TMC gene function in sensory hair cells

项目摘要 跨膜通道样基因1（TMC 1）的突变是显性进行性听力损失（DFNA 36）和 人类的隐性非综合征性听力损失（DFNB 7/B11）（Kurima等，2002年）。同样，半显性和 Tmc 1的隐性等位基因导致贝多芬（Bth）和耳聋（dn）突变小鼠的听力损失（Vreugde等，2002年; Kurima等人，2002年）。Tmc 1是Tmc基因家族的成员，该家族包括哺乳动物中的其他七个旁系同源物（Keresztes 例如，2003年）。Tmc 1和密切相关的Tmc 2在小鼠内耳的听觉和前庭毛细胞中表达 并且是机械感觉传导所必需的。我们最近证明了TMC 1是一种成孔蛋白， 它是毛细胞转导通道的一个亚基，含有四个跨膜结构域（S4-S7），排列在通道的两侧 孔（Pan等人，2018年）。 有了令人信服的证据证明TMC 1是该通道的主要组成部分，我们现在可以使用它 信息，以解决基础科学和转化研究的问题，以前是无法理解的。1)我们 假设可能有约40种TMC 1氨基酸排列在孔中，从而控制头发中的渗透特性 细胞机械感觉转导通道。我们最近鉴定了排列在孔中的11个氨基酸残基（Pan et 例如，2018），本文旨在鉴定剩余的约30个TMC 1残基。我们的方法将利用 TMEM 16 A-TMC 1同源性模型（Ballesteros等人，2018; Pan等人，2018; Corey等人，2018年，选择候选人 用于Tmc 1/Tmc 2双突变小鼠毛细胞的诱变和筛选的氨基酸。2)我们将调查 TMC 1的N末端结构域及其对生物病理学定义的门控弹簧有贡献的假设。我们将 设计并在Tmc 1/Tmc 2双突变小鼠的毛细胞中表达TMC 1 N末端突变，并测定其变化 门控动力学和灵敏度。3)我们将产生一种新的小鼠模型，它编码一种突变形式的TMC 1， 导致人类中度至重度听力损失。我们假设这种突变会导致通道功能低下， 不会导致毛细胞快速死亡。我们将使用这一小鼠品系在成熟小鼠中测试基因替代疗法。四、 我们将产生第二个小鼠品系，其编码显性的、进行性的TMC 1突变，作为大多数研究的模型。 在人类中常见的DFNA 36突变。我们将开发一种新的CRISPR/Cas9策略， 原型间隔区邻近基序（PAM）位点，选择性且有效地破坏突变体而不是野生型等位基因。 5)最后，我们将表征携带单个Tmc 1碱基突变的小鼠系作为体内碱基编辑的模型。 我们将使用第四代碱基编辑器来修复天然小鼠毛细胞DNA中的突变。如果成功，我们 假设Tmc 1 DNA修复将持久地恢复毛细胞感觉传导和听觉功能，这可能 为遗传性耳聋提供了第一个体内碱基编辑的例子。 根据有关TMC 1结构和功能的新信息，本提案中包含的项目将使我们能够 扩大我们对听觉毛细胞感觉转导的理解，并开发尖端的翻译 针对导致人类遗传性听力损失的常见TMC 1突变的方法。

PROJECT SUMMARY Mutations in transmembrane channel‐like gene 1 (TMC1) underlie dominant, progressive hearing loss (DFNA36) and recessive nonsyndromic hearing loss (DFNB7/B11) in humans (Kurima et al., 2002). Similarly, semidominant and recessive alleles of Tmc1 cause hearing loss in Beethoven (Bth) and deafness (dn) mutant mice (Vreugde et al.,2002; Kurima et al., 2002). Tmc1 is a member of the Tmc gene family that includes seven other paralogs in mammals (Keresztes et al., 2003). Tmc1 and closely related Tmc2 are expressed in auditory and vestibular hair cells of the mouse inner ear and are necessary for mechanosensory transduction. We have recently demonstrated that TMC1 is a pore‐forming subunit of the hair cell transduction channel and contains four transmembrane domains (S4‐S7) that line the channel pore (Pan et al., 2018). With compelling evidence in hand demonstrating that TMC1 is a major component of the channel, we can now use this information to tackle both basic science and translational research questions that were previously impenetrable. 1) We hypothesize that there may be ~40 TMC1 amino acids that line the pore and thus govern permeation properties in hair cell mechanosensory transduction channels. We recently identified 11 amino acid residues that line the pore (Pan et al., 2018) and herein aim to identify the remaining ~30 TMC1 residues. Our approach will take advantage of the TMEM16A‐TMC1 homology model (Ballesteros et al., 2018; Pan et al., 2018; Corey et al., 2018) to select candidate amino acids for mutagenesis and screening in hair cells of Tmc1/Tmc2 double mutant mice. 2) We will investigate the N‐terminal domain of TMC1 and the hypothesis that it contributes to the biophysically‐defined gating spring. We will design and express TMC1 N‐terminal mutations in hair cells of Tmc1/Tmc2 double mutant mice and assay for changes in gating kinetics and sensitivity. 3) We will generate a novel mouse model that encodes a mutant form of TMC1 which causes moderate to severe hearing loss in humans. We hypothesize this mutation leads to hypofunctional channels but does not cause rapid hair cell death. We will use this mouse line to test gene replacement therapies in mature mice. 4) We will generate a second mouse line that encodes a dominant, progressive TMC1 mutation as a model for the most commonly reported DFNA36 mutation in humans. We will develop a novel CRISPR/Cas9 strategy with an alternate protospacer adjacent motif (PAM) site that selectively and efficiently disrupts the mutant, but not the wild‐type, allele. 5) Lastly, we will characterize a mouse line that carries a single Tmc1 base mutation as model for in vivo base editing. We will use a fourth generation base editor to repair the mutation in native mouse hair cell DNA. If successful, we hypothesize that Tmc1 DNA repair will durably restore hair cell sensory transduction and auditory function, which may provide the first example of in vivo base editing for genetic deafness. Based on new information about the structure and function of TMC1, projects included in this proposal will allow us to expand our understanding of sensory transduction in auditory hair cells and develop cutting‐edge translational approaches for targeting common TMC1 mutations that cause genetic hearing loss in humans.