A Human-Derived Reporter Gene for Noninvasive Imaging in Humans: Mitochondrial Thymidine Kinase Type 2

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Abstract
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 (ΔhTK2), alone or fused with green fluorescent protein (GFP), was used for preclinical evaluation in a mouse model. The levels of enzymatic activity of ΔhTK2 and ΔhTK2 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 124I-2′-fluoro-2′-deoxy-1-β-d-β-arabinofuranosyl-5-iodouracil (FIAU), 18F-2′-fluoro-2′-deoxy-1-β-d-β-arabinofuranosyl-5-ethyluracil (FEAU), or 18F-9-(4-18F-fluoro-3-hydroxymethylbutyl)guanine (FHBG) uptake in tumors and biodistribution studies were performed. Results: ΔhTK2 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 ΔhTK2 and ΔhTK2 GFP were at least 10-fold higher than in wild-type cells in vitro and about 6 times higher in vivo. We determined that FEAU is a more specific reporter substrate for ΔhTK2 than FIAU, whereas FHBG is not phosphorylated by this enzyme. In addition, we showed that ΔhTK2 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 124I-FIAU, 18F-FIAU, or 18F-FEAU, it should be possible to image ΔhTK2 reporter gene expression with PET in preclinical and clinical studies.

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