Composition for treating hearing loss comprising viral vector

Abstract

It is an object of the present invention to provide a viral vector comprising a EF1a promoter and genes encoding neuronal differentiation proteins. Another object of the present invention is to provide a pharmaceutical composition for preventing or treating hearing loss, which comprises a viral vector comprising a EF1a promoter and neuronal differentiation protein-encoding genes.SOLUTION: [Technical Field] The present invention relates to a viral vector comprising a EF1a promoter and expressing MPZL2, and a pharmaceutical composition for preventing or treating hearing loss comprising the vector as an active ingredient. According to the present invention, genetic deafness caused by Mpzl2 mutation can be fundamentally treated by introducing a recombinant virus in the absence of a practical therapeutic agent.SELECTED DRAWING: Figure 2C

Description

[Technical Field]

[0001] The present invention relates to a composition for treating hearing loss that contains a viral vector, and more specifically to a viral vector that contains the EF1a promoter and expresses MPZL2, and a composition for treating hearing loss that contains the same. [Background technology]

[0002] Hearing loss can be divided into sensorineural hearing loss and conductive hearing loss depending on the structure of the auditory organ that causes it, and can also be classified based on whether it is hereditary or not.Hereditary hearing loss can be broadly divided into syndromic hearing loss and non-syndromic hearing loss, which can be further divided into autosomal dominant and autosomal recessive inheritance depending on the mode of inheritance.

[0003] MPZL2 mutations cause autosomal recessive nonsyndromic hearing loss (DFNB111), and because the disease results from loss of function of both alleles, gene replacement therapy can be applied to increase or replace the normal gene.

[0004] Currently, the only practical solutions for hearing loss due to hearing loss are hearing aids or cochlear implants, but no fundamental cure has been established. In this context, gene therapy has attracted much attention as a way to treat the cause of hereditary hearing loss. However, since problems can arise from in vivo overexpression during the delivery of exogenous genes, it is important to ensure gene expression is within a range that does not cause toxicity. The present inventors have conducted extensive research to develop a method for fundamentally treating hereditary hearing loss, particularly hearing loss caused by MPZL2 mutations. As a result, they discovered that injection of a vector containing the EF1a promoter increases gene expression within a range that does not cause toxicity, resulting in stable hearing recovery, leading to the completion of the present invention.

[0005] Numerous papers and patent documents are referenced throughout this specification and citations are provided, the disclosures of which are incorporated herein by reference in their entirety to more clearly describe the state of the art and the content of the present invention. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Korean Patent Registration Publication No. 10-2051393 Summary of the Invention [Problem to be solved by the invention]

[0007] An object of the present invention is to provide a viral vector comprising an EF1a promoter and a gene encoding a neuronal differentiation protein.

[0008] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating hearing loss, which comprises a viral vector containing an EF1a promoter and a gene encoding a neuronal differentiation protein.

[0009] The technical problems that the present invention aims to solve are not limited to those mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the following description. [Means for solving the problem]

[0010] Various embodiments described herein are described below with reference to the drawings. In the following description, various specific details, such as specific configurations, compositions, and processes, are set forth to provide a thorough understanding of the present invention. However, certain embodiments may be practiced with one or more of these specific details, without them, or with other known methods and configurations. In other instances, known processes and manufacturing techniques are not described in specific detail to avoid unnecessarily obscuring the present invention. Reference throughout this specification to "one embodiment" or "embodiment" means that a particular feature, configuration, composition, or characteristic described in connection with the embodiment is included in one or more embodiments of the present invention. Thus, the appearance of "in one embodiment" or "embodiment" in various places throughout this specification does not necessarily refer to the same embodiment of the present invention. Additionally, particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more embodiments.

[0011] Unless otherwise defined herein, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0012] As used herein, the term "EF1a promoter (Eukaryotic Translation Elongation Factor 1 Alpha promoter)" refers to a DNA sequence that regulates the expression of the EF1a gene, which plays an important role in the translation process. It is derived from the human EF1A1 gene, which encodes the eukaryotic elongation factor 1a (Ef-1a), a protein essential for cellular translation. The EF1a promoter can drive high levels of gene expression in many cell types and is particularly effective in cell lines such as immune cells and stem cells. This is because the EF1a promoter is relatively insensitive to silencing in these cell types. The EF1a promoter can drive high levels of gene expression in a variety of cell lines, operates in a variety of species, including mammalian, avian, and some plant cell lines, provides long-term, stable gene expression, and can regulate expression with a variety of regulatory elements. The EF1a promoter is widely used for research and therapeutic purposes due to its ability to drive significant gene expression.

