Profiling and Engineering the Ion Channel Transcriptome

About This Grant

Project Summary In the human brain, a large repertoire of ion channels regulates the excitability of neurons, circuits, and networks, generating complex human cognition and behavior. Most ion channels are encoded by long, multi- exon genes which undergo extensive alternative splicing. Dysregulated splicing can alter ion channel function and is implicated in disorders ranging from autism to epileptic encephalopathy. Specific splicing events in ion channels have been shown to be critical for brain development and homeostasis, such as the neonatal-to-adult splicing switch in voltage-gated sodium channels. Splice-modulating therapeutics, such as antisense oligonucleotides, have shown efficacy for multiple neurologic disorders including spinal muscular atrophy, Duchenne muscular dystrophy, and Dravet syndrome. As potential targets for both small molecules and RNA therapeutics, ion channels are particularly important candidates for the treatment of neurologic disorders. However, there has never been a systematic study of alternative splicing of ion channels to-date. This proposal applies two innovative methods to profile the ion channel transcriptome and study the functional impact of alternative splicing on neuronal physiology. Aim 1 combines long-read RNA-sequencing and transcript capture technology to comprehensively identify and annotate channel isoforms in the human cerebral cortex. Bioinformatic tools will be used to uncover ion channel isoforms which are differentially regulated during postnatal brain development. Aim 2 employs splice modulation technology to study the functional impact of alternative splicing on neuronal physiology, using the epilepsy-associated KCNMA1 gene as proof of concept. A Cas-based toolkit is developed for human stem cell-derived neurons to manipulate the splicing of KCNMA1, and whole-cell patch-clamp physiology measures the impact of splice modulation on neuronal excitability. Taken together, this proposal will generate the most comprehensive profile of ion channel isoforms to-date, uncover developmentally regulated splicing events that can be potentially targeted by RNA therapeutics, and demonstrate proof of concept for transcriptome engineering in human neurons. These skills and resources will catalyze my career as an independent researcher, which will focus on the study of alternative splicing and the development of splice-modulating therapeutics for epilepsy and other neurologic disorders.