Epigenetic Therapy and Prader-Willi Syndrome

总结 像大多数遗传性疾病一样，没有针对Prader-Willi的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是由染色体15 q11-q13区域中的基因的父系缺陷引起的。 母体染色体上相应的基因在结构上是完整的，但它们的转录受到抑制 表观遗传学表观遗传调控的参与使PWS成为探索 分子治疗最近的报道表明，SNORD 116，一个位于SNRPN和 UBE 3A基因负责PWS的关键特征。尽管DNA甲基化和染色质修饰 在PWS印记中心（PWS-IC）的基因被认为是调节PWS基因沉默表达的基因。 母亲的15 q11-q13区域，确切的机制仍然难以捉摸。因此，一种有吸引力的基于分子的， PWS的治疗策略是使父系表达的PWS基因的表达不沉默， SNORD 116，来自母体染色体。由于SNORD 116是从长非编码主机处理的， 针对从PWS-IC或Snrpn启动子起始的RNA，我们开发了使用小鼠的药物筛选系统， 来源于携带母体Snrpn-EGFP融合蛋白的小鼠的胚胎成纤维细胞（MEF）。合作 与Bryan Roth博士（该提案的顾问）一起，Jiang博士（PI）筛选了9200种小分子，并确定了 并验证了两种化合物可以在人PWS中解除Snrpn和Snord 116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（HMT）的选择性抑制剂， 如Jin博士（共同研究者）所定义的，他的研究小组是发现HMT选择性抑制剂的领导者。 有趣的是，与DNA甲基化抑制剂对SNRPN的再激活相反，这些化合物降低了SNRPN的活性。 H3 K9甲基化水平，但不改变PWS-IC的DNA甲基化。这些观察加在一起 为研究PWS印迹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，这些化合物通过修饰表观遗传来使PWS候选基因不沉默 这将在PWS小鼠模型中提供临床益处。我们提出一种染色质 通过H3 K9甲基化介导的扩散模型作为PWS基因的印记调节机制。我们 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。的 姜博士（PWS分子和人类遗传学）之间的互补专业知识和密切合作 和金博士（化学生物学的新型表观遗传药物开发）独特的位置，他们达到特定的 本研究的目的是了解这些化合物使PWS沉默的机制 候选印迹基因，以评估其功效和毒性，并优化其药物样性质。的 提出的研究是重要的，因为它将提供新的见解的分子机制， PWS中的基因组印记，并导致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.