Parry J J, Sharma V, Andrews R, Moros E G, Piwnica-Worms D, Rogers B E
Department of Radiation Oncology, Washington University School of Medicine, St Louis, MO 63108, USA.
Cancer Gene Ther. 2009 Feb;16(2):161-70. doi: 10.1038/cgt.2008.70. Epub 2008 Aug 29.
The ability to achieve tumor selective expression of therapeutic genes is an area that needs improvement for cancer gene therapy to be successful. One approach to address this is through the use of promoters that can be controlled by external means, such as hyperthermia. In this regard, we constructed a replication-deficient adenovirus that consists of a mutated herpes simplex virus 1 thymidine kinase (mTK) fused to enhanced green fluorescent protein (EGFP) under the control of the full-length human heat shock (HS) 70b promoter. The virus (AdHSmTK-EGFP) was evaluated both in vitro and in vivo in oral squamous cell carcinoma SCC-9 cells for expression of both mTK and EGFP. The in vitro expression of mTK-EGFP was validated using both (3)H-penciclovir and fluorescence-activated cell sorting assays. These studies show that specific expression could be achieved by heating the cells at 41 degrees C for 1 h, whereas little expression was observed using high doses of virus without hyperthermia. The vector was also evaluated in vivo by direct intratumoral injection into mice bearing SCC-9 xenografts. These studies demonstrated tumor expression of mTK-EGFP after ultrasound heating of the tumors by radioactive biodistribution assays, histology and microPET imaging. These in vivo results, which demonstrate HS-inducible transgene expression using PET imaging, provide a means for noninvasive monitoring of heat-induced gene therapy in local tumors, such as oral squamous cell carcinomas.
实现治疗性基因的肿瘤选择性表达的能力是癌症基因治疗取得成功所需改进的一个领域。解决这一问题的一种方法是使用可通过外部手段(如热疗)控制的启动子。在这方面,我们构建了一种复制缺陷型腺病毒,它由一个与增强型绿色荧光蛋白(EGFP)融合的突变单纯疱疹病毒1胸苷激酶(mTK)组成,该融合蛋白受全长人热休克(HS)70b启动子的控制。在体外和体内对该病毒(AdHSmTK-EGFP)在口腔鳞状细胞癌SCC-9细胞中mTK和EGFP的表达进行了评估。使用³H-喷昔洛韦和荧光激活细胞分选试验验证了mTK-EGFP的体外表达。这些研究表明,通过将细胞在41℃加热1小时可实现特异性表达,而在无热疗的情况下使用高剂量病毒几乎观察不到表达。还通过将载体直接瘤内注射到携带SCC-9异种移植瘤的小鼠体内进行了体内评估。通过放射性生物分布测定、组织学和微型PET成像,这些研究证明了在对肿瘤进行超声加热后mTK-EGFP在肿瘤中的表达。这些体内结果通过PET成像证明了HS诱导的转基因表达,为局部肿瘤(如口腔鳞状细胞癌)中热诱导基因治疗的无创监测提供了一种手段。