The molecular imaging of herpes simplex virus type-1 thymidine kinase reporter gene expression: Novel fluorine-18-labeled reporter probes

Gene therapy promises to be a new therapeutic avenue for a variety of diseases. However, one of the major limitations to this technology is the difficulty in achieving controlled and efficient delivery of therapeutic genes in vivo. Molecular imaging of reporter genes is a powerful method to monitor and evaluate the location, magnitude, and duration of gene expression following somatic DNA delivery in vivo. The herpes simplex virus type-1 thymidine kinase (HSV1-tk) gene, has evolved as the most investigated reporter gene for the repetitive and non-invasive in vivo imaging of therapeutic gene expression. At present, there remains a need for HSV1-TK enzyme reporter probes for accurately quantifying dynamic HSV1-tk reporter gene expression levels following gene delivery in vivo to correlate with the effectiveness of gene therapy. It is our hypothesis that enhanced HSV1-TK reporter probe efficacy may be attained by rational selection of chemical structures based on successful antiherpes agents that are selectively phosphorylated and trapped by HSV1-TK. Hence, the objective of this dissertation work was to design, synthesize, and biologically evaluate targeted fluorine-18 (18F)-labeled HSV1-TK reporter probes that can be detected by Positron Emission Tomography (PET). Towards this goal, we have developed new synthetic strategies to afford two series of nonradioactive and radioactive thymidine (pyrimidine) nucleoside analogs with (i) [19/18F]fluoroalkyl chains (three, four, or five carbon-unit length) introduced at the C-5 position of the nucleobase and (ii) a C-2\u27 hydrogen or fluorine in the arabino or up position of the ribose sugar. These compounds have never been synthesized nor have they been explored as PET imaging agents for assessing HSV1-tk gene expression in vivo. In vitro evaluations of the 5-[18F]fluoroalkyl nucleosides in a model cellular system demonstrated their sensitivity and selectivity for HSV1-tk expressing cells. Subsequent in vivo validation of these novel probes was done in tumor-bearing nude mice. Biodistribution studies revealed selective uptake of only two of six tracers into HSV1-tk expressing tumors compared to control tumors at 2 h post-injection. Although small animal PET imaging studies with the two candidates could visualize the HSV1-tk positive tumors (tumor to nontarget ratio 1.2-3), accumulation (sensitivity) was not very high as compared to surrounding background tissue, and there was consistent tumor washout over time. Potentially the very fast renal clearance of the radiotracers limits tracer availability to the target site. Studies thus far demonstrate the limited potential of these 5-[18F]fluoroalkyl nucleosides in evaluating HSV1-tk gene expression in vivo. Further studies in animal models and detailed metabolic studies are warranted to understand the contrasting in vitro and in vivo results with regards to sensitivity and selectivity