title: The human norepinephrine transporter in combination with 11C-m-hydroxyephedrine as a reporter gene/reporter probe for PET of gene therapy.
abstract: 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.
authors.affiliation: Department of Nuclear Medicine and Molecular Imaging, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands.
authors.forename: Anne Rixt
authors.initials: AR
authors.lastname: Buursma
authors.forename: Antoine M J
authors.initials: AM
authors.lastname: Beerens
authors.forename: Erik F J
authors.initials: EF
authors.lastname: de Vries
authors.forename: Aren
authors.initials: A
authors.lastname: van Waarde
authors.forename: Marianne G
authors.initials: MG
authors.lastname: Rots
authors.forename: Geke A P
authors.initials: GA
authors.lastname: Hospers
authors.forename: Willem
authors.initials: W
authors.lastname: Vaalburg
authors.forename: Hidde J
authors.initials: HJ
authors.lastname: Haisma
mesh_terms: 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
chemical_list: D002250:Carbon Radioisotopes; D003287:Contrast Media; D050484:Norepinephrine Plasma Membrane Transport Proteins; D049452:Green Fluorescent Proteins; C065363:3-hydroxyephedrine; D004809:Ephedrine; D003891:Desipramine