[0013] As used herein, the term "CAG promoter (CMV early enhancer / chicken beta-actin promoter)" refers to a synthetic promoter that combines the cytomegalovirus early enhancer and the chicken beta-actin promoter. It is particularly effective in neural cells and can be used with regulatory elements to achieve tissue- or time-specific expression. The CAG promoter can induce stable and strong gene expression in a variety of cell types and is widely used for research and therapeutic purposes.

[0014] As used herein, the term "viral vector" refers to a tool that utilizes the properties of viruses to deliver genetic material into cells, and refers to a virus that has been engineered to deliver a specific gene to a target cell using the viral infection mechanism.

[0015] As used herein, the term "AAV (Adeno-Associated Virus)" refers to a small, nonpathogenic virus often used as a vector for gene therapy. When a desired target gene sequence is inserted between the ITR sequences and injected, the target gene is expressed in vivo in an episomal form for a long period of time. The term "AAV" can also refer to the virus itself or its derivatives, such as AAV vectors, AAV viral particles, and AAV virions. The term "AAV" includes all subtypes, naturally occurring forms, and recombinant forms.

[0016] As used herein, the term "AVV-DJ (Adeno-Associated Virus-DJ)" refers to a modified AAV vector that has been engineered for a specific purpose, combining the capsid proteins of AAV2, AAV8, and AAV9 to enhance gene transfer efficiency and targeting efficiency to specific tissues.

[0017] As used herein, the term "AVV-PHP.eB (Adeno-Associated Virus-PHP.eB)" refers to a variant of an AAV vector that targets the central nervous system (CNS), and is particularly characterized by its ability to transfer genes to the brain and spinal cord with high efficiency in mouse models.

[0018] As used herein, the term "MPZ (Myelin Protein Zero)" refers to a neuronal differentiation protein associated with neurons and involved in the formation of myelin sheaths. "MPZL (Myelin Protein Zero-like)" refers to a protein that plays a functional role in cell-cell interactions and cell surface functions. Without limitation, MPZL2 is a member of the immunoglobulin superfamily (IgSF), which includes MPZL1 (Myelin Protein Zero-Like1), MPZL2, and MPZL3. The immunoglobulin superfamily (IgSF) is composed of a variety of cell surface molecules and performs functions such as immune response, cell adhesion, and signal transduction. Proteins in this superfamily share an immunoglobulin domain and play an important role in cell-cell interactions and signal transduction. MPZL2 (Myelin Protein Zero-Like2) is a membrane protein that plays an important role in various physiological processes, including cell-cell signaling, immune response regulation, cell adhesion and tissue formation, and the development and maintenance of the nervous and immune systems. MPZL2 is expressed in various organs and tissues. In particular, MPZL2 expression plays an important role in the maintenance and development of hearing function. It is expressed in the inner hair cells (IHCs), outer hair cells (OHCs), Deiter's cells, and pillar cells of the organ of Corti, and plays an essential role in the normal maintenance and development of hearing function. Mutations in the MPZL2 gene can cause neurodevelopmental, metabolic, and immune disorders, as well as DFNB111, a type of autosomal recessive non-syndromic hearing loss.In addition, diseases caused by mutations in the Mpzl2 gene may include, but are not limited to, metabolic disorders associated with abnormalities in membrane protein signaling functions, neurodegenerative diseases associated with abnormalities in neuronal signaling functions, and cancers, autoimmune diseases, and vascular diseases associated with abnormalities in cell adhesion functions (see Am J Hum Genet. 2018Jul5; 103(1):74-88. doi:10.1016 / j.ajhg.2018.05.011 and Hum Genet. 2018Jul; 137(6-7):479-486. doi:10.1007 / s00439-018-1901-4).

[0019] As used herein, the term "hearing loss" refers to a condition or disease in which hearing is impaired and sounds cannot be heard properly. Hearing loss can be divided into sensorineural hearing loss and conductive hearing loss depending on the structure of the auditory organ that causes it, and can also be classified based on whether it is hereditary or not. Hereditary hearing loss can be broadly divided into syndromic hearing loss and non-syndromic hearing loss, which can be further divided into autosomal dominant and autosomal recessive types depending on the mode of inheritance. Sensorineural hearing loss refers to hearing loss caused by damage to the inner ear or auditory nerve. Conductive hearing loss refers to hearing loss that does not reach the inner ear due to problems with the outer ear or middle ear. Syndromic hearing loss refers to hearing loss that occurs in conjunction with other symptoms or medical conditions. Non-syndromic hearing loss refers to hearing loss that occurs independently without other medical symptoms or conditions, and is the type to which most hereditary hearing loss belongs. As used herein, the term "hearing loss" includes, but is not limited to, sensorineural hearing loss, conductive hearing loss, syndromic hearing loss, and non-syndromic hearing loss.

