Epigenetic Therapy and Prader-Willi Syndrome

摘要 像大多数遗传性疾病一样，没有针对Prader-Willi分子缺陷的特定治疗干预 综合征(PWS)，一种显著影响生活质量的基因组印记和神经行为障碍 受影响的个人。PWS是由父亲在染色体15q11-q13区域的基因缺陷引起的。 母体染色体上的相应基因在结构上是完整的，但它们的转录受到抑制。 从表观遗传学上讲。表观遗传调控的参与使pws成为探索的最佳机会之一。 分子疗法。最近的报道表明，SNORD116是一个位于Snrpn和Snrpn之间的snoRNA簇 UBE3A基因，决定了PWS的关键特征。尽管DNA甲基化和染色质修饰 在PWS印迹中心(PWS-IC)被认为调节PWS基因在 母体的15q11-q13区域，其确切机制仍不清楚。因此，一种有吸引力的基于分子的， PWS的治疗策略是解除父系表达的PWS基因的表达，主要是 SNORD116，来自母体染色体。因为SNORD116是从长非编码主机处理的 PWS-IC或Snrpn启动子的RNAs，我们用小鼠建立了药物筛选系统 胚胎成纤维细胞(MEF)来自携带母体Snrpn-EGFP融合蛋白的小鼠。在协作中 在Bryan Roth博士(这项建议的顾问)的帮助下，姜博士(Pi)筛选了9200个小分子，并确定了 并验证了两个化合物，它们可以同时抑制人PWS中Snrpn和SNORD116的表达 细胞和PWS小鼠模型。这些化合物是组蛋白甲基转移酶(HMTs)的选择性抑制剂， 根据金博士(合作者)的定义，他的研究小组是发现HMTs选择性抑制剂的领先者。 有趣的是，与DNA甲基化抑制剂重新激活Snrpn相反，这些化合物减少了 H3K9甲基化水平，但不改变PWS-IC的DNA甲基化。这些观察结果加在一起 为研究PWS印记表达的机制提供了新的见解和机会 基因。我们的中心假设是，这些化合物通过改变表观遗传学，使PWS候选基因不再沉默 在PWS-IC中的复合体，这将在PWS小鼠模型中提供临床益处。我们提出了一种染色质 H3K9甲基化作为PWS基因印迹调控机制的传播模型我们的 长期目标是在人类PWS中使用这些化合物或其衍生物进行临床试验。这个 江博士(PWS分子与人类遗传学)的专业知识互补和密切合作 而金博士(新型表观遗传药物开发的化学生物学)独特地定位了它们，以达到特定的 这项研究的目的是了解这些化合物解除PWS沉默的机制 候选印迹基因，以评估其疗效和毒性，并优化其类药物特性。这个 提出的研究具有重要意义，因为它将为潜在的分子机制提供新的见解 在PWS中的基因组印记，并导致对该疾病的治疗干预的发展。

SUMMARY Like most genetic disorders, no specific therapeutic intervention targets the molecular defect of Prader-Willi syndrome (PWS), a genomic imprinting and neurobehavioral disorder that significantly affects the quality of life of affected individuals. PWS is caused by paternal deficiency of genes in the chromosome 15q11-q13 region. The corresponding genes on the maternal chromosome are structurally intact, but their transcription is repressed epigenetically. The involvement of epigenetic regulation renders PWS one of the best opportunities to explore molecular therapy. Recent reports indicate that SNORD116, a SnoRNA cluster located between the SNRPN and UBE3A genes, is responsible for key features of PWS. Although DNA methylation and chromatin modifications at the PWS imprinting center (PWS-IC) are believed to regulate the silent expression of PWS genes in the maternal 15q11-q13 region, the exact mechanism remains elusive. Thus, one attractive molecular-based, therapeutic strategy for PWS is to unsilence the expression of paternally expressed PWS genes, primarily SNORD116, from the maternal chromosome. Because SNORD116 is processed from the long noncoding host RNAs initiated from the PWS-IC or Snrpn promoter, we developed a drug screening system using mouse embryonic fibroblasts (MEFs) derived from mice carrying a maternal Snrpn-EGFP fusion protein. In collaboration with Dr. Bryan Roth (consultant for this proposal), Dr. Jiang (PI) screened 9200 small molecules and identified and validated two compounds that can unsilence the expression of both Snrpn and Snord116 in human PWS cells and a PWS mouse model. These compounds are selective inhibitors of histone methyltransferases (HMTs), as defined by Dr. Jin (co-PI), whose research group is a leader in discovering selective inhibitors of HMTs. Interestingly, in contrast with reactivation of SNRPN by DNA methylation inhibitors, these compounds reduced the H3K9 methylation level but did not change DNA methylation of the PWS-IC. These observations together offer new insights and opportunities to investigate the mechanism underlying the imprinted expression of PWS genes. Our central hypothesis is that these compounds unsilence PWS candidate genes by modifying epigenetic complexes in the PWS-IC, which will provide clinical benefits in PWS mouse models. We propose a Chromatin Spreading Model mediated by H3K9 methylation as a mechanism of imprinted regulation of PWS genes. Our long-term goal is to launch a clinical trial using these compounds or their derivatives in human PWS. The complementary expertise and close collaboration between Dr. Jiang (molecular and human genetics of PWS) and Dr. Jin (chemical biology of novel epigenetic drug development) uniquely position them to attain the specific objectives of this study, which are to understand the mechanism by which these compounds unsilence PWS candidate imprinted genes, to evaluate their efficacy and toxicity, and to optimize their drug-like properties. The proposed study is significant because it will provide novel insight into the molecular mechanism underlying genomic imprinting in PWS and lead to the development of a therapeutic intervention for the disease.