Micro-positron emission tomography imaging of cardiac gene expression in rats using bicistronic adenoviral vector-mediated gene delivery.
ABSTRACT 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).
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.
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.
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ABSTRACT: The evolution of positron emission tomography (PET) imaging for small animals has led to the development of dedicated PET scanner designs with high resolution and sensitivity. The animal PET scanner achieves these goals for imaging small animals such as mice and rats. The scanner uses a pixelated Anger-logic detector for discriminating 2 × 2 × 10 mm<sup>3</sup> crystals with 19-mm-diameter photomultiplier tubes. With a 19.7-cm ring diameter, the scanner has an axial length of 11.9 cm and operates exclusively in three-dimensional imaging mode, leading to very high sensitivity. Measurements show that the scanner design achieves a spatial resolution of 1.9 mm at the center of the field-of-view. Initially designed with gadolinium orthosilicate but changed to lutetium-yttrium orthosilicate, the scanner now achieves a sensitivity of 3.6% for a point source at the center of the field-of-view with an energy window of 250-665 keV. Iterative image reconstruction, together with accurate data corrections for scatter, random, and attenuation, are incorporated to achieve high-quality images and quantitative data. These results are demonstrated through our contrast recovery measurements as well as sample animal studies.IEEE Transactions on Medical Imaging 08/2005; · 3.64 Impact Factor
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ABSTRACT: Several methods are available for non-invasive imaging of gene delivery and transgene expression, including magnetic resonance imaging (MRI), single photon emission tomography (SPECT)/positron emission tomography (PET), and fluorescence and bioluminescence imaging. However, these imaging modalities differ greatly in terms of their sensitivity, cost, and ability to measure the signal. Whereas MRI can produce a resolution of approximately 50 mum, optical imaging achieves only 3-5 mm but outperforms MRI in terms of the cost of the imaging device. Similarly, SPECT and PET give a resolution of only 1-2 mm but provide for relatively easy quantitation of the signal and need only nanograms of probe, compared with the microgram or milligram levels required for MRI and optical imaging. To develop safer and more efficient gene delivery vectors, it is essential to perform rigorous in vivo experiments, to image particle biodistribution and transduction patterns, and to quantify the transgene expression profile. Differences between modalities have a significant effect on the resultant imaging resolution for gene therapy. This review describes the methodologies in use and highlights recent key approaches using the latest imaging modalities in gene therapy. Future trends in gene therapy imaging are also discussed.Molecular Therapy 10/2007; 15(9):1579-86. · 6.87 Impact Factor
Article: Positron emission tomography reporter genes and reporter probes: gene and cell therapy applications.[show abstract] [hide abstract]
ABSTRACT: Positron emission tomography (PET) imaging reporter genes (IRGs) and PET reporter probes (PRPs) are amongst the most valuable tools for gene and cell therapy. PET IRGs/PRPs can be used to non-invasively monitor all aspects of the kinetics of therapeutic transgenes and cells in all types of living mammals. This technology is generalizable and can allow long-term kinetics monitoring. In gene therapy, PET IRGs/PRPs can be used for whole-body imaging of therapeutic transgene expression, monitoring variations in the magnitude of transgene expression over time. In cell or cellular gene therapy, PET IRGs/PRPs can be used for whole-body monitoring of therapeutic cell locations, quantity at all locations, survival and proliferation over time and also possibly changes in characteristics or function over time. In this review, we have classified PET IRGs/PRPs into two groups based on the source from which they were derived: human or non-human. This classification addresses the important concern of potential immunogenicity in humans, which is important for expansion of PET IRG imaging in clinical trials. We have then discussed the application of this technology in gene/cell therapy and described its use in these fields, including a summary of using PET IRGs/PRPs in gene and cell therapy clinical trials. This review concludes with a discussion of the future direction of PET IRGs/PRPs and recommends cell and gene therapists collaborate with molecular imaging experts early in their investigations to choose a PET IRG/PRP system suitable for progression into clinical trials.Theranostics. 01/2012; 2(4):374-91.