Application of ECM related gene in treatment of hearing loss

Abstract

The invention discloses an application of an ECM related gene in treatment of hearing loss. Specifically, the invention provides an application of an active ingredient, and the active ingredient is used for preparing a composition for treating hearing loss. And the active ingredient comprises a down-regulating agent of an ECM-related gene selected from the group consisting of Col6a3, Ogn, Col3a1, or a combination thereof. The composition disclosed by the invention can be used for effectively treating sensorineural hearing loss caused by excessive ECM deposition.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of ECM-related genes in the treatment of hearing loss. Background Technology

[0002] Sensorineural hearing loss is the most common type of hearing impairment worldwide, primarily caused by damage or death of cochlear hair cells. While hearing aids and cochlear implants alleviate hearing loss to some extent, these methods cannot fundamentally repair damaged hair cells and often result in complications such as cochlear fibrosis, limiting long-term efficacy.

[0003] Recent studies have shown that excessive deposition of ECM within the cochlea is a significant contributing factor to sensorineural hearing loss. Abnormal accumulation of ECM components (such as the collagen network) increases the stiffness of the cochlear microenvironment, interfering with hair cell arrangement and mechanotransduction, ultimately leading to hearing loss.

[0004] However, there is currently no effective way to directly target excessive ECM deposition for treatment.

[0005] Therefore, developing new drugs that can effectively reduce ECM deposition and provide effective treatment for hearing loss that targets excessive ECM deposition is of great significance in this field. Summary of the Invention

[0006] This invention provides the application of ECM-related genes in the treatment of hearing loss.

[0007] In a first aspect of the invention, there is provided the use of an active ingredient for preparing a composition for treating hearing loss; and the active ingredient comprises:

[0008] Downregulators of ECM-related genes selected from the following group: Col6a3, Ogn, Col3a1, or combinations thereof.

[0009] In another preferred embodiment, the treatment includes:

[0010] (a) Reduce excessive ECM deposition in the cochlea;

[0011] (b) Restoring the morphology and function of cochlear hair cells; and

[0012] (c) Lower the hearing threshold.

[0013] In another preferred embodiment, the hearing loss is NSD2 low expression-induced hearing loss.

[0014] In another preferred embodiment, the hearing loss is hearing loss caused by excessive ECM deposition.

[0015] In another preferred embodiment, the hearing loss is sensorineural hearing loss.

[0016] In another preferred embodiment, the sensorineural hearing loss is selected from the group consisting of noise-induced hearing loss, drug-induced hearing loss, age-related hearing loss, sudden hearing loss, or a combination thereof.

[0017] In another preferred embodiment, the downregulator is a small chemical molecule antagonist or inhibitor targeting the gene; or the downregulator is a reagent that knocks out, silences, or knocks down the gene.

[0018] In another preferred embodiment, the agent for knocking out, silencing, or knocking down the gene is a reagent with the nucleotide sequence shown in SEQ ID NO:1.

[0019] In another preferred embodiment, the agent for knocking out, silencing, or knocking down the gene is a reagent with the nucleotide sequence shown in SEQ ID NO:2.

[0020] In another preferred embodiment, the reagent for knocking out, silencing, or knocking down the gene is a reagent with the nucleotide sequence shown in SEQ ID NO:1 and 2.

[0021] In another preferred embodiment, the downregulator is a reagent that knocks out, silences, or knocks down the gene (such as an interfering molecule or sgRNA), which is introduced into the target site (lesion, such as cochlear hair cells) through an expression construct (i.e., an expression vector); and the expression construct is a viral vector.

[0022] In another preferred embodiment, the viral vector is selected from the group consisting of adeno-associated virus vectors (AAV), adenovirus vectors (AdV), lentiviral vectors, retroviral vectors, or combinations thereof.

[0023] In another preferred embodiment, the inhibitor is selected from the group consisting of siRNA, mRNA, antisense nucleic acid, aptamer, antibody, gene editing reagent, small molecule compound, or a combination thereof.

[0024] In another preferred embodiment, the gene editing reagent is a CRISPR gene editing reagent.

[0025] In another preferred embodiment, the active ingredient accounts for 0.1-99.9 wt% of the total weight of the composition, more preferably 10-99.9 wt%, and even more preferably 70%-99.9 wt%.

[0026] In another preferred embodiment, the composition is also used for one or more uses selected from the group consisting of:

[0027] (a) Reduce excessive ECM deposition in the cochlea;

[0028] (b) Restoring the morphology and function of cochlear hair cells;

[0029] (c) Improve hearing threshold.

[0030] In another preferred embodiment, the composition is a pharmaceutical composition, a bioactive preparation composition, or a health product composition.

[0031] In another preferred embodiment, the composition is a pharmaceutical composition.

[0032] In another preferred embodiment, the composition is contained in a medicine box.

[0033] In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from the group consisting of: injections, lyophilized formulations, nebulized inhalers, and topical medications.

[0034] In a second aspect of the invention, a pharmaceutical composition for treating hearing loss is provided, comprising:

[0035] (a) Downregulators of ECM-related genes selected from the following group: Col6a3, Ogn, Col3a1, or combinations thereof; and

[0036] (b) Pharmaceutically acceptable carriers.

[0037] The downregulator is a small molecule chemical antagonist or inhibitor targeting the gene; or the downregulator is a reagent that knocks out or silences the gene.

