? PROJECT 2 Next generation DNA sequencing (NGS) has led to the rapid discovery of large numbers of epilepsy genes, and the list of epilepsy genes has grown well beyond ion channels to those that affect a wide array of cellular functions. Our understanding of how any specific gene mutation leads to epilepsy, however, increasingly lags behind gene discovery. Moreover, NGS has led to increased numbers of genetic variants of uncertain significance (VUS) that are difficult to interpret diagnostically. We lack the tools to assay VUS effects or effectively study pathogenic mechanisms for these epilepsy genes. To address these shortfalls, the EpiMVP will optimize cutting-edge multiplatform assays for epilepsy genes that include cell lines (Project 1), human pluripotent stem cells (hPSCs; Projects 1 and 2), human cortical organoids (hCOs; Project 2), and in vivo rodent and zebrafish models (Project 3). The Gene and Variant Curation Core (GVCC) will interact with the projects to select and refine specific genes and variants for testing as the VUS list is streamlined from Projects 1 to 2 to 3. Key to this effort is the Human Epilepsy Tools Core (HETC) which will provide cell lines (for Projects 1 and 2) and variant expression vectors (for all 3 projects). The long-term goal of our work is to deliver an in vitro testing pipeline in human neuronal models to assay clinically relevant VUS for all non-ion channel epilepsy genes. We have identified relevant morphological/functional 2-D or hCO phenotypes for 6 genes in the top 10 most commonly diagnosed non-ion channel genetic epilepsies, as well as reagents for several others, using: 1) 2-D hPSC cultures, including small molecule differentiation into excitatory or inhibitory cortical neurons, excitatory induced neurons and induced GABA neurons (iNeurons/iGNs) generated by transcription factor expression, and mixed cultures (iNeurons, iGNs and glia); and 2) 3-D hCO cultures, including multi-rosette, single rosette, excitatory, inhibitory and fusion hCOs. We will use these assays to test the hypothesis that our platforms will predict VUS pathogenicity and effectively prioritize variants for in vivo testing in Project 3. Our immediate goals are to optimize assays for 1-2 genes per year, determine VUS pathogenicity in vitro for these genes and, in concert with the VGCC, refine the VUS list for further in vivo testing in Project 3. The goals will be accomplished using 2-D hPSCs for Milestone 1 and 3-D hCOs in Milestone 2, and will include structural and functional assays for each model system. These studies will provide the following deliverables: 1) multiple optimized, cross-validated (between Parent and Ross labs) hPSC platforms to interrogate epilepsy genes; 2) determination of in vitro human neuronal VUS pathogenicity for at least 5 non-ion channel epilepsy genes; 3) human neuronal models for each epilepsy gene; and 4) optimized platforms for future mechanistic and precision therapeutic studies.
