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(Circulation. 2004;109:1415-1420.)
© 2004 American Heart Association, Inc.

Basic Science Reports

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

Ian Y. Chen, MSE; Joseph C. Wu, MD; Jung-Jun Min, MD; Gobalakrishnan Sundaresan, PhD; Xiaoman Lewis, MD; Qianwa Liang, PhD; Harvey R. Herschman, PhD; Sanjiv S. Gambhir, MD, PhD

From the Crump Institute for Molecular Imaging (I.Y.C., J.C.W., J.M., G.S., X.L., H.R.H., S.S.G.), Biomedical Physics Interdepartmental Graduate Program (I.Y.C.), Department of Molecular and Medical Pharmacology (J.C.W., J.M., G.S., X.L., H.R.H., S.S.G.), Department of Biochemistry/Molecular Biology Institute (Q.L., H.R.H.), and Department of Medicine, Division of Cardiology (J.C.W.), UCLA School of Medicine, Los Angeles, Calif.

Correspondence to Sanjiv S. Gambhir, MD, PhD, Stanford University, The James H. Clark Center, 318 Campus Dr, East Wing, First Floor, Stanford, CA 94305-5427. E-mail sgambhir{at}stanford.edu

Received July 11, 2003; de novo received October 2, 2003; accepted November 25, 2003.

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 2.5x10⁸ pfu) were assayed 48 hours later for reporter protein activities, which were found to correlate well with viral titer (r²=0.96, P<0.001 for D2R80A; r²=0.98, P<0.001 for HSV1-sr39TK) and each other (r²=0.97; P<0.001). Experimental (n=8) and control (n=6) athymic rats underwent intramyocardial injection of up to 2x10⁹ 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-[¹⁸F]fluoroethyl)spiperone ([¹⁸F]-FESP) and HSV1-sr39tk–dependent sequestration of 9-(4-[¹⁸F]fluoro-3-hydroxymethylbutyl)guanine ([¹⁸F]-FHBG) using microPET. Longitudinal [¹⁸F]-FESP and [¹⁸F]-FHBG imaging of experimental rats revealed a good correlation between the cardiac expressions of the 2 PET reporter genes (r²=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.

Key Words: gene therapy • imaging • nuclear medicine • myocardium

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