[0038] In another preferred embodiment, the agent for knocking out, silencing, or knocking down the gene includes (but is not limited to): an interfering molecule that specifically interferes with the expression of the gene encoding the gene; a CRISPR gene editing agent targeting the gene; or a homologous recombination agent or site-directed mutagenesis agent targeting the gene, wherein the homologous recombination agent or site-directed mutagenesis agent performs a loss-of-function mutation on the gene.

[0039] In another preferred embodiment, the interfering molecule includes (but is not limited to): shRNA, siRNA, miRNA, antisense nucleic acid, or a construct capable of forming the shRNA, siRNA, miRNA, or antisense nucleic acid.

[0040] In another preferred embodiment, the agent for knocking out, silencing, or knocking down the gene is an interfering molecule.

[0041] In another preferred embodiment, the agent for knocking out, silencing, or knocking down the gene is shRNA.

[0042] In another preferred embodiment, the sequence of the shRNA is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, or a combination thereof.

[0043] In a third aspect of the invention, a method for screening potential substances for treating hearing loss is provided, characterized in that it comprises:

[0044] (1) Treat an expression system with candidate substances, the system expressing genes selected from the group consisting of: Col6a3, Ogn, Col3a1, or combinations thereof; and

[0045] (2) Detect the expression or activity of the gene in the system; if the candidate substance statistically downregulates the expression or activity of the gene, then the candidate substance is a potential substance for treating hearing loss.

[0046] In another preferred embodiment, the system is selected from: cell systems (such as cells or cell cultures expressing the gene), subcellular (culture) systems, solution systems, tissue systems, organ systems, or animal systems.

[0047] In another preferred embodiment, the system is a cochlear hair cell system.

[0048] In another preferred embodiment, step (2) further includes: detecting the morphology and function of cochlear hair cells in the system; if the morphology and function of the cochlear hair cells are improved or restored, then the candidate substance is a potential substance for treating hearing loss.

[0049] In another preferred embodiment, step (2) further includes: detecting the deposition of ECM in the system; if the ECM deposition is significantly reduced, then the candidate substance is a potential substance for treating hearing loss.

[0050] In another preferred embodiment, the candidate substances include (but are not limited to): regulatory molecules or constructs thereof designed for the gene, its fragments or variants, its coding gene or its upstream or downstream molecules or signaling pathways (such as shRNA, siRNA, gene editing apparatus, expression vectors, recombinant viral or non-viral constructs, etc.), small chemical molecules (such as specific inhibitors or antagonists), interacting molecules, etc.

[0051] In another preferred embodiment, the method further includes conducting further cell experiments and / or animal experiments on the obtained potential substances to further select and identify substances useful for treating or improving hearing loss from the candidate substances.

[0052] In a fifth aspect of the invention, a diagnostic reagent for detecting hearing loss is provided, the diagnostic reagent comprising the expression or activity of the following ECM-related genes: Col6a3 and Ogn.

[0053] In another preferred embodiment, the detection reagent further contains a component for detecting the expression or activity of the following ECM-related gene: Col3a1.

[0054] In a sixth aspect of the invention, a kit is provided, the kit comprising the detection reagents described in the fourth aspect of the invention.

[0055] In a sixth aspect of the invention, there is provided the use of an injectable modeling composition for preparing a mouse animal model of hearing loss; wherein the modeling composition comprises a reagent that can be used to knock down NSD2 expression.

[0056] In a seventh aspect of the invention, a method for preparing a mouse animal model of hearing loss is provided, characterized by comprising the following steps:

[0057] (a) Provide an injectable modeling composition comprising an agent that can be used to knock down NSD2;

[0058] (b) The modeling composition is applied to mice to obtain a mouse model of hearing loss.

[0059] In another preferred embodiment, the reagent is CRISPR gene editing.

[0060] In an eighth aspect of the invention, a method for treating hearing loss is provided, comprising the steps of:

[0061] Administer downregulators of ECM-related genes selected from the following group to subjects in need: Col6a3, Ogn, Col3a1, or combinations thereof.

[0062] In another preferred embodiment, the subject is a person suffering from hearing loss.

[0063] In another preferred embodiment, the hearing loss is NSD2 low expression-induced hearing loss.

[0064] In another preferred embodiment, the hearing loss is hearing loss caused by excessive ECM deposition.

[0065] In another preferred embodiment, the hearing loss is sensorineural hearing loss.

[0066] In another preferred embodiment, the sensorineural hearing loss is selected from the group consisting of noise-induced hearing loss, drug-induced hearing loss, age-related hearing loss, sudden hearing loss, or a combination thereof.

[0067] In a ninth aspect of the invention, a method is provided to inhibit the increase in ECM-related gene expression in the NSD2 knockout trichomeoid cell line HEI-OC1 or to allow the cell line to grow normally, comprising the steps of:

[0068] The cells were treated with a downregulator of ECM-related genes selected from the group consisting of Col6a3, Ogn, Col3a1, or a combination thereof.

[0069] In another preferred embodiment, the ECM-related genes include: Col6a3, Ogn, Col3a1, and Ccdc80.

[0070] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0071] Figure 1 The process of creating the NSD2 knockout mouse model is shown.

[0072] Figure 2 The ABR test results showed that NSD2-deficient mice (Nsd2) Atoh1-KO Hearing threshold increases, resulting in hearing damage.

