PET of cardiac transgene expression: Comparison of 2 approaches based on herpesviral thymidine kinase reporter gene
Nuklearmedizinische Klinik und Poliklinik, Technische Universität München, 81675 Munich, Germany. Journal of Nuclear Medicine
(Impact Factor: 6.16).
PET of reporter gene expression holds promise for noninvasive monitoring of gene therapy. Previously, 2 approaches based on the herpes simplex virus type 1 thymidine kinase gene (HSV1-tk) have been successfully applied to the heart. Wild-type HSV1-tk was imaged with (124)I-labeled 2'-fluoro-2'-deoxy-5-iodo-1-beta-D-arabinofuranosyl-5-iodouracil (FIAU), and a mutant HSV1-tk (HSV1-sr39tk) was imaged with (18)F-labeled 9-[4-fluoro-3-(hydroxymethyl)butyl]guanine (FHBG). The aim of this study was to compare these 2 combinations with regard to specificity, imaging contrast, and reporter probe kinetics using dynamic PET in small and large animals.
Similar titers of adenovirus-expressing wild-type HSV1-tk (Ad(tk)), mutant HSV1-sr39tk (Ad(sr39tk)), or control genes were directly injected into the myocardium of 24 rats and 8 pigs. Two days later, dynamic PET was performed with a clinical scanner during the 120 min after injection of (124)I-FIAU (Ad(tk) animals and controls) or (18)F-FHBG (Ad(sr39tk) animals and controls). Imaging with (13)N-ammonia was performed to identify cardiac regions of interest.
In rats, significant cardiac (124)I-FIAU accumulation occurred in images obtained early (10-30 min) after Ad(tk) injection. Because of tracer washout, however, no difference between Ad(tk)-injected animals and controls was seen in the images obtained later. For (18)F-FHBG, specific myocardial accumulation greater than background levels was detected in Ad(sr39tk)-injected animals at early imaging and, in contrast to (124)I-FIAU accumulation, increased over time until the latest imaging (105-120 min). At maximum, cardiac (18)F-FHBG concentration showed a 4.15 +/- 1.65-fold increase compared with controls (105-120 min), and cardiac (124)I-FIAU concentration reached a maximal increase of 1.34 +/- 0.38-fold compared with controls (10-30 min, P = 0.0014). Global cardiac reporter probe kinetics in rats were confirmed by regional myocardial analysis in pig hearts. Transgene expression was specifically visualized by both approaches. The highest target-to-background ratio of (124)I-FIAU in Ad(tk)-infected pig myocardium was 1.50 +/- 0.20, versus 2.64 +/- 0.49 for (18)F-FHBG in Ad(sr39tk)-infected areas (P = 0.01). In vivo results were confirmed by ex vivo counting and autoradiography.
Both reporter gene/probe combinations were feasible for noninvasive imaging of cardiac transgene expression in different species. Specific probe kinetics suggest different myocardial handling of pyrimidine (FIAU) and acycloguanosine (FHBG) derivatives. The results favor (18)F-FHBG with mutant HSV1-sr39tk because of continuous accumulation over time and higher imaging contrast.
Available from: Frank C Marini
- "This reporter gene imaging approach with PET/CT has been used recently in clinical studies in human patients, including imaging of sr39HSV1-tk-based gene therapy of cancer  and track the localization of sr39HSV1-tk transduced T cells . We ,  and others  have shown the efficacy of PET imaging of HSV1-tk reporter gene–expressing stem cells in preclinical studies –, including studies of short-term stem cell fate after intramyocardial injection in rodent models of MI –, mesenchymal stem cells (MSCs) implanted in pig hearts , , and adenoviral-mediated HSV1-tk gene expression in pig hearts  demonstrating that this technology can be used in a large animal model. However, long-term (several months), repetitive imaging studies on the spatial and temporal dynamics of stem cells injected into the myocardium, their trafficking and engraftment, and persistence of stem cell–derived tissues have not been reported yet. "
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ABSTRACT: The long-term fate of stem cells after intramyocardial delivery is unknown. We used noninvasive, repetitive PET/CT imaging with [(18)F]FEAU to monitor the long-term (up to 5 months) spatial-temporal dynamics of MSCs retrovirally transduced with the sr39HSV1-tk gene (sr39HSV1-tk-MSC) and implanted intramyocardially in pigs with induced acute myocardial infarction. Repetitive [(18)F]FEAU PET/CT revealed a biphasic pattern of sr39HSV1-tk-MSC dynamics; cell proliferation peaked at 33-35 days after injection, in periinfarct regions and the major cardiac lymphatic vessels and lymph nodes. The sr39HSV1-tk-MSC-associated [(18)F]FEAU signals gradually decreased thereafter. Cardiac lymphography studies using PG-Gd-NIRF813 contrast for MRI and near-infrared fluorescence imaging showed rapid clearance of the contrast from the site of intramyocardial injection through the subepicardial lymphatic network into the lymphatic vessels and periaortic lymph nodes. Immunohistochemical analysis of cardiac tissue obtained at 35 and 150 days demonstrated several types of sr39HSV1-tk expressing cells, including fibro-myoblasts, lymphovascular cells, and microvascular and arterial endothelium. In summary, this study demonstrated the feasibility and sensitivity of [(18)F]FEAU PET/CT imaging for long-term, in-vivo monitoring (up to 5 months) of the fate of intramyocardially injected sr39HSV1-tk-MSC cells. Intramyocardially transplanted MSCs appear to integrate into the lymphatic endothelium and may help improve myocardial lymphatic system function after MI.
Available from: sciencedirect.com
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ABSTRACT: Genes encoding for intracellular enzymes or transmembrane proteins are suitable as reporters, but may differ in terms of applicability for cardiac imaging. The aim of this study was to compare the human sodium iodide symporter gene (hNIS) with the herpes simplex virus type 1 thymidine kinase gene (HSV1-tk) as the reporter gene in non-invasive imaging of cardiac transgene expression with positron emission tomography (PET).
Equal doses of adenoviral vectors encoding for hNIS, wild-type HSV1-tk, mutant HSV1-sr39tk or LacZ as the control gene were directly injected into the myocardium of 34 animals. Two days later, dynamic PET was performed with a clinical scanner, using reporter probes specific for the respective reporter gene. Imaging with (13)N-ammonia was also performed to identify cardiac regions of interest.
Kinetics differed significantly: (124)I as the probe for hNIS showed rapid early uptake, remaining stable over time. Maximal myocardial concentration was 3.61+/-1.15%. The nucleoside (18)F-FHBG, as the specific probe for HSV1-sr39tk, showed increasing uptake over time, but maximal accumulation was significantly lower (1.45+/-0.54%, P=0.0009). (124)I-FIAU, as the specific probe for wild-type HSV1-tk, showed early uptake with subsequent washout. Maximal accumulation was lowest (0.63+/-0.23%, P<0.0001). Post-mortem analysis by autoradiography and gamma counting confirmed the in vivo data.
Reporter genes encoding for transporter proteins such as hNIS are an attractive alternative to overexpression of intracellular enzymes for cardiac gene product imaging. hNIS yielded higher signal intensity and imaging contrast for PET than did HSV1-tk and HSV1-sr39tk. Therefore, this approach may be preferable for the future monitoring of cardiac gene- or cell-based therapy.
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