Dual vector system for treating hearing loss and its use

Abstract

The present invention relates to the field of gene therapy in the medical field, and in particular to the use of overexpressing normal genes to restore hearing in patients with hereditary hearing loss caused by gene mutations or deletions. The present invention relates to a dual vector system expressing an OTOF protein. The dual vector system comprises two segments of nucleotide sequence, the first segment comprising two ITR sequences and a gene expression cassette inserted between the ITR sequences, and the second segment comprising two ITR sequences and a gene expression cassette inserted between the ITR sequences. An adeno-associated virus packaged with the vector is also provided. The vector and virus can restore hearing in both ears by administering them to one ear, in the field of large-scale dual vector gene delivery for hearing loss gene therapy.

Description

[Technical Field]

[0001] The present invention belongs to the field of gene therapy in the medical field, and in particular relates to the use of overexpression of normal genes to restore hereditary hearing loss caused by gene mutations or deletions. [Background technology]

[0002] The ear is an important organ in the human body, consisting of the outer, middle, and inner ear. Its main function is to perceive sound and maintain balance. Abnormalities in ear function can lead to a range of physical disorders, including hearing loss, tinnitus, and dizziness.

[0003] Hearing loss is a common disorder characterized by abnormalities in auditory function. It can be divided into congenital and acquired hearing loss and is often associated with genetic and environmental factors. Two out of every 1,000 newborns are born with congenital hearing loss, and 50-60% of these patients suffer from hearing loss due to genetic mutations. Hearing loss caused by genetic mutations can be divided into dominant and recessive gene mutations. Dominant hearing loss genes include ACT1, CCDC50, CD164, CEACAM16, and DIAPH1. Recessive hearing loss genes include CLDN14, PJVK, GRXCR1, MYO7A, MYO6, MYO3A, MYO15A, OTOF, OTOG, OTOA, STRC, TMC1, SLC22A4, SLC26A4, SLC26A5, TECTA, GJB2, and GJB6. The discovery of these deafness genes provides potential targets for precise treatment of hereditary deafness, so if the above genes cause deafness, gene therapy may become the first-line strategy to cure deafness.

[0004] Gene therapy refers to a method of correcting, compensating, or suppressing abnormal nucleic acid sequence or expression in the body to achieve therapeutic goals. Currently, most gene therapies require vector delivery. Adeno-associated virus (AAV) is a safe and efficient delivery vector with a packaging capacity of approximately 4.7 kb. However, in the field of hearing loss, the coding regions of many genes are not suitable for AAV packaging. For example, the coding regions of BDP1, CDH23, COL11A2, LOXHD1, MET, MYO15A, MYO3A, MYO7A, OTOG, OTOF, OTOGL, PCDH15, PTPRQ, STRC, TECTA, and TARA exceed the packaging limit of AAV vectors, along with their associated regulatory elements. Currently, DNA recombination dual vectors are used to address this issue, but DNA recombination dual vector packaging has low in vivo recombination efficiency.

[0005] Among the hearing loss-related genes with coding sequences longer than 4 kb, OTOF plays an important role in hearing. OTOF protein is mainly expressed in the cochlear inner hair cells, and its main function is to transport calcium ions (Ca 2+ ) and initiate downstream neurotransmitter release. Deletion or loss-of-function mutations in the OTOF gene can cause DFNB9 hearing loss.

[0006] The OTOF gene (NCBI Gene ID: 9381) is transcribed into three distinct transcripts, including isoform 1 (NM_194248.3), isoform 2 (NM_004802.4), isoform 3 (NM_194322.3), isoform 4 (NM_194323.3), and isoform 5 (NM_001287489.2). All of these are derived from the same RNA by alternative splicing, and the splice variants associated with inner ear hair cell hearing are isoform 1 and isoform 5. The CDS regions of these two transcripts are 5994 bp long and encode a protein of 1997 amino acids.

[0007] For congenital hearing loss caused by OTOF gene mutations, AAV is the delivery vehicle of choice due to its non-integrated delivery, long expression time, and low immunogenicity. However, AAV's packaging capacity is less than 4.7 kb, which poses a packaging limitation that prevents the full packaging of the OTOF gene (the total length of the OTOF gene and regulatory sequences exceeds 7 kb). There are currently three main approaches to solving the OTOF packaging problem. The first is overload packaging. This method primarily involves packaging a 7.5 kb gene expression element into an AAV virus and injecting it into the mouse cochlea. After a period of time, approximately 30% of the inner ear hair cells were observed to express the OTOF protein, restoring the mouse's hearing to approximately 58 dB. However, overload packaging is not an optimal solution due to its low packaging efficiency, difficult product control, and low transfection efficiency. The second approach is to shorten the length of the coding sequence required for functional OTOF. OTOF is a C2 domain protein composed of six C2 domains, A, B, C, D, E, and F, and a TEM domain. Research has shown that mini-OTOFs composed of some of these domains can partially restore OTOF function but cannot restore hearing in animals. The third method is to use a dual-vector approach to perform DNA recombination to generate full-length mature OTOF mRNA and fully translate the protein. These methods can be divided into overlapping, trans-splicing, or a combination of overlapping and trans-splicing.

[0008] Although DNA recombination strategies can produce full-length, functional OTOF proteins (otoferlins), their recombination efficiency is not ideal, affecting the expression and accumulation of OTOF proteins. To overcome this issue, protein recombination is currently considered the best option. Intein-mediated protein trans-splicing, in particular, is characterized by rapid recombination and high recombination efficiency. Inteins were first discovered in fungi and yeast, and subsequently in various microorganisms, including bacteria, viruses, and archaea. Inteins can form both intramolecular and intermolecular protein bonds. Intermolecular intein bonds include naturally isolated inteins and artificially isolated intramolecular inteins. The N- and C-termini of an intein are connected to the C- and N-termini of two parts of a target protein, respectively, allowing the electrophilic group of the second part to attack the nucleophilic group of the first part to form a covalent bond, resulting in the formation of a complete two-part connexin through allostery. In molecular design, the only requirement is that the first amino acid in the C-terminal part be serine, threonine, or cysteine.

[0009] Therefore, considering the characteristics of protein recombination and the possibility of using dual vectors to improve protein recombination efficiency, the present invention employed a protein-level recombination strategy to recombine a hearing loss-related protein. In the method of the present invention, the OTOF protein is expressed using an AAV vector via intein recombination, thereby restoring abnormal gene function and achieving efficient expression. Summary of the Invention

[0010] Current methods using dual-vector AAV delivery and DNA recombination have low recombination efficiency in the OTOF expression system. Furthermore, current dual-vector delivery technology can only achieve OTOF expression in one cochlea and hearing restoration in one ear. If hearing restoration in both ears is required, cochlear injections must be administered in both ears. This incurs inconvenience and increased risk for patients, and to address these issues, the present invention provides a dual-vector system capable of expressing OTOF protein and the adeno-associated virus packaged by it. This method is useful in the field of large-scale gene dual-vector delivery for hearing loss gene therapy, enabling bilateral hearing restoration through single-ear administration.

[0011] The first technical solution provided by the present invention is a dual vector system for expressing OTOF protein, which comprises two segments of nucleotide sequences. The nucleotide sequence of the first segment includes two ITR sequences and an expression cassette inserted between the ITR sequences. The nucleotide sequence of the second segment comprises two ITR sequences and an expression cassette inserted between the mITR sequences. The expression cassette of the first segment of nucleotide sequence comprises a promoter, an N-terminal coding sequence of OTOF, an N-terminal coding sequence of Intein, and PolyA. The expression cassette of the second segment of nucleotide sequence comprises a promoter, a C-terminal coding sequence of Intein, a C-terminal coding sequence of OTOF and PolyA. The amino acid sequence of the OTOF is shown in SEQ ID NO: 1 or SEQ ID NO: 2 in the sequence listing. A division point is set on the OTOF amino acid sequence, and the nucleotide coding sequence from the N-terminus of the OTOF amino acid sequence to the division point is the N-terminal coding sequence of OTOF, and the nucleotide coding sequence from the amino acid next to the division point to the C-terminus of the OTOF amino acid sequence is the C-terminal coding sequence of OTOF. Furthermore, the division site of OTOF includes, but is not limited to, the amino acid immediately preceding serine, threonine, or cysteine ​​in the OTOF protein amino acid sequence, in order from the N-terminus to the C-terminus of OTOF. Examples of such promoters include, but are not limited to, a CAG promoter, a CMV promoter, a CBA promoter, a UbC promoter, an SFFV promoter, an EF1α promoter, a PGK promoter, or promoters encoding genes such as Myo7A, Myo15, Atoh1, POU4F3, Lhx3, Myo6, α9AchR, α10AchR, and OTOF. The PolyA is an adenine modification of the tail during mRNA maturation, which can further stabilize the mRNA. In the present invention, the PolyA sequence includes AATAAA and its variants, such as ATTAAA, AGTAAA, CATAAA, TATAAA, GATAAA, ACTAAA, AATATA, AAGAAA, AATAAT, AAAAAA, AATGAA, AATCAA, AACAAA, AATCAA, AATAAC, AATAGA, AATTAA, and AATAAG. Furthermore, the ITR sequences (inverted terminal repeats) are derived from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 or AAV9. The expression cassette may further comprise other expression elements, including, but not limited to, expression control elements and labeling elements. Furthermore, the expression regulatory elements include, but are not limited to, functional regulatory elements such as (1) elements for controlling the expression of target proteins, for example, IRES for initiating translation of downstream genes; (2) regulatory elements for expressing miRNA and siRNA sequences; (3) introns; (4) positioning sequences for localizing and expressing target proteins in the nucleus, cytoplasm, or various organelles and secreting them extracellularly; (5) sequences for promoting protein degradation (for example, PEST sequences); (6) some Kozak sequences (the Kozak sequence is GNCNCN, for example, GCCACC); (7) enhancers (enhancers may be derived from SV40 virus, CMV virus, adenovirus, etc.); and (8) WPRE.

[0012] Furthermore, the label element includes, but is not limited to, for example, FLAG, HA, MYC, fluorescent protein, luciferase, SUMO protein, ubiquitin protein, GST, and the like.

