| dc.description.abstract | Glioblastoma is a highly malignant, aggressive and most common primary brain tumor in adults. Tremendous research efforts have been conducted in the field of GBM treatment discovery and succcess has only been achieved at pre-clinical level. Despite numerous approaches used in the treatment of GBM, patients still experience tumor progression, show extremely poor prognosis, high mortality and barely 12-15 months of median survival. To a certain extent, the failure of current treatment modalities to provide durable responses is due to tumor heterogeneity, a hostile tumor microenvironment and the tumor’s unique ability to evade the host’s immune system. Suicide gene therapy strategies are among the ongoing clinical studies for the treatment of GBM. Previous work from our lab shows that treatment of CT2A and GL261 mouse glioma cell lines with HSV-TK GCV SGT induced the apoptotic mechanism of cell death. Apoptosis is known to be non-immunogenic, hence it does not stimulate antitumor immune responses. Therefore, in this study we aimed to block the apoptotic pathway and study potential activation of alternative cell death pathways following HSV-TK/GCV induced SGT, which could be more immunogenic. To achieve our aims, we blocked the apoptotic pathway by knocking out two crucial genes in the apoptotic pathway (gene x and y; due to patenting conflicts, the names are not revealed) by CRISPR/Cas9 technology. Thereafter, investigations were conducted to study potential activation of lytic mode(s) of cell death, which are known to be more immunogenic. Our results demonstrate that CRISPR/Cas9 effectively knocks out our genes of interest. Due to limited time, further studies could not be conducted using gene y KO cells. Further experiments conducted using gene x KO cells reveal effective inhibition of the apoptotic pathway with delay in cell death in both cell models. We found that apoptosis was not the dominant mode of cell death in both KO cell lines and, no enhanced lytic cell death was observed in both KO cell lines. However, in GL261 KO cells, our findings suggested possible activation of another mode of cell death which needs to be categorized in the future. We recommend further investigations to be conducted to understand mechanisms involved in this mode of cell death | |
