Dual-vector systems and their use for treating hearing impairment

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] This invention belongs to the field of gene therapy in the medical field, and in particular relates to a use in restoring hereditary hearing loss caused by gene mutations or deletions by utilizing the overexpression of normal genes. [Background technology]

[0002] The ear is a vital organ of the human body, composed of the outer ear, middle ear, and inner ear. Its primary functions are 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 hearing function, and is divided into congenital and acquired hearing loss, often related to 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 gene mutations. Hearing loss caused by gene mutations is 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 hearing loss genes provides potential targets for precisely treating hereditary hearing loss, meaning that if the aforementioned genes cause hearing loss, gene therapy could be the first-line strategy for curing it.

[0004] Gene therapy refers to methods of achieving therapeutic objectives by repairing diseases caused by abnormal nucleic acid sequences or expressions in the body through modification, compensation, or suppression at the DNA or RNA level. Currently, most gene therapies require delivery via vectors. Adeno-associated viruses (AAVs) are one of the safe and efficient delivery vectors, with a packaging capacity of approximately 4.7 kb. However, in the field of hearing loss, the coding regions of many genes are too long for adeno-associated virus packaging. For example, BDP1, CDH23, COL11A2, LOXHD1, MET, MYO15A, MYO3A, MYO7A, OTOG, OTOF, OTOGL, PCDH15, PTPRQ, STRC, TECTA, and TARA have coding regions longer than 4 kb, and together with their associated regulatory elements, they exceed the packaging limit of AAV vectors. Currently, this problem is being solved using DNA recombinant dual vectors, but DNA recombinant dual-vector packaging has low recombination efficiency in vivo.

[0005] Among hearing-related genes with coding sequences longer than 4kb, OTOF plays a crucial role in hearing. The OTOF protein is mainly expressed in the inner ear hair cells of the cochlea, and its primary function is the regulation of calcium ions (Ca 2+ It binds to () and initiates the release of downstream neurotransmitters. Deletions or loss-of-function mutations in the OTOF gene can cause DFNB9 hearing loss.

[0006] The OTOF gene (NCBI Gene ID: 9381) has different transcripts after transcription, 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 originate from different splicings of the same RNA, with isoform 1 or isoform 5 being the splicing variant associated with hearing in inner ear hair cells. The CDS region of these two transcripts is 5994 bp long and encodes a protein with a length of 1997 amino acids.

[0007] In cases of congenital hearing loss caused by OTOF gene mutations, AAV is the first-line delivery medium due to its non-integrated delivery, long expression duration, and low immunogenicity. However, AAV has a packaging limitation problem: its packaging capacity is less than 4.7kb, which is insufficient to package the OTOF gene (the total length of the OTOF gene and regulatory sequence exceeds 7kb). There are currently three main methods to solve the OTOF packaging problem. The first is overload packaging. This method primarily involves packaging a 7.5kb gene expression element into the AAV virus, injecting it into the mouse cochlea, and after a certain period of action, it is observed that approximately 30% of the inner ear hair cells express the OTOF protein, and the mice's hearing recovers to approximately 58dB. However, overload packaging has problems such as low packaging efficiency, difficulty in product control, and low transfection efficiency, making it not the optimal solution. The second method 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. Studies have shown that mini-OTOF, composed of some of these domains, can partially restore OTOF function, but cannot restore hearing in animals. The third method involves performing DNA recombination using a dual-vector method to generate full-length mature OTOF mRNA and fully translating the protein. Specifically, this can be divided into overlapping, trans-splicing, or a combination of overlapping and trans-splicing.

[0008] While DNA recombination strategies can yield full-length, functional OTOF proteins (otoferlins), their recombination efficiency is not ideal, affecting OTOF protein expression and accumulation. To overcome this problem, protein recombination is currently considered the best option, and intein-mediated protein transsplicing, in particular, is characterized by its rapid recombination and high efficiency. Intein was initially discovered in fungi and yeasts, and subsequently in various microorganisms such as bacteria, viruses, and archaea. Intein possesses both intramolecular and intermolecular protein bonds. Intermolecularly bonded intein includes naturally isolated intein and artificially isolated intramolecular intein. The N-terminus and C-terminus of intein connect to the C-terminus and N-terminus of two parts of the target protein, respectively. The electrophile of the second part attacks the nucleophile of the first part, forming a covalent bond and allosterially forming a complete connexin consisting of two parts. From a molecular design perspective, only the first amino acid of the C-terminus needs to be serine, threonine, or cysteine.

[0009] Therefore, in light of the characteristics of protein recombination, and because dual vectors have the potential to improve protein recombination efficiency, the present invention recombines hearing loss-related proteins using a protein-level recombination strategy. In the method of the present invention, by expressing the OTOF protein using the Intein recombination method with an AAV vector, the abnormal gene function is restored and efficient expression is achieved. [Overview of the Initiative]

[0010] Current methods using dual-vector AAV delivery and DNA recombination have low recombination efficiency in the OTOF expression system. Also, current dual-vector delivery technologies can only achieve OTOF expression in one-sided cochlea and hearing recovery in one ear. When hearing recovery in both ears is required, cochlear injection into both ears is necessary. Then, the second injection is inconvenient for patients and increases the risk. To solve the above problems, the present invention provides a dual-vector system capable of expressing OTOF protein and the adeno-associated virus packaged thereby. This method can restore hearing in both ears by unilateral administration in the field of large-scale gene dual-vector delivery for hearing loss gene therapy.

[0011] The first technical solution provided by the present invention is a dual-vector system that expresses OTOF protein. The dual-vector system includes 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 includes an expression cassette inserted between two ITR sequences and the mITR sequence. The expression cassette of the nucleotide sequence of the first segment includes a promoter, the N-terminal coding sequence of OTOF, the N-terminal coding sequence of Intein, and PolyA. The expression cassette of the nucleotide sequence of the second segment includes a promoter, the C-terminal coding sequence of Intein, the 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 splitting point is provided on the OTOF amino acid sequence. The nucleotide coding sequence from the N-terminal of the OTOF amino acid sequence to the splitting point is the N-terminal coding sequence of OTOF, and the nucleotide coding sequence from the amino acid after the splitting point to the C-terminal of the OTOF amino acid sequence is the C-terminal coding sequence of OTOF. Furthermore, the cleavage sites of OTOF include, but are not limited to, the amino acids immediately preceding serine, threonine, or cysteine ​​in the OTOF protein amino acid sequence; the order is from the N-terminus to the C-terminus of OTOF. The promoters include, but are not limited to, the CAG promoter, CMV promoter, CBA promoter, UbC promoter, SFFV promoter, EF1α promoter, PGK promoter, or promoters encoding genes such as Myo7A, Myo15, Atoh1, POU4F3, Lhx3, Myo6, α9AchR, α10AchR, and OTOF. The PolyA sequence is an adenine modification of the tail during the mRNA maturation process, which can further stabilize the mRNA. In the present invention, the PolyA sequence includes, but is not limited to, AATAAA and its variants, including ATTAAA, AGTAAA, CATAAA, TATAAA, GATAAA, ACTAAA, AATAT, AAGAAA, AATAAT, AAAAAA, AATGA, AATCA, AACAAA, AATCA, AATGA, AATTA, or AATAG. Furthermore, the ITR sequence (reverse terminal repeat sequence) is derived from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9. The expression cassette further includes, but is not limited to, other expression elements, expression regulatory elements, and label elements. Furthermore, the expression regulatory element includes, but is not limited to, functional regulatory elements such as (1) an element for controlling the expression of a target protein, for example, an IRES for initiating the translation of a downstream gene; (2) a regulatory element for expressing miRNA and siRNA sequences; (3) an intron; (4) a positioning sequence for localizing and expressing the target protein in the nucleus, cytoplasm, or various cell organelles and secreting it extracellularly; (5) a sequence that promotes protein degradation (e.g., a PEST sequence); (6) some kozak sequences (the kozak sequence is GNCNCN, for example, GCCACC); (7) an enhancer (the enhancer may be derived from the SV40 virus, CMV virus, adenovirus, etc.); (8) WPRE; and the like.

