Aim of the Study: (i) it should be established a suitable Glioblastoma (GBM) model for seRNA research in mice, (ii) to target GBM cells in vivo selectively without affecting the surrounding healthy brain tissue, and (iii) it should be assessed the therapeutic efficacy of seRNA treatment using multimodal non-invasive imaging with Magnet Resonance Imaging (MRI) and Positron Emission Tomography (PET). Introduction: Glioblastoma (GBM) is the most malignant primary brain tumor in adult humans. While not expressing steady surface markers, GBM cells express intracellular cytokeratin 13 (CK 13), an established tumor marker, which serves as a reliable target used for a newly developed selectively expressed RNA (seRNA). seRNA is a single-stranded RNA molecule that encodes selectable information and consists of an Antisense Sequence (AS; here = CK 13), IRES-Blocker (IB), RNAse-Inhibitor (RI), Internal Ribosomal Entry Sites (IRES) and an Effector Protein (EP; here = Caspase). seRNA binds intracellularly to CK-expressing tumor cells and, due to the special arrangement of its molecular elements, induces programmed cell death in the target tumor cells through selective expression of caspase. Healthy brain cells like neurons do not express CK and therefore seRNA has no impact on intact neuronal tissue. Preceding in vitro experiments had shown promising results. Animals, Materials and Methods: For GBM model establishment, 200,000 U87 cells were injected stereotactically into the striatum of male immunodeficient mice (NMRI-Foxn1nu/nu, n=23). Pain elimination was ensured before and after the procedure. Mice showed reliable tumor development as evaluated through MRI and validated ex vivo by histology. To assess the therapeutic efficacy of seRNA in vivo, GBM-bearing mice were injected with seRNA coupled to caspase 3 (n=10) in the treatment group, or either eGFP or sodium chloride in the control group (n=10). A further series of experiments was carried out with two consecutive injections of seRNA (n=10). MRI was obtained longitudinally at day three, seven and 14 for intraindividual read-outs of treatment effects. In addition, we performed PET imaging in a subset of animals (n=12). Results: Single intratumoral injection of seRNA-caspase resulted in significantly smaller tumor sizes (p = 0.0001, F = 17.45). Examination of individual tumor size developments over the study period allowed dichotomization of animals into responders and non-responders, and showed that mice treated with seRNA-caspase displayed a statistically significant shrinkage of tumor size over the observation period (p = 0.0253). Surprisingly, two consecutive injections of seRNA did not have any therapeutic effect, presumably caused by a technical issue. 18F-FET radiotracer uptake in the PET study was low for both groups and thus non-conclusive. Conclusion: Promising results for single treatment with seRNA-caspase show how essential further investigation of GBM treatment approaches are, as observed right now across the board in cancer research at RNA level. A follow-up study should include repetitive injections of seRNA as well as multi-modal imaging including MRI and PET imaging, in order to achieve translation into a clinical setting in the future. The current bivalent results once again emphasize how fundamental further research is in the highly demanding field of GBM treatment.
