Hackman Trevor, Doubrovin Michail, Balatoni Julius, Beresten Tatiana, Ponomarev Vladimir, Beattie Bradly, Finn Ronald, Bornmann William, Blasberg Ronald, Tjuvajev Juri Gelovani
Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, NY 10021, USA.
Mol Imaging. 2002 Jan-Mar;1(1):36-42. doi: 10.1162/15353500200200003.
Double prodrug activation gene therapy using the Escherichia coli cytosine deaminase (CD)-herpes simplex virus type 1 thymidine kinase (HSV1-tk) fusion gene (CD/TK) with 5-fluorocytosine (5FC), ganciclovir (GCV), and radiotherapy is currently under evaluation for treatment of different tumors. We assessed the efficacy of noninvasive imaging with [124I]FIAU (2'-fluoro-2'-deoxy-1-beta-D-arabinofuranosyl-5-iodo-uracil) and positron emission tomography (PET) for monitoring expression of the CD/TK fusion gene. Walker-256 tumor cells were transduced with a retroviral vector bearing the CD/TK gene (W256CD/TK cells). The activity of HSV1-TK and CD subunits of the CD/TK gene product was assessed in different single cell-derived clones of W256CD/TK cells using the FIAU radiotracer accumulation assay in cells and a CD enzyme assay in cell homogenates, respectively. A linear relationship was observed between the levels of CD and HSV1-tk subunit expression in corresponding clones in vitro over a wide range of CD/TK expression levels. Several clones of W256CD/TK cells with significantly different levels of CD/TK expression were selected and used to produce multiple subcutaneous tumors in rats. PET imaging of HSV1-TK subunit activity with [124I]FIAU was performed on these animals and demonstrated that different levels of CD/TK expression in subcutaneous W256CD/TK tumors can be imaged quantitatively. CD expression in subcutaneous tumor sample homogenates was measured using a CD enzyme assay. A comparison of CD and HSV1-TK subunit enzymatic activity of the CD/TK fusion protein in vivo showed a significant correlation. Knowing this relationship, the parametric images of CD subunit activity were generated. Imaging with [124I]FIAU and PET could provide pre- and posttreatment assessments of CD/TK-based double prodrug activation in clinical gene therapy trials.
使用大肠杆菌胞嘧啶脱氨酶（CD）-单纯疱疹病毒1型胸苷激酶（HSV1-tk）融合基因（CD/TK）与5-氟胞嘧啶（5FC）、更昔洛韦（GCV）以及放射疗法的双前药激活基因疗法目前正在针对不同肿瘤的治疗进行评估。我们评估了用[124I]FIAU（2'-氟-2'-脱氧-1-β-D-阿拉伯呋喃糖基-5-碘尿嘧啶）和正电子发射断层扫描（PET）进行无创成像以监测CD/TK融合基因表达的效果。用携带CD/TK基因的逆转录病毒载体转导Walker-256肿瘤细胞（W256CD/TK细胞）。分别使用细胞中的FIAU放射性示踪剂积累测定法和细胞匀浆中的CD酶测定法，在W256CD/TK细胞的不同单细胞衍生克隆中评估CD/TK基因产物的HSV1-TK和CD亚基的活性。在体外广泛的CD/TK表达水平范围内，在相应克隆中观察到CD和HSV1-tk亚基表达水平之间存在线性关系。选择了几个CD/TK表达水平明显不同的W256CD/TK细胞克隆，并用于在大鼠中产生多个皮下肿瘤。对这些动物进行了用[124I]FIAU对HSV1-TK亚基活性的PET成像，结果表明皮下W256CD/TK肿瘤中不同水平的CD/TK表达可以进行定量成像。使用CD酶测定法测量皮下肿瘤样品匀浆中的CD表达。体内CD/TK融合蛋白的CD和HSV1-TK亚基酶活性比较显示出显著相关性。了解这种关系后，生成了CD亚基活性的参数图像。用[124I]FIAU和PET成像可以在临床基因治疗试验中对基于CD/TK的双前药激活进行治疗前和治疗后评估。
