Biomarker for permanent threshold shift caused by noise and use thereof

Abstract

The present invention provides a composition for preventing or treating noise-induced hearing loss, the composition containing a CHOP protein inhibitor as an active ingredient. The present invention can achieve a fundamental treatment for permanent hearing loss, for which there is no realistic therapeutic agent, by reversibly restoring a permanent threshold shift (PTS) caused by noise. Furthermore, the present invention provides a screening method capable of rapidly searching for efficient therapeutic candidates for noise-induced hearing loss with high reliability by using the CHOP protein as a screening target. In addition, the present invention provides a composition for diagnosing noise-induced hearing loss, the composition containing an agent for measuring the expression level of the CHOP protein or a nucleic acid encoding same, and a method for diagnosing noise-induced hearing loss using the composition. The present invention predicts, with high reliability, whether a threshold shift caused by noise exposure is temporary or permanent, and thus can be useful for quickly identifying subjects in need of treatment and establishing treatment strategies early.

Description

Biomarkers of permanent noise-induced hearing threshold changes and their uses

[0001] The present invention relates to a diagnostic method using CHOP (C / EBP homologous protein), which is a marker of permanent threshold shift (PTS) caused by noise, as a diagnostic marker, and a method for treating the same by regulating the expression of CHOP protein.

[0002] Hearing threshold changes due to noise exposure can be broadly categorized into temporary threshold shift (TTS) and permanent threshold shift (PTS). Hearing damage caused by noise stimuli that induce TTS refers to cases where hearing returns to the original threshold level after a certain period of time after the stimulus is applied, whereas noise stimuli that induce PTS generally refer to cases where hearing is permanently lost due to exposure to high-decibel noise. Noises that induce TTS and PTS increase hearing threshold values ​​from immediately after noise exposure to one day later. After two weeks of noise exposure, the increased hearing threshold value due to TTS-induced noise returns to the level before noise exposure, whereas the hearing threshold value of noise exposure that induces PTS does not return even after two weeks and instead increases.

[0003] Meanwhile, the incidence of noise-induced hearing loss is increasing worldwide, regardless of age or gender. Currently, the best treatment for permanent hearing loss due to noise is prevention, and no established treatment exists. Therefore, there is a need to develop an effective treatment that is relatively accessible, safe, and offers significant therapeutic benefits.

[0004] To develop effective treatments, a treatment target for noise-induced hearing loss (PTS), which causes permanent hearing loss, is needed. Furthermore, because hearing returns to normal levels after a certain period of time after exposure to noise-induced hearing loss (TTS), the need for urgent treatment is low. Therefore, predicting the level of noise exposure and resulting changes in hearing thresholds is crucial for developing a rational treatment strategy for patients. However, no biomarkers have been reported to be specifically expressed by noise-induced hearing loss (PTS), compared to noise-induced hearing loss (TTS), which is a level of noise that can restore hearing.

[0005]

[0006] Numerous papers and patents are referenced and cited throughout this specification. The disclosures of these cited papers and patents are incorporated herein by reference in their entirety to provide a clearer understanding of the state of the art and the scope of the invention.

[0007] The present inventors have devoted extensive research efforts to develop an efficient biomarker that can reliably predict noise-induced hearing loss, particularly permanent hearing threshold changes caused by noise exposure. As a result, we discovered that exposure to high-decibel noise, which causes permanent hearing threshold changes, increases the expression of CHOP (C / EBP homologous protein) in the outer and inner hair cells of the cochlea. This biomarker can be used to clearly distinguish between temporary threshold shifts (TTS), which do not necessarily require treatment, and permanent threshold shifts (PTS), which require drug treatment. Furthermore, the present inventors discovered that suppressing the expression or activity of CHOP, the biomarker discovered in this way, reversibly restored hearing damage caused by high-decibel noise exposure. This discovery suggests that CHOP protein can serve as an efficient therapeutic target for fundamentally restoring permanent hearing loss caused by high-decibel noise exposure, thereby completing the present invention.

[0008] Therefore, the purpose of the present invention is to provide a composition for preventing or treating noise-induced hearing loss, which contains an inhibitor of CHOP protein as an active ingredient.

[0009] Another object of the present invention is to provide a method for screening a composition for preventing or treating noise-induced hearing loss.

[0010] Another object of the present invention is to provide a composition for diagnosing noise-induced hearing loss, comprising a preparation for measuring the expression level of CHOP protein or a nucleic acid encoding the same.

[0011]

[0012] Other objects and advantages of the present invention will become more apparent from the detailed description, claims and drawings below.

[0013] According to one aspect of the present invention, the present invention provides a composition for preventing or treating noise-induced hearing loss, comprising an inhibitor of CHOP (C / EBP homologous protein) protein as an active ingredient.

[0014] The present inventors have devoted extensive research efforts to develop an effective treatment method that can fundamentally restore noise-induced hearing loss, particularly permanent hearing loss caused by noise exposure. As a result, we discovered that exposure to high-decibel noise, which causes permanent hearing threshold changes, increases the expression of CHOP protein in outer and inner hair cells of the cochlea. Furthermore, inhibition of CHOP expression or activity significantly restores hearing loss. Therefore, we discovered that inhibition of CHOP protein can reversibly restore permanent hearing threshold changes caused by high-decibel noise.

[0015] As used herein, the term "noise-induced hearing loss (NIHL)" refers to a symptom in which hearing function is lost or deteriorated due to damage to the cochlea and auditory nerve caused by externally applied noise stimulation. Noise-induced hearing loss includes both temporary threshold shift (TTS) due to short-term low noise and permanent threshold shift (PTS) due to long-term high noise. According to a specific embodiment of the present invention, noise-induced hearing loss that can be prevented or treated by the composition of the present invention is permanent threshold shift (PTS) due to noise.

