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——包括自闭症和智力障碍（ID）——目前尚无有效的治疗方法。我们最近的工作已经确定了星形胶质细胞大规模突变的主要共同机制和表达编码 SLC6A1 的 GABA 转运蛋白 1 (GAT-1) 的抑制性神经元。建议 4-苯基丁酸 (PBA)，一种先前 FDA 批准用于儿科的伴侣诱导剂，显示出具体来说，PBA 似乎可以修复轻度错误折叠的蛋白质和此外，我们的成功还增加了所有测试突变中野生型等位基因的膜表达。进行了一项试点试验，取得了非常有希望的结果。这项研究旨在充分描述其效果并详细描述。我们的中心假设是蛋白质错误折叠和运输受损。是 SLC6A1 突变的标准机制，通过以下方式显示出救援潜力：作为我们研究的一部分，我们的实验室通过恢复有效的蛋白质活性进行了药物干预。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- 未来的研究将集中于这些模型系统。提供对疾病机制的重要见解，具有将研究结果转化为治疗的巨大潜力。展望未来，我们实验室的目标是 (1) 评估 PBA 对 20 名患者体外恢复 GAT-1 功能的影响 (2) 评估 PBA 对 Slc6a1 突变敲入小鼠体内恢复 GAT-1 功能的影响，以及 (3)阐明PBA拯救GAT-1功能的根本机制，为新型药物奠定基础我们将采用包含> 50 个的质粒库。从多种疾病表型的患者（两只敲入小鼠）中鉴定出 SLC6A1 突变，以及两系患者诱导多能干细胞 (iPSC) 衍生的神经元和星形胶质细胞，以确定 PBA 对突变体 GAT-1 运输和功能的影响我们将利用多学科方法，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所有携带 SLC6A1 突变的患者都是杂合子，这表明两者都有好处。增强剩余的野生型等位基因或拯救突变体副本或两者兼而有之。大规模研究将为我们提供对影响的广泛认识。相比之下，对小鼠和患者来源的细胞的深入研究将为我们提供重要的见解。 PBA 在部分和完全功能丧失突变中的救援机制我们建议检验这一假设。体外和体内高通量检测以及 iPSC 和突变敲入方面的尖端新技术我们相信，我们的目标是以 SLC6A1 为例子，确定遗传性癫痫的新治疗靶点。这项研究的影响是广泛的，因为它可以扩大到许多遗传性癫痫综合征和其他疾病。