Mutations in microRNA-96 ( MIR96 ) cause autosomal dominant deafness-50 (DFNA50), a form of delayed-onset hearing loss. Genome editing has shown efficacy in hearing recovery through intervention in neonatal mice, yet editing in the adult inner ear is necessary for clinical applications, which has not been done. Here, we developed a genome editing therapy for the MIR96 mutation 14C>A by screening different CRISPR systems and optimizing Cas9 expression and the sgRNA scaffold for efficient and specific mutation editing. AAV delivery of the KKH variant of Staphylococcus aureus Cas9 (SaCas9-KKH) and sgRNA to the cochleae of presymptomatic (3-week-old) and symptomatic (6-week-old) adult Mir96 14C>A/+ mutant mice improved hearing long term, with efficacy increased by injection at a younger age. Adult inner ear delivery resulted in transient Cas9 expression without evidence of AAV genomic integration, indicating the good safety profile of our in vivo genome editing strategy. We developed a dual-AAV system, including an AAV-sgmiR96-master carrying sgRNAs against all known human MIR96 mutations. Because mouse and human MIR96 sequences share 100% homology, our approach and sgRNA selection for efficient and specific hair cell editing for long-term hearing recovery lay the foundation for the development of treatment for patients with DFNA50 caused by MIR96 mutations.

中文翻译：

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靶向基因组编辑可恢复因人类 microRNA 突变导致进行性听力损失的成年小鼠的听觉功能


突变在微小RNA-96 （米尔96 ）导致常染色体显性耳聋 50 (DFNA50)，这是一种迟发性听力损失。通过对新生小鼠进行干预，基因组编辑已显示出对听力恢复的功效，但成人内耳的编辑对于临床应用来说是必要的，但目前尚未完成。在这里，我们开发了一种基因组编辑疗法米尔96通过筛选不同的 CRISPR 系统并优化 Cas9 表达和 sgRNA 支架，实现高效、特异的突变编辑，从而实现 14C>A 突变。 AAV 交付 KKH 变种金黄色葡萄球菌Cas9 (SaCas9-KKH) 和 sgRNA 对症状前（3 周龄）和有症状（6 周龄）成人耳蜗的影响和平号96 14C>A/+突变小鼠的听力长期得到改善，在年轻时注射可提高疗效。成人内耳递送导致短暂的 Cas9 表达，没有 AAV 基因组整合的证据，表明我们的体内基因组编辑策略具有良好的安全性。我们开发了一个双 AAV 系统，包括一个 AAV-sgmiR96-master，其携带针对所有已知人类的 sgRNA米尔96突变。因为老鼠和人类米尔96序列具有 100% 同源性，我们的方法和 sgRNA 选择用于高效和特异性毛细胞编辑以实现长期听力恢复，为开发由 DFNA50 引起的 DFNA50 患者的治疗奠定了基础米尔96突变。