[0016] As used herein, the term "inhibitor" refers to a substance that causes a decrease in the activity or expression of the CHOP protein, not only such that the activity or expression of the CHOP protein becomes undetectable or present at an insignificant level, but also such that endoplasmic reticulum (ER) stress mediated by the CHOP protein can be significantly inhibited. Therefore, the term "inhibitor of the CHOP protein" has the same meaning as "inhibitor of ER stress" or "inhibitor of ER stress mediated by the CHOP protein."

[0017] The term “reduction in expression” in this specification may mean a state in which the expression level of CHOP protein is reduced by, for example, 20% or more compared to the control group, more specifically, 30% or more, and even more specifically, 40% or more.

[0018] As used herein, the term "reduction in activity" refers to a measurable and significant decrease in the intrinsic function of the CHOP protein in vivo compared to a control, and specifically refers to a decrease in the activity of TRIM40 to the extent that the cytoskeleton and cell-to-cell contacts within the subject can be significantly improved or restored. The decrease in activity includes not only a simple decrease in function but also ultimate inhibition of activity due to a decrease in stability.

[0019] Inhibitors of the CHOP protein include, but are not limited to, shRNA, siRNA, miRNA, ribozyme, peptide nucleic acids (PNA), antisense oligonucleotides, CRISPR systems that suppress the expression of CHOP proteins, the amino acid sequences and encoding nucleotide sequences of which are already known in the art, at the gene level, guide RNAs that recognize target genes, antibodies or aptamers that suppress at the protein level, as well as small molecule compounds, peptides, and natural products that suppress their activity, and all possible suppression means at the gene and protein levels can be used.

[0020] According to a specific embodiment of the present invention, the CHOP protein inhibitor used in the present invention is selected from the group consisting of an antibody or an antigen-binding fragment thereof that specifically binds to the CHOP protein; a nucleic acid molecule that inhibits the expression of a gene encoding the CHOP protein; and a chemical chaperone.

[0021] According to the present invention, the CHOP protein inhibitor of the present invention may be a CHOP-specific antibody that inhibits the activity of the CHOP protein at the protein level. The antibody that specifically recognizes CHOP may be a polyclonal or monoclonal antibody, and is preferably a monoclonal antibody.

[0022] The antibodies of the present invention can be produced by methods commonly practiced in the art, for example, the fusion method (Kohler and Milstein, European Journal of Immunology, 6:511-519 (1976)), the recombinant DNA method (U.S. Patent No. 4,816,567), or the phage antibody library method (Clackson et al, Nature, 352:624-628 (1991) and Marks et al, J. Mol. Biol., 222:58, 1-597 (1991)). General procedures for antibody production are described in detail in Harlow, E. and Lane, D., Using Antibodies: A Laboratory Manual, Cold Spring Harbor Press, New York, 1999, etc.

[0023] As used herein, the term "antigen binding fragment" means a portion of a polypeptide in the overall structure of an immunoglobulin capable of binding an antigen, including, but not limited to, F(ab')2, Fab', Fab, Fv, and scFv.

[0024] As used herein, the term “specifically binding” is synonymous with “specifically recognizing” and means that an antigen and an antibody (or fragment thereof) specifically interact through an immunological reaction.

[0025] According to a specific embodiment of the present invention, the antibody or antigen-binding fragment thereof used in the present invention specifically binds to the amino acid sequence of the first sequence of the sequence listing.

[0026] According to the present invention, the inhibitor of the CHOP protein may be a nucleic acid molecule that inhibits the expression of a nucleotide encoding the CHOP protein.

[0027] In this specification, the term "nucleic acid molecule" has a meaning that comprehensively includes DNA (gDNA and cDNA) and RNA molecules, and nucleotides, which are the basic structural units in nucleic acid molecules, include not only natural nucleotides but also analogues in which the sugar or base portion is modified (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, 90:543-584 (1990)).

[0028] The term "nucleic acid molecule that suppresses expression" as used herein means a nucleic acid molecule that can specifically recognize a target gene by including a complementary nucleic acid sequence that can hybridize with the target gene and can cause a modification in the nucleotide structure that causes a decrease in its function, and includes, for example, shRNA, siRNA, miRNA, ribozyme, PNA, antisense oligonucleotide, and gRNA included in the CRISPR system that specifically recognize a nucleic acid sequence encoding the CHOP protein.

[0029] According to a specific embodiment of the present invention, the expression suppression nucleic acid molecule used in the present invention specifically recognizes the nucleotide sequence of the second sequence of the sequence list.

[0030] As used herein, the term "complementary" means that a nucleic acid molecule for suppressing expression is sufficiently complementary to a target nucleic acid sequence to selectively hybridize under certain annealing or hybridization conditions, and has a meaning that includes both substantially complementary and perfectly complementary, and preferably means perfectly complementary. As used herein, the term "substantially complementary sequence" includes not only a completely identical sequence, but also a sequence that is partially mismatched with the sequence to be compared, within the range where sequence-specific hybridization can occur by annealing to a specific sequence.

[0031] As used herein, the term "shRNA (small hairpin RNA)" refers to an RNA sequence that is a single strand consisting of 50-70 nucleotides that forms a stem-loop structure in vivo, and forms a tight hairpin structure to suppress the expression of a target gene through RNA interference. Typically, a long RNA of 19-29 nucleotides complementarily forms a double-stranded stem by base pairing on both sides of a loop region of 5-10 nucleotides, and is transduced into cells through a vector containing a U6 promoter to ensure constant expression, and is usually passed on to daughter cells to ensure heritable suppression of the expression of the target gene.

