Dual-vector system expressing otoferlin protein and use thereof

Abstract

A dual-vector system expressing Otoferlin protein, comprising a first nucleic acid vector and a second nucleic acid vector, the first nucleic acid vector comprising a first nucleotide sequence, and the second nucleic acid vector comprising a second nucleotide sequence; the first nucleotide sequence comprises an expression cassette inserted between two first ITR sequences; the second nucleotide sequence comprises an expression cassette inserted between two second ITR sequences; the expression cassette of the first nucleotide sequence comprises a promoter, an N-terminal coding sequence of Otoferlin, an N-terminal coding sequence of an intein, and polyA; the expression cassette of the second nucleotide sequence comprises a promoter, a C-terminal coding sequence of an intein, a C-terminal coding sequence of Otoferlin, and polyA, the first amino acid of the C-terminal coding sequence of Otoferlin being mutated to serine (S), threonine (T) or cysteine (C). An adeno-associated virus packaging vector system, an adeno-associated virus packaging method, and an adeno-associated virus obtained therefrom are also provided. The dual-vector system or adeno-associated virus for expressing Otoferlin protein can be used for gene therapy, particularly for treating hearing loss, such as for treating Otof gene mutation-related autosomal recessive nonsyndromic deafness 9 (DFNB9).

Description

Dual-vector system for expressing Otoferlin protein and its applications Technical Field

[0001] This invention relates to a dual-vector system for expressing Otoferlin protein and its application in gene therapy, particularly in the treatment of hearing loss. Background Technology

[0002] Hearing loss is one of the most common sensory impairments in humans, severely impacting daily life and placing a significant burden on society. According to the WHO, nearly 1.5 billion people worldwide suffer from varying degrees of hearing loss, with 466 million experiencing disabling hearing loss (moderate or severe hearing impairment), representing 5% of the total population, including 34 million children. It is projected that by 2050, over 700 million people globally may suffer from disabling hearing loss. In China, approximately 16% of the population has hearing impairment, and about 5% suffer from disabling hearing loss. The incidence of deafness in newborns in China ranges from 1% to 3.47%, meaning 1-3 deaf children are born out of every 1,000 newborns, with approximately 30,000 deaf children born each year.

[0003] Genetics and environment are the two major factors causing deafness. Environmental causes are mainly related to various environmental factors or certain complications, such as the use of ototoxic drugs, infections during pregnancy, neonatal hypoxia, and radiation exposure. Genetic factors are mainly due to individual defects in deafness genes, leading to varying degrees of hearing loss. Some people may be born with hearing loss, while others may gradually lose their hearing over time. Pathogenic genes are passed on to the next generation through different inheritance patterns and can occur at any age, with permanent effects. Heredity is a major cause of deafness, accounting for approximately 60% of cases. Currently, more than 200 genes have been identified as related to deafness, involving more than 1,500 pathogenic variants. To date, there are no drugs available clinically to treat hereditary deafness.

[0004] Based on the presence of clinical symptoms in organs other than the auditory system, hereditary hearing loss can be divided into syndromic hearing loss (SHL) and non-syndromic hearing loss (NSHL). Syndromic hearing loss (SHL) is often accompanied by clinical manifestations in other systems, including the eyes, heart, kidneys, nervous system, skin, and bones, accounting for 30% of hereditary hearing loss. Pendred syndrome, Usher syndrome (USH), and Waardenburg syndrome (WS) are the most well-known SHL types; while USH, Pendred syndrome, and Jervell and Lange-Nielsen syndrome (JLNS) have successfully achieved inner ear gene therapy in preclinical animal model studies. Non-syndromic hearing loss (NSHL) has four types: autosomal dominant (DFNA), autosomal recessive (DFNB), X-linked (DFNX), and mitochondrial non-syndromic hearing loss. Approximately 70% of hereditary hearing loss patients have non-syndromic hearing loss. The most common mode of inheritance for non-syndromic hearing loss (NSHL) is autosomal recessive (75%-80%), followed by autosomal dominant (20%), X-linked (<2%), and mitochondrial inheritance (<1%). To date, more than 120 genes have been reported to be associated with NSHL. Among them, auditory neuropathy spectrum disorder (ANSD) caused by mutations in the Otof gene is a common non-syndromic recessive inherited hearing loss condition, and more than 1,000 pathogenic mutations of this gene have been reported.

[0005] Each type of deafness is described according to the naming conventions of deafness genes. For example, DFNA1 was the first discovered type of autosomal dominant deafness. DFNB9 is the ninth described autosomal recessive nonsyndrome deafness. Patients with DFNB9 deafness usually have severe sensorineural hearing loss, caused by mutations in the Otof gene. The Otof gene is located on human chromosome 2p23.3, is 101496 bp in length, contains 48 exons, and encodes otoferlin (also known as "OTOF protein"). OTOF protein is a transmembrane protein belonging to the Ferlin protein family. It is a calcium ion sensor, containing 1997 amino acids, including 6 C2 domains that bind calcium ions, and mainly participates in calcium ion binding. 2+The Otof gene is involved in the synaptic vesicle fusion and neurotransmitter release of inner hair cells. Five transcripts of the Otof gene are expressed in both the inner ear and brain. Two OTOF transcripts are found in the brain, terminating at exon 47 or 48; only one transcript is found in the cochlea, terminating at exon 48. The OTOF protein, encoded by the Otof gene, is concentrated in the basolateral region of cochlear inner hair cells and is an important component of the presynaptic membrane structure of these cells. The OTOF protein triggers membrane fusion at the band-like synapse of the inner hair cell and plays a crucial role in the exocytosis of calcium-related synaptic vesicles.

[0006] The hearing loss phenotype caused by mutations in the Otof gene is auditory neuropathy (AN), also known as auditory neuropathy spectrum disorder (ANSD). In this disease, the survival and function of hair cells are unaffected except for the inability to release synaptic vesicles from the inner hair cells. Clinical manifestations include impaired language comprehension, and normal or severely impaired auditory brainstem response thresholds. Audiological testing shows undifferentiated or severely abnormal auditory brainstem responses, but because hair cell function is normal, otoacoustic emissions and cochlear microphonic potentials can be elicited normally.

[0007] OTOF protein is a crucial protein in the transmission of sound information at the synapses of inner hair cells. More than 56% of non-syndromic ANSD cases are caused by mutations in the Otof gene, affecting approximately 200,000 people worldwide. Currently, patients with Otof gene mutations can only benefit from traditional hearing aids, but clinical treatment outcomes are poor, and there are currently no approved drug treatments.

[0008] Otof gene mutations can cause congenital, prelingual, severe hereditary deafness, inherited in an autosomal recessive manner. Patients with Otof gene mutations often have severe bilateral deafness from birth, with absent or highly abnormal auditory brainstem response (ABR), making it the most common cause of hearing loss. Current treatments for deafness mostly involve physical methods such as hearing aids, vibrating sound bridges, and cochlear implants. While patients can achieve varying degrees of hearing improvement, there are significant individual differences and considerable limitations and weaknesses. These include limited treatment effectiveness, frequency sensitivity, difficulties in speech discrimination and perception in noisy environments, and the need for careful device use. Approximately 300,000 patients worldwide have received cochlear implants, but this represents only a small fraction of all deaf patients. For the majority of patients, there is still a pressing need for fundamental and effective drug treatments, and to date, no approved treatments exist, representing a severely unmet need. Summary of the Invention

[0009] The object of this invention is to provide a gene therapy for subjects suffering from hearing loss, such as DFNB9, thereby preventing and / or restoring the subject's hearing.

[0010] In a first aspect, the present invention provides a dual-vector system for expressing the Otoferlin protein, comprising a first nucleic acid vector and a second nucleic acid vector, wherein...

[0011] The first nucleic acid vector contains a first nucleotide sequence; and the second nucleic acid vector contains a second nucleotide sequence;

[0012] The first nucleotide sequence contains an expression cassette inserted between two first ITR sequences;

[0013] The second nucleotide sequence contains an expression cassette inserted between two second ITR sequences;

[0014] The expression cassette of the first nucleotide sequence includes a promoter, an N-terminal coding sequence for Otoferlin, an N-terminal coding sequence for an intended peptide, and polyA;

[0015] The expression cassette of the second nucleotide sequence includes a promoter, a C-terminal coding sequence for an intima-peptide, a C-terminal coding sequence for Otoferlin, and polyA; and

[0016] The Otoferlin amino acid sequence contains an Otoferlin cleavage site, for example, the Otoferlin amino acid sequence is as shown in the mutated SEQ ID NO:1 or its functional fragment, for example, an amino acid sequence with at least 80% sequence identity with SEQ ID NO:1, for example, an amino acid sequence with at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity with SEQ ID NO:1;

[0017] The N-terminal coding sequence of Otoferlin is the nucleotide coding sequence from the N-terminus of the Otoferlin amino acid sequence to the Otoferlin cleavage site; the C-terminal coding sequence of Otoferlin is the nucleotide coding sequence from the mutated amino acid one position after the Otoferlin cleavage site to the C-terminus of the Otoferlin amino acid sequence, wherein the mutated amino acid is the amino acid at the corresponding position of SEQ ID NO:1 that has been replaced with serine (S), threonine (T) or cysteine ​​(C).

[0018] In some embodiments, the Otoferlin cleavage site in the dual-vector system for expressing Otoferlin protein of the present invention is located at positions 782, 785, 796, 803, 810, 838, 855, 916, 945, 946, 1081, 1098, 1099, 1100, or 1109 in the Otoferlin amino acid sequence according to SEQ ID NO:1. For example, the mutant amino acid is selected from 783S, 786C, 797C, 804S, 811S, 839S, 856S, 917S, 946S, 947S, 1082S, 1099S, 1100S, 1101S, and 1110S.

[0019] In some embodiments, the promoter of the expression cassette of the first or second nucleotide sequence in the dual vector system for expressing Otoferlin protein of the present invention is selected from polynucleotides of the Otov-HC promoter region and truncated fragments thereof, CAG promoter, CMV promoter, CBA promoter, UbC promoter, SFFV promoter, EF1α promoter, PGK promoter, or promoters of genes encoding Myo7A, Myo15, Atoh1, POU4F3, Lhx3, Myo6, α9AchR, α10AchR, OTOF, and STRC.

[0020] In some embodiments, the polyA of the expression cassette of the first or second nucleotide sequence in the dual-vector system for expressing Otoferlin protein of the present invention comprises AATAAA (SEQ ID NO:24) and variants of AATAAA; said variants of AATAAA include ATTAAA (SEQ ID NO:25), AGTAAA (SEQ ID NO:26), CATAAA (SEQ ID NO:27), TATAAA (SEQ ID NO:28), GATAAA (SEQ ID NO:29), ACTAAA (SEQ ID NO:30), AATATA (SEQ ID NO:31), AAGAAA (SEQ ID NO:32), AATAT (SEQ ID NO:33), AAAAAA (SEQ ID NO:34), AATGA (SEQ ID NO:35), AATCA (SEQ ID NO:36), AACAAA (SEQ ID NO:37), AATCA (SEQ ID NO:38), AATAC (SEQ ID NO:39), AATGA (SEQ ID NO:40), AATTAAA (SEQ ID NO:39), and AATTAAA (SEQ ID NO:30). NO:41) or AATAAG (SEQ ID NO:42); for example, the polyA is a nucleotide sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the polyA signal sequence shown in SEQ ID NO:20 or SEQ ID NO:23.

[0021] In some embodiments, each of the two first ITR sequences and the two second ITR sequences in the dual-vector system for expressing Otoferlin protein of the present invention is derived from, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9.

[0022] In some embodiments, the expression cassette of the first or second nucleotide sequence in the dual-vector system for expressing Otoferlin protein of the present invention further comprises an expression regulatory element and / or a tag element, for example, the expression regulatory element being a marmot hepatitis post-transcriptional regulatory element (WPRE) or a variant thereof, preferably a truncated variant of WPRE, for example, a nucleotide sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the nucleotide sequence shown in SEQ ID NO:19, for example, the nucleotide sequence shown in SEQ ID NO:22; for example, the tag element is HA.

