Development of Advanced Oligonucleotides for Glioblastoma Therapeutics

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PROJECT SUMMARY Glioblastoma multiforme (GBM) is the most frequent and aggressive primary brain tumor in adults. Despite significant progress being made in characterizing the genetic, epigenetic, and molecular drivers of GBM, effective therapies remain limited. A considerable hurdle between GBM research and translation into efficacious treatment is the extensive infiltration and molecular heterogeneity of GBM tumors, both of which cause tumor recurrence after treatment. Consequently, the average survival expectancy for GBM patients is less than 15 months after diagnosis. For therapies to be effective in treating these lethal tumors, they must overcome both GBM infiltration and heterogeneity. Antisense oligonucleotides (ASOs) – compounds that can modulate the expression of virtually any RNA molecule – offer distinct advantages for combating GBM infiltration and heterogeneity. Following local delivery, ASOs distribute throughout the brain, a necessary feat to reach infiltrative GBM cells. Moreover, as sequence- programmable agents, ASOs possess the specificity and flexibility required to modulate expression of multiple gene targets – an effective strategy to characterize and combat GBM heterogeneity. In 2016, the ASO drug, nusinersen, was FDA approved to treat spinal muscular atrophy, establishing the clinical efficacy of ASOs in the central nervous system. However, several ASO drug candidates for GBM have failed in clinical trials due to high toxicity and low potency. Identifying potent, well-tolerated ASOs for gene modulation in brain tumors would open the door to developing effective GBM therapies. The Watts lab has developed chemically-optimized, non-toxic ASOs with enhanced distribution and potency in the brain following local CNS delivery. However, their effect on GBM is unknown. The goal of this proposal is to identify ASOs that potently and safely silence GBM drivers, and assess the impact on tumor progression and resistance in vivo. With support from Drs. Jonathan Watts (oligonucleotide chemistry), Richard Moser (neuro- oncology), Sunit Das (GBM mouse models), Manuel Garber (bioinformatics), and Michael Green (cancer biology & therapeutics), Aim 1 will test the ability of chemically-modified ASOs to silence a clinically-relevant GBM driver (ATF5), inhibit cell proliferation, and induce cell death in molecularly-distinct patient-derived GBM cell lines. Lead compounds will then be evaluated for therapeutic efficacy in a GBM mouse model by measuring ATF5 silencing, tumor growth, and mouse survival following treatment. In Aim 2, the consequences of ASO-mediated silencing on GBM tumor biology will be investigated. ASOs targeting ATF5 will be injected into GBM tumors of mice. After treatment response, residual GBM cells will be isolated for single-cell RNA sequencing to characterize the transcriptome and determine how ASO silencing perturbs functional heterogeneity. This aim will establish a rational framework for drug combinations to minimize GBM tumor resistance. Collectively, the proposed project will advance ASOs as a novel GBM therapeutic and as a tool to dissect GBM progression.