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.
We discovered that Transmembrane channel?like 1 (TMC1) functions as a key protein that converts sound information into electrical signals in sensory hair cells of the inner ear. Over forty different mutations have been identified in TMC1 that cause hereditary hearing loss in humans. Here we propose experiments to help better understand the structure and function of TMC1 and develop strategies to translate our discoveries into therapeutics for the treatment of hearing loss in patients who carry TMC1 mutations.
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