Development of Advanced Oligonucleotides for Glioblastoma Therapeutics

项目摘要 多形性胶质母细胞瘤（GBM）是成人中最常见和最具侵袭性的原发性脑肿瘤。尽管 在表征GBM的遗传、表观遗传和分子驱动因素方面取得了重大进展， 治疗仍然有限。GBM研究和转化为有效治疗之间存在相当大的障碍 是广泛的浸润和GBM肿瘤的分子异质性，这两者都导致肿瘤复发 治疗后因此，GBM患者的平均生存期预期不到15个月， 诊断.对于有效治疗这些致命肿瘤的疗法，它们必须克服GBM浸润和GBM浸润。 和异质性。 反义寡核苷酸（ASO）-可以调节几乎任何RNA分子表达的化合物 - 为防止GBM渗透和异质性提供了明显的优势。在当地交付后， 分布在整个大脑，这是到达浸润性GBM细胞的必要壮举。此外，作为序列- 作为可编程的试剂，ASO具有调节多种肿瘤细胞表达所需的特异性和灵活性。 基因靶点-一种有效的策略，以表征和打击GBM异质性。2016年，ASO药物， nusinersen被FDA批准用于治疗脊髓性肌萎缩症，确立了ASO在脊髓性肌萎缩症中的临床疗效 中枢神经系统然而，几种用于GBM的ASO候选药物在临床试验中失败，原因是高浓度的抗血小板药物。 毒性和低效力。确定有效的，耐受性良好的ASO用于脑肿瘤的基因调节将打开 开发有效的GBM疗法的大门。 Watts实验室已经开发出化学优化的无毒ASO，具有增强的分布和效力， 局部CNS递送后的脑。然而，它们对GBM的影响尚不清楚。这项提案的目的是 鉴定有效且安全地沉默GBM驱动因子的ASO，并评估对肿瘤进展的影响， 体内抗性。在Jonathan Watts博士（寡核苷酸化学）、Richard Moser博士（神经学）的支持下， 肿瘤学）、Sunit Das（GBM小鼠模型）、Manuel Garber（生物信息学）和Michael绿色（癌症生物学 Aim 1将测试化学修饰的ASO沉默临床相关GBM驱动因子的能力 在分子上不同的患者来源的GBM细胞系中，ATF5抑制细胞增殖并诱导细胞死亡。铅 然后通过测量ATF 5沉默来评价化合物在GBM小鼠模型中的治疗功效， 治疗后的肿瘤生长和小鼠存活率。在目标2中，ASO介导的沉默的后果是： 对GBM肿瘤生物学的影响。将靶向ATF 5的ASO注射到小鼠的GBM肿瘤中。后 治疗反应后，将分离残留的GBM细胞进行单细胞RNA测序，以表征 转录组和确定ASO沉默如何扰乱功能异质性。这一目标将建立一个 药物组合的合理框架，以最大限度地减少GBM肿瘤耐药性。总体而言，拟议项目 将推动ASO作为一种新型GBM治疗方法和一种分析GBM进展的工具。

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.