WO2025095735A1 - Pharmaceutical composition containing mitochondria as active ingredient for preventing or treating hereditary hearing impairment - Google Patents

Abstract

In the present invention process, the inventors have confirmed that the intracellular ATP and mitochondrial membrane potentials of cells isolated from hearing-impaired patients having mutations in the BCAP31 gene are significantly lower than those of normal persons. The finding indicates that the hearing-impaired patients having mutations in the BCAP31 gene have significantly lower-functioning mitochondria. In addition, susceptibility to cisplatin-induced cytotoxicity has also been confirmed to increase. On the other hand, treating lymphoid cell lines of the hearing-impaired patients with isolated mitochondria derived from stem cells has been confirmed to increase the activity of intracellular mitochondria and reduce cisplatin-induced cytotoxicity. Furthermore, the functioning of intracellular mitochondria has also been confirmed to recover when the isolated mitochondria derived from stem cells were applied to lymphocyte cell lines derived from hearing-impaired patients having mutations in the ferredoxin reductase (FDXR) gene, and fibroblasts derived from hearing-impaired patients having mitochondrial gene mutation (m.A1555G). Therefore, the mitochondria according to the present invention can effectively alleviate or treat hereditary hearing impairment.

Description

Pharmaceutical composition for preventing or treating hereditary hearing loss containing mitochondria as an active ingredient

The present invention relates to a pharmaceutical composition for preventing or treating hereditary hearing loss disease, which contains mitochondria as an active ingredient.

The ear is classified into the outer ear including the auricle and the external auditory canal, the middle ear including the tympanic membrane and the ossicles, and the inner ear including the cochlea and the auditory nerve. Sound is transmitted as acoustic energy through the auricle and the external auditory canal to vibrate the tympanic membrane, and the vibration of the tympanic membrane generates mechanical energy which is transmitted to the ossicles. The last bone of the ossicles, the stapes, is connected to the cochlea and transmits the transmitted energy to the lymph inside the cochlea. The energy transmitted to the lymph generates waves in the lymph, and these waves stimulate the hair cells inside the cochlea. As ionic changes occur due to the movement of the hair cells, neurotransmitters are transmitted to the auditory nerve attached to the hair cells, and sound energy is transmitted to the brain in the form of electrical energy from the auditory nerve.

Hearing loss is the most common sensorineural disease, occurring in 1 out of 1,000 newborns. Hearing loss is largely divided into hereditary and non-hereditary hearing loss, of which more than 50% are hereditary hearing loss. Hereditary hearing loss is further classified into syndromic and non-syndromic hearing loss, of which 30% are syndromic hearing loss with multiple symptoms, and the remaining 70% are non-syndromic hearing loss with no other symptoms in addition to hearing loss. In the case of non-syndromic hearing loss, autosomal recessive inheritance accounts for approximately 80%, autosomal dominant inheritance accounts for approximately 15-20%, and the remaining less than 2% is hearing loss caused by genes located on the X chromosome and mitochondria (Nance et al., Ment Retard Dev Disabil Res Rev, 9:109-119 (2003)).

Mitochondria are essential organelles for the survival of eukaryotic cells that are involved in the synthesis and regulation of adenosine triphosphate (ATP). Mitochondria are important organelles that are involved in various metabolic pathways in the body, such as cell signaling, cell differentiation, apoptosis, as well as the control of the cell cycle and cell growth. There has been no study on whether pharmaceutical compositions containing mitochondria as active ingredients may be related to the treatment of hereditary hearing loss.

Accordingly, the inventors of the present invention conducted research for the treatment of hearing loss and confirmed that isolated mitochondria increase mitochondrial activity of a lymphocyte cell line derived from a hearing-impaired patient and reduce cytotoxicity caused by cisplatin, thereby completing the present invention.

To achieve the above purpose, one aspect of the present invention provides a pharmaceutical composition for preventing or treating hereditary hearing loss disease, comprising isolated mitochondria as an active ingredient.

Another aspect of the present invention provides a diagnostic kit for hereditary hearing loss disease, a diagnostic composition and a diagnostic method using the same, comprising a polynucleotide comprising a contiguous nucleotide sequence selected from the nucleotide sequence of an exon region of a BCAP31 gene or a complementary polynucleotide thereof for detecting a c.397_398insGAG mutation of the BCAP31 gene.

Another aspect of the present invention provides a biomarker for diagnosing hereditary hearing loss disease of the BCAP31 gene comprising c.397_398insGAG mutation.

Another aspect of the present invention provides a use of the isolated mitochondria for preventing or treating hereditary hearing loss diseases.

Another aspect of the present invention provides a method for preventing or treating a hereditary hearing loss disease comprising administering the isolated mitochondria to a subject.

In the present invention, it was confirmed that the intracellular ATP and mitochondrial membrane potential of cells isolated from hearing-impaired patients with BCAP31 gene mutations were significantly lower than those of normal people. This means that the mitochondrial function of hearing-impaired patients with BCAP31 gene mutations was significantly reduced. In addition, it was confirmed that they were more sensitive to cytotoxicity by cisplatin. On the other hand, when the lymphoid cell line of the hearing-impaired patient was treated with isolated mitochondria derived from stem cells, it was confirmed that the activity of intracellular mitochondria increased and the cytotoxicity by cisplatin treatment decreased. Furthermore, it was confirmed that the function of the mitochondria within the cells was restored when the isolated mitochondria derived from stem cells were treated to a lymphocyte cell line derived from a hearing-impaired patient with a ferredoxin reductase (FDXR) gene mutation and a fibroblast derived from a hearing-impaired patient with a mitochondrial gene mutation (m.A1555G). Therefore, the mitochondria according to the present invention can effectively alleviate or treat hereditary hearing-impaired diseases.

Figure 1 is a pedigree diagram showing the results of identifying a mutation (c.397_398insGAG;p.Asp132_Ala133insGly) in the BCAP31 gene in a patient with non-syndromic hearing loss.

Figure 2 is a sequence chromatogram showing the results of comparing the BCAP31 gene sequences of normal individuals and hearing-impaired patients with BCAP31 gene mutations within family members with nonsyndromic hearing loss.

Figure 3 is a graph showing the results of measuring the pure tone audiogram of the proband of a hearing-impaired patient with a BCAP31 gene mutation.

Figures 4a and 4b are graphs comparing intracellular ATP (Figure 4a) and mitochondrial membrane potential (MMP) (Figure 4b) of lymphoblastoid cell lines (LCL) from normal subjects (C1, C2) and hearing-impaired patients with BCAP31 gene mutations (PB-1, PB-2), respectively. * p <0.05, ** p <0.01, *** p <0.001 vs C1.

Figure 5 is a graph showing the results of confirming apoptosis by cisplatin treatment in lymphoid cell lines from normal individuals (C1) and hearing-impaired patients (PB-1, PB-2) with BCAP31 gene mutations using annexin/PI staining. * p <0.05, ** p <0.01 vs C1

Figure 6 is a drawing showing an experimental method for confirming the intracellular transfer ability of mitochondria according to the present invention (left) and the results of confirming mitochondria transferred to a lymphoid cell line (right).

Figures 7a and 7b are graphs showing the results of comparing the intracellular ATP level (Figure 7a) and mitochondrial membrane potential (MMP) (Figure 7b) after treating the mitochondria (PN-101) of the present invention to lymphoid cell lines of a normal person (C1) and hearing-impaired patients (PB-1, PB-2) with BCAP31 gene mutation, respectively. * p <0.05, ** p <0.01, *** p <0.001 vs C1; # p <0.05, ### p <0.001 vs cisplatin.

Figure 8 is a graph showing the results of confirming the degree of cell death using annexin/PI staining after treating cisplatin and mitochondria (PN-101) of the present invention in lymphoid cell lines of each normal person (C1) and hearing-impaired patients (PB-1, PB-2) with BCAP31 gene mutation. *** p <0.001 vs C1; # p <0.05, ### p <0.001 vs cisplatin.

Figures 9a and 9b are drawings (Figure 9a) showing the expression of cyt C in the cytoplasm of lymphoid cell lines from a normal person (C1) and hearing-impaired patients (PB-1, PB-2) with a BCAP31 gene mutation, respectively, after treatment with cisplatin and treatment with mitochondria (PN-101) of the present invention, as confirmed by Western blot, and a graph (Figure 9b) showing the quantification of the results.