[0020] As used herein, the term "metabolic disorder" refers to a disease in which an abnormality occurs in the body's metabolic processes. Metabolism is a general term for chemical reactions that occur in the body, including processes that convert nutrients into energy and support cell growth and repair. Metabolic disorders occur when enzymes and proteins necessary for these metabolic processes are deficient or function abnormally. Examples of metabolic disorders include, but are not limited to, diabetes, hyperthyroidism, hypothyroidism, phenylketonuria, Gaucher disease, galactosemia, organic aciduria, lipid storage disorders, and anemia.

[0021] As used herein, the term "neurodegenerative disease" refers to a disease in which nerve cells in the central or peripheral nervous system are gradually damaged and lose function. Such diseases worsen over time, resulting in the loss of physical, cognitive, and functional abilities. Neurodegenerative diseases include, but are not limited to, Alzheimer's disease, Parkinson's disease, Lou Gehrig's disease, Huntington's disease, multiple sclerosis, and prion diseases.

[0022] As used herein, the term "cancer" refers to a disease that can disrupt or destroy the function of tissues or organs through the abnormal growth and division of normal cells, and abnormalities in cell adhesion proteins play an important role in the process of tumor formation and metastasis. The cancer may be, but is not limited to, breast cancer, ovarian cancer, colon cancer, gastric cancer, liver cancer, pancreatic cancer, cervical cancer, thyroid cancer, parathyroid cancer, lung cancer, non-small cell lung cancer, prostate cancer, gallbladder cancer, biliary tract cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, blood cancer, bladder cancer, kidney cancer, melanoma, colon cancer, bone cancer, skin cancer, head cancer, uterine cancer, rectal cancer, brain tumor, perianal cancer, fallopian tube carcinoma, endometrial carcinoma, vaginal cancer, vulva carcinoma, esophageal cancer, small intestine cancer, endocrine gland cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system (CNS) tumor, primary CNS lymphoma, spinal cord tumor, brain stem glioma, or pituitary adenoma.

[0023] As used herein, the term "autoimmune disease" refers to a condition in which the immune system attacks healthy tissues and cells, and abnormalities in cell adhesion function can affect the immune system. Autoimmune diseases may include, but are not limited to, lupus, rheumatoid arthritis, Crohn's disease, and peripheral vascular disease.

[0024] As used herein, the term "vascular disease" refers to a variety of diseases that can occur in the vascular system, and abnormalities in intravascular cell adhesion proteins can cause structural instability of blood vessels, resulting in a variety of diseases, including, but not limited to, arteriosclerosis, thrombosis, vascular artery disease, and hypertension.

[0025] In this specification, the term "Auditory Brainstem Response (ABR)" refers to a test method for measuring the auditory brainstem response, which measures how the auditory nerve transmits sound stimuli to the brain. The functional status of the auditory system is evaluated by measuring the electrical signals generated when sound stimuli pass through the inner ear canal.

[0026] In this specification, the term "DPOAE (Distortion Product Otoacoustic Emissions)" refers to a test method for measuring distortion-generated noises. It uses minute sounds to measure ultrasound waves generated by sound reflection in the inner ear canal. It is particularly useful for assessing the degree and type of hearing loss, and is generally used together with ABR to improve the completeness of hearing diagnosis.

[0027] As used herein, the term "express" refers to the artificial introduction of an exogenous gene into a subject's cells using a gene carrier to increase the natural expression level of an endogenous gene, thereby making the gene replicable in the subject's cells as an extrachromosomal element or by chromosomal integration. Therefore, the term "expression" has the same meaning as "transformation," "transfection," or "transduction."

[0028] As used herein, the term "prevention" 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.

[0029] As used herein, the term "treatment" refers to (a) inhibiting the development of a disease, disorder, or symptom; (b) alleviating a disease, disorder, or symptom; or (c) eliminating a disease, disorder, or symptom. The compositions of the present invention can inhibit, eliminate, or alleviate the development of hearing loss symptoms in patients with hearing loss caused by a mutation in the Mpzl2 gene by increasing expression of the Mpzl2 gene via a recombinant vector, ultimately reversibly restoring permanent hearing threshold changes. Therefore, the compositions of the present invention may be used alone to treat hereditary hearing loss, or may be administered together with other pharmacological ingredients to serve as a therapeutic adjunct. Therefore, as used herein, the terms "treatment" or "therapeutic agent" encompass the meaning of "therapeutic adjunct" or "therapeutic adjunct."

[0030] In addition, the composition of the present invention can suppress, eliminate, or alleviate the development of symptoms of metabolic disorders caused by mutations in the Mpzl2 gene by increasing the expression of the Mpzl2 gene via a recombinant vector.