[0023] In some embodiments, the integrin in the dual-carrier system for expressing Otoferlin protein of the present invention is derived from MxeGyrA, pabPolIII, MjaKlbA, SspDnaB, SceVMA, SspDnaE, ​​NpuDnaE, ​​AvaDnaE, ​​CraDnaE, ​​CspDnaE, ​​CwaDnaE, ​​MchtDnaE, ​​OliDnaE, ​​TerDnaE, ​​gp41-1, gp41-8, IMPDH-1, or RmaDnaB. For example, the integrin is derived from RmaDnaB, for instance, the N-terminus of the integrin is the N-terminus of the RmaDnaB integrin shown in SEQ ID NO:2, and the C-terminus of the integrin is the C-terminus of the RmaDnaB integrin shown in SEQ ID NO:3; or, the integrin is derived from NpuDnaE, ​​for instance, the N-terminus of the integrin is the N-terminus of the NpuDnaE integrin shown in SEQ ID NO:4, and the C-terminus of the integrin is the C-terminus of the RmaDnaB integrin shown in SEQ ID NO:3. NO:6 shows the C-terminus of the NpuDnaE peptide.

[0024] In some embodiments, in the dual-vector system for expressing Otoferlin protein of the present invention, the first nucleotide sequence is inserted into a plasmid containing two first ITR sequences, and the second nucleotide sequence is inserted into a plasmid containing two second ITR sequences. For example, the plasmid containing two first ITR sequences and the plasmid containing two second ITR sequences may be the same or different. For example, the plasmid is pAAV, pAAV-CMV, pX601, pX551, or pAAV-MCS plasmid.

[0025] In some embodiments, in the dual-vector system for expressing Otoferlin protein of the present invention, using the Otoferlin protein fragmentation scheme shown in Table 1, the N-terminal coding sequence of Otoferlin is ligated and fused with the N-terminal coding sequence of the RmaDnaB inteptide to construct the first nucleotide sequence; the C-terminal coding sequence of the RmaDnaB inteptide is ligated and fused with the C-terminal coding sequence of Otoferlin to construct the second nucleotide sequence, for example, using the pAAV-CMV plasmid as the vector; or using a plasmid in which the CMV promoter in the pAAV-CMV plasmid is replaced with a polynucleotide of the Otov-HC promoter region or a truncated fragment thereof as the vector.

[0026] In some embodiments, the N-terminal coding sequence of the RmaDnaB inteptide used in the dual-vector system for expressing Otoferlin protein of the present invention encodes the N-terminal portion of RmaDnaB shown in SEQ ID NO:2, and the C-terminal coding sequence of the RmaDnaB inteptide encodes the C-terminal portion of RmaDnaB shown in SEQ ID NO:3.

[0027] In some embodiments, in the dual-vector system of the present invention for expressing the Otoferlin protein,

[0028] The expression cassette of the first nucleotide sequence includes a promoter, the N-terminal coding sequence of Otoferlin as shown in SEQ ID NO:10, the N-terminal coding sequence of the inteptide, and polyA; the expression cassette of the second nucleotide sequence includes a promoter, the C-terminal coding sequence of the inteptide, the C-terminal coding sequence of Otoferlin as shown in SEQ ID NO:13, and polyA.

[0029] The expression cassette of the first nucleotide sequence includes a promoter, the N-terminal coding sequence of Otoferlin as shown in SEQ ID NO:11, the N-terminal coding sequence of the inteptide, and polyA; the expression cassette of the second nucleotide sequence includes a promoter, the C-terminal coding sequence of the inteptide, the C-terminal coding sequence of Otoferlin as shown in SEQ ID NO:14, and polyA; or

[0030] The expression cassette of the first nucleotide sequence includes a promoter, the N-terminal coding sequence of Otoferlin as shown in SEQ ID NO:12, the N-terminal coding sequence of the inteptide, and polyA; the expression cassette of the second nucleotide sequence includes a promoter, the C-terminal coding sequence of the inteptide, the C-terminal coding sequence of Otoferlin as shown in SEQ ID NO:15, and polyA.

[0031] Preferably, the promoter is a polynucleotide of the Otov-HC promoter region or a truncated fragment thereof, for example, the Otov-HC promoter shown in SEQ ID NO:8.

[0032] In some embodiments, the expression cassettes of the first and second nucleotide sequences in the dual-vector system for expressing Otoferlin protein of the present invention each contain a combination of a WPRE nucleotide sequence and an SV40 polyadenylated sequence at the N-terminus of the 3' ITR sequence, for example, having the nucleotide sequence shown in SEQ ID NO:18 or a nucleotide sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:18; or containing a combination of a WPRE3 nucleotide sequence and an SV40 late polyadenylated sequence, for example, having the nucleotide sequence shown in SEQ ID NO:21 or a nucleotide sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:21.

[0033] In a second aspect, the present invention provides a packaging vector system for adeno-associated virus, wherein the packaging vector system comprises the dual vector system of the present invention for expressing Otoferlin protein, a vector carrying AAV rep and cap genes, and a helper viral vector, packaged into an AAV vector, preferably wherein the amino acid sequence of the Otoferlin protein is as shown in the mutated SEQ ID NO:1.

[0034] In some embodiments, the vector carrying the AAV rep and cap genes in the adeno-associated virus packaging vector system of the present invention is selected from AAV1, AAV2, AAV5, AAV8, AAV9, Anc80, PHP.eB, AAV-DJ, and AAVrh.10 vectors; the auxiliary viral vector is the pHelper plasmid.

[0035] In a third aspect, the present invention provides a method for packaging adeno-associated virus, wherein the adeno-associated virus packaging vector system of the present invention is transferred into a host cell for packaging.

[0036] In some embodiments, the host cells used in the adeno-associated virus packaging method of the present invention are selected from Hela-S3 cells, HEK-293 cells, HEK-293T cells, HEK-293FT cells, A549 cells, and Sf9 cells.

[0037] In a fourth aspect, the present invention provides an adeno-associated virus obtained by using the packaging method of the present invention.

[0038] In a fifth aspect, the present invention provides the use of the dual-vector system of the present invention for expressing the Otoferlin protein or the adeno-associated virus of the present invention for preparing medicaments or formulations for treating deafness diseases or hearing loss or hearing dysfunction.

[0039] In a sixth aspect, the present invention provides a medicament or preparation for treating deafness, hearing loss, or hearing impairment, which is prepared from the dual-vector system for expressing Otoferlin protein of the present invention or the adeno-associated virus of the present invention, wherein the adeno-associated virus is obtained by packaging an adeno-associated virus packaging vector system into a host cell, the adeno-associated virus packaging vector system comprising a dual-vector system for expressing Otoferlin protein, a vector carrying the AAVrep and cap genes, and an auxiliary viral vector.

[0040] In some embodiments, the medicament or formulation of the present invention further comprises a neutral salt buffer, an acidic salt buffer, an alkaline salt buffer, glucose, mannose, mannitol, protein, polypeptide, amino acid, antibiotic, chelating agent, adjuvant, preservative, nanoparticles, liposomes, and positively charged lipid particles.

[0041] In some embodiments, the drug or formulation of the present invention is administered by injection through the round window, oval window, semicircular canals, or common canal of the cochlea; and may be administered once or multiple times throughout life, with a total dose of 1×10⁻⁶. 9 -1×10 13 Viral genome. Attached Figure Description

[0042] Figure 1 illustrates a schematic diagram of OTOF protein trans-splicing mediated by a double AAV intein using an inverted terminal repeat (ITR) sequence, a promoter, and a polyadenylated sequence. In the figure, "OTOF-N" represents the N-terminus of the full-length OTOF protein, encoded by the 5' coding region sequence (CDS) (5'OTOF), "OTOF-C" represents the C-terminus of the full-length OTOF protein, encoded by the 3' CDS (3'OTOF), "N-intein" represents the N-terminal intein, and "C-intein" represents the C-terminal intein.

[0043] Figure 2 illustrates a schematic diagram of the AAV dual-vector plasmid element. The pAAV-CMV-EGFP-WPRE-SV40 plasmid backbone sequence can be as shown in SEQ ID NO:17, wherein the 5' ITR sequence is located at 1bp-141bp, the CMV promoter sequence is located at 169bp-752bp, the Kozak sequence is located at 792bp-797bp, the EGFP sequence is located at 801bp-1517bp, the WPRE sequence is located at 1536bp-2124bp, the SV40 PolyA signal sequence is located at 2131bp-2252bp, the 3' ITR sequence is located at 2290bp-2430bp, the f1Ori sequence is located at 2505bp-2960bp, the kana resistance sequence is located at 3242bp-4156bp, and the Ori sequence is located at 4327bp-4515bp.

[0044] Figures 3-24 show the different cleavage sites of the OTOF full-length protein between the mutation site and the amino acid residue immediately adjacent to its N-terminus, which are then used for in vitro screening and verification.

[0045] Figure 25 shows the expression of full-length OTOF in HEK-293T cells co-transfected with both a plasmid vector encoding the N-terminal portion (N-Otof) and a plasmid vector encoding the C-terminal portion (C-Otof) of the Otoferlin protein. In Figure 25, lane "Ctrl" represents the protein control of HEK-293T cells without plasmid transfection; lane "FL" represents the molecular weight marker of full-length Otoferlin protein; lanes "1" to "22" correspond to the protein expression after transfecting 293T cells with the 22 pairs of first and second plasmid vectors shown in Table 2; and lane "wt" corresponds to the protein expression after transfecting 293T cells with the first and second plasmid vectors using the 866th position of the wild-type Otoferlin protein as the cleavage site.

[0046] Figure 26 shows the auditory brainstem response (ABR) results of treated adult Otof- / - knockout mouse models. It demonstrates that adult Otof- / - knockout mice treated with Otoferlin protein expressed at the newly segmented site showed significant recovery in low-frequency hearing, approaching the hearing threshold of wild-type mice. In the figure, "WT" represents wild-type mice; "KO" represents Otof- / - knockout mice; "16" represents mice treated with a mixture of N-terminal and C-terminal AAV viruses corresponding to segmentation scheme 16 shown in Table 2; "17" represents mice treated with a mixture of N-terminal and C-terminal AAV viruses corresponding to segmentation scheme 17 shown in Table 2; and "18" represents mice treated with a mixture of N-terminal and C-terminal AAV viruses corresponding to segmentation scheme 18 shown in Table 2.

[0047] Figure 27 shows the immunofluorescence results of adult Otof- / - knockout mouse models after treatment. Mice were sacrificed after hearing tests, and cochlear tissue was harvested for basilar membrane staining. Immunofluorescence results showed that the dual AAV system of this application achieved the expression of otoferlin protein in the mouse cochlea, restored the release of synaptic vesicles in the inner ear hair cells, and restored hearing in both ears. In the figure, the meanings of "WT", "KO", "16", "17", and "18" are the same as in Figure 26. Detailed Implementation

[0048] Unless otherwise defined below, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. Furthermore, the materials, methods, and examples described herein are illustrative only and are not intended to be limiting. Other features, objects, and advantages of the invention will become apparent from this specification and the accompanying drawings, and from the appended claims.

[0049] I. Definition

[0050] In this document, the term "about" when used in conjunction with a numeric value means to cover a range of numeric values ​​having a lower limit of 5% less than the specified numeric value and an upper limit of 5% greater than the specified numeric value. The term is also intended to cover values ​​within ±1%, ±0.5%, or ±0.1% of the specified numeric value.

[0051] In this document, the terms "comprising" or "including" mean including the mentioned elements, integers, or steps, or groups of elements, integers, or steps, but do not exclude any other elements, integers, or steps, or other groups of elements, integers, or steps. When the terms "comprising" or "including" are used herein, unless otherwise specified, they also cover situations consisting of the mentioned elements, integers, or steps. For example, when referring to a polynucleotide that "comprising" a specific sequence, it is also intended to cover polynucleotides consisting of that specific sequence.

[0052] In this document, the expression “and / or” when used in conjunction with two or more items is intended to mean any one of the listed related items, or any and all possible combinations of the listed related items.

[0053] In this document, "hearing loss" refers to hearing below the normal hearing threshold level as determined by audiometry, including mild, moderate, severe, and profound hearing loss, as well as deafness. Hearing loss can be described as a percentage of hearing loss, such as 30%, 60%, 80%, or even 100% hearing loss, or by a hearing loss grading. The hearing loss can be caused by or associated with a genetic defect, such as congenital deafness and prelingual deafness caused by genetic factors, or hearing loss associated with genetic factors but induced by environmental factors (e.g., aging, noise, drugs, or infection). Hearing loss can be asymptomatic (i.e., without associated visible abnormalities of the outer ear or other organs) or symptomatic. In some embodiments, the hearing loss is sensorineural hearing loss.

