Department of Otolaryngology-Head and Neck Surgery, Shanghai Ninth People's Hospital, Shanghai Jiaotong University School of Medicine, No.639, Zhizaoju Road, Shanghai, 200011, PR China; Ear Institute, Shanghai Jiaotong University School of Medicine, No.115, Jinzun Road, Shanghai, 200011, PR China; Shanghai Key Laboratory of Translation Medicine on Ear and Nose Disease, No.115, Jinzun Road, Shanghai, 200011, PR China.
Ear Institute, Shanghai Jiaotong University School of Medicine, No.115, Jinzun Road, Shanghai, 200011, PR China; Shanghai Key Laboratory of Translation Medicine on Ear and Nose Disease, No.115, Jinzun Road, Shanghai, 200011, PR China; Biobank of Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, No.115, Jinzun Road, Shanghai, 200011, PR China.
Hear Res. 2020 Sep 1;394:107999. doi: 10.1016/j.heares.2020.107999. Epub 2020 Jun 18.
CRISPR/Cas9 gene editing enables the treatment of hearing loss in congenitally deaf neonatal mice via both viral and non-viral delivery. While adeno-associated virus (AAV)-mediated gene delivery systems have been shown to be effective tools for gene replacement in the inner ear, application of the AAV-mediated CRISPR/Cas9 gene-editing approach for this purpose is yet to be documented. Based on our previous findings, we focused on the effects of several AAVs delivered via canalostomy injection in adult mice. Among the AAVs examined, AAV8 showed the greatest efficiency and specificity in transducing inner hair cells (IHC). The ability of Cre-expressing AAV8 to activate Cas9 in floxed-Cas9 knock-in (Cas9 KI) mice was further evaluated. We compared the effects of six different promoters (CMV, CAG, hSyn, CaMKIIa, GFAP, and ALB) of AAV8 delivered to the inner ear of adult Cas9 KI mice. Our findings showed that three AAV groups (CMV, CAG and hSyn promoters) infected the inner ear efficiently with different tropisms. Notably, AAVs with CMV, CAG, and hSyn promoters infected diverse cell types in mature murine cochleae, including IHCs. In particular, AAV8-hSyn showed high affinity to IHCs and spiral ganglion neurons (SGN). Neither the AAV8 virus itself (except AAV8-CAG) nor the surgical procedures used caused damage to HCs or impaired normal hearing. Our findings indicated that injection of AAV-Cre into mature inner ear efficiently induces Cas9 activation to achieve safe and efficient gene editing and different constituent promoters confer diverse infection patterns in cochlea, expanding the repertoire of gene-editing tools for regulating gene expression in target cells of the inner ear as part of the collective effort to rescue genetic hearing loss and develop effective gene therapy techniques.
CRISPR/Cas9 基因编辑通过病毒和非病毒传递使先天性耳聋的新生小鼠的听力损失得到治疗。虽然腺相关病毒 (AAV) 介导的基因传递系统已被证明是内耳基因替代的有效工具，但 AAV 介导的 CRISPR/Cas9 基因编辑方法在这方面的应用尚未得到记录。基于我们之前的发现，我们专注于通过耳道注射传递的几种 AAV 在成年小鼠中的作用。在研究的 AAV 中，AAV8 在转导内毛细胞 (IHC) 方面显示出最高的效率和特异性。进一步评估了表达 Cre 的 AAV8 在 floxed-Cas9 敲入 (Cas9 KI) 小鼠中激活 Cas9 的能力。我们比较了 AAV8 的六种不同启动子 (CMV、CAG、hSyn、CaMKIIa、GFAP 和 ALB) 在成年 Cas9 KI 小鼠内耳传递的效果。我们的研究结果表明，三种 AAV 组（CMV、CAG 和 hSyn 启动子）以不同的亲嗜性有效地感染内耳。值得注意的是，具有 CMV、CAG 和 hSyn 启动子的 AAV 感染成熟鼠耳蜗中的多种细胞类型，包括 IHC。特别是，AAV8-hSyn 对内耳的 IHC 和螺旋神经节神经元 (SGN) 具有高亲和力。AAV8 病毒本身（除 AAV8-CAG 外）或使用的手术程序都不会对内耳的 HC 造成损害或损害正常听力。我们的研究结果表明，将 AAV-Cre 注射到成熟的内耳中可以有效地诱导 Cas9 激活，从而实现安全有效的基因编辑，并且不同的组成启动子在耳蜗中赋予不同的感染模式，扩大了内耳靶细胞中基因表达调控的基因编辑工具的范围，这是挽救遗传性听力损失和开发有效的基因治疗技术的集体努力的一部分。
