Modeling gene-specific therapy of intractable childhood epileptic encephalopathy

项目摘要 早发性药物耐药性癫痫性脑病（EE）通常是由新生变异引起的， 神经元蛋白基因发动蛋白1（由DNM 1编码），一种关键的神经元特异性GT3，参与 内吞作用是疾病及其难治性的典型例子， 显性负性错义突变导致顽固性癫痫发作、发育迟缓和认知障碍。 损伤我们研究了DNM 1 EE的Dnm 1Ftfl（“fitful”）模型，该模型模拟了关键疾病特征， 为开发和测试基因疗法提供了一个引人注目的平台。 对于DNM 1和其他遗传EE，其中突变产生遗传显性效应，提供了 预期突变产物的野生型拷贝具有有限的益处（如果有的话）。因此，消除或改变 是一个更合理的方法我们已经开始建立显性负性基因沉默模型 通过微RNA穿梭于神经营养性自身互补腺相关病毒（AAV）的治疗 用于治疗遗传显性EE的载体，利用Dnm 1Ftfl小鼠模型。我们假设 这种方法将挽救核心疾病表型，包括致命性癫痫发作和主要合并症， 提供一种更持久的治疗选择 通过脑室内新生儿注射对纯合子间歇性小鼠进行初步治疗， 发育缺陷，降低严重的糖尿病相关的致死性，并延长寿命。为了推动这个 项目前进，我们的目标是：1）优化我们的拯救疾病定义的核心表型的断断续续的小鼠 2）识别在核心中达到高潮的神经元结构和功能缺陷 表型;和3）评估用scAAV 9-miDnm 1a拯救神经元结构和功能缺陷 治疗这些目标的成功完成将为癫痫发作的可能治疗提供信息。 表型和相关的发育迟缓，同时为其他类似的EE提供了一种可推广的方法 模型，为未来的临床应用提供信息。

Project Summary Early onset pharmacoresistant epileptic encephalopathies (EE) are often caused by de novo variants in neuronal protein genes. Dynamin 1 (encoded by DNM1), a key neuron-specific GTPase involved in endocytosis, is a prototypical example of both the disease and its intractable nature, whereby pathogenic dominant-negative missense mutations cause intractable seizures, developmental delays and cognitive impairment. We study the Dnm1Ftfl (“fitful”) model of DNM1 EE, which mimics key disease features and provides a compelling platform to develop and test gene therapies. For DNM1 and other genetic EE where the mutation exerts a genetically dominant effect, supplying the wildtype copy of the mutated product is expected to be of limited, if any, benefit. Thus, elimination or alteration of the mutated product is a more logical recourse. We have begun to model dominant negative gene silencing therapy via microRNAs shuttled through neurotrophic self-complementary adeno-associated viral (AAV) vectors for the treatment of genetically dominant EE, utilizing the Dnm1Ftfl mouse model. We hypothesize that this approach will rescue the core disease phenotypes, including lethal seizures and major comorbidities and provide a more permanent therapy option. Preliminary treatment of homozygous fitful mice via intracerebroventricular neonatal injection, remedied developmental deficits, decreased severe seizure-associated lethality, and extended lifespan. To propel this project forward, our aims are to: 1) optimize our rescue of the disease-defining core phenotypes of fitful mice towards full rescue; 2) identify neuronal structural and functional defects that culminates in the core phenotypes; and 3) assess rescue of neuronal structural and functional defects with scAAV9-miDnm1a treatment. Successful completion of these aims will inform on a possible treatment for both the seizure phenotype, and associated developmental delays while providing a generalizable approach for other similar EE models, informing future clinical applications.