Figures 10a and 10b are graphs showing the results of comparing the intracellular ATP level (Figure 10a) and mitochondrial membrane potential (MMP) (Figure 10b) after treating mitochondria (PN-101) of the present invention to a lymphoid cell line (PB-3) derived from a hearing-impaired patient with a ferredoxin reductase (FDXR) gene mutation. * p <0.05, *** p <0.001 vs C1; ### p <0.001 vs PB-3 untreated group.

Figure 11 is a graph showing the results of measuring the intracellular ATP concentration after treating mitochondria (PN-101) of the present invention to patient-derived fibroblast cell lines (PB-4, PB-5) having the m.A1555G mitochondrial mutation.

Figure 12 is a drawing showing the results of confirming the tissue location of MT ^dsRED through tissue immunostaining after treating the cochlea isolated from a mouse with MT ^dsRED for time periods (1 hour, 4 hours, 24 hours).

Pharmaceutical composition

One aspect of the present invention provides a pharmaceutical composition for preventing or treating hereditary hearing loss, comprising isolated mitochondria as an active ingredient.

As used herein, the term "mitochondria" is a double-membrane-bound organelle found in most eukaryotic organisms that produces most of the adenosine triphosphate (ATP) in the cell.

As used herein, the term "isolated mitochondria" refers to mitochondria obtained from an autologous, allogeneic, or xenogeneic source.

As used herein, the term "autologous mitochondria" refers to mitochondria obtained from the plasma, tissue, bone marrow or cells of the same individual. In addition, the term "allogeneic mitochondria" refers to mitochondria obtained from the plasma, tissue, bone marrow, platelets or cells of an individual belonging to the same species as the individual but having a different genotype for an allele. In addition, the term "heterologous mitochondria" refers to mitochondria obtained from the plasma, tissue, bone marrow or cells of an individual belonging to a different species from the individual.

At this time, the subject may be a mammal, and preferably a human.

The above mitochondria may be isolated from cells of the individual. The above mitochondria may be obtained from autologous or allogeneic cells cultured in vitro. In this case, the cells may have normal biological activity.

The term "cell" as used herein means a structural or functional unit that constitutes a living organism, consisting of cytoplasm surrounded by a cell membrane, and containing biomolecules such as proteins and nucleic acids. The cell refers to a cell that contains mitochondria inside the cell membrane.

Additionally, the mitochondria may be separated and used after concentrating and crushing the tissue or cells, or may be separated and crushed from a tissue or cell sample that has been frozen and then thawed.

The above mitochondria may be in an intact form, a fragmented form, or a combination thereof. In one specific example, the mitochondria may exhibit a pharmacological effect even if they are in a fragmented form, if they have mitochondrial activity.

In one specific example, the cell may be any one selected from the group consisting of stem cells, somatic cells, germ cells, and platelets.

The term "stem cell" as used herein refers to an undifferentiated cell that has the ability to differentiate into various types of tissue cells. The stem cell may be any one selected from the group consisting of mesenchymal stem cells, adult stem cells, induced pluripotent stem cells, embryonic stem cells, bone marrow stem cells, neural stem cells, and tissue-derived stem cells.

At this time, the mesenchymal stem cell may be any one selected from the group consisting of umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane, and placenta. Preferably, it may be derived from human umbilical cord.

The term "somatic cell" used in this specification refers to a cell excluding germ cells among the cells constituting an individual. The somatic cell may be one selected from the group consisting of muscle cells, hepatocytes, fibroblasts, epithelial cells, nerve cells, adipocytes, bone cells, periosteal cells, leukocytes, lymphocytes, and mucosal cells. Preferably, it may be obtained from muscle cells or hepatocytes with excellent mitochondrial activity. In addition, it may be obtained from autologous or allogeneic blood PBMC cells.

As used herein, the term "germ cell" means a cell that forms a zygote during reproduction in a sexually reproducing organism. The mitochondria may be obtained from autologous or allogeneic gametes. The gametes may be sperm or eggs.

As used herein, the term "platelet" refers to a solid component of blood that plays an important role in blood clotting by forming a clot by binding fibrin in the blood. The mitochondria may be obtained from autologous or allogeneic platelets.

In addition, the isolated mitochondria may have normal biological activity. Specifically, the mitochondria having normal biological activity may have one or more characteristics from the group consisting of (i) having a membrane potential, (ii) generating ATP within the mitochondria, and (iii) removing ROS or reducing the activity of ROS within the mitochondria.

The term "hearing loss" used in this specification refers to a disease that causes hearing impairment or hearing loss. Hearing loss can be classified into conductive and sensorineural hearing loss depending on the structure of the auditory organ. Conductive hearing loss occurs when sound does not reach the cochlea due to a deformity of the external auditory canal, an abnormality of the tympanic membrane or ossicles, etc. On the other hand, sensorineural hearing loss occurs when there is a problem with the function of the cochlea or an abnormality in the auditory nerve or central nervous system that transmits auditory stimuli to the brain.

In addition, depending on the time of onset of hearing loss, it can be divided into congenital (which appears from birth) and acquired (postnatal), or prelingual and postlingual. These can be classified into hereditary hearing loss and non-hereditary hearing loss, depending on whether it is hereditary. About 50% of congenital hearing loss is hereditary.

The term "genetic hearing loss" used in this specification refers to genetic hearing loss that can be largely divided into syndromic and non-syndromic. The syndromic hearing loss refers to hearing loss that accompanies approximately 300 types of syndromes registered in OMIM to date. Non-syndromic hearing loss is usually known to be caused by a single gene abnormality. The hereditary hearing loss follows various forms of inheritance. Autosomal recessive, autosomal dominant, sex-linked, mitochondrial, and recently, digenic inheritance due to mutations in two hearing loss genes have been reported. Most hereditary hearing loss (approximately 70%) is non-syndromic, and more than 75% of this non-syndromic hearing loss shows autosomal recessive inheritance. 12-24% is inherited through autosomal dominant, 1-3% through the X chromosome, and some through mitochondrial genes.

In the present invention, the hereditary hearing loss may include a mutation in an autosome or a sex chromosome.

Specifically, the hereditary hearing loss may include mutations in the BCAP31, ferredoxin reductase (FDRX), HDIA1, GJB3, KCNQ4, GJB2, GJB6, DFNA5, TECTA, COCH, EYA4, MYO7A, COL11A2, MYO15, TMPRSS3, OTOF, DFNB9 or DDP genes.

Additionally, the hereditary hearing loss may include genetic mutations associated with Pendred syndrome, Usher syndrome, Branchio-oto-renal syndrome, Waardenburg syndrome, Alport syndrome, Treacher collins syndrome, Stapes fixation/Gusher (DFN3) or Jervell and Lange-Nielsen syndrome.

In one specific example, the hereditary hearing loss in the present invention is caused by mutation of the BCAP31 gene. It may contain a mutation. At this time, the BCAP31 gene The mutation may be inherited in an X-linked recessive manner.

