In Project 2 we will determine the functional consequences of epilepsy-associated ion channel gene variants using human neurons differentiated from patient-specific induced pluripotent stem cells (iPSCs). We will initially focus on the SCN2A and KCNQ2 genes, which encode the voltage-gated Na+ (NaV1.2) and K+ (KV7.2) channels respectively. Mutations in SCN2A and KCNQ2 are responsible for monogenic early onset epileptic encephalopathy (EE) with overlapping clinical features and diverse severity. Collectively, variants in these two genes account for ~10% of all mutations identified in genetic epilepsy. The molecular pathogenic mechanisms responsible for the clinical manifestations of KCNQ2- and SCN2A-related epilepsies remain largely unknown. More importantly, no targeted therapeutic approach capable of diminishing seizure burden and improving developmental outcomes exists for these devastating neurological disorders.
In Aim 1, we will use patient- specific cortical neurons to elucidate the functional consequences of epilepsy-associated KCNQ2 and SCN2A variants. We will specifically examine cortical excitatory and inhibitory neurons derived from existing patient- specific iPSC lines with pathogenic variants and corresponding isogenic control lines. We will use a combination of transcriptional profiling (single-cell RNA-sequencing) with electrophysiological approaches (whole cell patch clamp recording and high-throughput optogenetic recordings) to determine the impact of mutations on neuronal function and excitability.
In Aim 2, we will assess the intrinsic excitability of patient neurons before and after treatment with NaV channel blockers and KV7 agonists that have clinical efficacy in the patients from whom the cells were derived. Our goal will be to rank the in vitro effectiveness of drugs in restoring normal neuron excitability for each genetic variant, and then to correlate the in vitro drug responses with the clinical responses to AEDs documented for these patients. This project entails a strategic collaboration between Dr. Kiskinis, whose lab focuses on using stem cell-based approaches to establish models of neurological disease, and Q-State Biosciences, Inc., which under the scientific leadership of Dr. McManus has been developing optogenetic technologies to enable high-throughput electrical recordings of human neurons and drug screening platforms for epilepsy syndromes. This project will work closely with the other Center teams, including Core A (Variant Prioritization and Curation Core), Project 1 (High-Throughput Functional Evaluation of Ion Channel Variants) and Project 3 (Development and Investigation of Murine Models of Channelopathy-associated Epilepsy). Core A is building tools to prioritize variants for experimental evaluation by the three Center projects. Correlation of findings from Project 2 with those of Projects 1 and 3 will help determine the reliability and accuracy of iPSC technology to predict in vivo physiology and pharmacology. Our findings will impact the field by demonstrating mechanistic effects of channelopathy-associated epilepsy variants, and by providing a systematic evaluation of human neuron platforms for precise drug selection.