[0013] Furthermore, the nucleotide sequence encoding the OTOF gene is shown in SEQ ID NO: 3 or SEQ ID NO: 4 in the sequence listing. Furthermore, examples of the intein include, but are not limited to, intein sequences in proteins such as MxeGyrA, pabPolIII, MjaKlbA, SspDnaB, SceVMA, SspDnaE, ​​NpuDnaE, ​​AvaDnaE, ​​CraDnaE, ​​CspDnaE, ​​CwaDnaE, ​​MchtDnaE, ​​OliDnaE, ​​TerDnaE, ​​gp41-1, gp41-8, IMPDH-1, and RmaDnaB. Furthermore, in the amino acid sequence of the OTOF protein shown in SEQ ID NO: 1 or 2, the division site includes, but is not limited to, amino acid residues at positions 827, 930, 954, and 1130. In addition, in the amino acid sequence of the OTOF protein shown in SEQ ID NO: 1 or 2, the division site is the amino acid residue at position 827. That is, the nucleotide coding sequence of amino acids 1-827 is the N-terminal coding sequence of OTOF, and the nucleotide coding sequence of amino acids 828-1997 is the C-terminal coding sequence of OTOF. In addition, in the amino acid sequence of the OTOF protein shown in SEQ ID NO: 1 or 2, the division site is the amino acid residue at position 930. That is, the nucleotide coding sequence of amino acids 1-930 is the N-terminal coding sequence of OTOF, and the nucleotide coding sequence of amino acids 931-1997 is the C-terminal coding sequence of OTOF. In addition, in the amino acid sequence of the OTOF protein shown in SEQ ID NO: 1 or 2, the division site is the amino acid residue at position 954. That is, the nucleotide coding sequence of amino acids 1-954 is the N-terminal coding sequence of OTOF, and the nucleotide coding sequence of amino acids 955-1997 is the C-terminal coding sequence of OTOF. In addition, in the amino acid sequence of the OTOF protein shown in SEQ ID NO: 1 or 2, the division site is the amino acid residue at position 1130. That is, the nucleotide coding sequence of amino acids 1-1130 is the N-terminal coding sequence of OTOF, and the nucleotide coding sequence of amino acids 1131-1997 is the C-terminal coding sequence of OTOF. Preferably, the amino acid sequence of said OTOF is shown in SEQ ID NO:2.

[0014] Preferably, the nucleotide sequence of the first segment is such that an expression cassette for the nucleotide sequence of the first segment is inserted between ITR sequences in a plasmid containing ITRs, and the nucleotide sequence of the second segment is such that an expression cassette for the nucleotide sequence of the second segment is inserted between ITR sequences in a plasmid containing ITRs. Furthermore, plasmids containing the ITRs include, but are not limited to, pAAV, pAAV-CMV, pX601, pX551, pAAV-MCS plasmids, etc.

[0015] The second technical solution provided by the present invention is an adeno-associated virus packaging vector system, which includes the dual vector system expressing the OTOF protein described in the first technical solution, a vector carrying AAV rep and cap genes, and a helper virus vector, and the vector becomes an AAV vector after packaging. Furthermore, the AAV rep and cap gene vectors include, but are not limited to, AAV1, AAV2, AAV5, AAV8, AAV9, Anc80, PHP.eB, AAV-DJ, or AAVrh.10 vectors. Furthermore, the helper virus vector is an adenovirus or herpesvirus helper virus vector, preferably a pHelper plasmid.

[0016] The third technical solution provided by the present invention is a method for packaging an adeno-associated virus, in which the adeno-associated virus packaging vector system described in the second technical solution is transfected into a host cell for packaging. Furthermore, the dual vector in the packaging vector system described in the second technical solution is transfected into host cells together with a vector carrying the AAV rep and cap genes and a helper virus vector, respectively, for packaging. Furthermore, the host cells are cell lines capable of viral replication and stable inheritance, including, but not limited to, Hela-S3, HEK-293, HEK-293T, HEK-293FT, A549, and Sf9 cells. Preferably, the host cells are HEK-293 or HEK-293T cells.

[0017] The fourth technical solution provided by the present invention is an adeno-associated virus obtained by the packaging method described in the third technical solution, which is a pair of viruses in which the N-terminal coding sequence of OTOF and the N-terminal coding sequence of Intein, and the C-terminal coding sequence of OTOF and the C-terminal coding sequence of Intein are packaged, respectively.

[0018] The fifth technical solution provided by the present invention is the use of the dual vector system described in the first technical solution or the adeno-associated virus described in the fourth technical solution, in particular in the preparation of drugs or preparations for treating hearing loss diseases, hearing impairments or hearing dysfunctions. Furthermore, the hearing loss disease, hearing impairment, or hearing dysfunction is caused by a genetic mutation involving the OTOF gene, including, but not limited to, base substitution, frameshift mutation, deletion mutation, and insertion mutation.

[0019] The sixth technical solution provided by the present invention is a preparation, formulation or drug prepared with the dual vector system described in the first technical solution or the adeno-associated virus described in the fourth technical solution. Furthermore, the formulation, formulation or drug may be in any dosage form, including, but not limited to, an injection dosage form and an ointment dosage form. Furthermore, in said formulation, formulation or medicament, said dual vector system or adeno-associated virus is the only active ingredient. Additionally, the formulation, formulation or drug may include commonly used solvents, buffers, such as commonly used drug vectors and adjuvants, including one or more of neutral salt buffer, acidic salt buffer, alkaline salt buffer, glucose, mannose, mannitol, proteins, polypeptides and amino acids, antibiotics, chelating agents, adjuvants or preservatives. Furthermore, the buffer solution is a phosphate buffer solution, a Tris buffer solution, a 0.01% Poloxamer PBS buffer solution, or a HEPES buffer solution. Furthermore, in said preparations, formulations or drugs, the active ingredient may be contained in other vectors, such as nanoparticles, liposomes and cationic lipid particles.

[0020] Furthermore, the method of administration of the formulation, formulation or drug is unilateral or bilateral administration. Preferably, the method of administration of the formulation, formulation or drug is unilateral administration. In addition, when administered unilaterally, the administration method is cochlear injection, including, but not limited to, cochlear round window injection, oval round window injection, semicircular canal injection, and utricle injection. It is administered once or multiple times throughout a lifetime, with a total dose of 1 × 10 9 -1×10 13 This is the viral genome. The beneficial effects are as follows:

[0021] In the present invention, the expression efficiency of the complete OTOF protein was significantly improved by using the AAV dual vector and Intein recombination method for OTOF expression. Furthermore, unilateral cochlear administration resulted in bilateral hearing recovery. After unilateral administration, Otof - / - Hearing in the injected ear of the mice was restored to the level of wild-type mice, and hearing in the contralateral uninjected ear was also improved by 60 decibels. [Brief explanation of the drawings]

[0022] [Figure 1] 1 is a pAAV-CMV plasmid map. [Figure 2] Figure 1 shows the pAAV-CMV-OTOF-N-S1-Npu-N-intein plasmid map. [Figure 3] Figure 1 shows the pAAV-CMV-Npu-C-intein-OTOF-C-S1 plasmid map. [Figure 4] Figure 1 shows the pAAV-CMV-OTOF-N-S2-Npu-N-intein plasmid map. [Figure 5] Figure 1 shows the pAAV-CMV-Npu-C-intein-OTOF-C-S2 plasmid map. [Figure 6]Figure 1 shows the pAAV-CMV-OTOF-N-S4-Npu-N-intein plasmid map. [Figure 7] Figure 1 shows the pAAV-CMV-Npu-C-intein-OTOF-C-S4 plasmid map. [Figure 8] Figure 1 shows the pAAV-CMV-OTOF-N-S1-Rma-N-intein plasmid map. [Figure 9] Figure 1 shows the pAAV-CMV-Rma-C-intein-OTOF-C-S1 plasmid map. [Figure 10] Figure 1 shows the pAAV-CMV-OTOF-N-S2-Rma-N-intein plasmid map. [Figure 11] Figure 1 shows the pAAV-CMV-Rma-C-intein-OTOF-C-S2 plasmid map. [Figure 12] Figure 1 shows the pAAV-CMV-OTOF-N-S3-Rma-N-intein plasmid map. [Figure 13] Figure 1 shows the plasmid map of pAAV-CMV-Rma-C-intein-OTOF-C-S3. [Figure 14] Figure 1 shows the pAAV-CMV-OTOF-N-S4-Rma-N-intein plasmid map. [Figure 15] Figure 1 shows the pAAV-CMV-Rma-C-intein-OTOF-C-S4 plasmid map. [Figure 16] 1 is a plasmid map of pAAV-CMV-OTOF-FL. [Figure 17] Figure 1 is the pAAV-CMV-OTOF-N-AK plasmid map. [Figure 18] 1 is a plasmid map of pAAV-AK-OTOF-C-PolyA. [Figure 19] Figure 1 is the pAAV-CMV-OTOF-N-AP plasmid map. [Figure 20] 1 is a map of the pAAV-AP-OTOF-C-PolyA plasmid. [Figure 21]Figure 1 is the pAAV-CMV-OTOF-N-TS plasmid map. [Figure 22] 1 is a map of the pAAV-TS-OTOF-C-PolyA plasmid. [Figure 23] 1 shows the imaging results of Example 6. [Figure 24] 10 shows the imaging results of Example 7. [Figure 25] 1 shows the imaging results of Example 8. [Figure 26] 10 shows the imaging results of Example 9. [Figure 27] 10 shows the imaging results of Example 10. [Figure 28] This is a comparison diagram of the results of OTOF Intein recombination and OTOF DNA recombination. [Figure 29] A comparison of the base pairs of Otof − / − mutant mice and wild-type mice. [Figure 30] This is a comparison of hearing ability between Otof- / - mutant mice and wild-type mice. [Figure 31] 1 shows the state of hearing recovery after one month in Example 13. [Figure 32] 11 shows the state of hearing recovery after 2 months in Example 13. [Figure 33] 1 shows the state of hearing recovery after one month in Example 14. [Figure 34] 1 shows the state of hearing recovery after two months in Example 14. [Figure 35] Hearing recovery effect in mice in the low dose group. [Figure 36] OTOF expression status in wild-type and gene-deficient mice. [Figure 37] OTOF expression in the treated ear and the contralateral ear of treated mice. [Figure 38] FIG. 1 is a statistical diagram showing the expression status of OTOF in mice. [Figure 39] This shows the recovery of hearing in mice by AAV1 serotype OTOF. DETAILED DESCRIPTION OF THE INVENTION

[0023] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with specific examples, which are merely for the purpose of illustrating the present invention and are not intended to limit the present invention.