The term "BCAP31 (B-cell receptor-associated protein 31)" or "BAP31" used in this specification is a protein that binds to the ER membrane, and is involved in the transport of proteins secreted from the ER, protein folding quality control, and caspase-8 dependency. It is known that dysfunction of BCAP31 is related to various diseases including tinnitus, paroxysmal movement disorder, central hypotonic hypotonic syndrome (DDCH), cancer, metabolic syndrome, cystic fibrosis, and neurodegenerative diseases. In the present invention, the BCAP31 may include a nucleic acid sequence of SEQ ID NO: 4. The BCAP31 may include an amino acid sequence of SEQ ID NO: 5. The nucleic acid sequence and amino acid sequence are shown in Table 1.

division order Sequence number BCAP31 cDNA
order ATGGGTGCCGAGGCGTCCTCCTCTTGGTGCCCTGGCACTGCTCTTCCCGAAGAACGCCTTTCAGTTAAACGGGCGTCGGAAATCTCGGGCTTCCTGGGGCAGGGATCGTCGGGAGAGGCCGCTCTGGACGTGTTGACACACGTGCTGGAGGGGGCAGGAAACAAGCTCACATCTTCCTGTGGGGAAACCTTCTAGCAAC AGGATGAGTCTGCAGTGGACTGCAGTTGCCACCTTCCTCTATGCGGAGGTCTTTGTTGTGTTGCTTCTCTGCATTCCCTTCATTTCTCCTAAAAGATGGCAGAAGATTTTCAAGTCCCGGCTGGTGGAGTTGTTAGTGTCCTATGGCAACACCTTCTTTGTGGTTCTCATTGTCATCCTTGTGCTGTTGGTCATCGAT GC 4 BCAP31 amino acid sequence MGAEASSSWCPGTALPEERLSVKRASEISGFLGQGSSGEAALDVLTHVLEGAGNKLTSSCGKPSSNRMSLQWTAVATFLYAEVFVVLLLCIPFISPKRWQKIFKSRLVELLVSYGNTFFVVLIVILVLLVID A VREIRKYDDVTEKVNLQNNPGAMEHFHMKLFRAQRNLYIAGFSLLLSFLLRRLVTLISQQATLLASNEAFKKQAESASEAAKKYMEENDQLKKGAAVDGGKLDVGNAEVKLEEENRSLKADLQKLKDELASTKQKLEKAENQVLAMRKQSEGLTKEYDRLLEEHAKLQAAVDGPMDKKEE 5 BCAP31 mutant cDNA sequence ATGGGTGCCGAGGCGTCCTCCTCTTGGTGCCCTGGCACTGCTCTTCCCGAAGAACGCCTTTCAGTTAAACGGGCGTCGGAAATCTCGGGCTTCCTGGGGCAGGGATCGTCGGGAGAGGCCGCTCTGGACGTGTTGACACACGTGCTGGAGGGGGCAGGAAACAAGCTCACATCTTCCTGTGGGGAAACCTTCTAGCAAC AGGATGAGTCTGCAGTGGACTGCAGTTGCCACCTTCCTCTATGCGGAGGTCTTTGTTGTGTTGCTTCTCTGCATTCCCTTCATTTCTCCTAAAAGATGGCAGAAGATTTTCAAGTCCCGGCTGGTGGAGTTGTTAGTGTCCTATGGCAACACCTTCTTTGTGGTTCTCATTGTCATCCTTGTGCTGTTGGTCATCGAT GGAGC 6 BCAP31 mutant amino acid sequence MGAEASSSWCPGTALPEERLSVKRASEISGFLGQGSSGEAALDVLTHVLEGAGNKLTSSCGKPSSNRMSLQWTAVATFLYAEVFVVLLLCIPFISPKRWQKIFKSRLVELLVSYGNTFFVVLIVILVLLVID GA VREIRKYDDVTEKVNLQNNPGAMEHFHMKLFRAQRNLYIAGFSLLLSFLLRRLVTLISQQATLLASNEAFKKQAESASEAAKKYMEENDQLKKGAAVDGGKLDVGNAEVKLEEENRSLKADLQKLKDELASTKQKLEKAENQVLAMRKQSEGLTKEYDRLLEEHAKLQAAVDGPMDKKEE 7

In this specification, the normal BCAP31 gene or the wild-type BCAP31 gene may mean a gene comprising a nucleic acid sequence of SEQ ID NO: 4, and the amino acid sequence of the normal BCAP31 protein or the amino acid sequence of the wild-type BCAP31 protein may mean a gene comprising an amino acid sequence of SEQ ID NO: 5. In this case, “normal” or “wild-type” means a typical phenotype found in a species existing in nature, and may be used interchangeably.

In the present invention, the mutation of the BCAP31 gene may have a mutation in the nucleotide sequence with respect to the nucleic acid sequence of the normal BCAP31 gene (the BCAP31 gene of the standard genomic DNA). Specifically, the mutation of the BCAP31 gene may include an insertion, deletion, substitution or translocation of one or more nucleotide sequences with respect to the normal BCAP31 gene.

The above "insertion" or "deletion" means insertion or deletion of a nucleotide sequence that can change the number of nucleic acids in a gene. The above "translocation" means a phenomenon in which a part of a chromosome is broken and the fragment binds to another part of the same chromosome or to another chromosome, thereby changing the shape of the chromosome.

More specifically, the mutation of the BCAP31 gene in the present invention may include an insertion of three nucleotide sequences with respect to the normal BCAP31 gene. More specifically, the mutation of the BCAP31 gene may be a mutation in which guanine (G), adenine (A), and guanine (G) are sequentially inserted between the 397th and 398th base sequences of SEQ ID NO: 4. At this time, the mutation of the BCAP31 gene may be a mutation in which glycine (Gly) is inserted between the 132nd amino acid residue and the 133rd amino acid residue in the amino acid sequence of the normal BCAP31 protein (SEQ ID NO: 5). In one embodiment, the mutation of the BCAP31 gene may include the nucleic acid sequence of SEQ ID NO: 6 or the amino acid sequence of SEQ ID NO: 7.

In another specific example of the present invention, the hereditary hearing loss in the present invention is caused by a mutation in the ferredoxin reductase (FDXR) gene. It may contain mutations.

The term "ferredoxin reductase (FDXR)" used in this specification refers to one of the flavoproteins located in the inner membrane of mitochondria. It is also known as adrenodoxin reductase (ADXR). The FDXR transfers electrons obtained from NADPH (nicotinamide adenine dinucleotide phosphate) to the ferredoxin (FDX) protein to regulate steroid production, Fe-S homeostasis, and energy metabolism. In the present invention, the FDXR gene is a nucleotide sequence of SEQ ID NO: 8 (NCBI Reference Sequence NM_024417.5) or a nucleotide sequence of SEQ ID NO: 9 (NCBI Reference Sequence NP_077728.3) may contain the amino acid sequence.

In general, mutations in the FDXR gene are known to be associated with auditory neuropathy spectrum disorder (ANSD). This neuropathy spectrum disorder is a unique form of hearing loss characterized by the absence of auditory brainstem response (ABR) and normal evoked otoacoustic emissions. ANSD is known to be characterized by reduced speech discrimination ability compared to hearing thresholds on pure tone audiometry.

The above "FDXR mutation" may have a mutation in a nucleotide sequence with respect to the nucleic acid sequence of a normal FDXR gene (FDXR gene of a standard genome DNA). Specifically, the mutation in the FDXR gene may include an insertion, deletion, substitution or translocation of one or more nucleotide sequences with respect to the normal FDXR gene. Specifically, the FDXR mutation may be a mutation in which one or more nucleotide sequences are substituted with respect to the normal FDXR gene. In this case, the "substitution" means a phenomenon in which a part of a nucleic acid sequence is changed to a different nucleic acid sequence.

In one embodiment of the present invention, the mutation of the FDXR gene may be a mutation in which the 940th base sequence of SEQ ID NO: 8 is substituted with T instead of G. At this time, the mutation of the FDXR gene may be a mutation in which valine (Val), which is the 314th amino acid residue in the amino acid sequence of the normal FDXR protein (SEQ ID NO: 9), is substituted with leucine (Leu). Specifically, the mutation of the FDXR gene may include the nucleic acid sequence of SEQ ID NO: 10 or the amino acid sequence of SEQ ID NO: 11.

In the present invention, the hereditary hearing loss may include a mutation in mitochondria.

Specifically, the hereditary deafness may involve mutations in the mitochondrial 12S rRNA gene or the Ser(UCN)-tRNA gene.

In one specific example, the hereditary hearing loss in the present invention may include a mutation in the mitochondrial 12S rRNA gene.

As used herein, the term "12S ribosomal RNA (12S rRNA or 12S)" refers to the rRNA encoded in mitochondria, namely SSU rRNA of mitochondrial ribosomes. Human 12S rRNA is encoded by the MT-RNR1 gene.

Specifically, the mitochondrial mutation may include an insertion, deletion, substitution or translocation of one or more nucleotide sequences relative to a normal 12S rRNA gene. Specifically, the mitochondrial 12S rRNA gene mutation may be a substitution of one or more nucleotide sequences relative to a normal 12S rRNA gene. In this case, the substitution is the same as described above.

More specifically, it may be that A in some of the nucleic acid sequences of the normal 12S rRNA gene is substituted with C, G, or T. More specifically, it may be that A at the 1555th base sequence of the nucleic acid sequence described in GenBank: FM865407.1 is substituted with T.

