[18F]pFBC, a Covalent CLIP-Tag Radiotracer for Detection of Viral Reporter Gene Transfer in the Murine Brain,Bioconjugate Chemistry

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[18F]pFBC, a Covalent CLIP-Tag Radiotracer for Detection of Viral Reporter Gene Transfer in the Murine Brain
Bioconjugate Chemistry ( IF 4.0 ) Pub Date : 2024-02-03 , DOI: 10.1021/acs.bioconjchem.3c00551
Gregory D Bowden _{1,

2} , Sophie Stotz _{1,

2} , Gina Dunkel _{1,

2} , Sabrina Haas ₁ , Elena Kimmerle ₁ , Martin Schaller ₃ , Bettina Weigelin _{1,

2} , Kristina Herfert ₁ , Bernd J Pichler _{1,

2} , Andreas Maurer _{1,

2}

Affiliation

Preclinical models of neurological diseases and gene therapy are essential for neurobiological research. However, the evaluation of such models lacks reliable reporter systems for use with noninvasive imaging methods. Here, we report the development of a reporter system based on the CLIP-tag enzyme and [¹⁸F]pFBC, an ¹⁸F-labeled covalent CLIP-tag-ligand synthesized via a DoE-optimized and fully automated process. We demonstrated its specificity using a subcutaneous xenograft model and a model of viral vector-mediated brain gene transfer by engineering HEK293 cells and striatal neurons to express membrane-tethered CLIP-tag protein. After in vitro characterization of the reporter, mice carrying either CLIP-tag expressing or control subcutaneous xenografts underwent dynamic [¹⁸F]pFBC PET imaging. The CLIP-tag expressing xenografts showed a significantly higher uptake than control xenografts (tumor-to-muscle ratio 5.0 vs 1.7, p = 0.0379). In vivo, metabolite analysis by radio-HPLC from plasma and brain homogenates showed only one radio-metabolite in plasma and none in the brain. In addition, [¹⁸F]pFBC showed fast uptake and rapid clearance from the brain in animals injected with adeno-associated virus (AAV)-CLIP in the right striatum but no right-to-left (R-L) uptake difference in the striata in the acquired PET data. In contrast, autoradiography showed a clear accumulation of radioactivity in the AAV-CLIP-injected right striatum compared to the sham-injected left striatum control. CLIP-tag expression and brain integrity were verified by immunofluorescence and light sheet microscopy. In conclusion, we established a novel reporter gene system for PET imaging of gene expression in the brain and periphery and demonstrated its potential for a wide range of applications, particularly for neurobiological research and gene therapy with viral vectors.

中文翻译：

[18F]pFBC，一种共价 CLIP 标签放射性示踪剂，用于检测小鼠大脑中的病毒报告基因转移

神经系统疾病和基因治疗的临床前模型对于神经生物学研究至关重要。然而，此类模型的评估缺乏用于非侵入性成像方法的可靠报告系统。在这里，我们报告了基于 CLIP 标签酶和 [ ¹⁸ F] p FBC 的报告系统的开发，[ 18 F] p FBC 是通过 DoE 优化的全自动流程合成的¹⁸ F 标记共价 CLIP 标签配体。我们通过改造 HEK293 细胞和纹状体神经元来表达膜束缚的 CLIP 标签蛋白，使用皮下异种移植模型和病毒载体介导的脑基因转移模型证明了其特异性。在对报告基因进行体外表征后，携带 CLIP 标签表达或对照皮下异种移植物的小鼠进行了动态 [ ¹⁸ F] p FBC PET 成像。表达 CLIP 标签的异种移植物显示出比对照异种移植物显着更高的摄取（肿瘤与肌肉的比率 5.0 vs 1.7，p = 0.0379）。在体内，通过放射性 HPLC 对血浆和脑匀浆进行的代谢物分析显示，血浆中仅存在一种放射性代谢物，而大脑中则没有。此外，[ ¹⁸ F] p FBC 在右侧纹状体中注射腺相关病毒 (AAV)-CLIP 的动物中显示出快速摄取和从大脑中快速清除，但纹状体中没有从右到左 (RL) 摄取差异在获取的 PET 数据中。相反，放射自显影显示，与假注射的左侧纹状体对照相比，注射 AAV-CLIP 的右侧纹状体中放射性明显积累。通过免疫荧光和光片显微镜验证 CLIP 标签表达和大脑完整性。总之，我们建立了一种新型报告基因系统，用于大脑和外周基因表达的 PET 成像，并证明了其广泛应用的潜力，特别是在神经生物学研究和病毒载体基因治疗方面。

更新日期：2024-02-03

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