[0024] In the present invention, the combination of dual vector (or AAV dual vector) delivery and Intein recombination enables efficient expression of OTOF protein in the host, thereby restoring hearing loss diseases, hearing impairments or hearing dysfunction, and achieving the technical effect of restoring hearing in both ears by administration to one ear.

[0025] In the present invention, OTOF proteins are expressed by intein recombination. The OTOF regions are isoform 5 (NM_001287489.2) (the translated amino acid sequence is NP_001274418.1) and isoform 1 (NM_194248.3) (the translated amino acid sequence is NP_919224.1).

[0026] In some embodiments of the present invention, the protein required for the restoration or amelioration of hearing loss is the OTOF set forth in SEQ ID NO: 1 or SEQ ID NO: 2. The protein of interest may have a sequence that is 65%-100% identical to SEQ ID NO: 1 or 2, for example, 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75%, 70%, or 65% identical to SEQ ID NO: 1 or 2. Suitable truncated forms of the protein may also exist, for example, proteins that are 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, or 60% in length of the full-length protein of SEQ ID NO: 1 or 2. Proteins may have suitable insertions, including proteins that are 101%, 102%, 103%, 104%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, and 140% of the length of the full-length protein. The relevant sequences are shown in Table 1.

[0027] Table 1: OTOF sequences and correspondences JPEG2025530726000002.jpg30170

[0028] For the amino acid sequence of SEQ ID NO: 1 or 2, in addition to the corresponding nucleotide sequence of SEQ ID NO: 3 or 4, the codons may be optimized, and the corresponding CAI may be 0.65-1.0, for example, 1.0, 0.99, 0.98, 0.97, 0.96, 0.95, 0.90, 0.85, 0.80, 0.75, 0.70 or 0.65.

[0029] In addition to OTOF protein sequences from humans, OTOF protein sequences can be selected from other animals, such as mouse (protein sequence: NP_001273350.1, NP_001300696.1, NP_001093865.1 or NP_114081.2), rat (protein sequence: NP_001263649.1), pig (protein sequence: XP_020943388.1), monkey (protein sequence: XP_0149 67378.2, XP_014967379.2, XP_028687700.1, XP_014967380.2 or XP_028687701.1) and sequences having 65%-100%, for example, 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75%, 70%, 65% homology thereto.

[0030] Inteins can perform protein splicing functions and function during or after protein translation to covalently link two different proteins. Inteins were first discovered in yeast and fungi, and through alignment comparison and analysis, the number of intein residues present in viruses, bacteria, archaea, and eukaryotic microorganisms is predicted to exceed 600. Most inteins are complete proteins, while some have separate N- and C-termini, each of which binds to a portion of the protein and can be reassembled after protein translation to form the complete protein through nucleophilic chemical reactions and allostery. In the present invention, inteins with separate N- and C-termini are preferred. The intein can be selected from MxeGyrA, pabPolIII, MjaKlbA, SspDnaB, SceVMA, SspDnaE, ​​NpuDnaE, ​​AvaDnaE, ​​CraDnaE, ​​CspDnaE, ​​CwaDnaE, ​​MchtDnaE, ​​OliDnaE, ​​TerDnaE, ​​gp41-1, gp41-8, IMPDH-1, RmaDnaB, etc. Some of the intein amino acid sequences used in the examples of the present invention are shown in Table 2.

[0031] Table 2: Partial Intein Amino Acid Sequences JPEG2025530726000003.jpg34170

[0032] In the present invention, when assembling a target protein into two vectors using the intein recombination method, OTOF is first divided into two segments, the N-terminal and the C-terminal, at the division point. The conditions for division are: 1) it must be capable of being packaged into an AAV virus, and 2) the first amino acid at the C-terminus must be serine, threonine, or cysteine. The N-terminus of the intein is fused to the C-terminus of the N-terminus of the OTOF protein, and the C-terminus of the intein is fused to the N-terminus of the C-terminus of the OTOF protein, and then expressed.

[0033] Preferably, the OTOF protein is divided into two segments with approximately equal N- and C-terminal lengths, or the N-terminal / C-terminal length is 0.3-3, for example, N-terminal length / C-terminal length is 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.5, 1.7, 1.8, 2.0, 2.0, 2.5 or 3.0.

[0034] More preferably, the division schemes for the N-terminus and C-terminus of the OTOF protein include, but are not limited to, those shown in Table 3 of Example 1.

[0035] More preferably, for the amino acid sequence of the OTOF protein shown in SEQ ID NO: 1 or 2, the N-terminus and C-terminus are divided into amino acids 1-827 and 828-1997, amino acids 1-930 and 931-1997, amino acids 1-954 and 955-1997, and amino acids 1-1130 and 1131-1997.

[0036] In the present invention, a dual vector expressing an OTOF protein is constructed by assembling two vectors containing the nucleotide sequences of two segments of a target protein using the intein method. The nucleotide sequence of the first segment includes two ITR sequences and an expression cassette inserted between the ITR sequences. The nucleotide sequence of the second segment includes two ITR sequences and an expression cassette inserted between the ITR sequences. The ITRs are used by adeno-associated proteins to recognize and package DNA, and are also involved in the repair and replication of the adeno-associated viral genome. Preferred ITR sequences are derived from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9, and ITR sequences having 65%-100%, e.g., 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75%, 70%, or 65% homology to these sequences.

[0037] The N-terminus of the Intein is fused to the C-terminus of the N-terminus of the OTOF protein and expressed, and the resulting sequence is constructed into an expression cassette for the nucleotide sequence of the first segment, which includes a promoter, an N-terminal coding sequence of OTOF, an N-terminal coding sequence of the Intein, and PolyA. The C-terminus of the Intein is fused to the N-terminus of the C-terminus of the OTOF protein, and the resulting sequence is constructed into an expression cassette for the nucleotide sequence of the second segment, which includes a promoter, a C-terminal coding sequence for the Intein, a C-terminal coding sequence for OTOF, and PolyA. The nucleotide sequence of the first segment and the nucleotide sequence of the second segment can be in any order and can constitute either the N-terminal or C-terminal sequence of OTOF.

[0038] In the present invention, the above-mentioned promoter refers to a related sequence capable of initiating transcription of a downstream target protein. It is typically used to recruit transcription factors and express target proteins in a specific space and time. Examples of promoters include, but are not limited to, the following: The promoter may be an RNA polymerase II promoter or an RNA polymerase III promoter. Promoters may be broadly expressed promoters, such as the CMV promoter and the CAG promoter. The promoter may be a tissue-specific promoter, with preferred promoters highly expressed in the ear, additional promoters highly expressed in the cochlea or vestibule, and additional promoters highly expressed in the cochlea. Tissue-specific promoters may be derived from partial or complete sequences 1 to 10,000 bp upstream of the OTOF gene transcription start site and sequences with 65%-100% sequence identity, e.g., 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75%, 70%, or 65% sequence identity. Preferably, promoters applicable to the present invention include, but are not limited to, CAG promoter, CMV promoter, CBA promoter, UbC promoter, SFFV promoter, EF1α promoter, PGK promoter, or promoters corresponding to genes such as Myo7A, Myo15, Atoh1, POU4F3, Lhx3, Myo6, α9AchR, α10AchR, and OTOF.

[0039] PolyA refers to the modification of the tail with adenine during the maturation process of mRNA, which can further stabilize the mRNA. In the present invention, the PolyA sequence includes AATAAA and its variants, such as ATTAAA, AGTAAA, CATAAA, TATAAA, GATAAA, ACTAAA, AATATA, AAGAAA, AATAAT, AAAAAA, AATGAA, AATCAA, AACAAA, AATCAA, AATAAC, AATAGA, AATTAA, and AATAAG.

[0040] The nucleotide sequence of the first segment or the second segment may include one or more (e.g., 2, 3, 4, 5, etc.) regulatory elements. The regulatory elements of the first segment and the second segment may be the same or different. These regulatory elements include, but are not limited to, the following: (1) regulatory elements used to regulate the expression of a target protein. For example, an IRES is used to initiate translation of a downstream gene. An IRES is a sequence that can initiate protein translation within an mRNA. For example, an IRES may be derived from a virus such as FMDV, EMCV, HRV, HIV, HAV, HCV, or PV. (2) regulatory elements used to express miRNA and siRNA sequences. (3) introns. (4) positioning sequences that localize and express a target protein in the nucleus, cytoplasm, or various organelles and secrete it extracellularly. (5) sequences that promote protein degradation (e.g., PEST sequences). (6) The regulatory element may be a Kozak sequence, such as GNCNCN, e.g., GCCACC. (7) Enhancer refers to a sequence that can enhance gene expression, such as a CMV enhancer, an SV40 virus enhancer, or an adenovirus enhancer, and may be located upstream of a target gene, downstream of a target gene, or within a gene, and may or may not have tissue specificity, and may or may not transcribe eRNA. (8) The regulatory element may be a WPRE.

[0041] The nucleotide sequence of the first segment or the second segment may further include one or more (e.g., 2, 3, 4, 5, etc.) tags. The tags of the first segment and the second segment may be the same or different. For example, FLAG, HA, MYC, fluorescent proteins, luciferase, etc. can also be used to improve the properties of proteins such as SUMO proteins, ubiquitin proteins, and GST.

[0042] The nucleotide sequence of the first or second segment constructed above may be a plasmid or other form of linear or circular nucleic acid, and both the N-terminus and C-terminus of the target protein can be expressed in cells, tissues, organs, and individuals through appropriate expression and regulatory elements. The preferred scope herein is expression in the cochlea. Preferably, in the present invention, a target gene expression cassette is integrated into a plasmid containing ITRs (including pAAV, pAAV-CMV, pX601, pX551, pAAV-MCS, etc.), followed by AAV packaging.

[0043] When the nucleotide sequences of the first and second segments constructed above are used in a viral vector, they can be applied to any type of AAV packaging. The nucleotide sequences of the first and second segments are introduced into host cells together with a vector carrying the AAV rep and cap genes, respectively, and a helper virus vector, and then packaged to obtain an adeno-associated virus. The host cells are cell lines that allow viral replication and stable inheritance, including, but not limited to, Hela-S3, HEK-293, HEK-293T, HEK-293FT, A549, or Sf9 cells. Preferably, the host cells are HEK-293 or HEK-293T cells. AAV virus samples obtained after packaging may contain empty capsid viruses. The content of empty capsid virus may be 0%-99%, for example, 0%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, preferably 0%-50%.