The A1555G mutation (hereinafter, mixed with m.A1555G) of the mitochondrial 12S rRNA gene has been reported to be associated with many hearing loss conditions, including aminoglycoside-induced hearing loss and maternally inherited non-syndromic hearing loss. In particular, the m.A1555G mutation has been reported to have a prevalence of 2.4% in European sensorineural hearing loss patients and 3.2% in Chinese sensorineural hearing loss patients.

Alternatively, the mutation in the Ser(UCN)-tRNA gene may be the A7445G mutation.

The term "treatment" used in this specification can be used to mean both therapeutic treatment and preventive treatment. In this case, "prevention" can be used to mean alleviating or reducing a pathological condition or disease of an individual. In one specific example, the isolated mitochondria as the effective ingredient can suppress hearing loss due to damage.

The term "active ingredient" as used herein refers to an ingredient that is active on its own or together with an excipient (carrier) that is inactive on its own.

When the mitochondria are separated from a specific cell, they can be separated by various known methods, such as using a specific buffer solution or using a potential difference and a magnetic field. In addition, the separation of the mitochondria can include a step of centrifuging and filtering the plasma to remove all cellular components, and a step of centrifuging the filtered plasma.

The above mitochondrial separation can be obtained by disrupting and centrifuging tissues or cells in terms of maintaining mitochondrial activity. In one specific example, the method can be performed by culturing cells, performing a first centrifugation on a composition containing the cells to generate a pellet, resuspending the pellet in a buffer solution and homogenizing it, performing a second centrifugation on the homogenized solution to produce a supernatant, and performing a third centrifugation on the supernatant to purify mitochondria. At this time, it is preferable that the time for performing the second centrifugation be shorter than the time for performing the first and third centrifugations in terms of maintaining cell activity, and the speed can be increased from the first centrifugation to the third centrifugation.

Specifically, the first to third centrifugations may be performed at a temperature of about 0° C. to about 10° C., preferably about 3° C. to about 5° C. In addition, the time for performing the centrifugation may be performed for about 1 minute to 50 minutes, and may be appropriately adjusted depending on the number of centrifugations and the content of the sample.

In addition, the first centrifugation can be performed at a speed of about 100xg to about 1,000xg, about 200xg to about 700xg, or about 300xg to about 450xg. In addition, the second centrifugation can be performed at a speed of about 1xg to about 2,000xg, about 25xg to about 1,800xg, or about 500xg to about 1,600xg. In addition, the third centrifugation can be performed at a speed of about 100xg to about 20,000xg, about 500xg to about 18,000xg, or about 800xg to about 15,000xg.

In addition, a pharmaceutically acceptable sugar may be used to stabilize the obtained mitochondria. Specifically, the sugar may be, but is not limited to, sucrose, mannitol, trehalose, etc. In addition, a pharmaceutically acceptable sugar may be used as a pH buffering agent, but is not limited to, tris, HEPES, phosphate, etc.

Meanwhile, additives such as chelators and antioxidants can be used to remove damage caused by ion outflow after obtaining the mitochondria and to suppress oxidative stress, and these can include, without limitation, reagents widely known in the art. For example, these can be EDTA, EGTA, citrate, glycine, taurine, ATP, etc., but are not limited thereto.

In addition, the above mitochondrial separation can be obtained by thawing, crushing, and centrifuging frozen cells or tissues in terms of maintaining mitochondrial activity. The above method for obtaining mitochondria can be performed by the steps of freezing cells or tissues, thawing the cells or tissues, and crushing the thawed cells and tissues.

The freezing may be, but is not limited to, a temperature of -1° C. or lower. Specifically, the freezing may be performed in a temperature range of about -5° C. to about -200° C., about -15° C. to about -180° C., about -25° C. to about -160° C., about -40° C. to about -140° C., about -55° C. to about -120° C., about -60° C. to about -100° C., or about -70° C. to about -90° C.

The above freezing can be performed using liquid nitrogen (LN ₂ ) or a refrigeration device. Specifically, the refrigeration or freezing process can be performed by rapid freezing using LN ₂ , or by using a refrigeration device such as a deep freezer, a freezer, or a freezing container. The above freezing is not particularly limited as long as it is a method capable of freezing or freezing mitochondria.

The above freezing time is not particularly limited, as long as the frozen mitochondria have normal activity.

The membrane protein of the separated mitochondria can be quantified to quantify the mitochondria. Specifically, the separated mitochondria can be quantified using the BCA (bicinchoninic acid assay) analysis method. At this time, the mitochondria in the pharmaceutical composition can be included at a concentration of about 0.1 ㎍/㎖ to about 1,000 ㎍/㎖, about 0.5 ㎍/㎖ to about 750 ㎍/㎖, about 1 ㎍/㎖ to about 500 ㎍/㎖, about 2 ㎍/㎖ to about 150 ㎍/㎖, about 3 ㎍/㎖ to about 100 ㎍/㎖, about 4 ㎍/㎖ to about 50 ㎍/㎖, or about 5 ㎍/㎖ to about 10 ㎍/㎖. In one embodiment of the present invention, the mitochondria can be used at a concentration of about 0.5 ㎍/㎖ to about 5 ㎍/㎖.

Additionally, the number of separated mitochondria can be measured using a particle counter (Multisizer 4e, Beckman Coulter).

In one specific example, the mitochondria in the pharmaceutical composition may be included in an amount of about 1×10 ⁵ /mL to about 9×10 ⁹ /mL. Specifically, the mitochondria in the pharmaceutical composition may be included in an amount of about 1×10 ⁵ /mL to about 5×10 ⁹ /mL, about 2×10 ⁵ /mL to about 2×10 ⁹ /mL, about 5×10 ⁵ /mL to about 1× ^{10 9 /} mL, about 1×10 ⁶ /mL to about 5×10 ⁸ /mL, about 2×10 ⁶ /mL to about 2×10 ⁸ /mL, about 5×10 ⁶ /mL to about 1×10 ⁸ /mL, or about 1×10 7 /mL to about 5×10 ⁷ /mL.

At this time, the pharmaceutical composition may further include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be any non-toxic substance suitable for delivery to a patient. Distilled water, alcohol, fat, wax, and inert solids may be included as carriers. A pharmaceutically acceptable adjuvant (buffer, dispersant) may also be included in the pharmaceutical composition.

Specifically, the pharmaceutical composition may be prepared as a parenteral formulation according to the route of administration by a conventional method known in the art, including a pharmaceutically acceptable carrier in addition to the active ingredient. Here, the meaning of "pharmaceutically acceptable" means that it does not inhibit the activity of the active ingredient and does not have toxicity exceeding the adaptability of the application (prescription) target.

When the pharmaceutical composition of the present invention is prepared as a parenteral formulation, it can be formulated in the form of injections, transdermal administration agents, and nasal inhalers according to a method known in the art together with a suitable carrier. When formulated as an injection, suitable carriers include sterile water, ethanol, polyols such as glycerol or propylene glycol, or mixtures thereof, and preferably, Ringer's solution, PBS (phosphate buffered saline) containing triethanolamine, sterile water for injection, and isotonic solutions such as 5% dextrose can be used.

The pharmaceutical composition of the present invention may be an injectable preparation. Therefore, the pharmaceutical composition according to the present invention can be manufactured into an injectable preparation that is very stable both physically and chemically by adjusting the pH using a buffer solution such as an acid solution or phosphate that can be used as an injectable preparation, in order to secure product stability according to the distribution of the injection prescription.

Specifically, the pharmaceutical composition of the present invention may include water for injection.

The above-mentioned water for injection is distilled water prepared for dissolving solid injections or diluting water-soluble injections, and may be glucose injection, xylitol injection, D-mannitol injection, fructose injection, saline solution, dextran 40 injection, dextran 70 injection, amino acid injection, Ringer's solution, lactated-Ringer's solution, or a phosphate buffer solution having a pH range of about 3.5 to about pH 7.5, or a sodium dihydrogen phosphate-citrate buffer solution.

The pharmaceutical composition of the present invention may further comprise a stabilizer or a solubilizer. For example, the stabilizer may be pyrosulfite or ethylene diaminetetraacetic acid, and the solubilizer may be hydrochloric acid, acetic acid, potassium phosphate hydroxide, potassium bicarbonate, potassium carbonate, or tris. In one specific example, the pharmaceutical composition may comprise a mixed preservative solution such as a TTG (trehalose-tris-glycine) solution, which may be commonly used in pharmaceutically acceptable drug preparations.

