EXploring novEl Molecular Determinants of DRAvet Syndrome Phenotype Heterogeneity

Overview

Dravet syndrome is characterized as a developmental encephalopathy resulting from mutations of SCN1A, the gene encoding the alpha subunit of the voltage-gated sodium channel Nav1.1. The syndrome typically presents with drug-resistant epilepsy and varying degrees of cognitive disorders. Current treatment efficacy may be hindered by insufficient knowledge of undiscovered molecular determinants of the disease and its heterogeneous nature. Utilizing induced pluripotent stem cells (iPSCs) derived from skin biopsies, accessibility to patients' brain neurons has enabled successful modeling of various genetic neurological diseases. Neurons and brain organoids will be obtained from Dravet syndrome patients exhibiting diverse phenotypic severities, encompassing behavioral and developmental delays, to discern the molecular determinants of phenotypic diversity. Specifically, emphasis will be placed on investigating cellular and molecular mechanisms linking altered neuronal excitability with synaptic dysfunction.The study will focus on exploring the expression of newly identified modifiers potentially associated with neuronal excitability and synaptic function in iPSC-derived human neurons. This aims to establish correlations between the severity of epileptic and cognitive phenotypes and the altered expression of these proteins, whose functions are not fully understood.In the mid to long term, efforts will be directed towards overcoming the limitations of conventional therapeutic approaches for Dravet syndrome. This will involve attempting to reverse the observed morphological and functional alterations in Dravet syndrome neurons using viral vectors to promote overexpression/downregulation of identified modifiers correlated with disease severity. The anticipated outcomes of this project are expected to unveil novel molecular mechanisms underlying the pathophysiology of this severe neurogenetic disease, characterized by varying degrees of cognitive impairment. Moreover, these findings may pave the way for the discovery of innovative therapeutic strategies.

Dravet Syndrome (DS) is characterized as both an epileptic encephalopathy, where seizures contribute to phenotype severity, and a developmental encephalopathy, where genetic background directly impacts developmental delay independently from seizure activity. The syndrome exhibits significant heterogeneity in phenotype severity, with polymorphic seizures typically occurring before age 12 months and progressing with varying severity. Additionally, cognitive and behavioral impairments become apparent during the second year of life or later, worsening with age and seizure frequency. Factors limiting the efficacy of current treatments likely include insufficient knowledge of unknown molecular determinants of the disease and its heterogeneity. To address this, novel molecular modifiers linked to excitability and synaptic function are hypothesized to unveil the pathophysiology of DS phenotype heterogeneity. The study aims to investigate hiPSCs-derived neurons from DS patients compared with age-matched healthy subjects. Specifically, skin biopsies will be obtained from DS patients and healthy controls to generate neurons and brain organoids. These models will be utilized to understand how changes in excitability and synaptic function affect phenotype severity in DS and to identify new molecular determinants associated with altered neuronal excitability and synaptic dysfunction. Aims of the study include classifying the epileptic and cognitive/behavioral phenotype severity of DS patients, characterizing neurons and brain organoids obtained from DS patients, and identifying potential phenotype modifiers. Additionally, the study aims to provide proof of concept for the development of novel modifier-related pharmacogenetic strategies to alleviate or rescue DS phenotype. Recruitment of DS patients and healthy controls will be conducted, and various assessments will be performed to classify phenotype severity. Functional, morphological, and molecular alterations in human neurons derived from DS patients will be investigated using a range of experimental techniques. Ultimately, the study seeks to deepen understanding of DS pathophysiology, identify potential therapeutic targets, and pave the way for personalized medicine approaches tailored to individual patients' molecular profiles.