[0044] The viruses obtained by this packaging method are a pair of adeno-associated viruses packaged with the N-terminal coding sequence of OTOF, the N-terminal coding sequence of Intein, the C-terminal coding sequence of Intein, and the C-terminal coding sequence of OTOF, respectively. This pair of adeno-associated viruses can express both the N-terminus and C-terminus of a target protein in cells, tissues, organs, and individuals. The Intein protein performs protein splicing, playing a role during or after protein translation, covalently linking two different proteins to obtain a full-length, functional OTOF protein. It can be applied to cells of various mammals (e.g., humans, mice, dogs, pigs, rabbits, hamsters, sheep, cats, horses, or non-human primates). It can be applied to the ear, inner ear, cochlea, and inner ear hair cells, as well as cells unrelated to the ear. The cells can be in vitro, in vivo, or ex vivo.

[0045] Therefore, the dual vector or adeno-associated virus can be used to prepare a drug for treating hearing loss, hearing impairment, or hearing dysfunction caused by a genetic mutation involving the OTOF gene. Such mutations include, but are not limited to, base substitutions, frameshift mutations, deletion mutations, and insertion mutations.

[0046] The formulations prepared using the dual vector or adeno-associated virus may be in the form of a powder or solution. They may contain common solvents and buffers, such as common drug vectors and auxiliary agents. They may also contain neutral salt buffers, acidic salt buffers, alkaline salt buffers, glucose, mannose, mannitol, proteins, polypeptides, and amino acids, antibiotics, chelating agents, auxiliary agents, and preservatives. Buffers include phosphate buffer, Tris buffer, and HEPES buffer. Other vectors, such as nanoparticles, liposomes, and cationic lipid particles, may also be included. The solution may mimic the components of perilymph. The NaCl concentration is 20-200 mM, the KCl concentration is 1-5 mM, the CaCl2 concentration is 0.1-10 mM, the glucose concentration is 1-10 mM, the HEPES concentration is 2-50 mM, and the pH is 6-9. The final solution is sterile and may be dissolved in water, glycerin, ethanol, polyols, oils, etc. The solution may be in a ready-to-use form or may be diluted before use.

[0047] Administration Routes Used in the Present Invention: In the present invention, dual vectors or the final packaged adeno-associated viruses can be delivered to the ear by various methods, including through the round window (a circular foramen on the medial wall of the middle ear cavity, also known as the "cochlear window"), the round window oval (also known as the "oval window" or "vestibular window"; it refers to the oval foramen located posterior-superior to the medial wall of the middle ear cavity), the semicircular canals, and the utricle, with or without the use of auxiliary tools. Administration can be a single dose or multiple doses based on protein expression and hearing recovery. The dose can range from 1 to 200 μL, containing an AAV dose of 1×10 9 -1×10 13 For example, in a mouse, 1-2 μL (1 × 10 9 , 1×10 10 or 1 x 10 11 In humans, the AAV virus is administered in a volume of 10-100 μL (1 × 109 -1×10 13 The virus (containing one viral genome) may be administered to one ear or both ears, but in the present invention, administration to one ear is preferred, as it can ultimately achieve the effect of restoring hearing in both ears.

[0048] The present invention will be described in more detail below with reference to specific examples.

[0049] Example 1: Selection of Intein Cleavage Sites in OTOF (SEQ ID NO: 1 or 2) A division point was set in the OTOF amino acid sequence, and the nucleotide coding sequence from the N-terminus of the OTOF amino acid sequence to the division point was defined as the N-terminal coding sequence of OTOF. The nucleotide coding sequence from the position next to the division point to the C-terminus of the OTOF amino acid sequence was defined as the C-terminal coding sequence of OTOF. The N-terminus of OTOF was fused with the N-terminus of Intein, and the C-terminus of Intein was fused with the C-terminus of OTOF. There are several options for the division site of OTOF, some of which are shown in Table 3.

[0050] Table 3: Some division sites applicable to OTOF shown in SEQ ID NO: 1 or 2 JPEG2025530726000004.jpg205170JPEG2025530726000005.jpg255160JPEG2025530726000006.jpg255158JPEG2025530726000007.jpg51170

[0051] Example 2: Construction of the nucleotide sequences of the first segment / second segment using pAAV-CMV plasmid containing ITR sequences In the amino acid sequence of the OTOF protein shown in SEQ ID NO: 2, the 827th amino acid residue was used as the division point (S1), the 930th amino acid residue as the division point (S2), and the 1130th amino acid residue as the division point (S4), and NpuDnaE was used as the intein. Using the pAAV-CMV plasmid (Figure 1; sequence shown in SEQ ID NO: 9) as the vector, the nucleotide sequences of the first segment / second segment, i.e., a dual vector expressing the OTOF protein, was constructed. Specifically, the procedure is as follows.

[0052] The pAAV-CMV plasmid was digested with BstBI and HindIII, and the resulting plasmid fragments were recovered from agarose gel. Gene fragment 1 (encoding OTOF amino acids 1-827 and the N-terminus of the NpuDnaE intein), gene fragment 2 (encoding the C-terminus of the NpuDnaE intein and amino acids 828-1997 of the OTOF), gene fragment 3 (encoding OTOF amino acids 1-930 and the N-terminus of the NpuDnaE intein), gene fragment 4 (encoding the C-terminus of the NpuDnaE intein and amino acids 931-1997 of the OTOF), gene fragment 5 (encoding OTOF amino acids 1-1130 and the N-terminus of the NpuDnaE intein), and gene fragment 6 (encoding the C-terminus of the NpuDnaE intein and amino acids 1131-1997 of the OTOF) were synthesized by gene synthesis and digested with BstBI and HindIII. Furthermore, the enzymatically digested pAAV-CMV plasmid was ligated to each of the enzymatically digested gene fragments 1-6 using the T4 ligase system. After ligation, 2 μL of the ligation product was added to 50 μL of DH5α competent cells, incubated in an ice bath for 30 minutes, heat-stimulated for 1 minute, and then immediately placed on ice for 1 minute. 200 μL of LB liquid medium was added and incubated at 37°C for 30 minutes. 200 μL of the bacterial solution was then evenly spread onto ampicillin-resistant solid LB medium and incubated overnight at 37°C. Five monoclonal strains were selected and accurately sequenced. The following six plasmids were obtained and used in subsequent experiments. pAAV-CMV-OTOF-N-S1-Npu-N-intein plasmid (see Figure 2 for plasmid map) pAAV-CMV-Npu-C-intein-OTOF-C-S1 plasmid (see Figure 3 for plasmid map) pAAV-CMV-OTOF-N-S2-Npu-N-intein plasmid (see Figure 4 for plasmid map) pAAV-CMV-Npu-C-intein-OTOF-C-S2 plasmid (see Figure 5 for plasmid map) pAAV-CMV-OTOF-N-S4-Npu-N-intein plasmid (see Figure 6 for plasmid map) pAAV-CMV-Npu-C-intein-OTOF-C-S4 plasmid (see Figure 7 for plasmid map)

[0053] pAAV-CMV-OTOF-N-S1-Npu-N-intein,pAAV-CMV-Npu-C-intein-OTOF-C-S1; pAAV-CMV-OTOF-N-S2-Npu-N-intein,pAAV-CMV-Npu-C-intein-OTOF-C-S2; pAAV-CMV-OTOF-N-S4-Npu-N-intein,pAAV-CMV-Npu-C-intein-OTOF-C-S4; The transcripts of the plasmids all contain a covalently linked OTOF (part) and NpuDnaE Intein (part). The transcripts are shown in Table 4.

[0054] Table 4: Transcription and translation products of different N- and C-termini of OTOF fused to Npu JPEG2025530726000008.jpg36170

[0055] Example 3: Construction of the nucleotide sequences of the first segment / second segment using pAAV-CMV plasmid containing ITR sequences The nucleotide sequences of the first and second segments, i.e., a dual vector expressing the OTOF protein, were constructed using the pAAV-CMV plasmid (Figure 1; sequence shown in SEQ ID NO: 9) as a vector, with the amino acid residue at positions 827, 930, 954, and 1130 of the amino acid sequence of the OTOF protein shown in SEQ ID NO: 2 as division points (S1), S2, S3, and S4, respectively.

