Inducing H3F3A exon skipping with antisense oligonucleotides as an approach to treat diffuse intrinsic pontine glioma

项目摘要 儿童高级别胶质瘤（pHGG）占儿童诊断的所有脑肿瘤的10-15%。一种亚型 pHGG的一种，弥漫性内在脑桥胶质瘤（DIPG），尤其致命，5年生存率<1%。 目前的治疗选择是非治愈性的;手术切除，局部放疗和化疗是共同的。 肿瘤的脑干位置和伴随的负面副作用使其复杂化。因此，更有效 迫切需要战略。大约70-80%的DIPG肿瘤以显性杂合子为标志， H3 F3 A中的gous点突变，其编码非经典组蛋白H3.3。这种毒性的功能获得性μ- 用甲硫氨酸（K27 M）取代赖氨酸27，防止赖氨酸27（H3 K27 me 3）的三甲基化。H3K27M 也被证明干扰Polycomb阻遏复合物2（PRC 2），导致二- 和三甲基化。这种突变的H3.3组蛋白被预测为致瘤因子的主要驱动因素。 在H3 K27 M突变的DIPG中通过破坏正常的神经分化来表达。反义寡核苷酸 提供了一种独特的高特异性、低毒性的Watson-Crick碱基配对靶向mRNA的方法。 在最近提交的手稿中，我的实验室开发了一种“gapmer”阿索，它靶向H3 F3 A的RNaseH度。 辐射，诱导神经分化，延长存活时间。为了进一步取得这些成果， 在阿索模式中，我将开发一种剪接阿索，通过诱导突变H3 K27 M RNA的翻译来减少突变H3 K27 M RNA的翻译。 H3 F3 A外显子2的跳跃，其含有H3 F3 A和K27 M突变的唯一框内起始密码子。这 剪接转换模态是重要，因为均匀修饰的ASO与“缺口体”ASO不同， 半衰期和降低中枢神经系统的脱靶倾向，这种化学物质的ASO已经 FDA批准的治疗方法。此外，H3 F3 A及其同源基因H3 F3 B编码相同的H3.3 组蛋白H3 F3 B足以补偿H3 F3 A的损失，并且H3 F3 B应保持不受组蛋白H3 F3 B的影响。 我的阿索策略，因为序列不同。我假设H3 K27 M的减少将限制DIPG 通过促进肿瘤细胞的分化来抑制肿瘤生长并延长存活。我的目标是亲- 确定一种减少H3 K27 M并恢复H3 K27 me 3的导线拼接开关阿索， 证实了RNA结合蛋白RBFOX 3在增强神经分化和H3 F3 A外显子2跳跃中的作用。 阿索注射后ping。实现的方法将包括建立临床前体内小鼠模型， H3.3K27M DIPG异种移植物，通过RT-PCR、蛋白表达和免疫组化研究H3 F3 A和H3 F3 B的剪接变化。 H3 K27 M和H3 K27 me 3通过免疫印迹的锡永，以及神经细胞通过免疫荧光的分化。 注射阿索后出现充血。此外，将使用剪接和分析性RNA-蛋白质结合试验， 表征RBFOX 3与H3 F3 A RNA的作用和结合动力学。所提出的研究是有意义的 因为它将：（i）帮助识别和提供用于药物开发的新的临床候选物的机理解释， opment;和（ii）提供使用ASO治疗神经肿瘤疾病的进一步理由。

PROJECT SUMMARY Pediatric high grade gliomas (pHGGs) represent 10-15% of all brain tumors diagnosed in children. One subtype of pHGGs, diffuse intrinsic pontine glioma (DIPG), is especially deadly, with a five-year survival rate of < 1%. Current treatment options are non-curative; surgical resection, localized radiation, and chemotherapy are com- plicated by the brainstem location of the tumor and accompanying negative side-effects. Thus, more effective strategies are urgently needed. Approximately 70-80% of all DIPG tumors are marked by a dominant heterozy- gous point mutation in H3F3A, which codes for the non-canonical histone H3.3. This toxic gain-of-function mu- tation replaces lysine 27 with methionine (K27M), preventing trimethylation of lysine 27 (H3K27me3). H3K27M has also been shown to interfere with Polycomb repressive complex 2 (PRC2), leading to global reduction of di- and tri-methylation on histone proteins. This mutant H3.3 histone is predicted to be a major driver of tumorigen- esis in H3K27M-mutated DIPG by disrupting normal neural differentiation. Antisense oligonucleotides (ASOs) offer a unique method to target mRNA through Watson-Crick base pairing with high specificity and low toxicity. In a recently submitted manuscript, my lab developed a “gapmer” ASO which targets H3F3A for RNaseH deg- radation, inducing neural differentiation and prolonging survival. To further these results by pursuing a parallel ASO modality, I will develop a splice-switching ASO that reduces translation of mutant H3K27M RNA by inducing skipping of H3F3A exon 2, which contains the only in-frame start codon for H3F3A and the K27M mutation. This splice-switching modality is significant because uniformly-modified ASOs, unlike “gapmer” ASOs, exhibit longer half-lives and reduced off-target liability in the central nervous system, and ASOs of this chemistry are already FDA-approved treatments. Furthermore, H3F3A and its paralogous gene H3F3B both encode identical H3.3 histone proteins, H3F3B is sufficient to compensate for loss of H3F3A, and H3F3B should remain unaffected by my ASO strategy due to sequence dissimilarities. I hypothesize that the reduction of H3K27M will limit DIPG tumor growth and prolong survival by promoting differentiation of the tumor cells. The aims of my pro- posal are to identify a lead splice-switching ASO that reduces H3K27M and restores H3K27me3 and to charac- terize the role of the RNA-binding protein RBFOX3 in reinforcing neural differentiation and H3F3A exon 2 skip- ping following ASO injection. Methods to achieve will include establishing a preclinical in vivo mouse model with H3.3K27M DIPG xenografts to study splicing changes of H3F3A and H3F3B through RT-PCR, protein expres- sion of H3K27M and H3K27me3 through immunoblot, and neural cell differentiation through immunofluores- cence following ASO injection. Additionally, splicing and analytical RNA-protein binding assays will be used to characterize the role and binding kinetics of RBFOX3 with H3F3A RNA. The proposed research is significant because it will: (i) help identify and provide mechanistic explanation for a new clinical candidate for drug devel- opment; and (ii) provide further justification for use of ASOs in the treatment of neuro-oncological disorders.