[0032] The term "siRNA" in this specification refers to a short double-stranded RNA that can induce RNAi (RNA interference) by cleavage of a specific mRNA. It is composed of a sense RNA strand having a sequence homologous to the mRNA of a target gene and an antisense RNA strand having a sequence complementary thereto. The total length is 10 to 100 bases, preferably 15 to 80 bases, and most preferably 20 to 70 bases, and either blunt-ended or cohesive-ended is possible as long as it can suppress the expression of the target gene through the RNAi effect. The cohesive-ended structure can be either a 3-terminal protruding structure or a 5-terminal protruding structure.

[0033] In this specification, the term "miRNA (microRNA)" refers to a single-stranded RNA molecule that is an oligonucleotide that is not expressed in cells and has a short stem-loop structure and suppresses target gene expression through complementary binding to the mRNA of the target gene.

[0034] As used herein, the term "ribozyme" refers to an RNA molecule that functions like an enzyme, recognizing a specific base sequence in RNA and cleaving it on its own. A ribozyme consists of a region that specifically binds to a complementary base sequence of a target mRNA strand and a region that cleaves the target RNA.

[0035] As used herein, the term "PNA (Peptide Nucleic Acid)" refers to a molecule that possesses properties of both nucleic acids and proteins and can complementarily bind to DNA or RNA. PNA is not found in nature and is artificially synthesized through chemical methods. It forms a double strand through hybridization with a natural nucleic acid of complementary base sequence, thereby regulating the expression of target genes.

[0036] As used herein, the term "antisense oligonucleotide" refers to a nucleic acid molecule that is a nucleotide sequence complementary to a sequence of a specific mRNA and binds to the complementary sequence in the target mRNA, thereby inhibiting its translation into protein, translocation into the cytoplasm, maturation, or any other essential activity for its overall biological function. Antisense oligonucleotides can be modified at one or more base, sugar, or backbone positions to enhance their potency (De Mesmaeker et al., Curr Opin Struct Biol., 5(3):343-55, 1995). The oligonucleotide backbone can be modified with phosphorothioates, phosphotriesters, methyl phosphonates, short-chain alkyls, cycloalkyls, short-chain heteroatoms, heterocyclic sugar sulphonates, etc.

[0037] As used herein, the term "gRNA (guideRNA)" refers to an RNA molecule used in a gene editing system that recognizes a target gene and induces a nuclease to specifically cleave the recognized fragment. A representative example of such gene editing systems is the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) system.

[0038] The nucleic acid molecule of the present invention described above can suppress the expression of CHOP at the genetic level by expressing it in a subject suffering from noise-induced hearing loss, specifically, a subject in whom permanent hearing threshold changes have occurred due to exposure to high noise.

[0039] As used herein, the term "express" means artificially introducing a gene using a gene vector to cause a subject to express an exogenous gene or to increase the natural expression level of an endogenous gene, thereby making the gene replicable within the subject's cells as an extrachromosomal element or through chromosomal integration. Accordingly, the term "expression" has the same meaning as "transformation," "transfection," or "transduction."

[0040] As used herein, the term "gene delivery system" refers to any means of transporting genes into cells, and gene delivery is synonymous with transduction of genes into cells. At the tissue level, the term "gene delivery" is synonymous with gene spread. Therefore, the gene delivery system of the present invention can be described as both a gene penetration system and a gene spread system.

[0041] As used herein, the term "chemical chaperone" refers to a low-molecular-weight compound that functions to promote protein folding and / or stability and to alleviate endoplasmic reticulum stress caused by unfolded or misfolded proteins. Chemical chaperones are a broad and diverse group of small molecules that enhance protein stability through a variety of mechanisms, including, for example, osmotic pressure regulators, hydrophobic compounds, hydrophilic compounds, and pharmacological chaperones.

[0042] Osmoregulators are small polar molecules synthesized endogenously by cells or absorbed externally to maintain the integrity of cellular components during osmotic stress. Examples include glycerol, trehalose, trimethylamine n-oxide (TMAO), and glycine. Hydrophobic compounds act to prevent aggregation of unfolded or misfolded proteins by binding to solvent-exposed hydrophobic sites in an aqueous environment, and include 4-phenylbutyric acid (PBA) and lysophosphatidic acid. Hydrophilic compounds include taurourusodeoxycholic acid (TUDCA), a hydrophilic bile that regulates Akt phosphorylation. Pharmacological chaperones are protein ligands, cofactors, competitive inhibitors, and low-molecular-weight compounds that bind specifically to specific proteins. Because a molecule is active only for a specific protein, they are called pharmacological chaperones.

[0043] According to a more specific embodiment of the present invention, the chemical chaperone used in the present invention is selected from the group consisting of a compound represented by the following chemical formula 1, a compound represented by the following chemical formula 2, and pharmaceutically acceptable salts thereof:

[0044] Chemical Formula 1

[0045]

[0046] In the above chemical formula 1, n is an integer from 2 to 4.

[0047] Chemical Formula 2

[0048]

[0049] In the above chemical formula 2, R1 to R3 are each independently C1-C3 alkyl.

[0050] As used herein, the term "alkyl" means a straight-chain or branched saturated hydrocarbon group, and includes, for example, methyl, ethyl, propyl, isopropyl, etc. C1-C3 alkyl means an alkyl group having an alkyl unit having 1 to 3 carbon atoms, and when C1-C3 alkyl is substituted, the carbon number of the substituent is not included.

[0051] According to a specific embodiment of the present invention, n is 3. The compound of chemical formula 1 in which n is 3 is 4-PBA (4-Phenylbutyric acid, C6H5(CH2)3COOH).

[0052] According to a specific embodiment of the present invention, R1 to R3 are C1 alkyl (methyl). The compound of formula 2 in which R1 to R3 are methyl is TUDCA (Tauroursodeoxycholic acid, C 26 H 45 NO6S).