[0012] Furthermore, the label element includes, but is not limited to, for example, FLAG, HA, MYC, fluorescent proteins, luciferase, SUMO proteins, ubiquitin proteins, 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, the Intein includes, but is 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, RmaDnaB, and the like. Furthermore, in the amino acid sequence of the OTOF protein shown in SEQ ID NO: 1 or 2, the cleavage sites include, but are not limited to, the amino acid residues at positions 827, 930, 954, and 1130. Furthermore, in the amino acid sequence of the OTOF protein shown in Sequence ID No. 1 or 2, the splitting 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. Furthermore, in the amino acid sequence of the OTOF protein shown in SEQ ID NO: 1 or 2, the splitting 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. Furthermore, in the amino acid sequence of the OTOF protein shown in SEQ ID NO: 1 or 2, the splitting 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. Furthermore, in the amino acid sequence of the OTOF protein shown in Sequence ID No. 1 or 2, the splitting site is the amino acid residue at position 1130. That is, the nucleotide coding sequence of amino acid sequence 1-1130 is the N-terminal coding sequence of OTOF, and the nucleotide coding sequence of amino acid sequence 1131-1997 is the C-terminal coding sequence of OTOF. Preferably, the amino acid sequence of OTOF is shown in SEQ ID NO: 2.

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

[0015] A second technical solution provided in the present invention is an adeno-associated virus packaging vector system. The packaging vector system comprises a dual vector system expressing the OTOF protein described in the first technical solution, a vector carrying the AAV rep and cap genes, and a helper virus vector, the vector becoming an AAV vector upon 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] A third technical solution provided in the present invention is a method for packaging adeno-associated viruses. The adeno-associated virus packaging vector system described in the second technical solution is transferred into a host cell for packaging. Furthermore, the dual vectors in the packaging vector system described in the second technical solution are imported into host cells together with a vector carrying the AAV rep and cap genes, and a helper virus vector, respectively, and packaged. Furthermore, the host cells are cell lines capable of viral replication and stable inheritance, and include, but are not limited to, cells such as Hela-S3, HEK-293, HEK-293T, HEK-293FT, A549, and Sf9. Preferably, the host cells are HEK-293 or HEK-293T cells.

[0017] A fourth technical solution provided in the present invention is an adeno-associated virus obtained by the packaging method described in the third technical solution. The virus 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] A fifth technical solution provided in 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, particularly in the preparation of drugs or formulations for the treatment of hearing disorders, hearing impairments or auditory dysfunctions. Furthermore, the aforementioned hearing loss, hearing impairment, or hearing dysfunction is caused by a gene mutation, including the OTOF gene. The mutation includes, but is not limited to, base substitutions, frameshift mutations, deletion mutations, and insertion mutations.

[0019] The sixth technical solution provided in the present invention is a formulation, prescription, 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 aforementioned preparations, prescriptions, or drugs may be in any dosage form, including but not limited to injectable and ointment forms. Furthermore, in the aforementioned formulation, prescription, or drug, the dual vector system or adeno-associated virus is the sole active ingredient. Furthermore, the formulation, prescription, or drug may include commonly used solvents, buffers, such as commonly used drug vectors and adjuvants, and may include one or more of the following: neutral salt buffers, acidic salt buffers, alkaline salt buffers, glucose, mannose, mannitol, proteins, polypeptides and amino acids, antibiotics, chelating agents, adjuvants, or preservatives. Furthermore, the buffer solution may be phosphate buffer, Tris buffer, 0.01% poloxamer PBS buffer, or HEPES buffer. Furthermore, in the aforementioned formulation, prescription, or drug, the active ingredient may be contained in other vectors, such as nanoparticles, liposomes, and positive lipid particles.

[0020] Furthermore, the method of administering the aforementioned formulation, prescription, or drug may be administration to one ear or both ears. Preferably, the formulation, prescription, or method of administering the drug is administered to one ear. Furthermore, when administering to one ear, the administration method is cochlear injection, and includes, but is not limited to, round window injection into the cochlea, round window injection into the oval, semicircular canal injection, and utricle injection. Furthermore, it is administered as a single dose or multiple doses throughout one's life, with a total dose of 1 × 10⁶ 9 -1 × 10 13 This is the genome of a single virus. The beneficial effects are as follows:

[0021] In this invention, the expression efficiency of the complete OTOF protein was significantly improved by using an AAV dual vector and an Intein recombination method for OTOF expression. Furthermore, the effect of restoring bilateral hearing was obtained by unilateral cochlear administration. After administration to one ear, Otof - / - Hearing in the injected ear of mice could be restored to the level of wild-type mice, and hearing in the non-injected ear could also be improved to 60 decibels. [Brief explanation of the drawing]

[0022] [Figure 1] This is a pAAV-CMV plasmid map. [Figure 2] This is a pAAV-CMV-OTOF-N-S1-Npu-N-intein plasmid map. [Figure 3] This is a pAAV-CMV-Npu-C-intein-OTOF-C-S1 plasmid map. [Figure 4] This is a pAAV-CMV-OTOF-N-S2-Npu-N-intein plasmid map. [Figure 5] This is a pAAV-CMV-Npu-C-intein-OTOF-C-S2 plasmid map. [Figure 6]This is a pAAV-CMV-OTOF-N-S4-Npu-N-intein plasmid map. [Figure 7] This is a pAAV-CMV-Npu-C-intein-OTOF-C-S4 plasmid map. [Figure 8] This is a pAAV-CMV-OTOF-N-S1-Rma-N-intein plasmid map. [Figure 9] This is a pAAV-CMV-Rma-C-intein-OTOF-C-S1 plasmid map. [Figure 10] This is a pAAV-CMV-OTOF-N-S2-Rma-N-intein plasmid map. [Figure 11] This is a pAAV-CMV-Rma-C-intein-OTOF-C-S2 plasmid map. [Figure 12] This is a pAAV-CMV-OTOF-N-S3-Rma-N-intein plasmid map. [Figure 13] This is a pAAV-CMV-Rma-C-intein-OTOF-C-S3 plasmid map. [Figure 14] This is a pAAV-CMV-OTOF-N-S4-Rma-N-intein plasmid map. [Figure 15] This is a pAAV-CMV-Rma-C-intein-OTOF-C-S4 plasmid map. [Figure 16] This is a pAAV-CMV-OTOF-FL plasmid map. [Figure 17] This is a pAAV-CMV-OTOF-N-AK plasmid map. [Figure 18] This is a pAAV-AK-OTOF-C-PolyA plasmid map. [Figure 19] This is a pAAV-CMV-OTOF-N-AP plasmid map. [Figure 20] This is a pAAV-AP-OTOF-C-PolyA plasmid map. [Figure 21]This is a pAAV-CMV-OTOF-N-TS plasmid map. [Figure 22] This is a pAAV-TS-OTOF-C-PolyA plasmid map. [Figure 23] This is the imaging result for Example 6. [Figure 24] This is the imaging result for Example 7. [Figure 25] This is the imaging result for Example 8. [Figure 26] These are the imaging results for Example 9. [Figure 27] This is the imaging result for Example 10. [Figure 28] This is a comparison chart of OTOF Intein recombination and OTOF DNA recombination results. [Figure 29] This is a nucleotide comparison chart of Otof- / - gene mutant mice and wild-type mice. [Figure 30] This chart compares the hearing of Otof- / - gene mutant mice and wild-type mice. [Figure 31] This shows the hearing recovery status one month after the case in Example 13. [Figure 32] This shows the hearing recovery status after 2 months in Example 13. [Figure 33] This shows the hearing recovery status one month after the case in Example 14. [Figure 34] This shows the hearing recovery status after 2 months in Example 14. [Figure 35] This shows the hearing recovery effect in mice in the low-dose group. [Figure 36] This shows the expression status of OTOF in wild-type and genetically defective mice. [Figure 37] This shows the expression status of OTOF in the administered ear and the opposite ear of the administered mouse. [Figure 38] This is a statistical diagram showing the expression status of OTOF in mice. [Figure 39] This shows the recovery of hearing in mice treated with AAV1 serotype OTOF. [Modes for carrying out the invention]

[0023] To further clarify the object, technical solutions, and advantages of the present invention, the present invention will be described in more detail below with reference to specific examples. Note that the following specific examples are for interpretation purposes only and do not limit the present invention.

[0024] In this invention, by combining dual-vector (or AAV dual-vector) delivery with Intein recombination, efficient expression of the OTOF protein in the host is achieved, enabling recovery from hearing loss, hearing impairment, or hearing dysfunction, and achieving the technical effect of restoring hearing in both ears through administration to one ear.

