耳聋是人类最常见的感官或功能缺陷性疾病，单基因突变导致的遗传性聋占语前聋的一半以上。申请人前期与美国Emory大学的林曦课题组的合作研究成果表明，通过中阶显微注射将腺相关病毒（AAV）包装的正常基因导入相应基因纯合缺失小鼠模型的耳蜗中，可以观察到正常基因的表达，并部分恢复小鼠听力，表明腺相关病毒可以作为一种合适的载体进行基因治疗。本项目中我们将采用AAV为载体，通过CRISPR/Cas9基因组定点编辑技术在显性突变的遗传性聋小鼠模型中敲除具有显性负效应的突变等位基因，探索遗传性迟发型聋的基因治疗策略。建立适用于耳蜗各种细胞基因组定点编辑的AAV-CRISPR/Cas9基因治疗系统；对采用AAV-CRISPR/Cas9系统敲除显性突变的遗传性聋小鼠模型进行耳蜗细胞形态和功能研究以及小鼠听力测试；为建立把腺相关病毒转染和CRISPR/Cas9技术有机结合的内耳基因治疗系统奠定理论和实验基础。

Hearing loss is the most common human sensory disorder; more than half cases of pre-lingual hearing loss are caused by monogenic mutations. Joint studies of professor Xi Lin from Emory University and the applicant found that inoculation of modified adeno-associated virus (AAV) construct carrying a normal gene expression cassette into the scala media resulted in efficient on-target expression in the cochlea and a preserved hearing of the gene knockout mice. These results suggested that AAV could serve as a potent tool for developing gene therapy for genetic deafness in humans. In this project, we planned to develop a novel gene therapy approach for dominant-negative mutations associated with age related genetic hearing loss combining AAV and CRISPR/Cas9 genome editing techniques. We will first develop an AAV-CRISPR/Cas9 gene therapy system targeting different cell types in the cochlea. And then we will evaluate the specificity and efficiency of this AAV-CRISPR/Cas9 gene therapy system. Last, we will apply this AAV-CRISPR/Cas9 system to further investigate the gene therapy strategy for the treatment of monogenic mutation induced pre-lingual age related hearing loss by functional study of the treated cochlea and by testing hearing preservation of treated mice. This project will establish theoretical basis and provide experimental data that promote gene therapy for age related hearing loss.