Modeling gene-specific therapy of intractable childhood epileptic encephalopathy

项目摘要 早发性耐药性癫痫脑病(EE)通常由新基因变异引起 神经元蛋白基因。Dynamin 1(由DNM1编码)，一个关键的神经元特异性GTP酶，参与了 内吞是该病及其难治性的典型例子，由此引起的 显性-负性错义突变导致难治性癫痫发作、发育迟缓和认知 减损。我们研究了DNM1EE的Dnm1Ftfl模型，它模拟了关键的疾病特征和 为开发和测试基因疗法提供了一个引人注目的平台。 对于DNM1和其他基因EE来说，突变发挥基因显性效应，提供 突变产物的野生型拷贝预计益处有限(如果有的话)。因此，取消或更改 是一种更合乎逻辑的方法。我们已经开始模拟显性负基因沉默 穿梭于神经营养自补充腺相关病毒(AAV)中的microRNA治疗 利用Dnm1Ftfl小鼠模型，治疗遗传显性EE的载体。我们假设 这种方法将挽救核心疾病的表型，包括致命的癫痫发作和主要的合并症以及 提供更持久的治疗选择。 脑室注射新生小鼠治疗纯合子小鼠阵发性发作的初步治疗 发育缺陷，减少与癫痫相关的严重致命性，并延长寿命。来推动这一进程 在项目的推进中，我们的目标是：1)优化我们对阵发性小鼠疾病定义的核心表型的救援 走向全面抢救；2)确定最终导致核心的神经元结构和功能缺陷 表型；以及3)评估scAAV9-miDnm1a对神经元结构和功能缺陷的挽救 治疗。这些目标的成功完成将为这两种癫痫的可能治疗提供信息 表型和相关的发育延迟，同时为其他类似的EE提供了一种普遍的方法 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.