# Proteins to Cell Systems

> **NIH NIH U54** · UNIVERSITY OF MICHIGAN AT ANN ARBOR · 2020 · $464,012

## Abstract

PROJECT SUMMARY – 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.

## Key facts

- **NIH application ID:** 10003682
- **Project number:** 1U54NS117170-01
- **Recipient organization:** UNIVERSITY OF MICHIGAN AT ANN ARBOR
- **Principal Investigator:** Jack M Parent
- **Activity code:** U54 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $464,012
- **Award type:** 1
- **Project period:** 2020-09-15 → 2025-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10003682

## Citation

> US National Institutes of Health, RePORTER application 10003682, Proteins to Cell Systems (1U54NS117170-01). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10003682. Licensed CC0.

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