[0053] As used herein, the term "pharmaceutically acceptable salt" includes salts derived from pharmaceutically acceptable inorganic acids, organic acids, or bases. Examples of suitable acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluene-p-sulfonic acid, tartaric acid, acetic acid, trifluoroacetic acid, citric acid, methanesulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, and the like. Salts derived from suitable bases may include alkali metals such as sodium, alkaline earth metals such as magnesium, and ammonium.

[0054] The term "prevention" as used herein means inhibiting the occurrence of a disease or condition in a subject who has not been diagnosed as having the disease or condition but is susceptible to such disease or condition.

[0055] As used herein, the term "treatment" means (a) inhibiting the development of a disease, condition, or symptom; (b) alleviating the disease, condition, or symptom; or (c) eliminating the disease, condition, or symptom. The composition of the present invention can inhibit the development of, eliminate, or alleviate the symptoms of hearing loss by alleviating endoplasmic reticulum stress mediated by the CHOP protein in an individual who has experienced a permanent hearing threshold change due to exposure to high noise, thereby ultimately reversibly restoring the permanent hearing threshold change. Therefore, the composition of the present invention can be a composition for treating noise-induced hearing loss on its own, or can be used as a therapeutic adjuvant by being administered together with other pharmacological ingredients. Accordingly, the terms "treatment" or "therapeutic agent" as used herein include the meaning of "therapeutic adjuvant" or "therapeutic adjuvant."

[0056] As used herein, the term “administration” or “administering” refers to directly administering a therapeutically effective amount of the composition of the present invention to a subject so that the same amount is formed in the body of the subject.

[0057] In the present invention, the term "therapeutically effective amount" means the content of a composition containing a pharmacological ingredient in the composition sufficient to provide a therapeutic or preventive effect to a subject to whom the pharmaceutical composition of the present invention is to be administered, and includes a "prophylactically effective amount".

[0058] The term "subject" as used herein includes, without limitation, a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, monkey, chimpanzee, baboon, or rhesus macaque. Specifically, the subject of the present invention is a human.

[0059] When the composition of the present invention is prepared as a pharmaceutical composition, the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier.

[0060] Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are those commonly used in formulations, and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition to the above components, the pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweetening agents, flavoring agents, emulsifiers, suspending agents, preservatives, and the like. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

[0061] The pharmaceutical composition of the present invention can be administered orally or parenterally, and specifically can be administered subcutaneously, transdermally, intravenously, or intratympanic.

[0062] The appropriate dosage of the pharmaceutical composition of the present invention may be prescribed in various ways depending on factors such as the formulation method, administration method, patient age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and response sensitivity. The preferred dosage of the pharmaceutical composition of the present invention is within the range of 0.001-100 mg / kg for adults.

[0063] The pharmaceutical composition of the present invention can be manufactured in a unit dose form or can be manufactured by inserting it into a multi-dose container by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method that can be easily performed by a person having ordinary skill in the art to which the present invention pertains, and the like. In this case, the formulation may be in the form of a solution, suspension, syrup or emulsion in an oil or aqueous medium, or in the form of an extract, powder, granule, tablet or capsule, and may additionally include a dispersing agent or stabilizer.

[0064] According to another aspect of the present invention, the present invention provides a method for preventing or treating noise-induced hearing loss, comprising administering to a subject an inhibitor of CHOP protein.

[0065] The CHOP protein inhibitor used in the present invention and the noise-induced hearing loss to be prevented or treated using the same have already been described above, so description thereof is omitted to avoid excessive duplication.

[0066] According to another aspect of the present invention, the present invention provides a method for screening a composition for preventing or treating noise-induced hearing loss, comprising the following steps:

[0067] (a) a step of contacting a candidate substance with a biological sample containing cells expressing CHOP (C / EBP homologous protein) protein; and

[0068] (b) a step of measuring the activity or expression level of CHOP protein in the sample;

[0069] If the activity or expression level of the CHOP protein is reduced, the candidate substance is determined to be a composition for preventing or treating noise-induced hearing loss.

[0070] In the present invention, the term "biological sample" refers to any sample containing cells expressing CHOP protein obtained from a mammal, including a human, including, but not limited to, tissues, organs, cells, or cell cultures.

[0071] The term "candidate substance" used in referring to the screening method of the present invention refers to an unknown substance used in the screening to examine whether it affects the activity or expression level of the CHOP protein when added to a sample containing cells expressing the CHOP protein. The test substance includes, but is not limited to, compounds, nucleotides, peptides, and natural extracts. The step of measuring the expression level or activity of the CHOP protein in a biological sample treated with the test substance can be performed using various expression level and activity measurement methods known in the art. If the measurement result shows a decrease in the expression level or activity of the CHOP protein, the test substance can be determined as a composition for preventing or treating epithelial barrier dysfunction.

[0072] As used herein, the term "reduction in activity or expression" means a decrease in the expression level or intrinsic function of the CHOP protein in vivo to the extent that the endoplasmic reticulum stress mediated by the CHOP protein, which is highly expressed in permanent hearing loss, is significantly alleviated, and hearing thresholds are restored to a measurable level. The decrease in activity includes not only a simple decrease in function but also ultimate inhibition of activity due to a decrease in stability. Specifically, it may mean a state in which the activity or expression level is decreased by 20% or more compared to the control group, more specifically, a state in which the activity or expression level is decreased by 40% or more, and even more specifically, a state in which the activity or expression level is decreased by 60% or more.

[0073] According to a specific embodiment of the present invention, the cell expressing the CHOP protein is an outer hair cell or an inner hair cell.

[0074] According to a specific embodiment of the present invention, the noise-induced hearing loss is a permanent threshold shift (PTS) caused by noise.

