Method for screening drug for treating or preventing age-related hearing loss

Abstract

The present invention provides a means or a system for screening a drug for treating or preventing age-related hearing loss. Specifically, the present invention provides a method for screening a drug for treating or preventing age-related hearing loss, said method comprising: a step for preparing a pluripotent stem cell that has a genetic variant correlating with age-related hearing loss; a step for inducing the differentiation of the pluripotent stem cell into an inner ear cell; a step for applying cell stress to the inner ear cell in the presence of a factor to be tested; and a step for evaluating the survival of the inner ear cell.

Description

Screening method for therapeutic or preventive drugs for age-related hearing loss

[0001] The present invention relates to methods and means for screening drugs for treating or preventing hearing loss, particularly age-related hearing loss.

[0002] With the advent of a super-aging society, progressive hearing loss (age-related hearing loss) is steadily increasing. Hearing loss (deafness) is the most common disability in daily life worldwide and is also the biggest risk factor for the development of dementia, with hearing loss accounting for as much as 9% of all dementia cases. Therefore, preventing progressive hearing loss is an urgent issue not only to prevent a decline in quality of life caused by hearing loss, but also to extend healthy life expectancy and reduce social security burdens.

[0003] Age-related hearing loss is a progressive hearing loss caused by multiple factors, including environmental and genetic factors. Most cases of progressive hearing loss result from damage to the inner ear, the organ that receives sound and converts it into neural activity. However, this organ is located deep within the temporal bone, making biopsy virtually impossible and histological and cell biological analysis difficult. Furthermore, hearing loss itself does not cause death, and pathological findings are scarce. For these reasons, analysis of the mechanisms underlying hearing loss progression must rely on animal models. However, creating animal models is time-consuming and labor-intensive, and species differences between animals and humans often result in a disconnect between the animal models and the actual disease.

[0004] As a research tool to solve these problems, differentiated cells derived from pluripotent stem cells, such as human embryonic stem cells (ES cells) and human induced pluripotent stem cells (iPS cells), have attracted attention. Our laboratory has established several methods for inducing differentiation of inner ear cell-like cells from human iPS cells (Patent Documents 1 and 2, Non-Patent Documents 1 and 2), and is using these methods to search for therapeutic drugs for hereditary cochlear hearing loss. Furthermore, the efficacy of some of the candidate drugs obtained as a result of this search has been confirmed in investigator-initiated clinical trials (Non-Patent Document 3).

[0005] Patent No. 6218152 International Publication No. WO2023 / 033149

[0006] Okura, S. et al., Stem Cell Res. 67:103017, 2023Hosoya, M. et al., Cell Reports 18(1):68-81, 2017Fujioka, M. et al., Medicine 99(19):e19763, 2020

[0007] An object of the present invention is to establish a means and system for screening therapeutic or preventive agents for age-related hearing loss, and to provide effective therapeutic or preventive agents for age-related hearing loss.

[0008] As a result of intensive research to solve the above problems, the present inventors have found that inner ear cells carrying genetic variants found in hereditary progressive hearing loss can be produced using pluripotent stem cells, and that drugs for treating or preventing age-related hearing loss can be screened in a system in which the produced inner ear cells are subjected to oxidative stress. Furthermore, through screening, they have been able to identify substances that can serve as drugs for treating or preventing age-related hearing loss.

[0009] Thus, the present invention encompasses, for example, the following: [1] A method for screening for a therapeutic or preventive agent for age-related hearing loss, comprising the steps of: preparing pluripotent stem cells having a genetic variant correlated with age-related hearing loss; inducing differentiation of inner ear cells from the pluripotent stem cells; applying cellular stress to the inner ear cells in the presence of a test factor; and evaluating survival of the inner ear cells. [2] The method described in [1], wherein the pluripotent stem cells comprise induced pluripotent stem cells (iPS cells) or embryonic stem cells (ES cells). [3] The method described in [1], wherein the pluripotent stem cells having a genetic variant correlated with age-related hearing loss are prepared from somatic cells of a patient with hereditary hearing loss. [4] The method described in [3], wherein the hereditary hearing loss is early-onset bilateral hearing loss. [5] The method described in [1], wherein the pluripotent stem cells having a genetic variant correlated with age-related hearing loss are prepared by introducing at least one of the genetic variants into healthy pluripotent stem cells. [6] The method according to [1], wherein the genetic variant comprises a mutation in the EYA4 gene. [7] The method according to [1], wherein the application of cellular stress is application of oxidative stress. [8] The method according to [7], wherein the application of oxidative stress comprises treatment with hydrogen peroxide.

