Children with catastrophic epilepsies develop intractable forms of epilepsy with severe intellectual and behavioral disabilities, including autism. Many of these children are diagnosed, early in life, with genetic forms of epilepsy. With recent advances in gene sequencing approximately 60 loss-of-function (LOF) single-gene mutations have been identified in this patient population. Zebrafish LOF mutants designed to represent these human genetic conditions would provide valuable tools for elucidating basic disease mechanisms and drug discovery. Methods such as the CRISPR/Cas9 system (clustered regularly interspaced short palindromic repeat/Cas9) now allow for rapid and efficient modification of endogenous genes in a range of animal models, including zebrafish (Danio rerio). Using this technique, we recently developed and characterized a stxbp1 mutant zebrafish line representing children with early infantile epileptic encephalopathy with burst suppression. In this proposal, we will generate pre-clinical mutant zebrafish lines for all known human LOF mutations using CRISPR/Cas9 gene editing techniques (Aim I). To determine whether these mutants exhibit functional phenotypes (including epilepsy), we will carefully monitor behavior and neural activity using unique assays and methodologies developed in our laboratory (Aim II). Our strategy includes (i) detailed analysis of behavior using automated locomotion tracking and high-speed imaging, (ii) recording brain activity using in vivo electrophysiology techniques and (iii) brain-wide calcium imaging of neural networks. Zebrafish lines with epileptic phenotypes will also be screened against a small panel of existing antiepileptic drugs. All zebrafish mutant lines generated and carefully characterized here will be made available to the scientific community via the NINDS-supported Zebrafish International Resource Center in Oregon. Both as a resource and offering insight into functional consequences of these single gene mutations, our proposal establishes a comprehensive zebrafish-based precision medicine approach targeted to monogenic epilepsy disorders seen primarily in children.
With nearly 68% of all epilepsies now thought to have a genetic cause, including the catastrophic epilepsies of childhood that are in desperate need of more effective therapies, our proposal is squarely focused on a timely and significant unmet need in the epilepsy community. While it is possible to generate mutant mice for all known human epilepsy genes, doing so would be time consuming, expensive and labor intensive. An alternative pre-clinical animal model already recognized in fields like cancer, diabetes, and cardiovascular disease is the genetically modified zebrafish. Zebrafish offer significant advantages for in vivo analysis of gene function and whole-brain imaging of neural networks.
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