[0073] Figure 3 The study showed the ECM deposition after successful modeling, including Masson staining and immunofluorescence staining of collagen3.

[0074] Figure 4 The results showed that after NSD2 knockout, qPCR analysis of mRNA levels revealed a significant upregulation of multiple ECM-related genes, including Col6a3 and Ogn.

[0075] Figure 5 A schematic diagram of AAV vector construction is shown.

[0076] Figure 6 The study demonstrated that continued culture of the hairy cell line HEI-OC1(Sg2) after Nsd2 gene knockout revealed cell growth defects. Cv2 served as the control group, transfected only with the empty vector CRISPR v2.

[0077] Figure 7 The results show the expression levels of ECM-related genes in Sg2 after knocking out Col6a3 or Ogn genes, as detected by qPCR.

[0078] Figure 8 The study showed that knocking out Col6a3 and Ogn genes in Sg2 cells restored cell growth compared to the control group Sg2.

[0079] Figure 9RNA was extracted from the cochlea of ​​NSD2 gene-deficient mice after knocking out ECM-related genes (Col6a3 or Ogn), and the expression of ECM-related genes was identified by qPCR. It was found that the AAV vector was successfully delivered to the cochlear hair cells and significantly reduced the expression of Col6a3, Ogn, Col3a1 and Ccdc80.

[0080] Figure 10 Masson staining revealed changes in ECM deposition within the cochlea.

[0081] Figure 11 The ABR test results of mice in the experimental group and the control group are shown. Detailed Implementation

[0082] Through extensive and in-depth research, the inventors have discovered for the first time that NSD2 deficiency leads to a decrease in H2K36me2 levels, which in turn activates the transcription of ECM-related genes (such as Col6a3, Ogn, and Col3a1) through abnormally open chromatin structures, ultimately causing excessive ECM deposition and hearing loss. Based on this, the present invention finds that targeted knockdown or silencing of ECM-related genes (such as Col6a3, Ogn, and Col3a1) can effectively reduce excessive ECM deposition in the cochlea, restore the morphology and function of encapsulated cells, and effectively improve the hearing threshold. Based on this, the present invention was completed.

[0083] Furthermore, based on the findings of this invention, ECM-related genes can be used to effectively screen candidate drugs for the treatment of hearing loss.

[0084] the term

[0085] To facilitate a clearer understanding of this disclosure, certain terms are first defined. As used herein, unless otherwise expressly specified herein, each of the following terms shall have the meaning given below. Other definitions are set forth throughout the application.

[0086] The term “about” can refer to a value or composition within an acceptable margin of error for a particular value or composition as determined by a person skilled in the art, depending in part on how the value or composition is measured or determined. For example, as used herein, the expression “about 100” includes all values ​​between 99 and 101.

[0087] As used herein, the terms “containing” or “including (comprise)” can be open-ended, semi-closed, or closed. In other words, the terms also include “consistently made of” or “composed of”.

[0088] As used herein, unless otherwise stated, any concentration range, percentage range, proportion range, or integer range shall be understood to include any integer value within the range and, where appropriate, its fractional value (e.g., one-tenth and one-hundredth of an integer).

[0089] As used herein, the term “and / or” refers to and covers any and all possible combinations of one or more of the related listed items.

[0090] As used in this article, the terms “hearing loss,” “hearing impairment,” “hearing disorder,” and “deafness” have the same meaning and can be used interchangeably.

[0091] As used in this article, “NSD2” f / f ;Atoh1-cre”“Nsd2” Atoh1-KO "The meanings are the same, both referring to NSD2 knockout mice."

[0092] NSD2

[0093] Histone methyltransferase NSD2 (Nuclear receptor binding SET domain protein 2) is a member of the NSD family. It mainly catalyzes the methylation modification of histone H3K36me2 and participates in gene transcription regulation, cell differentiation, and other processes. Existing research shows that NSD2 plays an important role in the development of the nervous system, but there are no reports on its role in maintaining the function of cochlear hair cells.

[0094] Hearing loss

[0095] Hearing loss, also commonly known as hearing impairment, auditory disturbance, or deafness, refers to a physiological disorder in which a person's ear experiences a partial or complete reduction in its ability to perceive or understand sound. It is not a single disease, but rather a general term encompassing a wide range of symptoms, from mild to profound.

[0096] Hearing loss includes conductive hearing loss, sensorineural hearing loss, and mixed hearing loss (the first two coexisting). Among them, sensorineural hearing loss is caused by damage to the hair cells of the inner ear or the auditory nerve (such as aging, noise, genetics, drug toxicity), which leads to the failure of sound conversion nerve signals. It is mostly permanent and is often accompanied by speech discrimination difficulties ("can hear but cannot understand").

[0097] ECM, Col6a3, Ogn, Col3a1

[0098] The extracellular matrix (ECM) is a complex, dynamic network structure secreted and assembled by cells, existing both around and between cells. It provides physical scaffolding, mechanical support, and biochemical signaling for tissues and organs, and is a key microenvironment for maintaining cell survival, proliferation, migration, differentiation, and function.

[0099] The main components of the ECM include: structural fibrous proteins (such as collagen and elastin, which provide strength and elasticity); adhesion glycoproteins (such as fibronectin and laminin, which mediate cell adhesion to the ECM); proteoglycans and glycosaminoglycans (such as core proteoglycans and multifunctional proteoglycans, which bind water molecules like a "sponge" and provide stress resistance and signal regulation functions), etc.