[0010] [9] A kit for screening for a therapeutic or preventive drug for age-related hearing loss, comprising pluripotent stem cells having a genetic variant correlated with age-related hearing loss, or inner ear cells differentiated from the pluripotent stem cells, and hydrogen peroxide as oxidative stress.

[10] The kit according to [9], which is for carrying out the screening method according to [1].

[0011] The present invention provides a method and kit for screening for therapeutic or preventive drugs for age-related hearing loss. Because the method and kit of the present invention are an effective model that reflects the pathological condition of human hearing loss, they can be used to screen and evaluate therapeutic or preventive drugs for age-related hearing loss, and are useful for drug development, clinical research, etc.

[0012] This graph shows the cell viability of inner ear cells derived from iPS cells of a patient with hereditary hearing loss (EYA4 gene mutation case) and inner ear cells derived from iPS cells of a healthy individual (WD39) after applying oxidative stress by adding H2O2. This graph shows the cell viability of inner ear cells derived from iPS cells of a patient with hereditary hearing loss (EYA4 gene mutation case) and inner ear cells derived from iPS cells of a healthy individual (WD39) after applying proteasome inhibition stress by adding epoxomicin. This graph shows the cell viability of inner ear cells derived from iPS cells of a patient with hereditary hearing loss (EYA4 gene mutation case) and inner ear cells derived from iPS cells of a healthy individual (WD39) after applying endoplasmic reticulum stress by adding tunicamycin. This graph shows the cell viability of inner ear cells derived from iPS cells of a patient with hereditary hearing loss (EYA4 gene mutation case) and inner ear cells derived from iPS cells of a healthy individual (WD39) after applying oxidative stress by adding H2O2, as observed visually, and the viability index analyzed using ImageJ after cell photography. 1 shows an example of the Viability Index after oxidative stress was applied to inner ear cells derived from iPS cells of a patient with hereditary hearing loss (EYA4 gene mutation case) in the presence of compounds included in the library. The structures of example compounds that reduce oxidative stress on inner ear cells and may serve as therapeutic or preventive agents for age-related hearing loss are shown.

[0013] The present invention is described in detail below. This specification includes the disclosure of Japanese Patent Application No. 2024-079943, filed on May 16, 2024, from which the present application claims priority.

[0014] The present inventors have focused on the fact that highly pathogenic mutations in hearing loss genes (e.g., the EYA4 gene) correlated with age-related hearing loss result in hereditary hearing loss (i.e., the causative gene), whereas the presence of relatively less pathogenic single nucleotide polymorphisms (SNPs) indicates a predisposition to age-related hearing loss. Based on this finding, the present inventors have discovered that inner ear cells can be differentiated from pluripotent stem cells carrying genetic variants correlated with age-related hearing loss, and that factors that affect the survival of inner ear cells after application of oxidative stress can be selected as therapeutic or preventive agents for age-related hearing loss. Furthermore, the inventors have discovered that the compounds and catechins listed in Table 2, described below, particularly reduce oxidative stress on inner ear cells, and have selected them as therapeutic or preventive agents for age-related hearing loss.

[0015] Therefore, in one aspect, the present invention relates to a method for screening for a therapeutic or preventive agent for age-related hearing loss. As shown in the Examples below, inner ear cells used as a hearing loss model have a reduced cell viability under oxidative stress. Therefore, a factor that improves the survival of inner ear cells in the presence of cellular stress, including oxidative stress, may be a therapeutic or preventive agent for age-related hearing loss.