[0056] The pAAV-CMV plasmid was digested with BstBI and HindIII enzymes, and the plasmid fragment was recovered from an agarose gel. Gene fragment 7 (a gene encoding OTOF amino acids 1-827 and the N-terminus of RmaDnaB Intein as shown in SEQ ID NO:5 (further containing a partial sequence bound to the plasmid)), gene fragment 8 (a gene encoding the C-terminus of RmaDnaB Intein and OTOF amino acids 828-1997 as shown in SEQ ID NO:6 (further containing a partial sequence bound to the plasmid)), gene fragment 9 (a gene encoding OTOF amino acids 1-930 and the N-terminus of RmaDnaB Intein as shown in SEQ ID NO:7 (further containing a partial sequence bound to the plasmid)), gene fragment 10 (a gene encoding the C-terminus of RmaDnaB Intein and OTOF amino acids 931-1997 as shown in SEQ ID NO:8 (further containing a partial sequence bound to the plasmid)), gene fragment 11 (a gene encoding OTOF amino acids 1-954 and the N-terminus of RmaDnaB Intein), gene fragment 12 (a gene encoding the RmaDnaB Intein Fragment 1 (encoding the C-terminus of OTOF and amino acids 955-1997), gene fragment 13 (encoding the C-terminus of OTOF and amino acids 1-1130 of the RmaDnaB intein and the N-terminus of the RmaDnaB intein), and gene fragment 14 (encoding the C-terminus of RmaDnaB intein and amino acids 1131-1997 of the OTOF) were synthesized and digested with BstBI and HindIII. The digested pAAV-CMV plasmid was then ligated to the digested gene fragments 7-14 using the T4 ligase system. After ligation, 2 μL of the ligated product was added to 50 μL of DH5α competent cells and incubated in an ice bath for 30 minutes. After 1 minute of heat stimulation, the cells were immediately placed on ice for 1 minute. 200 μL of LB liquid medium was added and incubated at 37°C for 30 minutes. 200 μL of the bacterial suspension was then evenly plated onto solid LB medium with ampicillin resistance and incubated overnight at 37°C. Five monoclonal strains were selected and accurately sequenced. The following eight plasmids were obtained and used in the following examples: pAAV-CMV-OTOF-N-S1-Rma-N-intein plasmid (see Figure 8 for plasmid map) pAAV-CMV-Rma-C-intein-OTOF-C-S1 plasmid (see Figure 9 for plasmid map) pAAV-CMV-OTOF-N-S2-Rma-N-intein plasmid (see Figure 10 for plasmid map) pAAV-CMV-Rma-C-intein-OTOF-C-S2 plasmid (see Figure 11 for plasmid map) pAAV-CMV-OTOF-N-S3-Rma-N-intein plasmid (see Figure 12 for plasmid map) pAAV-CMV-Rma-C-intein-OTOF-C-S3 plasmid (see Figure 13 for plasmid map) pAAV-CMV-OTOF-N-S4-Rma-N-intein plasmid (see Figure 14 for plasmid map) pAAV-CMV-Rma-C-intein-OTOF-C-S4 plasmid (see Figure 15 for plasmid map) pAAV-CMV-OTOF-N-S1-Rma-N-intein,pAAV-CMV-Rma-C-intein-OTOF-C-S1 pAAV-CMV-OTOF-N-S2-Rma-N-intein,pAAV-CMV-Rma-C-intein-OTOF-C-S2 pAAV-CMV-OTOF-N-S3-Rma-N-intein,pAAV-CMV-Rma-C-intein-OTOF-C-S3 pAAV-CMV-OTOF-N-S4-Rma-N-intein,pAAV-CMV-Rma-C-intein-OTOF-C-S4 The plasmid transcripts all contain a covalently linked OTOF (part) and RmaDnaB Intein (part). The transcripts are shown in Table 5.

[0057] Table 5: Transcription and translation products of different N- and C-termini of OTOF fused to Rma JPEG2025530726000009.jpg45170

[0058] Example 4: Construction of different recombinant plasmids using OTOF shown in SEQ ID NO: 2 Using the same method as in Examples 2 and 3, a full-length plasmid expressing the OTOF protein: pAAV-CMV-OTOF-FL plasmid (containing a CMV promoter, OTOF isoform 5 (sequence number 4), and bGH PolyA; see Figure 16 for a map) was constructed.

[0059] A plasmid used for DNA recombination was constructed in the same manner as in Examples 2 and 3. pAAV-CMV-OTOF-N-AK plasmid (containing a CMV promoter, an OTOF 1-930 amino acid coding sequence, an SD sequence, and an AK sequence. See Figure 17 for a map), and pAAV-AK-OTOF-C-PloyA plasmid (containing an AK sequence, an SA sequence, an OTOF 931-1997 amino acid coding sequence, and a bGH polyA sequence. See Figure 18 for a map); pAAV-CMV-OTOF-N-AP plasmid (containing a CMV promoter, an OTOF 1-930 amino acid coding sequence, an SD sequence, and an AP sequence. See Figure 19 for a map), and pAAV-AP-OTOF-C-PloyA plasmid (containing an AP sequence, an SA sequence, an OTOF 931-1997 amino acid coding sequence, and a bGH polyA sequence. See Figure 20 for a map); pAAV-CMV-OTOF-N-TS plasmid (containing a CMV promoter, an OTOF 1-930 amino acid coding sequence, and an SD sequence; see Figure 21 for a map), and pAAV-TS-OTOF-C-PloyA plasmid (containing an SA sequence, an OTOF 931-1997 amino acid coding sequence, and a bGH polyA sequence; see Figure 22 for a map).

[0060] Here, AK and AP are sequences that undergo DNA recombination after the adeno-associated virus enters a cell. After DNA recombination, the SA-SD sequence cleaves the adeno-associated virus in trans to form a complete mRNA, which expresses a full-length, functional OTOF protein. The pAAV-CMV-OTOF-N-TS and pAAV-TS-OTOF-C-PolyA adeno-associated viruses undergo ITR recombination followed by trans cleavage by SA-SD to form a complete, mature mRNA, which expresses a full-length, functional OTOF protein.

[0061] Example 5: Preparation of adeno-associated virus The eight plasmids constructed by the method of the present invention in Example 3 and the six plasmids constructed in Example 4 were co-transfected into HEK-293T cells with the pHelper plasmid and the PHP.eB pRC plasmid at a molar ratio of 1:1:1 using a PEI transfection reagent (approximately 1 μg of plasmid was added per million cells). After culturing in a 10% fetal bovine serum-containing DMEM medium in an incubator at 37°C and 5% carbon dioxide for 3 days, the cells were washed once with PBS buffer, collected, and then freeze-thawed five times. Solid NaCl was added to a final concentration of 500 mM. The cells were then centrifuged at 10,000 g for 0.5 hours. The supernatant was filtered through a 0.45 μm filter membrane and purified by the iodixanol method. A portion of the sample was concentrated to obtain a virus titer of 1±0.2×10 13 The resulting adeno-associated virus had a viral genome count per ml, a solvent of 0.01% poloxamer PBS buffer, and an empty shell ratio of approximately 50%. The specific experimental system is shown in Table 6 below. Hereinafter, viruses will be named by their plasmid names. For example, the adenovirus packaged with pAAV-CMV-OTOF-N-S1-Rma-N-intein, pHelper plasmid, and PHP.eB (number 1) is named pAAV-CMV-OTOF-N-S1-Rma-N-intein adeno-associated virus.

[0062] Preparation of 0.01% poloxamer in PBS buffer (1) Preparation of PBS buffer: 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, the remainder being water. (2) Poloxamer F68 was added to the PBS buffer solution prepared in Preparation (1) in a mass-to-volume ratio to a final concentration of 0.01%. The 0.01% poloxamer PBS buffer solution according to the present invention was prepared by this method.

[0063] JPEG2025530726000010.jpg83170

[0064] Example 6: Intracellular recombination of the plasmids constructed in Examples 2 and 3 (plasmids 1:1) The seven pairs of plasmids constructed in Examples 2 and 3 and pAAV-CMV-OTOF-FL constructed in Example 4 were transfected into HEK-293T cells cultured in a 6-well plate (the number of cells in each well was approximately 1 × 10 62 μg of plasmid was mixed with 100 μL of Opti-MEM to obtain a plasmid premix. 4 μL of PEI was mixed with 100 μL of Opti-MEM to obtain a PEI premix. The plasmid premix and PEI premix were mixed, left to stand for 10 minutes, and then added dropwise to cultured HEK-293T cells. After 48 hours of culture, the cell medium was aspirated and 200 μL of cell lysis solution (1% Triton-X 100, 50 mM Tris-HCl, 1 mM PMSF, pH 7.4) was added. The cells were then left on ice for 10 minutes. The cell lysate was collected and centrifuged at 12,000 g for 10 minutes at 4°C, and the supernatant was retained. A quarter volume of 5x loading buffer (0.25M Tris-HCl, 10% SDS, 0.05% bromophenol blue, 50% glycerol, 0.25M DTT) was added to the supernatant and heated at 90°C for 10 minutes to obtain protein samples. A 50µL sample was then subjected to polyacrylamide gel electrophoresis. After transfer to a PVDF membrane, the sample was incubated with OTOF N-terminal antibody (Part Number A20266, abclonal) or C-terminal antibody (Part Number PA5-52935, Invitrogen) at room temperature for 2 hours, followed by incubation with the corresponding HRP-conjugated secondary antibody for 1 hour. ECL reagent was added and imaging was performed. The results are shown in Figure 23. NpuS1, NpuS2, RmaS1, RmaS2, and RmaS4 all exhibited relatively high recombination efficiencies. Among these, NpuS2 and RmaS2 showed the highest efficiencies.

[0065] The doses of transfection plasmids are shown in Table 7.

[0066] JPEG2025530726000011.jpg86170

[0067] Example 7: Recombination of S2 plasmids in cells at a ratio of 1:2, etc. The pAAV-CMV-OTOF-N-S2-Rma-N-intein plasmid and pAAV-CMV-Rma-C-intein-OTOF-C-S2 plasmid were transfected into HEK-293T cells cultured in a 6-well plate with 2 μg of pAAV-CMV-OTOF-FL at a mass ratio of 1:1 (1 μg, 1 μg), 1:2 (0.65 μg, 1.35 μg), 1:3 (0.5 μg, 1.5 μg), and 1:4 (0.4 μg, 1.6 μg), respectively (approximately 1 × 10 cells per well). 6 The total number of cells was 1. 2 μg of plasmid was added to 100 μL of Opti-MEM and mixed to obtain a plasmid premix. 4 μL of PEI was added to 100 μL of Opti-MEM and mixed to obtain a PEI premix. The plasmid premix and PEI premix were mixed, left to stand for 10 minutes, and then added dropwise to the cultured HEK-293T cells. After 48 hours of culture, the cell medium was aspirated and 200 μL of cell lysis solution (1% Triton-X 100, 50 mM Tris-HCl, 1 mM PMSF, pH 7.4) was added. The cells were then left on ice for 10 minutes. The cell lysate was then collected and centrifuged at 12,000 g for 10 minutes at 4°C, and the supernatant was retained. A quarter volume of 5x loading buffer (0.25 M Tris-HCl, 10% SDS, 0.05% bromophenol blue, 50% glycerol, 0.25 M DTT) was added to the supernatant and heated at 90°C for 10 minutes to obtain protein samples. A 50 μL sample was then subjected to polyacrylamide gel electrophoresis and transferred to a PVDF membrane. The membrane was then incubated with an N-terminal antibody (A20266, abclonal) or a C-terminal antibody (PA5-52935, Invitrogen) against OTOF for 2 hours at room temperature, followed by incubation with the corresponding HRP-conjugated secondary antibody for 1 hour. ECL reagent was added and imaging was performed. The results are shown in Figure 24. For RmaS2, transfection efficiency was found to be highest when the N:C ratio was 1:2.