[0025] In this invention, the OTOF protein is expressed by the Intein recombination method. The OTOF regions are isoform 5 (NM_001287489.2) (post-translational amino acid sequence is NP_001274418.1) and isoform 1 (NM_194248.3) (post-translational amino acid sequence is NP_919224.1).

[0026] In some embodiments of the present invention, the protein necessary for the recovery or improvement of hearing loss is OTOF as shown in SEQ ID NO: 1 or SEQ ID NO: 2. The target protein may have sequences with 65%-100% homology to SEQ ID NO: 1 or 2, for example, 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75%, 70%, and 65% homology. Furthermore, suitable truncated forms of the protein may exist, for example, proteins whose lengths are 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, and 60% of the full-length protein of SEQ ID NO: 1 or 2. Suitable insertions may exist in proteins, for example, proteins whose length is 101%, 102%, 103%, 104%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, and 140% of the total length. Relevant sequences are shown in Table 1.

[0027] Table 1: OTOF sequences and their corresponding relationships JPEG0007897422000001.jpg30170

[0028] For the amino acid sequence of SEQ ID NO: 1 or 2, in addition to the nucleotide sequence of the corresponding SEQ ID NO: 3 or 4, the codon may be optimized, and the corresponding CAI may be between 0.65 and 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 human-derived OTOF protein sequences, OTOF protein sequences can be selected from other animals. For example, mice (protein sequences: NP_001273350.1, NP_001300696.1, NP_001093865.1 or NP_114081.2), rats (protein sequence: NP_001263649.1), pigs (protein sequence: XP_020943388.1), and monkeys (protein sequence: XP_0149 Examples include sequences such as 67378.2, XP_014967379.2, XP_028687700.1, XP_014967380.2, or XP_028687701.1) and sequences having 65%-100% homology to them, for example, 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75%, 70%, and 65%.

[0030] Inteins can perform protein splicing functions, acting post-translation or during translation to covalently bond two different proteins. Inteins were first discovered in yeast and fungi, and through alignment comparisons and analyses, the number of intein residues present in viruses, bacteria, archaea, and eukaryotic microorganisms is estimated to exceed 600. Most inteins are complete proteins, while some inteins have separated N-terminates and C-terminates, each binding to a portion of a protein and being reconstituted post-translation to form a complete protein through nucleophilic reactions and allostery. In this invention, inteins with separated N-terminates and C-terminates are preferred. Intein can be selected from inteins such as MxeGyrA, pabPolIII, MjaKlbA, SspDnaB, SceVMA, SspDnaE, ​​NpuDnaE, ​​AvaDnaE, ​​CraDnaE, ​​CspDnaE, ​​CwaDnaE, ​​MchtDnaE, ​​OliDnaE, ​​TerDnaE, ​​gp41-1, gp41-8, IMPDH-1, and RmaDnaB. Some 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 JPEG0007897422000002.jpg34170

[0032] In this invention, when assembling the target protein into two vectors by the Intein recombination method, first, OTOF is divided into two segments, the N-terminus and the C-terminus, with the division point as the boundary. The division conditions are: 1) it must be packageable in the AAV virus, and 2) the first amino acid of the C-terminus must be serine, threonine, or cysteine. The N-terminus of Intein is fused to the C-terminus of the N-terminus of the OTOF protein, and then the C-terminus of Intein is fused to the N-terminus of the C-terminus of the OTOF protein.

[0033] Preferably, the OTOF protein is divided into two segments with approximately equal N-terminus and C-terminus lengths, or divided such that the N-terminus / C-terminus length is 0.3-3, for example, 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 methods for splitting the OTOF protein into its N-terminus and C-terminus include, but are not limited to, those shown in Table 3 of Example 1.

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

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

[0037] The N-terminus of Intein is fused to the C-terminus of the N-terminus of the OTOF protein, and the resulting sequence is constructed into an expression cassette for the nucleotide sequence of the first segment. As a result, the expression cassette for the nucleotide sequence of the first segment contains the promoter, the N-terminal coding sequence of OTOF, the N-terminal coding sequence of Intein, and PolyA. The C-terminus of 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. As a result, the expression cassette for the nucleotide sequence of the second segment contains the promoter, the C-terminal coding sequence of Intein, the C-terminal coding sequence of OTOF, and PolyA. The nucleotide sequences of the first segment and the second segment can be used in any order to construct the N-terminal or C-terminal sequence of OTOF.

[0038] In the present invention, the above-mentioned promoter refers to an associated sequence capable of initiating the transcription of a downstream target protein, is typically used to recruit transcription factors, and can express the target protein in a specific space and time, and includes, but is not limited to, the following promoters. The promoter may be an RNA polymerase II promoter or an RNA polymerase III promoter. The promoter may be divided into broadly expressing promoters, such as the CMV promoter, the CAG promoter, etc. The promoter may also be a tissue-specific promoter, where a preferred promoter is highly expressed in the ear, a further promoter is highly expressed in the cochlea or vestibule, and a further promoter is highly expressed in the cochlea. The tissue-specific promoter may be derived from partial and whole sequences 1 to 10,000 bp upstream of the OTOF gene transcription start site, as well as sequences with sequence homology of 65%-100%, for example, 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75%, 70%, and 65%. Preferably, the promoters applied to the present invention include, but are not limited to, the 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 adenine modification of the tail during mRNA maturation, which can further stabilize mRNA. In this invention, the PolyA sequence includes AATAAA and its variants, including ATTAAA, AGTAAA, CATAAA, TATAAA, GATAAA, ACTAAA, AATAT, AAGAAA, AATAAT, AAAAAA, AATGA, AATCA, AACAAA, AATCA, AATGA, AATTA, or AATAG.

[0040] The nucleotide sequence of the first or second segment may contain one or more (e.g., two, three, four, five, etc.) regulatory elements. The regulatory elements of the first and second segments may be the same or different. These include, but are not limited to, the following regulatory elements: (1) Used to regulate the expression of the target protein; for example, IRES is used to initiate translation of downstream genes, and IRES is a sequence that can initiate protein translation within mRNA. For example, IRES may be derived from viruses such as FMDV, EMCV, HRV, HIV, HAV, HCV, PV, etc. (2) Regulatory elements used to express miRNA and siRNA sequences. (3) Introns. (4) Positioning sequences that localize and express the 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 elements may be some kozak sequences, where kozak sequences are GNCNCN, for example, GCCACC. (7) An 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 target gene, may or may not be tissue-specific, may or may not transcribe eRNA. (8) The regulatory element may also be a WPRE.

[0041] The nucleotide sequence of the first or second segment may further include one or more tags (e.g., two, three, four, or five). The tags of the first and second segments may be the same or different. For example, FLAG, HA, MYC, fluorescent proteins, luciferases, etc., can also be used to improve the properties of proteins such as SUMO proteins, ubiquitin proteins, and GSTs.

[0042] The nucleotide sequences of the first or second segment constructed above may be plasmids, but may also be linear or circular nucleic acids of other forms, and both the N terminus and C terminus of the target protein can be expressed in cells, tissues, organs, and organisms through appropriate expression and regulatory elements. A preferred range in this specification is cochlear expression. Preferably, in the present invention, a target gene expression cassette is incorporated using an ITR-containing plasmid (including pAAV, pAAV-CMV, pX601, pX551, pAAV-MCS, etc.), followed by AAV packaging.

[0043] If the nucleotide sequences of the first and second segments constructed above are viral vectors, they can be applied to any type of AAV packaging. Adeno-associated virus is obtained by transferring the nucleotide sequences of the first and second segments together with a vector carrying the AAV rep, cap gene, and a helper virus vector into a host cell and packaging them. The host cell is a cell line that enables viral replication and stable inheritance, and includes, but is not limited to, cells such as Hela-S3, HEK-293, HEK-293T, HEK-293FT, A549, or Sf9. Preferably, the host cell is a HEK-293 or HEK-293T cell. The AAV virus sample obtained by packaging may contain empty capsid viruses. The empty capsid virus content 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%, and preferably 0%-50%.

