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Project 2 - Investigation of human neuron models of channelopathy-associated epilepsy

项目 2 - 通道病相关癫痫的人类神经元模型的研究

基本信息

批准号：
10247557
负责人：
Evangelos Kiskinis
金额：
$ 36.84万
依托单位：
NORTHWESTERN UNIVERSITY AT CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-09-30 至 2023-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10247557
关键词：
Aftercare Agonist Antiepileptic Agents Biological Models Biological Sciences Biophysics CRISPR/Cas technology Cell Line Cells Clinical Collaborations Collection Coupled Defect Development Disease Drug Screening Effectiveness Electrophysiology (science)Epilepsy Evaluation Functional disorder Gene Expression Profiling Generations Genes Genetic Glutamates Goals Human In Vitro Induced pluripotent stem cell derived neurons Interneurons Investigation Ion Channel Leadership Link Measures Methods Modeling Molecular Mutation Neonatal Neurologic Neurological Models Neuronal Differentiation Neurons Optics Outcome Pathogenicity Patient Recruitments Patient Selection Patients Persons Pharmaceutical Preparations Pharmacology Pharmacology Study Pharmacotherapy Physiology Positioning Attribute Property Refractory Regimen Research Seizures Severities Syndrome Technology Variant Work channel blockers clinical efficacy early onset epileptic encephalopathies excitatory neuron experimental study genetic variant genome editing improved in vivo induced pluripotent stem cell induced pluripotent stem cell technology inhibitory neuron innovation mouse model nervous system disorder neurophysiology optimal treatments optogenetics patch clamp prevent response single-cell RNA sequencing stem cell based approach success targeted treatment tool voltage voltage clamp

项目摘要

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.

在项目2中，我们将确定癫痫相关离子通道基因变异的功能后果