Development of Advanced Oligonucleotides for Glioblastoma Therapeutics

项目总结 多形性胶质母细胞瘤(GBM)是成人最常见、最具侵袭性的原发脑肿瘤。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，有效 治疗方法仍然有限。GBM研究和转化为有效治疗之间的相当大障碍 基底膜肿瘤的广泛浸润性和分子异质性，两者都会导致肿瘤复发。 治疗后。因此，GBM患者的平均预期寿命不到15个月 诊断。为了有效地治疗这些致命的肿瘤，它们必须克服两种基底膜的浸润性。 和异质性。 反义寡核苷酸(ASO)--几乎可以调节任何RNA分子表达的化合物 -为抗击GBM渗透和异质性提供明显优势。在本地交付后，ASOS 分布在整个大脑中，这是到达浸润性GBM细胞的必要壮举。此外，作为序列- 可编程试剂，ASO具有调节多种基因表达所需的特异性和灵活性 基因靶点--表征和对抗GBM异质性的有效策略。2016年，ASO药物， Nusinesen，被FDA批准用于治疗脊髓性肌萎缩症，确立了ASOS在 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 毒性和低效性。发现有效的、耐受性良好的ASO用于脑瘤的基因调节将是开放的 开发有效的GBM疗法的大门。 美国瓦茨实验室已经开发出经过化学优化的无毒ASO，其分布和效力在 局部中枢神经系统传递后的大脑。然而，它们对GBM的影响尚不清楚。这项提议的目标是 确定有效且安全地使GBM驱动程序沉默的ASO，并评估其对肿瘤进展和 体内抗药性。在乔纳森·瓦茨博士(寡核苷酸化学)的支持下，理查德·莫泽(Neuro- 肿瘤学)、Sunit Das(GBM小鼠模型)、Manuel Garber(生物信息学)和Michael Green(癌症生物学 和治疗学)，Aim 1将测试化学修饰的ASO使临床相关的GBM司机沉默的能力 (ATF5)，抑制细胞增殖，并在分子上不同的患者来源的GBM细胞系中诱导细胞死亡。铅 然后将通过测量ATF5沉默来评估化合物在GBM小鼠模型中的治疗效果， 肿瘤生长和治疗后的小鼠存活。在目标2中，ASO介导的沉默的后果 对基底膜的肿瘤生物学进行研究。靶向ATF5的ASO将被注射到小鼠的GBM肿瘤中。之后 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 转录组学和确定ASO沉默如何扰乱功能异质性。这一目标将建立一个 合理的药物组合框架，以最大限度地减少对GBM肿瘤的耐药性。总的来说，拟议的项目 将ASOS作为一种新的GBM治疗方法和作为解剖GBM进展的工具来推进。

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.