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

项目总结/摘要 消失性白色物质病（VWMD）是一种常见的遗传性脑白质营养不良，发病率约为1：15，000 活产VWMD给患者带来了巨大的、往往是致命的负担。需要新的治疗方法。 我们的目标是产生和验证一种新的小型脊椎动物模型（斑马鱼）消失的白色 物质病（VWMD）。真核细胞起始因子EIF 2B（1-5）的五个不同亚基的突变是 已知引起VWMD，但VWMD治疗的发现受到疾病进展缓慢的限制 在小鼠模型中，大规模筛选困难，成本高。斑马鱼（Danio rerio）提供关键 规避这些问题的好处：髓鞘发育开始于受精后的前3天; 髓鞘发育基因的保守性;低成本和小尺寸便于筛选不可能 在其他系统中。我们为该项目的R21和R33阶段制定了四个具体目标。在R21阶段 我们将首先利用CRISPR/Cas9基因， 靶向eif 2B 5的外显子1和2，获得eif 2B 2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。我们将进行免疫组化， 髓鞘，少突胶质细胞和轴突的完整性;幼虫运动（游泳）行为的测定;和存活 曲线.对于R33阶段的进展，Go/No-Go里程碑将是生成和提高 eif 2B 2和eif 2B 5中的突变体;用PCR和基因组突变测序的证明; RNA 转录物和蛋白质产物丢失或减少;少突胶质细胞和髓鞘发育被 受损;生存和运动能力受到影响。在R33阶段，我们将首先描述 斑马鱼VWMD突变体表型与人VWMD的比较。我们将测试斑马鱼 突变体具有人类VWMD的标志性特征，包括死亡率增加;诱导性髓鞘丢失;髓鞘 MRI变化;体细胞生长受损; CSF甘氨酸水平升高。接下来，我们将测试 斑马鱼VWMD疾病的病理学通过人类基因的表达得以拯救， 遗传和生化病理生理学的保守性。其次，我们将确定斑马鱼VWMD 表型范围以及疾病和规模参数。我们将检查产生足够的 用于药物筛选的动物数量。我们将测试是否游泳行为或荧光髓鞘GFP 突变体中的报告基因可用于筛选，并测定信噪比;通量 筛选能力;与基因拯救相比的预期效应大小。总之，工作 本提案中所描述的将建立和验证一个小型脊椎动物模型的VWMD。这项工作是 在体内进行并利用最先进的技术。这一提议解决了一个尚未满足的重大需求， 使用独特的试剂，并为治疗筛选管道提供了巨大的潜力。

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.