Promoting Protein Trafficking with 4-phenylbutyrate to Treat Genetic Epilepsy

尽管发现了数百种与癫痫和神经发育有关的基因突变 共病--包括自闭症和智力残疾(ID)--目前还没有有效的治疗方法。 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 以及表达编码SLC6A1的GABA转运蛋白1(GAT-1)的抑制神经元。初步调查结果 建议的4-苯丁酸酯(PBA)，一种先前FDA批准的儿科伴侣诱导剂，显示出 在这些突变中进行基于机制的拯救。具体地说，PBA似乎修复轻微错误折叠的蛋白质和 在所有测试的突变中增加野生型等位基因的膜表达。此外，我们的成功已经 促使进行了试点试验，取得了非常有希望的结果。这项研究旨在充分表征这一效果并详细说明 PBA救援机制。我们的中心假说是蛋白质错误折叠并阻碍运输 是SLC6A1突变的标准机制，通过 通过恢复有效的蛋白质活性进行药物干预。作为我们研究的一部分，我们的实验室 开发了高度相关的临床前模型系统，包括SLC6A1突变、突变- 承载患者细胞系，并敲打小鼠模型。未来专注于这些模型系统的研究将 提供对疾病机制的关键见解，具有将发现转化为治疗的高潜力。 展望未来，我们实验室的目标是(1)评估PBA在体外恢复20例患者GAT-1功能的效果 突变，(2)评估PBA在SLC6a1突变敲击小鼠体内恢复GAT-1功能的作用，以及 (3)阐明PBA拯救GAT-1的潜在机制，为以后的研究奠定基础 治疗方法正在接近。研究设计与方法。我们将使用包含&gt；50的质粒库 SLC6A1突变从多种疾病表型的患者，两只敲门小鼠， 和两株患者诱导的多能干细胞(IPSCs)来源的神经元和星形胶质细胞来确定 PBA对突变型GAT-1转运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 诺克金老鼠。所有携带SLC6A1突变的患者都是杂合子，这表明无论是哪种情况都有好处 增强剩余的野生型等位基因或挽救突变副本，或两者兼而有之。在任何一种情况下，总体GABA 患者的摄取活动应得到改善。这项大规模的研究将使我们对影响有一个广泛的了解。 PBA的。相比之下，对小鼠和患者来源细胞的深入研究将提供对 PBA在部分和完全功能丧失突变中的挽救机制。我们建议检验这一假设 IPSCs和突变敲击的高通量检测和前沿新技术在体外和体内的应用 老鼠。我们的目标是以SLC6A1为例确定遗传性癫痫的新治疗靶点。我们相信 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。

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.