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千分之一的新生儿，并对普通人群的听力损失造成重大影响。 超过 130 个耳聋基因座已被绘制，超过 80 个已被克隆。尽管取得了巨大进步 耳聋基因的发现，目前还没有治疗遗传性听力损失的药物。有紧急且未得到满足的医疗 需要开发针对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，并有望成为新的研究领域 治疗疾病。它使得核酸酶与指导RNA的应用能够与DNA永久配对和修改， 它可以开发成针对多种疾病的新疗法。我们已经成功地使用了瞬态和体内 局部递送编辑剂来治疗人类显性听力损失 DFNA36 的贝多芬小鼠模型。在这个 应用中，我们提出了两个广泛的目标，即进一步开发用于内耳编辑的 CRISPR 技术并将其应用于治疗 遗传性听力损失。首先，我们将通过直接表征用于毛细胞编辑的 CRISPR 核酸酶变体 SaCas9 和 Cpf1 RNP（核糖核蛋白）递送。这项研究将扩大基因组编辑针对的耳聋突变，因为 额外的 PAM（原型间隔子相邻基序）序列和核酸酶活性，有可能改进 编辑效率和特异性。我们将使用纯化的毛细胞进行全基因组应用（WGA），以实现高 通量测序 (HTS) 并识别毛细胞中的插入和缺失 (indel)，并将毛细胞关联起来 编辑效率与听力救援的结果。我们将评估通过 RNP 传递对成熟毛细胞进行的编辑， 由于人类内耳已经完全成熟，这与患者的治疗相关。我们将筛选新的纳米颗粒基 脂质体为有效的内耳递送和编辑提供选择。第二个目标，我们将进行听证会 通过 RNP 传递编辑剂来破坏人类显性耳聋小鼠模型中的突变进行救援研究， 包括1）。通过编辑影响外毛细胞的 Pmca2 基因中的 Oblivion (Obl) 突变来挽救听力； 2）。 通过编辑 microRNA 96 (Mir96) 中的突变来挽救听力，该突变会导致延迟性进行性听力损失。 这些模型中的听力挽救将展示针对显性基因组编辑的一般治疗应用 毛细胞起源的突变。该研究将描绘编辑效率、特异性和脱靶之间的关系 听力抢救效果与听力抢救程度的关系，评价远期抢救效果和后期干预效果。 该提案有可能被开发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着大量儿童，并导致成人耳聋，且无法治疗。我们 正在开发 CRISPR/Cas9 介导的基因组编辑作为新的治疗平台，通过以下方式靶向显性突变： 瞬时局部传输到内耳，有可能治疗多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.