A Novel Positron Emission Tomography Reporter Gene/Reporter Probe For The Central Nervous System

78 Objectives: Gene therapy is limited by a lack of appropriate imaging techniques that can successfully monitor delivery/expression of the therapy. We set out to develop a novel positron emission tomography (PET) reporter gene system for monitoring gene therapy specifically within the central nervous system (CNS). In this study, we have over-expressed an isoform of pyruvate kinase (PKM2) in the CNS of mice, delivered by an associated-adeno virus (AAV9). The expression of PKM2 was imaged using [18F]DASA-23 PET. PKM2 is particularly suited as a reporter gene in the CNS as it has very low endogenous expression in the healthy brain and its corresponding reporter probe ([18F]DASA-23) has been shown to freely cross the blood brain barrier (BBB) allowing imaging in the CNS. Methods: The utility of PKM2 as a PET reporter gene was first validated in cell culture by overexpressing PKM2 in HeLa cells by transfecting with varying levels of a PKM2 containing plasmid. Following transfection, analysis by [18F]DASA-23 uptake and qPCR was performed. Moving to in vivo studies, mice were infected with AAV9 containing the PKM2 gene via stereotactic injection into one side of the brain. Dynamic small animal PET/CT imaging of [18F]DASA-23 was serially carried out over a period of 2 months to observe the increase in PKM2 expression over time. After 2 months, the mouse brains were excised for analysis of PKM2 expression, including PKM2 mRNA levels determined by qPCR, autoradiography, and immunohistochemistry (IHC) on adjacent sections of excised brains. Results: Cell culture studies showed that increasing the concentration of PKM2 containing plasmid resulted in an increase in [18F]DASA-23 uptake where plasmid concentrations of 0.625, 1.5 and 2.5 μg/mL gave 145 ± 9, 201 ± 12 and 302 ± 31 %uptake/mg protein respectively (R2=0.92, P