[0031] The composition of the present invention can suppress, eliminate, or alleviate the development of symptoms of neurodegenerative diseases caused by mutations in the Mpzl2 gene by increasing the expression of the Mpzl2 gene via a recombinant vector.

[0032] The composition of the present invention can suppress, eliminate, or alleviate the development of symptoms of neurodegenerative diseases caused by mutations in the Mpzl2 gene by increasing the expression of the Mpzl2 gene via a recombinant vector.

[0033] The composition of the present invention can suppress, eliminate or alleviate the development of cancer caused by mutations in the Mpzl2 gene by increasing the expression of the Mpzl2 gene via a recombinant vector.

[0034] The composition of the present invention can suppress, eliminate, or alleviate the development of symptoms of autoimmune diseases caused by mutations in the Mpzl2 gene by increasing the expression of the Mpzl2 gene via a recombinant vector.

[0035] The composition of the present invention can suppress the development of, eliminate, or alleviate the symptoms of vascular disease caused by mutations in the Mpzl2 gene by increasing the expression of the Mpzl2 gene via a recombinant vector.

[0036] As used herein, the term "administration" or "administering" refers to administering a therapeutically effective amount of a composition of the present invention directly to a subject, thereby allowing the same amount to be formed in the subject's body.

[0037] In the present invention, the term "therapeutically effective amount" means the content of the composition in an amount sufficient to provide a therapeutic or prophylactic effect to an individual to whom the pharmaceutical composition of the present invention is to be administered, and therefore includes a "prophylactically effective amount."

[0038] As used herein, the term "subject" includes, without limitation, a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, monkey, chimpanzee, baboon, or rhesus monkey. Specifically, the subject of the present invention is a human.

[0039] When the composition of the present invention is prepared as a pharmaceutical composition, the pharmaceutical composition of the present invention contains a pharmaceutically acceptable carrier.

[0040] Pharmaceutically acceptable carriers contained in the pharmaceutical compositions of the present invention are those commonly used in formulations, including, but not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition to the above ingredients, the pharmaceutical compositions of the present invention may additionally contain lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

[0041] The pharmaceutical compositions of the present invention can be administered orally or parenterally, specifically by subcutaneous, transdermal, intravenous or intratympanic administration.

[0042] The suitable dosage of the pharmaceutical composition of the present invention can be variously formulated depending on factors such as formulation method, administration method, age, weight, sex, pathological condition, food, administration time, administration route, excretion rate and reaction sensitivity of the patient, etc. The preferred dosage of the pharmaceutical composition of the present invention is within the range of 0.001-100 mg / kg for adults.

[0043] The pharmaceutical compositions of the present invention may be prepared in unit dose form or in multi-dose containers by formulating them with pharmaceutically acceptable carriers and / or excipients in a manner easily understood by those skilled in the art to which this invention pertains. In this case, the dosage form may be in the form of a solution, suspension, syrup, or emulsion in an oily or aqueous medium, or in the form of an extract, powder, granule, tablet, or capsule, and may additionally contain a dispersing agent or stabilizer.

[0044] In one embodiment of the present invention, a viral vector is provided that includes an EF1a promoter and a gene encoding a neuronal differentiation protein. In the above embodiment, the viral vector is an adeno-associated virus (AAV), the viral vector is AAV-DJ, the neuronal differentiation protein is an Mpzl protein, the Mpzl protein is Mpzl1, Mpzl2, or Mpzl3, the viral vector is for use in treating or preventing diseases caused by Mpzl2 mutations, and the viral vector targets hair cells or supporting cells (Deiter's cells, Pillar cells).

[0045] In one embodiment of the present invention, there is provided a pharmaceutical composition for preventing or treating hearing loss, which comprises the vector as an active ingredient.

[0046] In one embodiment of the present invention, there is provided a pharmaceutical composition for preventing or treating metabolic disorders, which comprises the vector as an active ingredient.

[0047] In one embodiment of the present invention, there is provided a pharmaceutical composition for preventing or treating neurodegenerative diseases, comprising the vector as an active ingredient.

[0048] In one embodiment of the present invention, there is provided a pharmaceutical composition for preventing or treating cancer, which comprises the vector as an active ingredient.

[0049] In one embodiment of the present invention, there is provided a pharmaceutical composition for preventing or treating autoimmune diseases, which comprises the vector as an active ingredient.

[0050] In one embodiment of the present invention, there is provided a pharmaceutical composition for preventing or treating vascular diseases, which comprises the vector as an active ingredient.