[0016] In the context of the present invention, "age-related hearing loss" refers to a hearing impairment characterized by a progressive age-related decline in auditory function, which has several causes, primarily a decline in inner ear function associated with a decrease in sensory cells in the inner ear. Furthermore, "treatment or prevention of age-related hearing loss" refers to preventing the onset or progression of age-related hearing loss, delaying the onset or progression, or alleviating symptoms. Hearing loss can be assessed by methods known in the art, such as the average hearing level (sound becomes difficult to hear at 25 dB or above) expressed as the average of hearing levels between 500 Hz and 2000 Hz in a pure-tone audiometry test, or the maximum speech intelligibility in a speech audiometry test.

[0017] The screening method for a therapeutic or preventive agent for age-related hearing loss according to the present invention (hereinafter also referred to as "the screening method") comprises the steps of: preparing pluripotent stem cells having a genetic variant correlated with age-related hearing loss; inducing differentiation of inner ear cells from the pluripotent stem cells; applying cellular stress to the inner ear cells in the presence of a test factor; and evaluating the survival of the inner ear cells.

[0018] Pluripotent stem cells refer to stem cells that have the ability to differentiate into various cells that make up the body. In the present invention, not only stem cells that can differentiate into any cell type can be used, but also stem cells that can differentiate into inner ear cells. Examples of such pluripotent stem cells include induced pluripotent stem cells (iPS cells) and embryonic stem cells (ES cells). iPS cells are cells with pluripotency that can be obtained by initializing a cell through the introduction of a specific group of factors called reprogramming factors. ES cells are cells with pluripotency that can be obtained by extracting and culturing cells from inside an embryo.

[0019] In the present invention, pluripotent stem cells are prepared that have a genetic variant correlated with age-related hearing loss. Here, "genetic variants correlated with age-related hearing loss" refer to mutations in genes or proteins known to cause age-related progressive decline in hearing function, but are not limited to these. Examples include mutations in the EYA4 gene, GJB2 gene, KCNQ4 gene, COCH gene, TECTA gene, WFS1 gene, CRYM gene, MYO7A gene, TMPRSS1 gene, TMPRSS3 gene, SLC26A4 gene, ACTG1 gene, DIAPH1 gene, GRHL2 gene, P2RX2 gene, MYH9 gene, MYH14 gene, and POU4F3 gene.

[0020] In one embodiment, pluripotent stem cells containing a genetic variant correlated with age-related hearing loss are generated from somatic cells of a patient with hereditary hearing loss. Hereditary hearing loss includes non-syndromic and syndromic hearing loss, and is not limited to hearing loss caused by genetic factors. For example, early-onset bilateral hearing loss is known, and a variant of this condition with a pathogenic variant in the EYA4 gene is called DFNA10. Methods for generating pluripotent stem cells from patients with hereditary hearing loss are known in the art, and can be generated, for example, by introducing reprogramming factors into patient-derived cells (e.g., Non-Patent Document 2: Hosoya et al., Cell Reports 18(1):68-81, 2017). If necessary, the somatic cells of the patient with hereditary hearing loss and / or the generated pluripotent stem cells may be confirmed to contain the genetic variant.

[0021] In another embodiment, pluripotent stem cells having a genetic variant correlated with age-related hearing loss are generated by introducing at least one genetic variant into healthy pluripotent stem cells. Healthy pluripotent stem cells can be obtained and prepared according to methods known in the art (e.g., Okura, S. et al., Stem Cell Res. 67:103017, 2023), or commercially available or publicly available cells can be used. At least one genetic variant correlated with age-related hearing loss (e.g., a mutation in the EYA4 gene) is introduced into such healthy pluripotent stem cells. Introduction of the mutation into cells can be performed using conventional genetic recombination methods (e.g., homologous recombination, transfection, etc.) in the art.

[0022] In this screening method, the obtained pluripotent stem cells are induced to differentiate into inner ear cells. In the present invention, cells present in the inner ear and the cochlea are referred to as "inner ear cells," and inner ear cells include, for example, cells of the inner ear sensory epithelium (including supporting cells and hair cells), cochlear ganglion cells (including neurons and glial cells), outer spiral sulcus cells (OSCs), and cells of the stria vascularis of the inner ear (including marginal cells of the stria vascularis of the inner ear).