[0054] In this article, "hearing loss-related genes" refers to genes whose variations can cause hearing loss or create susceptibility to hearing loss by altering the inner ear's ability to function normally. Such genes are also referred to as "hearing loss genes" in this article. More than 100 genes have been identified as being associated with hearing loss (see Hereditary Hearing Loss Homepage, https: / / hereditaryhearingloss.org / , which lists the known locations and identification data of single-gene asymptomatic hearing loss). In cases of susceptibility to hearing loss, individuals carrying variations in these hearing loss genes are more likely to experience hearing loss due to environmental factors such as aging, noise, medications, or infections compared to healthy individuals.

[0055] In this article, "cochlear hair cells" refers to isolated or in vitro cochlear hair cells, cell lines, or cell populations from mammals, or hair cells in the cochlea of ​​mammals.

[0056] In this article, "cochlear outer hair cells" refers to cochlear outer hair cells, cell lines, or cell populations derived from mammals, either isolated or in vitro, or to outer hair cells in the cochlea of ​​mammals.

[0057] In this document, "isolated" nucleic acid means a nucleic acid molecule that is artificially synthesized or isolated from at least some components of its natural environment containing it. For example, an isolated nucleic acid may be part of a larger nucleic acid, or part of a carrier or composition of substances, or may be contained within a cell and remain "isolated" provided that the larger nucleic acid, carrier, composition of substances, or particular cell is not the natural environment of the nucleic acid.

[0058] In this paper, the term "operable link," also known as "effective link" or "functional link," refers to a relationship in which two or more polynucleotide (e.g., DNA) segments are positioned to allow them to function in the intended manner. For example, if a promoter sequence stimulates or regulates transcription of a coding sequence in a suitable host cell or other expression system, then the promoter sequence is effectively linked to that coding sequence. Generally, promoters effectively linked to a transcribed sequence are contiguous to that transcribed sequence; that is, they are cis-acting. However, some transcriptional regulatory sequences (such as enhancers) do not need to be physically adjacent to or closely proximate to the coding sequence that enhances transcription.

[0059] The "Otof gene" encodes otoferlin (also known as "OTOF protein"), a protein in the same family as dysferlin and myoferlin. These proteins share homology with FER-1 (sperm-forming factor-1) from nematodes, suggesting that OTOF protein may be involved in vesicle membrane fusion. OTOF protein is expressed in trace amounts in the spiral ganglion cells and inner and outer hair cells of the cochlea in late embryonic and newborn mice. In adult mice, it is expressed only in inner hair cells, mainly concentrated at the base of the inner hair cells. The basal region of the inner hair cells contains numerous band-like synapses that continuously release large amounts of neurotransmitters. Previous studies have suggested that OTOF protein is a calcium receptor in the exocytosis of inner hair cells and an important component of afferent synapses. The C2 domain of OTOF protein can bind Ca2+. 2+ And accompanied by Ca 2+ OTOF protein exhibits dependence on phospholipids and proteins, playing a crucial role in cell membrane transport and signal transduction. It can also bind to cytoplasmic phospholipase A2, synaptic protein I, and Ras-associated GTP-binding proteins, participating in neurotransmitter release. Therefore, it can be inferred that OTOF protein is related to calcium-related synaptic vesicle fusion and neurotransmitter release. - / - The mice showed no obvious abnormalities in their band-like synaptic structure and Ca 2+While OTOF proteins can be secreted, complete hearing loss is observed, accompanied by abnormal synaptic vesicle release, indicating that OTOF proteins are crucial components mediating synaptic vesicle release. Since neurotransmitters nourish the postsynaptic region, a lack of nutrition can lead to postsynaptic degeneration, which may explain the progressive hearing loss observed in human cases.

[0060] The Otof gene is expressed in the cochlea, vestibular system, and brain tissue. It has five transcripts, two of which are expressed in the brain. The encoded protein exists in both long and short forms, terminating at exon 47 and exon 48, respectively. The short OTOF protein has three C2 domains and one carboxyl transmembrane domain; this form is found only in humans. The long OTOF protein has six C2 domains and one transmembrane domain; it is expressed in both humans and mice, but only the transcript terminating at exon 48 exists in the human cochlea. In some embodiments, a modified transcript variant 5 of the human Otof gene (NM_001287489.1) is used for gene therapy in the cochlea. In one embodiment, the amino acid sequence of OTOF isoform 5 encoded by transcript variant 5 of the Otof gene is shown in SEQ ID NO:1. Previous studies have found that different domains of the OTOF protein (C2A-F) exhibit varying mutation rates, with the C2F domain showing the highest mutation frequency. It is currently believed that the differences in mutation rates are due to variations in the conformation of the different C2 domains or the different substances they bind during auditory function development.

[0061] The inteins, or protein inteins, described in this article are polypeptide chains within immature precursor proteins. Through a series of self-catalyzed reactions such as rearrangement, transesterification, and cyclization, they can be cleaved from the precursor protein, linking their two exopeptide segments (protein exteins) together via a natural peptide bond. This process, known as protein self-splicing, achieves a rearrangement of the protein structure. Broken inteins are a structural type of intein. Structurally, their N-terminal and C-terminal regions are separate. When the two segments containing the N-terminal and C-terminal regions of an intein are joined, the exopeptides at both ends can be spliced ​​together according to the standard intein splicing pathway.

[0062] Most inteins consist of terminal splicing regions at both ends and a homing endonuclease domain or linker domain in the middle. Inteins can be classified into three types: canonical inteins, miniinteins, and split inteins. Both canonical and miniinteins contain terminal splicing domains and a middle region. The difference lies in the middle region: canonical inteins have a homing endonuclease domain, while miniinteins have a linker domain. The length of the linker domain varies among different miniinteins. Split inteins, on the other hand, break at a specific site in the middle region, forming an N-terminal and a C-terminal fragment, located on two relatively distant genes in the genome. During the translation and maturation of precursor proteins, these two intein fragments recognize each other and restore endonuclease activity, mediating protein transsplicing. In this paper, a dual AAV vector system can be used to deliver nucleic acids containing nucleic acids encoding fragmented integrins.

[0063] Typically, an intipeptide consists of 10 modules, ordered from the N-terminus as A, N2, B, N4, C, D, E, H, F, and G. A, N2, B, and N4 are N-terminal splicing regions, F and G are C-terminal splicing regions, and C, D, E, and H are self-guided endonuclease active regions or linker domains. The motifs in modules A, B, F, and G involved in intipeptide splicing have highly conserved amino acid residues at the splice sites, essential for the affinity substitution reaction during intipeptide splicing. The motifs in module A usually contain amino acids with hydroxyl or thiol groups, such as Ser and Cys. The motifs in module B contain the highly conserved Thr-XX-His amino acid sequence, which is also present in serine proteases. The conserved amino acid residues in the motifs in module G involved in the splicing reaction are Asn, Ser, Cys, Thr, and His. In addition, conserved sites in the motif of module A (such as Ser and Cys) can be replaced by Ala, Gln, or Pro in some integrins, and the same is true in the motif of module G.

[0064] The term "full-length Otoferlin protein" refers to wild-type or functional human Otoferlin protein. The amino acid sequence of wild-type human Otoferlin protein and the polynucleotide sequence encoding it are known in the art (see, for example, GenBank accessions NP_000251 and U39226.1). In some specific embodiments, the full-length Otoferlin protein is the full-length Otoferlin protein shown in SEQ ID NO:1 or a functional derivative thereof (e.g., the mutant Otoferlin protein of the present invention) or a functional fragment thereof.

[0065] As used in this article, the term "adeno-associated virus (AAV)" is named after its discovery in adenovirus products. AAV is a member of the Parvovirus family, which includes multiple serotypes, and its genome is single-stranded DNA.

[0066] AAV is a dependent virus, requiring other viruses such as adenovirus, herpes simplex virus, human papillomavirus, or cofactors to provide auxiliary functional proteins in order to replicate.

[0067] The earliest isolated AAV virus was serotype 2 AAV (AAV2). The AAV2 genome is approximately 4.7 kb long, with 145 bp inverted terminal repeats (ITRs) at both ends, exhibiting a palindromic-hairpin structure. The genome contains two large open reading frames (ORFs), encoding the rep and cap genes, respectively.

[0068] ITRs are cis-acting elements of the AAV vector genome, playing a crucial role in AAV virus integration, rescue, replication, and genome packaging. ITR sequences contain a Rep protein binding site (RBS) and a terminal resolution site (trs), enabling them to be recognized by Rep protein binding and creating a nick at the trs. ITR sequences can also form a unique "T"-shaped secondary structure, playing a vital role in the AAV virus life cycle.

[0069] The rest of the AAV2 genome can be divided into two functional regions: the rep gene region and the cap gene region.

[0070] The rep gene region encodes four Rep proteins: Rep78, Rep68, Rep52, and Rep40. Rep proteins play crucial roles in AAV viral replication, integration, rescue, and packaging. Rep78 and Rep68 specifically bind to the terminal unwinding sites trs and GAGY repeat motifs in the ITR, initiating the AAV genome replication process from single-stranded to double-stranded. The trs and GAGC repeat motifs and / or GAGY repeat motifs in the ITR are central to AAV genome replication; therefore, although the ITR sequences differ across AAV serotypes, they all form hairpin structures and contain Rep binding sites. The p19 promoter is located at position 19 on the AAV2 genome map, initiating the expression of Rep52 and Rep40, respectively. Rep52 and Rep40 possess ATP-dependent DNA helicase activity but lack DNA-binding function.

[0071] The cap gene encodes the capsid proteins VP1, VP2, and VP3 of the AAV virus. VP3 has the smallest molecular weight but is the most abundant; in mature AAV particles, the ratio of VP1, VP2, and VP3 is approximately 1:1:10. VP1 is essential for the formation of infectious AAV; VP2 assists VP3 in entering the cell nucleus; VP3 is the main protein constituting the AAV particle.

[0072] As used in this article, the term "AAV vector" refers to a highly efficient exogenous gene transfer tool created by modifying wild-type AAV viruses, based on our understanding of the AAV virus life cycle and related molecular biological mechanisms. The modified AAV vector genome contains only the AAV virus's ITR sequence and the exogenous sequence to be transferred. The Rep and Cap proteins required for AAV virus packaging are provided trans-associated through other exogenous plasmids, thereby reducing the potential harm caused by packaging the Rep and Cap genes into the AAV vector. Furthermore, the AAV virus itself is non-pathogenic, making AAV vectors one of the most recognized safest viral vectors.

[0073] AAV virus has many serotypes, and different serotypes have different tissue tropisms. Therefore, AAV vectors can be used to transport exogenous genes to specific organs and tissues.

[0074] Existing technologies have relatively mature packaging systems for AAV carriers, which facilitates the large-scale production of AAV carriers.

[0075] The term "vector genome (vg)" refers to the nucleic acid sequence packaged within the rAAV capsid to form the rAAV vector.

[0076] In this article, "individual" and "subject" are used interchangeably and refer to mammals. Examples of mammals include, but are not limited to, humans, non-human primates (e.g., cynomolgus monkeys, rhesus monkeys), rodents, and other mammals such as cattle, pigs, horses, and dogs. In this article, mammals include individuals at all stages of development (including embryonic and fetal stages).

[0077] In this article, the term "treatment" refers to a clinical intervention intended to alter the natural course of a disease in an individual receiving treatment. Desired therapeutic effects include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or mitigating the disease state, and alleviating or improving prognosis. The term "treatment" also encompasses modifying or improving at least one bodily parameter, including those that the patient may not be aware of.

[0078] In this article, the term "prevention" refers to preventing or delaying the onset, development, or progression of a disease or condition. In this article, "prevention" generally refers to hospital intervention implemented before at least one symptom of a disease occurs.

[0079] The various aspects of the present invention will now be described.

[0080] II. Dual-carrier system

[0081] Gene therapy refers to methods that correct, compensate for, or inhibit defective genes at the DNA or RNA level, thereby enabling subjects to recover from diseases caused by abnormal nucleic acid sequences or expression, achieving the goal of disease treatment. Gene therapy, as an emerging treatment strategy, shows great promise for the application of hereditary hearing loss. Currently, most gene therapies require vector delivery. Adeno-associated virus (AAV) is one safe and efficient delivery vector, characterized by delivery non-integration, long expression time, and low immunogenicity, making it the preferred delivery tool. However, AAV's packaging capacity is less than 4.7kb, presenting packaging limitations; a single AAV vector cannot meet the packaging requirements of the Otof gene (the Otof gene plus regulatory sequences exceeds 7kb in length).

