TMC gene function in sensory hair cells

项目总结 跨膜通道样基因1(TMC1)突变是导致遗传性进行性听力损失(DFNA36)和 人类隐性非综合征性听力损失(DFNB7/B11)(Kurima等人，2002年)。类似地，半恒等式和 TMC1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等人，2002年； Kurima等人，2002年)。TMC1是TMC基因家族中的一员，该家族包括哺乳动物(Keresztes)的其他七个近亲 等人，2003年)。TMC1和TMC2在小鼠内耳的听觉和前庭毛细胞中表达 是机械感觉转导所必需的。我们最近证明了TMC1是一种成孔剂 毛细胞转导通道的亚基，包含四个跨膜结构域(S4-S7)，排列在通道上 孔洞(潘等人，2018年)。 有了令人信服的证据证明TMC1是渠道的主要组成部分，我们现在可以使用 解决基础科学和翻译研究问题的信息，这些问题以前是无法理解的。1)我们 假设可能有~40个TMC1氨基酸排列在毛孔内，从而控制头发的渗透特性 细胞机械感觉转导通道。我们最近发现了11个排列在孔洞中的氨基酸残基(Panet 等，2018年)，这里的目的是鉴定剩余的~30个TMC1残基。我们的方法将利用 TMEM16A-TMC1同源模型(Ballesteros等人，2018年；潘等人，2018年；Corey等人，2018年)以选择候选 TMC1/TMC2双突变小鼠毛细胞的氨基酸诱变及筛选。2)我们将调查 TMC1的N-末端结构域以及它对生物物理定义的门控弹簧的贡献的假设。我们会 TMC1/TMC2双突变小鼠毛细胞TMC1 N端突变的设计、表达及变化检测 在选通动力学和灵敏度方面。3)我们将产生一种新的小鼠模型，编码TMC1的突变形式，该突变形式 会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，但 不会导致毛细胞迅速死亡。我们将利用这一小鼠品系在成熟小鼠身上测试基因替代疗法。4) 我们将产生第二个小鼠系，它编码一个显性、进行性TMC1突变，作为大多数 常见的人类DFNA36突变报告。我们将开发一种新的CRISPR/CAS9策略 保护邻近基序(PAM)位点，选择性和有效地干扰突变的等位基因，而不是野生型的等位基因。 5)最后，我们将鉴定携带单个TMC1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自然小鼠毛细胞DNA中的突变。如果成功，我们将 假设TMC1 DNA修复将持久地恢复毛细胞的感觉转导和听觉功能，这可能 提供了第一个针对遗传性耳聋的体内碱基编辑的例子。 根据关于TMC1结构和功能的新信息，本提案中包括的项目将使我们能够 扩大我们对听觉毛细胞感觉传导的理解，开发尖端的翻译 靶向导致人类遗传性听力损失的常见TMC1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.