TMC gene function in sensory hair cells

TMC 基因在感觉毛细胞中的功能

• 批准号: 10451576
• 负责人: JEFFREY R HOLT
• 金额: $ 49.42万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-07-10 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10451576
• 关键词: Alleles; Amino Acid Substitution; Amino Acids; Auditory; Base Pairing; Base Sequence; Basic Science; Binding; Biological Assay; Biophysics; CRISPR/Cas technology; Cell Death; Cell membrane; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Code; DNA; DNA Repair; Development; Family; Funding; Gene Family; Generations; Genes; Genome; Guide RNA; Hair Cells; Hand; Homology Modeling; Human; In Vitro; Inner Hair Cells; Ion Channel; Ions; Kinetics; Labyrinth; Lead; Mammals; Membrane; Modeling; Molecular; Mus; Mutagenesis; Mutant Strains Mice; Mutation; N-terminal; Outer Hair Cells; Patients; Physiological; Point Mutation; Positioning Attribute; Property; Proteins; Recovery; Reporting; Sensory; Sensory Hair; Signal Transduction; Site; Structural Models; Structure; Structure-Activity Relationship; Techniques; Testing; Therapeutic; Translating; Translational Research; Translations; Transmembrane Domain; Vestibular Hair Cells; Viral Vector; Work; base; base editing; base editor; cellular transduction; clinical application; deaf; deafness; design; effectiveness evaluation; experimental study; gene function; gene replacement; gene replacement therapy; gene therapy; genetic deafness; genome editing; hearing impairment; hearing loss treatment; hereditary hearing loss; in vivo; in vivo Model; innovation; interest; mechanotransduction; member; mouse model; mutant; novel; novel strategies; paralogous gene; prevent; progressive hearing loss; repaired; restoration; screening; sound; success; tool; translational approach; vector

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.

项目概要 跨膜通道样基因 1 (TMC1) 的突变是显性进行性听力损失 (DFNA36) 和 人类隐性非综合征性听力损失 (DFNB7/B11)（Kurima 等，2002）。同样，半主导和 Tmc1 的隐性等位基因导致贝多芬 (Bth) 和耳聋 (dn) 突变小鼠听力损失 (Vreugde et al.,2002; Kurima 等人，2002）。 Tmc1 是 Tmc 基因家族的成员，该家族还包括哺乳动物中的其他七个旁系同源物 (Keresztes 等人，2003）。 Tmc1 和密切相关的 Tmc2 在小鼠内耳的听觉毛细胞和前庭毛细胞中表达 并且是机械感觉转导所必需的。我们最近证明TMC1是一种成孔剂 毛细胞转导通道的亚基，包含四个排列在通道内的跨膜结构域 (S4-S7) 孔隙（Pan et al., 2018）。 手头有令人信服的证据表明 TMC1 是该通道的主要组成部分，我们现在可以使用它 解决以前难以理解的基础科学和转化研究问题的信息。 1）我们 假设毛孔内可能有约 40 个 TMC1 氨基酸，从而控制头发的渗透特性 细胞机械感觉转导通道。我们最近鉴定了排列在孔内的 11 个氨基酸残基（Pan 等 al., 2018)，本文旨在鉴定剩余的约 30 个 TMC1 残基。我们的方法将利用 TMEM16A-TMC1 同源模型（Ballesteros et al., 2018; Pan et al., 2018; Corey et al., 2018）来选择候选 用于 Tmc1/Tmc2 双突变小鼠毛细胞诱变和筛选的氨基酸。 2）我们将调查 TMC1 的 N 端结构域及其有助于生物物理定义的门控弹簧的假设。我们将 在 Tmc1/Tmc2 双突变小鼠的毛细胞中设计和表达 TMC1 N 端突变并分析变化 门控动力学和灵敏度。 3) 我们将生成一个新的小鼠模型，该模型编码 TMC1 的突变形式， 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，但是 不会导致毛细胞快速死亡。我们将使用该小鼠品系在成熟小鼠中测试基因替代疗法。 4） 我们将生成第二个小鼠品系，该品系编码显性、进行性 TMC1 突变，作为大多数小鼠的模型。 人类中常见的 DFNA36 突变。我们将开发一种新的 CRISPR/Cas9 策略和替代方案 原型间隔子相邻基序 (PAM) 位点，选择性且有效地破坏突变体，但不破坏野生型等位基因。 5）最后，我们将表征携带单个 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天然小鼠毛细胞 DNA 中的突变。如果成功的话，我们 假设 Tmc1 DNA 修复将持久恢复毛细胞感觉转导和听觉功能，这可能 提供了第一个用于遗传性耳聋体内碱基编辑的例子。 基于有关 TMC1 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 TMC1 突变的方法。