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 DNM1 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, Dnm1-specific interfering RNA (RNAi) bivalently in tail-to-tail configuration with a neuron-specific, RNAi-resistant, codon-optimized Dnm1 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 DNM1 and related genetic neurodevelopmental disease.

中文翻译：

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DNM1 发育性和癫痫性脑病新型小鼠模型中的有效敲低替代基因治疗


针对功能获得性或显性阴性疾病突变的有效基因治疗可能需要消除突变拷贝的表达以及野生型替换。我们在 DNM1 疾病（一种使人衰弱且顽固的神经发育性癫痫）的小鼠模型中评估了这种敲低替代策略。为了有力地挑战该方法，我们在 GABA 能神经元中表达了一种基于患者的变异——这导致出生后第 3 周出现明显的生长延迟和致命性癫痫发作——并将基于 AAV9 的载体传递给新生幼仔，该载体编码普遍表达的 Dnm1 特异性干扰 RNA （RNAi） 以尾对尾配置二价与神经元特异性、RNAi 抗性、密码子优化的 Dnm1 cDNA。单独接受 RNAi 或 cDNA 的幼仔并不比未经治疗的幼仔好，而绝大多数接受适度剂量的突变体存活下来，几乎完全恢复了生长。来自治疗幼崽的皮层神经元的突触记录显示，二价载体应用纠正了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。突变体在高度显著和相关的功能簇中表现出 1,000 多个基因的异常表达，这些簇被治疗消除。总之，这些结果表明敲低替代是治疗 DNM1 和相关遗传性神经发育疾病的潜在有效策略。