Specifically, the pharmaceutical composition of the present invention may include an injectable liquid composition in addition to isolated mitochondria. At this time, the isolated mitochondria are the same as described above. Since the pharmaceutical composition of the present invention includes an injectable liquid composition, it is a composition for preventing or treating a disease related to mitochondrial function, and can suppress thrombosis that may be caused by aggregation of mitochondria, reduction and aggregation of platelets, etc. when administering mitochondria through injection, and can maintain and/or strengthen the stability of mitochondria, and can also stably maintain the activity of mitochondria.

Here, the liquid composition may include glycine, sugar or a buffer.

The glycine above is not limited thereto, but may be present in the injectable liquid composition at a concentration of about 15 mM or more, and specifically, may be present at a concentration of about 15 mM to about 150 mM, about 17 mM to about 130 mM, about 20 mM to about 120 mM, about 22 mM to about 110 mM, or about 25 mM to about 100 mM. In addition, the glycine may be used together with one or more amino acids selected from the group consisting of, but not limited to, histidine, isoleucine, leucine, lysine acetate, methionine, phenylalanine, threonine, tryptophan, valine, alanine, arginine, aspartic acid, cysteine, glutamic acid, proline, serine, and tyrosine.

In addition, the saccharide included in the injectable liquid composition may be at least one selected from the group consisting of sucrose, trehalose, mannitol, sorbitol, glucose, fructose, mannose, maltose, lactose, isomaltose, dextran, and dextrin, but is not limited thereto. In particular, the saccharide may be trehalose, mannitol, or sucrose. Preferably, the saccharide may be trehalose.

The buffer contained in the above injectable liquid composition may be selected from the group consisting of, but not limited to, tris buffer, HEPES buffer, MOPS buffer, and acetate or phosphate containing buffer. Preferably, the buffer may be an injectable tris buffer.

At this time, the pH of the buffer is not limited thereto, but may be in a range of about 7.0 to about pH 7.8, a range of about pH 7.2 to about pH 7.6, or a range of about pH 7.3 to about pH 7.5.

Additionally, the buffer may be present in the injectable liquid composition at a concentration of, but is not limited to, about 5 mM to about 50 mM, about 8 mM to about 40 mM, about 10 mM to about 35 mM, about 13 mM to about 30 mM, or about 15 mM to about 25 mM.

The above-described liquid composition for injection may have an osmolarity in a range of about 200 to about 400 mOsm, about 230 to about 380 mOsm, about 250 to about 350 mOsm, about 260 to about 320 mOsm, about 270 to about 330 mOsm, or about 280 to about 300 mOsm. The osmolarity in the above range facilitates long-term storage at a temperature of 2°C to 8°C or higher, while making the composition suitable for parenteral administration, for example, intravascular, intramuscular, or subcutaneous injection, without causing side effects in a subject.

The term "osmolarity" as used herein refers to the moles of solute contributing to the osmotic pressure of a solution per kilogram of solvent, and osmolarity is determined by measuring the freezing point depression of a sample using an osmometer.

Additionally, the injectable liquid composition may further contain a chelating agent.

The chelating agent may be, but is not limited to, one or more selected from the group consisting of injectable grades of EGTA, EDTA and BAPTA. The chelating agent may eliminate damage caused by ion outflow after obtaining mitochondria included in the injectable liquid composition.

In addition, the pharmaceutical composition may contain, as additives, antioxidants, ATP, magnesium, etc., which are effective in maintaining the function and activity of mitochondria.

The pharmaceutical composition of the present invention may be stored in a container selected from the group consisting of a vial, a cartridge, a syringe, and an autoinjector. In addition, the container in which the pharmaceutical composition is stored may be stored at room temperature, a refrigerated temperature of about 2° C. to about 8° C., or a temperature of about 25° C. to about 40° C. until it is administered to a subject in need of treatment.

The above entity may be a mammal, such as, but not limited to, a human, a dog, a cow, a horse, a pig, a sheep, a goat, a cat, a mouse, a rabbit, a rat, and preferably a human.

Meanwhile, the pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. The term "therapeutically effective amount" or "pharmaceutically effective amount" refers to an amount of a compound or composition that is effective in preventing or treating a hearing loss disease, which is sufficient to treat the disease at a reasonable benefit/risk ratio applicable to medical treatment and does not cause side effects. The level of the effective amount can be determined based on factors including the patient's health condition, the type and severity of the disease, the activity of the drug, the sensitivity to the drug, the administration method, the administration time, the administration route and the excretion rate, the treatment period, the drug used in combination or simultaneously, and other factors well known in the medical field. In one specific embodiment, the therapeutically effective amount refers to an amount of a drug that is effective in treating a genetic hearing loss disease.

As used herein, the term "administration" means introducing a given substance into a subject in an appropriate manner, and the route of administration of the composition may be administered through any common route as long as it can reach the target tissue. The pharmaceutical composition may be administered intratympanically, intraperitoneally, intravenously, intramuscularly, subcutaneously, intradermally, topically, intranasally, or rectally, but is not limited thereto. The intratympanic administration may be by direct injection into the tympanic cavity or by administration through surgery (e.g., tympanostomy).

The preferred dosage of the pharmaceutical composition of the present invention is about 0.01 mg/kg to about 5 mg/kg, about 0.1 mg/kg to about 4 mg/kg, or about 0.25 mg/kg to about 2.5 mg/kg of mitochondria per dose, based on the body weight of the subject to be administered, but is not limited thereto. That is, in terms of cell activity, it is most preferable that the isolated mitochondria of the pharmaceutical composition are administered in the above range of amounts based on the body weight of the subject suffering from hearing loss. In addition, the pharmaceutical composition can be administered 1 to 10 times, 3 to 8 times, or 5 to 6 times, and preferably 5 times. At this time, the administration interval can be 1 to 7 days or 2 to 5 days, and preferably 3 days. Such dosage should not be construed as limiting the scope of the present invention in any aspect.

The above term "subject" means a subject to which the composition of the present invention can be applied (prescribed), and may be a subject suffering from hearing loss. In addition, the subject may be a mammal such as a rat, mouse, or livestock, including a human, but is preferably a human.

In one specific embodiment, the pharmaceutical composition may additionally comprise an agent for the prevention or treatment of a known hereditary hearing loss disease, and administration of the pharmaceutical composition may additionally be concurrently administered with treatment of the hereditary hearing loss disease.

Another aspect of the present invention provides the use of isolated mitochondria for the prevention or treatment of hereditary hearing loss disorders.

Another aspect of the present invention provides a method of treating and/or preventing a hereditary hearing loss disorder comprising administering isolated mitochondria to a subject.

In addition, the pharmaceutical composition may additionally include an agent for preventing or treating a known hereditary hearing loss disease, and administration of the pharmaceutical composition may additionally be performed in parallel with treatment of the hereditary hearing loss disease. In this case, the isolated mitochondria, hereditary hearing loss, preventive treatment, entity, and administration are the same as described above.

Diagnostic Kit

Another aspect of the present invention provides a diagnostic kit for hereditary hearing loss disease, comprising a polynucleotide comprising a contiguous nucleotide sequence selected from the nucleotide sequence of an exon region of a BCAP31 gene or a complementary polynucleotide thereof for detecting c.397_398insGAG mutation of the BCAP31 gene.

The above BCAP31 and hereditary hearing loss are the same as described above.

The above mutation may be an insertion of a nucleotide sequence into the normal BCAP31 gene.

The c.397_398insGAG mutation of the above BCPA31 gene refers to a mutation in which guanine (G), adenine (A), and guanine (G) are inserted between the 397th and 398th base sequences in the BCAP31 gene. Preferably, it may be a mutation in which guanine (G), adenine (A), and guanine (G) are sequentially inserted between the 397th and 398th base sequences of sequence number 4. In this case, the mutation of the BCAP31 gene may be a mutation in which glycine (Gly) is inserted between the 132nd amino acid residue and the 133rd amino acid residue in the amino acid sequence of the normal BCAP31 protein (SEQ ID NO: 5).

