UC Irvine

Over 1250 mutations in SCN1A, the Nav1.1 voltage-gated sodium channel gene, are associated with a variety of seizure disorders including Dravet syndrome (DS) and genetic epilepsy with febrile seizure plus (GEFS+). Individuals with SCN1A genetic epilepsy exhibit a broad range of seizure phenotypes and many of them are not well controlled by conventional anti-convulsant therapy. How specific mutations alter sodium channel function in a way that contributes to seizure generation is still largely unexplored. An understanding of cellular mechanisms is important for future studies targeted at developing more effective patient-specific therapies.

Previous studies in models including Xenopus oocytes, human embryonic kidney cells, mouse, Drosophila and zebrafish have provided some important insights into functional changes in sodium currents and firing properties associated with a number of SCN1A mutations. However, they also reveal that the same mutation can have distinct effects in the different models. By combining recent advances in stem cell reprogramming technology and gene editing tools it is now possible explore how specific gene mutations alter activity in human neurons. To evaluate changes in neuronal activity associated with a specific mutation, independent of genetic background, we generated two pairs of isogenic human iPSC lines by CRISPR/Cas9 editing. One pair is a control line from an unaffected sibling, and the mutated control homozygous for the GEFS+ K1270T SCN1A mutation. The second pair is a GEFS+ patient line heterozygous for the K1270T mutation, and the corrected patient line.

To detect mutation-associated changes by comparing electrophysiological properties between cell lines, it is necessary to differentiate iPSC into neurons that could both fire action potentials and form synaptic connections. While there were a number of protocols for differentiation of iPSCs into neurons in the literature, there was a lot of variability in the time course and degree of differentiation even from plating to plating within one cell line. Starting with the published protocols we identified conditions that support robust reproducible differentiation from plating to plating and between iPSCs lines into cultures with GABAergic and glutamatergic neurons. The iPSC-derived neuronal cultures from both isogenic pairs of iPSCs that we generated contained a similar proportion of GABAergic and glutamatergic neurons. By comparing the electrophysiological properties in inhibitory and excitatory iPSC-derived neurons from these pairs, we found the K1270T mutation causes gene dosage-dependent and cell type-specific alterations in evoked firing and sodium currents that result in hyperactive neural networks. We also identified differences associated with genetic background and interaction between the mutation and genetic background. Dual isogenic iPSC-derived neuronal cultures provide an efficient strategy to evaluate the causality of a single gene mutation independent of genetic background and to develop patient-specific anti-seizure therapies.