Establishment of biological and pharmacokinetic assays of telomerase-specific replication-selective adenovirus
Establishment of biological and pharmacokinetic assays of telomerase-specific replication-selective adenovirus
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DOI：
10.1111/j.1349-7006.2007.00665.x
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发表时间：
2008-02-01
期刊：
影响因子：
5.7
通讯作者：
Fujiwara, Toshiyoshi
中科院分区：
文献类型：
--
作者：
Hashimoto, Yuuri;Watanabe, Yuichi;Fujiwara, Toshiyoshi
使用复制选择性肿瘤特异性病毒代表了治疗肿瘤疾病的一种新方法。我们构建了一种减毒腺病毒，端粒酶特异性复制选择性腺病毒（TRAD），其中人端粒酶逆转录酶启动子元件驱动与内部核糖体进入位点（IRES）连接的E1A和E1B基因的表达。 TRAD以1.0的感染复数感染后48小时，H1299人肺癌细胞的细胞活力始终低于50%，因此该程序可用作评估TRAD生物活性的效价测定。我们还利用腺病毒 E1A 或 IRES 序列的共有引物建立了定量实时聚合酶链反应 (PCR) 分析。血浆中 E1A 和 IRES 引物定量的线性范围分别为 10(3)-10(8) 和 10(2)-10(8) 噬菌斑形成单位/mL。 PCR分析表明，瘤内注射后28天，正常组织中E1A的水平比nu/n mu小鼠A549人肺肿瘤异种移植物的肿瘤中低10(3)以上。我们的结果表明，针对 H1299 细胞的细胞杀伤试验和实时 PCR 可用于评估临床试验中 TRAD 的生物活性和生物分布。
The use of replication-selective tumor-specific viruses represents a novel approach for the treatment of neoplastic disease. We constructed an attenuated adenovirus, telomerase-specific replication-selective adenovirus (TRAD), in which the human telomerase reverse transcriptase promoter element drives the expression of the E1A and E1B genes linked with an internal ribosome entry site (IRES). Forty-eight hours after TRAD infection at a multiplicity of infection of 1.0, the cell viability of H1299 human lung cancer cells was consistently less than 50% and therefore this procedure could be used as a potency assay to assess the biological activity of TRAD. We also established a quantitative real-time polymerase chain reaction (PCR) analysis with consensus primers for either the adenovirus E1A or IRES sequence. The linear ranges of quantitation with E1A and IRES primers were 10(3)-10(8) and 10(2)-10(8) plaque-forming units/mL in the plasma, respectively. The PCR analysis demonstrated that the levels of E1A in normal tissues were more than 10(3) lower than in the tumors of A549 human lung tumor xenografts in nu/n mu mice at 28 days after intratumoral injection. Our results suggest that the cell-killing assay against H1299 cells and real-time PCR can be used to assess the biological activity and biodistribution of TRAD in clinical trials.