[0068] Example 8: Recombination of S2 plasmids in cells at different ratios such as 1:2.5 The pAAV-CMV-OTOF-N-S2-Rma-N-intein plasmid and pAAV-CMV-Rma-C-intein-OTOF-C-S2 plasmid were transfected into HEK-293T cells cultured in 6-well plates with 2 μg of pAAV-CMV-OTOF-FL at a mass ratio of 1:2 (0.65 μg, 1.35 μg), 1:2.5 (0.55 μg, 1.45 μg), 1:3 (0.5 μg, 1.5 μg), and 1:3.5 (0.4 μg, 1.6 μg). The number of cells in each well was approximately 1 × 10 6 The total number of cells was 100. Two μg of plasmid was added to 100 μL of Opti-MEM and mixed to obtain a plasmid premix. Four μL of PEI was added to 100 μL of Opti-MEM and mixed to obtain a PEI premix. The plasmid premix and PEI premix were mixed, left to stand for 10 minutes, and then added dropwise to the cultured HEK-293T cells. After 48 hours of culture, the cell medium was aspirated and 200 μL of cell lysis solution (1% Triton-X 100, 50 mM Tris-HCl, 1 mM PMSF, pH 7.4) was added. The cells were then left on ice for 10 minutes. The cell lysate was then collected and centrifuged at 12,000 xg for 10 minutes at 4°C, and the supernatant was retained. A quarter volume of 5x loading buffer (0.25M Tris-HCl, 10% SDS, 0.05% bromophenol blue, 50% glycerol, 0.25M DTT) was added to the supernatant and heated at 90°C for 10 minutes to obtain protein samples. A 50µL sample was then subjected to polyacrylamide gel electrophoresis. After transfer to a PVDF membrane, the sample was incubated with either the N-terminal (A20266, abclonal) or C-terminal antibody (PA5-52935, Invitrogen) of OTOF at room temperature for 2 hours, followed by incubation with the corresponding HRP-conjugated secondary antibody for 1 hour. ECL reagent was added and imaging was performed. The results are shown in Figure 25. The highest transfection efficiency was observed when the RmaS2 ratio was adjusted to N:C = 1:2.5.

[0069] Example 9: Recombination of S1 plasmids in cells at a ratio of 1:2, etc. The pAAV-CMV-OTOF-N-S1-Rma-N-intein plasmid and pAAV-CMV-Rma-C-intein-OTOF-C-S1 plasmid were transfected into HEK-293T cells cultured in 6-well plates with 2 μg of pAAV-CMV-OTOF-FL at mass ratios of 1:1 (1 μg, 1 μg), 1:2 (0.65 μg, 1.35 μg), 1:3 (0.5 μg, 1.5 μg), and 1:4 (0.4 μg, 1.6 μg), respectively (approximately 1 × 10 cells per well). 6 The N-terminal and C-terminal plasmids were added to 100 μL of Opti-MEM and mixed in the appropriate ratio to obtain a plasmid premix. 4 μL of PEI was added to 100 μL of Opti-MEM and mixed to obtain a PEI premix. The plasmid premix and PEI premix were mixed, left to stand for 10 minutes, and then added dropwise to cultured HEK-293T cells. After 48 hours of culture, the cell medium was aspirated and 200 μL of cell lysis solution (1% Triton-X 100, 50 mM Tris-HCl, 1 mM PMSF, pH 7.4) was added. The cells were then left on ice for 10 minutes. The cell lysate was collected and centrifuged at 12,000 xg for 10 minutes at 4°C, and the supernatant was retained. A quarter volume of 5x loading buffer (0.25 M Tris-HCl, 10% SDS, 0.05% bromophenol blue, 50% glycerol, 0.25 M DTT) was added to the supernatant and heated at 90°C for 10 minutes to obtain protein samples. A 50 μL sample was then subjected to polyacrylamide gel electrophoresis and transferred to a PVDF membrane. After incubation with OTOF N-terminal (A20266, abclonal) or C-terminal antibody (PA5-52935, Invitrogen) at room temperature for 2 hours, the corresponding HRP-conjugated secondary antibody was incubated for 1 hour. ECL reagent was added and imaging was performed. The results are shown in Figure 26. For RmaS1, optimizing the N-terminal and C-terminal transfection ratio revealed that transfection at a ratio of N:C = 1:2 yielded the highest transfection efficiency.

[0070] Example 10: Transfection efficiency at different incubation times The pAAV-CMV-OTOF-N-S2-Rma-N-intein plasmid and the pAAV-CMV-Rma-C-intein-OTOF-C-S2 plasmid were transfected into HEK-293T cells cultured in a 6-well plate at a mass ratio of 1:2.5 (0.55 μg, 1.45 μg) (the number of cells in each well was approximately 1 × 10 6 The results were as follows: 2 μg of plasmid was added to 100 μL of Opti-MEM and mixed to obtain a plasmid premix. 4 μL of PEI was added to 100 μL of Opti-MEM and mixed to obtain a PEI premix. The plasmid premix and PEI premix were mixed, left to stand for 10 minutes, and then added dropwise to cultured HEK-293T cells. After culturing for 2, 4, 6, 8, 12, 24, 48, and 72 hours, the cell medium was aspirated and 200 μL of cell lysis solution (1% Triton-X 100, 50 mM Tris-HCl, 1 mM PMSF, pH 7.4) was added. The cells were then left on ice for 10 minutes. The cell lysates were collected and centrifuged at 12,000×g for 10 minutes at 4°C, and the supernatants were retained. A quarter volume of 5x loading buffer (0.25M Tris-HCl, 10% SDS, 0.05% bromophenol blue, 50% glycerol, 0.25M DTT) was added to the supernatant and heated at 90°C for 10 minutes to obtain protein samples. A 50µL sample was then subjected to polyacrylamide gel electrophoresis and transferred to a PVDF membrane. The membrane was then incubated with an N-terminal antibody (A20266, abclonal) or a C-terminal antibody (PA5-52935, Invitrogen) against OTOF at room temperature for 2 hours, followed by incubation with the corresponding HRP-conjugated secondary antibody for 1 hour. ECL reagent was added and imaging was performed. The results are shown in Figure 27. The recombination rate of OTOF using the intein method was very fast, and the ratio of fragment to full-length OTOF was nearly the same at different time points.

[0071] Example 11: Comparison of OTOF Intein Recombination and OTOF DNA Recombination Two aliquots of the virus packaged in Example 5 were added to 400 μL of serum-free DMEM medium (as shown in Table 8), mixed evenly, and then inoculated into HEK-293T cells (approximately 1 × 106 The cells were added to the tubes (the medium was aspirated and removed before adding the virus) and incubated for 4 hours, after which 1.6 mL of 10% fetal bovine serum-containing DMEM medium was added. After 2 days of culture, the cell medium was aspirated and 200 μL of cell lysis solution (1% Triton-X 100, 50 mM Tris-HCl, 1 mM PMSF, pH = 7.4) was added. The cells were then left on ice for 10 minutes, after which the cell lysate was collected and centrifuged at 12,000 x g for 10 minutes at 4 °C. The supernatant was then retained. A quarter volume of 5x loading buffer (0.25 M Tris-HCl, 10% SDS, 0.05% bromophenol blue, 50% glycerol, 0.25 M DTT) was added to the supernatant and heated at 90 °C for 10 minutes to obtain a protein sample. A 50 μL protein sample was taken and subjected to polyacrylamide gel electrophoresis. After transfer to a PVDF membrane, the sample was incubated with OTOF antibody (product number A20266, abclonal) diluted 1:3000 and then incubated with the corresponding HRP-conjugated secondary antibody for 1 hour. ECL reagent was added and imaging was performed. The experiment was performed three times, and grayscale statistics were performed on the full-length band. The results are shown in Figure 28. The recombination efficiency of the protein recombined with the OTOF Intein used in this invention was found to be significantly higher than that of OTOF DNA recombination.

[0072] Table 8: Protein and nucleic acid recombination systems JPEG2025530726000012.jpg74170

[0073] Example 12: Construction of Otof gene-deficient mice Otof - / - The genetically modified mice were constructed using the CRISPR / Cas9 method based on the 129S2 / SvPasCrl mouse strain. - / - The model mice carry the Otof gene NM_001100395.1:c.2503_2504insA, which causes a homozygous frameshift mutation in the Otof gene. The results are shown in Figure 29. - / -The mutant mice have a single base insertion, which causes a frameshift in the reading frame. - / - The hearing of mutant and wild-type mice was compared using ABR, and the mutant mice showed complete hearing loss in both ears.

[0074] Example 13: Unilateral administration of S1 Intein to young mice and its effects The plasmids pAAV-CMV-OTOF-N-S1-Rma-N-intein and pAAV-CMV-Rma-C-intein-OTOF-C-S1 were co-transfected with the pHelper plasmid and the PHP.eB pRC plasmid at a molar ratio of 1:1:1 into HEK-293T cells (approximately 1 μg of plasmid was added per million cells) using a PEI transfection reagent. The cells were cultured in a 10% fetal bovine serum-containing DMEM medium at 37°C in a 5% carbon dioxide incubator for 3 days. The cells were washed once with PBS buffer, harvested, and then freeze-thawed five times. Solid NaCl was added to a final concentration of 500 mM, and the cells were centrifuged at 10,000 g for 0.5 hours. The supernatant was filtered through a 0.45 μm filter membrane. A gradient iodixanol solution was prepared and added to a centrifuge tube (5 mL 60% iodixanol, 5 mL The sample was added to the top layer and centrifuged at 350,000 g for 1 hour. The virus layer at the 40% and 60% interfaces was aspirated. The tube was centrifuged at 10,000 g using a 50 Kda ultrafiltration tube, and the solution was exchanged five times with 0.01% poloxamer PBS buffer solution. The virus titer was measured by qPCR, and the titer was adjusted to a final viral titer of 1 ± 0.2 × 10. 13 pAAV-CMV-OTOF-N-S1-Rma-N-intein PHP.eB AAV and pAAV-CMV-Rma-C-intein-OTOF-C-S1 PHP.eB AAV were obtained at a viral genome count / ml in 0.01% poloxamer PBS buffer solution.

