Despite significant research and clinical efforts, survival of patients with most common and aggressive primary brain tumor glioblastoma (GBM) has only marginally changed over the past 25 years and is only around fifteen months. There is, therefore, an urgent need for new molecular targets, concepts and approaches to treating GBM. In the recent years, microRNA (miRNA) emerged as a new and promising class of targetable molecules that holds great potential in oncology. Our work on oncogenic miRNAs led us to focus on miR-10b, a unique miRNA highly expressed in all GBM subtypes but absent in normal neuroglial cells of the brain. miR-10b controls the growth of malignant gliomas;; moreover, it appears essential for viability of heterogeneous glioma cells and glioma-initiating stem cells (GSC). Inhibition or loss of miR-10b is, therefore, lethal for glioma. Our preliminary data suggests that, among several targeting approaches, CRISPR-Cas9- based miR-10b gene ablation eradicates established intracranial GBM in mice more effectively. Importantly, despite the fundamental addictive role of miR-10b in GBM, and the potential of its targeting, molecular mechanisms underlying miR-10b activity are poorly understood and largely unconventional. The overall goal of this proposal is, therefore, two-fold: first, to investigate molecular mechanisms underlying GBM dependence on miR-10b, and second, develop efficient therapeutic strategies based on miR-10b gene editing. Elucidation of the signaling pathways regulated by miR-10b, and development of approaches for its therapeutic targeting will have an impact on both basic and clinical neuro-oncology, and cancer more generally. The work for Specific Aim 1 will characterize endogenous miR-10b interactome and identify principal miR-10b targets in glioma and glioma stem cells. Using a combination of functional assays, CLEAR- CLIP and CRISPR-Cas9-based technologies, and quantitative proteomics, we will define physiologically important protein-coding and non-coding targets underlying GBM addiction to miR-10b. Specific Aim 2 will investigate the effects of CRISPR-Cas9-based miR-10b gene editing on growth and invasion of orthotopic glioma. Using human GSC-derived xenografts and immunocompetent GL261 orthotopic GBM mouse models, as well as primary normal neuroglial cells, we will examine efficacy and safety of various engineered CRISPR-Cas9 systems and viral vehicles for targeted miR-10b ablation in GBM. We will further compare off- target effects of antisense oligonucleotide based and gene editing based targeting strategies. The proposed work promises to yield significant new insights into the biology of malignant gliomas and...