[0100] Col6a3, short for collagen type VI α3 chain, is the α3 chain encoding type VI collagen. Type VI collagen is a unique microfibrillary collagen with a beaded structure. Mutations in the Col6a1 / 2 / 3 genes can lead to collagen VI-related myopathy, such as Bethlem myopathy and Ullrich's congenital muscular dystrophy, characterized by progressive muscle weakness and joint contractures.

[0101] Ogn, short for bone glycine / bone factor, encodes the Osteoglycin protein, which belongs to the leucine-rich small proteoglycan (SLRP) family. It is associated with processes such as myocardial fibrosis, atherosclerotic plaque stability, corneal transparency, and abnormal bone metabolism.

[0102] Col3a1, short for Collagen Type III α1 Chain, is the core gene encoding type III collagen. Type III collagen is a crucial fiber-forming collagen. Mutations in the COL3A1 gene are a major cause of vascular Ehlers-Danlos syndrome (vEDS), a serious genetic disorder of connective tissue characterized by ruptured aorta and intestines, because defects in type III collagen directly affect the integrity of the blood vessel walls.

[0103] Col6a3 encodes the α3 chain of type VI collagen, a key anchoring molecule connecting cells to the extracellular matrix (ECM). Ogn encodes bone glycine, a key regulatory molecule for finely controlling collagen fiber assembly and ECM tissue structure. Col3a1 encodes type III collagen, one of the main fibrous skeletal components of the ECM. These three are indispensable components in constructing, connecting, and optimizing the supramolecular network structure of the ECM. They work synergistically to determine the mechanical properties and biochemical signaling environment of the ECM, thereby affecting the health and function of the entire tissue.

[0104] However, there are currently no reports on the correlation between Col6a3, Ogn, and Col3a1 and ECM deposition or hearing loss.

[0105] Downregulators and their applications

[0106] The research of this invention shows that knocking out NSD2 leads to excessive accumulation of ECM, affecting the morphology and function of cochlear hair cells, thereby causing hearing loss. However, targeted delivery of gene-editing reagents that knock down extracellular matrix (ECM)-related genes (such as Col6a3, Ogn, and Col3a1) inhibits the excessive accumulation of ECM caused by NSD2 deficiency, restores cochlear hair cell function, and improves hearing.

[0107] Based on the above-mentioned new discovery of the inventors, the present invention provides a use of an active ingredient, wherein the active ingredient is used to prepare a composition for treating hearing loss; and the active ingredient comprises: downregulators of ECM-related genes selected from the group consisting of Col6a3, Ogn, Col3a1, or combinations thereof.

[0108] As used herein, the term "gene downregulators" includes inhibitors, antagonists, blockers, occluders, degraders, etc., and these terms are used interchangeably.

[0109] The gene downregulator refers to any substance that can reduce the activity of the gene, reduce the stability of the gene or its encoding gene, downregulate the expression of the gene, reduce the effective duration of the gene's action, or inhibit the transcription and translation of the gene. These substances can all be used in this invention as substances useful for downregulating the gene, thereby reducing ECM deposition, improving the morphology and function of cochlear hair cells, lowering the hearing threshold, and thus effectively improving or treating hearing loss. For example, the downregulator includes interfering RNA molecules or antisense nucleotides that specifically interfere with the expression of the gene; antibodies or ligands that specifically bind to the protein encoded by the gene, etc.

[0110] As another particularly preferred embodiment of the present invention, the downregulator is a gene-specific interfering RNA molecule (such as siRNA, shRNA, miRNA, etc.). Such interfering RNA molecules can be prepared based on the sequence information of the aforementioned genes known in the prior art. There are no particular limitations on the preparation method of the interfering RNA molecule, including but not limited to: chemical synthesis, in vitro transcription, etc. The interfering RNA can be delivered into cells using appropriate transfection reagents, or it can be delivered into cells using various techniques known in the art.

[0111] In a preferred embodiment of the present invention, the downregulator can be a small molecule compound targeting the gene. Those skilled in the art can use conventional screening methods to screen for such small molecule compounds. For example, in the embodiments of the present invention, several optional screening methods are provided in conjunction with the regulatory mechanism disclosed herein.

[0112] As an optional approach of this invention, a CRISPR / Cas (e.g., Cas9) system can be used for targeted gene editing to knock out the gene in the target disease region. Common methods for knocking out the gene include co-transferring sgRNA or a nucleic acid capable of forming sgRNA, Cas9 mRNA or a nucleic acid capable of forming Cas9 mRNA to the target region or target cells. After identifying the target site, known methods can be used to introduce sgRNA and Cas9 into the cell. The nucleic acid capable of forming sgRNA is a nucleic acid construct or expression vector, or the nucleic acid capable of forming Cas9 mRNA is a nucleic acid construct or expression vector. These expression vectors are introduced into the cell, thereby forming active sgRNA and Cas9 mRNA within the cell.

[0113] As an optional approach of this invention, homologous recombination can be used to specifically target the gene, causing expression defects or deletions. Alternatively, Cre and Loxp methods can be applied to selectively knock out, reduce, or inactivate related genes in the cell's genome.

[0114] The above are some representative or preferred methods for downregulating the genes. After those skilled in the art understand the overall scheme of this invention, other methods known in the art can also be used to regulate the genes, and these methods are also included in this invention.