[0044] The viruses obtained by the packaging method described above are a pair of adeno-associated viruses, each containing 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. The pair of adeno-associated viruses can express both the N-terminus and C-terminus of the target protein in cells, tissues, organs, and organisms. The Intein protein performs protein splicing, playing a role post-translation or during translation to covalently bond two different proteins, thereby obtaining 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 ear, inner ear, cochlea, and inner ear hair cells, and may also be cells unrelated to the ear. The cells may be in vitro, in vivo, or ex vivo cells.

[0045] Therefore, the dual vector or adeno-associated virus can be used in the preparation of drugs for treating hearing disorders, hearing impairments, or auditory dysfunctions. These hearing disorders, hearing impairments, or auditory dysfunctions are caused by gene mutations, including those involving the OTOF gene. These mutations include, but are not limited to, base substitutions, frameshift mutations, deletion mutations, and insertion mutations.

[0046] The preparation or formulation prepared using the dual vector or adeno-associated virus may be a powder or a solution. The preparation or formulation may contain common solvents and buffers, for example, common drug vectors and adjuvants. It may contain neutral salt buffers, acidic salt buffers, alkaline salt buffers, glucose, mannose, mannitol, proteins, polypeptides and amino acids, antibiotics, chelating agents, adjuvants and preservatives. The buffers include phosphate buffer, Tris buffer and HEPES buffer. It may also be included in other vectors such as nanoparticles, liposomes and positive lipid particles. The solution may mimic the components of the perilymph. The concentration of NaCl is 20 - 200 mM, the concentration of KCl is 1 - 5 mM, the concentration of CaCl2 is 0.1 - 10 mM, the concentration of glucose is 1 - 10 mM, the concentration of HEPES is 2 - 50 mM, and the pH is 6 - 9. The solution is ultimately sterile and may be dissolved in water or may be dissolved in glycerin, ethanol, polyols and oils. The solution may be in a form that can be used directly or may be used after dilution.

[0047] Routes of administration used in the present invention: In the present invention, the dual vector or the adeno-associated virus finally obtained by packaging can be delivered to the ear by various methods. Regardless of the use of auxiliary tools, it can be delivered to the ear from regions such as the round window (a circular hole on the inner wall of the middle ear tympanic cavity, also called the "cochlear window"), the oval round window (also called the "oval window", "vestibular window", referring to an oval hole located in the upper rear of the inner wall of the middle ear tympanic cavity), the semicircular canals, the utricle, etc. The administration may be a single administration or may be multiple administrations based on protein expression and hearing recovery. The dosage may be 1 - 200 μL, and the amount of AAV contained is 1×10 9 -1×10 13 viral genomes. For example, in mice, 1 - 2 μL (1×10 9 , 1×10 10 or 1×10 11 containing) of AAV virus is administered, and in humans, 10 - 100 μL (1×109 -1 × 10 13 The virus (containing a viral genome) is administered. It may be administered to one or both ears, but in this invention, administration to one ear is preferred, and ultimately the effect of restoring hearing in both ears can be achieved.

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

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

[0050] Table 3: Partial divisions applicable to OTOF as shown in Sequence ID No. 1 or 2 JPEG0007897422000003.jpg205170JPEG0007897422000004.jpg255160JPEG0007897422000005.jpg255158JPEG0007897422000006.jpg51170

[0051] Example 2: Construction of nucleotide sequences of the first and second segments using a pAAV-CMV plasmid containing an ITR sequence. For the amino acid sequence of the OTOF protein shown in Sequence ID No. 2, a dual vector expressing the nucleotide sequences of the first and second segments, i.e., the OTOF protein, was constructed using the 827th amino acid residue as the splitting point (S1), the 930th amino acid residue as the splitting point (S2), and the 1130th amino acid residue as the splitting point (S4), with NpuDnaE as the intein and the pAAV-CMV plasmid (Figure 1; sequence shown in Sequence ID No. 9) as the vector. Specifically, the details are as follows.

[0052] The pAAV-CMV plasmid was enzymatically cleaved using BstBI and HindIII, and plasmid fragments were recovered from the agarose gel. Gene synthesis was performed to synthesize gene fragment 1 (coding gene at OTOF amino acids 1-827 and N-terminus of NpuDnaE Intein), gene fragment 2 (coding gene at NpuDnaE Intein C-terminus and OTOF amino acids 828-1997), gene fragment 3 (coding gene at OTOF amino acids 1-930 and N-terminus of NpuDnaE Intein), gene fragment 4 (coding gene at NpuDnaE Intein C-terminus and OTOF amino acids 931-1997), gene fragment 5 (coding gene at OTOF amino acids 1-1130 and N-terminus of NpuDnaE Intein), and gene fragment 6 (coding gene at NpuDnaE Intein C-terminus and OTOF amino acids 1131-1997), and these were enzymatically cleaved using BstBI and HindIII. Furthermore, the pAAV-CMV plasmids, after enzymatic cleavage using the T4 ligase system, were conjugated to the respective gene fragments 1-6, also after enzymatic cleavage. After conjugation, 2 μL of the conjugation product was taken and added to 50 μL of DH5α-competent cells, treated with an ice bath for 30 minutes, subjected to heat stimulation for 1 minute, immediately left on ice for 1 minute, 200 μL of LB liquid medium was added, and the cells were incubated at 37°C for 30 minutes. Then, 200 μL of bacterial suspension was taken and uniformly 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 the subsequent examples. 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 aforementioned plasmids all contain covalently bound OTOF (part) and NpuDnaE Intein (part). The transcripts are shown in Table 4.

[0054] Table 4: Transcriptional and translational products in which Npu is fused with different N-terminuses and C-terminuses of OTOF JPEG0007897422000007.jpg36170

[0055] Example 3: Construction of nucleotide sequences of the first and second segments using a pAAV-CMV plasmid containing an ITR sequence. For the amino acid sequence of the OTOF protein shown in Sequence ID No. 2, the 827th amino acid residue was designated as the splitting point (S1), the 930th amino acid residue as the splitting point (S2), the 954th amino acid residue as the splitting point (S3), and the 1130th amino acid residue as the splitting point (S4). Using RmaDnaB Intein and the pAAV-CMV plasmid (Figure 1; sequence shown in Sequence ID No. 9) as the vector, a dual vector expressing the nucleotide sequences of the first and second segments, i.e., the OTOF protein, was constructed. Specifically, the details are as follows.

[0056] The pAAV-CMV plasmid was enzymatically cleaved with BstBI and HindIII, and plasmid fragments were recovered from the agarose gel. Gene synthesis yielded gene fragment 7 (coding gene for OTOF amino acid positions 1-827 and RmaDnaB Intein N-terminus as shown in SEQ ID NO: 5 (including further plasmid-bound sequences)), gene fragment 8 (coding gene for RmaDnaB Intein C-terminus and OTOF amino acid positions 828-1997 as shown in SEQ ID NO: 6 (including further plasmid-bound sequences)), gene fragment 9 (coding gene for OTOF amino acid positions 1-930 and RmaDnaB Intein N-terminus as shown in SEQ ID NO: 7 (including further plasmid-bound sequences)), gene fragment 10 (coding gene for RmaDnaB Intein C-terminus and OTOF amino acid positions 931-1997 as shown in SEQ ID NO: 8 (including further plasmid-bound sequences)), gene fragment 11 (coding gene for OTOF amino acid positions 1-954 and RmaDnaB Intein N-terminus), gene fragment 12 (RmaDnaB Intein Gene fragments 13 (coding gene at the C-terminus and OTOF amino acid positions 955-1997), 14 (coding gene at OTOF amino acid positions 1-1130 and RmaDnaB Intein N-terminus), and 15 (coding gene at the RmaDnaB Intein C-terminus and OTOF amino acid positions 1131-1997) were synthesized and enzymatically cleaved with BstBI and HindIII, respectively. Furthermore, the enzymatically cleaved pAAV-CMV plasmids were conjugated to the enzymatically cleaved gene fragments 7-14, respectively, using the T4 ligase system. After conjugation, 2 μL of the conjugation product was taken and added to 50 μL of DH5α-competent cells, treated with an ice bath for 30 minutes, subjected to thermal stimulation for 1 minute, immediately left on ice for 1 minute, 200 μL of LB liquid medium was added, and the cells were incubated at 37°C for 30 minutes. Then, 200 μL of bacterial suspension was taken and uniformly spread onto ampicillin-resistant solid LB medium and incubated overnight at 37°C. Five monoclonal strains were selected and accurately sequenced. The following eight plasmids were obtained and used in the subsequent 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 plasmid map in Figure 14) 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 covalently bound OTOF (part) and RmaDnaB Intein (part). The transcripts are shown in Table 5.

