The use of reporter genes to non-invasively image molecular processes inside cells has significant translational potential, particularly in the context of systemically administered gene therapy vectors and adoptively administered cells such as immune or stem cell based therapies. Bacterial nitroreductase enzymes possess ideal properties for reporter gene imaging applications, being of non-human origin and possessing the ability to metabolize a range of clinically relevant nitro(hetero)cyclic substrates./nMethods: A library of eleven Escherichia coli nitroreductase candidates were screened for the ability to efficiently metabolize 2-nitroimidazole based positron emission tomography (PET) probes originally developed as radiotracers for hypoxic cell imaging. Several complementary methods were utilized to detect formation of cell-entrapped metabolites, including various in vitro and in vivo models to establish the capacity of the 2-nitroimidazole PET agent EF5 to quantify expression of a nitroreductase candidate. Proof-of-principle PET imaging studies were successfully conducted using ¹⁸F-HX4./nResults: Recombinant enzyme kinetics, bacterial SOS reporter assays, anti-proliferative assays and flow cytometry approaches collectively identified the major oxygen-insensitive nitroreductase NfsA from E. coli (NfsA_Ec) as the most promising nitroreductase reporter gene. Cells expressing NfsA_Ec were demonstrably labelled with the imaging agent EF5 in a manner that was quantitatively superior to hypoxia, in monolayers (2D), multicellular layers (3D), and in human tumor xenograft models. EF5 retention correlated with NfsA_Ec positive cell density over a range of EF5 concentrations in 3D in vitro models and in xenografts in vivo and was predictive of in vivo anti-tumor activity of the cytotoxic prodrug PR-104. Following PET imaging with ¹⁸F-HX4, a significantly higher tumor-to-blood ratio was observed in two xenograft models for NfsA_Ec expressing tumors compared to the parental tumors thereof, providing verification of this reporter gene imaging approach./nConclusion: This study establishes that the bacterial nitroreductase NfsA_Ec can be utilized as an imaging capable reporter gene, with the ability to metabolize and trap 2-nitroimidazole PET imaging agents for non-invasive imaging of gene expression.

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大肠杆菌硝基还原酶NfsA是在癌症基因治疗应用中用于非侵入性PET成像的报告基因。

使用报告基因对细胞内部的分子过程进行非侵入性成像具有显着的翻译潜力，尤其是在系统施用基因治疗载体和过继施用细胞（例如基于免疫或干细胞的疗法）的情况下。细菌硝基还原酶具有报告基因成像应用的理想特性，它是非人类来源的，并且具有代谢一系列临床相关硝基（杂环）底物的能力。/n方法： 11种大肠杆菌的文库筛选硝基还原酶候选物有效代谢基于2-硝基咪唑的正电子发射断层扫描（PET）探针的能力，这些探针最初是作为低氧细胞成像的放射性示踪剂而开发的。几种互补的方法被用于检测细胞捕获的代谢物的形成，包括各种体外和体内模型，以建立2-硝基咪唑PET试剂EF5量化硝基还原酶候选物表达的能力。使用¹⁸ F-HX4 / n成功地进行了原理证明PET成像研究。重组酶动力学，细菌SOS报告基因分析，抗增殖分析和流式细胞术方法共同确定了来自大肠杆菌的主要的氧敏感性氮还原酶NfsA （NfsA_Ec）是最有希望的氮还原酶报告基因。在单层（2D），多细胞层（3D）和人肿瘤异种移植模型中，表达的NfsA_Ec细胞已用显像剂EF5​​进行了定量定量，其效果优于缺氧。在3D体外模型和体内异种移植物中，EF5保留与一系列EF5浓度下的NfsA_Ec阳性细胞密度相关，并预测了细胞毒性前药PR-104的体内抗肿瘤活性。进行PET成像后¹⁸ F-HX4，在两个表达NfsA_Ec的异种移植模型中，与亲本肿瘤相比，在肿瘤模型中观察到明显更高的肿瘤血比，从而证实了该报告基因成像方法。/n结论：这项研究确定了细菌硝基还原酶NfsA_Ec可用作具有显像能力的报告基因，具有代谢和捕获2-硝基咪唑PET显像剂的能力，可用于基因表达的非侵入性显像​​。