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 基因疗法是一种新兴癫痫的治疗策略，有望在特定方面抑制活性 癫痫发作相关的电路以防止或减少癫痫发作的强度。找到适当的目标 对于递送，代表了临床前癫痫模型的重大挑战。为了优化交付 必须比较参数，多种策略，位置和剂量。我们已经开发了一个新颖的筛选 使用光遗传强度响应曲线来精确确定人口排出阈值的工具 （又名插座尖峰），opdt。一旦知道了这个阈值 长度可用于确定出院阈值，这是癫痫发作敏感性的量度。这两个 可以在同一动物中收集指标，并进行比较和跟踪。阈值随着阈值而异 行为状态（睡眠/唤醒），但随着时间的流逝稳定，可以进行多个主题实验。一个 海马和连接结构中的慢性多站点阵列可检测网络范围 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 使用我们的光遗传学阈值技术，非癫痫动物中的有前途的基因治疗策略， 为了评估治疗潜力。 KV1.1神经元中的过表达可通过提高兴奋性来降低兴奋性 激活和爆发产生减少的功能阈值。 Kir4.1星形胶质细胞的过表达 提高了它们吸收细胞外K+的能力，以防止K+堆积并产生的刻有生成。目标 1，我们将定位有效的目标区域并优化KV1.1的剂量以平衡疗效 正常功能受损。由我们的合作者爱德华·佩雷斯·里耶斯（Edward Perez-Reyes）开发的AAV矢量 将用于交付KV1.1。基线活动，OPDT和OADT将随着时间的流逝而随着多个 在表达之前进行的测量（<2周），而表达构建（2-6周），当 表达水平稳定（> 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过表达的电路水平及其生存能力作为癫痫的治疗方法。在AIM 2中，我们 将确定星形胶质细胞中的Kir4.1是否足以减少癫痫发作 令人震惊的性。特定于星形胶质细胞的AAV矢量（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目标，评估 治疗窗口，并提供有关人口排出和癫痫发作性质的关键线索 一代。