[0115] Applications related to diagnosis and prognostic assessment

[0116] This invention discloses targets that play a crucial regulatory role in hearing loss. Based on this new discovery, ECM-related genes (e.g., Col6a3, Ogn, Col3a1, or combinations thereof) can be used as targets for diagnosing or predicting hearing loss recovery: (i) for the classification and differential diagnosis of hearing loss; (ii) for evaluating treatment drugs, drug efficacy, prognosis, and selecting appropriate treatment methods in relevant populations (e.g., those with sensorineural hearing loss). For example, individuals with abnormal gene expression can be isolated, allowing for more targeted therapy.

[0117] The prognosis of the subject providing the sample can be predicted by assessing the expression or activity of the gene in the sample, allowing for the selection of appropriate drugs for treatment. Typically, a threshold for gene expression can be defined; when the gene expression exceeds this threshold, treatment to inhibit the gene is considered. This threshold is easily determined by those skilled in the art; for example, it can be obtained by comparing the gene expression in normal human cells or tissues with that in the subject's cells or tissues. The specific value of the threshold may vary depending on the measurement parameters and instruments used.

[0118] The presence and expression of the gene can be detected using various techniques known in the art, all of which are included in this invention. For example, existing techniques such as Southern blotting, Western blotting, DNA sequencing, and PCR can be used, and these methods can be used in combination.

[0119] The present invention also provides reagents for detecting the presence and expression of the gene in an analyte. Preferably, when performing gene-level detection, primers that specifically amplify the gene or probes that specifically recognize the gene can be used to determine the presence or absence of the gene; when performing protein-level detection, antibodies or ligands that specifically bind to the protein encoded by the gene can be used to determine the expression of the gene.

[0120] The method of using antibodies that specifically bind to the gene to detect the expression of the gene in an analyte is a technique well known to those skilled in the art.

[0121] The design of gene-specific probes is a technique well known to those skilled in the art. For example, a probe can be prepared that can specifically bind to a specific site on the gene, but not specifically bind to other genes besides the gene, and the probe carries a detectable signal.

[0122] The present invention also provides a kit for detecting the presence and expression of the gene in an analyte, the kit comprising: primers for specifically amplifying the gene; probes for specifically recognizing the gene; or antibodies or ligands for specifically binding to the protein encoded by the gene.

[0123] In addition, the kit may also include various reagents required for DNA extraction, PCR, hybridization, color development, etc., including but not limited to: extraction buffer, amplification buffer, hybridization buffer, enzyme, control solution, color development solution, washing solution, etc.

[0124] In addition, the kit may also include instructions for use and / or nucleic acid sequence analysis software.

[0125] Drug screening

[0126] Having established the close relationship between ECM-related genes (Col6a3, Ogn, Col3a1, or combinations thereof) and hearing loss, substances that inhibit the expression or activity of these genes can be screened based on this characteristic. Drugs that are truly useful for improving or treating hearing loss can then be found among these substances.

[0127] Therefore, this invention provides a method for screening potential substances (candidate substances or candidate drugs) for improving or treating hearing loss. The method includes: treating a system expressing ECM-related genes (Col6a3, Ogn, Col3a1, or combinations thereof) with a candidate substance; and detecting the expression or activity of the gene in the system. If the candidate substance inhibits the expression or activity of the gene, it indicates that the candidate substance is a potential substance for improving or treating hearing loss. Preferably, the system expressing the gene is a cell (or cell culture) system, and the cells may be cells endogenously expressing the gene or cells recombinantly expressing the gene. Furthermore, the usefulness of the potential substance can also be assessed by observing the interaction between the gene and its upstream and downstream proteins.

[0128] Based on the inventors' research results, as a preferred method of the screening method of the present invention, the effectiveness of the potential substance (candidate substance or candidate drug) can be further determined by analyzing the morphology and function of cochlear hair cells and / or ECM deposition. This can typically be performed using a cochlear hair cell culture screening system that culture cells expressing the gene. Observable improvements in the morphology and function of cochlear hair cells or a significant reduction in ECM deposition predict the effectiveness of the potential substance.

[0129] In a preferred embodiment of the present invention, during screening, a control group may be set up to facilitate observation of changes in gene expression or activity. The control group can be a system expressing the gene without the addition of the candidate substance. The control group includes, but is not limited to, a blank control without the candidate substance, an empty plasmid control, etc.

[0130] As a preferred embodiment of the invention, the method further includes: conducting further cell experiments and / or animal experiments on the obtained potential substances to further select and identify substances that are truly useful for improving or treating hearing loss.

[0131] On the other hand, the present invention also provides potential substances for improving or treating hearing loss obtained using the aforementioned screening method. These initially screened substances can constitute a screening library, from which substances useful for inhibiting the expression and activity of the aforementioned genes, thereby improving or treating hearing loss, can ultimately be selected.

[0132] Pharmaceutical Compositions and Administration

[0133] The present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and an effective amount of the following active ingredient: a downregulator of an ECM-related gene selected from the group consisting of Col6a3, Ogn, Col3a1, or a combination thereof.

[0134] As used herein, the term “effective amount” or “effective dose” means an amount that is functional or active in humans and / or animals and is acceptable to humans and / or animals.