[0051] In one embodiment of the present invention, there is provided a method for preventing or treating hearing loss, comprising administering to an individual a pharmaceutical composition containing, as an active ingredient, a viral vector comprising an EF1a promoter and a gene encoding a neuronal differentiation protein.

[0052] In the specific example, the viral vector is an adeno-associated virus (AAV), the neuronal differentiation protein is an MPZ or MPZL protein, and the hearing loss is autosomal recessive non-syndromic hearing loss caused by a mutation.

[0053] In one embodiment of the present invention, there is provided a method for preventing or treating metabolic disorders caused by mutations in the Mpzl gene, comprising administering to an individual a pharmaceutical composition containing as an active ingredient a viral vector comprising the EF1a promoter and a gene encoding a neuronal differentiation protein.

[0054] In the above-mentioned specific example, the metabolic disorder is any one selected from the group consisting of diabetes, hyperthyroidism, hypothyroidism, phenylketonuria, Gaucher disease, galactosemia, organic aciduria, fat storage disease, anemia, and the like.

[0055] In one embodiment of the present invention, there is provided a method for preventing or treating a neurodegenerative disease caused by a mutation in the Mpzl gene, comprising administering to an individual a pharmaceutical composition containing as an active ingredient a viral vector comprising the EF1a promoter and a gene encoding a neuronal differentiation protein.

[0056] In the specific example, the neurodegenerative disease is any one selected from the group consisting of Alzheimer's disease, Parkinson's disease, Lou Gehrig's disease, Huntington's disease, multiple sclerosis, and prion disease.

[0057] In one embodiment of the present invention, a method for preventing or treating cancer caused by a mutation in the Mpzl gene is provided, comprising administering to an individual a pharmaceutical composition containing as an active ingredient a viral vector comprising the EF1a promoter and a gene encoding a neuronal differentiation protein.

[0058] In the above embodiment, the cancer is any one selected from the group consisting of breast cancer, ovarian cancer, colon cancer, gastric cancer, liver cancer, pancreatic cancer, cervical cancer, thyroid cancer, parathyroid cancer, lung cancer, non-small cell lung cancer, prostate cancer, gallbladder cancer, biliary tract cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, blood cancer, bladder cancer, kidney cancer, melanoma, colon cancer, bone cancer, skin cancer, head cancer, uterine cancer, rectal cancer, brain tumor, perianal cancer, fallopian tube carcinoma, endometrial carcinoma, vaginal cancer, vulva carcinoma, esophageal cancer, small intestine cancer, endocrine gland cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system (CNS) tumor, primary CNS lymphoma, spinal cord tumor, brain stem glioma, or pituitary adenoma.

[0059] In one embodiment of the present invention, a method for preventing or treating an autoimmune disease caused by a mutation in the Mpzl gene is provided, comprising administering to an individual a pharmaceutical composition containing, as an active ingredient, a viral vector comprising the EF1a promoter and a gene encoding a neuronal differentiation protein.

[0060] In the above embodiment, the method is provided wherein the autoimmune disease is any one selected from the group consisting of lupus, rheumatoid arthritis, Crohn's disease, marginalized disease, and the like.

[0061] In one embodiment of the present invention, a method for preventing or treating vascular diseases caused by mutations in the Mpzl gene is provided, comprising administering to an individual a pharmaceutical composition containing as an active ingredient a viral vector comprising the EF1a promoter and a gene encoding a neuronal differentiation protein.

[0062] In the above-mentioned specific example, the method is provided wherein the vascular disease is any one selected from the group consisting of arteriosclerosis, thrombosis, vascular artery disease, hypertension, and the like.

[0063] [Array List]

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[0064] The viral vector containing the EF1a promoter and a gene encoding a neuronal differentiation protein of the present invention, or a composition for treating hearing loss containing the viral vector as an active ingredient, can restore hearing loss caused by MPZL2 mutation through gene replacement therapy, thereby achieving a fundamental treatment for hereditary hearing loss, for which no therapeutic agent is currently available.

[0065] Furthermore, in the present invention, the use of the EF1a promoter can achieve stable gene expression within a range that does not cause toxicity, in response to the problem of in vivo overexpression that can occur during the process of transferring an exogenous gene. [Brief explanation of the drawings]

[0066] [Figure 1A]FIG. 1 shows hearing tests of mice injected with AAV-PHP.eB-hMPZL2 CAG promoter at 4 weeks of age, measured by ABR threshold (FIG. 1A). [Figure 1B] FIG. 1 shows hearing tests of mice injected with AAV-PHP.eB-hMPZL2 CAG promoter at 4 weeks of age, measured by DPOAE thresholds (FIG. 1B).

