Micro–Positron Emission Tomography Imaging of Cardiac Gene Expression in Rats Using Bicistronic Adenoviral Vector-Mediated Gene Delivery

Abstract

Background— We have previously validated the use of micro-positron emission tomography (microPET) for monitoring the expression of a single PET reporter gene in rat myocardium. We now report the use of a bicistronic adenoviral vector (Ad-CMV-D2R80a-IRES-HSV1-sr39tk) for linking the expression of 2 PET reporter genes, a mutant rat dopamine type 2 receptor (D2R80a) and a mutant herpes simplex virus type 1 thymidine kinase (HSV1-sr39tk), with the aid of an internal ribosomal entry site (IRES). Methods and Results— Rat H9c2 cardiomyoblasts transduced with increasing titers of Ad-CMV-D2R80a-IRES-HSV1-sr39tk (0 to 2.5×10 8 pfu) were assayed 48 hours later for reporter protein activities, which were found to correlate well with viral titer ( r 2 =0.96, P <0.001 for D2R80A; r 2 =0.98, P <0.001 for HSV1-sr39TK) and each other ( r 2 =0.97; P <0.001). Experimental (n=8) and control (n=6) athymic rats underwent intramyocardial injection of up to 2×10 9 pfu of Ad-CMV-D2R80a-IRES-HSV1-sr39tk and saline, respectively. Forty-eight hours later and weekly thereafter, rats were assessed for D2R80a-dependent myocardial accumulation of 3-(2-[ 18 F]fluoroethyl)spiperone ([ 18 F]-FESP) and HSV1-sr39tk–dependent sequestration of 9-(4-[ 18 F]fluoro-3-hydroxymethylbutyl)guanine ([ 18 F]-FHBG) using microPET. Longitudinal [ 18 F]-FESP and [ 18 F]-FHBG imaging of experimental rats revealed a good correlation between the cardiac expressions of the 2 PET reporter genes ( r 2 =0.73; P <0.001). The location of adenovirus-mediated transgene expression, as inferred from microPET images, was confirmed by ex vivo gamma counting of explanted heart. Conclusions— The IRES-based bicistronic adenoviral vector can potentially be used in conjunction with PET for indirect imaging of therapeutic gene expression by replacing 1 of the 2 PET reporter genes with a therapeutic gene of choice.