Epigenome editing of the Prader-Willi syndrome imprinted domain with CRISPR/Cas9

项目摘要 Prader-Willi综合征(PWS)是一种复杂的神经发育疾病，据推测，每15,000人中就有1人受到影响 活产。受PWS影响的婴儿最初表现为肌肉张力低，喂养不良，无法茁壮成长。一次 婴儿长到18个月大时，会出现无法控制的食欲(吞噬过多)。目前， 这是一种最常见的遗传性疾病，会导致儿童患上危及生命的肥胖。除了……之外 由于超重，患有PWS的儿童通常患有智力残疾、运动发育迟缓、 身材矮小，性发育不完全。虽然PWS的确切遗传基础尚不清楚， 患者的突变特征表明15号染色体上有一个基因座(15q11-13)。有趣的是，这个轨迹是 母性印记，意味着母性等位基因因DNA甲基化的存在而在表观遗传上沉默 和抑制性的组蛋白修饰。因此，当PWS相关基因发生突变或缺失时 父亲的等位基因，对应的母体副本存在，但无法补偿基因的丢失 表情。虽然目前还没有治愈PWS的方法，但之前的研究表明，母体PWS- 相关基因可以在用DNA和组蛋白甲基转移酶抑制剂治疗后激活，导致 减少与PWS相关的病理改变。然而，表观遗传修饰酶影响许多其他基因。 在整个基因组中，它们通常在广泛的组织中表达，增加了脱靶效应的风险 是由于全球酶活性的丧失造成的。尽管如此，这一发现表明，母体的重新激活 15q11-13为治疗干预恢复PWS相关基因的表达提供了机会。 拟议项目的目标是使用CRISPR/Cas9表观基因组编辑技术来开发 针对PWS的靶向分子治疗。在这里，我们将使用高通量、无偏见的CRISPR/Cas9筛查 一种确定控制白粉病相关关键基因表达的基因组调控元件的方法 可作为表观基因组编辑靶点的小鼠神经前体细胞。我们还将确定 是否通过靶向去甲基化PWS印迹中心实现了整个基因座的重新激活，a 被认为控制整个域印记的区域。最后，我们将测试其安全性和有效性。 在体内靶向这些元件以及通过传递 CRISPR/dCas9通过腺相关病毒感染PWS小鼠模型。这项研究的成功不仅将 有助于理解PWS的监管机制，但也将奠定基础 用于这种疾病的分子治疗。此外，该项目将提供对设计和 基于CRISPR/Cas9的体内治疗策略对其他遗传性和表观遗传性疾病的疗效。

Project Summary Prader-Willi Syndrome (PWS) is a complex neurodevelopmental disease that is speculated to affect 1 in ~15,000 live births. Infants affected by PWS initially display low muscle tone, poor feeding and a failure to thrive. Once infants reach ~18 months of age, the child experiences an uncontrollable desire to eat (hyperphagia). Currently, PWS it the most common genetic disease that leads to life-threatening obesity in children. In addition to being overweight, children with PWS typically suffer from intellectual disabilities, delays in motor development, short stature, and an incomplete sexual development. While the exact genetic basis of PWS remains unclear, patient mutation profiles have implicated a locus on chromosome 15 (15q11-13). Interestingly, this locus is maternally imprinted, meaning that the maternal allele is epigenetically silenced by presence of DNA methylation and repressive histone modifications. Therefore, when PWS-associated genes harbor mutations or deletions on the paternal allele, the corresponding maternal copy is present but unable to compensate for loss of gene expression. While there is currently no cure for PWS, previous studies have shown that maternal PWS- associated genes can be activated upon treatment with DNA and histone-methyltransferase inhibitors, leading to reduction in PWS-associated pathologies. However, epigenetic modifying enzymes affect many other genes across the genome and are typically expressed in a broad range of tissues, raising the risk of off-target effects resulting from a global loss of enzymatic activity. Nonetheless, this finding suggests that reactivation of maternal 15q11-13 provides an opportunity for therapeutic intervention to restore expression of PWS-associated genes. The goal of the proposed project is to use CRISPR/Cas9 epigenome editing technologies to develop a targeted molecular therapy for PWS. Here, we will use a high-throughput, unbiased CRISPR/Cas9 screening approach to identify genomic regulatory elements that control expression of key PWS-associated genes in mouse neural progenitor cells that can be used as the targets for epigenome editing. We will also determine whether reactivation of the whole locus is achieved by targeted demethylation of the PWS imprinting center, a region that is thought to control the imprinting of the entire domain. Finally, we will test the safety and efficacy of targeting these elements in vivo and the ability to ameliorate PWS-associated phenotypes by delivering CRISPR/dCas9 by adeno-associated virus to a mouse model of PWS. The success of this study will not only contribute to the understanding of the regulatory mechanisms of PWS, but will also lay the groundwork for a molecular therapy of this disease. Furthermore, this project will provide insight into the design and efficacy of in vivo CRISPR/Cas9-based therapeutic strategies for other genetic and epigenetic disorders.