As used herein, the term "polynucleotide" means a nucleotide polymer of any length, and as used herein, polynucleotide can be used interchangeably with nucleic acid or oligonucleotide.

The polynucleotide may be, for example, 10 to 100 nucleotides. If the polynucleotide has a size of 10 or less, the accuracy of capturing the target region is low, and if the polynucleotide has a size of 100 or more, the synthesis cost increases. Therefore, the polynucleotide is economical and has a size optimized for mutation detection of genomic DNA.

In the present invention, the hereditary hearing loss may specifically be genetically caused sensorineural hearing loss, and more specifically may be non-syndromic sensorineural hearing loss (NS-SNHL).

The above sensorineural hearing loss is the same as described above.

The above non-syndromic sensorineural hearing loss refers to hearing loss that does not show any abnormal symptoms or signs of organs other than inner ear dysfunction.

The polynucleotide of the present invention can specifically bind to the sequence of a target gene. By utilizing the specific binding characteristics of this polynucleotide, the target gene or a fragment thereof can be effectively separated from a mixture of mixed samples. Therefore, the polynucleotide can be called a probe. The term "probe" means a substance that specifically detects a specific substance, site, state, etc.

The kit may further comprise known materials required for hybridization of the polynucleotide with the genomic nucleic acid. For example, it may further comprise reagents, buffers, buffers, cofactors, and/or substrates required for hybridization of the nucleic acid. In addition, when the kit is applied to a PCR amplification process, it may optionally comprise reagents required for PCR amplification, such as buffers, DNA polymerase, DNA polymerase cofactors, and dNTPs, and when the kit is applied to an immunoassay, the diagnostic kit of the present invention may optionally comprise a secondary antibody and a substrate for a label. In addition, the kit may further comprise instructions for use for amplifying the target nucleic acid, and may be manufactured into a plurality of separate packages or compartments containing the reagent components described above.

Diagnostic composition

Another aspect of the present invention provides a composition for diagnosing hereditary hearing loss disease, comprising a polynucleotide comprising a contiguous nucleotide sequence selected from the nucleotide sequence of an exon region of a BCAP31 gene or a complementary polynucleotide thereof, for detecting c.397_398insGAG mutation of the BCAP31 gene.

The above BCAP31, c.397_398insGAG mutation of the BCAP31 gene and hereditary hearing loss, and the polynucleotide or the complementary polynucleotide thereof are the same as described above.

How to provide information

Another aspect of the present invention provides a method for providing information for diagnosing a hereditary hearing loss disease, comprising the steps of: 1) contacting a diagnostic composition or kit and genomic DNA isolated from an individual to obtain a hybridization product; 2) identifying a nucleotide sequence of genomic DNA isolated from the individual among the hybridization products; and 3) comparing the identified nucleotide sequence of the genomic DNA with a standard nucleotide sequence to identify a c.397_398insGAG mutation of the BCAP31 gene of the genomic DNA.

At this time, the mutation of the BCAP31 gene may include an insertion of nucleotides with respect to the normal BCAP31 gene.

The diagnostic composition, diagnostic kit, BCAP31, c.397_398insGAG mutation of the BCAP31 gene, and hereditary hearing loss are the same as described above.

In the above method, the genomic DNA isolated from the subject may be genomic DNA or a fragment thereof isolated from a biological sample. For example, the sample may be at least one selected from the group consisting of blood, saliva, urine, feces, tissues, cells, and biopsies. In addition, the sample may include a stored biological sample or genomic DNA isolated therefrom. The storage may be stored by a known method. The genomic DNA may be DNA or RNA derived from tissue that has been frozen or formalin-fixed paraffin-embedded tissue stored at room temperature. Methods for isolating genomic DNA from biological samples are well known. Accordingly, the sample may be isolated from cells, tissues, organs, or body fluids of a patient with hereditary hearing loss, and in this case, the sample may be obtained by a conventional method, for example, a biopsy using a method well known to those skilled in the art in the relevant medical techniques.

In one specific example, the genomic DNA included in the sample may be fragmented into arbitrary sizes. The fragmentation may be performed by a method well known to those skilled in the art. For example, the genomic DNA may be fragmented by using ultrasound. The method may include a step of ligating a sequence for amplification to both ends of the fragmented genomic DNA after fragmentation of the genomic DNA. A method for ligating the sequence for amplification (e.g., a paired-end tag, a universal tag) may be performed by a person skilled in the art by appropriately selecting a known technique.

In the above method, the hybridization can be performed by a known method. For example, it can be performed by incubating the polynucleotide and the genomic DNA in a buffer known to be suitable for hybridization of nucleic acids. The hybridization can be performed at an appropriate temperature. A temperature suitable for hybridization can be, for example, about 40° C. to about 80° C., about 50° C. to about 75° C., about 60° C. to about 70° C., or about 62° C. to about 67° C. In addition, the hybridization temperature is not limited thereto and can be appropriately selected depending on the sequence and length of the polynucleotide included in the composition. The hybridization time can be, for example, 1 hour to 12 hours (overnight).

The method may further include, prior to the step of confirming the nucleotide sequence of the genomic DNA in the hybridization product, a step of separating a hybridization product of the genomic DNA and the polynucleotide from the contact product obtained in the contacting step. The separation may be by using a moiety for separation or purification attached to the polynucleotide. The separation or purification may be achieved by a substance or a magnetic field that specifically binds to the moiety.

In addition, the method may further include a step of amplifying the genomic DNA by PCR using the separated hybridization product or the genomic DNA as a template and a universal primer complementary to a sequence for amplification attached to each of the genomic DNAs as a primer. The nucleotide sequence can be confirmed using the amplified genomic DNA.

The above nucleotide sequence can be confirmed, for example, by a sequencing method, and specifically, by next generation sequencing (NGS).

The above "Next-generation sequencing (NGS) method can analyze the entire genome spanning the DNA level, RNA level, and epigenetic level. To this end, it includes various analysis platforms such as whole genome sequencing (WGS), whole exome sequencing (WES), and whole transcriptome sequencing (WTS). Single nucleotide variants (SNVs), insertions/deletions, and copy number alterations in DNA can be identified through whole genome sequencing or exome sequencing, and changes in mRNA expression levels, gene fusions, and alternative splicing in RNA can be identified through transcriptome sequencing.

The method comprises a step of comparing the identified nucleotide sequence of the genomic DNA with a reference nucleotide sequence. The term "reference nucleotide sequence" may mean a human genome sequence that does not contain a mutation and serves as a reference for mutation identification. For example, the human gene base sequence published in the database of the National Center for Biotechnology Information (NCBI) of the National Institutes of Health in the United States may be used as a reference nucleotide sequence.

Comparison between the base sequence of the above genomic DNA and the standard base sequence can be performed using various known sequence comparison analysis programs, such as Maq, Bowtie, SOAP, and GSNAP.

The term "subject" as used herein refers to any animal classified as a mammal that is afflicted with or suspected of being afflicted with hereditary deafness, and may include a human, a non-human primate, a cow, a horse, a pig, a sheep, a goat, a dog, a cat or a rodent. Preferably, it may be a human. The terms "subject" and "patient" may be used interchangeably herein.

In the step of obtaining genomic DNA from the subject, the genomic DNA may be obtained from the blood of the subject. The obtaining method may utilize a method known to those skilled in the art for isolating genomic DNA from tissues or cells.

If a mutation is confirmed in the BCAP31 gene through the above method, the subject can be diagnosed as having a hereditary hearing loss disease. The above method can be used to diagnose hereditary hearing loss in a subject without additional experiments under in vitro and in vivo conditions.

Biomarker

Another aspect of the present invention provides a biomarker for diagnosing hereditary hearing loss disease of the BCAP31 gene comprising c.397_398insGAG mutation.

The c.397_398insGAG mutation, BCAP31, and hereditary hearing loss are the same as described above.

All technical terms used in the present invention, unless otherwise defined, have the same meaning as commonly understood by those skilled in the art in the relevant field of the present invention.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are only intended to illustrate the present invention, and the scope of the present invention is not limited to these examples.

Manufacturing Example 1. Manufacturing of Fluorescently Conjugated Mitochondria

MT ^GFP and MT ^dsRED were prepared by introducing nucleic acids encoding fusion proteins bound to TOM20, a mitochondria targeting sequence (MTS), and GFP or dsRED into HEK293 cells, and then isolating mitochondria bound to GFP or dsRED.

