嵌合型腺病毒的人血管内皮生长因子启动子，制备了条件复制型腺病毒Ad5VEGFE1和Ad5/3VEGFE1。其次，将Ad5VEGFE1和Ad5/3VEGFE1共感染HEK293细胞，得到在同一病毒粒子上具有两种血清型腺病毒纤维的嵌合型腺病毒。血清5型与血清5/3混合感染比例为7：3的嵌合型腺病毒的感染率最高。在大量制备病毒后，按既定规则在CsCl2溶液中进行超速离心浓缩。随后对病毒溶液进行透析提纯。采集的病毒溶液浓度在10~10 pfu/mL时产量较好。试管试验结果已在2004财年报告中公布。采用C33A、NCI-H157和SKOV三种肿瘤细胞系进行体内实验。这些细胞系已经检查了它们在裸鼠皮下空间的肿瘤形成能力。肿瘤形成后，分别注射109pfu的Ad5VEGFE1、Ad5/3VEGFE1和AdmsVEGFE1。测量肿瘤直径，评价治疗后肿瘤的增殖情况。结果表明，Ad5VEGFE1和AdmsVEGFE1对C33A和NCI-H157肿瘤有较高的抗肿瘤作用，体外实验结果表明，Ad5VEGFE1和AdmsVEGFE1对C33A和NCI-H157肿瘤有较好的抑制作用。Ad5/3VEGFE1和AdmsVEGFE1对SKOV瘤的生长抑制作用也较好。而AdmsVEGFE1的作用位于Ad5VEGFE1和Ad5/3VEGFE1的中间。据认为，在体内的肿瘤中没有产生足够的马赛克病毒。基因修饰的腺病毒被认为有助于增强复制型腺病毒的抗肿瘤作用。

Conditionally replicative Adenoviruses, Ad5VEGFE1 and Ad5/3VEGFE1, were made using human VEGF promoter based on serotyp 5 adenovirus and 5/3 chimeric adenovirus according to the research plan. Next, mosaic adnovirus possessing both serotype adenovirus fibers on the same virion was made from co-infection of Ad5VEGFE1 and Ad5/3VEGFE1 into the HEK293 cell. Co-infection ratio of serotype 5 and serotype 5/3 of 7:3 showed the highest value to make a mosaic adenovirus. It concentrated by the ultracentrifugation in the CsCl_2 solution according to the established rule after a large amount of viral preparation. The virus solution was refined by the dialysis afterwards. Yield of the density of the collected virus solution was comparatively excellent in 10^9 to 10^<10> pfu/mL. In vitro experimet results was reported in 2004 fiscal year report. Three cancer cell lines, C33A, NCI-H157, and SKOV were used for the in vivo experiment. These cell lines already checked for their tumor formation ability in the subcutaneous space of the nude mouse. After tumor formation of these cell lines, each tumor was injected by 10^9 pfu of Ad5VEGFE1, Ad5/3VEGFE1, and AdmsVEGFE1 respectively. The tumor diameter was measured to evaluate the tumor proliferation after treatment. As a result, the antitumor effects of Ad5VEGFE1 and AdmsVEGFE1 are higher for C33A and NCI-H157 tumors as well as the in vitro experiment result. Also, the growth suppressive effect of Ad5/3VEGFE1 and AdmsVEGFE1 was excellent for the SKOV tumor. However, the effect of AdmsVEGFE1 was located in the middle in the effect of Ad5VEGFE1 and Ad5/3VEGFE1. It was thought that an enough mosaic virus generation did not happen in the tumor in vivo. Genetically modified adenovirus was thought to be useful for the reinforcement of the antitumor effect in the replicative adenovirus.