[0075] The adeno-associated virus constructed above was used as the P0 / P1 Otof - / - The mutant mice were administered 2 × 10 β-glucan-1-phosphate dehydrogenase (G1)-10 β-glucan via round window injection into the right cochlea. 10 The number of viral genomes (1 × 10 for each of the two types of AAV) 10 A single dose of 1000 viral genomes was administered to the mice, and the ABR index of the mice was detected to confirm the state of hearing recovery. The results after one month are shown in Figure 31. In the figure, the circle marks indicate the non-administered group (n = 8), the square marks indicate the contralateral ear (n = 10) of the administered ear, the triangle marks indicate the administered ear (n = 10), and the diagonal square marks indicate the wild-type animal group (n = 20). The results after two months are shown in Figure 32. In the figure, the circle marks indicate the non-administered group (n = 8), the square marks indicate the contralateral ear (n = 3), the triangle marks indicate the administered ear (n = 3), and the diagonal square marks indicate the wild-type animal group (n = 11). As can be seen from the results, Otof was successfully administered using the AAV constructed in the present invention. - / - After administration into one cochlea of ​​a model mouse, hearing was not only restored in the injected ear, but also in the contralateral uninjected ear.

[0076] Example 14: Unilateral administration of S2 Intein to young mice and its effects The plasmids pAAV-CMV-OTOF-N-S2-Rma-N-intein and pAAV-CMV-Rma-C-intein-OTOF-C-S2 were co-transfected into HEK-293T cells using a PEI transfection reagent at a molar ratio of 1:1:1 with the pHelper plasmid and the PHP.eB pRC plasmid (approximately 1 μg of plasmid was added per million cells). The cells were cultured in a 10% fetal bovine serum-containing DMEM medium at 37°C in a 5% carbon dioxide incubator for 3 days. The cells were washed once with PBS buffer, harvested, and then freeze-thawed five times. Solid NaCl was added to a final concentration of 500 mM. The cells were then centrifuged at 10,000 g for 0.5 hours. The supernatant was filtered through a 0.45 μm filter membrane. A gradient iodixanol solution was prepared and added to a centrifuge tube (5 mL 60% iodixanol, 5 mL The sample was added to the top layer and centrifuged at 350,000 g for 1 hour. The virus layer at the 40% and 60% interfaces was aspirated. The sample was centrifuged at 10,000 g using a 50 Kda ultrafiltration tube. The solution was exchanged five times with 0.01% poloxamer PBS buffer solution. The virus titer was measured by qPCR, and the titer was adjusted until the final virus titer was 1 ± 0.2 × 10. 13 The resulting adeno-associated viruses, pAAV-CMV-OTOF-N-S2-Rma-N-intein PHP.eB AAV and pAAV-CMV-Rma-C-intein-OTOF-C-S2 PHP.eB AAV, had a viral genome count of 1000 / ml and an empty shell ratio of approximately 50%. The solvent was a 0.01% poloxamer PBS buffer solution.

[0077] The adeno-associated virus constructed above was used as the P0 / P1 Otof - / - The mutant mice were administered 2 × 10 β-glucan-1-phosphate dehydrogenase (G1)-10 β-glucan via round window injection into the right cochlea. 10 The number of viral genomes (1 × 10 for each of the two types of AAV) 10The mice were administered 1000 ribosomal RNA (1000 ribosomal RNA) containing 1000 ribosomal RNA (1000 ribosomal RNA) and the mouse ABR index was detected to confirm the hearing recovery status of the mice. The results after one month are shown in Figure 33. In the figure, the circle marks represent the non-administered group (n = 10), the square marks represent the contralateral ear (n = 33) of the administered virus, the triangle marks represent the administered ear (n = 33), and the diagonal square marks represent the wild-type animal group (n = 20). The results after two months are shown in Figure 34. In the figure, the circle marks represent the non-administered group (n = 8), the square marks represent the contralateral ear (n = 27) of the administered virus, the triangle marks represent the administered ear (n = 27), and the diagonal square marks represent the wild-type animal group (n = 11). As can be seen from the results, Otof was successfully transduced using the AAV constructed in the present invention. - / - After administration to one cochlea of ​​the mutant mice, hearing was not only restored in the injected ear (triangle mark), but also in the contralateral uninjected ear (square mark).

[0078] Example 15: Safety observation of administration groups The AAV constructed in Examples 13 and 14 of the present invention was administered to 6-8 week-old wild-type CD-1 mice via round window injection into the cochleae. Observations of the mice's daily activities, hair smoothness, and dietary habits over the three months following administration revealed no significant differences compared with a control group that did not receive AAV injection, and there was no difference in hearing ability between the treatment and control groups. The mouse cochleae were dissected on days 28 and 91, and immunofluorescent staining of inner ear hair cells revealed no significant differences in hair cell number between the treatment and control groups.

[0079] Example 16: Unilateral administration of low-dose drugs and their effects The plasmids pAAV-CMV-OTOF-N-S2-Rma-N-intein and pAAV-CMV-Rma-C-intein-OTOF-C-S2 were co-transfected into HEK-293T cells using a PEI transfection reagent at a molar ratio of 1:1:1 with the pHelper plasmid and the PHP.eB pRC plasmid (approximately 1 μg of plasmid was added per million cells). The cells were cultured in a 10% fetal bovine serum-containing DMEM medium at 37°C in a 5% carbon dioxide incubator for 3 days. The cells were washed once with PBS buffer, harvested, and then freeze-thawed five times. Solid NaCl was added to a final concentration of 500 mM. The cells were then centrifuged at 10,000 g for 0.5 hours. The supernatant was filtered through a 0.45 μm filter membrane. A gradient iodixanol solution was prepared and added to a centrifuge tube (5 mL 60% iodixanol, 5 mL The sample was added to the top layer and centrifuged at 350,000 g for 1 hour. The virus layer at the 40% and 60% interfaces was aspirated. The sample was centrifuged at 10,000 g using a 50 Kda ultrafiltration tube. The solution was exchanged five times with 0.01% poloxamer PBS buffer solution. The virus titer was measured by qPCR, and the titer was adjusted until the final virus titer was 1 ± 0.2 × 10. 13 pAAV-CMV-OTOF-N-S2-Rma-N-intein PHP.eB AAV and pAAV-CMV-Rma-C-intein-OTOF-C-S2 PHP.eB AAV were obtained at viral genome counts / ml in a 0.01% poloxamer PBS buffer solution.

[0080] The adeno-associated virus constructed above was used as the Otof virus constructed in Example 12. - / - The mutant mice were administered 5 × 10 round window injections into the right cochlea. 9 The number of viral genomes (2.5 × 10 for each of the two AAVs) 9The mice were administered a single dose of 1000 ribosomal RNA (1000 ribosomal RNA; ... - / - After administration into one cochlea of ​​a model mouse, hearing was not only restored in the injected ear, but also in the contralateral uninjected ear.

[0081] Example 17: In vivo protein immunofluorescence detection after administration One month after administration of Example 14, mice were euthanized, and cochlear tissue was dissected and immersed in 4% paraformaldehyde overnight at 4°C. It was then decalcified in 10% EDTA solution for 3 days. Prior to staining, the tissue was incubated in 0.3% Triton X-100 PBS buffer for 10 minutes and blocked with 10% donkey blood at room temperature for 1 hour. The tissue was then incubated overnight at 4°C with OTOF antibody PA5-52935 (C-terminus) or ab53233 (N-terminus) and washed three times with 0.1% Triton X-100 PBS (10 minutes each time). The tissue was then incubated with the corresponding fluorescent secondary antibody and DAPI for 1 hour, followed by three washes with 0.1% Triton X-100 PBS (10 minutes each time). Finally, the tissue was imaged using a confocal fluorescence microscope. The results are as follows:

[0082] Figure 36 shows wild-type mice and Otof - / - This is a diagram of the expression of OTOF in the cochlea of ​​the mutant mice. As can be seen from the figure, the single row of DAPI-stained cells is the cochlea hair cells. In wild-type mice, OTOF was clearly expressed in the cochlea hair cells, but Otof - / - In the mutant mice, OTOF was completely absent.

[0083] Figure 37 shows the expression status of OTOF in the injected ear and the contralateral ear. - / - The left panel shows the whole mount of the cochlea, and the right panel shows a local enlargement of the left panel. - / -The figure shows the expression of OTOF protein in the contralateral ear of mice treated with OTOF. The Apex, Middle, and Base regions in the figure represent different regions of the cochlear hair cells. As can be seen from the figure, OTOF was expressed in most of the cochlear hair cells in the treated ear, and OTOF was also significantly expressed in the contralateral ear.

[0084] Figure 38 shows the Otof - / - Statistical graphs of OTOF expression in mice. Figure 38A shows the statistical status of OTOF-expressing cochlear hair cells in the injected ear, indicating that the number of cochlear hair cells expressing OTOF protein exceeded 60%. Figure 38B shows the statistical status of OTOF-expressing cochlear hair cells in the contralateral injected ear, indicating that the number of cochlear hair cells expressing OTOF protein exceeded 40%. The apical turn, middle turn, and basal turn in the graph indicate different regions of the cochlea. The ordinate indicates the infection rate of cochlear hair cells.

[0085] Example 18: Unilateral administration to 4-week-old mice and its effects The plasmids pAAV-CMV-OTOF-N-S2-Rma-N-intein and pAAV-CMV-Rma-C-intein-OTOF-C-S2 were co-transfected into HEK-293T cells using a PEI transfection reagent at a molar ratio of 1:1:1 with the pHelper plasmid and the AAV1 pRC plasmid (approximately 1 μg of plasmid was added per million cells). After culturing for 3 days in a 5% carbon dioxide incubator at 37°C using DMEM medium containing 10% fetal bovine serum, the cells were washed once with PBS buffer, harvested, and then freeze-thawed five times. Solid NaCl was added to a final concentration of 500 mM, and the cells were centrifuged at 10,000 g for 0.5 hours. The supernatant was removed and filtered through a 0.45 μm filter membrane. A gradient iodixanol solution was prepared and added to a centrifuge tube (5 mL 60% iodixanol, 5 mL The sample was added to the top layer and centrifuged at 350,000 g for 1 hour. The virus layer at the 40% and 60% interfaces was aspirated. The sample was centrifuged at 10,000 g using a 50 Kda ultrafiltration tube. The solution was exchanged five times with 0.01% poloxamer PBS buffer solution. The virus titer was measured by qPCR, and the titer was adjusted until the final virus titer was 1 ± 0.2 × 10. 13 The adeno-associated viruses obtained were pAAV-CMV-OTOF-N-S2-Rma-N-intein AAV1 and pAAV-CMV-Rma-C-intein-OTOF-C-S2 AAV1, with a viral genome count of 100 / ml and an empty shell rate of approximately 50%. The solvent was a 0.01% poloxamer PBS buffer solution.

