弥漫性中线胶质瘤（DMGs）是儿童高级别的脑肿瘤，位于丘脑、中脑或脑桥；后一类亚组被称为弥漫性内皮脑桥胶质瘤（DIPG）。这些肿瘤的脑干位置限制了DIPG的临床治疗，导致患者预后不佳。大多数DIPG肿瘤中存在一个非规范组蛋白H3.3编码基因的杂合性体点突变，该H3-3A基因中的优势性突变导致赖氨酸27被甲硫氨酸取代（K27M），并在所有野生型组蛋白H3蛋白上全局降低K27的三甲基化，被认为是胶质母细胞瘤发生的驱动事件。在这项研究中，我们设计并系统筛选了2'-O-甲氧基乙基磷酸硫酯反义寡核苷酸（ASO），以引导RNase H介导的H3-3A mRNA敲下。我们发现了一种能有效降低患者来源的神经球中H3-3A mRNA和H3.3K27M蛋白、并恢复全局H3K27三甲基化的ASO。然后，我们在两个DIPG小鼠模型中测试了主导的ASO：一个使用转导突变人H3-3A cDNA的免疫竞争性小鼠模型和一个使用患者来源细胞的原位移植模型。在这两个模型中，ASO治疗恢复了组蛋白H3蛋白的K27三甲基化，并减少了肿瘤生长，在神经干细胞分化为星形胶质细胞、神经元和少突胶质细胞方面有所促进，并延长了小鼠的存活时间。这些结果证明了H3.3K27M肿瘤组蛋白在肿瘤维持中的作用，确认了它促进的异常表观遗传改变的可逆性，并提供了DMG反义治疗的临床前概念证明。

Diffuse midline gliomas (DMGs) are pediatric high-grade brain tumors in the thalamus, midbrain, or pons; the latter subgroup are termed diffuse intrinsic pontine gliomas (DIPG). The brain stem location of these tumors limits the clinical management of DIPG, resulting in poor outcomes for patients. A heterozygous, somatic point mutation in one of two genes coding for the noncanonical histone H3.3 is present in most DIPG tumors. This dominant mutation in the H3-3A gene results in replacement of lysine 27 with methionine (K27M) and causes a global reduction of trimethylation on K27 of all wild-type histone H3 proteins, which is thought to be a driving event in gliomagenesis. In this study, we designed and systematically screened 2'-O-methoxyethyl phosphorothioate antisense oligonucleotides (ASOs) that direct RNase H-mediated knockdown of H3-3A mRNA. We identified a lead ASO that effectively reduced H3-3A mRNA and H3.3K27M protein and restored global H3K27 trimethylation in patient-derived neurospheres. We then tested the lead ASO in two mouse models of DIPG: an immunocompetent mouse model using transduced mutant human H3-3A cDNA and an orthotopic xenograft with patient-derived cells. In both models, ASO treatment restored K27 trimethylation of histone H3 proteins and reduced tumor growth, promoted neural stem cell differentiation into astrocytes, neurons, and oligodendrocytes, and increased survival. These results demonstrate the involvement of the H3.3K27M oncohistone in tumor maintenance, confirm the reversibility of the aberrant epigenetic changes it promotes, and provide preclinical proof of concept for DMG antisense therapy.