[0135] As used herein, the term “therapeutic effective dose” means any amount of a drug as described below, which, when used alone or in combination with another therapeutic agent, promotes disease remission, manifested as a reduction in the severity of disease symptoms, an increase in the frequency and duration of symptom-free periods, or prevention of impairment or disability caused by the disease.

[0136] The "therapeutic effective dose" of the drug of the present invention also includes the "preventive effective dose". The "preventive effective dose" is any amount of the drug as described below, which, when administered alone or in combination with another therapeutic agent, can inhibit the occurrence or recurrence of the disease in a subject at risk of developing the disease or suffering from a recurrence of the disease.

[0137] As used herein, a "pharmaceuticalally acceptable" ingredient is a substance suitable for human and / or mammalian use without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., a substance with a reasonable benefit / risk ratio. The term "pharmaceuticalally acceptable carrier" refers to a carrier used for the administration of therapeutic agents, including various excipients and diluents.

[0138] The pharmaceutical compositions of the present invention contain a safe and effective amount of the active ingredient of the present invention and a pharmaceutically acceptable carrier. Such carriers include (but are not limited to): saline, buffer solutions, glucose, water, glycerol, ethanol, and combinations thereof. Generally, pharmaceutical formulations should be matched to the route of administration; the dosage forms of the pharmaceutical compositions of the present invention are injections, oral formulations (tablets, capsules, oral liquids), transdermal formulations, and sustained-release formulations. They are prepared, for example, using physiological saline or an aqueous solution containing glucose and other excipients by conventional methods. The pharmaceutical compositions are preferably manufactured under aseptic conditions.

[0139] The effective amount of the active ingredient described in this invention can vary depending on the administration method and the severity of the disease to be treated. A preferred effective amount can be determined by those skilled in the art based on various factors (e.g., through clinical trials). These factors include, but are not limited to: pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, and half-life; the severity of the disease to be treated, the patient's weight, the patient's immune status, and the route of administration. Generally, satisfactory results are obtained when the active ingredient of this invention is administered daily at a dose of approximately 0.00001 mg to 50 mg / kg animal body weight (preferably 0.0001 mg to 10 mg / kg animal body weight). For example, due to the urgency of the treatment condition, several separate doses may be administered daily, or the dose may be reduced proportionally.

[0140] The pharmaceutically acceptable carriers described in this invention include (but are not limited to): water, saline, liposomes, lipids, proteins, protein-antibody conjugates, peptides, cellulose, nanogels, or combinations thereof. The choice of carrier should be matched to the route of administration, as is well known to those skilled in the art.

[0141] The active ingredient of this invention can be administered alone or in combination with other pharmaceutically acceptable therapeutic agents. In another preferred embodiment, the pharmaceutical composition further comprises one or more drugs or preparations for treating hearing loss.

[0142] The pharmaceutical compositions of the present invention can be formulated into dosage forms such as injections, lyophilized formulations, nebulized inhalers, and topical preparations. The pharmaceutical compositions of the present invention can be delivered (administered) by any suitable method, including oral, parenteral, and topical methods. The pharmaceutical compositions of the present invention can also be administered by injection, i.e., intravenous, intratumoral, intramuscular, intradermal, subcutaneous, or intraperitoneal injection. Furthermore, the pharmaceutical compositions of the present invention can be administered transdermally. Transdermal administration via a local route can be formulated into medicated sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes, gels, paints, powders, and aerosols. In addition, the pharmaceutical compositions of the present invention can be actively administered to intradermal, subcutaneous, intramuscular, tumor, tissue, organ, and central nervous system sites via electrodes / electric fields / potential differences.

[0143] The pharmaceutical compositions of the present invention can be co-administered with another active agent. Co-administration includes administering the compound and active agent of the present invention within 0.5, 1, 2, 4, 6, 8, 10, 12, 16, 20, or 24 hours of each other. Co-administration also includes administering the compound and active agent of the present invention simultaneously, substantially simultaneously (e.g., within about 1, 5, 10, 15, 20, or 30 minutes of each other), or sequentially in any order. In some embodiments, co-administration can be accomplished by co-formulation, i.e., preparing a single pharmaceutical composition comprising both the active ingredient (a downregulator of ECM-related genes) and the active agent of the present invention. In other embodiments, the active ingredient and active agent of the present invention can be formulated separately.

[0144] medicine box

[0145] This document also describes a medicine box comprising a therapeutic agent (e.g., downregulators of ECM-related genes selected from the group consisting of Col6a3, Ogn, Col3a1, or combinations thereof; and optionally other medications for the treatment of hearing loss) and its formulation, as well as instructions for use. The medicine box also contains a diagnostic reagent for detecting the expression levels of the following genes: Col6a3, Ogn, Col3a1, or combinations thereof. The medicine box typically includes a label indicating the intended use of the contents. Terminology labels include any written or recorded material provided on, with, or additionally accompanying the medicine box.

[0146] In addition to the components described above, the kit of the present invention also includes (in some embodiments) instructions for practicing the subject method. These instructions may exist in various forms within the subject kit, one or more of these forms. One form of these instructions may be printed information on a suitable medium or substrate in the packaging, insert, etc., of the kit, for example, one or more sheets of paper on which information is printed. Another form of these instructions is a computer-readable medium on which information is recorded, such as a floppy disk, CD, flash drive, etc. Yet another form of these instructions may be a URL used to access the information remotely via the Internet. Preferably, the instructions indicate that if a significant change in the expression level of the gene occurs when the test reagent is used to test the in vitro sample, the test subject should be treated with the therapeutic agent.

