Development and Validation of a Zebrafish Model for Vanishing White Matter Disease

白质消失病斑马鱼模型的开发和验证

• 批准号: 10532469
• 负责人: Josh Leitch Bonkowsky
• 金额: $ 37.94万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-12-15 至 2023-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10532469
• 关键词: Address; Adult; Affect; Alleles; Animals; Astrocytes; Axon; Behavior; Biochemical; Biological Assay; CRISPR/Cas technology; Cessation of life; Clustered Regularly Interspaced Short Palindromic Repeats; Data; Demyelinations; Development; Disease; Disease Progression; Drug Screening; EIF2B2 gene; Eukaryotic Initiation Factors; Exons; Fertilization; Functional disorder; Future; Generations; Genes; Genetic; Genomics; Genotype; Germ Cells; Glycine; Growth; Guanine Nucleotide Exchange Factors; Hepatic; Human; Immunohistochemistry; Impairment; Inherited; International; Kidney; Knock-in; Knock-out; Link; Live Birth; Magnetic Resonance Imaging; Mammals; Modeling; Molecular; Molecular Disease; Motor; Mutagenesis; Mutation; Myelin; Noise; Oligodendroglia; Pathology; Patients; Pediatric Hospitals; Phase; Phenotype; Production; Proteins; RNA; RNA Splicing; Reagent; Reporter; Research; Signal Transduction; Site; Stains; Swimming; System; Techniques; Testing; Therapeutic; Transcript; Universities; Utah; Validation; White Matter Disease; Work; Zebrafish; behavior test; clinical center; cost; disability; disease phenotype; drug discovery; experience; gene conservation; genomic RNA; in vivo; leukodystrophy; mortality; mouse model; mutant; myelin degeneration; nervous system disorder; novel; screening; tool; validation studies; white matter

Project Summary/Abstract Vanishing white matter disease (VWMD) is a common inherited leukodystrophy affecting almost ~1:15,000 live births. VWMD imposes tremendous and often lethal burdens on patients. New treatments are needed. Our objective is to generate and validate a novel small vertebrate model (zebrafish) for vanishing white matter disease (VWMD). Mutations in five different subunits of the eukaryotic initiation factor EIF2B (1-5) are known to cause VWMD, but discovery of treatments for VWMD has been limited by slow disease progression in mouse models, difficulty in large-scale screening, and high costs. Zebrafish (Danio rerio) offers key benefits to circumvent these issues: myelin development begins in the first 3 days after fertilization; there is conservation of genes for myelin development; and low costs and small size facilitate screening not possible in other systems. We have four specific aims for the R21 and R33 phases of this project. In the R21 phase we will first, generate zebrafish mutants for vanishing white matter disease, using CRISPR/Cas9 targeting of exons 1 and 2 of eif2B5, and obtaining an eif2B2 splice-site mutant. Second, we will validate zebrafish VWMD molecular and biochemical phenotypes. We will perform immunohistochemistry for myelin, oligodendrocytes, and axonal integrity; assays of larval motor (swimming) behavior; and survival curves. For progression to the R33 phase, Go/No-Go milestones will be the generation and raising of mutants in eif2B2 and eif2B5; demonstration with PCR and sequencing of the genomic mutations; that RNA transcript and protein product are lost or diminished; that oligodendrocyte and myelin development is impaired; and that survival and motor ability are affected. In the R33 phase we will first, characterize zebrafish VWMD mutant phenotypes compared to human VWMD. We will test whether the zebrafish mutants have hallmark features of human VWMD, including increased mortality; inducible myelin loss; myelin changes on MRI; impaired somatic growth; and increased CSF glycine levels. Next, we will test whether zebrafish VWMD disease pathology is rescued by expression of the human gene, demonstrating conservation of the genetic and biochemical pathophysiology. Second, we will determine zebrafish VWMD phenotype range and disease and scale parameters. We will examine ability to generate sufficient numbers of animals for drug screening. We will test whether swimming behavior or a fluorescent-myelin GFP reporter in the mutants could be used for screening, and determine the signal-to-noise ratio; throughput capacity for screening; and expected effect size in comparison to a genetic rescue. In summary, the work described in this proposal will establish and validate a small vertebrate model for VWMD. This work is carried out in vivo and utilizes state-of-the-art techniques. This proposal addresses a significant unmet need, uses unique reagents, and offers significant potential for a therapeutics screening pipeline.

项目摘要/摘要 消失的白质疾病（VWMD）是一种常见的遗传性白细胞营养不良，影响近1：15,000 活产。 VWMD对患者施加了巨大且经常致命的负担。需要新的治疗方法。 我们的目标是生成和验证一种新型的小脊椎动物模型（斑马鱼），以消失白色 物质疾病（VWMD）。真核开始因子EIF2B（1-5）的五个不同亚基的突变是 已知会导致VWMD，但是发现VWMD的治疗已受到疾病进展缓慢的限制 在鼠标模型中，大规模筛选的难度以及高成本。斑马鱼（Danio Rerio）提供关键 避免这些问题的好处：髓磷脂的发育始于受精后的前三天；有 髓磷脂发育的基因保护；低成本和小尺寸可有助于筛查 在其他系统中。我们对该项目的R21和R33阶段有四个具体目标。在R21阶段 首先，我们将使用CRISPR/CAS9产生斑马鱼突变体，以消除白质疾病 靶向EIF2B5的外显子1和2，并获得EIF2B2剪接位点突变体。第二，我们将验证 斑马鱼VWMD分子和生化表型。我们将执行免疫组织化学 髓磷脂，少突胶质细胞和轴突完整性；幼虫运动（游泳）行为的测定；和生存 曲线。为了发展到R33阶段，GO/No-Go里程碑将是一代和升高 EIF2B2和EIF2B5中的突变体；用PCR和基因组突变的测序进行演示；那个RNA 转录本和蛋白质产物丢失或减少；那些少突胶质细胞和髓磷脂的发育是 受损；并且这种生存和运动能力受到影响。在R33阶段，我们将首先表征 与人VWMD相比，斑马鱼VWMD突变体表型。我们将测试斑马鱼是否 突变体具有人类VWMD的标志性特征，包括死亡率增加；诱导髓磷脂损失；髓线 MRI的变化；躯体增长受损；并提高了CSF甘氨酸水平。接下来，我们将测试是否 斑马鱼VWMD疾病病理是通过人类基因的表达挽救的，证明 遗传和生化病理生理学的保护。第二，我们将确定斑马鱼VWMD 表型范围和疾病和比例参数。我们将研究产生足够的能力 药物筛查的动物数量。我们将测试游泳行为还是荧光膜蛋白GFP 突变体中的记者可用于筛选，并确定信噪比。吞吐量 筛选的能力；与遗传救援相比，预期效应大小。总而言之 该提案中描述的将建立并验证VWMD的小脊椎动物模型。这项工作是 在体内进行并利用最先进的技术。该提案解决了一个巨大的未满足需求， 使用独特的试剂，并为治疗筛查管道提供了巨大的潜力。