[0075] According to another aspect of the present invention, the present invention provides a composition for diagnosing noise-induced hearing loss, comprising as an active ingredient an agent for measuring the expression level of CHOP (C / EBP homologous protein) protein or a nucleic acid encoding the same.

[0076] The present inventors have conducted extensive research to develop an effective biomarker that can reliably predict noise-induced hearing loss, particularly permanent hearing threshold shifts due to noise exposure. As a result, we discovered that exposure to high-decibel noise, which causes permanent hearing threshold shifts, increases CHOP protein expression in outer and inner hair cells of the cochlea. Furthermore, measuring CHOP protein expression levels allows us to clearly distinguish between temporary threshold shifts (TTS) and permanent threshold shifts (PTS), providing useful clinical information for selecting patients requiring treatment and early formulation of treatment strategies.

[0077] The term "diagnosis" as used herein includes determining an individual's susceptibility to a particular disease, determining whether an individual currently has a particular disease, and determining a prognosis for an individual with a particular disease.

[0078] The term "diagnostic composition" as used herein means an integrated mixture or device including a means for measuring the expression level of CHOP protein or a gene encoding it for determining whether or not a subject has noise-induced hearing loss, specifically, permanent hearing threshold change due to high noise, or predicting the possibility of occurrence, and may also be expressed as a "diagnostic kit."

[0079] According to a specific embodiment of the present invention, the agent for measuring the expression level of the CHOP protein is an antibody or an antigen-binding fragment thereof that specifically binds to the CHOP protein; or an aptamer that specifically binds to the CHOP protein.

[0080] According to the present invention, the CHOP protein of the present invention can be used to analyze whether an individual has lost epithelial barrier function by detecting it according to an immunoassay method using an antigen-antibody reaction. This immunoassay can be performed according to various immunoassay or immunostaining protocols developed in the past. For example, when the method of the present invention is performed according to a radioimmunoassay method, a radioactive isotope (e.g., C) 14 , I 125 , P 32 and S 35 ) can be used.

[0081] By analyzing the intensity of the final signal from the immunoassay process described above, epithelial barrier function can be predicted. Specifically, if the CHOP protein signal is stronger in an individual's sample than in a normal sample, it is believed to indicate noise-induced hearing loss, specifically, a permanent change in hearing threshold caused by noise.

[0082] The meaning of the antibody that specifically recognizes the CHOP protein of the present invention is as described above. Similarly, the meanings of the terms "antigen binding fragment" and "specifically binding" in the specific embodiment are also as described above.

[0083]

[0084] The present invention can also utilize an aptamer that specifically binds to the CHOP protein instead of an antibody. As used herein, the term "aptamer" refers to a single-stranded nucleic acid (RNA or DNA) molecule or peptide molecule that binds to a specific target substance with high affinity and specificity. General information on aptamers is described in detail in Hoppe-Seyler F, Butz K, "Peptide aptamers: powerful new tools for molecular medicine". J Mol Med. 78(8):426-30 (2000); Cohen BA, Colas P, Brent R, "An artificial cell-cycle inhibitor isolated from a combinatorial library". Proc Natl Acad Sci USA. 95(24):14272-7 (1998).

[0085] According to a specific embodiment of the present invention, the antibody or aptamer used in the present invention specifically binds to the amino acid sequence of the first sequence of the sequence list.

[0086]

[0087] According to a specific embodiment of the present invention, the agent for measuring the expression level of the gene encoding the CHOP protein is a primer or probe that specifically binds to a nucleic acid molecule of the gene.

[0088] The term "primer" as used herein refers to an oligonucleotide that acts as an initiation point for synthesis under conditions that induce the synthesis of a primer extension product complementary to a nucleic acid chain (template), i.e., the presence of nucleotides and a polymerization agent such as DNA polymerase, and conditions of suitable temperature and pH. Specifically, the primer is a single chain of deoxyribonucleotides. The primer used in the present invention may include naturally occurring dNMPs (i.e., dAMP, dGMP, dCMP, and dTMP), modified nucleotides, or non-natural nucleotides, and may also include ribonucleotides.

[0089] The primer of the present invention may be an extension primer that anneals to a target nucleic acid and forms a sequence complementary to the target nucleic acid by a template-dependent nucleic acid polymerase, which extends to a position to which an immobilized probe is annealed and occupies a portion to which the probe is annealed.

[0090] The extension primer used in the present invention includes a hybridizing nucleotide sequence complementary to a specific base sequence of a target nucleic acid, for example, the DDIT3 gene.

[0091] The primer must be sufficiently long to prime the synthesis of the extension product in the presence of a polymerization agent. The appropriate primer length depends on several factors, such as temperature, pH, and the primer source, but is typically 15-30 nucleotides. Shorter primer molecules generally require lower temperatures to form sufficiently stable hybrid complexes with the template. The design of such primers can be readily accomplished by those skilled in the art, using the target nucleotide sequence as a reference, and can be accomplished, for example, using a primer design program (e.g., PRIMER 3).

[0092] As used herein, the term "probe" refers to a linear oligomer having a natural or modified monomer or linkage comprising deoxyribonucleotides and ribonucleotides that can hybridize to a specific nucleotide sequence. Specifically, the probe is single-stranded for maximum efficiency in hybridization, and more specifically, is a deoxyribonucleotide. As the probe used in the present invention, a sequence perfectly complementary to a specific base sequence of the DDIT3 gene may be used, but a sequence substantially complementary to the extent that it does not interfere with specific hybridization may also be used. In general, since the stability of a duplex formed by hybridization tends to be determined by the identity of the terminal sequence, it is preferable to use a probe complementary to the 3'-end or 5'-end of the target sequence.