Experimental Example 1. Recruitment of Test Participants

This study was conducted with approval from the Institutional Review Board of Seoul National University Bundang Hospital (IRB-B-1007-105-402).

First, nine participants were recruited from a family with X-linked recessive hearing loss (Provand: 4 years old). In particular, among the above participants, Provand had normal hearing as a newborn, but his hearing threshold decreased by 5 dB every year, and his language identification score deteriorated rapidly from 70% to 50% during a 3-year follow-up.

The above participants underwent a comprehensive characterization including medical history, physical examination, imaging and audiological evaluation. The results of the auditory brainstem response test (ABR test) confirmed that the participants with hearing loss had progressive sensorineural hearing loss in both ears.

Experimental Example 2. Molecular Genetic Diagnosis

Exome sequencing was performed by extracting genomic DNA samples from peripheral blood of the participants in Experimental Example 1 above. The sequencing results were filtered and analyzed as follows.

Non-synonymous SNPs were filtered based on dept≥15. The MAF of the variants was checked using databases such as ExAC, 1000 Genomes, TOPMED, and GnomAD. Variants with MAF values ≥0.5% were excluded from the results if they were not reported as pathogenic in the literature, ClinVar, or DVD. In silico tests were performed to evaluate the pathogenicity of candidate variants using the scores of SIFT, PolyPhen2, GERP, CLINVAR, and CADD. Segregation analysis was then performed.

The pathogenic potential of the above-mentioned selected novel variants was evaluated according to the American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP).

As a result, as shown in Figs. 1 to 3, a new hemizygous mutation (c.397_398insGAG; p.Asp132_Ala133insGly) was found in the BCAP31 gene associated with the onset of hearing loss. The mutation was confirmed to be inherited in an X-linked recessive manner. Hearing-impaired patients with the mutation in the gene exhibited bilateral symmetrical non-syndromic sensorineural hearing loss (SNHL) with an average hearing threshold of 53 dB HL.

Experimental Example 3. Isolation of mouse cochlea and tissue immunostaining

Mice (C57BL/6, 1 month old) were sacrificed, the scalp was removed, and the cochlea on both sides was extracted. The extracted cochlea was treated with MT ^dsRED prepared by the method of Manufacturing Example 1 at a concentration of 4 μg. Tissues were sampled 1 hour, 4 hours, and 24 hours after MT ^dsRED treatment, and tissue slides were prepared and tissue immunostaining was performed. Anti-MTCO2 antibody was used as the primary antibody, and the nucleus was stained using DAPI and observed.

As a result, as shown in Fig. 12, mitochondria (MT ^dsRED ) labeled with dsRED fluorescent protein were located inside the cochlea, and it was confirmed that the mitochondria increased as the treatment time passed.

Experimental Example 4. Isolation of mitochondria from stem cells

Umbilical cord mesenchymal stem cells (US-MSC) (1×10 ⁷ cells/㎖) of passage 7 were physically disrupted and homogenized by pressurization, and then centrifuged for the first time at 1,100Хg. Thereafter, the supernatant was re-centrifuged at 12,000Хg for 15 minutes at 4℃, and the obtained pellet was used as a mitochondrial sample. The sample was quantified using the BCA (bicinchoninic acid) assay method and used. In this specification, the stem cell-derived mitochondria were named "PN-101" and are described interchangeably with mitochondria.

Experimental Example 5. Measurement of ATP and mitochondrial membrane potential in lymphoid cell lines derived from normal individuals and hearing-impaired patients with BCAP31 mutations

Intracellular ATP concentrations in lymphoblastoid cell lines (LCLs) from normal individuals (C1, C2) and hearing-impaired patients with BCAP31 gene mutations (PB-1, PB-2) were measured using CellTiterGlo Luminescent reagent (Promega) according to the manufacturer's method.

Additionally, the mitochondrial membrane potential (MMP) of each cell was measured using tetramethylrhodamine ethyl ester (TRME), a fluorescent membrane potential marker.

Specifically, lymphoblastoid cell lines derived from normal or hearing-impaired patients were seeded in a 6-well plate at a concentration of 1 × 10 ⁶ cells/well, cultured overnight, and then treated with PN-101 at a concentration of 10 ㎍ each. After 24 hours of reaction, 500 nM TMRE was treated, stained at 37°C for 30 minutes, and then measured using flow cytometry (CytoFLEX LX).

As a result, as shown in Figures 4a and 4b, it was confirmed that intracellular ATP and mitochondrial membrane potential were significantly reduced in the hearing-impaired patient group with BCAP31 mutations compared to normal people.

Experimental Example 6. Measurement of cytotoxicity by cisplatin treatment on lymphoid cell lines derived from normal individuals and hearing-impaired patients with BCAP31 mutations

After treating lymphoblastoid cell lines (LCLs) from normal individuals (C1) and hearing-impaired patients with BCAP31 mutations (PB-1, PB-2) with cisplatin at various concentrations, the degree of cell death was confirmed using annexin/PI staining.

Specifically, lymphoblastoid cell lines derived from normal or hearing-impaired patients were seeded in a 6-well plate at a concentration of 1× ¹⁰⁶ cells/well and cultured overnight. The cells prepared as described above were treated with cisplatin at various concentrations (3.125 uM to 100 uM) for 6 hours, and then washed twice with PBS to completely remove cisplatin. The cells were cultured for an additional 24 hours, stained with annexin/PI, and measured using flow cytometry (CytoFLEX LX).

As a result, as shown in Fig. 5, it was confirmed that apoptosis induced by cisplatin was more significantly increased in LCLs of hearing-impaired patients with BCAP31 mutations compared to normal subjects.

Experimental Example 7. Western Blot

Expression of cytochrome C (cyt C) was confirmed in lymphoblastoid cell lines (LCLs) derived from normal individuals (C1) and hearing-impaired patients with BCAP31 mutations (PB-1, PB-2).

Specifically, lymphoblastoid cell lines derived from normal people or hearing-impaired patients were seeded in a 6-well plate at a concentration of 1 × 10 ⁶ cells/well and cultured overnight. The cells prepared as described above were pretreated with cisplatin (25 uM) for 6 hours. Then, the cells were washed twice with PBS to completely remove cisplatin, and PN-101 was treated at a concentration of 10 ㎍ each and cultured for an additional 24 hours. Then, cell lysates were obtained using a lysis buffer, and the expression of cytochrome c and β-actin was confirmed using SDS-PAGE. At this time, anti-cyt C antibody (Santa Cruz, SC-13156) and anti-β-actin antibody were used as primary antibodies.

As a result, as shown in Figures 9a and 9b, it was confirmed that the expression of cyt C was increased in the cytosolic fraction of LCLs derived from hearing-impaired patients compared to the normal group.

Experimental Example 8. Confirmation of the improvement effect by mitochondrial treatment in a lymphoid cell line derived from a hearing-impaired patient with a BCAP31 mutation

Experimental Example 8.1. Recovery of intracellular mitochondrial function by mitochondrial treatment

Stem cell-derived mitochondria (PN-101) isolated using the same method as in Experimental Example 4 were treated to lymphoid cell lines (LCL) derived from normal individuals (C1) and hearing-impaired patients with BCAP31 mutations (PB-1, PB-2), and then the intracellular ATP concentration and membrane potential were measured.

Specifically, lymphoid cell lines derived from normal or hearing-impaired patients were seeded in a 6-well plate at a concentration of 1× ¹⁰⁶ cells/well, cultured overnight, and then treated with PN-101 at a concentration of 10 ㎍ each. After reacting for 24 hours, the intracellular ATP concentration and membrane potential were measured using the same method as in Experimental Example 5.

As a result, as shown in Figs. 7a and 7b, it was confirmed that the ATP concentration and membrane potential of intracellular mitochondria increased by PN-101 treatment compared to the untreated group in the lymphoid cell line derived from hearing-impaired patients (membrane potential increased only in PB-2).

Experimental Example 8.2. Confirmation of the effect of mitochondria on cytotoxicity induced by cisplatin treatment

The degree of cell death was determined in lymphoblastoid cell lines (LCLs) derived from normal individuals (C1) and hearing-impaired patients with BCAP31 mutations (PB-1, PB-2) treated with cisplatin and PN-101.