[0067] [Figure 2A] FIG. 2 shows the hearing test results of mice injected with AAV-DJ-hMPZL2 CAG promoter and EF1a promoter at 4 weeks of age (FIGS. 2A and 2B) by measuring ABR and DPOAE thresholds. [Figure 2B] FIG. 2 shows the hearing test results of mice injected with AAV-DJ-hMPZL2 CAG promoter and EF1a promoter at 4 weeks of age (FIGS. 2A and 2B) by measuring ABR and DPOAE thresholds. [Figure 2C] FIG. 2 shows the hearing test results of mice injected with AAV-DJ-hMPZL2 CAG promoter and EF1a promoter at 8 weeks of age (FIGS. 2C and 2D), measured by ABR and DPOAE threshold measurements. [Figure 2D] FIG. 2 shows the hearing test results of mice injected with AAV-DJ-hMPZL2 CAG promoter and EF1a promoter at 8 weeks of age (FIGS. 2C and 2D), measured by ABR and DPOAE threshold measurements. [Figure 2E] FIG. 2 shows the hearing test results of mice injected with AAV-DJ-hMPZL2 CAG promoter and EF1a promoter at 12 weeks of age (FIGS. 2E and 2F) using ABR and DPOAE threshold measurements. [Figure 2F] FIG. 2 shows the hearing test results of mice injected with AAV-DJ-hMPZL2 CAG promoter and EF1a promoter at 12 weeks of age (FIGS. 2E and 2F) using ABR and DPOAE threshold measurements.

[0068] [Figure 3] Figure 3 shows the expression level of hMPZL2 in the cochlea of ​​4-week-old mice injected with AAV-DJ-hMPZL2 CAG promoter and EF1a promoter. It also shows a graph comparing the expression levels of actin, which is regulated by the EF1a and CAG promoters, normalized to the EF1a promoter and the CAG promoter.

[0069] [Figure 4] FIG. 4 shows the results of immunofluorescence staining in the cochleae of 4-week-old mice injected with AAV-DJ-hMPZL2 CAG promoter and EF1a promoter. DETAILED DESCRIPTION OF THE INVENTION

[0070] Example 1. Creation of an MPZL2 mutation deafness mouse model We generated an MPZL2 mutation deafness mouse model using the CRSPR-CAS9 system at Cyagen Biosciences. Cas9 mRNA, one donor oligo, and two gRNAs [AATTTCCGACCTCGAGATGG-GGG (forward) and TGCTCACGACCCCCATCTCG-AGG (reverse)] were microinjected into fertilized eggs of C57BL / 6N mice. The Mpzl2 mutation was confirmed by Sanger sequencing and genotyping.

[0071] Example 2. AAV generation and injection The hair cell-specific AAV-PHP.eB serotype and the Deiter's cell- and pillar cell-specific AAV-DJ serotype were used. The AAV-PHP.eB and AAV-DJ viral vectors were engineered to deliver the coding sequence of the human MPZL2 gene, optimized for mouse codons, and carry the CAG and EF1a promoters. AAV-PHP.eB-CAG-MPZL2 and AAV-PHP.eB-EF1a-MPZL2 were used in 80 × 10 13 , 1.32 × 10 13 AAV-DJ-CAG-MPZL2 and AAV-DJ-EF1a-MPZL2 were produced with Vector Builder at titers of 2.03 x 10 genome copies per milliliter. 13 , 1.23×10 13 AAV-PHP.eB-CAG-MPZL2, AAV-PHP.eB-EF1a-MPZL2, AAV-DJ-CAG-MPZL2, and AAV-DJ-EF1a-MPZL2 were produced with a vector builder at titers of 1 genome copy per milliliter and stored at -80°C until thawed prior to in vivo injection.

[0072] Mice carrying the Mpzl2 p.Q74* mutation were anesthetized with ice for approximately 1 minute between days 0 and 3 after birth. AAV-PHP.eB-CAG-hMPZL2, AAV-PHP.eB-EF1a-hMPZL2, AAV-DJ-CAG-hMPZL2, and AAV-DJ-EF1a-hMPZL2 were then injected in vivo through the round window membrane (RWM) to deliver the vectors from the scala tympani to the scala media, targeting hair cells and supporting cells (Deiter's cells, Pillar cells).

[0073] Example 3. Hearing measurement Hearing tests were performed on mice injected with the viral vector of Example 2 and mice not injected with it (control group, Mpzl2-p.Q74*) at 4, 8, and 12 weeks of age using brainstem response (ABR) and distortion product otoacoustic emissions (DPOAE).

