Micro-positron emission tomography imaging of cardiac gene expression in rats using bicistronic adenoviral vector-mediated gene delivery.
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).


RESULTS
Rat H9c2 cardiomyoblasts transduced with increasing titers of Ad-CMV-D2R80a-IRES-HSV1-sr39tk (0 to 2.5x10(8) 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 2x10(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-[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.
Chen
Ian Y
IY
Crump Institute for Molecular Imaging, UCLA School of Medicine, Los Angeles, Calif 94305-5427, USA.
Wu
Joseph C
JC
Min
Jung-Jun
JJ
Sundaresan
Gobalakrishnan
G
Lewis
Xiaoman
X
Liang
Qianwa
Q
Herschman
Harvey R
HR
Gambhir
Sanjiv S
SS
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D002478:Cells, Cultured / Q000378:metabolism; D003673:Defective Viruses / Q000235:genetics; D005462:Fluorine Radioisotopes; D015967:Gene Expression Regulation, Viral; D017930:Genes, Reporter; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000008:administration & dosage / Q000235:genetics*; D006147:Guanine / Q000031:analogs & derivatives*; D006321:Heart / Q000000981:diagnostic imaging*; D018259:Herpesvirus 1, Human / Q000235:genetics; D007267:Injections; D009206:Myocardium / Q000378:metabolism; D032383:Myocytes, Cardiac / Q000378:metabolism; D011877:Radionuclide Imaging; D019275:Radiopharmaceuticals; D051381:Rats; D011923:Rats, Nude; D017207:Rats, Sprague-Dawley; D017448:Receptors, Dopamine D2 / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D013134:Spiperone / Q000031:analogs & derivatives*; D013937:Thymidine Kinase / Q000235:genetics; D014161:Transduction, Genetic
C113308:9-(4-fluoro-3-hydroxymethylbutyl)guanine; D005462:Fluorine Radioisotopes; D019275:Radiopharmaceuticals; D017448:Receptors, Dopamine D2; D011993:Recombinant Fusion Proteins; C051175:3-N-(2-fluoroethyl)spiperone; D013134:Spiperone; D006147:Guanine; D013937:Thymidine Kinase