[0147] The main advantages of this invention include:

[0148] (a) The present invention uses an AAV vector to achieve specific delivery to cochlear hair cells, avoiding non-targeting effects.

[0149] (b) The drugs or compositions of the present invention can directly inhibit the expression of ECM-related genes and significantly reduce excessive ECM deposition.

[0150] (c) The drugs or compositions of the present invention are applicable to a variety of sensorineural hearing loss (such as noise-induced hearing loss, drug-induced hearing loss, and age-related hearing loss).

[0151] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0152] Example 1: NSD2 defects lead to ECM deposition and hearing loss

[0153] 1.1 Experimental Materials

[0154] Nsd2 f / f Mice (constructed according to existing methods) and Atoh1-cre mice were both C57BL / 6J background and were housed in a specific pathogen-free (SPF) environment.

[0155] Instruments: Confocal electron microscope (for observing hair cell morphology), ABR tester (for detecting hearing threshold), and real-time quantitative PCR instrument (Applied Biosystems) (for detecting gene expression).

[0156] 1.2 Experimental Methods

[0157] Constructing a hearing loss model: using Atoh1-cre mice and Nsd2 f / f Mice are mated to produce offspring, which are NSD2-specific knockout mice (Nsd2). Atoh1-KO The modeling process is as follows: Figure 1 As shown in the figure. Hearing tests were performed on the mice after modeling, and the results are as follows. Figure 2 As shown, Nsd2 was found Atoh1-KO The hearing threshold of mice increased significantly, Nsd2 Atoh1-KO The mice have hearing loss.

[0158] The control group mice were Nsd2. f / f Mice.

[0159] The gene expression of offspring mice was examined, especially the expression of ECM-related genes, ECM deposition, and hearing loss.

[0160] 1.3 Experimental Results

[0161] Experimental results are as follows Figure 3 As shown.

[0162] Masson staining can be used to observe ECM expression. A major component of the ECM is collagen fibers, which are stained red by Ponceau S. Figure 3 The top left image shows that the red color is more intense in the cochlear hair cells after NSD2 knockout, indicating increased collagen fiber expression.

[0163] The inventors performed immunofluorescence staining using type III collagen (collagen 3) as a representative protein, and the results are as follows: Figure 3 The image in the upper right corner and Figure 3 As shown in the figure below, increased collegen3 expression was observed in cochlear hair cells after NSD2 knockout. Therefore, it can be concluded that ECM expression increases after NSD2 knockout.

[0164] like Figure 4 As shown, NSD2 deficiency leads to a decrease in H3K36me2 levels, which in turn activates the transcription of ECM-related genes (such as Col6a3, Ogn, Angpt1, Col3a1, Serpinb1a, and lgf1) through abnormally open chromatin structures, resulting in a significant increase in the expression of these genes, ultimately leading to excessive ECM deposition and hearing loss.

[0165] Example 2

[0166] 2.1 Experimental Methods

[0167] 2.1 Experimental Methods

[0168] (1) Constructing AAV carriers

[0169] like Figure 5 As shown.

[0170] Serotype: Use AAV2 / 8 or AAV2 / 9, as they have a high affinity for cochlear hair cells;

[0171] Promoter design: Carrying the Atoh1 promoter to ensure specific expression in cochlear hair cells;

[0172] Nucleic acid sequence insertion:

[0173] The shRNA sequence targets the mRNA regions of Col6a3 and Ogn to knock down the Col6a3 or Ogn gene; the specific sequence is shown below:

[0174] Col6a3 shRNA:

[0175] AGTTCTATTTAAATTCCTAttcaagagaTAGGAATTTAAATAGAACT(SEQ ID NO:1);

[0176] Ogn shRNA:

[0177] CGTGTAATTCACCTTCAGTTTctcgagAAACTGAAGGTGAATTACACG (SEQ ID NO: 2).

[0178] (2) Cell model and grouping

[0179] Cellular material: The hairy cell line HEI-OC1 was used. The Nsd2 gene was knocked out in this cell line using a lentiviral CRISPRv2 plasmid, and the knockout cell line was named Sg2. Cv2 served as the control group, which was transfected only with the empty CRISPRv2 vector.

[0180] The hair-like cell line HEI-OC1, after the Nsd2 gene was knocked out, was further cultured, and the results were as follows: Figure 6 As shown in the figure. The results showed that, compared with the control group, cell growth was defective after knocking out the Nsd2 gene.

[0181] Experimental groups: Using a CRISPR / Cas9 system containing sgRNA and SpCas9 coding sequences targeting the above genes (Col6a3 or Ogn), the Col6a3 or Ogn genes were knocked out or knocked down. Cell experimental groups 2 (Sg2) with Col6a3 knockout or knockdown were obtained. Col6a3-KO ), and Ogn knockout or knockdown cell experimental group 1 (Sg2) Ogn-KO ).

[0182] (3) Animal models and grouping

[0183] Mouse strain: Nsd2 conditional knockout mice (Atoh1-Cre mice and Nsd2) f / f The F2 generation mice after mating, i.e., NSD2 Atoh1-KO (Mice); and the test revealed that the hearing threshold of the mice was significantly increased, indicating that the model was successfully established.

