dCas9-Based Scn1a Gene Activation Restores Inhibitory Interneuron Excitability and Attenuates Seizures in Dravet Syndrome Mice
dCas9-Based Scn1a Gene Activation Restores Inhibitory Interneuron Excitability and Attenuates Seizures in Dravet Syndrome Mice
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DOI：
10.1016/j.ymthe.2019.08.018
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发表时间：
2020-01-08
影响因子：
12.4
通讯作者：
Broccoli, Vania
中科院分区：
文献类型：
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作者：
Colasante, Gaia;Lignani, Gabriele;Broccoli, Vania
Dravet综合征（DS）是一种严重的癫痫性脑病，主要由SCN 1A基因的杂合性功能丧失突变引起，表明单倍不足是致病机制。在这里，我们测试了催化死亡的Cas9（dCas 9）介导的Scn 1a基因激活是否可以在小鼠DS模型中拯救Scn 1a单倍不足，并恢复其基因产物Nav1.1电压门控钠通道的生理水平。我们筛选了单向导RNA（sgRNA），以确定它们刺激Scn 1a转录与dCas 9激活系统相关的能力。我们鉴定了一种特异性sgRNA，其以高特异性增加细胞系和原代神经元中的Scn 1a基因表达水平。Na（v）1.1蛋白水平增加，野生型未成熟GABA能中间神经元激发动作电位的能力也增加。在成熟的DS中间神经元中实现了类似的Scn 1a转录增强，挽救了它们的放电能力。为了测试这种方法的治疗潜力，我们使用腺相关病毒将Scn 1a-dCas 9激活系统递送给DS幼仔。小白蛋白中间神经元恢复其放电能力，热性惊厥明显减弱。我们的研究结果为利用基于dCas 9的基因激活作为治疗DS和其他由基因剂量改变引起的疾病的有效和靶向方法铺平了道路。
Dravet syndrome (DS) is a severe epileptic encephalopathy caused mainly by heterozygous loss-of-function mutations of the SCN1A gene, indicating haploinsufficiency as the pathogenic mechanism. Here we tested whether catalytically dead Cas9 (dCas9)-mediated Scn1a gene activation can rescue Scn1a haploinsufficiency in a mouse DS model and restore physiological levels of its gene product, the Nav1.1 voltage-gated sodium channel. We screened single guide RNAs (sgRNAs) for their ability to stimulate Scn1a transcription in association with the dCas9 activation system. We identified a specific sgRNA that increases Scn1a gene expression levels in cell lines and primary neurons with high specificity. Na(v)1.1 protein levels were augmented, as was the ability of wild-type immature GABAergic interneurons to fire action potentials. A similar enhancement of Scn1a transcription was achieved in mature DS interneurons, rescuing their ability to fire. To test the therapeutic potential of this approach, we delivered the Scn1a-dCas9 activation system to DS pups using adeno-associated viruses. Parvalbumin interneurons recovered their firing ability, and febrile seizures were significantly attenuated. Our results pave the way for exploiting dCas9-based gene activation as an effective and targeted approach to DS and other disorders resulting from altered gene dosage.