A human-derived reporter gene for noninvasive imaging in humans: mitochondrial thymidine kinase type 2.
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 (DeltahTK2), alone or fused with green fluorescent protein (GFP), was used for preclinical evaluation in a mouse model. The levels of enzymatic activity of DeltahTK2 and DeltahTK2 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 (124)I-2'-fluoro-2'-deoxy-1-beta-D-beta-arabinofuranosyl-5-iodouracil (FIAU), (18)F-2'-fluoro-2'-deoxy-1-beta-D-beta-arabinofuranosyl-5-ethyluracil (FEAU), or (18)F-9-(4-(18)F-fluoro-3-hydroxymethylbutyl)guanine (FHBG) uptake in tumors and biodistribution studies were performed.


RESULTS
DeltahTK2 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 DeltahTK2 and DeltahTK2 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 DeltahTK2 than FIAU, whereas FHBG is not phosphorylated by this enzyme. In addition, we showed that DeltahTK2 transduced cells can be eliminated by treatment with d-arabinofuranosyl-cytosine.


CONCLUSIONS
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 (124)I-FIAU, (18)F-FIAU, or (18)F-FEAU, it should be possible to image DeltahTK2 reporter gene expression with PET in preclinical and clinical studies.
Ponomarev
Vladimir
V
Department of Radiology, Memorial Sloan-Kettering Cancer Center, New York, NY, USA.
Doubrovin
Michael
M
Shavrin
Aleksander
A
Serganova
Inna
I
Beresten
Tatiana
T
Ageyeva
Ludmila
L
Cai
Changde
C
Balatoni
Julius
J
Alauddin
Mian
M
Gelovani
Juri
J
D045744:Cell Line, Tumor; D017930:Genes, Reporter / Q000235:genetics; D005910:Glioma / Q000000981:diagnostic imaging* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008928:Mitochondria / Q000201:enzymology*; D011877:Radionuclide Imaging; D013937:Thymidine Kinase / Q000235:genetics / Q000378:metabolism*
C105339:thymidine kinase 2; D013937:Thymidine Kinase