[0086] The adeno-associated virus constructed above was used as the Otof virus constructed in Example 12. - / - The transgenic mice (4 weeks old) were administered 2 × 10 β-glucan monophosphate (GMO) by posterior semicircular canal injection into the right cochlea. 10 The number of viral genomes (1 × 10 for each of the two types of AAV) 10The mice were administered a total of 10 viral genomes (each containing 10 viral genomes), and the mouse ABR index was detected to confirm the hearing recovery status of the mice. The results after one month are shown in Figure 39. In the figure, the circle marks represent the non-administered group (n = 8), the square marks represent the contralateral ear (n = 6) that received the administration, the triangle marks represent the administered ear (n = 6), and the diagonal square marks represent the wild-type animal group (n = 8). As can be seen from the results, the adeno-associated virus obtained by packaging in the present invention is highly effective in restoring hearing in adult mice, and is capable of restoring hearing in both ears with just one ear injection.

[0087] The above examples only describe some embodiments of the present invention, and although the descriptions are relatively specific and detailed, they do not limit the scope of the present invention. It should be noted that those skilled in the art may make some modifications, combinations, and improvements to each of the above embodiments without departing from the spirit of the present invention, and all of them fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A dual vector system expressing an OTOF protein, comprising: The dual vector system comprises two segments of nucleotide sequences, the nucleotide sequence of the first segment comprises two ITR sequences and an expression cassette inserted between the ITR sequences; the nucleotide sequence of the second segment comprises two ITR sequences and an expression cassette inserted between the ITR sequences; The expression cassette of the nucleotide sequence of the first segment comprises a promoter, an N-terminal coding sequence of OTOF, an N-terminal coding sequence of Intein, and PolyA; The expression cassette of the nucleotide sequence of the second segment comprises a promoter, a C-terminal coding sequence of Intein, a C-terminal coding sequence of OTOF, and PolyA; A dual vector system characterized in that a division point is provided in the OTOF amino acid sequence, the nucleotide coding sequence from the N-terminus of the OTOF amino acid sequence to the division point is the N-terminal coding sequence of OTOF, and the nucleotide coding sequence from the amino acid at the position next to the division point to the C-terminus of the OTOF amino acid sequence is the C-terminal coding sequence of OTOF.

2. The dual vector system according to claim 1, wherein the amino acid sequence of OTOF is set forth in SEQ ID NO: 1 or SEQ ID NO: 2 in the sequence listing.

3. The dual vector system of claim 1, wherein the OTOF division site includes, but is not limited to, the amino acid immediately preceding serine, threonine, or cysteine ​​in the OTOF protein amino acid sequence.

4. The promoter includes, but is not limited to, a CAG promoter, a CMV promoter, a CBA promoter, a UbC promoter, a SFFV promoter, an EF1α promoter, a PGK promoter, or a promoter encoding a gene such as Myo7A, Myo15, Atoh1, POU4F3, Lhx3, Myo6, α9AchR, α10AchR, or OTOF; the PolyA includes AATAAA and variants thereof, including, but not limited to, ATTAAA, AGTAAA, CATAAA, TATAAA, GATAAA, ACTAAA, AATATA, AAGAAA, AATAAT, AAAAAAA, AATGAA, AATCAA, AACAAA, AATCAA, AATAAC, AATAGA, AATTAA or AATAAG; 2. The dual vector system of claim 1, wherein the ITR sequences are derived from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9.

5. The dual vector system according to claim 1 , wherein the expression cassette further comprises an expression regulatory element or a label element.

6. The dual vector system according to claim 1, wherein the Intein is selected from the Intein sequences of MxeGyrA, pabPolIII, MjaKlbA, SspDnaB, SceVMA, SspDnaE, ​​NpuDnaE, ​​AvaDnaE, ​​CraDnaE, ​​CspDnaE, ​​CwaDnaE, ​​MchtDnaE, ​​OliDnaE, ​​TerDnaE, ​​gp41-1, gp41-8, IMPDH-1, and RmaDnaB.

7. the nucleotide sequence of the first segment is located on a plasmid containing an ITR, and an expression cassette of the nucleotide sequence of the first segment is inserted between the ITR sequences; The dual vector system of claim 1, wherein the nucleotide sequence of the second segment is located on a plasmid containing an ITR, and an expression cassette of the nucleotide sequence of the second segment is inserted between the ITR sequences.

8. 2. The dual vector system according to claim 1, wherein the plasmid containing ITR is pAAV, pAAV-CMV, pX601, pX551, or pAAV-MCS plasmid.

9. Using the 827th amino acid of OTOF shown in SEQ ID NO: 2 as a division point, NpuDnaE Intein is used, the N-terminal coding sequence of OTOF is fused to the N-terminal coding sequence of NpuDnaE Intein, and then the nucleotide sequence of the first segment is constructed using a pAAV-CMV plasmid as a vector; and the C-terminal coding sequence of NpuDnaE Intein is fused to the C-terminal coding sequence of OTOF, and then the nucleotide sequence of the second segment is constructed using a pAAV-CMV plasmid as a vector; or Using the 930th amino acid of OTOF shown in SEQ ID NO: 2 as a division point, NpuDnaE Intein is used, the N-terminal coding sequence of OTOF is fused to the N-terminal coding sequence of NpuDnaE Intein, and then the nucleotide sequence of the first segment is constructed using a pAAV-CMV plasmid as a vector; and the C-terminal coding sequence of NpuDnaE Intein is fused to the C-terminal coding sequence of OTOF, and then the nucleotide sequence of the second segment is constructed using a pAAV-CMV plasmid as a vector; or Using the amino acid at position 1130 of OTOF as set forth in SEQ ID NO: 2 as a division point, NpuDnaE Intein is used, the N-terminal coding sequence of OTOF is fused to the N-terminal coding sequence of NpuDnaE Intein, and then the nucleotide sequence of a first segment is constructed using a pAAV-CMV plasmid as a vector; and the C-terminal coding sequence of NpuDnaE Intein is fused to the C-terminal coding sequence of OTOF, and then the nucleotide sequence of a second segment is constructed using a pAAV-CMV plasmid as a vector; or Using the 827th amino acid of OTOF shown in SEQ ID NO: 2 as a division point, RmaDnaB Intein is used, and the N-terminal coding sequence of OTOF is fused to the N-terminal coding sequence of RmaDnaB Intein, and then the nucleotide sequence of the first segment is constructed using a pAAV-CMV plasmid as a vector; and the C-terminal coding sequence of RmaDnaB Intein is fused to the C-terminal coding sequence of OTOF, and then the nucleotide sequence of the second segment is constructed using a pAAV-CMV plasmid as a vector; or Using the 930th amino acid of OTOF shown in SEQ ID NO: 2 as a division point, RmaDnaB Intein is used, the N-terminal coding sequence of OTOF is fused to the N-terminal coding sequence of RmaDnaB Intein, and then the nucleotide sequence of the first segment is constructed using a pAAV-CMV plasmid as a vector; the C-terminal coding sequence of RmaDnaB Intein is fused to the C-terminal coding sequence of OTOF, and then the nucleotide sequence of the second segment is constructed using a pAAV-CMV plasmid as a vector; or Using the 954th amino acid of OTOF shown in SEQ ID NO: 2 as a division point, RmaDnaB Intein is used, and the N-terminal coding sequence of OTOF is fused to the N-terminal coding sequence of RmaDnaB Intein, and then the nucleotide sequence of the first segment is constructed using a pAAV-CMV plasmid as a vector; and the C-terminal coding sequence of RmaDnaB Intein is fused to the C-terminal coding sequence of OTOF, and then the nucleotide sequence of the second segment is constructed using a pAAV-CMV plasmid as a vector; or The dual vector system of claim 1, characterized in that the amino acid at position 1130 of OTOF shown in SEQ ID NO: 2 is used as a division point, and the N-terminal coding sequence of OTOF is fused to the N-terminal coding sequence of RmaDnaB Intein using RmaDnaB Intein, and the nucleotide sequence of the first segment is constructed using a pAAV-CMV plasmid as a vector, and the C-terminal coding sequence of RmaDnaB Intein is fused to the C-terminal coding sequence of OTOF, and the nucleotide sequence of the second segment is constructed using a pAAV-CMV plasmid as a vector.

10. 10. An adeno-associated virus packaging vector system comprising: the dual vector system of claim 1; a vector carrying AAV rep and cap genes; and a helper virus vector, wherein the vector becomes an AAV vector upon packaging.

11. 11. The adeno-associated virus packaging vector system of claim 10, wherein the vector carrying the AAV rep and cap genes is selected from the group consisting of AAV1, AAV2, AAV5, AAV8, AAV9, Anc80, PHP.eB, AAV-DJ, and AAVrh.10 vectors, and the helper virus vector is a pHelper plasmid.

12. A method for packaging an adeno-associated virus, comprising transfecting the adeno-associated virus packaging vector system according to claim 10 into a host cell and packaging the virus.

13. 13. The method for packaging an adeno-associated virus according to claim 12, wherein the host cells are selected from Hela-S3, HEK-293, HEK-293T, HEK-293FT, A549, and Sf9 cells.

14. An adeno-associated virus obtained by packaging using the method of claim 12.

15. Use of a dual vector system expressing the OTOF protein of claim 1 or the adeno-associated virus of claim 14 in the preparation of a drug or formulation for treating hearing loss diseases, hearing impairment or hearing dysfunction.

16. A drug or formulation for treating hearing loss diseases, hearing impairments or hearing dysfunction, prepared using a dual vector system expressing the OTOF protein described in claim 1 or the adeno-associated virus described in claim 14.

17. 17. The drug or formulation of claim 16, further comprising one or more of the following: a neutral salt buffer, an acidic salt buffer, an alkaline salt buffer, glucose, mannose, mannitol, proteins, polypeptides and amino acids, antibiotics, chelating agents, adjuvants, preservatives, nanoparticles, liposomes or positive lipid particles.

18. It is administered by injection, and the injection routes include round window injection in the cochlea, round window injection in the ovary, semicircular canal injection, and utricle injection; It is administered once or multiple times throughout a lifetime, with a total dose of 1 x 10 ９ -1 x 10 １３ The drug or formulation according to claim 16, characterized in that it is a viral genome.

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