[0074] At 4 weeks of age, AAV-PHP.eB-CAG-hMPZL2-injected mice showed worse hearing than uninjected mice, as confirmed by ABR and DPOAE tests (Figure 1). AAV-DJ-CAG-hMPZL2-injected mice showed complete loss of hearing, as confirmed by ABR and DPOAE tests (Figure 2). However, AAV-DJ-EF1a-hMPZL2-injected mice showed better hearing than uninjected mice (control group) from 4 to 8 and 12 weeks of age, as confirmed by ABR tests. DPOAE tests also showed better hearing in AAV-DJ-EF1a-hMPZL2-injected mice than uninjected mice (control group) from 4 to 8 and 12 weeks of age (Figure 2).

[0075] This is summarized in Table 1:

[0076] [Table 1]

[0077] In the control group, which did not receive a viral vector, the threshold (decibels, dB) for the Click stimulus was 43.333. The threshold indicates sensitivity to sound; a lower value indicates that softer sounds can be heard more clearly. In the AAV-PHP.eB-CAG-hMPZL2 vector-injected group, the Click (dB) was 60, indicating a worsening of hearing compared to the control group (Figure 1). In the AAV-DJ-CAG-hMPZL2 vector-injected group, the Click (dB) was 86.818, indicating a loss of hearing. In the AAV-DJ-EF1a-hMPZL2 vector-injected group, the Click (dB) was 31.428, indicating an improvement in hearing compared to the control group (Figure 2).

[0078] Example 4. Western blot Two cochlear tissue samples were used for each sample. Cochlear tissue was isolated from mice injected with AAV-DJ-CAG-hMPZL2 and AAV-DJ-EF1a-hMPZL2 and from uninjected mice. The tissue was lysed using a tissue lyser in a cell lysis buffer containing a protease and phosphatase inhibitor cocktail (Thermo Scientific, cat. 78440). The lysed tissue was then centrifuged at 15,000 rpm for 20 minutes at 4°C, and the supernatant was separated. The protein concentration of the lysate was measured using the Bradford method (Bio-Rad, cat. 5000006). For electrophoresis, 20 μg of cochlear lysate was added to 2X SDS loading buffer and boiled at 100°C for 10 minutes to prepare a sample. SDS-PAGE was performed on a 4-12% gradient mini protein TGX gel (Bio-Rad), and proteins were transferred to a nitrocellulose membrane using a Bio-Rad Trans-Blot Turbo transfer system (250 mA, 100 min). The membrane was then incubated in 5% skim milk (Biopure, cat. 8110s) for 1 hour at room temperature and treated overnight at 4°C with a primary antibody (MPZL2 Polyclonal antibody, Proteintech, cat. 11787-1-AP) at a 1:1000 ratio. The membrane was washed three times with 1X TBST and then treated with anti-rabbit IgG-HRP (Enzo, cat. ADI-SAB-300-J) in 5% skim milk at a 1:1000 ratio for 1 hour at room temperature. After subsequent washing with 1X TBST, the membrane was transferred to a Pierce TM Antibody-antigen complexes were detected using ECL Western Blotting Substrate (Thermo, cat. 32209), and protein bands were detected in the dark using CP-BU NEW XRAY FILM BLUE (AGFA, cat. CP-BU). Protein bands were then quantified using the ImageJ program.

[0079] When comparing the expression levels of hMPZL2 in the cochlea of ​​4-week-old mice injected with AAV-DJ-CAG-hMPZL2 and those injected with AAV-DJ-EF1a-hMPZL2, we found that the expression levels were approximately 23-fold higher in the CAG promoter-injected mice than in the EF1a promoter-injected mice (Figure 3).

[0080] Example 5. Immunofluorescence staining Cochlear tissue was isolated from mice injected with AAV-DJ-CAG-hMPZL2 and AAV-DJ-EF1a-hMPZL2 at 4 weeks of age. The tissue was fixed in 4% paraformaldehyde (PFA) solution at 4°C overnight. The fixed tissue was then treated with 5M EDTA (Intron, cat. IBS-BE002) for 3 days. The cochlear tissue was then blocked in 0.1% Triton X-100 solution prepared using donkey serum and Triton X-100 at room temperature for 1 hour to allow for membrane permeabilization. The sample was then incubated overnight at 4°C with primary antibody (MPZL2 Polyclonal antibody, Proteintech, cat. 11787-1-AP) in 0.1% Triton X-100 solution at a ratio of 1:200. After washing three times with 1X PBS, the samples were incubated with Alexa Fluor 594-conjugated secondary antibodies (Invitrogen, Alexa Fluor™ 594 donkey anti-rabbit IgG (H+L)) in 0.1% Triton X-100 at 1:200, 1:100, and 1:400 concentrations, respectively, along with DAPI and Alexa Fluor-conjugated phalloidin 488 (Thermo Fisher Scientific, Rockford, IL, USA) for 90 minutes at room temperature. After washing three times with 1X PBS for 15 minutes each, the samples were treated with antifade reagent (Thermo Fisher Scientific, Rockford, IL, USA) and mounted on glass slides. Images were captured using an LSM780 confocal microscope (Carl Zeiss, Jena, Germany). Images were processed using ZEN (Blue edition) software.

