Development of Genome Editing as Treatment for Genetic Hearing Loss

项目摘要和相关性 遗传性听力损失影响每1000名新生儿中就有一名，对一般听力损失人群的影响很大。 目前已经定位了130多个耳聋基因座，克隆了80多个耳聋基因座。尽管在这方面取得了巨大进展 耳聋基因的发现，目前还没有针对遗传性耳聋的药物治疗。有一个紧急的和未得到满足的医疗 需要开发遗传性听力损失的治疗方法。 CRISPR/Cas9介导的基因组编辑正在改变生物医学研究，并有望成为新的 疾病的治疗。它使核酸酶和引导RNA能够永久地与DNA配对和修饰， 它可以被开发成治疗各种疾病的新疗法。我们已经成功地使用了瞬变和活体 为治疗人类遗传性听力损失的小鼠模型贝多芬DFNA36，在当地交付了编辑剂。在这 应用方面，我们提出了进一步开发CRISPR内耳编辑技术并将其应用于治疗的两个大目标 遗传性听力损失。首先，我们将鉴定用于毛细胞编辑的CRISPR核酸酶变体SaCas9和Cpf1 RNP(核糖核蛋白)传递。这项研究将扩大耳聋突变，使其成为基因组编辑的目标 额外的PAM(Protspacer相邻基序)序列和核酸酶活性，有可能在 编辑的效率和专业性。我们将使用纯化的毛细胞进行全基因组应用(WGA)。 吞吐量测序(HTS)和识别毛细胞中的插入和缺失(INDel)，并将毛细胞关联起来 编辑效率与听力救助结果的关系。我们将通过RNP传递来评估成熟毛细胞中的编辑， 这与患者的治疗有关，因为人类内耳已经完全成熟。我们将筛选新的纳米颗粒 脂质体为高效的内耳输送和编辑提供选项。在第二个目标中，我们将进行听证 通过RNP递送编辑剂来破坏人类显性耳聋小鼠模型中的突变的救援研究， 包括1)。2)通过编辑影响外毛细胞的Pmca2基因中的遗忘(Obl)突变来挽救听力； 通过编辑microRNA96(Mir96)的突变来挽救听力，这会导致延迟性进行性听力损失。 在这些模型中的听力挽救将展示针对显性基因的基因组编辑的一般治疗应用 毛细胞起源的突变。这项研究将勾勒出编辑效率、专用性和偏离目标之间的关系 以听力抢救的程度评价远期抢救效果及晚期干预效果。 该提案有可能成为基于基因组编辑的遗传性耳聋治疗的新平台。 遗传性听力损失影响了大量儿童，并导致成人耳聋，目前尚无治疗方法。我们 正在开发CRISPR/Cas9介导的基因组编辑作为一种新的治疗平台，通过以下方式针对显性突变 短暂的局部注射到内耳，有可能治疗各种形式的遗传性听力损失。

Project Summary and Relevance Genetic hearing loss affects one in 1000 newborns and contributes significantly to general populations with hearing loss. Over 130 deafness loci have been mapped and more than 80 have been cloned. Despite the tremendous progress in deafness gene discovery, there is no medical treatment for genetic hearing loss. There is an urgent and unmet medical need to develop treatment for genetic hearing loss. CRISPR/Cas9-mediated genome editing is transforming biomedical research and with a promise of becoming new treatment of disease. It enables the application of nuclease with guide RNA to pair with and modify DNA permanently, which can be developed into new therapies for wide range of diseases. We have successfully used transient and in vivo local delivery of editing agents to treat a mouse model, Beethoven, of human dominant hearing loss DFNA36. In this application, we propose two broad aims to further develop CRISPR technology for inner ear editing and to apply it to treat genetic hearing loss. First, we will characterize CRISPR nuclease variants SaCas9 and Cpf1 for hair cell editing by direct RNP (ribonucleoprotein) delivery. This study will expand deafness mutations to be targeted by genome editing due to additional PAM (protospacer adjacent motif) sequences and nuclease activities, with a possibility of improvement in editing efficiency and specificity. We will apply whole-genome application (WGA) using purified hair cells for high- throughput sequencing (HTS) and to identify insertions and deletions (indels) in hair cells, and will correlate hair cell editing efficiency with the outcome of hearing rescue. We will evaluate editing in mature hair cells by RNP delivery, which is relevant to treatment in patients as human inner ears are fully mature. We will screen new nanoparticle based liposomes to provide the options for efficient inner ear delivery and editing. In second aim, we will perform hearing rescue studies by RNP delivery of editing agents to disrupt mutations in mouse models of human dominant deafness, including 1). Hearing rescue by editing Oblivion (Obl) mutation in the Pmca2 gene that affects outer hair cells; 2). Hearing rescue by editing a mutation in the microRNA 96 (Mir96) that results in delayed onset progressive hearing loss. Hearing rescue in those models will demonstrate general therapeutic application of genome editing targeting dominant mutations of hair cell origin. The study will delineate the relationship between editing efficiency, specificity and off-target effect with the extent of hearing rescue and evaluate long-term rescue effect and the outcome of intervention at late stages. The proposal has potential to be developed as new platform for genome editing based therapy for genetic deafness. Genetic hearing loss affects large number of children and contributes to adult deafness with no treatment available. We are developing CRISPR/Cas9-mediated genome editing as a new treatment platform to target dominant mutations by transient local delivery into inner ear, with the potential to treat diverse forms of genetic hearing loss.