[0184] Experimental Groups:

[0185] Experimental group: To NSD2 Atoh1-KO The AAV vectors constructed in step (1) of local cochlear injection in mice, namely the AAV vector containing Col6a3 shRNA and the AAV vector containing Ogn shRNA, were used to obtain Col6a3 knockdown mouse NSD2. Atoh1-KO Col6a3, and Ogn knockdown mouse NSD2Atoh1-KO Ogn.

[0186] Control group: NSD2 Atoh1-KO Mouse cochlea was locally injected with empty AAV carrier or physiological saline.

[0187] (4) Detection indicators

[0188] Hearing function: ABR test (8-32kHz frequency range);

[0189] Histological analysis: Masson trichrome staining was used to detect ECM deposition;

[0190] Molecular level: qPCR detection of ECM-related gene expression levels.

[0191] 2.2 Experimental Results

[0192] like Figure 7 As shown, in Sg2, knocking out or downsampling the Ogn gene significantly inhibited the expression of ECM-related genes (Col6a3, Ogn, Col3a1, Ccdc80); furthermore, knocking out or downsampling the Col6a3 gene also significantly inhibited the expression of ECM-related genes (Col6a3, Ogn, Col3a1, Ccdc80). This effectively reduces ECM deposition.

[0193] like Figure 8 As shown, the inventors cultured Sg2 cells after Col6a3 and Ogn knockout and found that, compared with the control group Sg2, knocking out the Col6a3 and Ogn genes could restore cell growth. This indicates that knocking out Col6a3 and / or Ogn can salvage the cell growth defects caused by Nsd2 deficiency.

[0194] like Figure 9 As shown, RNA was extracted from the cochlea of ​​NSD2 gene-deficient mice after ECM-related genes (Col6a3 or Ogn) were knocked out or knocked down, and the expression of ECM-related genes was identified by qPCR. It was found that the AAV vector was successfully delivered to cochlear hair cells and significantly reduced the expression of Col6a3, Ogn, Col3a1 and Ccdc80 in Col6a3 knockdown mice and Ogn knockdown mice.

[0195] like Figure 10 As shown, Masson staining revealed a significant reduction in ECM deposition in the cochlea of ​​the animal experimental group.

[0196] like Figure 11As shown, after targeted knockdown of two ECM-related genes (Ogn or Col6a3) in NSD2 knockout mice, significant recovery of hearing thresholds was observed to varying degrees in both groups. Specifically, the two experimental groups of mice (Col6a3 knockdown group and NSD2 knockdown group) showed significant recovery of hearing thresholds to varying degrees. Atoh1-KO Col6a3 and Ogn knockdown group NSD2 Atoh1-KO The Ogn hearing threshold was significantly lower than that of the control group, with a reduction of approximately 5-40 dB (P<0.01).

[0197] In summary, the above results indicate that knocking down Ogn and / or Col6a3 expression can effectively reduce ECM deposition and treat hearing loss. Similarly, knocking down Col3a1 expression can achieve the same or similar effects.

[0198] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. The use of an active ingredient, characterized in that, The active ingredient is used to prepare a composition for treating hearing loss; and the active ingredient comprises: Downregulators of ECM-related genes selected from the following group: Col6a3, Ogn, Col3a1, or combinations thereof.

2. The use as described in claim 1, characterized in that, The treatment includes: (a) Reduce excessive ECM deposition in the cochlea; (b) Restoring the morphology and function of cochlear hair cells; and (c) Lower the hearing threshold.

3. The use as described in claim 1, characterized in that, The hearing loss mentioned is sensorineural hearing loss.

4. The use as described in claim 1, characterized in that, The hearing loss mentioned is caused by excessive ECM deposition.

5. The use as described in claim 1, characterized in that, The downregulator is a small molecule chemical antagonist or inhibitor targeting the gene; or the downregulator is a reagent that knocks out or knocks down the gene.

6. The use as described in claim 1, characterized in that, The reagents used to knock out or silence the gene are selected from the following group: (a) A reagent containing the nucleotide sequence shown in SEQ ID NO:1; (b) A reagent containing the nucleotide sequence shown in SEQ ID NO:2; (c) A combination of (a) and (b) above.

7. A pharmaceutical composition for treating hearing loss, characterized in that, include: (a) Downregulators of ECM-related genes selected from the following group: Col6a3, Ogn, Col3a1, or combinations thereof; as well as (b) Pharmaceutically acceptable carriers.

8. The pharmaceutical composition according to claim 7, characterized in that, The downregulator is a small molecule chemical antagonist or inhibitor targeting the gene; or the downregulator is a reagent that knocks out or knocks down the gene.

9. The pharmaceutical composition according to claim 8, characterized in that, The reagents for knocking out or knocking down the gene include: interfering molecules that specifically interfere with the expression of the gene encoding the gene; CRISPR gene editing reagents targeting the gene; and homologous recombination reagents or site-directed mutagenesis reagents targeting the gene, wherein the homologous recombination reagents or site-directed mutagenesis reagents induce loss-of-function mutations in the gene.

10. A method for screening potential substances for treating hearing loss, characterized in that, include: (1) Treat an expression system with a candidate substance, the system expressing genes selected from the group consisting of Col6a3, Ogn, Col3a1, or combinations thereof; and (2) Detect the expression or activity of the gene in the system; if the candidate substance statistically downregulates the expression or activity of the gene, then the candidate substance is a potential substance for treating hearing loss.

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