[0093] Suitable conditions for hybridization can be determined by referring to those disclosed in Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY (2001) and Haymes, BD, et al., Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington, DC (1985).

[0094] According to a specific embodiment of the present invention, the primer or probe used in the present invention specifically binds to the nucleotide sequence of the second sequence of the sequence list.

[0095] According to the present invention, the nucleotide sequence of the second sequence of the sequence list is a nucleotide sequence encoding the amino acid sequence of the first sequence of the sequence list.

[0096] According to a specific embodiment of the present invention, the noise-induced hearing loss is a permanent threshold shift (PTS) caused by noise.

[0097] Since the present invention specifically detects the occurrence of PTS, which causes permanent hearing loss by exposure to high-decibel noise, the “composition for diagnosing permanent hearing threshold change” of the present invention has the same meaning as “composition for distinguishing permanent hearing threshold change from temporary hearing threshold change.”

[0098] In addition, the present invention provides a method for providing information necessary for diagnosing noise-induced hearing loss, including a step of measuring the expression level of CHOP (C / EBP homologous protein) protein or a gene encoding the same in a biological sample isolated from an individual.

[0099] The CHOP protein of the present invention, the gene encoding it, and noise-induced hearing loss that can be diagnosed using it have already been described above, so they are omitted to avoid excessive duplication.

[0100] The present inventors have discovered for the first time a positive correlation between CHOP protein expression and permanent noise-induced hearing threshold changes. Therefore, if CHOP protein or the gene encoding it is highly expressed in an individual, the individual is considered to have permanent hearing threshold changes requiring treatment.

[0101] The term "high expression" in this specification means that the expression level of the CHOP protein or the DDIT3 gene encoding it is significantly higher than that of an individual with normal hearing or experiencing a temporary change in hearing threshold, and specifically means that the expression level is 130% or more of that of a normal individual, more specifically means 150% or more, and most specifically means 170% or more.

[0102] The term "subject" as used herein refers to a subject that provides a sample for measuring the expression level of the CHOP protein or DDIT3 gene and is ultimately the subject of analysis of epithelial barrier function. The subject includes, without limitation, a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, monkey, chimpanzee, baboon, or rhesus macaque, and is specifically a human.

[0103] The features and advantages of the present invention are summarized as follows:

[0104] (a) The present invention provides a composition for preventing or treating noise-induced hearing loss, comprising an inhibitor of CHOP protein as an active ingredient.

[0105] (b) The present invention can achieve a fundamental treatment for permanent hearing loss for which there is no realistic treatment by reversibly restoring permanent threshold shift caused by noise.

[0106] (c) The present invention also provides a screening method capable of rapidly and reliably searching for an effective therapeutic agent candidate for noise-induced hearing loss using the CHOP protein as a screening target.

[0107] (d) The present invention provides a composition for diagnosing noise-induced hearing loss, including a preparation for measuring the expression level of CHOP protein or a nucleic acid encoding the same, and a method for diagnosing noise-induced hearing loss using the same.

[0108] (e) The present invention can be usefully utilized to quickly distinguish subjects requiring treatment and to establish treatment strategies early by predicting with high reliability whether changes in hearing threshold due to noise exposure are temporary or permanent.

[0109] Figure 1 shows the results of hearing tests of mice 2 hours, 1 day, and 2 weeks after exposure to TTS and PTS noise.

[0110] Figure 2 is a schematic diagram of the experimental procedure for transcriptome analysis over time after exposure to TTS and PTS noise.

[0111] Figure 3 shows the changes in the expression levels of endoplasmic reticulum stress (ER stress) (Figure 3a) and unfolded protein response (UPR) (Figure 3b) transcripts in the mouse cochlea one day after exposure to TTS and PTS-induced noise, respectively.

[0112] Figure 4 is a graph showing the results of a Western blot analysis showing that the expression level of CHOP protein increases specifically in response to PTS-induced noise exposure in the mouse cochlea at 3 days and 2 weeks after TTS and PTS-induced noise exposure (Figure 4a) and quantitatively showing the changes in the expression levels of p-PERK protein and CHOP protein (Figure 4b).

[0113] Figure 5 is a drawing showing the results of immunofluorescence staining measuring the expression level of CHOP protein, which changes specifically in response to PTS-induced noise exposure, in mouse cochlear hair cells at 3 days and 2 weeks after TTS and PTS-induced noise exposure (Figure 5a) and the results of quantitatively evaluating the ratio of CHOP-positive cells (Figure 5b), respectively.

[0114] Figure 6 is a drawing showing the hearing protection effect of chemical chaperone drugs administered after PTS-induced noise exposure through measurements of DPOAE and ABR threshold values.

[0115] Figure 7 is an immunofluorescence staining result (Figure 7a) and a quantified graph (Figure 7b) showing a decrease in the expression of CHOP, an apoptotic factor, in the cochlea of ​​mice treated with a chemical chaperone drug after exposure to PTS-induced noise.

[0116] Figure 8 is an immunofluorescence staining result (Figure 8a) and a quantified graph (Figure 8b) showing a reduction in aggresome structures in the cochlea of ​​mice treated with a chemical chaperone drug after PTS-induced noise exposure.

[0117]

[0118] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples, in accordance with the gist of the present invention.