Specifically, lymphoblastoid cell lines derived from normal people or hearing-impaired patients with BCAP31 mutations were seeded at a concentration of 1× ¹⁰⁶ cells/well in a 6-well plate and cultured overnight. Then, the cells were treated with cisplatin (25 uM) and reacted for 6 hours, and then treated with PN-101 at a concentration of 10 ㎍. After 24 hours of reaction, the degree of cell death (cytotoxicity) was confirmed. The cell death (cytotoxicity) was confirmed through annexin/PI staining in the same manner as in Experimental Example 6.

As a result, as shown in Fig. 8, it was confirmed that the increased apoptosis by cisplatin treatment in LCLs of hearing-impaired patients with BCAP31 mutations was significantly reduced by PN-101 treatment compared to the untreated group.

In addition, under the same conditions, the expression of cytochrome C in the cytosolic fraction was confirmed using the same method as in Experimental Example 7, and this was quantified and presented in a graph.

As a result, as shown in Fig. 9a and Fig. 9b, cytochrome C in the cytosolic fraction was significantly increased by cisplatin in LCLs of hearing-impaired patients with BCAP31 mutations compared to the untreated group. On the other hand, it was confirmed that the expression of cytochrome C increased by cisplatin treatment was significantly decreased in the PN-101 treated group.

Through the above results, it was confirmed that the pathogenesis of hearing loss caused by BCAP31 mutation is related to mitochondrial dysfunction and increased sensitivity to ototoxicity. In addition, it was confirmed that when mitochondria (PN-101) were administered to lymphoblastoid cell lines (LCL) derived from hearing-impaired patients, mitochondrial abnormalities were restored and sensitivity to cisplatin was reduced. Therefore, the above results suggest that the administration of functionally normal mitochondria can be a potential method for treating hearing loss caused by mitochondrial dysfunction.

Experimental Example 9. Recovery of impaired mitochondrial function in a lymphoid cell line derived from a hearing-impaired patient with a mutation in the ferredoxin reductase gene.

After PN-101 treatment of a normal human (C1) and a lymphoid cell line (PB-3) derived from a hearing-impaired patient with a ferredoxin reductase (FDXR) mutation, intracellular ATP concentration and mitochondrial membrane potential were measured.

Specifically, lymphoblastoid cell lines derived from normal individuals or hearing-impaired patients with FDXR mutations were seeded in 6-well plates at a concentration of 1× ¹⁰⁶ cells/well and cultured overnight. Then, the cells were treated with PN-101 at a concentration of 10 ㎍ and reacted for 2 days, after which the intracellular ATP concentration and mitochondrial membrane potential were measured. At this time, the ATP concentration and membrane potential were measured in the same manner as in Experimental Example 5.

As a result, as shown in Fig. 10a and Fig. 10b, it was confirmed that the ATP concentration and membrane potential in the lymphoid cell line (PB-3) derived from a hearing-impaired patient with an FDXR mutation were decreased compared to the normal person (C1). On the other hand, in the case of the lymphoid cell line (PB-3) derived from a hearing-impaired patient, it was confirmed that the ATP concentration and mitochondrial membrane potential were significantly increased in the PN-101 treated group compared to the untreated group.

Experimental Example 10. Recovery of impaired mitochondrial function in a fibroblast cell line derived from a hearing-impaired patient with the m.A1555G mitochondrial mutation

PN-101 was treated at a concentration of 0.15 ㎍ for 24 hours to fibroblast cell lines (PB-4, PB-5) derived from hearing-impaired patients with mitochondria in which adenine at position 1555 of the mitochondrial gene was mutated to guanine (m.A1555G) from each normal person (C1), and the intracellular ATP concentration was measured. At this time, the fibroblast cell lines were seeded at a concentration of 5 × 10 ³ cells/well in a 96-well plate and then cultured overnight to prepare. The intracellular ATP concentration was measured by the method of Experimental Example 5.

As a result, as shown in Fig. 11, it was confirmed that the ATP concentration in the fibroblast cell lines (PB-4, PB-5) derived from hearing-impaired patients with the m.A1555G mutation was decreased compared to normal subjects. On the other hand, in the case of the fibroblast cell lines derived from the hearing-impaired patients, it was confirmed that the decreased ATP concentration was increased in the PN-101 treatment group compared to the untreated group.

Claims (20)

A pharmaceutical composition for preventing or treating hereditary hearing loss disease, comprising isolated mitochondria as an active ingredient. In the first paragraph, A pharmaceutical composition for preventing or treating hereditary hearing loss, wherein mitochondria are isolated from a cell. In the second paragraph, A pharmaceutical composition for preventing or treating hereditary hearing loss disease, wherein the cell is a somatic cell, a germ cell, a stem cell, a blood cell or a combination thereof. In the third paragraph, A pharmaceutical composition for preventing or treating hereditary hearing loss, wherein the stem cells are mesenchymal stem cells, induced pluripotent stem cells, embryonic stem cells or a combination thereof. In paragraph 4, A pharmaceutical composition for preventing or treating hereditary hearing loss, wherein the mesenchymal stem cells are umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane, placenta, synovial fluid, testis, periosteum or a combination thereof. In the first paragraph, A pharmaceutical composition for preventing or treating hereditary hearing loss, wherein the isolated mitochondria have normal activity. In the first paragraph, The above hereditary hearing loss is caused by a gene called BCAP31. A pharmaceutical composition for preventing or treating hereditary hearing loss, comprising a mutation. In Article 7, A pharmaceutical composition for preventing or treating hereditary hearing loss, wherein the mutation in the BCAP31 gene comprises an insertion of a nucleotide sequence relative to the nucleic acid sequence of a normal BCAP31 gene. In Article 8, A pharmaceutical composition for preventing or treating hereditary hearing loss, wherein the mutation in the BCAP31 gene is such that guanine (G), adenine (A), and guanine (G) are sequentially inserted between the 397th and 398th base sequences of sequence number 4. In Article 9, A pharmaceutical composition for preventing or treating hereditary hearing loss, wherein the mutation of the above BCAP31 gene is such that glycine (Gly) is inserted between the 132nd amino acid residue and the 133rd amino acid residue in the amino acid sequence of sequence number 5. In Article 7, A pharmaceutical composition for preventing or treating hereditary hearing loss, wherein the above BCAP31 gene mutation is an X-chromosome-linked recessive inheritance. A diagnostic kit for hereditary hearing loss disease, comprising a polynucleotide comprising a contiguous nucleotide sequence selected from the nucleotide sequence of an exon region of a BCAP31 gene or a complementary polynucleotide thereof, for detecting c.397_398insGAG mutation of the BCAP31 gene. In Article 12, A diagnostic kit for hereditary hearing loss disease, wherein the mutation is an insertion of a nucleotide sequence into a normal BCAP31 gene. In Article 12, A diagnostic kit for hereditary hearing loss disease, wherein the polynucleotide has a length of 10 to 100 nucleotides. In Article 12, A diagnostic kit for hereditary hearing loss, wherein the hereditary hearing loss is non-syndromic sensorineural hearing loss (NS-SNHL). A composition for diagnosing hereditary hearing loss disease, comprising a polynucleotide comprising a contiguous nucleotide sequence selected from the nucleotide sequence of an exon region of a BCAP31 gene or a complementary polynucleotide thereof, for detecting c.397_398insGAG mutation of the BCAP31 gene. 1) A step of contacting the diagnostic composition of Article 16 with genomic DNA isolated from an individual to obtain a hybridization product; 2) a step of confirming the nucleotide sequence of the genomic DNA separated from the individual among the hybridization products; and 3) A method for detecting a mutation in genomic DNA to provide information for diagnosing a hereditary hearing loss disease, comprising: a step of comparing the identified nucleotide sequence of the genomic DNA with a standard nucleotide sequence to identify a c.397_398insGAG mutation in the BCAP31 gene of the genomic DNA. Biomarker for diagnosing hereditary hearing loss disease of the BCAP31 gene containing c.397_398insGAG mutation. Use of isolated mitochondria for the prevention or treatment of hereditary deafness. A method for preventing or treating hereditary hearing loss, comprising the step of administering isolated mitochondria to a subject.

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