Non-invasive imaging of cell type-specific gene activation using HSV-tk as positron emission tomography reporter gene in combination with the SLC43A3 nucleobase transporter

• Applicants: Dr. Heiko Backes; Professor Dr. Bernd Neumaier
• Subject Area: Medical Physics, Biomedical Technology ; Molecular Biology and Physiology of Neurons and Glial Cells
• Term: from 2019 to 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 433048408

Expression of genes leads to the biosynthesis of proteins that fulfil crucial functions, such as transport of molecules through cellular membranes. Gene expression depends on internal and external factors and varies in time. Expression of a specific gene within a living cell can be assessed with the help of reporter genes. For this purpose the reporter gene is inserted into the DNA at a position that is controlled by the same promoter as the target gene, of which the expression is to be measured. Translation of the target gene is then accompanied by translation of the reporter gene. In our project we will use a gene reporter system that allows for the assessment of gene expression with positron emission tomography (PET). The reporter gene consists of the Herpes Simplex Virus-1 Thymidine Kinase (HSV-tk) gene. The enzyme HSV-tk phosphorylates the PET tracer molecule [18F]Fluoro-3-hydroxymethylbutylguanine ([18F][FHBG) and leads to accumulation of [18F]FHBG in HSV-tk positive cells. Thereby, target gene expression can be measured non-invasively and quantitatively using PET. In vivo monitoring of HSV-tk expression with [18F]FHBG is limited by the inability of [18F]FHBG to enter certain cell types. [18F]FHBG, for instance, fails to cross the intact blood brain barrier. Recently, a group from Japan discovered a gene, SLC43A3, that is capable of selectively transporting nucleobases, such as [18F]FHBG, through cellular membranes. Based on these results we hypothesize that the limitations can be solved when HSV-tk is combined with the nucleobase transporter SLC43A3. To this end, we have generated a mouse line that expresses the reporter gene HSV-tk under control of the cfos promoter and additionally expresses ubiquitously the nucleobase transporter SLC43A3. cfos belongs to the class of immediate early genes and is the most prominent marker for cellular activation. [18F]FHBG, applied to the awake mouse, accumulates while the mouse performs a behavioural task. The sub-sequent PET measurement will show task-dependent cfos expression in the living animal and thereby introduce the first PET gene reporter system that works in the central nervous system. Furthermore, by crossing with the DAT-Cre and the AgRP-Cre mouse line, we will generate mouse lines with HSV-tk expression under the control of the cfos promoter exclusively in dopaminergic cells or AgRP cells, respectively. In these mice [18F]FHBG accumulation shows task-dependent cfos expression exclusively in dopaminergic cells or AgRP cells, respectively. Thereby we will introduce the first cell type-specific PET gene reporter system.

• DFG Programme: Research Grants
• Co-Investigator: Professor Dr. Frank Thomas Wunderlich