CRISPRa-mediated Kcna1 upregulation decreases neuronal excitability and suppresses seizures in a rodent model of temporal lobe epilepsy [I]

Epilepsy is a major health burden, calling for new mechanistic and therapeutic insights. CRISPR–mediated gene editing shows promise to cure genetic pathologies, although hitherto it has mostly been applied ex-vivo. Its translational potential for treating non-genetic pathologies is still unexplored. Furthermore, neurological diseases represent an important challenge for the application of CRISPR, because of the need in many cases to manipulate gene function of neurons in situ. A variant of CRISPR, CRISPRa, offers the possibility to modulate the expression of endogenous genes by directly targeting their promoters. We asked if this strategy can be effective to treat acquired focal epilepsy. We applied a doxycycline-inducible CRISPRa technology to increase the expression of the potassium channel gene Kcna1 (encoding Kv1.1) in mouse hippocampal excitatory neurons. CRISPRa-mediated Kv1.1 upregulation led to a substantial decrease in neuronal excitability. Continuous video-EEG telemetry showed that AAV9-mediated delivery of CRISPRa, upon doxycycline administration, decreased spontaneous generalized tonic-clonic seizures in a model of temporal lobe epilepsy. The focal treatment minimizes concerns about off-targets effects in other organs and brain areas. This study provides the proof of principle for a translational CRISPR-based approach to treat neurological diseases characterized by abnormal circuit excitability. Overall design: 6x Epileptic treated with sgLacZ, 6x Epileptic treated with sgKcna1, and 5x Control treated with sgLacZ adult mouse hippocampi

癫痫是一类重大的健康负担，亟需在疾病机制与治疗策略层面获得新的认知与突破。成簇规律间隔短回文重复序列（CRISPR）介导的基因编辑技术在治愈遗传性病理状态方面展现出巨大潜力，但迄今为止其应用大多局限于离体（ex-vivo）操作。该技术在治疗非遗传性疾病领域的转化潜力尚未得到探索。此外，神经系统疾病是CRISPR应用的重要挑战，因为多数情况下需要对神经元的基因功能进行原位（in situ）调控。CRISPR的一个变体——CRISPR激活系统（CRISPRa）——可通过直接靶向内源基因的启动子来调控其表达水平。我们旨在探究该策略是否可有效治疗获得性局灶性癫痫。

我们采用多西环素（doxycycline）诱导型CRISPRa技术，在小鼠海马兴奋性神经元中提升钾通道基因Kcna1（编码蛋白Kv1.1）的表达量。CRISPRa介导的Kv1.1上调可显著降低神经元兴奋性。连续视频脑电图（video-EEG）遥测结果显示，在颞叶癫痫模型中，通过腺相关病毒9型（AAV9）递送CRISPRa系统并给予多西环素后，自发性全面性强直-阵挛发作的发生率显著降低。该局域性治疗策略可最大程度降低其他器官与脑区出现脱靶效应的风险。本研究为基于CRISPR的转化疗法治疗以异常神经元环路兴奋性为特征的神经系统疾病提供了原理验证。

整体实验设计：6只经sgLacZ处理的癫痫模型小鼠、6只经sgKcna1处理的癫痫模型小鼠，以及5只经sgLacZ处理的对照成年小鼠的海马组织样本