Promoting Protein Trafficking with 4-phenylbutyrate to Treat Genetic Epilepsy

用 4-苯基丁酸酯促进蛋白质运输来治疗遗传性癫痫

基本信息

批准号：
10579318
负责人：
Jing-Qiong Kang
金额：
$ 43.25万
依托单位：
VANDERBILT UNIVERSITY MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-03-01 至 2027-02-28
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10579318
关键词：
Acids Address Affect Alleles Astrocytes Biological Models Brain Calnexin Cell Line Cell model Cell surface Cells Child Childhood Clinical Trials Collaborations DNA Sequence Alteration Data Disease Disease Outcome Dominant-Negative Mutation Electroencephalography Epilepsy FDA approved Foundations Functional disorder Future GABA Transporter 1 Genes Genetic Goals Heterozygote Impairment In Vitro Induced pluripotent stem cell derived neurons Intellectual functioning disability Intervention Investigation Knock-in Mouse Letters Libraries Measures Mediating Membrane Microdialysis Molecular Chaperones Mus Mutation Neurodevelopmental Disorder Neurons Nonsense Mutation Pathway interactions Patients Pattern Phase Phenotype Phenylbutyrates Plasmids Pre-Clinical Model Proteins Public Health Research Research Design Research Methodology Seizures Surface Synaptosomes Syndrome Techniques Testing Translating Work autism spectrum disorder comorbidity design disease mechanisms study disease phenotype effective therapy endoplasmic reticulum stress extracellular gamma-Aminobutyric Acid high throughput screening improved in vivo induced pluripotent stem cell inhibitor inhibitory neuron insight interdisciplinary approach loss of function loss of function mutation misfolded protein mouse model mutant new therapeutic target novel pharmacologic phenotypic data pilot trial programs protein misfolding protein transport receptor repaired scale up small molecule success tiagabine trafficking uptake

项目摘要

Despite the discovery of hundreds of genetic mutations associated with epilepsy and neurodevelopmental comorbidities – including autism and intellectual disability (ID) – no effective treatments are currently available. Our recent work has identified the primary common mechanisms across mutations at a large scale in astrocytes and inhibitory neurons that express the GABA transporter 1 (GAT-1) encoding SLC6A1. Preliminary findings suggest 4-phenylbutyrate (PBA), a previously FDA-approved chaperone inducer for pediatric use, displays a mechanism-based rescue in those mutations. Specifically, PBA appears to repair mildly misfolded proteins and increase membrane expression of the wildtype allele across all tested mutations. Moreover, our success has prompted a pilot trial with very promising results. This study aims to fully characterize the effect and detailed mechanisms of PBA rescue. Our central hypothesis states that protein misfolding and impaired trafficking are standard mechanisms for SLC6A1 mutations, which display rescue potential through pharmacological intervention by restoring effective protein activity. As a part of our research, our lab has developed highly relevant preclinical model systems, including a plasmid library of SLC6A1 mutations, mutation- bearing patient cell lines, and knockin mouse models. Future studies focused on these model systems will provide critical insights into disease mechanisms with high potential of translating the findings to treatment. Moving forward, our lab aims to (1) evaluate the effect of PBA on restoring GAT-1 function in vitro for 20 patient mutations, (2) gauge the effect of PBA on restoring GAT-1 function in vivo in Slc6a1 mutation knockin mice, and (3) elucidate the underlying mechanism of PBA rescue on GAT-1 functioning to establish a foundation for novel treatment approaches. Research design and methods. We will employ a plasmid library containing >50 SLC6A1 mutations identified from patients across a wide spectrum of disease phenotypes, two knockin mice, and two lines of patient induced pluripotent stem cells (iPSCs) derived neurons and astrocytes to determine the impact of PBA on the mutant GAT-1 trafficking and function. We will utilize a multidisciplinary approach, including in vivo microdialysis, to determine the dynamic interplay of GAT-1 inhibition, GABA levels, and seizures in knockin mice. All patients carrying SLC6A1 mutations are heterozygous, which suggests there is benefit in either boosting the remaining wildtype allele or rescuing the mutant copy or both. In either case, the overall GABA uptake activity should be improved in patients. The large-scale study will provide us a broad view of the impact of PBA. In contrast, the in-depth investigation of mice and patient-derived cells will provide critical insights into PBA's rescue mechanism in partial and complete loss-of-function mutations. We propose to test this hypothesis in vitro and in vivo with high-throughput assays and cutting-edge new techniques in iPSCs and mutation knockin mice. Our goal is to identify a novel treatment target for genetic epilepsy using SLC6A1 as example. We believe the impact of this study is broad as it can be scaled up for many genetic epilepsy syndromes and others.

尽管发现了数百种与癫痫和神经发育相关的基因突变合并症 - 包括自闭症和智力残疾（ID） - 目前尚无有效治疗。我们最近的工作已经确定了大规模星形胶质细胞中突变的主要常见机制和表达编码SLC6A1的GABA转运蛋白1（GAT-1）的抑制性神经元。初步发现建议4-苯基丁酸（PBA），这是一种先前由FDA批准的伴侣诱导剂用于儿科使用，显示出A 这些突变中基于机制的救援。具体而言，PBA似乎可以修复轻度折叠的蛋白质和在所有测试突变中增加野生型等位基因的膜表达。而且，我们的成功已经促使试点审判具有非常有希望的结果。这项研究旨在充分表征效果和详细的效果 PBA救援机制。我们的中心假设指出，蛋白质折叠式错误和贩运障碍是SLC6A1突变的标准机制，通过通过恢复有效的蛋白质活性，药理干预。作为我们研究的一部分，我们的实验室有开发了高度相关的临床前模型系统，包括SLC6A1突变的质粒库，突变 - 轴承患者细胞系和敲击小鼠模型。这些模型系统的未来研究将提供对将发现转化为治疗的高潜力的疾病机制的关键见解。向前迈进，我们的实验室旨在（1）评估PBA对20名患者在体外恢复GAT-1功能的影响突变，（2）衡量PBA对SLC6A1突变敲击蛋白小鼠体内恢复GAT-1功能的影响，并且（3）在GAT-1上阐明PBA救援的基本机制，以建立新颖的基础治疗方法。研究设计和方法。我们将采用一个包含> 50的质粒库从各种疾病表型，两只敲门蛋白小鼠的患者中鉴定出的SLC6A1突变，两条患者诱导的多能干细胞（IPSC）衍生的神经元和星形胶质细胞确定 PBA对突变GAT-1运输和功能的影响。我们将利用一种多学科的方法，包括体内微透析，以确定GAT-1抑制，GABA水平和癫痫发作的动态相互作用敲击小鼠。所有携带SLC6A1突变的患者都是杂合的，这表明这两个患者都有好处增强剩余的野生型等位基因或营救突变副本或两者兼而有之。无论哪种情况，总体GABA 患者应改善摄取活性。大规模研究将为我们提供对影响的广泛看法 PBA。相反，对小鼠和患者衍生细胞的深入研究将为您提供关键的见解 PBA的救援机制是部分和完全功能丧失的突变。我们建议检验这一假设体外和体内具有高通量测定和IPSC中最尖锐的新技术和突变敲击素老鼠。我们的目标是确定使用SLC6A1作为例如遗传癫痫的新型治疗靶标。我们相信这项研究的影响很大，因为它可以针对许多遗传癫痫综合征和其他人进行扩展。