[0057] Table 5: Transcriptional and translational products in which Rma is fused with different N-terminuses and C-terminuses of OTOF JPEG0007897422000008.jpg45170

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

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

[0060] Here, AK and AP are sequences that perform DNA recombination after adeno-associated viruses enter cells. After DNA recombination, they are trans-cleaved by the SA-SD sequence to form complete mRNA, which then expresses a full-length, functional OTOF protein. pAAV-CMV-OTOF-N-TS and pAAV-TS-OTOF-C-PolyA adeno-associated viruses undergo ITR recombination, then trans-cleaved by SA-SD to form complete, mature mRNA, which then expresses a full-length, functional OTOF protein.

[0061] Example 5: Preparation of adeno-associated virus Eight plasmids constructed by the method of the present invention in Example 3 and six plasmids constructed in Example 4 were co-transfected into HEK-293T cells with pHelper plasmid and PHP.eB pRC plasmid, respectively, in a molar ratio of 1:1:1 using PEI transfection reagent (approximately 1 μg of plasmid was added per 1 million cells). After culturing in a 37°C, 5% carbon dioxide incubator using 10% fetal bovine serum-containing DMEM medium for 3 days, the cells were washed once with PBS buffer, collected, and subjected to 5 freeze-thaw cycles. Solid NaCl was added to a final concentration of 500 mM, and the cells were centrifuged at 10000 g for 0.5 hours. The supernatant was taken and filtered through a 0.45 μm filtration membrane. The cells were then purified by the iodixanol method, and a portion of the sample was concentrated to obtain a viral titer of 1 ± 0.2 × 10⁶. 13 Adeno-associated viruses were obtained with a viral genome count / ml, using a 0.01% poloxamer PBS buffer solution as the solvent, and an empty shell rate of approximately 50%. The specific experimental setup is shown in Table 6 below. Hereafter, viruses will be named by their plasmid names. For example, the adenovirus packaged with pAAV-CMV-OTOF-N-S1-Rma-N-intein, indicated by number 1, and the pHelper plasmid PHP.eB will be named pAAV-CMV-OTOF-N-S1-Rma-N-intein adeno-associated virus.

[0062] Method for preparing a 0.01% poloxamer PBS buffer solution (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 prepared in preparation (1) by mass-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] JPEG0007897422000009.jpg83170

[0064] Example 6: Intracellular recombination of plasmids constructed in Examples 2 and 3 (plasmid ratio 1:1) The seven pairs of plasmids constructed in Examples 2 and 3 and the 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 × 10⁶). 6A plasmid premix was obtained by mixing 2 μg plasmid with 100 μL of Opti-MEM. A PEI premix was obtained by mixing 4 μL of PEI with 100 μL of Opti-MEM. After mixing the plasmid premix and the PEI premix, the mixture was allowed to stand for 10 minutes and then added to cultured HEK-293T cells. After culturing for 48 hours, the cell medium was aspirated and removed, and 200 μL of cell lysate (1% triton-X 100, 50 mM Tris-HCl, 1 mM PMSF, pH=7.4) was added. After standing on ice for 10 minutes, the cell lysate was collected, and the mixture was centrifuged at 4°C and 12000 g for 10 minutes, and the supernatant was retained. To the supernatant, 1 / 4 volume of 5× loading buffer (0.25M Tris-HCl, 10% SDS, 0.05% bromophenol blue, 50% glycerin, 0.25M DTT) was added, and the mixture was heated at 90°C for 10 minutes to obtain a protein sample. 50 μL of the protein sample was taken and subjected to polyacrylamide gel electrophoresis. After transfer to a PVDF membrane, the OTOF N-terminal antibody (Catalog No. A20266, abclonal) or C-terminal antibody (Catalog No. PA5-52935, Invitrogen) was incubated 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 showed relatively high recombination efficiencies. Of these, NpuS2 and RmaS2 showed the highest efficiencies.

[0065] The transfection plasmid dosages are shown in Table 7.

[0066] JPEG0007897422000010.jpg86170

[0067] Example 7: Intracellular recombination of S2 plasmid in a ratio such as 1:2 HEK-293T cells cultured in a 6-well plate with 2 μg of pAAV-CMV-OTOF-FL were transfected with the pAAV-CMV-OTOF-N-S2-Rma-N-intein plasmid and the pAAV-CMV-Rma-C-intein-OTOF-C-S2 plasmid in 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 (the number of cells in each well was approximately 1 × 10⁶). 6 The sample size was [number]. A 2 μg plasmid was added to 100 μL of Opti-MEM and mixed to obtain a plasmid premix. A 4 μL PEI was added to 100 μL of Opti-MEM and mixed to obtain a PEI premix. After mixing the plasmid premix and the PEI premix, the mixture was allowed to stand for 10 minutes and then added to cultured HEK-293T cells. After culturing for 48 hours, the cell medium was aspirated and removed, and 200 μL of cell lysate (1% triton-X 100, 50 mM Tris-HCl, 1 mM PMSF, pH=7.4) was added. After standing on ice for 10 minutes, the cell lysate was collected, and the mixture was centrifuged at 4°C and 12000 g for 10 minutes, and the supernatant was retained. To the supernatant, 1 / 4 volume of 5× loading buffer (0.25 M Tris-HCl, 10% SDS, 0.05% bromophenol blue, 50% glycerin, 0.25 M DTT) was added, and the mixture was 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 translocation to a PVDF membrane, the OTOF N-terminal antibody (A20266, abclonal) or C-terminal antibody (PA5-52935, Invitrogen) was incubated 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 24. For RmaS2, it was found that the transfection efficiency was highest when transfecting with an N:C ratio of 1:2.

[0068] Example 8: Intracellular recombination of S2 plasmid in different ratios, such as 1:2.5. 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 with 2 μg of pAAV-CMV-OTOF-FL in mass ratios 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 × 10⁶). 6 The sample size was [number]. A 2 μg plasmid was added to 100 μL of Opti-MEM and mixed to obtain a plasmid premix. A 4 μL PEI was added to 100 μL of Opti-MEM and mixed to obtain a PEI premix. After mixing the plasmid premix and the PEI premix, the mixture was allowed to stand for 10 minutes and then added dropwise to cultured HEK-293T cells. After culturing for 48 hours, the cell medium was aspirated and removed, and 200 μL of cell lysate (1% triton-X 100, 50 mM Tris-HCl, 1 mM PMSF, pH=7.4) was added. After standing on ice for 10 minutes, the cell lysate was collected, and the mixture was centrifuged at 4°C and 12000 g for 10 minutes, and the supernatant was retained. To the supernatant, 1 / 4 volume of 5× loading buffer (0.25M Tris-HCl, 10% SDS, 0.05% bromophenol blue, 50% glycerin, 0.25M DTT) was added, and the mixture was heated at 90°C for 10 minutes to obtain a protein sample. 50 μL of the protein sample was taken and subjected to polyacrylamide gel electrophoresis. After transfer to a PVDF membrane, the N-terminal (A20266, abclonal) or C-terminal antibody (PA5-52935, Invitrogen) of OTOF was incubated 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. It was found that the transfection efficiency was highest when the ratio of RmaS2 was adjusted to N:C = 1:2.5.

[0069] Example 9: Intracellular recombination of S1 plasmid in a ratio such as 1:2 The pAAV-CMV-OTOF-N-S1-Rma-N-intein plasmid and the pAAV-CMV-Rma-C-intein-OTOF-C-S1 plasmid were transfected into HEK-293T cells cultured in a 6-well plate with 2 μg of pAAV-CMV-OTOF-FL in 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 (the number of cells in each well was approximately 1 × 10⁶). 6 The N-terminal plasmid and C-terminal plasmid were added in a specific ratio 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. After mixing the plasmid premix and the PEI premix, they were allowed to stand for 10 minutes and then added dropwise to cultured HEK-293T cells. After culturing for 48 hours, the cell medium was aspirated and removed, and 200 μL of cell lysate (1% triton-X 100, 50 mM Tris-HCl, 1 mM PMSF, pH=7.4) was added. After standing on ice for 10 minutes, the cell lysate was collected, and the mixture was centrifuged at 4°C and 12000 g for 10 minutes, and the supernatant was retained. To the supernatant, 1 / 4 volume of 5× loading buffer (0.25M Tris-HCl, 10% SDS, 0.05% bromophenol blue, 50% glycerin, 0.25M DTT) was added, and the mixture was 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 N-terminal (A20266, abclonal) or C-terminal antibody (PA5-52935, Invitrogen) of OTOF was incubated 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 26. For RmaS1, it was found that the transfection efficiency was highest when transfecting at an N:C ratio of 1:2 by optimizing the transfection ratio of the N-terminus and C-terminus.