[0082] For congenital deafness caused by Otof gene mutations, there are currently three main methods to address the Otof gene packaging issue. The first is overpackaging, which involves packaging a 7.5kb Otof gene expression cassette into an AAV viral vector and injecting it into the cochlea of ​​mice. After a period of time, approximately 30% of the inner ear hair cells express OTOF protein, restoring hearing to around 58dB in the mice. However, overpackaging suffers from low packaging efficiency, difficulty in product control, and low transfection efficiency, making it not the optimal solution. The second method involves shortening the coding sequence length required for functional OTOF. OTOF is a C2 domain protein composed of six C2 domains (A, B, C, D, E, and F domains) and a TEM domain. Studies have shown that mini-OTOF proteins composed of only a few of these domains can partially restore the function of the full-length OTOF protein, but cannot restore complete hearing in animals. Thirdly, DNA recombination is performed using a dual AAV vector to generate full-length mature OTOF mRNA and complete protein translation. Specifically, this can be divided into overlapping, trans-splicing, or a combination of overlapping and trans-splicing recombination.

[0083] Although the strategy of DNA recombination using dual AAV vectors can yield full-length, functional OTOF proteins, the recombination efficiency of this strategy is not ideal, affecting the expression and accumulation of OTOF proteins. To overcome this problem, protein recombination at the protein level may be the optimal choice, especially intein-mediated trans-splicing, which features rapid and efficient recombination. Inteins were first discovered in molds and yeasts, and have since been found in various microorganisms such as bacteria, viruses, and archaea. Inteins exist as intramolecular and intermolecular protein linkages. Intermolecular inteins can be naturally separated or artificially separated intramolecular inteins. When the N-terminus and C-terminus of an intein are linked to the N-terminal and C-terminal portions of the target protein, respectively, the electrophilic group at the C-terminus of the intein attacks the nucleophilic group at the N-terminus, forming a covalent link and, through conformational changes, forming a complete N-terminal and C-terminal linker protein of the target protein. In molecular design, the first amino acid of the C-terminal portion of the target protein should be serine, threonine, or cysteine, etc. However, the efficacy shown by the existing natural cleavage sites of the OTOF protein has not reached the expected level, and it is necessary to try to find new cleavage sites.

[0084] Intended peptides

[0085] Inteins can splice proteins and function by covalently linking two different proteins, either post-translational or concurrently. The earliest inteins were discovered in fungi. Through comparison and analysis of intein sequences, the number of intein genes predicted to exist in viruses, bacteria, archaea, and eukaryotic microorganisms exceeds 600. Most inteins are complete proteins, but a small subset are N-terminal and C-terminal fragments, each linked to a portion of a protein. These fragments then recombine post-translationally, producing complete proteins through nucleophilic chemistry and allosteric transformation.

[0086] In this invention, preferably, the integrins are separated at the N-terminus and C-terminus. The integrins may be derived from MxeGyrA, pabPolIII, MjaKlbA, SspDnaB, SceVMA, SspDnaE, ​​NpuDnaE, ​​AvaDnaE, ​​CraDnaE, ​​CspDnaE, ​​CwaDnaE, ​​MchtDnaE, ​​OliDnaE, ​​TerDnaE, ​​gp41-1, gp41-8, IMPDH-1, or RmaDnaB.

[0087] In some embodiments, the integrin is an RmaDnaB integrin, for example, having the N-terminal portion of the RmaDnaB integrin shown in SEQ ID NO:2 and the C-terminal portion of the RmaDnaB integrin shown in SEQ ID NO:3. In some embodiments, the integrin is an NpuDnaE integrin, for example, having the N-terminal portion of the NpuDnaE integrin shown in SEQ ID NO:4 and the C-terminal portion of the NpuDnaE integrin shown in SEQ ID NO:5.

[0088] OTOF protein cleavage

[0089] In some specific implementations, the pre-mutant wild-type human OTOF protein contains or is composed of the amino acid sequence of SEQ ID NO:1.

[0090] This invention generates new C / S / T sites by mutating the wild-type human OTOF protein sequence shown in SEQ ID NO:1. These mutated sites are used as the first amino acid of the C-terminal portion of the OTOF protein, and the portion preceding them is the N-terminal portion. This divides the OTOF protein into the N-terminal portion (also referred to herein as the "N-terminus of OTOF") and the C-terminal portion (also referred to herein as the "C-terminus of OTOF," the first amino acid residue of which is the mutated C, S, or T). The N-terminus of OTOF is the sequence from the N-terminus of the OTOF amino acid sequence to the cleavage site, and the C-terminus of OTOF is the sequence from the mutated amino acid residue C, S, or T immediately adjacent to the cleavage site to the C-terminus of the OTOF amino acid sequence. The N-terminus of OTOF is then linked and fused to the N-terminus of an inteptide, and the C-terminus of the inteptide is linked and fused to the C-terminus of OTOF.

[0091] vector plasmid

[0092] The vector plasmid of the present invention can be any plasmid capable of replicating and expressing the corresponding polypeptide in a host cell.

[0093] In some implementations, the vector plasmid contains two ITR sequences, namely a 5' inverted terminal repeat (5'ITR) sequence and a 3' inverted terminal repeat (3'ITR) sequence.

[0094] In some embodiments, in the dual-vector system for expressing Otoferlin protein of the present invention, a first nucleotide sequence is inserted into a plasmid containing two first ITR sequences, and a second nucleotide sequence is inserted into a plasmid containing two second ITR sequences. For example, the plasmid containing the two first ITR sequences and the plasmid containing the two second ITR sequences may be the same or different. For example, the plasmid is pAAV, pAAV-CMV, pX601, pX551, or pAAV-MCS plasmid.

[0095] promoter

[0096] Currently, the vectors commonly used in inner ear gene therapy to promote the expression of specific transgenes in hair cells are generally broad and cannot target specific cell types.

[0097] To address the shortcomings of existing technologies, this invention provides an expression cassette containing a specific promoter. In one embodiment, this invention provides a polynucleotide (SEQ ID NO:45) containing the Otov-HC promoter region and truncated fragments thereof (e.g., SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:8, or their functional equivalents, e.g., nucleotide sequences having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or greater identity with SEQ ID NO:46, SEQ ID NO:47, or SEQ ID NO:8), and an expression cassette containing this promoter region, which can be used to promote the expression of a target transgene in inner hair cells. The polynucleotide described herein is operatively linked to a transgene, such as a transgene encoding a therapeutic protein, thereby promoting inner hair cell-specific expression of the transgene. The operative linking of the polynucleotide described herein to a therapeutic transgene provides a theoretical basis for clinical targeted therapy of hereditary hearing loss.

[0098] This invention provides the application of polynucleotides in the Otov-HC promoter region and truncated fragments therein in the treatment of sensorineural hearing loss. Studies have shown that the application of this promoter can promote the expression of the target transgene in cochlear hair cells, providing a reference for clinical application.

[0099] In one implementation, the Otov-HC promoter, as shown in SEQ ID NO:8, is used.

[0100] Dual-carrier system

[0101] This invention provides a dual-vector system comprising a first nucleic acid vector and a second nucleic acid vector, wherein:

[0102] The first nucleic acid vector contains, in the 5'-3' direction: a 5' inverted terminal repeat (5'ITR) sequence, a nucleic acid sequence encoding the N-terminal portion of the Otoferlin protein, a nucleic acid sequence encoding the N-terminal portion of the inteptide, and a 3' inverted terminal repeat (3'ITR) sequence;

[0103] The second nucleic acid vector contains, in the 5'-3' direction: a 5' ITR sequence, a nucleic acid sequence encoding the C-terminal portion of an intapeptide, a nucleic acid sequence encoding the C-terminal portion of an Otoferlin protein, and a 3' ITR sequence.

[0104] Optionally, after the first nucleic acid vector and the second nucleic acid vector are introduced into a host cell, the N-terminal portion and the C-terminal portion of the Otoferlin protein are effectively linked to generate the Otoferlin protein.

[0105] In some embodiments, the present invention provides a dual-vector system comprising a first nucleic acid vector and a second nucleic acid vector, wherein:

[0106] The first nucleic acid vector contains, in the 5'-3' direction: a 5' inverted terminal repeat (5'ITR) sequence, a nucleic acid sequence encoding the N-terminal portion of the Otoferlin protein, a nucleic acid sequence encoding the N-terminal portion of the inteptide, and a 3' inverted terminal repeat (3'ITR) sequence;

[0107] The second nucleic acid vector contains, in the 5'-3' direction: a 5' ITR sequence, a nucleic acid sequence encoding the C-terminal portion of an intapeptide, a nucleic acid sequence encoding the C-terminal portion of an Otoferlin protein, and a 3' ITR sequence, as well as...

[0108] The Otoferlin protein has an Otoferlin cleavage site in its amino acid sequence. For example, the amino acid sequence of the Otoferlin protein is as shown in the mutated SEQ ID NO:1 or its functional fragment, for example, an amino acid sequence with at least 80% sequence identity with SEQ ID NO:1, for example, an amino acid sequence with at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity with SEQ ID NO:1.

[0109] The N-terminal portion of the Otoferlin protein is the sequence from the N-terminus of the Otoferlin amino acid sequence to the Otoferlin cleavage site;

[0110] The C-terminal portion of the Otoferlin protein is the sequence from the mutated amino acid one position after the Otoferlin cleavage site to the C-terminus of the Otoferlin amino acid sequence.

[0111] Optionally, after the first nucleic acid vector and the second nucleic acid vector are introduced into cells, the N-terminal portion and the C-terminal portion of the Otoferlin protein are effectively linked to generate a full-length Otoferlin protein.

[0112] In some embodiments, the nucleotide sequences of the ITRs in the dual-vector system are derived from the same AAV serotype or different AAV serotypes, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9 serotypes. In some embodiments, the 5'-ITR and 3'-ITR of the first nucleic acid vector and the 5'-ITR and 3'-ITR of the second nucleic acid vector are derived from the same AAV serotype. In some embodiments, the 5'-ITR and 3'-ITR of the first nucleic acid vector and the 5'-ITR and 3'-ITR of the second nucleic acid vector are derived from different AAV serotypes.

[0113] In some implementations, tissue-specific promoters are used in the dual-vector system. For example, promoters that mediate expression in the ear, such as synaptic protein promoters, GFAP promoters, polynucleotides in the Otov-HC promoter region, and truncated fragments thereof.

[0114] In some implementations, the dual-vector system uses any of the following promoters: cytomegalovirus (CMV) promoter, SV40 promoter, Rous sarcoma virus (RSV) promoter, CAG promoter, chimeric CMV / chicken β-actin (CBA) promoter, truncated CBA (smCBA) promoter, UbC promoter, SFFV promoter, EF1α promoter, PGK promoter, or promoters encoding genes of Myo7A, Myo15, Atoh1, POU4F3, Lhx3, Myo6, α9AchR, α10AchR, OTOF, and STRC.

[0115] The dual-vector system of the present invention may also contain one or more other regulatory sequences that can function before or after transcription. The regulatory sequences may be part of a natural transgenic locus or may be heterologous regulatory sequences. The dual-vector system of the present invention may contain a portion of the 5'UTR or 3'UTR of a natural transgenic transcript.

[0116] Regulatory sequences can be any sequence that promotes transgene expression, i.e., it is used to increase transcript expression, improve nuclear export of mRNA, or enhance its stability. Such regulatory sequences include, for example, enhancer elements, post-transcriptional regulatory elements, and polyadenylation sequences.

[0117] Enhancers are cis-regulatory elements that affect the transcription of genes on the same DNA molecule. Enhancers can be located upstream, downstream, within introns, or even relatively far from the genes they regulate.

[0118] The preferred post-transcriptional regulatory element used in the dual-vector system of this invention is the marmot hepatitis post-transcriptional regulatory element (WPRE) or a variant thereof. Compared to AAV vectors without WPRE or its variants, AAV vectors containing WPRE or its variants increase the expression of Otoferlin protein.

[0119] In one embodiment, the dual-vector system of the present invention comprises the WPRE nucleotide sequence shown in SEQ ID NO:19. In another embodiment, the dual-vector system of the present invention comprises a post-transcriptional regulatory element having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the WPRE nucleotide sequence shown in SEQ ID NO:19, wherein the nucleotide sequence substantially preserves the functional activity of the post-transcriptional regulatory element as shown in SEQ ID NO:19, for example, a truncated variant of WPRE. Reducing the size of the AAV genome allows for increased flexibility in introducing other regulatory elements into the vector besides transgenes. In one embodiment, the truncated variant of WPRE has the WPRE3 nucleotide sequence shown in SEQ ID NO:22.

