Neural stem cell-mediated enhanced gene therapy of malignant gliomas using a novel plant thymidine kinase

Sammanfattning: Malignant gliomas are the most common brain tumors in adults. Approximately, 190,000 new patients are diagnosed with malignant gliomas worldwide every year. Unfortunately, the prognosis of malignant gliomas still remains very poor, despite advances in management. One of the major complicating factors in managing malignant gliomas is their invasiveness, which leads to formation of multiple tumor satellites and thereby inevitable recurrence. Therefore, it is imperative to develop new treatment modalities which effectively address this issue. Suicide gene therapy strategies have been applied against gliomas, but their success have been limited due to inefficient gene transfer technologies, inappropriate enzymatic activities, limited prodrug penetration into the brain, or low bystander effects. The studies presented in this thesis identify a novel suicide gene which can circumvent gene and prodrug related limitations, utilize neural stem cells as gene delivery vehicle and use the histone deacetylase inhibitor 4-phenylbutyrate (4-PB) as modulators of the gene therapy. First, using the herpes simplex virus 1 thymidine kinase/ ganciclovir (HSV-tk/GCV) gene therapy approach, we investigated 4-PB for its effect on HSV-tk gene expression and the bystander effect in glioma cell cultures. The bystander effect is to a great extent mediated by gap junction communication (GJC). Gap junctions are intercellular channels that permit small water soluble molecules to be exchanged between cells. Each cell contributes half of the channel composed of connexin proteins (Cxs). Cx43 is shown to be involved in facilitating the bystander effect. We found that 4-PB upregulated the Cx43 level, amplified the GJC, as well as potentiated the recombinant HSV-tk gene expression in rat glioma cells stably expressing HSV-tk. As a result, a dramatic enhancement of bystander effect by administration of the HSV-tk prodrug GCV together with 4-PB was found. Neural stem cells show extensive tropism for brain tumors and could be exploited to carry and deliver suicide genes. We characterized the human embryonic midbrain stem cell line NGC-407 and found that these cells possessed migratory capacity toward intracranial glioblastoma xenografts. Interestingly, 4-PB was also found to enhance functional GJC between NGC-407 cells and human glioma cells. This finding was supported by upregulation, increased membranous deposition, as well as decreased phosphorylation of Cx43 in NGC-407 cells. 4-PB effect on the differentiation of NGC-407 cells is also needed to be considered, as epigenetic mechanisms are known to play a pivotal role in embryonic development. We found that 4-PB inhibited both astrocytic and neuronal differentiation of NGC-407 cells and preserved their immature phenotype. How the enhancement of GJC together with the differentiation inhibitory effect of 4-PB in NGC-407 cells influence the stem cell-mediated suicide gene therapy in vivo remains to be investigated. Finally and most importantly, we present here, the tomato thymidine kinase 1 (ToTK1) from its identification and characterization to its in vitro and in vivo testing as a suicide gene. Deposited GenBank sequences were searched for human thymidine kinase 1 homologs in plants and ToTK1 was identified. ToTK1 was found specific for the nucleoside analog azidothymidine (AZT), known to successfully pass the blood-brain barrier. ToTK1 efficiently phosphorylated AZT and surprisingly also phosphorylated AZT monophosphate into AZT diphosphate, an additional rate limiting step for AZT activation. The kinetic analysis and in vitro testing in human glioma cells indicated that ToTK1/AZT combination is superior to HSV-tk/GCV approach as a suicide gene therapy strategy. When NGC-407 cells expressing ToTK1 were implanted into nude rat brain along with U87MG human glioblastoma cells, the glioblastoma xenograft growth was substantially attenuated upon exposure to AZT as indicated by magnetic resonance imaging and autopsy analysis. Significantly improved survival was also observed for the treated animals, showing that the ToTK1/AZT suicide gene prodrug combination with stem cell-mediated gene delivery represents a promising new treatment of malignant gliomas.