[0119]

[0120] Example

[0121] Creation of a noise-induced hearing loss mouse model

[0122] Eight-week-old mice were exposed to noises that induced temporary threshold shift (TTS) (105 dB, 30 min) and permanent threshold shift (PTS) (110 dB, 2 h), respectively, and then audiometric tests were performed using the TDT System-3 (Tucker Davis Technologies, Gainesville, FL, USA). In brief, auditory brainstem responses (ABR) tests, which measure inner hair cell function (left), and distortion product otoacoustic emissions (DPOAE) tests, which measure outer hair cell function (right), were performed on mice 2 h, 1 day, and 2 weeks after TTS- and PTS-induced noise exposure, including the control group not exposed to noise. As a result, as shown in Fig. 1, the ABR and DPOAE measurement threshold values ​​increased 2 hours and 1 day after TTS and PTS noise exposure, and 2 weeks after noise exposure, the ABR and DPOAE threshold values ​​increased by TTS-induced noise exposure recovered to the level of the control group not exposed to noise, whereas the ABR and DPOAE threshold values ​​increased by PTS-induced noise exposure did not recover but remained increased. Through this hearing recovery, it was confirmed that the noise-induced hearing loss model caused by TTS and PTS was well established.

[0123]

[0124] RNA sequencing and analysis

[0125] Bulk RNA-sequencing was performed on cochlear tissues from mice at 2 hours, 1 day, and 2 weeks after TTS and PTS-induced noise exposure, respectively (Fig. 2). Differential expression analysis was performed using CLC Genomics Workbench and R software. DEGs were considered statistically significant if their fold change in gene expression was >1.5 and their p value was <0.05.

[0126]

[0127] Western blot

[0128] Each Western blot sample used four cochlear tissues from two mice each. Cochlear tissues were isolated from mice at 3 days and 2 weeks after TTS and PTS-induced noise exposure, and homogenized in cell lysis buffer containing a protease and phosphatase inhibitor cocktail (Thermo Scientific, cat. 78440) using a homogenizer. The tissues were centrifuged at 15,000 rpm for 20 minutes at 4°C, and only the supernatant was separated. A small amount of the lysate was used to measure the protein concentration using the Bradford method (Bio-Rad, cat. 5000006). Samples for electrophoresis were prepared by adding 5 X SDS loading buffer to 40 μg of cochlear lysate and boiling it at 100°C for 10 minutes. SDS-PAGE was run on 4–15% gradient mini protein TGX gels (Bio-Rad), and proteins were transferred to nitrocellulose membranes using the Bio-Rad Trans-Blot Turbo transfer system (250 mA – 100 min). The membranes were then incubated with 5% blocking reagent (Biopure, cat. 8110s) for 1 h at room temperature and then incubated with primary antibodies (1:1000) overnight at 4°C. The membranes were washed three times with TBST and incubated with secondary antibodies (1:1000) for 1 h at room temperature, either anti-rabbit IgG-HRP (Enzo, cat# ADI-SAB-300-J) or anti-mouse IgG-HRP (Enzo, cat# ADI-SAB-100-J). After washing with TBST, antibody-antigen complexes were captured using SuperSignal West Femto chemiluminescent substrate (Thermo, cat# 34096), and protein bands were detected using the ImageQuant 800 System (Cytiva, Korea). Protein band quantification was performed using the ImageJ program.

[0129]

[0130] Immunofluorescence staining

[0131] Cochlear tissues from mice were isolated at 3 days and 2 weeks after PTS-induced noise exposure and fixed overnight in 4% paraformaldehyde (PFA) at 4°C. The fixed cochleae were blocked with goat serum and permeabilized with 10% goat serum dissolved in 0.5% Triton X-100 for 1 h at room temperature. The obtained samples were reacted overnight at 4°C with primary antibody (1:500) (CHOP, Cell signal, T2895S) diluted in 3% goat serum in 0.5% Triton X-100. After washing three times with 1X PBS, the samples were reacted with Alexa Fluor 488-conjugated secondary antibody (1:500) (Thermo Fisher Scientific, Rockford, IL, USA) diluted in 3% goat serum in 0.5% Triton X-100 for 90 min at room temperature. After additional staining with DAPI and Alexa Fluor-conjugated Phalloidin 594 (Thermo Fisher Scientific, Rockford, IL, USA), the cells were washed three times with 1 × PBS, mounted on glass slides using an antifade reagent (Thermo Fisher Scientific, Rockford, IL, USA), and images were captured using an LSM 780 confocal microscope (Carl Zeiss, Jena, Germany). Images were processed using ZEN (Blue edition) software, and Image J program was used for quantification.

[0132]

[0133] Comparison of transcriptome expression in response to noise exposure

[0134] Comparison of the expression levels of transcripts related to endoplasmic reticulum stress (ER stress) (Fig. 3a) and unfolded protein response (UPR) (Fig. 3b) one day after TTS and PTS-induced noise exposure with that of mice not exposed to noise (Control) revealed that the expression levels of genes related to ER stress and unfolded protein response increased in both the TTS and PTS groups one day after noise exposure, and that the expression of these genes was increased by PTS-induced noise exposure compared to the TTS-induced noise exposure group.

[0135] In addition, when the expression levels of PERK pathway proteins and CHOP proteins in the unfolded protein response in the mouse cochlea 3 days and 2 weeks after TTS and PTS-induced noise exposure were compared using Western blot with respect to mice not exposed to noise, it was found that the p-PERK signaling pathway was activated 3 days after TTS and PTS noise exposure, and the expression level of p-PERK activated by TTS noise exposure returned to the control level 2 weeks after noise exposure, whereas the expression of p-PERK activated by PTS noise exposure increased to the level of 3 days of noise exposure (Fig. 4). In contrast, the expression of CHOP, an apoptosis factor known as a downstream signaling pathway of p-PERK, was specifically expressed by PTS-induced noise exposure from 3 days after noise exposure, and the expression level continuously increased until 2 weeks after noise exposure (Fig. 4).

[0136] In addition, the expression level of CHOP in the mouse cochlea 3 days and 2 weeks after TTS and PTS-induced noise exposure was compared using immunofluorescence staining based on mice not exposed to noise. As a result, it was found that the expression of CHOP in the outer and inner hair cells of the cochlea increased from 3 days after PTS-induced noise exposure compared to TTS-induced noise exposure, and the expression level increased until 2 weeks after noise exposure (Fig. 5).

