A human-derived reporter gene for noninvasive imaging in humans: Mitochondrial thymidine kinase type 2
A human-derived reporter gene for noninvasive imaging in humans: Mitochondrial thymidine kinase type 2
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DOI：
10.2967/jnumed.106.036962
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发表时间：
2007-05-01
影响因子：
9.3
通讯作者：
Gelovani, Juri
中科院分区：
文献类型：
--
作者：
Ponomarev, Vladimir;Doubrovin, Michael;Gelovani, Juri
一个人源性的固有的非免疫原性报告基因进行了测试的PET成像的不同分子遗传过程的潜在临床用途。研究方法：N末端截短的人线粒体胸苷激酶2型（hTK 2）报告基因（Delta hTK 2）单独或与绿色荧光蛋白（GFP）融合，用于小鼠模型的临床前评估。使用放射性示踪剂累积和前药活化测定法在体外和在裸鼠中从相应细胞系生长的皮下肿瘤中评估Δ hTK 2和Δ hTK 2 GFP蛋白的酶活性水平。对肿瘤中的I-124- 2 '-氟-2'-脱氧-1-β-D-β-阿拉伯呋喃糖基-5-碘尿嘧啶（FIAU）、F-18- 2 '-氟-2'-脱氧-1-β-D-β-阿拉伯呋喃糖基-5-乙基尿嘧啶（FEAU）或F-18-9-（4-F-18-氟-3-羟甲基丁基）鸟嘌呤（FHBG）摄取进行了动力学分析，并进行了生物分布研究。结果：Δ hTK 2在转染细胞的胞浆中成功表达。本文报道了一种新的抗hTK 2单克隆抗体8 G2。表达Δ hTK 2和Δ hTK 2 GFP的细胞中FIAU和FEAU积累的水平在体外比野生型细胞高至少10倍，在体内高约6倍。我们确定FEAU是比FIAU更特异的Delta hTK 2报告底物，而FHBG不被这种酶磷酸化。此外，我们表明Δ hTK 2转导的细胞可以通过用D-阿拉伯呋喃糖基-胞嘧啶处理来消除。结论：我们已经测试了一个人源性的报告基因，可能是nonimmunogenic和潜在的允许长期监测不同的分子遗传过程中的核成像技术在人类。使用I-124-FIAU、F-18-FIAU或F-18-FEAU，应该可以在临床前和临床研究中使用PET对Delta hTK 2报告基因表达进行成像。
A human-derived intrinsically nonimmunogenic reporter gene was tested for PET imaging of different molecular-genetic processes for potential clinical use. Methods: The human mitochondrial thymidine kinase type 2 (hTK2) reporter gene truncated at the N terminus (Delta hTK2), alone or fused with green fluorescent protein (GFP), was used for preclinical evaluation in a mouse model. The levels of enzymatic activity of Delta hTK2 and Delta hTK2 GFP proteins were assessed using radiotracer accumulation and prodrug activation assays in vitro and in subcutaneous tumors grown from the corresponding cell lines in nude mice. Kinetic analyses of I-124-2'-fluoro-2'-deoxy-1-beta-D-beta-arabinofuranosyl-5-iodouracil (FIAU), F-18-2'-fluoro-2'-deoxy-1-beta-D-beta-arabinofuranosyl-5-ethyluracil (FEAU), or F-18-9-(4-F-18-fluoro-3-hydroxymethylbutyl)guanine (FHBG) uptake in tumors and biodistribution studies were performed. Results: Delta hTK2 was successfully expressed in the cytoplasm of transduced cells. A new anti-hTK2 monoclonal antibody 8G2 was developed. The levels of FIAU and FEAU accumulation in cells expressing Delta hTK2 and Delta hTK2 GFP were at least 10-fold higher than in wildtype cells in vitro and about 6 times higher in vivo. We determined that FEAU is a more specific reporter substrate for Delta hTK2 than FIAU, whereas FHBG is not phosphorylated by this enzyme. In addition, we showed that Delta hTK2 transduced cells can be eliminated by treatment with D-arabinofuranosyl-cytosine. Conclusion: We have tested a human-derived reporter gene that is likely to be nonimmunogenic and potentially allows for long-term monitoring of different molecular-genetic processes by nuclear imaging techniques in humans. Using I-124-FIAU, F-18-FIAU, or F-18-FEAU, it should be possible to image Delta hTK2 reporter gene expression with PET in preclinical and clinical studies.