Effective gene therapy for gain-of-function or dominant-negative disease mutations may require eliminating expression of the mutant copy together with wild-type replacement. We evaluated such a knockdown-replace strategy in a mouse model of disease, a debilitating and intractable neurodevelopmental epilepsy. To challenge the approach robustly, we expressed a patient-based variant in GABAergic neurons—which resulted in growth delay and lethal seizures evident by postnatal week three—and delivered to newborn pups an AAV9-based vector encoding a ubiquitously expressed, -specific interfering RNA (RNAi) bivalently in tail-to-tail configuration with a neuron-specific, RNAi-resistant, codon-optimized cDNA. Pups receiving RNAi or cDNA alone fared no better than untreated pups, whereas the vast majority of mutants receiving modest doses survived with almost full growth recovery. Synaptic recordings of cortical neurons derived from treated pups revealed that significant alterations in transmission from inhibitory to excitatory neurons were rectified by bivalent vector application. To examine the mutant transcriptome and impact of treatment, we used RNA sequencing and functional annotation clustering. Mutants displayed abnormal expression of more than 1,000 genes in highly significant and relevant functional clusters, clusters that were abrogated by treatment. Together these results suggest knockdown-replace as a potentially effective strategy for treating and related genetic neurodevelopmental disease.

中文翻译：

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在新型 DNM1 发育性和癫痫性脑病小鼠模型中进行有效的敲除替换基因治疗


针对功能获得或显性失活疾病突变的有效基因治疗可能需要消除突变体副本的表达以及野生型替代。我们在小鼠疾病模型（一种使人衰弱且顽固的神经发育性癫痫）中评估了这种敲低替代策略。为了有力地挑战这种方法，我们在 GABA 能神经元中表达了一种基于患者的变异（这会导致出生后第三周出现明显的生长延迟和致命性癫痫发作），并向新生幼犬提供基于 AAV9 的载体，该载体编码普遍表达的特异性干扰 RNA (RNAi) 以尾对尾结构与神经元特异性、RNAi 抗性、密码子优化的 cDNA 进行二价连接。单独接受 RNAi 或 cDNA 的幼崽表现并不比未治疗的幼崽好，而接受适度剂量的绝大多数突变体都存活了下来，并且几乎完全恢复了生长。来自接受治疗的幼崽的皮层神经元的突触记录显示，通过二价载体应用可以纠正从抑制性神经元到兴奋性神经元的传递的显着变化。为了检查突变转录组和治疗的影响，我们使用了 RNA 测序和功能注释聚类。突变体在高度显着且相关的功能簇中表现出 1,000 多个基因的异常表达，这些簇通过治疗而被消除。总之，这些结果表明敲除替换是治疗相关遗传性神经发育疾病的潜在有效策略。