[0023] Pluripotent stem cells can be differentiated into inner ear cells by appropriate methods known in the art, depending on the type of pluripotent stem cells used and the type of inner ear cells to be differentiated. Specifically, pluripotent stem cells can be cultured in the presence of appropriate differentiation factors to induce differentiation into the desired inner ear cells. For example, pluripotent stem cells can be cultured in a serum-free medium (e.g., mTeSR medium) containing a suitable ROCK inhibitor (e.g., Y27632) on an appropriately coated culture dish, followed by culture in a serum-free medium without a ROCK inhibitor, followed by culture in a serum-free medium (e.g., DMEM / F12 medium) for growth factor treatment, followed by culture in a serum-free medium supplemented with an appropriate combination of growth factors (e.g., bFGF / FGF2, FGF3, FGF9, FGF10, FGF19, FGF20, BMP, EGF, IGF1, etc.). The degree of differentiation into inner ear cells can be confirmed using the expression of markers that vary depending on the type of inner ear cells to be differentiated. For example, PAX2, PAX8, FOXG1, GATA3, TFAP2A, ECAD, SOX10, JAG1, SOX2, SIX1, and the like, which are known as markers of inner ear progenitor cells, can be used.

[0024] Next, in the screening method, cellular stress is applied to inner ear cells in the presence of a test factor, and survival of the inner ear cells is assessed.

[0025] The type of test factor that can be used in this screening method is not particularly limited. For example, test factors can include any substance, specifically naturally occurring molecules such as amino acids, peptides, oligopeptides, polypeptides, proteins, nucleic acids, lipids, carbohydrates (such as sugars), steroids, glycopeptides, glycoproteins, and proteoglycans; synthetic analogs or derivatives of naturally occurring molecules such as peptidomimetics, nucleic acid molecules (such as aptamers, antisense nucleic acids, and double-stranded RNA (RNAi)); non-naturally occurring molecules such as small organic compounds (such as inorganic and organic compound libraries or combinatorial libraries) prepared using combinatorial chemistry techniques; and mixtures thereof. The test factor can be a single substance, a complex composed of multiple substances, or a transcription factor. Furthermore, the test factor can be an environmental factor such as radiation, ultraviolet light, carbon dioxide concentration, or temperature.

[0026] Furthermore, a single test factor may be tested independently, or a mixture (including a library) of several candidate test factors may be tested. Examples of libraries containing multiple test factors include synthetic compound libraries (e.g., combinatorial libraries) and peptide libraries (e.g., combinatorial libraries).

[0027] When placing inner ear cells in the presence of a test factor, the conditions vary depending on the type of factor, but can be easily determined by one skilled in the art. For example, inner ear cells can be cultured in a medium containing the test factor, by immersing the inner ear cells in a solution containing the test factor, by layering the test factor on the inner ear cells, or by culturing the inner ear cells in the presence of the test factor.

[0028] The effect and efficacy of the test factor can also be examined under several conditions. Such conditions include the time or duration, amount (large or small), and number of times of exposure to the test factor. For example, multiple doses can be established by preparing a dilution series of the test factor. The treatment period of the test factor can also be appropriately determined, and can range from one day to several weeks, months, or years.

[0029] Furthermore, when examining the additive or synergistic effects of multiple factors, test factors may be used in combination.

[0030] In the presence of such a test factor, a cellular stress is applied to the inner ear cells. "Application of cellular stress" means exposing the cells to some kind of cellular stress, and includes, for example, application of oxidative stress. "Application of oxidative stress" means exposing the cells to excess reactive oxygen species (ROS), such as hydrogen peroxide (H2O2) and superoxide anion (O2 - In one embodiment, the application of oxidative stress includes treatment of cells with hydroxyl radicals (OH). In one embodiment, the application of oxidative stress includes treatment with hydrogen peroxide. For example, after adding a test factor to the culture medium of inner ear cells and culturing them, oxidative stress can be applied by adding hydrogen peroxide to the culture medium or by changing the culture medium to one containing hydrogen peroxide.

