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

用反义寡核苷酸诱导 H3F3A 外显子跳跃作为治疗弥漫性内源性脑桥胶质瘤的方法

基本信息

批准号：
10677284
负责人：
Lucia Yang
金额：
$ 4万
依托单位：
STATE UNIVERSITY NEW YORK STONY BROOK
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-06-01 至 2027-02-28
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10677284
关键词：
Antibodies Antisense Oligonucleotide Therapy Antisense Oligonucleotides Base Pairing Binding Biological Assay Brain Neoplasms Brain Stem Cell Differentiation process Cells Central Nervous System Chemistry Child Childhood Brain Neoplasm Childhood Glioma Clinical Drug Development Code Codon Nucleotides Compensation Complex Data Differentiation Antigens Diffuse intrinsic pontine glioma Disease Electrophoretic Mobility Shift Assay Excision Exhibits Exons FDA approved Genetic Diseases Glial Fibrillary Acidic Protein Glioma Goals H3 K27M mutation Heterozygote Histone H3 Histone H3.3 Histones Human Immunofluorescence Immunologic Initiator Codon Injections Kinetics Knowledge Lead Local Therapy Location Lysine Manuscripts Mediating Messenger RNA Methionine Methods Methylation Mitochondria Modality Molecular Target Mutate Mutation Nature Neoplasms Neuroglia Neuronal Differentiation Neurons Oncology Operative Surgical Procedures Pathologic Patients Pediatric Neoplasm Point Mutation Polycomb Proliferating Proteins Protocols documentation RNA RNA Splicing RNA-Binding Proteins Radiation therapy Research Reverse Transcriptase Polymerase Chain Reaction Role Site Site-Directed Mutagenesis Specificity Survival Rate Therapeutic Toxic effect Translations Tumor Burden Tumor Volume Tumor-Derived Visualization Western Blotting Xenograft procedure chemotherapy clinical candidate curative treatments design drug development effective therapy exon skipping gain of function mutation in vivo mouse model mutant neoplastic cell neural novel therapeutics overexpression paralogous gene pre-clinical prevent protein H(3)protein expression restoration side effect tumor tumor diagnosis tumor growth tumorigenesis

项目摘要

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.

项目概要儿童高级别胶质瘤 (pHGG) 占儿童诊断的所有脑肿瘤的 10-15%。 pHGG 的弥漫性内质性脑桥胶质瘤 (DIPG) 尤其致命，五年生存率 < 1%。目前的治疗选择是非治愈性的；手术切除、局部放疗和化疗都是有效的。通过肿瘤的脑干位置复制并伴随负面副作用，因此更有效。大约 70-80% 的 DIPG 肿瘤以显性杂合为标志。 H3F3A 中的 gous 点突变，编码非典型组蛋白 H3.3，这种有毒的功能获得性 mu-。用蛋氨酸 (K27M) 替换赖氨酸 27，防止赖氨酸 27 (H3K27me3) 发生三甲基化。也已被证明可以干扰 Polycomb 抑制复合物 2 (PRC2)，从而导致全球性减少组蛋白上的三甲基化预计是肿瘤发生的主要驱动因素。通过破坏正常的神经分化（ASO）来抑制 H3K27M 突变的 DIPG 的发生。提供了一种通过 Watson-Crick 碱基配对靶向 mRNA 的独特方法，具有高特异性和低毒性。在最近提交的手稿中，我的实验室开发了一种“gapmer”ASO，它针对 RNaseH 脱氢酶 H3F3A 辐射，诱导神经分化并延长生存期。 ASO 模式，我将开发一种剪接转换 ASO，通过诱导减少突变 H3K27M RNA 的翻译跳过 H3F3A 外显子 2，其中包含 H3F3A 的唯一框内起始密码子和 K27M 突变。剪接转换模式非常重要，因为与“gapmer”ASO 不同，一致修饰的 ASO 表现出更长的半衰期和减少中枢神经系统的脱靶倾向，并且这种化学的 ASO 已经此外，H3F3A 及其旁系同源基因 H3F3B 均编码相同的 H3.3。组蛋白，H3F3B 足以补偿 H3F3A 的损失，并且 H3F3B 应保持不受由于序列差异，我的 ASO 策略我认为 H3K27M 的减少将限制 DIPG。通过促进肿瘤细胞的分化来促进肿瘤生长并延长生存期。 posal 的目标是确定一个可减少 H3K27M 并恢复 H3K27me3 的先导剪接转换 ASO 以及特征确定 RNA 结合蛋白 RBFOX3 在增强神经分化和 H3F3A 外显子 2 跳跃中的作用 ASO 注射后的 ping 实现方法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研究H3F3A和H3F3B的剪接变化，蛋白表达通过免疫印迹区分 H3K27M 和 H3K27me3，并通过免疫荧光区分神经细胞此外，将使用剪接和分析 RNA-蛋白质结合测定。表征 RBFOX3 与 H3F3A RNA 的作用和结合动力学本研究具有重要意义。因为它将：（i）帮助识别新的临床候选药物并为其提供机制解释 (ii) 为使用 ASO 治疗神经肿瘤疾病提供进一步的理由。