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.
Vanishing White Matter Disease (VWMD) impairs normal development of the white matter (myelin) and leads to catastrophic myelin degeneration with resultant disability and eventual death. The work described here generates and validates a novel small vertebrate model for VWMD in zebrafish (Danio rerio). We will generate zebrafish VWMD alleles; validate molecular and biochemical effects in the mutant alleles; validate homology of the zebrafish alleles compared to human VWMD; and determine scalability and screenability of the model.