[0120] In one embodiment, the dual-vector system of the present invention comprises a polyadenylated sequence, such as a bovine growth hormone polyadenylated sequence, an SV40 polyadenylated sequence, and / or an SV40 late polyadenylated sequence. In one embodiment, the dual-vector system of the present invention comprises an SV40 polyadenylated sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the nucleotide sequence shown in SEQ ID NO:20. In one embodiment, the dual-vector system of the present invention comprises an SV40 late polyadenylated sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the nucleotide sequence shown in SEQ ID NO:23.

[0121] In some embodiments, the dual-vector system of the present invention comprises a combination of a WPRE nucleotide sequence and an SV40 polyadenylated sequence. For example, the dual-vector system of the present invention has the nucleotide sequence shown in SEQ ID NO:18 or a nucleotide sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:18. The combination of the WPRE nucleotide sequence and the SV40 polyadenylated sequence enables high-level expression of transgenes.

[0122] In some embodiments, the dual-vector system of the present invention comprises a combination of the WPRE3 nucleotide sequence and the SV40 late polyadenylation sequence. For example, the dual-vector system of the present invention has the nucleotide sequence shown in SEQ ID NO:21 or a nucleotide sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:21. The combination of the WPRE3 nucleotide sequence and the SV40 late polyadenylation sequence (also referred to as "W3SL") can efficiently express larger exogenous genes and occupy less AAV packaging capacity.

[0123] This invention utilizes a dual-vector system to deliver the Otoferlin protein gene in two parts to inner ear cells, inner hair cells, or outer hair cells. There, the N-terminal and C-terminal portions of the expressed Otoferlin protein undergo trans-splicing to form the full-length Otoferlin protein. This invention demonstrates that the dual-vector system for expressing Otoferlin protein can effectively transduce the targeted inner ear cells, inner hair cells, or outer hair cells, producing Otoferlin protein in these cells and persistently restoring hearing loss caused by Otoferlin gene knockout.

[0124] In a preferred embodiment, the dual-vector system of the present invention allows the expression of homologous polypeptides with an amino acid sequence having at least 70% identity and / or similarity to SEQ ID NO:1. The homologous sequence more preferably has at least 75%, even more preferably at least 80%, or at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 99%, or at least 99% identity and / or similarity to SEQ ID NO:1. When the homologous polypeptide is much shorter than SEQ ID NO:1, local alignment can be considered.

[0125] In another embodiment, the dual-vector system of the present invention can allow expression of functional fragments of the Otoferlin protein polypeptide. The term "functional fragment" herein refers to any fragment that retains at least one biological function of the target Otoferlin protein polypeptide.

[0126] The full-length Otoferlin protein can be obtained by transforming host cells using the dual-vector system described in this invention. In some embodiments, the host cells are selected from HeLa-S3 cells, HEK-293 cells, HEK-293T cells, HEK-293FT cells, A549 cells, and Sf9 cells.

[0127] III. Applications of Dual-Carrier Systems

[0128] The dual-vector system of the present invention is intended for use in patients with DFNB9-induced hearing loss. "Patients with DFNB9-induced hearing loss" refers to such patients, particularly human patients, who are believed to have (or have been diagnosed with) a mutation in the gene encoding the OTOF protein, which triggers abnormal expression, abnormal function, or both of the OTOF protein.

[0129] In some embodiments, the dual-carrier system of the present invention is a dual AAV carrier system. In some embodiments, the first AAV carrier and the second AAV carrier in the dual AAV carrier system are carriers each having a capsid with the same or different AAV sources. For example, the first AAV carrier and the second AAV carrier in the dual AAV carrier system are carriers each having a capsid with AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV-Anc80, or AAV carriers with interlocking capsids, particularly AAV carriers with an AAV-Anc80 capsid.

[0130] In some embodiments, the capsid protein of the AAV vector of the present invention is the capsid protein VP1 of the adeno-associated virus isolate Anc80L65 used in the treatment of sensorineural hearing loss, the amino acid sequence of the Anc80L65 protein capsid being shown in SEQ ID NO:9.

[0131] By using a dual AAV vector strategy to deliver a healthy copy of the Otof gene to the sensory cells of the cochlea, and then injecting the gene therapy into the cochlea, the aim is to restore long-term physiological hearing by promoting the expression of normal functional OTOF protein in the affected cochlear cells after drug administration.

[0132] The dual AAV vector system treats OTOF mutation-associated autosomal recessive deafness 9 (DFNB9) disease by increasing the expression of functional OTOF protein or by providing functional OTOF protein to the subject after administration.

[0133] When administered to a subject, the dual-carrier system of the present invention can trigger the expression of a functional full-length OTOF protein polypeptide or a functional fragment thereof in inner ear cells, inner hair cells or outer hair cells.

[0134] The patients to whom the dual-carrier system of the present invention is administered are preferably newborn infants, typically less than 6 months of age, or even less than 3 months of age (if they are diagnosed with DFNB9 deafness in early childhood). These infants are more preferably between 3 months and 1 year of age.

[0135] The dual-carrier system of the present invention can also be applied to, for example, toddlers (2-6 years old), children (6-12 years old), adolescents (12-18 years old) or adults (18 years old and above).

[0136] As used herein, the term "treatment" is intended to refer to the application of a therapeutically effective amount of the dual-carrier system of the present invention to a patient with DFNB9-induced hearing loss in order to partially or completely restore the patient's hearing. This restoration can be assessed by testing the auditory brainstem response (ABR) using electrophysiological equipment. "Treatment for OTOF mutation-induced hearing loss" specifically refers to complete restoration of hearing function. The term "prevention" refers to reducing or delaying hearing loss within the auditory frequency range.

[0137] Example

[0138] Example 1: Designing mutations in the amino acid sequence of the full-length Otoferlin protein

[0139] In this embodiment, a new C / S / T site was generated by designing mutations in the human wild-type Otof amino acid sequence (see column 4 of Table 1) for integrity splicing.

[0140] Figure 1 illustrates the intein-mediated expression of the full-length Otoferlin protein. The N-terminal coding sequence of Otoferlin is the nucleotide coding sequence from the N-terminus of the Otoferlin amino acid sequence to the cleavage site, and the C-terminal coding sequence is the nucleotide coding sequence from the amino acid following the cleavage site to the C-terminus. The N-terminal sequence of Otoferlin (OTOF-N) is fused with the N-terminal sequence of the intein (N-intein), and the C-terminal sequence of the intein (C-intein) is fused with the C-terminal sequence of Otoferlin (OTOF-C). A scheme was designed to mutate the first amino acid residue at the C-terminus of Otoferlin to Cys, Ser, or Thr, as shown in column 4 of Table 1.

[0141] Table 1. Otoferlin protein cleavage scheme designed based on SEQ ID NO:1.

[0142] Figures 3-24 show the various segmentation schemes of the Otoferlin protein designed based on SEQ ID NO:1 in Table 1.

[0143] Example 2. Construction of a dual-plasmid vector using a plasmid containing an ITR sequence

[0144] Using pAAV-CMV-EGFP-WPRE-SV40 plasmid (synthesized by Nanjing GenScript, Figure 2) as the plasmid backbone, a first plasmid vector expressing the N-terminus of Otoferlin protein and a second plasmid vector expressing the C-terminus of Otoferlin protein were constructed.

[0145] Specifically, the CMV promoter sequence of the pAAV-CMV-EGFP-WPRE-SV40 plasmid was replaced with the Otov-HC promoter sequence shown in SEQ ID NO:8 by double digestion with MluI and HindIII, and the EGFP reporter gene sequence of the pAAV-CMV-EGFP-WPRE-SV40 plasmid was replaced with the desired target sequence by double digestion with EcoRI and EcoRV. The synthesis of the target sequence and the construction of the vector were commissioned to Nanjing GenScript Biotech Co., Ltd., resulting in the first and second plasmid vectors.

[0146] Figures 3-24 show the expression cassettes in the first and second plasmid vectors constructed in various Otoferlin protein splitting schemes, respectively.

[0147] For example, for segmentation scheme 1 in Table 1, the expression cassette in the first plasmid vector contains the element shown in the small figure in Figure 3, wherein an N-intein coding sequence is connected after the N-Otof (i.e., the N-terminal part of Otof, which can also be represented as 5'Otof) coding sequence.

[0148] The expression cassette in the second plasmid vector contains the elements shown in the lower inset of Figure 3, wherein a C-Otof (i.e., the C-terminal portion of Otof, also represented as 3'Otof) coding sequence is attached after the C-intein coding sequence. An HA tag (HA tag sequence YPYDVPDYA (SEQ ID NO:16)) is used to verify in vitro expression.

[0149] Twenty-two pairs of dual-plasmid vectors corresponding to the various segmentation schemes in Table 1 were obtained (Table 2).

[0150] Table 2. Names of 22 pairs of dual-plasmid vectors and their expected translation products.

[0151] In addition, using the 866th position of the wild-type Otoferlin protein as the cleavage site and the 867th position of the wild-type Otoferlin protein as Ser, the first plasmid vector pAAV-Otov-HC-Otof-wt-N-inteptide plasmid was prepared similarly, with the expected translation product being Otof 1-866-N-terminal inteptide; and the second plasmid vector pAAV-Otov-HC-C-inteptide-Otof-wt plasmid, with the expected translation product being C-terminal inteptide-Otof 867-1997.

[0152] Example 3. Screening for mutated Otoferlin protein cleavage sites

[0153] The 22 pairs of first and second plasmid vectors obtained in Example 2, as well as a pair of first and second plasmid vectors with the 866th position of wild-type Otoferlin protein as the cleavage site, were transfected into HEK-293T cells (cells purchased from ATCC). Forty-eight hours after transfection, the expression of full-length Otoferlin protein was analyzed by Western blotting. The specific experimental methods are as follows.

[0154] Cell transfection: Seed HEK-293T cells (Human Embryonic Kidney 293T cells, hereinafter abbreviated as "293T cells") to a density of 70-90% for transfection. Prepare tube A: 125 μL serum-free DMEM medium + 8 μL Lipofectamine 3000 reagent (Invitrogen, catalog number: L3000015), mix thoroughly. Prepare tube B: 125 μL serum-free DMEM medium + 2 μg of the first plasmid vector + 2 μg of the second plasmid vector + 8 μL of P3000 reagent (Invitrogen, catalog number: L3000015), mix thoroughly. Add the mixture from tube B to tube A, gently mix thoroughly, and incubate at room temperature for 10-15 min to obtain the DNA-liposome complex. Tube A contains a mixture of culture medium and Lipofectamine 3000 transfection reagent, while tube B contains a mixture of culture medium, plasmid vector DNA, and transfection enhancer P3000. The obtained DNA-liposome complex was added to 293T cells for transfection. The cells were incubated at 37°C, 95% air, and 5% CO2 for 48 hours. After transfection, the culture medium was removed, and the cells were washed 2-3 times with 1 mL of PBS. The cells were then gently dispersed with 1 mL of PBS, and the cell suspension was collected in a centrifuge tube. After centrifugation at 700g for 5 minutes, the supernatant was discarded, and the cells were harvested by centrifugation.

[0155] Protein extraction: After transfection, the cell pellet harvested by centrifugation was resuspended in 120 μL of RIPA lysis buffer (Thermo Fisher Scientific, catalog number: 89900) (containing a 1% protease inhibitor cocktail (Thermo Fisher Scientific, catalog number: 87786), 1% PMSF) for 30 min on ice, vortexing every 10 min to ensure complete resuscitation. Centrifuge at 12000 rpm for 15 min and collect the supernatant (avoiding aspirating the pellet). Add 5X loading buffer (GenScript), incubate in a 75°C metal bath for 15 min, cool on ice, centrifuge at 12000 rpm for 2 min, and then collect the supernatant for Western blot analysis. The loading volume was 15 μL per 15-well gel.

[0156] Western blot detection of Otoferlin protein expression: Wash the glass plates and fix them flat on the rack, clamping them with the concave side facing inward, placing the two plates symmetrically. Prepare the separating gel, seal with isopropanol, and after 0.5 h, pour off the isopropanol and aspirate dry with a pump while the plate is placed on its side. Prepare the stacking gel, adding until overflowing, and insert the comb. After 45 min, remove the gel plate and attach it to the clamp of the electrophoresis tank. Add running buffer from the center of the tank until it overflows to 1 / 2 of the tank volume, and carefully pull out the comb. Load the sample and perform electrophoresis at a constant voltage of 100V for 1 h (Beyotime Biotechnology, 4-12%, Hepes system). After electrophoresis, remove the gel, separate the two glass plates with a spatula, and cut off the upper gel layer.

