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Vectors derived from herpes simplex virus type 1 (HSV-1) have great potential for transducing therapeutic genes into the central nervous system; however, inefficient distribution of vector particles in vivo may limit their therapeutic potential in patients with gliomas. This study was performed to investigate the extent of HSV-1 amplicon vector–mediated gene expression in a three-dimensional glioma model of multicellular spheroids by imaging highly infectious HSV-1 virions expressing green fluorescent protein (HSV-GFP). After infection or microscopy-guided vector injection of glioma spheroids at various spheroid sizes, injection pressures and injection times, the extent of HSV-1 vector–mediated gene expression was investigated via laser scanning microscopy. Infection of spheroids with HSV-GFP demonstrated a maximal depth of vector-mediated GFP expression at 70 to 80 μm. A > 80% transduction efficiency was reached only in small spheroids with a diameter of < 150 μm. Guided vector injection into the spheroids showed transduction efficiencies ranging between < 10 and > 90%. The results demonstrated that vector-mediated gene expression in glioma spheroids was strongly dependent on the mode of vector application—injection pressure and injection time being the most important parameters. The assessment of these vector application parameters in tissue models will contribute to the development of safe and efficient gene therapy protocols for clinical application.

For clinical application of stem cell–based therapies, noninvasive detection of applied stem cells is of high importance. We report on the feasibility of detecting implanted neural progenitor cells (NPCs) noninvasively and follow their fate and functional status by sequential multimodal molecular imaging and reporter gene technology. We investigated C17.2 cells stably expressing herpes simplex virus type 1–thymidine kinase (HSV-1- tk ) and green fluorescent protein ( gfp ) (C17.2- tk IRES gfp = C17.2-TIG) or HSV-1- tk , gfp , and firefly luciferase ( luc ) (C17.2- luc IRES tkgfp = C17.2-LITG) and determined the detection sensitivity of positron emission tomography (PET) and bioluminescence imaging (BLI) for these cells in culture and in vivo in subcutaneous and intracranial glioma models. In addition, PET and BLI were used to further investigate and follow the fate of implanted C17.2-LITG cells in an intracranial glioma model. We show that both imaging modalities are sensitive in detecting reporter gene expressing NPCs; however, PET, by the use of 9-[4-[ 18 F]fluoro-3-hydroxymethyl)butyl]guanine ([ 18 F]FHBG), detects NPCs only at sites of disrupted blood-brain barrier. Furthermore, both imaging modalities can be used to detect stem cell fate and migration and indicate excessive proliferation and aberrant migration. In conclusion, multimodal imaging can be used for longitudinal noninvasive monitoring of grafted NPCs in rodents.

To develop efficient and safe gene therapy approaches, the herpes simplex virus type 1 thymidine kinase gene (HSV-1- tk ) has been shown to function as a marker gene for the direct noninvasive in vivo localization of thymidine kinase (TK) expression by positron emission tomography (PET) using radiolabeled nucleoside analogues as specific TK substrates. Moreover, the gene encoding dopamine type 2 receptor ( d2r ) could be used as a PET marker gene using specific radiolabeled receptor binding compounds. Here we describe the quantitative colocalization of d2r and HSV-1- tk gene expression mediated from a universal HSV-1 amplicon vector in a subcutaneous human Gli36dEGFR glioma model by PET. The HSV-1 amplicon vector was constructed using a bicistronic gene cassette to contain (1) the d2r80A mutant, which is able to bind its ligand racloprid but unable to activate downstream signal transduction pathways, and (2) the tk39 mutant with enhanced enzymatic activity toward guanosine analogues fused to the green fluorescent protein gene ( tk39gfp ) serving as a marker gene in cell culture. After infection of human Gli36dEGFR glioma cells with the HSV- d2r80A IRES tk39gfp (HSV-DITG) amplicon vector in cell culture, D2 receptor expression and its targeting to the cell surface were determined by Western blotting and immunolabeling. Vector application in vivo served for quantitative colocalization of d2r80A - and tk39gfp -derived PET signals employing the specific D2 receptor binding compound [ 11 C]racloprid and the specific TK39 substrate 9-(4-[ 18 F]fluoro-3-hydroxymethylbutyl)guanine. Our results demonstrate that for the range of gene expression studied in vivo, both enzymatic and receptor binding assays give comparable quantitative information on the level of vector-mediated gene expression in vivo. The d2r80A in combination with a specific binding compound passing the intact blood-brain barrier might be an alternative marker gene for the noninvasive assessment of vector-mediated gene expression in the brain using PET.

Gliomas are the most common types of brain tumors. Although sophisticated regimens of conventional therapies are being carried out to treat patients with gliomas, the disease invariably leads to death over months or years. Before new and potentially more effective treatment strategies, such as gene- and cell-based therapies, can be effectively implemented in the clinical application, certain prerequisites have to be established. First of all, the exact localization, extent, and metabolic activity of the glioma must be determined to identify the biologically active target tissue for a biological treatment regimen; this is usually performed by imaging the expression of up-regulated endogenous genes coding for glucose or amino acid transporters and cellular hexokinase and thymidine kinase genes, respectively. Second, neuronal function and functional changes within the surrounding brain tissue have to be assessed in order to save this tissue from therapy-induced damage. Third, pathognomonic genetic changes leading to disease have to be explored on the molecular level to serve as specific targets for patient-tailored therapies. Last, a concerted noninvasive analysis of both endogenous and exogenous gene expression in animal models as well as the clinical setting is desirable to effectively translate new treatment strategies from experimental into clinical application. All of these issues can be addressed by multimodal radionuclide and magnetic resonance imaging techniques and fall into the exciting and fast growing field of molecular and functional imaging. Noninvasive imaging of endogenous gene expression by means of positron emission tomography (PET) may reveal insight into the molecular basis of pathogenesis and metabolic activity of the glioma and the extent of treatment response. When exogenous genes are introduced to serve for a therapeutic function, PET imaging may reveal the assessment of the “location,” “magnitude,” and “duration” of therapeutic gene expression and its relation to the therapeutic effect. Detailed reviews on molecular imaging have been published from the perspective of radionuclide imaging (Gambhir et al., 2000; Blasberg and Tjuvajev, 2002) as well as magnetic resonance and optical imaging (Weissleder, 2002). The present review focuses on molecular imaging of gliomas with special reference on the status and perspectives of imaging of endogenous and exogenously introduced gene expression in order to develop improved diagnostics and more effective treatment strategies of gliomas and, in that, to eventually improve the grim prognosis of this devastating disease.