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Preliminary development of a zebrafish platform for screening MeCP2 therapeutics


Rett syndrome is a severe neurodevelopmental disorder predominantly caused by mutations in the Methyl-CpG-binding protein 2 (MECP2). Four out of the eight common mutations in MECP2 account for ~35% of RTT cases. A method for overcoming the phenotypes found in these nonsense mutations is treatment with nonsense suppression therapy. Nonsense suppression therapy are small molecules that suppress premature termination codons that would allow for full length readthrough of MeCP2.However, the ability screen for therapeutics has been limited due to the low throughput of RTT animal disease models. Zebrafish have been shown to be a complementary model translatable for neuroscience research and for high throughput drug screenings. Here, I describe the applications and importance of zebrafish as a preliminary model for MECP2 therapeutics. I re-capitulated the behavioral and molecular phenotypes characterized in zebrafish containing the mecp2Q63x mutation, successfully showing expression of Mecp2 in the brain of 6dpf zebrafish. Using a custom video-based activity capture system, I demonstrated a decrease in total distance travelled, percentage of swimming activity, and decrease in anxiety-like behavior in the mecp2Q63x fish. These findings reproduce previous reports demonstrating dysfunction in motor circuitry seen in RTT patients. I also found increased astrogenesis, as measured by expression of GFAP, in the mecp2Q63x fish. Finally, I generated a novel mecp2 zebrafish mutant with a complete excision of the mecp2 locus using CRISPR/Cas9 system to demonstrate the ability to engineer this locus and paving the way for future generation of fish engineered to contain human disease-causing nonsense mutations. In summary, this works provides the foundation and a platform to use genetically engineered zebrafish to perform high-throughput screening of nonsense suppression therapeutics.

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