[0031] In the Examples described below, because inner ear cells subjected to cellular stress (e.g., oxidative stress) have a low survival rate, a test factor that increases or improves the survival rate is thought to reduce cellular stress (e.g., oxidative stress) on inner ear cells, thereby having therapeutic or preventive activity for age-related hearing loss. Therefore, in this screening method, inner ear cells are subjected to cellular stress (e.g., oxidative stress) in the presence of a test factor, and then the survival of the inner ear cells is evaluated.

[0032] Cell viability can be determined by methods known in the art, for example, using commercially available staining reagents (Hoechst staining). After determining cell viability, the cell viability is compared with a control to evaluate whether cell viability has improved. Controls that can be used include cell viability in the absence of the test factor, cell viability without cell stress, and cell viability in the presence of a positive control (a drug previously known to reduce cell stress). A test factor that improves cell viability (e.g., a test factor that increases cell viability compared to the absence of the test factor, a test factor that results in cell viability comparable to that of cells without cell stress or a positive control) is selected as a drug that reduces cell stress on inner ear cells and as a drug for treating or preventing age-related hearing loss.

[0033] In addition, in the present invention, a test factor that showed improvement in the survival of inner ear cells treated with oxidative stress may be selected as a primary screening, and then, as a secondary screening, the selected test factor may be evaluated in a different screening system.

[0034] Furthermore, in screening for therapeutic or preventive agents, the selected test factor may be administered to an animal (e.g., a patient with age-related hearing loss, a hearing loss model animal, etc.) to determine whether the test factor affects age-related hearing loss in the animal. Those skilled in the art can appropriately determine whether a test factor affects age-related hearing loss in an animal. For example, hearing acuity measurement, electroencephalogram measurement, etc. can be performed. Generally, after the efficacy and safety of the test factor are confirmed in a model animal, the efficacy and safety are evaluated in humans, for example, through clinical trials.

[0035] As described above, this screening method makes it possible to identify therapeutic or prophylactic agents for treating or preventing age-related hearing loss, and further to confirm the effectiveness of the therapeutic or prophylactic agents.

[0036] This screening method can be easily and conveniently performed by using a kit. That is, in a further aspect, the present invention relates to a kit for screening for a therapeutic or preventive agent for age-related hearing loss, comprising pluripotent stem cells having a genetic variant correlated with age-related hearing loss, or inner ear cells differentiated from the pluripotent stem cells, and hydrogen peroxide as oxidative stress. Preferably, the kit according to the present invention is a kit for performing the above-described screening method.

[0037] In addition to the above-mentioned components, the kit of the present invention may also include a culture medium for maintaining the cells, a reagent for determining cell viability, a standard sample for calibration or control, an instruction manual (description of procedures for carrying out the screening method, evaluation criteria, etc.), etc. By providing the kit, screening of therapeutic or prophylactic agents can be carried out more quickly and easily.

[0038] The present invention will be described in more detail below with reference to examples and drawings, but the present invention is not limited to these examples.

[0039] Example 1 In this example, inner ear cells that can serve as a model for age-related hearing loss were established.

[0040] Establishment of EYA4 mutant iPSCs. A 48-year-old woman (45 years old at the time of initial consultation) had a heterozygous mutation in the EYA4 gene (c.960_693del:p.P323Efs*80) and presented with a hearing loss of 72.5 dB in the right ear and 70.0 dB in the left ear (based on a quadrant test) at initial consultation. Mononuclear cells isolated from peripheral blood were electroporated with episomal plasmids encoding OCT3 / 4, SOX2, KLF4, LIN28, L-MYC, and dominant-negative mutant p53 (Hosoya et al., Cell Reports 18(1):68-81, 2017).

