Therapeutic potential for Prader-Willi syndrome

描述（由申请人提供）：与大多数遗传性疾病一样，没有针对普拉德-威利综合征（PWS）（一种基因组印记和神经行为障碍）的特定分子缺陷的特定治疗干预。PWS是由染色体15 q11-q13区域中的基因的父系缺陷引起的。母体染色体中相应的基因在结构上是完整的，但它们的转录在表观遗传学上受到抑制。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。最近的报告表明SnoRNA，包括SNRPN和UBE 3A基因之间的HBII-85（SNORD 116）簇，负责PWS的关键特征，包括儿童肥胖，性腺功能减退，摄食过多和发育迟缓。表观遗传机制包括DNA甲基化和PWS印记中心区域（PWS-ICR）的染色质修饰，参与调节15 q11-q13区域的基因（包括SNRPN和SnoRNA）的父系特异性表达。小鼠15 q11-q13区域的印迹结构域高度保守。SnoRNA簇的表达是从SNRPN的PWS-ICR/启动子结合的CpG岛起始的连续转录物加工的。DNA甲基化可以激活PWS患者和小鼠模型培养细胞中沉默母体染色体的SNRPN基因的表达，但其临床应用受到这些药物的全基因组效应的限制。然而，这些观察结果强烈支持通过表观遗传机制来解除/激活来自母体染色体的SnoRNA表达的方法。由于SnoRNA是非编码的，我们建议使用来自携带Snrpn-EGFP融合蛋白的小鼠的胚胎成纤维细胞（MEFs）作为筛选工具。预期激活Snrpn-EGFP的药物对SnoRNA具有相同的影响。在与Bryan Roth博士（NIMH精神活性药物筛选项目主任）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。我们已经表明，组蛋白甲基转移酶抑制剂激活的Snrpn和SnoRNA的表达从培养的PWS细胞的母体染色体。使用相同的策略，Roth博士的团队还确定了一种FDA批准的药物，可以在体内激活来自父亲染色体的Angelman综合征Ube 3a基因。我们项目的长期目标是开发针对PWS特定表观遗传缺陷的治疗干预。中心假设是小分子可以调节PWS-ICR中的表观遗传修饰，并导致来自母体染色体的沉默PWS候选基因的激活。具体目标是通过使用母体Snrpn-EGFP作为标记物进行高含量小分子筛选来鉴定和表征激活来自母体染色体的Snrpn和SnoRNA表达的小分子。这项研究具有重要意义，因为它将导致PWS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.