[0157] Prepare 1x transfer buffer and pre-cool. Equilibrate the lower gel layer in the 1x transfer buffer; soak the PVDF membrane in methanol (activate for 3 min) and place it in the 1x transfer buffer. With the black side of the transfer clamp facing down, arrange the membrane in the following order: sponge—1 layer of filter paper—gel—PVDF membrane—1 layer of filter paper—sponge, expel air with a spatula, and clamp tightly (avoid air bubbles); after assembling the transfer apparatus, pour the 1x transfer buffer into the electrophoresis tank; transfer at a constant current of 300mA for 90 min on ice.

[0158] After transfer, the PVDF membrane was removed and incubated for 1 hour at room temperature on a shaker using blocking buffer (5% skim milk powder, prepared with TBST buffer). The blocked PVDF membrane was then incubated overnight at 4°C with the primary antibody (anti-otoferlin (SC-271092) mouse IgG1 antibody (Santa Cruz, 1:500); β-Actin mouse mAb, Cell Signaling Technology (1:20000), catalog number: 8H10D10). Anti-otoferlin was used as the primary antibody to detect the expression of full-length Otoferlin protein, and β-actin antibody was used as the primary antibody to detect the level of β-actin, the internal control protein in Western blotting. The protein level of β-actin typically does not change, therefore it can be used to detect whether the loading amount is consistent during Western blotting.

[0159] The next day, remove the incubated PVDF membrane, rinse three times with 1X TBST for 10-15 minutes each time, and incubate it on a shaker with the secondary antibody (HRP-conjugated Affinipure Goat Anti-Mouse IgG (H+L), Proteintech, catalog number: SA00001-1) (1:5000) for 1-2 hours at room temperature, rinsing three times with 1X TBST for 10-15 minutes each time. Then, develop the membrane with Novizan developing solution, observe the results and take pictures in a Western spectroscopy imaging system.

[0160] Table 2 shows the target protein expression results after transfecting 293T cells with 22 pairs of first and second plasmid vectors, as illustrated in Figure 25. In Figure 25, lane "Ctrl" represents the HEK-293T cell protein control without plasmid transfection; lane "FL" represents the molecular weight marker of full-length Otoferlin protein; lanes "1" to "22" correspond to the protein expression after transfecting 293T cells with 22 pairs of first and second plasmid vectors as shown in Table 2, respectively; and lane "wt" corresponds to the protein expression after transfecting 293T cells with the first and second plasmid vectors using the 866th position of wild-type Otoferlin protein as the cleavage site.

[0161] Western blot analysis in Figure 25 shows that lanes 1, 4, 5, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, and wt express full-length otoferlin protein. Lanes 16, 17, and 18 show significantly higher levels of full-length otoferlin protein expression, indicating that the splitting schemes 16, 17, and 18 enable efficient recombination of the corresponding paired plasmids in cells, demonstrating excellent application potential and serving as candidate sites for dual AAV vector therapy.

[0162] Example 4. Preparation of dual AAV carriers

[0163] Prepare HEK-293T cells: passage at a ratio of 1:3 and culture in 150mm sterile dishes up to 8 plates, with a cell density of 80%-90%.

[0164] Prepare DMEM, PEI, N-Otof and C-Otof plasmids from Example 2, pRC plasmid (containing the VP1 gene of adeno-associated virus Anc80L65, GenBank: KT235804.1), and pHelper plasmid (GenBank: AF369965.1).

[0165] Prepare three sterile 15ml EP tubes. Label the first tube A and add 3.444ml of DMEM and 0.756ml of PEI (1μg / μl) to tube A. Let it stand for 5 minutes. Label the remaining two tubes B1 and B2. Add 7μg of N-Otof plasmid or C-Otof plasmid, 9μg of Anc80L65 plasmid, and 14μg of pHelper plasmid to each tube, respectively. Add DMEM to a final volume of 2.1ml.

[0166] Add the liquid from tube A to tubes B1 and B2 dropwise, 2.1 ml per tube. Incubate at room temperature for 20-25 minutes, then add the mixture to the prepared dish of HEK-293T cells. After 12 hours, discard the culture medium and add 20 ml of 293T cell transfection medium to each dish. After 48 hours, collect the supernatant into a sterile bottle and store at 4°C. Add 20 ml of 293T transfection medium to each dish. After another 48 hours of culture, collect the cells and supernatant into a sterile bottle for virus purification and titer detection. The titer was determined by virus inverted terminal repeat-specific qPCR.

[0167] The virus purification steps are as follows:

[0168] 1) Place the collected supernatant and cells in a 50 mL centrifuge tube (chloroform resistant) and centrifuge for 7 min at 11000 r / min, 4℃. Aspirate the supernatant and resuspend a portion of the cell pellet. Add 1 / 10 volume of chloroform and shake at 37℃, 220 r / min for 3 h.

[0169] 2) At 11000 r / min, 4℃, for 20 min, collect the supernatant and mix it with the supernatant collected in the previous step.

[0170] 3) Add solid NaCl to a concentration of 1 mol / L, add PEG8000 to a final concentration of 10% (w / v), dissolve completely, and let stand overnight at 4°C.

[0171] 4) Centrifuge at 11000 r / min, 4℃ for 20 min, remove most of the supernatant, leave 1-2 mL to resuspend all the precipitate, transfer the remaining precipitate to several new 2 mL EP tubes, and then centrifuge the 2 mL EP tubes containing the precipitate at 12000 r / min for 7 min.

[0172] 5) Add 700 μl of enzymatic hydrolysate (containing Dnase I and Rnase) to the precipitate, and incubate in a water bath at 37°C for 1 hour. During this time, you can mix the mixture by blowing and stirring multiple times.

[0173] 6) Add an equal volume of chloroform, shake vigorously, centrifuge at 12000 r / min for 7 min, and collect the supernatant.

[0174] 7) Add an equal volume of virus lysis buffer (20% PEG8000 + 2M NaCl + 0.002% F68 + PBS) and incubate overnight at 4°C.

[0175] 8) Centrifuge at 12000 r / min for 7 min, discard the supernatant, then centrifuge for another 3 min, and discard the supernatant.

[0176] 9) Dissolve the precipitate in a solution containing Dnase I and Rnase, add an appropriate amount of enzyme hydrolysate to dissolve it, and then pass it through chloroform to obtain the supernatant, which is the final virus.

[0177] 10) After leaving the 1.5ml EP tube containing the virus at room temperature for 30 minutes, it can be temporarily placed in a 4℃ refrigerator for subsequent titer testing.

[0178] The titer detection steps are as follows:

[0179] 1) Digest genomic DNA and plasmid DNA, incubate at 37°C for 20 min; then at 95°C for 10 min, using the following system:

[0180] (Genomic DNA and plasmid DNA were digested at 37°C; DNase I was inactivated by heat treatment at 95°C)

[0181] 2) Add an equal volume of lysis buffer (10 μl) and 2 μl of 20 mg / ml proteinase K, and incubate at 55°C for 30 min; then at 95°C for 10 min. (Lyslysis buffer: 10 mM Tris-HCl pH 8.0 + 5 mM EDTA + 100 μg / ml proteinase K) (store at room temperature). Take 4 μl of supernatant and dilute it 100-fold to 400 μl with ddH2O. Use 2 μl as a template for RT-qPCR. The primers for qPCR are designed on the ITR sequence. The primer sequences are: upstream primer F: 5'-GGAACCCCTAGTGATGGAGTT-3' (SEQ ID NO:43); downstream primer R: 5'-CGGCCTCAGTGAGCGA-3' (SEQ ID NO:44).

[0182] The PCR system is as follows:

[0183] The PCR program was as follows: 95℃, 5 min; 95℃, 10 s; 58℃, 30 s (40X); 95℃, 15 s; 60℃, 1 min; 95℃, 15 s.

[0184] Titer calculation formula: Titer = 1,000,000 * power(10) x )

[0185] The formula for calculating x is: x = (38.71 - y) / 3.54

[0186] Where y is the CT value, that is, the threshold cycle (Ct) in qPCR. The CT value can be derived after qPCR is completed. Substitute the CT value into x = (38.71-y) / 3.54 to calculate the x value.

[0187] For example, if the CT value of the qPCR result is 13.925 = y, substituting it into the above formula, then x = (38.71 - 13.925) / 3.54 = 7. Therefore, the viral titer value is = 1000000 * power(10 7 That is, 1.00E+13.

[0188] The adeno-associated virus (AAV) is named according to the name of the plasmid. For example, an adenovirus packaged using the pAAV-Otov-HC-Otof-N16-N-include peptide plasmid, pHelper plasmid, and pRC plasmid is named pAAV-Otov-HC-Otof-N16-N-include peptide plasmid AAV; an adenovirus packaged using the pAAV-Otov-HC-C-include peptide-Otof-C16 plasmid, pHelper plasmid, and pRC plasmid is named pAAV-Otov-HC-C-include peptide-Otof-C16 plasmid AAV.

[0189] Example 5: Recovery of deafness in Otof gene knockout mice by dual-vector AAV delivery

[0190] 5.1 Detection of hearing-related indicators in Otof gene knockout mouse model

[0191] OtofQ939* / Q939* mice were ordered from Cyagen Biosciences for breeding. To ensure that the Otof protein in the inner ear of the Otof knockout mice was indeed knocked out, homozygous and heterozygous Otof knockout mice were first screened by genotyping and compared with wild-type mice.

[0192] To explore the role of the Otof gene in the development of the inner ear of wild-type mice, the expression of Otof in the inner ear of wild-type mice was first examined. The results showed that Otof was abundantly expressed in the inner ear. To further investigate the specific expression sites and time spectrum of Otof in the basilar membrane of the inner ear, immunofluorescence staining was performed on wild-type mice at different time points after birth. Immunofluorescence staining was used to locate Otof expression in the inner ear using cochlear basilar membrane flaps. Myosin7a was used as a marker for hair cells. Four developmental time points were selected: day 0 (P0), day 7 (P7), day 14 (P14), and day 30 (P30). At the hair cell level, OTOF co-localized with Myosin7a in the basilar membrane flaps at all four developmental time points, indicating that OTOF is expressed in the cytoplasm of hair cells. These experiments demonstrate that OTOF is abundantly expressed in the inner ear, primarily in the inner hair cells.

[0193] 5.1.1 Immunofluorescence detection of Otof gene knockout mice

[0194] 1) Cut off the entire ear tissue of the mouse and put it into 1×HBSS. Dissect the temporal bone under a microscope, put it into 4% PFA, gently punch a hole at the top of the temporal bone with forceps, and repeatedly blow PFA into it with a syringe. Fix at room temperature for 1-2 hours.

[0195] 2) Soak the temporal bone in 0.5M EDTA for decalcification: P0-P7, decalcification for 3-4 hours; P8-P15, decalcification for 1 day; P15-P60, decalcification for 2 days.

[0196] 3) Rinse repeatedly with 1×PBST 3 times.

[0197] 4) Place the temporal bone in a 1×HBSS and dissect the cochlea under a microscope. Divide the entire sensory epithelial cell into three equal segments, label them as the top, middle and bottom of the cochlea, and preserve them in HBSS until staining.

[0198] 5) After coating the coverslip with Cell-tak, place it in the dish containing the cochlea and attach the cochlea to the coverslip (face up).

[0199] 6) Place the adhered slides into a 4-well dish (3 mL of PBS has been added beforehand). After all the adhered slides are in the 4-well dish, permeate them with PBS containing 1% Triton X-100 and 10% donkey serum at room temperature and block them for 1 hour.

[0200] 7) Dilute the primary antibody, otoferlin antibody (ab53233, 1:200, Abcam), and Myosin7a antibody (256790, 1:1000, Proteus) with PBT-1 according to the ratio, 80-100 μl per well, and incubate overnight at 4°C.

[0201] 8) Wash 3 times with 1×PBST, 5 min each time.

[0202] 9) Dilute the corresponding secondary antibody with PBT-2 according to the ratio, 80-100 μl per well, and incubate at room temperature in the dark for 1 h.

[0203] 10) Wash 3 times with 1×PBST, 5 min each time.

[0204] 11) Remove the slides and place them on a slide. Add 6 μl of DAKO to each slide, cover with a new slide, seal with nail polish, observe and take pictures.

[0205] 5.1.2 Auditory function testing in Otof gene knockout mice

[0206] The overall auditory function differences between Otof knockout mice (ordered from Cyagen Biosciences) and their littermate wild-type mice were detected using auditory brainstem response (ABR) and aberration product otoacoustic emission (DPOAE).