[0041] Differentiation of iPS cells into inner ear-like cells Differentiation of iPS cells into outer spiral sulcus cell (OSC)-like cells of the inner ear was performed as previously reported (Non-Patent Document 2). Specifically, iPS cells in the feeder-free culture established as described above were detached using Accutase, passed through a 40 μm cell strainer to obtain single cells, and then plated onto a Matrigel-coated dish at a density of 15,000 to 25,000 cells / cm. 2Cells were seeded at a density of 10 μM (Day 0) and cultured in mTeSR medium containing 10 μM Y27632 for 24 hours, after which the medium was replaced with mTeSR medium without Y27632 (Day 1). The medium was replaced every 24 hours until Day 11. From Day 2 to Day 5, cells were cultured in DMEM / F12 medium containing 1% N2 supplement, 2% B27 supplement, 1× non-essential amino acids, 1× Glutamax, 0.1 mM 2-mercaptoethanol, and 100 μg / mL ampicillin (SF medium). From Day 5 to Day 8, cells were cultured in SF medium supplemented with 25 ng / mL FGF2, 25 ng / mL FGF3, 25 ng / mL FGF10, 25 ng / mL FGF19, and 10 ng / mL BMP4. From Day 8 to Day 11, cells were cultured in SF medium supplemented with 25 ng / mL FGF2, 25 ng / mL FGF3, 25 ng / mL FGF10, and 25 ng / mL FGF19. On Day 11, cells were detached using Accutase and plated on Poly-L-ornithine and fibronectin-coated dishes. They were cultured in DMEM / F12 medium containing 1% N2 supplement, 2% B27 supplement, 25 ng / mL FGF2, 25 ng / mL FGF3, 25 ng / mL FGF10, and 25 ng / mL FGF19. The next day, the medium was replaced with DMEM medium containing 4 ng / mL FGF2, 10 mM HEPES, 6% fetal bovine serum (FBS), and 100 μg / mL ampicillin. The cells were cultured for 2 weeks to 1 month, with medium changes every 2–3 days. If the cells reached confluence, they were detached and reseeded at a 1:3 ratio. The medium was then replaced with DMEM containing 10% FBS, 0.75% NaHCO3, and 50 U / mL penicillin / streptomycin. After culturing for one week with medium changes every other day, the medium was replaced with DMEM containing 10% FBS, 0.375% NaHCO3, and 50 U / mL penicillin / streptomycin. OSC-like cells were obtained after another week of culturing with medium changes every other day.The obtained OSC-like cells were seeded at a density of 10,000 cells / well onto a 96-well plate coated with poly-L-ornithine and fibronectin and used for screening of antioxidants.

[0042] Example 2 In this example, the cellular stress that affects inner ear cells was investigated.

[0043] Various cell stresses were applied to outer spiral sulcus cell (OSC)-like cells induced from iPS cells established from EYA4 patients, as described in Example 1, and the degree of cell death induction was analyzed. Specifically, OSC-like cells induced from iPS cells established from healthy individuals and EYA4 patients were subjected to oxidative stress by adding H2O2, proteasome inhibitory stress by adding epoxomicin, or endoplasmic reticulum stress by adding tunicamycin.

[0044] The results are shown in Figures 1–3. In Figures 1–3, cell viability after each stress is expressed as 1, with the survival rate without stress set at 1. WD39 inner ear cells derived from iPS cells from healthy individuals were not sensitive to oxidative stress induced by the addition of H2O2 (Figure 1), proteasome inhibition stress induced by the addition of epoxomicin (Figure 2), or endoplasmic reticulum stress induced by the addition of tunicamycin (Figure 3). On the other hand, EYA4 mutant inner ear cells (EYA cases) were not sensitive to proteasome inhibition or endoplasmic reticulum stress (Figures 2 and 3), but specifically induced cell death in response to oxidative stress (Figure 1), demonstrating vulnerability to oxidative stress in EYA4 mutant inner ear cells. Therefore, we decided to screen for antioxidants that could reduce oxidative stress induced by the addition of H2O2 to inner ear cells.

[0045] [Example 3] In this example, antioxidants that reduce oxidative stress on inner ear cells caused by the addition of H2O2 were screened. The compounds screened are shown in Table 1.