[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), respectively (the number of cells in each well was approximately 1 × 10⁶). 6 The sample size was [number]. A 2 μg plasmid was added to 100 μL of Opti-MEM and mixed to obtain a plasmid premix. A 4 μL PEI was added to 100 μL of Opti-MEM and mixed to obtain a PEI premix. After mixing the plasmid premix and the PEI premix, they were allowed to stand for 10 minutes and then added to cultured HEK-293T cells. Each cell was cultured for 2, 4, 6, 8, 12, 24, 48, and 72 hours, respectively. The cell medium was aspirated and removed, and 200 μL of cell lysate (1% triton-X 100, 50 mM Tris-HCl, 1 mM PMSF, pH=7.4) was added. After standing on ice for 10 minutes, the cell lysate was collected, and the mixture was centrifuged at 4°C and 12000 g for 10 minutes. The supernatant was retained. To the supernatant, 1 / 4 volume of 5× loading buffer (0.25M Tris-HCl, 10% SDS, 0.05% bromophenol blue, 50% glycerin, 0.25M DTT) was added, and the mixture was 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 N-terminus (A20266, abclonal) or C-terminus antibody (PA5-52935, Invitrogen) of OTOF was incubated 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 found to be very fast, and the ratio of fragments to full-length OTOF was almost the same at different time points.

[0071] Example 11: Comparison of OTOF Intein Recombination and OTOF DNA Recombination Two viruses packaged in Example 5 were added to 400 μL of serum-free DMEM medium (as shown in Table 8), and after homogeneous mixing, HEK-293 T cells (approximately 1 × 10⁶) were added to one well of a 6-well plate.6 Individual cells (the culture medium was aspirated and removed before adding to the virus) were added and incubated for 4 hours, then 1.6 mL of DMEM medium containing 10% fetal bovine serum was added. After culturing for 2 days, the cell medium was aspirated and removed, 200 μL of cell lysate (1% Triton-X 100, 50 mM Tris-HCl, 1 mM PMSF, pH=7.4) was added, and the mixture was left on ice for 10 minutes. The cell lysate was collected and centrifuged at 12000 g at 4°C for 10 minutes, and the supernatant was retained. 1 / 4 volume of 5× loading buffer (0.25 M Tris-HCl, 10% SDS, 0.05% bromophenol blue, 50% glycerin, 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 OTOF antibody (catalog number A20266, abclonal) was diluted 1:3000 and incubated, followed by incubation with the corresponding HRP-conjugated secondary antibody for 1 hour. ECL reagent was added and imaging was performed. The experiment was recombined three times, and grayscale statistics were performed on the full-length bands. The results are shown in Figure 28. The recombination efficiency of proteins recombined with OTOF Intein used in this invention was found to be significantly higher than that of OTOF DNA recombination.

[0072] Table 8: Proteins and nucleic acid recombination systems JPEG0007897422000011.jpg74170

[0073] Example 12: Construction of Otof gene-deficient mice Otof - / - The genetically modified mouse was constructed using the CRISPR / Cas9 method based on the 129S2 / SvPasCrl mouse strain. Otof - / - The model mouse possesses the Otof gene NM_001100395.1:c.2503_2504insA, which induces a homozygous frameshift mutation in the Otof gene. The results are shown in Figure 29, and Otof - / -The mutant mouse has a single base insertion, which causes a frame shift in the read frame. As shown in Figure 30, Otof - / - Hearing ability in mutant mice and wild-type mice was compared using ABR (Acoustic Brain Testing). The mutant mice had complete hearing loss in both ears.

[0074] Example 13: Administration of S1 Intein to one ear of 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 pHelper plasmid and PHP.eB pRC plasmid, respectively, in a molar ratio of 1:1:1 using PEI transfection reagent into HEK-293T cells (approximately 1 μg of plasmid added per 1 million cells). The cells were cultured for 3 days in a 37°C, 5% carbon dioxide incubator using DMEM medium containing 10% fetal bovine serum. After washing the cells once with PBS buffer and collecting them, the cells were frozen and thawed 5 times. Solid NaCl was added to a final concentration of 500 mM, and the cells were centrifuged at 10000 g for 0.5 hours. The supernatant was taken and filtered through a 0.45 μm filtration membrane. A gradient iodixanol solution was prepared and added to a centrifuge tube (5 mL 60% iodixanol, 5 mL 40% iodixanol (6 ml), 25% iodixanol (8 ml), 15% iodixanol (6 ml), sample added to the top layer, centrifuged at 350,000 g for 1 hour, and the virus layers at the 40% and 60% interfaces were aspirated. Centrifuged at 10,000 g using a 50 kDa ultrafiltration tube, the solution was changed 5 times with 0.01% poloxamer PBS buffer, and the viral titer was measured by qPCR. After adjusting the titer, the final viral titer was 1 ± 0.2 × 10⁻⁶. 13 We obtained viruses with a viral genome count of 1 / ml, namely pAAV-CMV-OTOF-N-S1-Rma-N-intein PHP.eB AAV and pAAV-CMV-Rma-C-intein-OTOF-C-S1 PHP.eB AAV. The solvent was 0.01% poloxamer PBS buffer solution.

[0075] The adeno-associated virus constructed above was used to construct the P0 / P1 Otof virus in Example 12. - / - The drug was administered to genetically mutant mice. Specifically, it was administered to the right cochlea via a round window injection, with each mouse receiving 2 × 10⁶ doses. 10 The number of viral genomes (1 x 10 for each of the two types of AAV) 10 A single dose of the virus genome was administered to mice, and the ABR index was detected to confirm the degree of hearing recovery. The results after one month are shown in Figure 31. In the figure, circles indicate the non-administered group (n=8), squares indicate the ear opposite the administration (n=10), triangles indicate the administered ear (n=10), and diagonal rectangles indicate the wild-type group (n=20). The results after two months are shown in Figure 32. In the figure, circles indicate the non-administered group (n=8), squares indicate the ear opposite the administration (n=3), triangles indicate the administered ear (n=3), and diagonal rectangles indicate the wild-type group (n=11). As can be seen from the results, Otof was used with the AAV constructed in this invention. - / - After administration to one cochlea of ​​model mice, hearing not only recovered in the injected ear, but also in the non-injected ear.

[0076] Example 14: Administration of S2 Intein to one ear of 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 PEI transfection reagent with pHelper plasmid and PHP.eB pRC plasmid, respectively, in a molar ratio of 1:1:1 (approximately 1 μg of plasmid was added per 1 million cells). After culturing in 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, collected, and subjected to 5 freeze-thaw cycles. Solid NaCl was added to a final concentration of 500 mM, and the cells were centrifuged at 10000 g for 0.5 hours. The supernatant was collected and filtered through a 0.45 μm filtration membrane. A gradient iodixanol solution was then prepared and added to a centrifuge tube (5 mL 60% iodixanol, 5 mL 40% iodixanol, 6 ml; 25% iodixanol, 8 ml; 15% iodixanol). The sample was added to the top layer and centrifuged at 350,000 g for 1 hour. The virus layers at the 40% and 60% interfaces were aspirated. The mixture was centrifuged at 10,000 g using a 50 kD ultrafiltration tube, and the solution was changed 5 times with 0.01% poloxamer PBS buffer. The viral titer was measured by qPCR, and the titer was adjusted to a final viral titer of 1 ± 0.2 × 10⁶. 13 Adeno-associated viruses with a viral genome count of 1 / ml and an empty shell rate of approximately 50%, namely 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. The solvent used was 0.01% poloxamer PBS buffer solution.