[0207] 1) Weigh the mouse and inject it intraperitoneally with 1% sodium pentobarbital according to the appropriate mass-to-volume ratio.

[0208] 2) After anesthetizing the mouse, place it in the soundproof enclosure of the TDT workstation. Insert the positive electrode subcutaneously along the midline of the mouse's brain, the negative electrode subcutaneously behind the ear, and the ground wire into the thigh muscle. Check the electrode indicator lights to confirm that the connection is normal.

[0209] 3) Open the test file on the computer, select the test frequency as 4-32kHz, and record the critical threshold above the noise level.

[0210] The hearing thresholds of mice to short pure tones (tone bursts) at 4kHz, 8kHz, 12kHz, 16kHz, 24kHz, and 32kHz, with a duration of 10ms, were measured. This allowed for the analysis of the mice's auditory sensitivity and a comprehensive assessment of whether the mice possessed normal auditory function from the hair cells to the cerebral cortex. ABR and DPOAE effectively reflect the integrity of cochlear hair cells and cochlear function.

[0211] 5.1.3 Electrophysiological function detection of Otof gene knockout mice

[0212] Using patch-clamp technology and whole-cell extracellular recording, electrochemical changes in hair cells in the cochlea of ​​Otof gene knockout mice were recorded, thereby detecting changes in the electrophysiological function of hair cells in Otof gene knockout mice.

[0213] 5.1.4 Otof gene knockout mice: Detection of hair cell count in Otof gene knockout mice

[0214] Otof protein is highly expressed in the inner hair cells of the mouse cochlea but lowly expressed in the outer hair cells. The number of surviving inner ear hair cells from Otof gene knockout mice at different time points (P0, P7, P14, P30) was detected by immunofluorescence staining.

[0215] 1) Cut off the entire ear tissue of the mouse and put it into 1×HBSS. Dissect the temporal bone under a microscope, put it into 4% PFA, gently punch a hole at the top of the temporal bone with forceps, and repeatedly blow PFA into it with a syringe. Shake on a shaker for 1-2 hours.

[0216] 2) Decalcify the temporal bone in 0.5M EDTA: P0-P7, decalcification for 3-4 hours; P8-P15, decalcification for 1 day; P15-P30, decalcification for 2 days.

[0217] 3) Rinse repeatedly with 1×PBST 3 times.

[0218] 4) The temporal bone was placed in a 1×HBSS and the cochlea was dissected under a microscope.

[0219] 5) After coating the coverslip with Cell-tak, place it in the dish containing the cochlea and attach the cochlea to the coverslip (face up).

[0220] 6) Place the adhered slides into a 4-well dish (3 mL of PBS has been added previously). After all the adhered slides are in the 4-well dish, add 1 mL of 16% PFA.

[0221] 7) Fix at room temperature for 1 hour, then wash 3 times with PBST.

[0222] 8) Add 100 μL of sealing solution to each well and seal for 1 hour.

[0223] 9) Dilute the primary antibody with PBT-1 proportionally, 80-100 μL per well, and incubate overnight at 4°C.

[0224] 10) Wash 3 times with 1×PBST, 5 min each time.

[0225] 11) Dilute the secondary antibody with PBT-2 according to the ratio, 80-100 μL per well, and incubate at room temperature in the dark for 1 h.

[0226] 12) Wash 3 times with 1×PBST, 5 min each time.

[0227] 13) Remove the slides and place them on a slide. Add 6 μL of DAKO to each slide, cover with a new slide, seal with nail polish, observe and take pictures.

[0228] The above experimental results show that, using WT and Otof- / - mice at different time points (P0, P7, P14, P30, P30, P40), immunofluorescence staining revealed abundant Otof expression in the hair cell epidermal plate layer of wild-type mice, but undetectable OTOF expression in the inner ear hair cell epidermal plate layer of Otof- / - mice. This indicates that the OTOF protein in the inner ear of the Otof- / - mice used in this embodiment has been completely knocked out.

[0229] 5.2 Effects of AAV virus on Otof gene knockout mice

[0230] Otof- / - mice prepared in Example 5.1 were injected into the posterior semicircular canal (PSCC) at P30 (i.e., 30 days after birth). Specifically, mice were anesthetized with 1% sodium pentobarbital, and after anesthesia, the hair around the ear was removed, and an incision was made behind the ear to expose the semicircular canal. After the semicircular canal was perforated, manual injection was performed using a glass micropipette. 2 μL of a mixture of N-terminal AAV virus and C-terminal AAV virus prepared in Example 4 corresponding to each cleavage site was injected into the left cochlea of ​​each mouse (N-terminal virus:C-terminal virus = 1:1 for each cleavage site, N-terminal virus titer was 8.5 e). 12 vg / mL, C-terminal viral titer is 8.5e 12 The viral load was vg / mL, with a total viral load of 3.4 x 10^9 viruses injected into each mouse. 10 (vg). After injection, the skin incision was sealed with tissue glue. The injected mice were then placed on a 37°C heating pad for recovery. The mice fully recovered approximately 30 minutes post-surgery and were returned to their cages. Standard post-operative care was provided. Auditory brainstem response (ABR) was used to assess the recovery of auditory function in the gene-treated mice. Wild-type C57BL / 6 mice were used as control mice for auditory function analysis.

[0231] Next, the hearing status of Otof knockout mice was tested. First, the hearing of homozygous Otof knockout mice and wild-type control mice from the same littermate were tested using the ABR (auditory test). ABR is mainly used to detect whether the function of all parts during sound transmission is normal.

[0232] Experimental results show that the Otof gene plays an important regulatory role in the hearing of mice, especially in the function of inner hair cells.

[0233] To determine whether and to what extent hearing loss could be restored using the dual AAV vector system, auditory brainstem response (ABR) was used to assess the recovery of auditory function in Otof- / - mice after gene therapy two weeks after dual AAV vector injection. Furthermore, the cochlear sensory epithelium on the virus-injected side of the Otof- / - mice was microsurgically dissected after audiometry, and otoferlin expressed in inner hair cells and Myosin7a expressed in hair cells were immunolabeled to investigate the recombinant expression of the exogenous Otof gene in the cochlear cells of Otof gene knockout (ko) mice.

[0234] The auditory response threshold, latency, and inter-wave period were measured using ABR (Audio-Resonance Detection). Short sounds (clicks), especially short sounds of different frequencies (4kHz, 8kHz, 12kHz, 16kHz, 24kHz, and 32kHz), were used as stimuli to detect the hearing threshold of mice to different frequencies. The auditory sensitivity of the mice was analyzed to determine whether the mice had normal auditory function from hair cells to the cerebral cortex as a whole.

[0235] A higher ABR threshold indicates more severe hearing loss in mice with the Otof gene mutation. Conversely, a lower ABR threshold indicates better efficacy of gene therapy.

[0236] As shown in Figure 26, in the treated Otof- / - knockout mouse model, the ABR threshold decreased and hearing in some frequency bands was significantly restored two weeks after dual AAV vector injection. In the control Otof- / - knockout mouse model, the hearing thresholds in the low, mid, and high frequencies were significantly increased, indicating that Otof plays an important role in mouse hearing. The dual AAV system prepared in Example 4 achieved the expression of otoferlin protein in the mouse cochlea, restored the release of synaptic vesicles in the inner ear hair cells, and restored hearing in both ears.

[0237] Two weeks after treatment, cochlear tissue was harvested from Otof- / - knockout mice for basilar membrane smear staining, as shown in Figure 27. Immunofluorescence results showed that both AAV systems successfully expressed otoferlin protein in the mouse cochlea.

[0238] The foregoing describes exemplary embodiments of the present invention. Those skilled in the art should understand that these disclosures are merely exemplary, and various other substitutions, adaptations, and modifications can be made within the scope of the present invention. Therefore, the present invention is not limited to the specific embodiments listed herein.

[0239] Exemplary sequence

[0240] The amino acid sequence of wild-type Otoferlin (SEQ.ID NO.1)

[0241] The amino acid sequence of the N-terminal portion of the RmaDnaB inteptide (SEQ.ID NO:2)

[0242] The amino acid sequence of the C-terminal portion of the RmaDnaB inteptide (SEQ.ID NO:3)

[0243] The amino acid sequence of the N-terminal portion of the NpuDnaE intima-peptide (SEQ ID NO:4)

[0244] The nucleotide sequence of the N-terminal portion of the NpuDnaE peptide is SEQ ID NO:5

[0245] The amino acid sequence of the C-terminal portion of the NpuDnaE intima-peptide is SEQ ID NO:6

[0246] The nucleotide sequence of the C-terminal portion of the NpuDnaE intima-peptide is SEQ ID NO:7

[0247] The nucleotide sequence of the Otov-HC promoter (SEQ.ID NO.8)

[0248] The amino acid sequence of the capsid protein VP1 of adeno-associated virus isolate Anc80L65 (SEQ.ID NO.9)

[0249] The amino acid sequence of OTOFERLIN protein from position 1 to 782 (SEQ.ID NO:10)

[0250] The amino acid sequence of OTOFERLIN protein from position 1 to 1081 (SEQ.ID NO:11)

[0251] The amino acid sequence of OTOFERLIN protein from position 1 to 1098 (SEQ.ID NO:12)

[0252] The amino acid sequence of OTOFERLIN protein from position 783 to 1997 (SEQ.ID NO:13, Gly783Ser)

[0253] The amino acid sequence of OTOFERLIN protein from position 1082 to 1997 (SEQ.ID NO:14, Asn1082Ser)

[0254] The amino acid sequence of OTOFERLIN protein from position 1099 to 1997 (SEQ.ID NO:15, Pro1099Ser)

[0255] HA-tagged amino acid sequence SEQ ID NO:16

[0256] The nucleotide sequence of the vector pAAV-CMV-EGFP-WPRE-SV40 plasmid shown in Figure 2 is as shown in SEQ ID NO:17.

[0257] Nucleotide sequence of WPRE+SV40poly(A)signal (717bp): SEQ ID NO:18

[0258] Nucleotide sequence of WPRE (589bp): SEQ ID NO:19

[0259] Nucleotide sequence of SV40poly(A) signal (122 bp): SEQ ID NO:20

[0260] Nucleotide sequence of WPRE3-SV40late poly(A) (432bp): SEQ ID NO:21

[0261] Nucleotide sequence of WPRE3: SEQ ID NO:22

[0262] Nucleotide sequence of the SV40late poly(A) signal: SEQ ID NO:23

[0263] Sequences in polyA

[0264] AATAAA (SEQ ID NO: 24), ATTAAA (SEQ ID NO: 25), AGTAAA (SEQ ID NO: 26), CATAAA (SEQ ID NO: 27), TATAAA (SEQ ID NO: 28), GATAAA (SEQ ID NO: 29), ACTAAA (SEQ ID NO: 30), AATATA (SEQ ID NO: 31), AAGAAA (SEQ ID NO: 32), AATAAT (SEQ ID NO: 33), AAAAAA (SEQ ID NO: 34), AATGAA (SEQ ID NO: 35), AATCAA (SEQ ID NO: 36), AACAAA (SEQ ID NO: 37), AATCAA (SEQ ID NO: 38), AATAAC (SEQ ID NO: 39), AATAGA (SEQ ID NO: 40), AATTAA (SEQ ID NO: 41) or AATAAG (SEQ ID NO: 42)

[0265] Upstream primer sequence for qPCR (SEQ ID NO:43)

[0266] Downstream primer sequence for qPCR (SEQ ID NO:44)

[0267] Downstream primer R: 5'-CGGCCTCAGTGAGCGA-3'

[0268] Nucleotide sequence of the Otov-HC promoter region (SEQ.ID NO.45)

[0269] The truncated nucleotide sequence 1 of the Otov-HC promoter region (SEQ.ID NO.46)

[0270] 2. Truncated nucleotide sequence of the Otov-HC promoter region (SEQ.ID NO.47)

Claims

1. A dual vector system for expressing Otoferlin protein, comprising a first nucleic acid vector and a second nucleic acid vector, wherein the first nucleic acid vector comprises a first nucleotide sequence; and the second nucleic acid vector comprises a second nucleotide sequence; the first nucleotide sequence comprises an expression cassette inserted between two first ITR sequences; the second nucleotide sequence comprises an expression cassette inserted between two second ITR sequences; the expression cassette of the first nucleotide sequence comprises a promoter, an N-terminal coding sequence of Otoferlin, an N-terminal coding sequence of an intein, and a polyA; the expression cassette of the second nucleotide sequence comprises a promoter, a C-terminal coding sequence of an intein, a C-terminal coding sequence of Otoferlin, and a polyA; and an Otoferlin cleavage site is provided in an Otoferlin amino acid sequence, e.g., the Otoferlin amino acid sequence is set forth in mutated SEQ ID NO: 1 or a functional fragment thereof, e.g., an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 1; the N-terminal coding sequence of Otoferlin is a nucleotide coding sequence from the N-terminus of the Otoferlin amino acid sequence to the Otoferlin cleavage site; and the C-terminal coding sequence of Otoferlin is a nucleotide coding sequence from a mutated amino acid one position after the Otoferlin cleavage site to the C-terminus of the Otoferlin amino acid sequence, wherein the mutated amino acid is an amino acid at the corresponding position of SEQ ID NO: 1 replaced with a serine (S), a threonine (T), or a cysteine (C).

