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

基因治疗中转基因腺病毒增强抗癌效果

• 批准号: 16590751
• 负责人: TAKAYAMA Koichi
• 金额: $ 2.3万
• 依托单位: KYUSHU UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2004
• 资助国家: 日本
• 起止时间: 2004 至 2005
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-16590751/
• 关键词: gene therapy; cancer treatment; adenovirus; conditionally replicative virus; mosaic adenovirus; VEGF; VEGF; 制限増殖型ウィルス

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.

根据研究计划，基于血清型5腺病毒和5/3嵌合腺病毒，利用人VEGF启动子制备了条件复制型腺病毒Ad5VEGFE1和Ad5/3VEGFE1。接下来，通过将 Ad5VEGFE1 和 Ad5/3VEGFE1 共感染 HEK293 细胞，制备在同一病毒粒子上具有两种血清型腺病毒纤维的嵌合腺病毒。血清型5和血清型5/3的共感染比为7:3，显示出制作花叶腺病毒的最高值。大量病毒制备后，按既定规则在CsCl_2溶液中超速离心浓缩。随后通过透析精制病毒溶液。收集的病毒溶液的密度的收率在10^9至10^10pfu/mL之间是比较好的。 2004财年报告中报告了体外实验结果。体内实验使用了三种癌细胞系：C33A、NCI-H157 和 SKOV。这些细胞系已经在裸鼠皮下空间中检查了它们的肿瘤形成能力。这些细胞系形成肿瘤后，每个肿瘤分别注射10^9 pfu的Ad5VEGFE1、Ad5/3VEGFE1和AdmsVEGFE1。测量肿瘤直径以评估治疗后肿瘤增殖情况。因此，无论是体外实验结果还是Ad5VEGFE1和AdmsVEGFE1对C33A和NCI-H157肿瘤的抗肿瘤作用均较高。此外，Ad5/3VEGFE1和AdmsVEGFE1对SKOV肿瘤的生长抑制作用也非常好。然而，AdmsVEGFE1的作用位于Ad5VVEGFE1和Ad5/3VVEGFE1的作用的中间。人们认为体内肿瘤中没有产生足够的花叶病毒。转基因腺病毒被认为可用于增强复制腺病毒的抗肿瘤作用。