[0077] The adeno-associated virus constructed above was used in the P0 / P1 Otof constructed in Example 12. - / - The drug was administered to genetically mutant mice. Specifically, it was administered to the right cochlea via a round window injection, with each mouse receiving 2 × 10⁶ doses. 10 The number of viral genomes (1 x 10 for each of the two types of AAV) 10The mice were administered a viral genome (one of which is one), and the degree of hearing recovery was confirmed by detecting the mouse ABR index. The results after one month are shown in Figure 33. In the figure, circles represent the unadministered group (n=10), squares represent the ear opposite to the administered ear (n=33), triangles represent the administered ear (n=33), and diagonal squares represent the wild-type group (n=20). The results after two months are shown in Figure 34. In the figure, circles represent the unadministered group (n=8), squares represent the ear opposite to the administered ear (n=27), triangles represent the administered ear (n=27), and diagonal squares represent the wild-type group (n=11). As can be seen from the results, Otof was used with the AAV constructed in this invention. - / - After administration to one cochlea of ​​genetically mutant mice, not only did hearing in the injected ear (triangle mark) recover, but hearing in the opposite, non-injected ear (square mark) also recovered.

[0078] Example 15: Safety observation of the treatment group After administering AAV, constructed in Examples 13 and 14 of the present invention, to the cochlea of ​​6-8 week old wild-type CD-1 mice via round window injection, the mice's daily activity, hair smoothness, and dietary status were observed for 3 months. No significant differences were observed between the administered group and the control group that did not receive AAV injection, and there was no difference in hearing between the administered group and the control group. The cochlea of ​​the mice was dissected on days 28 and 91, and immunofluorescence staining of inner ear hair cells was performed. The number of hair cells did not differ significantly between the administered group and the control group.

[0079] Example 16: Administration of low-dose drug to one ear and its effect 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 PEI transfection reagent with pHelper plasmid and PHP.eB pRC plasmid, respectively, in a molar ratio of 1:1:1 (approximately 1 μg of plasmid was added per 1 million cells). After culturing in 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, collected, and subjected to 5 freeze-thaw cycles. Solid NaCl was added to a final concentration of 500 mM, and the cells were centrifuged at 10000 g for 0.5 hours. The supernatant was collected and filtered through a 0.45 μm filtration membrane. A gradient iodixanol solution was then prepared and added to a centrifuge tube (5 mL 60% iodixanol, 5 mL 40% iodixanol, 6 ml; 25% iodixanol, 8 ml; 15% iodixanol). The sample was added to the top layer and centrifuged at 350,000 g for 1 hour. The virus layers at the 40% and 60% interfaces were aspirated. The mixture was centrifuged at 10,000 g using a 50 kD ultrafiltration tube, and the solution was changed 5 times with 0.01% poloxamer PBS buffer. The viral titer was measured by qPCR, and the titer was adjusted to a final viral titer of 1 ± 0.2 × 10⁶. 13 We obtained viruses with a viral genome count of 1 / ml, namely pAAV-CMV-OTOF-N-S2-Rma-N-intein PHP.eB AAV and pAAV-CMV-Rma-C-intein-OTOF-C-S2 PHP.eB AAV. The solvent was 0.01% poloxamer PBS buffer solution.

[0080] The adeno-associated virus constructed above was used in the Otof virus constructed in Example 12. - / - It was administered to genetically mutant mice. Specifically, it was administered to the right cochlea via a round window injection, with 5 × 10⁶ doses given to each mouse. 9 The number of viral genomes (2.5 × 10 for each of the two types of AAV) 9A single dose of (number of viral genomes) was administered, and the hearing recovery status of mice was confirmed by detecting the mouse ABR index. The results after one month are shown in Figure 35. As can be seen from the results, the AAV constructed in this invention was used in Otof - / - After administration to one cochlea of ​​model mice, hearing not only recovered in the injected ear, but also in the non-injected ear.

[0081] Example 17: In vivo protein immunofluorescence detection after administration Mice in Example 14 were euthanized one month after administration, their cochlear tissue was removed, immersed overnight in 4% paraformaldehyde at 4°C, and decalcified with 10% EDTA solution for 3 days. Before 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 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). Subsequently, the tissue was incubated with the corresponding fluorescent secondary antibody and DAPI for 1 hour, and then washed three times with 0.1% Triton X-100 PBS (10 minutes each time). Finally, imaging was performed using a confocal fluorescence microscope. The results are as follows.

[0082] Figure 36 shows wild-type mice and Otof - / - This figure shows the expression status of OTOF in the cochlea of ​​genetically mutant mice. As can be seen from the figure, the cells stained with single row DAPI are inner ear hair cells, and in wild-type mice, OTOF was significantly expressed in inner ear hair cells, but Otof - / - In mutant mice, it was observed that OTOF was not fully expressed.

[0083] Figure 37 shows the expression status of OTOF in the injection ear and the opposite ear. Figure 37A shows Otof - / - This shows the expression status of OTOF protein in the ear of a mouse that has been administered the protein. The left side is a whole mount of the cochlea, and the right side is a localized magnified view of the area in the left frame. Figure 37B shows the expression of Otof - / -The figure shows the expression of OTOF protein in the ear opposite to the one administered in mice. In the figure, Apex, Middle, and Base represent different regions of cochlear hair cells. As can be seen from the figure, OTOF is expressed in most inner ear hair cells in the administered ear, and OTOF is also significantly expressed in the ear opposite the one administered.

[0084] Figure 38 shows Otof after administration. - / - This is a statistical diagram of OTOF expression in mice. Figure 38A shows the statistical status of inner ear hair cells expressing OTOF in the administered ear, indicating that the number of inner ear hair cells expressing the OTOF protein exceeded 60%. Figure 38B shows the statistical status of inner ear hair cells expressing OTOF in the ear opposite the administered ear, indicating that the number of inner ear hair cells expressing the OTOF protein exceeded 40%. In the figures, apical turn, middle turn, and basal turn indicate different parts of the cochlea. The vertical axis indicates the infection rate of inner ear hair cells.

[0085] Example 18: Administration to one ear of 4-week-old mice and its effect 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 with the pHelper plasmid and the AAV1 pRC plasmid, respectively, in a molar ratio of 1:1:1 using PEI transfection reagent (approximately 1 μg of plasmid was added per 1 million cells). After culturing in 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, collected, and subjected to 5 freeze-thaw cycles. Solid NaCl was added to a final concentration of 500 mM, and the cells were centrifuged at 10000 g for 0.5 hours. The supernatant was collected and filtered through a 0.45 μm filtration membrane. A gradient iodixanol solution was then prepared and added to a centrifuge tube (5 mL 60% iodixanol, 5 mL 40% iodixanol, 6 ml; 25% iodixanol, 8 ml; 15% iodixanol). The sample was added to the top layer and centrifuged at 350,000 g for 1 hour. The virus layers at the 40% and 60% interfaces were aspirated. The mixture was centrifuged at 10,000 g using a 50 kD ultrafiltration tube, and the solution was changed 5 times with 0.01% poloxamer PBS buffer. The viral titer was measured by qPCR, and the titer was adjusted to a final viral titer of 1 ± 0.2 × 10⁶. 13 Adeno-associated viruses with a viral genome count of 1 / ml and an empty shell rate of approximately 50%, namely pAAV-CMV-OTOF-N-S2-Rma-N-intein AAV1 and pAAV-CMV-Rma-C-intein-OTOF-C-S2 AAV1, were obtained. The solvent used was 0.01% poloxamer PBS buffer solution.

[0086] The adeno-associated virus constructed above was used in the Otof virus constructed in Example 12. - / - The drug was administered to genetically mutant mice (4 weeks old). Specifically, it was administered via injection into the posterior semicircular canal of the right cochlea, with each mouse receiving 2 × 10⁶ doses. 10 The number of viral genomes (1 x 10 for each of the two types of AAV) 10The mice were administered with a single viral genome, and their hearing recovery was confirmed by detecting the mouse ABR index. The results after one month are shown in Figure 39. In the figure, circles represent the untreated group (n=8), squares represent the ear opposite to the treated ear (n=6), triangles represent the treated ear (n=6), and diagonal squares represent the wild-type group (n=8). As can be seen from the results, the adeno-associated virus obtained by packaging according to the present invention has a high efficacy in restoring hearing in adult mice, and it is also effective in restoring hearing in both ears with only one ear injection.