[0046]

[0047] The day after seeding the inner ear cells prepared in Example 1 onto a 96-well plate, 10 μM of compounds from a library (Table 1) created using Selleck Biotech's compound library creation service (https: / / www.selleck.co.jp / screening-libraries.html) were added and incubated for 2 hours. Then, 200 μM HO was added and incubated for 24 hours. The medium was then removed, and the cells were stained for 10 minutes with PBS containing 1.25 μM Hoechst 33372 and 1 μg / μL 7-amino actinomycin D (7-AAD). After staining, the medium was replaced with dye-free PBS, and blue fluorescent images derived from Hoechst staining and red fluorescent images derived from 7-AAD staining were captured using a Keyence BZ-X810. These images were binarized using the image analysis software ImageJ, and the number of Hoechst-positive cells (total cells) and 7-AAD-positive cells (dead cells) were counted to determine cell viability.

[0048] First, to confirm whether this evaluation system was functioning properly, OSC-like cells were treated with hydrogen peroxide (HO) at concentrations between 0 mM and 1,000 mM, and fluorescent images were taken and analyzed. Visual observation under a fluorescent microscope confirmed that almost all cells were dead at HO concentrations above 250 mM (Figure 4). Images of these cells were then analyzed using ImageJ. Although the values ​​tended to be lower in areas with low cell viability compared to visual viability analysis, the results were generally consistent with visual analysis (Figure 4). Because the analysis using ImageJ differs from the actual viability, we defined the Viability Index (VI).

[0049] Next, we analyzed whether the library compounds inhibited cell death induced by hydrogen peroxide, and each compound inhibited cell death to various degrees (Figure 5). Examples of compounds with a high Viability Index (VI) are shown in Table 2 below: compounds with a VI greater than 0.90, greater than 0.80, greater than 0.70, and greater than 0.60.

[0050]

[0051] Among these compounds, catechins such as (+)-catechin (VI = 0.281), (-)-epicatechin (VI = 0.887), methyl protocatechuate (VI = 0.830), and (-)-epigallocatechin gallate (VI = 0.494) exhibited relatively high VI values. These catechins were selected as potential therapeutic or preventative agents for age-related hearing loss. The structural formulas of these catechins are shown in Figure 6.

[0052] In addition, compounds other than catechins in Table 2, particularly ferulic acid methyl ester (compound ID 234) and 7,8-dihydroxyflavone (compound ID 292), also have the effect of reducing oxidative stress on inner ear cells and can be used as therapeutic or preventive agents for age-related hearing loss.

[0053] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

Claims

1. A method for screening for therapeutic or preventive agents for age-related hearing loss, comprising the steps of: preparing pluripotent stem cells having a genetic variant correlated with age-related hearing loss; inducing differentiation of inner ear cells from the pluripotent stem cells; applying cellular stress to the inner ear cells in the presence of a test factor; and evaluating the survival of the inner ear cells.

2. The method of claim 1, wherein the pluripotent stem cells comprise induced pluripotent stem cells (iPS cells) or embryonic stem cells (ES cells).

3. The method of claim 1, wherein the pluripotent stem cells having a genetic variant correlated with age-related hearing loss are generated from somatic cells of a patient with hereditary hearing loss.

4. The method of claim 3, wherein the hereditary hearing loss is early-onset bilateral hearing loss.

5. The method of claim 1, wherein the pluripotent stem cells having a genetic variant correlated with age-related hearing loss are generated by introducing at least one of the genetic variants into healthy pluripotent stem cells.

6. The method of claim 1, wherein the genetic variant comprises a mutation in the EYA4 gene.

7. The method of claim 1, wherein the application of cellular stress is the application of oxidative stress.

8. The method of claim 7, wherein the application of oxidative stress comprises treatment with hydrogen peroxide.

9. A kit for screening therapeutic or preventive drugs for age-related hearing loss, comprising pluripotent stem cells having a genetic variant correlated with age-related hearing loss, or inner ear cells induced to differentiate from the pluripotent stem cells, and hydrogen peroxide as oxidative stress.

10. A kit according to claim 9, for carrying out the screening method according to claim 1.

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