2. The dual vector system for expressing Otoferlin protein according to claim 1, wherein, the Otoferlin cleavage site is at position 782, 785, 796, 803, 810, 838, 855, 916, 945, 946, 1081, 1098, 1099, 1100, or 1109 of the Otoferlin amino acid sequence according to the numbering of SEQ ID NO: 1, and the mutated amino acid is selected from 783S, 786C, 797C, 804S, 811S, 839S, 856S, 917S, 946S, 947S, 1082S, 1099S, 1100S, 1101S, and 1110S.

3. The dual vector system for expressing Otoferlin protein according to claim 1, wherein, the promoter of the expression cassette of the first nucleotide sequence or the second nucleotide sequence is selected from a polynucleotide of the Otov-HC promoter region and a truncated fragment thereof, a CAG promoter, a CMV promoter, a CBA promoter, a UbC promoter, an SFFV promoter, an EF1a promoter, a PGK promoter, or a promoter of a Myo7A, Myo15, Atoh1, POU4F3, Lhx3, Myo6, a9AchR, a10AchR, OTOF, and STRC encoding gene. the polyA of the expression cassette of the first nucleotide sequence or the second nucleotide sequence comprises AATAAA (SEQ ID NO: 24) and a variant of AATAAA; the variant of AATAAA comprises ATTAAA (SEQ ID NO: 25), AGTAAA (SEQ ID NO: 26), CATAAA (SEQ ID NO: 27), TATAAA (SEQ ID NO: 28), GATAAA (SEQ ID NO: 29), ACTAAA (SEQ ID NO: 30), AATATA (SEQ ID NO: 31), AAGAAA (SEQ ID NO: 32), AATAAT (SEQ ID NO: 33), AAAAAA (SEQ ID NO: 34), AATGAA (SEQ ID NO: 35), AATCAA (SEQ ID NO: 36), AACAAA (SEQ ID NO: 37), AATCAA (SEQ ID NO: 38), AATAAC (SEQ ID NO: 39), AATAGA (SEQ ID NO: 40), AATTAA (SEQ ID NO: 41), or AATAAG (SEQ ID NO: 42); for example, the polyA is a nucleotide sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the polyA signal sequence set forth in SEQ ID NO: 20 or SEQ ID NO: 23; and each of the two first ITR sequences and the two second ITR sequences is derived from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9.

4. The dual vector system for expressing Otoferlin protein according to claim 1, wherein, the expression cassette of the first nucleotide sequence or the second nucleotide sequence further comprises an expression regulatory element and / or a tag element, for example, the expression regulatory element is a woodchuck hepatitis post-transcriptional regulatory element (WPRE) or a variant thereof, preferably a WPRE truncated variant, for example, a nucleotide sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the nucleotide sequence set forth in SEQ ID NO: 19, for example, the nucleotide sequence set forth in SEQ ID NO: 22; for example, the tag element is HA.

5. The dual vector system for expressing Otoferlin protein according to claim 1, wherein, The intein is derived from Mxe GyrA, pab PolIII, Mja KlbA, Ssp DnaB, Sce VMA, Ssp DnaE, Npu DnaE, Ava DnaE, Cra DnaE, Csp DnaE, Cwa DnaE, Mcht DnaE, Oli DnaE, Ter DnaE, gp41-1, gp41-8, IMPDH-1 or Rma DnaB, for example, the intein is derived from Rma DnaB, for example, the N-terminus of the intein is the N-terminus of the Rma DnaB intein as shown in SEQ ID NO: 2, and the C-terminus of the intein is the C-terminus of the Rma DnaB intein as shown in SEQ ID NO: 3; or, the intein is derived from Npu DnaE, for example, the N-terminus of the intein is the N-terminus of the Npu DnaE intein as shown in SEQ ID NO: 4, and the C-terminus of the intein is the C-terminus of the Npu DnaE intein as shown in SEQ ID NO:

6.

6. The dual vector system for expressing Otoferlin protein according to claim 1, wherein, The first nucleotide sequence is inserted into a plasmid comprising two first ITR sequences, and the second nucleotide sequence is inserted into a plasmid comprising two second ITR sequences, for example, the plasmid comprising two first ITR sequences and the plasmid comprising two second ITR sequences are the same or different, for example, the plasmid is a pAAV, pAAV-CMV, pX601, pX551 or pAAV-MCS plasmid.

7. The dual vector system for expressing Otoferlin protein according to any one of claims 1-6, wherein, The first nucleotide sequence is constructed by connecting and fusing the N-terminal coding sequence of Otoferlin with the N-terminal coding sequence of the Rma DnaB intein using the Otoferlin protein cleavage scheme shown in Table 1, and the second nucleotide sequence is constructed by connecting and fusing the C-terminal coding sequence of the Rma DnaB intein with the C-terminal coding sequence of Otoferlin, for example, using a pAAV-CMV plasmid as the vector; or using a plasmid in which the CMV promoter in the pAAV-CMV plasmid is replaced by a polynucleotide or a truncated fragment thereof of the Otov-HC promoter region as the vector; For example, the N-terminal coding sequence of the Rma DnaB intein encodes the Rma DnaB N-terminal portion as shown in SEQ ID NO: 2, and the C-terminal coding sequence of the Rma DnaB intein encodes the Rma DnaB C-terminal portion as shown in SEQ ID NO:

3.

8. The dual vector system for expressing an Otoferlin protein according to any one of claims 1-7, wherein, The expression cassette of the first nucleotide sequence comprises a promoter, the coding sequence of the N-terminus of Otoferlin as shown in SEQ ID NO: 10, the N-terminal coding sequence of the intein, and a polyA; and the expression cassette of the second nucleotide sequence comprises a promoter, the C-terminal coding sequence of the intein, the coding sequence of the C-terminus of Otoferlin as shown in SEQ ID NO: 13, and a polyA. The expression cassette of the first nucleotide sequence comprises a promoter, the coding sequence of the N-terminus of Otoferlin as shown in SEQ ID NO: 10, the N-terminal coding sequence of the intein, and a polyA; and the expression cassette of the second nucleotide sequence comprises a promoter, the C-terminal coding sequence of the intein, the coding sequence of the C-terminus of Otoferlin as shown in SEQ ID NO: 13, and a polyA. the expression cassette of the first nucleotide sequence comprises a promoter, a coding sequence of an N-terminus of Otoferlin as shown in SEQ ID NO: 11, a coding sequence of an N-terminus of an intein, and a polyA; and the expression cassette of the second nucleotide sequence comprises a promoter, a coding sequence of a C-terminus of an intein, a coding sequence of a C-terminus of Otoferlin as shown in SEQ ID NO: 14, and a polyA; or the expression cassette of the first nucleotide sequence comprises a promoter, a coding sequence of an N-terminus of Otoferlin as shown in SEQ ID NO: 12, a coding sequence of an N-terminus of an intein, and a polyA; and the expression cassette of the second nucleotide sequence comprises a promoter, a coding sequence of a C-terminus of an intein, a coding sequence of a C-terminus of Otoferlin as shown in SEQ ID NO: 15, and a polyA. Preferably, the promoter is a polynucleotide of the Otov-HC promoter region and truncated fragments thereof, for example, a polynucleotide as shown in SEQ ID NO: 45 or a truncated fragment thereof, for example, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 8, or a functional equivalent thereof, for example, a nucleotide sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or greater identity to SEQ ID NO: 46, SEQ ID NO: 47, or SEQ ID NO: 8; more preferably, the promoter is the Otov-HC promoter as shown in SEQ ID NO:

8.

9. The dual vector system for expressing an Otoferlin protein according to any one of claims 1-8, wherein the expression cassette of the first nucleotide sequence and the expression cassette of the second nucleotide sequence each comprises a combination of a WPRE nucleotide sequence and a SV40 polyadenylation sequence at the N-terminus of a 3’ITR sequence, for example, having a nucleotide sequence as shown in SEQ ID NO: 18 or a nucleotide sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 18; or a combination of a WPRE3 nucleotide sequence and a SV40 late polyadenylation sequence, for example, having a nucleotide sequence as shown in SEQ ID NO: 21 or a nucleotide sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:

21.

10. A packaging vector system for adeno-associated virus, wherein, The packaged vector system comprises the dual vector system for expressing an Otoferlin protein according to any one of claims 1-9, a vector carrying AAV rep and cap genes, and a helper virus vector, packaged as an AAV vector, preferably, wherein the amino acid sequence of the Otoferlin protein is as shown in mutated SEQ ID NO:

1.

11. The packaging vector system of adeno-associated virus according to claim 10, wherein, The vector carrying AAV rep and cap genes is selected from 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 of packaging an adeno-associated virus, wherein, The packaging vector system of the adeno-associated virus according to claim 10 or 11 is transferred into a host cell for packaging.

13. The method of packaging an adeno-associated virus of claim 12, wherein, The host cell is selected from Hela-S3 cells, HEK-293 cells, HEK-293T cells, HEK-293FT cells, A549 cells and Sf9 cells.

14. An adeno-associated virus obtained by the packaging method according to claim 12 or 13.

15. Use of the double vector system for expressing Otoferlin protein according to any one of claims 1-9 or the adeno-associated virus according to claim 14 for the preparation of a medicament or preparation for treating a deafness disease or hearing impairment or hearing dysfunction.

16. A medicament or preparation for the treatment of a deafness disease or a hearing impairment or a hearing dysfunction, prepared from the dual vector system for the expression of the Otoferlin protein according to any one of claims 1-9 or the adeno-associated virus according to claim 14, wherein, The adeno-associated virus is obtained by transferring the packaging vector system of the adeno-associated virus into a host cell for packaging, wherein the packaging vector system of the adeno-associated virus comprises the double vector system for expressing Otoferlin protein, a vector carrying AAV rep and cap genes, and a helper virus vector.

17. The medicament or preparation of claim 16, wherein, The medicament or preparation further comprises a neutral salt buffer, an acidic salt buffer, an alkaline salt buffer, glucose, mannose, mannitol, a protein, a polypeptide, an amino acid, an antibiotic, a chelating agent, an adjuvant, a preservative, a nanoparticle, a liposome and a positive lipid particle. The vector carrying AAV rep and cap genes is selected from AAV1, AAV2, AAV5, AAV8, AAV9, Anc80, PHP.eB, AAV-DJ and AAVrh.10 vectors; and the helper virus vector is a pHelper plasmid. The packaging vector system of the adeno-associated virus according to claim 10 or 11 is transferred into a host cell for packaging. The host cell is selected from Hela-S3 cells, HEK-293 cells, HEK-293T cells, HEK-293FT cells, A549 cells and Sf9 cells.

14. An adeno-associated virus obtained by the packaging method according to claim 12 or 13.

15. Use of the double vector system for expressing Otoferlin protein according to any one of claims 1-9 or the adeno-associated virus according to claim 14 for the preparation of a medicament or preparation for treating a deafness disease or hearing impairment or hearing dysfunction. The adeno-associated virus is obtained by transferring the packaging vector system of the adeno-associated virus into a host cell for packaging, wherein the packaging vector system of the adeno-associated virus comprises the double vector system for expressing Otoferlin protein, a vector carrying AAV rep and cap genes, and a helper virus vector. The medicament or preparation further comprises a neutral salt buffer, an acidic salt buffer, an alkaline salt buffer, glucose, mannose, mannitol, a protein, a polypeptide, an amino acid, an antibiotic, a chelating agent, an adjuvant, a preservative, a nanoparticle, a liposome and a positive lipid particle.

18. The medicament or preparation of claim 16 or 17, wherein, Injection administration through the round window, oval window, semicircular canal, common canal of the cochlea; and single or multiple administration for life, total dose is 1 x 10 9 -1 x 10 13 viral genome.

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