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

使用 CRISPR/Cas9 对 Prader-Willi 综合征印迹域进行表观基因组编辑

• 批准号: 10410369
• 负责人: Sara Alexandra Garcia-Moreno
• 金额: $ 7.23万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-02-01 至 2024-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10410369
• 关键词: Affect; Age-Months; Alleles; Body Weights and Measures; CRISPR screen; CRISPR therapeutics; CRISPR/Cas technology; Candidate Disease Gene; Catalytic Domain; Child; Chromatin; Chromatin Remodeling Factor; Chromosome 15; Chromosomes; Clinical; Clinical Trials; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Control Locus; DNA; DNA Methylation; DNA Modification Methylases; Data; Defect; Dependovirus; Development; Developmental Delay Disorders; Diabetes Mellitus; Disease; Disease model; Eating; Elements; Enzymes; Epigenetic Process; Failure to Thrive; Foundations; Gene Expression; Genes; Genetic; Genetic Diseases; Genetic Transcription; Genome; Genomic Imprinting; Genomics; Goals; Growth Factor; Hormones; Human; Hunger; Hyperphagia; Impairment; Individual; Infant; Insulin; Intellectual functioning disability; Life; Link; Live Birth; Maintenance; Methyltransferase; Molecular; Motor; Mus; Muscle Tonus; Muscle hypotonia; Mutation; Neurodevelopmental Disorder; Obesity; Other Genetics; Outcome; Parents; Pathology; Patients; Phenotype; Prader-Willi Syndrome; Process; Regulatory Element; Risk; Role; SETDB1 gene; Sexual Development; Technology; Testing; Therapeutic; Therapeutic Intervention; Tissues; Transcription Coactivator; Transcription Repressor; United States; Viral; Work; adeno-associated viral vector; base; chromatin modification; clinically relevant; demethylation; design; epigenetic silencing; epigenetic therapy; epigenome; epigenome editing; experience; feeding; genome editing; ghrelin; high risk; histone methylation; histone methyltransferase; histone modification; imprint; improved; in vivo; induced pluripotent stem cell; inhibitor; insight; maternal imprint; molecular targeted therapies; mouse model; neonate; nerve stem cell; nervous system disorder; neurobehavioral disorder; novel; obesity in children; overweight child; reduced muscle mass; safety testing; screening; success; vector

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.

项目摘要 Prader-Willi综合征（PWS）是一种复杂的神经发育疾病，据推测约15，000人中有1人患病 活产受PWS影响的婴儿最初表现出肌肉张力低，喂养不良和发育不良。一旦 婴儿达到~18个月大时，儿童会出现无法控制的进食欲望（食欲过盛）。目前， PWS是最常见的遗传性疾病，导致儿童肥胖危及生命。除了 由于超重，PWS儿童通常患有智力障碍，运动发育迟缓， 身材矮小性发育不全虽然PWS的确切遗传基础尚不清楚， 患者突变谱涉及染色体15（15 q11 -13）上的基因座。有趣的是，这个位点 母系印记，意味着母系等位基因由于DNA甲基化的存在而在表观遗传学上沉默 和抑制性组蛋白修饰。因此，当与PWS相关的基因在其上发生突变或缺失时， 父方等位基因，相应的母方拷贝存在，但不能补偿基因的丢失 表情虽然目前还没有治愈PWS的方法，但以前的研究表明，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被激活， 减少与PWS相关的病理。然而，表观遗传修饰酶影响许多其他基因 通常在广泛的组织中表达，增加了脱靶效应的风险 这是由于酶活性的全面丧失。尽管如此，这一发现表明，重新激活母亲的 15 q11 -13为治疗干预提供了恢复PWS相关基因表达的机会。 该项目的目标是利用CRISPR/Cas9表观基因组编辑技术开发一种新的基因组编辑技术。 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筛选， 一种鉴定控制PWS相关基因表达的基因组调控元件的方法， 小鼠神经祖细胞，可用作表观基因组编辑的靶点。我们还将确定 是否通过PWS印迹中心的靶向去甲基化实现整个基因座的再活化， 被认为控制整个域的印记的区域。最后，我们将测试 在体内靶向这些元件，并通过递送 CRISPR/dCas 9通过腺相关病毒应用于PWS小鼠模型。这项研究的成功不仅 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，该项目将提供深入的设计和 基于CRISPR/Cas9的体内治疗策略对其他遗传和表观遗传疾病的疗效。