RESEARCH ARTICLE Non-invasive gamma camera imaging of gene transfer using an adenoviral vector encoding an epitope-tagged receptor as a reporter BE Rogers 1,5,6 , TR Chaudhuri 2 , PN Reynolds 3,4 , D Della Manna 1 and KR Zinn 2,5 1 Departments of Radiation Oncology, University of Alabama at Birmingham, Birmingham, AL, USA; 2 Department of Radiology, University of Alabama at Birmingham, Birmingham, AL, USA; 3 Department of Medicine, University of Alabama at Birmingham, Birmingham, AL, USA; and 4 Division of Human Gene Therapy, University of Alabama at Birmingham, Birmingham, AL, USA A model epitope-tagged receptor was constructed by fusing the hemagglutinin (HA) sequence on the extracellular N- terminus of the human somatostatin receptor subtype 2 (hSSTr2) gene. This construct was placed in an adenoviral (Ad-HAhSSTr2) vector. This study evaluated Ad-HAhSSTr2 in vitro and in vivo using FACS, fluorescent microscopy, radioactive binding assays, and gamma camera imaging techniques. Infection of A-427 non-small cell lung cancer cells with Ad-HAhSSTr2 or Ad-hSSTr2 resulted in similar expression of hSSTr2 by FACS analysis and binding assays using a 99m Tc-labeled somatostatin analogue ( 99m Tc-P2045). HAhSSTr2 expression in A-427 cells was specific for infection with Ad-HAhSSTr2. FITC-labeled anti-HA antibody (FITC-HA) confirmed surface expression in live A-427 cells and the absence of internalization. Gamma camera imaging and gamma counter analysis of normal mice showed significantly greater (Po0.05) liver uptake of 99m Tc-labeled anti-HA antibody ( 99m Tc-anti-HA) in mice injected i.v. 48 h earlier with Ad-HAhSSTr2 (53.6 7 6.9% ID/g) as compared to mice similarly injected with Ad-hSSTr2 (9.0 7 1.3% ID/g). In a mouse tumor model, imaging detected increased tumor localization of 99m Tc-anti-HA due to direct intratumor injection Ad-HAhSSTr2. Gamma counter analysis confirmed signifi- cantly greater (Po0.05) uptake of 99m Tc-anti-HA in tumors injected with Ad-HAhSSTr2 (12.5 7 4.1% ID/g) as compared to Ad-hSSTr2-infected tumors (5.1 7 1.5% ID/g). These studies demonstrate the feasibility of using an epitope- tagged reporter receptor for non-invasively imaging gene transfer. Gene Therapy (2003) 10, 105–114. doi:10.1038/sj.gt.3301853 Keywords: imaging; 99m Tc; gene transfer; hemagglutinin; somatostatin receptor Introduction There are currently more than 2000 patients enrolled in Phase I and Phase II clinical gene therapy trials. 1 A majority of these patients are being treated for neoplastic disease and require tumor and tissue biopsies to confirm gene transfer by assaying for mRNA or protein products. In vivo imaging of gene transfer using reporter genes and reporter substrates should have a tremendous impact on clinical gene therapy trials due to the ability to non- invasively quantitate the level of gene transfer over time. In this regard, our group and several others have utilized optical, magnetic, or nuclear imaging techniques to evaluate gene transfer in animal models. Optical imaging has focused on green fluorescent protein (GFP) and luciferase as reporter genes. 2–7 These techniques offer high spatial resolution, but may be difficult to quantitate and detect depending upon the depth issue in animal models. The transferrin receptor and b-galactosidase have been used as reporter genes for magnetic resonance imaging with iron oxide nanoparti- cles and a gadolinium chelate, respectively. 8,9 Magnetic resonance spectroscopy has been used to detect gene transfer using cytosine deaminase, arginine kinase, and the murine creatine kinase as reporter genes. 10–12 These studies observed the changes in chemical shift of the 19 F or the 31 P isotopes. However, these magnetic strategies have limited temporal resolution and sensitivity. In general, the most common nuclear imaging techni- ques have used either enzymes or receptors as reporter genes. The herpes simplex virus type 1 thymidine kinase (HSV1-tk) has been used extensively as a reporter enzyme with radiolabeled 2 0 -fluoro-2 0 deoxy-1-b-D-arabi- nofuranosyl-5-iodouracil, 8-fluoro-ganciclovir, 8-fluoro- penciclovir, 9-[(3-fluoro-1-hydroxy-2-propoxy)-methyl] guanine or 9-(4-fluoro-3-hydroxymethylbutyl)guanine as reporter substrates for gamma camera or positron- emission tomography (PET) imaging. 13–21 This system has been improved with the use of a mutant HSV1-tk reporter gene (HSV1-sr39tk) that enhances the intracel- lular trapping of reporter substrates. The type 2 dopamine receptor (D 2 R) and the human somatostatin receptor subtype 2 (hSSTr2) have also been used as reporter genes. 3-(2 0 - 18 F-fluoroethylspiperone and 11 C- raclopride have been used to image D 2 R by PET, 18,22,23 while 99m Tc-P829, 188 Re-P829, 99m Tc-P2045, and 111 In- Received 14 December 2001; accepted 27 June 2002 Correspondence: BE Rogers, 1824 6th Ave. South, WTI 674, Birmingham, AL 35294, USA 5 The first and the last authors contributed equally 6 Current Address: Department of Radiation Oncology, Radiation and Cancer Biology Division, 4511 Forest Park Blrd., Suite 411, St. Louis, MO, 43108 Gene Therapy (2003) 10, 105–114 & 2003 Nature Publishing Group All rights reserved 0969-7128/03 $25.00 www.nature.com/gt