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相关的基因突变， 合并症-包括自闭症和智力残疾（ID）-目前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 和表达编码SLC 6A 1的GABA转运蛋白1（GAT-1）的抑制性神经元。初步调查结果 建议4-苯基丁酸（PBA），一种以前FDA批准用于儿科的伴侣诱导剂， 在这些突变中进行基于机制的拯救。具体来说，PBA似乎修复轻度错误折叠的蛋白质， 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 促使进行了一项试验性试验，取得了非常有希望的结果。本研究旨在充分描述效果，并详细 PBA拯救机制我们的中心假设指出，蛋白质错误折叠和受损的运输 是SLC 6A 1突变的标准机制，其通过以下途径显示出拯救潜力： 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SLC 6A 1突变的质粒文库，突变- 携带患者细胞系和敲入小鼠模型。未来的研究将集中在这些模型系统， 为疾病机制提供重要的见解，具有将发现转化为治疗的高潜力。 下一步，我们的实验室旨在（1）评估PBA对20例患者体外恢复GAT-1功能的作用 （2）测量PBA对Slc 6a 1突变敲入小鼠体内恢复GAT-1功能的作用，和 (3)阐明PBA对GAT-1功能的拯救的潜在机制，为新的 治疗方法。研究设计和方法。我们将使用含有>50个的质粒文库， 从广泛的疾病表型患者中鉴定出SLC 6A 1突变，两只敲入小鼠， 以及两个患者诱导的多能干细胞（iPSC）衍生的神经元和星形胶质细胞系，以确定神经元和星形胶质细胞的分化。 PBA对突变型GAT-1运输和功能的影响。我们将采用多学科方法，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， 敲入老鼠所有携带SLC 6A 1突变的患者都是杂合子，这表明在任何一种情况下都有益处。 加强剩余的野生型等位基因或拯救突变体拷贝或两者。无论哪种情况， 应改善患者的摄取活性。这项大规模的研究将为我们提供一个广泛的看法， 的PBA。相比之下，对小鼠和患者源性细胞的深入研究将为以下方面提供重要见解： PBA在部分和完全丧失功能突变中的拯救机制。我们建议检验这一假设 在体外和体内的高通量测定和尖端的新技术在iPSCs和突变敲入 小鼠我们的目标是以SLC 6A 1为例，确定一个新的遗传性癫痫治疗靶点。我们认为 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综合征。

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.