Evaluating Gene Therapy Strategies to Treat Epilepsy Using a Novel Optogenetic Measure of Network Excitability and Seizure Susceptibility

使用网络兴奋性和癫痫易感性的新型光遗传学测量方法评估治疗癫痫的基因治疗策略

• 批准号: 10057595
• 负责人: DWAYNE W GODWIN
• 金额: $ 20.21万
• 依托单位: WAKE FOREST UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-15 至 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10057595
• 关键词: Ablation; Action Potentials; Animals; Area; Astrocytes; Behavior; Behavioral; Brain; Buffers; Cells; Chronic; Cognitive; Consequentialism; Custom; Detection; Diagnosis; Dose; Epilepsy; Epileptogenesis; Equilibrium; Excision; Gap Junctions; Generations; Glial Fibrillary Acidic Protein; Hippocampus (Brain); Impairment; Implant; Individual; Intervention; Knowledge; Length; Lesion; Location; Measurement; Measures; Methods; Mission; Modeling; Monitor; Mus; Nature; Neurons; Organism; Outcome; Pharmacology; Physiology; Pilocarpine; Population; Potassium; Potassium Channel; Predisposition; Prevention; Probability; Production; Public Health; Recurrence; Research; Response to stimulus physiology; Screening procedure; Seizures; Site; Sleep; Speed; Status Epilepticus; Stimulus; Structure; Supporting Cell; Synapses; System; Techniques; Testing; Therapeutic; Therapeutic Intervention; Time; Training; United States National Institutes of Health; Voltage-Gated Potassium Channel; Work; adeno-associated viral vector; base; clinical application; design; experimental study; extracellular; functional disability; gene therapy; improved; nervous system disorder; novel; optogenetics; overexpression; pre-clinical; pre-clinical research; prevent; promoter; recruit; relating to nervous system; response; side effect; success; targeted delivery; targeted treatment; therapeutic gene; treatment optimization; treatment strategy; uptake

Project Summary Gene therapy is an emerging treatment strategy for epilepsy that promises to dampen activity in specific seizure related circuitry in order to prevent or lessen the intensity of seizures. Finding the appropriate target for delivery represents a significant challenge for preclinical seizure models. In order to optimize delivery parameters, multiple strategies, locations, and doses must be compared. We have developed a novel screening tool that uses optogenetic intensity-response curves to precisely determine thresholds for population discharge (aka. interictal spikes), the oPDT. Once this threshold is known, suprathreshold stimulus trains of varying length can be used to determine an after discharge threshold, a measure of seizure susceptibility. These two metrics can be collected in the same animals, compared, and tracked. Thresholds vary predictably with behavioral state (sleep/wake), but are stable over time allowing for multiple within subject experiments. A chronic multi-site array in hippocampus and connected structures allows for detection of network wide stimulus responses and also continuous monitoring of normal activity. We propose to test and optimize two promising gene therapy strategies using our optogenetic thresholding technique, in non-epileptic animals, in order to assess therapeutic potential. Kv1.1 overexpression in neurons reduces excitability by raising the functional threshold for activation and decreasing burst production. Kir4.1 overexpression in astrocytes improves their ability to absorb extracellular K+, preventing K+ build up and the resulting ictogenesis. In Aim 1, we will locate effective target areas and optimize the dose of Kv1.1 in order to balance efficacy with impairment of normal function. An AAV vector developed by our collaborator Edward Perez-Reyes will be used to deliver Kv1.1. Baseline activity, the oPDT, and the oADT will be tracked over time with multiple measurements taken before expression occurs (<2 weeks), while expression builds (2-6 weeks), and when expression levels stabilize (>6 weeks). Changes in these metrics over time will reveal important information about the circuit level effects of Kv1.1 overexpression and its viability as a treatment for epilepsy. In Aim 2, we will determine if Kir4.1 overexpression in astrocytes is sufficient to reduce seizure suseptability. An AAV vector specific for astrocytes (using the GFAP promoter), will be used to overexpress Kir4.1. Success in these experiments will help to identify potential targets for therapeutic intervention, assess the therapeutic window, and provide critical clues about the nature of population discharge and seizure generation.

项目摘要 基因治疗是一种新兴的癫痫治疗策略，有望抑制特定疾病的活动。 癫痫发作相关的回路，以防止或减轻癫痫发作强度。找到合适的目标 对于临床前癫痫模型来说，这是一个巨大的挑战。为了优化交付 必须对参数、多种策略、位置和剂量进行比较。我们已经开发出一种新的筛选器 使用光遗传强度-响应曲线精确确定种群放电阈值的工具 (又名。发作间期棘波)，OPDT。一旦知道这个阈值，各种不同的阈值上的刺激序列 长度可以用来确定后放电阈值，这是一种衡量癫痫敏感度的指标。这两个 可以在相同的动物身上收集、比较和跟踪指标。阈值可预测地随 行为状态(睡眠/清醒)，但随着时间的推移保持稳定，允许在受试者内进行多次实验。一个 海马体和连接结构中的慢性多点阵列允许检测整个网络 刺激反应以及对正常活动的持续监测。我们建议测试和优化两个 在非癫痫动物中，使用我们的光遗传阈值技术进行基因治疗的策略前景看好 以评估治疗潜力。Kv1.1在神经元中的过表达通过增加 激活和减少突发性生产的功能阈值。KIR4.1在星形胶质细胞中的过表达 提高他们吸收细胞外K+的能力，防止K+积聚和由此导致的细胞分裂。在AIM 1、我们将定位有效靶区，优化Kv1.1的剂量，以平衡疗效 并有正常功能损害。由我们的合作者Edward Perez-Reyes开发的AAV载体 将用于交付Kv1.1。基准活动、oPDT和oADT将随着时间的推移通过 在表达发生前(&lt；2周)、在表达建立时(2-6周)以及何时进行测量 表达水平稳定(&gt；6周)。随着时间的推移，这些指标的变化将揭示重要信息 关于Kv1.1过表达的电路水平效应及其作为癫痫治疗的可行性。在目标2中，我们 将确定Kir4.1在星形胶质细胞中的过度表达是否足以减少癫痫发作 疑虑重重。针对星形胶质细胞的AAV载体(使用GFAP启动子)将用于过表达 KIR4.1。这些实验的成功将有助于确定治疗干预的潜在靶点，评估 治疗窗口，并提供有关人群放电和癫痫发作性质的关键线索 一代。