Probing the myopathic activity of DUX4 using isogenic telomere-edited iPS cells

使用同基因端粒编辑的 iPS 细胞探讨 DUX4 的肌病活性

基本信息

批准号：
10000825
负责人：
Natalya Aleksandra Goloviznina
金额：
$ 3.11万
依托单位：
UNIVERSITY OF MINNESOTA
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-09-01 至 2021-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10000825
关键词：
4q35 Affect Alleles Biological Assay Blood Cells Cell Differentiation process Cell Line Cells Chromosome 4 Chromosome Deletion Coculture Techniques D4Z4 Development Diagnosis Diagnostic Procedure Direct Lytic Factors Disease EP300 gene Elements Endothelial Cells Endothelium Engraftment Epigenetic Process Facioscapulohumeral Muscular Dystrophy Future Gene Expression Genes Genetic Genetic Transcription Genome engineering Genomic DNA Global Change Head Hematopoietic Histone Acetylation Homeobox Genes Homeodomain Proteins Human Immunodeficient Mouse In Vitro Individual Inflammatory Injury Lead Light Maintenance Mediating Mesenchymal Methodology Methods Modeling Motor Muscle Muscle Cells Muscle function Muscle satellite cell Muscular Atrophy Muscular Dystrophies Mutation Myoblasts Myopathy Noise Pathogenesis Pathogenicity Pathologic Pathology Patients Physiological Play Primates Property Public Health Regulation Reproducibility Role Site Skeletal Muscle Supporting Cell Techniques Testing Transplantation Work Xenograft Model Xenograft procedure base cell type cytotoxic demethylation derepression effective therapy experimental study gain of function gene therapy homeodomain in vivo in vivo Model induced pluripotent stem cell innovation insight molecular modeling muscle degeneration mutant myogenesis novel novel therapeutics nuclease placental mammal progenitor recruit satellite cell self-renewal telomere therapeutic genome editing tool transcription factor

项目摘要

Project Summary Facioscapulohumeral Muscular Dystrophy (FSHD) is a dominant degenerative muscle disease with no cure or treatment. The genetic cause of FSHD reduction of copy number of subtelomeric D4Z4 repeats at 4q35 encoding DUX4 homeobox protein, which is a potent transcription factor that is toxic to the cell. Contractions lead to loss of repeat-induced silencing, allowing for transcription of DUX4. Interestingly, the complete absence of D4Z4 repeats mimics the WT allele in that there is no pathologic consequence. Since D4Z4 is the last element on chr4 before the telomere, deleting the end of chr4 effectively corrects the mutant allele. In this study, I propose to use a genome engineering approach mediated by site-specific nucleases to delete the 4q35 telomeric region containing D4Z4 repeats, thus specifically eliminating DUX4. Targeted integration of an artificial telomere to generate terminal chromosomal deletions has never been demonstrated before, and has a unique application in FSHD, a dominant disease whose gene is the terminal gene on chromosome 4. A common diagnostic method for FSHD shows demethylation of D4Z4 using gDNA of blood cells, and our lab has shown that D4Z4 is demethylated in FSHD iPS cells, meaning that any cell type in an FSHD patient could be subject to deregulation by DUX4. We have previously shown that DUX4 recruits p300 to induce global changes in histone acetylation, massively disrupting gene expression, thus pathology is not obviously muscle-specific. Why the disease is muscle-specific and which cells in muscle may be deregulated by DUX4 are unknowns. Because there is no evidence that DUX4 expression is myoblast or myofiber-specific, I hypothesize that DUX4 might be misexpressed in non-myogenenic cells that are involved in myogenic support, which would lead to FSHD pathogenic progression. These cell types have been largely overlooked in the field. I will therefore generate a panel of isogenic pairs of corrected/diseased iPS lines, and study the presence and consequence of DUX4 expression in myogenic as well as supportive cell types (obtained by differentiating the iPS cells). Aim 1 will focus on the myogenic lineage and make use of xenograft models pioneered by our collaborators, the Perlingeiro Lab. Aim 2 will focus on the supportive cell types and evaluate cell non-autonomous effects on myogenesis. These studies will develop a unique and innovative method of genome engineering and shed light on the pathophysiological mechanism of muscle degeneration in FSHD.

项目概要面肩肱型肌营养不良症 (FSHD) 是一种显性退行性肌肉疾病，不伴有任何症状。治愈或治疗。 FSHD 4q35 亚端粒 D4Z4 重复拷贝数减少的遗传原因编码 DUX4 同源框蛋白，这是一种对细胞有毒的有效转录因子。宫缩导致重复诱导沉默的丧失，从而允许 DUX4 转录。有趣的是，完全缺席 D4Z4 重复序列模仿 WT 等位基因，因为没有病理后果。由于D4Z4是最后一个端粒之前的 chr4 上的元件，删除 chr4 的末端可有效纠正突变等位基因。在这个研究中，我建议使用位点特异性核酸酶介导的基因组工程方法来删除 4q35 端粒区域含有 D4Z4 重复序列，从而特异性消除 DUX4。有针对性的整合人工端粒产生末端染色体缺失以前从未被证实过，并且具有在 FSHD 中的独特应用，FSHD 是一种显性疾病，其基因是 4 号染色体上的末端基因。 FSHD 的常见诊断方法显示使用血细胞 gDNA 进行 D4Z4 去甲基化，并且我们的实验室已表明 D4Z4 在 FSHD iPS 细胞中去甲基化，这意味着 FSHD 中的任何细胞类型患者可能会受到 DUX4 的放松管制。我们之前已经表明，DUX4 招募 p300 来诱导组蛋白乙酰化的整体变化，大规模破坏基因表达，因此病理学不是明显是肌肉特异性的。为什么这种疾病是肌肉特异性的以及肌肉中的哪些细胞可能失调 DUX4 的情况未知。因为没有证据表明 DUX4 表达具有成肌细胞或肌纤维特异性，我推测 DUX4 可能在参与肌源性的非肌源性细胞中错误表达。支持，这将导致 FSHD 致病进展。这些细胞类型在很大程度上被忽视了领域。因此，我将生成一组校正/患病 iPS 系的同基因对，并研究生肌细胞和支持细胞类型中 DUX4 表达的存在和后果（通过分化 iPS 细胞）。目标 1 将重点关注肌源谱系并利用异种移植模型由我们的合作者 Perlingeiro 实验室首创。目标 2 将重点关注支持细胞类型并评估细胞对肌生成的非自主效应。这些研究将开发一种独特且创新的方法基因组工程并揭示 FSHD 肌肉退化的病理生理机制。