[0087] The above embodiments illustrate only a few embodiments of the present invention, and while the descriptions are relatively specific and detailed, they do not limit the scope of the invention. Those skilled in the art will know that several modifications, combinations, and improvements can be made to each of the above embodiments, without departing from the spirit of the invention, and all of these fall within the scope of protection of the invention. Therefore, the scope of protection of the invention should be determined by the claims.

Claims

1. A dual vector system expressing the OTOF protein, The dual vector system comprises nucleotide sequences of two segments, 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 expression cassette for the nucleotide sequence of the first segment includes a promoter, the N-terminal coding sequence of OTOF, the N-terminal coding sequence of Intein, and PolyA. The expression cassette for the nucleotide sequence of the second segment includes a promoter, the C-terminal coding sequence of Intein, the C-terminal coding sequence of OTOF, and PolyA. The amino acid sequence of the aforementioned OTOF is shown in sequence number 1 or sequence number 2 in the sequence listing. A dual vector system characterized in that a splitting point is provided in the OTOF amino acid sequence, the splitting point is at the 827th, 930th, or 1130th amino acid residue, the nucleotide coding sequence from the N-terminus of the OTOF amino acid sequence to the splitting point is the N-terminal coding sequence of OTOF, and the nucleotide coding sequence from the amino acid at the position following the splitting 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, characterized in that the cleavage 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.

3. The promoters include, but are not limited to, the CAG promoter, CMV promoter, CBA promoter, UbC promoter, SFFV promoter, EF1α promoter, PGK promoter, or promoters encoding genes such as Myo7A, Myo15, Atoh1, POU4F3, Lhx3, Myo6, α9AchR, α10AchR, and OTOF. The aforementioned PbllyA includes AATAAAA and its variants, the variants including, but not limited to, ATTAAA, AGTAAA, CATAAAA, TATAAA, GATAAA, ACTAAAA, AATATA, AAGAAAA, AATAAT, AAAAAA, AATGAA, AATCAA, AACAAAA, AACAAA, AATAAC, AATAGA, AATTAA, or AATAAG. The dual-vector system according to claim 1, characterized in that the ITR sequence is derived from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9.

4. The dual vector system according to claim 1, characterized in that the expression cassette further comprises an expression regulatory element or a label element.

5. The dual-vector system according to claim 1, characterized in that 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, or RmaDnaB.

6. The nucleotide sequence of the first segment is located in a plasmid containing ITRs, and an expression cassette of the nucleotide sequence of the first segment is inserted between the ITR sequences. The dual vector system according to claim 1, characterized in that the nucleotide sequence of the second segment is located in a plasmid containing ITRs, and an expression cassette of the nucleotide sequence of the second segment is inserted between the ITR sequences.

7. The dual vector system according to claim 6, characterized in that the plasmid containing the ITR is pAAV, pAAV-CMV, pX601, pX551, or pAAV-MCS plasmid.

8. Using the 827th amino acid of OTOF shown in Sequence ID No. 2 as the splitting point, NpuDnaE Intein is used to fused the N-terminal coding sequence of OTOF with the N-terminal coding sequence of NpuDnaE Intein, and then the nucleotide sequence of the first segment is constructed using the pAAV-CMV plasmid as a vector. Then, the C-terminal coding sequence of NpuDnaE Intein is fused with the C-terminal coding sequence of OTOF, and then the nucleotide sequence of the second segment is constructed using the pAAV-CMV plasmid as a vector, or Using the 930th amino acid of OTOF shown in Sequence ID No. 2 as the splitting point, NpuDnaE Intein is used to fused the N-terminal coding sequence of OTOF with the N-terminal coding sequence of NpuDnaE Intein, and then the nucleotide sequence of the first segment is constructed using the pAAV-CMV plasmid as a vector. Then, the C-terminal coding sequence of NpuDnaE Intein is fused with the C-terminal coding sequence of OTOF, and then the nucleotide sequence of the second segment is constructed using the pAAV-CMV plasmid as a vector, or Using the 1130th amino acid of OTOF shown in Sequence ID No. 2 as the splitting point, NpuDnaE Intein is used to fused the N-terminal coding sequence of OTOF with the N-terminal coding sequence of NpuDnaE Intein, and then the nucleotide sequence of the first segment is constructed using the pAAV-CMV plasmid as a vector. Then, the C-terminal coding sequence of NpuDnaE Intein is fused with the C-terminal coding sequence of OTOF, and then the nucleotide sequence of the second segment is constructed using the pAAV-CMV plasmid as a vector, or Using the 827th amino acid of OTOF shown in Sequence ID No. 2 as the splitting point, RmaDnaB Intein is used to fused the N-terminal coding sequence of OTOF with the N-terminal coding sequence of RmaDnaB Intein, and then the nucleotide sequence of the first segment is constructed using the pAAV-CMV plasmid as a vector. Then, the C-terminal coding sequence of RmaDnaB Intein is fused with the C-terminal coding sequence of OTOF, and then the nucleotide sequence of the second segment is constructed using the pAAV-CMV plasmid as a vector, or Using the 930th amino acid of OTOF shown in Sequence ID No. 2 as the splitting point, RmaDnaB Intein is used to fused the N-terminal coding sequence of OTOF with the N-terminal coding sequence of RmaDnaB Intein, and then the nucleotide sequence of the first segment is constructed using the pAAV-CMV plasmid as a vector. Then, the C-terminal coding sequence of RmaDnaB Intein is fused with the C-terminal coding sequence of OTOF, and then the nucleotide sequence of the second segment is constructed using the pAAV-CMV plasmid as a vector, or The dual vector system according to claim 1, characterized in that the 1130th amino acid of OTOF shown in Sequence ID No. 2 is used as the splitting point, RmaDnaB Intein is used to conjugate and fuse the N-terminal coding sequence of OTOF with the N-terminal coding sequence of RmaDnaB Intein, and then the nucleotide sequence of the first segment is constructed using the pAAV-CMV plasmid as a vector, and then the C-terminal coding sequence of RmaDnaB Intein is conjugated and fused with the C-terminal coding sequence of OTOF, and then the nucleotide sequence of the second segment is constructed using the pAAV-CMV plasmid as a vector.

9. An adeno-associated virus packaging vector system comprising a dual vector system according to any one of claims 1 to 8, a vector carrying the AAV rep, cap gene, and a helper virus vector, wherein the vector becomes an AAV vector upon packaging.

10. The adeno-associated virus packaging vector system according to claim 9, characterized in that the vector carrying the AAV rep,cap gene is selected from AAV1, AAV2, AAV5, AAV8, AAV9, Anc80, PHP.eB, AAV-DJ, or AAVrh.10 vectors, and the helper virus vector is a pHelper plasmid.

11. A method for packaging adeno-associated viruses, characterized by transferring the adeno-associated virus packaging vector system described in claim 9 into a host cell and packaging it.

12. The method for packaging adeno-associated viruses according to claim 11, characterized in that the host cells are selected from Hela-S3, HEK-293, HEK-293T, HEK-293FT, A549, or Sf9 cells.

13. Adeno-associated virus obtained by packaging according to the method of claim 11.

14. Use of a dual vector system expressing the OTOF protein according to claim 1 in the preparation of a drug or formulation for treating hearing loss, hearing impairment, or hearing dysfunction.

15. Use of adeno-associated virus according to claim 13 in the preparation of a drug or formulation for treating hearing loss, hearing impairment, or hearing dysfunction.

16. A drug or formulation for treating hearing loss, hearing impairment, or hearing dysfunction, prepared in a dual-vector system expressing the OTOF protein described in claim 1.

17. A drug or preparation prepared with the adeno-associated virus described in Claim 13 for the treatment of hearing loss, hearing impairment, or hearing dysfunction.

18. The drug or formulation according to claim 16, characterized in that the drug or formulation further comprises one or more of a neutral salt buffer, an acidic salt buffer, an alkaline salt buffer, glucose, mannose, mannitol, protein, polypeptide and amino acid, antibiotic, chelating agent, adjuvant, preservative, nanoparticle, liposome or positive lipid particle.

19. It is administered by injection, and the injection routes include round window injection into the enclosed cochlea, round window injection into the oval, semicircular canal injection, and utricle injection. It is administered as a single dose or multiple doses throughout one's life, with a total dose of 1 x 10⁶ ９ -1 x 10 １３ The drug or preparation according to claim 16, characterized in that it is a single viral genome.

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