[0137]

[0138] Treatment with chemical chaperones

[0139] Two types of chemical chaperones, 4-PBA (Medchem, HY-A0281) and TUDCA (Millipore, 14605-22-2), were used. 4-PBA was dissolved in DMSO as a stock solution at 100 mg / ml, and the 4-PBA stock solution was diluted to the full volume in filtered 1X PBS containing 10% DMSO before use. TUDCA was dissolved in filtered 1X PBS as a 100 mg / ml stock solution, and the TUDCA stock solution was diluted to the same volume as 4-PBA in 1X PBS before use. Chemical chaperones were injected intraperitoneally at a dose of 300 mg / kg once daily for up to 2 weeks, starting immediately after exposure to PTS-induced noise, while control mice were injected with the same volume of vehicle solution.

[0140]

[0141] Comparison of chemical chaperone treatment groups and control groups

[0142] When audiometry was performed two weeks after exposure to PTS-induced noise for the group that was not treated with chemical chaperone drugs (Vehicle) and the group treated with chemical chaperones TUDCA and 4-PBA, it was confirmed that the TUDCA and 4-PBA treated groups showed a protective effect against noise-induced hearing loss, which was maintained for up to two weeks (Fig. 6).

[0143] In addition, the expression level of CHOP in the cochlea of ​​mice treated with TUDCA and 4-PBA and the untreated control group (Vehicle) 2 weeks after PTS-induced noise exposure was compared through immunofluorescence staining. It was confirmed that the expression of CHOP was increased in the outer and inner hair cells of the cochlea after PTS-induced noise exposure, and was significantly reduced in both outer and inner hair cells by treatment with TUDCA and 4-PBA (Fig. 7).

[0144] The expression levels of aggresome structures in the cochlea of ​​mice treated with TUDCA and 4-PBA and the untreated control group (Vehicle) 2 weeks after PTS-induced noise exposure were compared through immunofluorescence staining. The results showed that the expression of aggresomes increased in the outer and inner hair cells of the cochlea after PTS-induced noise exposure, and that the group treated with chemical chaperone drugs showed a significant decrease in the expression of aggresome structures in both outer and inner hair cells compared to the untreated control group (Fig. 8).

[0145]

[0146] While specific aspects of the present invention have been described in detail above, it should be apparent to those skilled in the art that these specific descriptions are merely preferred embodiments and do not limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A composition for preventing or treating noise-induced hearing loss, comprising an inhibitor of CHOP (C / EBP homologous protein) protein as an active ingredient.

2. A composition according to claim 1, wherein the CHOP protein inhibitor is selected from the group consisting of an antibody or an antigen-binding fragment thereof that specifically binds to the CHOP protein; a nucleic acid molecule that inhibits the expression of a gene encoding the CHOP protein; and a chemical chaperone.

3. A composition according to claim 2, characterized in that the nucleic acid molecule that suppresses the expression of the gene encoding the CHOP protein is selected from the group consisting of shRNA, siRNA, miRNA, ribozyme, PNA, antisense oligonucleotide, and guide RNA that specifically recognize a nucleic acid sequence encoding the CHOP protein.

4. In the second paragraph, the chemical chaperone is a composition characterized in that it is selected from the group consisting of a compound represented by the following chemical formula 1, a compound represented by the following chemical formula 2, and a pharmaceutically acceptable salt thereof: Chemical Formula 1 In the above chemical formula 1, n is an integer from 2 to 4. Chemical Formula 2 In the above chemical formula 2, R1 to R3 are each independently C1-C3 alkyl.

5. A composition according to claim 1, wherein the noise-induced hearing loss is a permanent threshold shift (PTS) caused by noise.

6. A method for screening a composition for preventing or treating noise-induced hearing loss, comprising the following steps: (a) a step of contacting a candidate substance with a biological sample containing cells expressing CHOP (C / EBP homologous protein) protein; and (b) a step of measuring the activity or expression level of CHOP protein in the sample; If the activity or expression level of the CHOP protein is reduced, the candidate substance is determined to be a composition for preventing or treating noise-induced hearing loss.

7. A method according to claim 6, characterized in that the cell expressing the CHOP protein is an outer hair cell or an inner hair cell.

8. A method according to claim 6, wherein the noise-induced hearing loss is a permanent threshold shift (PTS) caused by noise.

9. A composition for diagnosing noise-induced hearing loss, comprising as an active ingredient a preparation for measuring the expression level of CHOP (C / EBP homologous protein) protein or a nucleic acid encoding the same.

10. A composition characterized in that, in claim 9, the agent for measuring the expression level of the CHOP protein is an antibody or an antigen-binding fragment thereof that specifically binds to the CHOP protein; or an aptamer that specifically binds to the CHOP protein.

11. A composition according to claim 10, wherein the antibody or aptamer specifically binds to the amino acid sequence of the first sequence of the sequence list.

12. A composition according to claim 9, wherein the agent for measuring the expression level of the gene encoding the CHOP protein is a primer or probe that specifically binds to a nucleic acid molecule of the gene.

13. A composition according to claim 12, wherein the primer or probe specifically binds to a nucleotide sequence in the second sequence of the sequence list.

14. A composition according to claim 9, wherein the noise-induced hearing loss is a permanent threshold shift (PTS) caused by noise.

15. A method for providing information necessary for diagnosing noise-induced hearing loss, comprising a step of measuring the expression level of CHOP (C / EBP homologous protein) protein or a gene encoding the same in a biological sample isolated from an individual.

16. A method according to claim 15, wherein the noise-induced hearing loss is a permanent threshold shift (PTS) caused by noise.

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