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.