Gene$c

zebrash model of Dravet syndrome

1Laboratory for Molecular Biodiscovery, Department of Drug Design and Development, University of Leuven, Leuven, Belgium

Angela Kecskes1, Peter A. M. de WiBe1, Alexander D. Crawford1, Camila V. Esguerra1*

*camila.esguerra@pharm.kuleuven.be

Introduc)on
Dravet syndrome (DS) is rare but severe myoclonic epilepsy in infancy that persists throughout adulthood. Developmental delay starts in the second year of life and is followed by cogni$ve impairment. Since DS is markedly pharmacoresistant, new, eec$ve an$epilep$c drugs would be of signicant value for the expansion of current treatment possibili$es. The muta$ons in the voltage-gated sodium-channel gene SCN1A and protocadherin 19 (PCDH19) are known to be at the origin of Dravet syndrome. Loss-of-func$on studies will be carried out in zebrash embryos by targe$ng scn1lab and pcdh19 through morpholino-mediated knockdown. The phenotypes will be characterized by analysis of locomotor and seizure behavior in knockdown zebrash larvae both through direct visual observa$on of seizing embryos and larvae as well as quan$ta$ve analysis of locomotor ac$vity using automated video-tracking devices. In order to assess whether par$cular neuronal popula$ons are altered in the brains of knockdown zebrash larvae, histopathological analysis of zebrash larvae will be carried out. To conrm and characterize seizure ac$vity induced by these gene knockdowns, we will also perform local eld poten$al recordings in zebrash larvae as an electrophysiological measurement and quan$ca$on of epilep$form ac$vity. Two photon and epi-uorescence calcium imaging will be performed during epilepsy in zebrash larvae. Intracellular recordings of dis$nct popula$ons of neurons in larval tectum and other brain areas will be carried out during epilep$c seizures that arise as a result of neurodevelopmental defects caused by scn1lab and pcdh19 knockdown. Ul$mately, stable gene knockouts for scn1laa, san1lab and pcdh19 will be generated in zebrash using the zinc nger nuclease (ZFN) technology based on validated zebrash morpholino knockdown models of Dravet syndrome.

Aims

The primary aim of this project is to model and validate Dravet syndrome in zebrash. All analyses in zebrash will be carried with the human and mouse muta$on phenotypes in mind in order to determine to what extent or which aspects of this disorder can be phenocopied well in the zebrash. The long-term goal of this project is the development of drug-like lead compounds for the treatment of this disease. For that purpose, a small-molecule compound library will be tested in the future in order to iden$fy compounds that decrease seizure frequency or preferably inhibit seizure progression

Objec)ves

1. Modeling Dravet Syndrome through an$sense-mediated morpholino knockdown of scn1a and pcdh19 in zebrash 2. Assessment of locomotor behaviors, seizure liability and underlying neuropathology in zebrash Dravet models 3. Electrophysiological recordings and imaging analysis of zebrash Dravet models 4. Genera$on of stable gene knockouts in zebrash.

Methods
1. MODELING DRAVET SYNDROME THROUGH ANTISENSE-MEDIATED MORPHOLINO KNOCKDOWN OF SCN1LAB AND PCDH19 IN ZEBRAFISH The design of MO will be tailored according to the type of causa$ve gene muta$ons reported for Dravet syndrome pa$ents. Of the SCN1A muta$ons iden$ed, 50% are trunca$ng and the other 50% are missense (most likely leading to loss-of-func$on). Therefore, scn1lab MO designed against the transla$onal start should mimic loss-of- func$on alleles of these target genes through abroga$on of protein synthesis. In addi$on, splice-site targeted MOs will also be designed to truncate target proteins. In the case of pcdh19 for which the pathological manifesta$on appears to be dependent on the co-expression of wild-type and mutant alleles Thus, it may be necessary to phenocopy Dravet syndrome in zebrash by simultaneous co-injec$on of pcdh19 MO to induce haploinsuciency together with mRNA encoding pcdh19 muta$on/s.

3. ELECTROPHYSIOLOGICAL RECORDINGS AND IMAGING ANALYSIS OF ZEBRAFISH DRAVET MODELS

SP-scn1lab MO ATG-scn1lab MO

scn1lab mRNA
Exon 2 Intron 2 Exon 3

scn1lab mRNA: ATG

5-UTR Exon 1 SP-pcdh19 MO

pcdh19 mRNA
Exon 2 Intron 2 Exon 3

Transla$on blocked by a Morpholino oligo

Splicing blocked by a Morpholino oligo

2. ASSESSMENT OF LOCOMOTOR BEHAVIORS, SEIZURE LIABILITY AND UNDERLYING NEUROPATHOLOGY IN ZEBRAFISH DRAVET MODELS The phenotypes will be characterized further by in-depth analysis of locomotor and seizure behavior in knockdown zebrash larvae both through direct visual observa$on of seizing embryos and larvae as well as quan$ta$ve analysis of locomotor ac$vity using automated video-tracking devices. All locomotor and seizure behaviors exhibited by knockdown larvae will be carefully scored in order to assign unique behavioral ngerprints to each gene$c model.

Automated tracking scheme of zebrash larval locomotor ac)vity. Lek: Viewpoint ZebraboxTM system for monitoring up to 96 individual larvae in parallel. Upper right: Enlarged view of 7-day old larva in well. .