Project 3: Cell Systems to Preclinical Models

Figure 1. Project 3, in concert with GVCC and Projects 1 and 2, will test the pathogenicity of selected Variant of Unknown Significance (VUS) using in vivo rodent and zebrafish models. The goal is to incorporate in vivo functional assays into the EpiMVP pipeline to arrive at a decisive probability of VUS pathogenicity. Project 3 will take advantage of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) gene editing methods to generate predicted loss-of-function (LOF) epilepsy models in rodent and zebrafish as a null background on which to evaluate human VUS. Data generated in Project 3 will be shared with Projects 1 and 2 and the GVCC in an iterative fashion with an overall deliverable to develop EpiPred (Epilepsy Variant Prediction), a machine learning model that will allow accurate classification of missense epilepsy gene variants as likely pathogenic or benign.

Milestone 1: To interrogate selected VUS in rodents using IUE-mediated mutagenesis.

1. During the first year, we will perform CRISPR-in utero electroporation (IUE) to model Stxbp1 LOF and provide a positive control for restoring function by expressing the WT allele and known benign variants (BVs), while expressing known pathogenic variants (PVs) serves as a negative control.
2. We will utilize advanced technologies in molecular genetics and electrophysiology to analyze the neurodevelopmental and functional impact of Stxbp1 LOF at molecular, cellular and circuitry levels.
3. We will perform functional rescue experiments by co-transfecting CRISPR with constructs expressing a VUS to evaluate its pathogenicity. In the first year, we will evaluate 1~2 human VUS for functional rescue. In subsequent years, we will complete 3~4 VUS assays for 1-2 new genes per year.

Figure 2. a) The diagram shows the IUE system. On the right, an epifluorescence image shows a brain with GFP-IUE over the right hemisphere (RH). b) Significantly increased pS6 immunoreactivity (red) is seen in the Depdc5 CRISPR-IUE transfected cortex, suggesting mTOR hyperactivation due to Depdc5 LOF. c) Epileptiform discharges are highly similar to those recorded in human Depdc5-related epilepsies. PFA: paroxysmal fast activity; BRDs: brief rhythmic discharges; PEDs: periodic epileptiform discharges. d) A representative seizure arises from the hemisphere with dysplastic cortex.

Milestone 2: To interrogate selected VUS in zebrafish CRISPR LOF epilepsy models.

1. Using CRISPR/Cas9 genome editing, the Baraban laboratory recently engineered zebrafish LOF lines for 38 human epilepsy genes. Highly advanced technologies will be utilized to examine neurodevelopment and behavioral phenotypes and assess network dynamics in LOF zebrafish lines.
2. Zebrafish LOF mutants designed to represent human gene mutations provide a valuable null background on which to evaluate human VUS. During the first year, we will evaluate 3~4 human VUS for STXBP1 in zebrafish CRISPR LOF models that have already been generated. Two common BVs and PVs are chosen as positive controls and negative controls, respectively.

Figure 3. Representative examples of the electrophysiology (iZAP based multi-fluidic system to recording electrical seizure activity in intact zebrafish larvae), imaging (light-sheet microscopy to analyze neuronal densities and development in intact zebrafish larvae) and behavior (locomotion-based tracking assays to evaluate spontaneous swim behavior and response to manipulations such as startle in intact zebrafish larvae) approaches for Project 3.