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

项目摘要 基因治疗是一种新兴的癫痫治疗策略，有望抑制特定的 癫痫发作相关的电路，以防止或减轻癫痫发作的强度。寻找合适的目标 对于临床前癫痫发作模型来说，递送是一个重大挑战。为了优化交付 参数、多种策略、位置和剂量必须进行比较。我们开发了一种新的筛选方法 一种使用光遗传学强度响应曲线来精确确定种群释放阈值的工具 （又名。发作间期尖峰），oPDT。一旦这个阈值是已知的，阈上刺激序列的变化 长度可用于确定后放电阈值，即癫痫发作敏感性的量度。这两 可以在相同的动物中收集度量、比较和跟踪。可预见的是， 行为状态（睡眠/清醒），但随时间稳定，允许受试者实验内的多次。一 海马和连接结构中的慢性多位点阵列允许检测网络范围内的 刺激反应以及持续监测正常活动。我们建议测试和优化两个 使用我们的光遗传学阈值技术，在非癫痫动物中， 以评估治疗潜力。Kv1.1在神经元中的过表达通过提高神经元的兴奋性来降低兴奋性。 激活和减少爆发产生的功能阈值。星形胶质细胞中Kir4.1的过度表达 提高其吸收细胞外K+的能力，防止K+积聚和导致的癫痫发作。在Aim中 1，我们将定位有效靶区，优化Kv1.1剂量，以平衡疗效 正常功能受损。我们的合作者Edward Perez-Reyes开发了一种AAV载体， 将被用于传输Kv1.1。基线活动、oPDT和oADT将随着时间的推移进行跟踪， 在表达发生前（<2周）、表达建立时（2-6周）和表达完成后（<2周）进行测量。 表达水平稳定（>6周）。这些指标随时间的变化将揭示重要信息 关于Kv1.1过表达的回路水平效应及其作为癫痫治疗的可行性。在目标2中， 将确定星形胶质细胞中Kir4.1的过度表达是否足以减少癫痫发作 易受感染性将使用对星形胶质细胞特异性的AAV载体（使用GFAP启动子）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， 治疗窗口，并提供有关人口放电和癫痫发作的性质的关键线索 一代

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.