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TMC gene function in sensory hair cells

TMC 基因在感觉毛细胞中的功能

基本信息

批准号：
10652441
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
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-07-10 至 2025-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10652441
关键词：
Acceleration 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 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 candidate selection cellular transduction clinical application deaf deafness design dominant genetic mutation 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 translational potential vector

项目摘要

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，二○ ○二年; 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，毛细胞转导通道的亚基，并含有四个跨膜结构域（S4-S7），排列在通道上孔（Pan等人，2018年）。有了令人信服的证据表明TMC 1是通道的主要组成部分，我们现在可以使用这个信息，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 TMEM16A-TMC 1同源性模型（Ballesteros等人，2018年; 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DFNA36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。

项目成果

期刊论文数量（10）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

TMC function in hair cell transduction.

DOI：
10.1016/j.heares.2014.01.001
发表时间：
2014-05
期刊：
HEARING RESEARCH
影响因子：
2.8
作者：
Holt, Jeffrey R.;Pan, Bifeng;Koussa, Mounir A.;Asai, Yukako
通讯作者：
Asai, Yukako

Function and Dysfunction of TMC Channels in Inner Ear Hair Cells.

DOI：
10.1101/cshperspect.a033506
发表时间：
2019-10-01
期刊：
Cold Spring Harbor perspectives in medicine
影响因子：
5.4
作者：
Corey DP;Akyuz N;Holt JR
通讯作者：
Holt JR

The Mechanosensory Transduction Machinery in Inner Ear Hair Cells.

DOI：
10.1146/annurev-biophys-062420-081842
发表时间：
2021-05-06
期刊：
Annual review of biophysics
影响因子：
12.4
作者：
Zheng W;Holt JR
通讯作者：
Holt JR

Response to Letter to the Editor Regarding "Hearing Preservation and Spatial Hearing Outcomes after Cochlear Implantation in Children with TMPRSS3 Mutations".

回复关于“TMPRSS3 突变儿童人工耳蜗植入后的听力保护和空间听力结果”的致编辑信。

DOI：
10.1097/mao.0000000000003946
发表时间：
2023
期刊：
Otology & neurotology : official publication of the American Otological Society, American Neurotology Society [and] European Academy of Otology and Neurotology
影响因子：
0
作者：
Peng,ZEllen;Garcia,Alejandro;Godar,ShellyP;Holt,JeffreyR;Lee,DanielJ;Litovsky,RuthY
通讯作者：
Litovsky,RuthY

Transmembrane channel-like (TMC) genes are required for auditory and vestibular mechanosensation.

DOI：
10.1007/s00424-014-1582-3
发表时间：
2015-01
期刊：
PFLUGERS ARCHIV-EUROPEAN JOURNAL OF PHYSIOLOGY
影响因子：
4.5
作者：
Kawashima, Yoshiyuki;Kurima, Kiyoto;Pan, Bifeng;Griffith, Andrew J.;Holt, Jeffrey R.
通讯作者：
Holt, Jeffrey R.