Abstract
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<0.01). An increase in plasmid concentration also showed an increase in PKM2 mRNA expression by qPCR, where 0.625, 1.5 and 2.5 μg/mL gave 455 ± 21, 793 ± 347 and 1953 ± 167 relative normalized expression (R2=0.87, P<0.01). When PKM2 mRNA expression was plotted against radiotracer uptake, a good correlation (R2=0.69, P<0.05) was observed (Fig. 1). In vivo PET imaging was carried out at 4-time points post infection, 2, 15, 35 and 57 days. At 2 days, there was no increase in signal when compared to control at 30 min post injection. At 15 days, a small increase in signal was visible in some of the infected mice (0.12 %ID/g, n=3). At 35 days a clear increase in uptake of [18F]DASA-23 was observed in the majority of mice (0.63 %ID/g, n=5). At 57 days, the signal was further clearly defined with a 1.14 %ID/g (P<0.001) increase in infected over controls, infected region 2.64 ± 0.15 %ID/g, control region 1.30 ± 0.04 %ID/g and contralateral region 1.22 ± 0.25 %ID/g. Brains sectioned and analyzed by autoradiography and IHC showed distinct accumulation of radiotracer and PKM2 antibody respectively in regions consistent with the PET images. mRNA analysis by qPCR showed an increase in PKM2 expression in all mice which were infected with the PKM2 containing AAV from 0.07-1.7 (n=6) fold change in expression. PKM2 was so low in control animals that no PKM2 mRNA was detected (n=4) by pPCR. Conclusion: Increasing plasmid concentration during transfection showed an increase in PKM2 expression, confirmed by [18F]DASA-23 uptake studies and mRNA analysis. In vivo imaging showed an increase in [18F]DASA-23 uptake in the CNS of infected mice over 2 months. Further analysis of the infected brains by qPCR, autoradiography and IHC showed an increase in PKM2 expression in infected mice when compared to controls. This encouraging data suggests PKM2 has the potential to be further developed into a PET reporter gene system for the imaging of gene therapy in the CNS.
In this issue
Jump to section
Related Articles
Cited By...
- No citing articles found.