Imaging-guided gene therapy of experimental gliomas.

To further develop gene therapy for patients with glioblastomas, an experimental gene therapy protocol was established comprising a series of imaging parameters for (i) noninvasive assessment of viable target tissue followed by (ii) targeted application of herpes simplex virus type 1 (HSV-1) amplicon vectors and (iii) quantification of treatment effects by imaging. We show that viable target tissue amenable for application of gene therapy vectors can be identified by multitracer positron emission tomography (PET) using 2-(18)F-fluoro-2-deoxy-D-glucose, methyl-(11)C-L-methionine, or 3'-deoxy-3'-(18)F-fluoro-L-thymidine ([(18)F]FLT). Targeted application of HSV-1 amplicon vectors containing two therapeutic genes with synergistic antitumor activity (Escherichia coli cytosine deaminase, cd, and mutated HSV-1 thymidine kinase, tk39, fused to green fluorescent protein gene, gfp) leads to an overall response rate of 68%, with 18% complete responses and 50% partial responses. Most importantly, we show that the "tissue dose" of HSV-1 amplicon vector-mediated gene expression can be noninvasively assessed by 9-[4-(18)F-fluoro-3-(hydroxymethyl)butyl]guanine ([(18)F]FHBG) PET. Therapeutic effects could be monitored by PET with significant differences in [(18)F]FLT accumulation in all positive control tumors and 72% in vivo transduced tumors (P = 0.01) as early as 4 days after prodrug therapy. For all stably and in vivo transduced tumors, cdIREStk39gfp gene expression as measured by [(18)F]FHBG-PET correlated with therapeutic efficiency as measured by [(18)F]FLT-PET. These data indicate that imaging-guided vector application with determination of tissue dose of vector-mediated gene expression and correlation to induced therapeutic effect using multimodal imaging is feasible. This strategy will help in the development of safe and efficient gene therapy protocols for clinical application.

[18F]FLT PET for non-invasive monitoring of early response to gene therapy in experimental gliomas.

The purpose of this study was to investigate the potential of 3'-deoxy-3'-[¹8F]fluorothymidine ([¹8F]FLT) positron emission tomography (PET) to detect early treatment responses in gliomas. Human glioma cells were stably transduced with genes yielding therapeutic activity, sorted for different levels of exogenous gene expression, and implanted subcutaneously into nude mice. Multimodality imaging during prodrug therapy included (a) magnetic resonance imaging, (b) PET with 9-(4-[¹8F]fluoro-3-hydroxymethylbutyl)guanine assessing exogenous gene expression, and (c) repeat [¹8F]FLT PET assessing antiproliferative therapeutic response. All stably transduced gliomas responded to therapy with significant reduction in tumor volume and [¹8F]FLT accumulation within 3 days after initiation of therapy. The change in [¹8F]FLT uptake before and after treatment correlated to volumetrically calculated growth rates. Therapeutic efficacy as monitored by [¹8F]FLT PET correlated to levels of therapeutic gene expression measured in vivo. Thus, [¹8F]FLT PET assesses early antiproliferative effects, making it a promising radiotracer for the development of novel treatments for glioma.

Multitracer positron emission tomographic imaging of exogenous gene expression mediated by a universal herpes simplex virus 1 amplicon vector.

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-d2r80AIREStk39gfp (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.