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: 10550159
• 负责人: Sara Alexandra Garcia-Moreno
• 金额: $ 7.63万
• 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/10550159
• 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; Compensation; 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; Mutation; Neurodevelopmental Disorder; Obesity; Other Genetics; Outcome; Overweight; 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; chromatin modification; clinically relevant; demethylation; design; 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; 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.

项目概要 普瑞德威利综合症 (PWS) 是一种复杂的神经发育疾病，据推测影响大约 15,000 分之一的人 活产数。受 PWS 影响的婴儿最初表现出肌张力低下、喂养不良和发育迟缓。一次 婴儿长到约 18 个月大时，孩子会出现无法控制的进食欲望（食欲过盛）。现在， PWS 是最常见的遗传病，会导致儿童出现危及生命的肥胖症。此外 由于体重超重，患有 PWS 的儿童通常会出现智力障碍、运动发育迟缓、 身材矮小，性发育不完全。虽然 PWS 的确切遗传基础仍不清楚， 患者突变谱表明 15 号染色体 (15q11-13) 上有一个位点。有趣的是，这个轨迹是 母系印记，意味着母系等位基因因 DNA 甲基化的存在而被表观遗传沉默 和抑制性组蛋白修饰。因此，当 PWS 相关基因发生突变或缺失时 父系等位基因，相应的母系拷贝存在，但无法补偿基因的损失 表达。虽然目前尚无治愈 PWS 的方法，但之前的研究表明，产妇 PWS- DNA 和组蛋白甲基转移酶抑制剂治疗后可以激活相关基因，从而导致 减少 PWS 相关病症。然而，表观遗传修饰酶影响许多其他基因 跨基因组，通常在广泛的组织中表达，增加了脱靶效应的风险 由于酶活性的整体丧失。尽管如此，这一发现表明母体的重新激活 15q11-13 提供了治疗干预的机会，以恢复 PWS 相关基因的表达。 该项目的目标是利用 CRISPR/Cas9 表观基因组编辑技术来开发 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 识别控制关键 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标。我们还将确定 整个基因座的重新激活是否是通过 PWS 印记中心的定向去甲基化来实现的，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 体内靶向这些元素以及通过传递改善 PWS 相关表型的能力 通过腺相关病毒对 PWS 小鼠模型进行 CRISPR/dCas9。这项研究的成功不仅 有助于理解 PWS 的监管机制，同时也奠定基础 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和表观遗传疾病的疗效。