The human norepinephrine transporter in combination with 11C-m-hydroxyephedrine as a reporter gene/reporter probe for PET of gene therapy.
Although the herpes simplex virus thymidine kinase gene has been frequently applied as a reporter gene for monitoring gene transfection in animals, it has some intrinsic limitations for use in humans. In our search for a reporter gene that lacks these limitations, we have evaluated the feasibility of the human norepinephrine transporter (hNET) as a reporter gene in combination with the reporter probe 11C-m-hydroxyephedrine (mHED) for PET.


METHODS
An adenoviral vector (AdTrack-hNET) containing the hNET gene as reporter gene and the enhanced green fluorescent protein (EGFP) as a substitute for a therapeutic gene was constructed. After COS-7, A2780, and U373 cells were transiently transduced with AdTrack-hNET, hNET protein expression, EGFP fluorescence, and cellular uptake of 11C-mHED were determined. In rats, U373 tumor xenografts were grown and transiently transduced with either AdTrack-hNET or an AdTrack-Luc control adenovirus. Intratumoral accumulation of 11C-mHED was determined by PET and ex vivo biodistribution. The tumors were subsequently examined for EGFP fluorescence.


RESULTS
11C-mHED uptake was positively correlated with AdTrack-hNET viral titer and hNET protein expression. However, large differences in transfection efficiency between cell lines were observed. The highest 11C-mHED uptake was found in hNET transfected U373 cells, in which tracer uptake was >70-fold higher than that in control cells. 11C-mHED accumulation could be inhibited by desipramine, a potent inhibitor of hNET. In all cell lines, 11C-mHED uptake was positively correlated with EGFP fluorescence, implying that imaging of hNET with 11C-mHED would enable monitoring of a coexpressed therapeutic gene. In the animal model, gene transfection efficiencies were very low, as determined by EGFP fluorescence. Still, a significantly higher 11C-mHED uptake in hNET transduced tumors than that in control tumors was demonstrated by ex vivo biodistribution studies. PET with a clinical camera could visualize 1 of 3 hNET transduced tumors, indicating that the transfection efficiency was near the detection limit.


CONCLUSIONS
These results indicate that monitoring of gene therapy using the hNET/11C-mHED reporter gene/probe is feasible, but further investigation with regard to the sensitivity of the technique is required.
Buursma
Anne Rixt
AR
Department of Nuclear Medicine and Molecular Imaging, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands.
Beerens
Antoine M J
AM
de Vries
Erik F J
EF
van Waarde
Aren
A
Rots
Marianne G
MG
Hospers
Geke A P
GA
Vaalburg
Willem
W
Haisma
Hidde J
HJ
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D019556:COS Cells; D002250:Carbon Radioisotopes / Q000494:pharmacology*; D002460:Cell Line; D045744:Cell Line, Tumor; D002522:Chlorocebus aethiops; D003287:Contrast Media / Q000494:pharmacology*; D003891:Desipramine / Q000494:pharmacology; D004809:Ephedrine / Q000031:analogs & derivatives* / Q000494:pharmacology; D017930:Genes, Reporter; D015316:Genetic Therapy / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D008956:Models, Chemical; D009368:Neoplasm Transplantation; D050484:Norepinephrine Plasma Membrane Transport Proteins / Q000235:genetics* / Q000378:metabolism; D049268:Positron-Emission Tomography / Q000379:methods*; D051381:Rats; D012680:Sensitivity and Specificity; D013997:Time Factors; D014162:Transfection
D002250:Carbon Radioisotopes; D003287:Contrast Media; D050484:Norepinephrine Plasma Membrane Transport Proteins; D049452:Green Fluorescent Proteins; C065363:3-hydroxyephedrine; D004809:Ephedrine; D003891:Desipramine