Therapeutic potential for Prader-Willi syndrome

描述(申请人提供)：与大多数遗传疾病一样，没有针对Prader-Willi综合征(PWS)特定分子缺陷的特定治疗干预措施，PWS是一种基因组印记和神经行为障碍。PWS是由父亲在染色体15q11-q13区域的基因缺陷引起的。母体染色体中的相应基因在结构上是完整的，但它们的转录受到表观遗传的抑制。表观遗传抑制的参与使PWS成为探索分子治疗的最佳机会之一。最近的报道表明，snoRNA是包括Snrpn和UBE3A基因之间的HBII-85(SNORD116)在内的簇，与PWS的关键特征有关，包括儿童肥胖、性腺功能低下、吞噬功能亢进和发育迟缓。表观遗传机制，包括PWS印记中心区(PWS-ICR)的DNA甲基化和染色质修饰，参与调节15q11-q13区域的Snrpn和snoRNAs等基因的父系特异性表达。15q11-q13区域的印迹结构域在小鼠中高度保守。SnoRNA簇的表达是从Snrpn的PWS-ICR/启动子结合的CpG岛开始的连续转录产物。DNA甲基化可以在PWS患者和小鼠模型的培养细胞中激活沉默的母体染色体中Snrpn基因的表达，但这些药物的全基因组效应限制了其临床应用。然而，这些观察结果有力地支持了一种方法，即通过表观遗传机制取消/激活母体染色体上snoRNAs的表达。由于snoRNAs是非编码的，我们建议使用携带Snrpn-EGFP融合蛋白的小鼠胚胎成纤维细胞(MEF)作为筛选工具。激活Snrpn-EGFP的药物预计也会对snoRNAs产生同样的影响。在与NIMH精神活性药物筛选计划主任Bryan Roth博士的合作下，我们已经完成了第一阶段的筛选，并确定了几种候选药物。我们已经证明，组蛋白甲基转移酶抑制剂激活了培养的PWS细胞母体染色体上Snrpn和snoRNAs的表达。使用同样的策略，罗斯博士的团队还发现了FDA批准的一种药物，该药物可以在体内激活父亲染色体中的安杰曼综合症Ube3a基因。我们项目的长期目标是开发一种针对PWS特定表观遗传缺陷的治疗干预措施。核心假设是，一个小分子可以调节PWS-ICR的表观遗传修饰，并导致母体染色体上沉默的PWS候选基因的激活。其具体目标是通过使用母体Snrpn-EGFP作为标记进行高含量小分子筛选，来鉴定和表征激活母体染色体中Snrpn和snoRNAs表达的小分子。这项拟议的研究具有重要意义，因为它将导致对PWS的治疗干预的发展，并为基因组印迹的分子机制提供新的见解。

DESCRIPTION (provided by applicant): Like most genetic disorders, no specific therapeutic intervention targets the specific molecular defect of Prader- Willi syndrome (PWS), a genomic imprinting and neurobehavioral disorder. PWS is caused by paternal deficiency of genes in the chromosome 15q11-q13 region. The corresponding genes in the maternal chromosome are structurally intact, but their transcription is repressed epigenetically. The involvement of epigenetic repression renders PWS as one of the best opportunities to explore molecular therapy. Recent reports indicate SnoRNA, clusters including HBII-85 (SNORD116) between the SNRPN and UBE3A genes, is responsible for key features of PWS including childhood obesity, hypogonadism, hyperphagia, and developmental delay. Epigenetic mechanisms, including DNA methylation and chromatin modifications at the PWS imprinting center region (PWS-ICR), are involved in regulating the paternal-specific expression of genes including SNRPN and SnoRNAs in the 15q11-q13 region. The imprinting domain in the 15q11-q13 region is highly conserved in mice. The expression of SnoRNA clusters is processed from continuous transcripts initiated from the PWS-ICR/promoter bound CpG island of SNRPN. DNA methylation can activate the expression of the SNRPN gene from the silent maternal chromosome in cultured cells of PWS patients and mouse models, but its clinical utility is limited by the concern of the genome wide effect of these drugs. These observations, however, strongly support a approach to unsilence/activate the expression of SnoRNAs from maternal chromosome through an epigenetic mechanism. Because the SnoRNAs are non-coding, we propose to use embryonic fibroblasts (MEFs) from mice carrying Snrpn-EGFP fusion protein as screening tool. Drugs that activate the Snrpn-EGFP are expected to have the same impact on the SnoRNAs. In collaboration with Dr. Bryan Roth (Director of NIMH Psychoactive Drug Screening Program), we have completed the first phase screen and identified several candidate drugs. We have showed that a histone methyltransferase inhibitor activated the expression of Snrpn and SnoRNAs from the maternal chromosome in cultured PWS cells. Using the same strategy, Dr. Roth's team also identified a FDA approved drug that activates the Angelman syndrome Ube3a gene from paternal chromosome in vivo. The long term goal of our project is to develop a therapeutic intervention targeted to the specific epigenetic defects of PWS. The central hypothesis is that a small molecule can modulate the epigenetic modification in the PWS-ICR and lead to the activation of the silenced PWS candidate genes from the maternal chromosome. The specific objective is to identify and characterize small molecules that activate the expression of Snrpn and SnoRNAs from the maternal chromosome by performing high-content small molecule screening using maternal Snrpn-EGFP as a marker. The proposed study is significant because it will lead to the development of a therapeutic intervention in PWS and provide the novel insight for molecular mechanism underlying the genomic imprinting.