[0081] In 4-week-old mice injected with AAV-DJ-CAG-hMPZL2, hair cells remained intact compared to the control group (Mpzl2 p.Q74*), whereas in 4-week-old mice injected with AAV-DJ-EF1a-hMPZL2, all hair cells were found to have died (Figure 4).

[0082] Although certain parts of the present invention have been described in detail above, it will be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the present invention. Therefore, the true scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A viral vector comprising the EF1a promoter and a gene encoding a neuronal differentiation protein.

2. The viral vector of claim 1 , wherein the viral vector is an adeno-associated virus (AAV).

3. The viral vector according to claim 1, wherein the viral vector is AAV-DJ.

4. The vector of claim 1 , wherein the neuronal differentiation protein is an MPZ or MPZL protein.

5. The vector of claim 4 , wherein the MPZL protein is MPZL1, MPZL2, or MPZL3.

6. The viral vector of claim 1 , wherein the viral vector is for use in treating or preventing a disease caused by an Mpzl2 mutation.

7. The vector according to claim 1 , wherein the viral vector targets hair cells or supporting cells (Deiter's cells, Pillar cells).

8. A pharmaceutical composition for preventing or treating hearing loss, comprising the vector according to any one of claims 1 to 7 as an active ingredient.

9. The pharmaceutical composition for preventing or treating hearing loss according to claim 8, wherein the hearing loss is symptomatic or non-syndromic.

10. The pharmaceutical composition for preventing or treating hearing loss according to claim 8, wherein the hearing loss is autosomal recessive non-syndromic hearing loss caused by a mutation.

11. A pharmaceutical composition for preventing or treating a metabolic disorder caused by a mutation in the Mpzl gene, comprising the vector according to any one of claims 1 to 7 as an active ingredient.

12. 12. The pharmaceutical composition according to claim 11, wherein the metabolic disorder is any one selected from the group consisting of diabetes, hyperthyroidism, hypothyroidism, phenylketonuria, Gaucher disease, galactosemia, organic aciduria, lipid accumulation disease, and anemia.

13. A pharmaceutical composition for preventing or treating a neurodegenerative disease caused by a mutation in the Mpzl gene, comprising the vector according to any one of claims 1 to 7 as an active ingredient.

14. 14. The pharmaceutical composition according to claim 13, wherein the neurodegenerative disease is any one selected from the group consisting of Alzheimer's disease, Parkinson's disease, Lou Gehrig's disease, Huntington's disease, multiple sclerosis, and prion disease.

15. A pharmaceutical composition for preventing or treating cancer caused by a mutation in the Mpzl gene, comprising the vector according to any one of claims 1 to 7 as an active ingredient.

16. The cancers include breast cancer, ovarian cancer, colon cancer, stomach cancer, liver cancer, pancreatic cancer, cervical cancer, thyroid cancer, parathyroid cancer, lung cancer, non-small cell lung cancer, prostate cancer, gallbladder cancer, biliary tract cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, blood cancer, bladder cancer, kidney cancer, melanoma, colon cancer, bone cancer, skin cancer, head cancer, uterine cancer, rectal cancer, brain tumor, anal cancer, fallopian tube carcinoma, endometrial carcinoma, vaginal cancer, vulva carcinoma, esophageal cancer, small intestine cancer, endocrine gland cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, and central nervous system (CNS) cancer.

16. The pharmaceutical composition of claim 15, wherein the tumor is any one selected from the group consisting of a primary CNS lymphoma, a spinal cord tumor, a brainstem glioma, or a pituitary adenoma.

17. A pharmaceutical composition for preventing or treating an autoimmune disease caused by a mutation in the Mpzl gene, comprising the vector according to any one of claims 1 to 7 as an active ingredient.

18. The pharmaceutical composition according to claim 17, wherein the autoimmune disease is any one selected from the group consisting of lupus, rheumatoid arthritis, Crohn's disease, and marginalized arthritis.

19. A pharmaceutical composition for preventing or treating vascular diseases caused by mutations in the Mpzl gene, comprising the vector according to any one of claims 1 to 7 as an active ingredient.

20. The pharmaceutical composition according to claim 19, wherein the vascular disease is any one selected from the group consisting of arteriosclerosis, thrombosis, vascular artery disease, and hypertension.

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