Research team
Expertise
This research requires the culturing of human pluripotent stem cells (hPSCs) and differentiation into excitatory cortical-like neurons. Spontaneous, electrical- and drug-induced neuronal activity is recorded using a high-density microelectrode array (MEA). Outputs of MEA experiments are analyzed with statistical programs in GraphPad Prism and R scripts. Antisense oligonucleotide are designed using online primers design tools including IDT OligoAnalyzer™ Tool and manually curated in SnapGene. Knockdown of hPSC KCNQ2 is measured by SYBRgreen and Taqman probe qPCR. Live imaging hPSC neuron experiments are performed on an Incucyte S3.
A novel in vitro antisense oligonucleotide neurotoxicity screen.
Abstract
Antisense oligonucleotide (ASO) gene therapy is increasingly being investigated for treatment of neurological disorders. However, recently, an acute in vivo neurotoxicity has been described in a subset of treatments. The mechanism of this toxicity is poorly understood but in vitro studies on rodent neurons implicates ASO-mediated AMPA receptor inhibition causing a reduction in intracellular free calcium. My proposal aims to address the knowledge gap connecting reduced intracellular calcium to in vivo toxicity, in a first human neuron (iPSC) model. Based on preliminary data I have generated, I have identified ASO toxicity in synchronized neuronal network bursting patterns measured using a cutting edge high-density microelectrode array. My proposal will generate much needed replicates describing in vitro, intracellular calcium reduction and provide a first assay that can longitudinally measure high-resolution timecourse of neuronal function when exposed to known toxic ASOs. Understanding the mechanism of toxicity is an important step towards improving future drug designs and developing high-resolution in vitro assays will be an important toxicity screening tool for selecting lead compounds for in vivo testing.Researcher(s)
- Promoter: Kaji Marcus
Research team(s)
Project type(s)
- Research Project
Electrophysiological and pharmacological characterization of human iPSC derived neuronal networks using a high-density microelectrode array.
Abstract
Pathogenic variants of KCNQ2 and KCNQ3 (KCNQ2/3) leads to a spectrum of disorders ranging from temporary neonatal seizures to epilepsy with lifelong severe intellectual disability. These genes encode voltage-gated potassium channels (Kv7.2/3) expressed in the central nervous system that regulate neuronal excitability. Over 190 variants have been identified, and channel function in non-neuronal heterologous systems has been described for the most clinically relevant of these variants. However, there is a knowledge gap between Kv7.2/3 dysfunction and the mechanisms by which they affect neurodevelopment. I propose to use human induced pluripotent stem cell-derived neurons carrying gain of function (GoF) and loss of function (LoF) KCNQ2/3 variants to test the hypothesis that pathogenic variants in KCNQ2/3 lead to aberrant neuronal network development. Using a state-of-the-art high-density microelectrode array (MEA), I will compare the electrophysiology between KCNQ2/3 variants in exclusively excitatory, and mixed excitatory:inhibitory neuronal networks. To test the ability of this in vitro MEA system for screening drugs, I will a) assess efficacy of channel activators and blockers and b) design novel antisense oligonucleotides as a targeted knockdown approach for GoF channelopathy. This proposal will provide a tunable system to study neurodevelopment, validate its potential for high-throughput drug screens, and develop lead compounds for treating Kv7.2/3 channelopathy.Researcher(s)
- Promoter: Weckhuysen Sarah
- Fellow: Kaji Marcus
Research team(s)
Project type(s)
- Research Project