Dual-vector system for expressing STRC protein and use thereof

Abstract

A dual-vector system for expressing STRC protein is provided, which comprises 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, and 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 STRC, 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 the intein, a C-terminal coding sequence of STRC, and polyA. Also provided are an adeno-associated virus packaging vector, an adeno-associated virus packaging method, and an adeno-associated virus obtained thereby. The dual-vector system or adeno-associated virus for expressing STRC protein can be used for gene therapy, especially for treating hearing loss, such as for treating STRC mutation-associated autosomal recessive nonsyndromic DFNB16 deafness disorder.

Description

Dual vector system for expressing STRC protein and its use Technical Field

[0001] The present invention relates to a dual vector system for expressing STRC protein and its application in gene therapy, especially in the treatment of hearing loss. Background Art

[0002] Deafness is the most common disabling disease in clinical practice, severely impacting normal human life and placing a significant burden on society. According to the WHO, nearly 1.5 billion people worldwide have varying degrees of hearing loss, and 466 million, or 5% of the total population, suffer from disabling hearing loss, including 34 million children. It is estimated that by 2050, 2.5 billion people worldwide will suffer from varying degrees of hearing loss, with 700 million suffering from disabling hearing loss.

[0003] Heredity and environment are the two main contributing factors to deafness. Environmental factors are primarily related to the use of ototoxic drugs, infections during pregnancy, neonatal hypoxia, radiation exposure, and other environmental factors or certain complications. Heredity is the primary cause of deafness, accounting for approximately 60% of deafness cases. Genetic factors primarily involve defects in an individual's deafness-causing gene, leading to varying degrees of hearing loss. The pathogenic genes are passed on to the next generation through various genetic pathways, can occur at any age, and the effects are permanent. Currently, over 120 genes associated with deafness have been identified, involving over 1,500 pathogenic variants. To date, no medications are clinically available to treat hereditary deafness.

[0004] Based on the presence of clinical symptoms affecting 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, and accounts for 30% of hereditary hearing loss. Among SHL, Pendred syndrome, Usher syndrome (USH), and Waardenburg syndrome (WS) are the most well-known. In preclinical animal models, USH, Pendred syndrome, and Jervell and Lange-Nielsen syndrome (JLNS) have been successfully treated with inner ear gene therapy. There are four types of non-syndromic hearing loss (NSHL): autosomal dominant (DFNA), autosomal recessive (DFNB), X-linked (DFNX), and mitochondrial non-syndromic hearing loss. Approximately 70% of patients with hereditary hearing loss have the non-syndromic type. The most common mode of inheritance for NSHL is autosomal recessive (75%-80%), followed by autosomal dominant (20%), X-linked (<2%), and mitochondrial inheritance (<1%). To date, over 120 genes have been reported to be associated with NSHL. Among them, 51 deafness genes are associated with DFNA, 78 with DFNB, and 10 with both DFNA and DFNB: CLOL11A2, GJB2, GJB6, MYO3A, MYO6, MYO7A, PTPRQ, TCB1D24, TECTA, and TMC1. Five deafness genes are associated with DFNX. GJB2 is the most common causative gene (21.6%), followed by STRC (16.1%), SLC26A4 (6.6%), and TECTA (5.2%).

[0005] Each deafness type is described according to the nomenclature of the deafness gene. For example, DFNA1 was the first autosomal dominant deafness type to be discovered. DFNB16 is the sixteenth autosomal recessive nonsyndromic deafness type to be described. It is a monogenic, nonsyndromic, recessive hearing loss caused by mutations in the Strc gene, which is located on chromosome 15q15.3 and is part of a tandem duplication of chromosome 15; the second copy is a pseudogene. Approximately 40 different mutations (mostly recessive) have been identified in the Strc gene, most of which result in the synthesis of a defective STRC protein or completely prevent its synthesis.

[0006] Mutations in the Strc gene are believed to be the primary cause of mild to moderate autosomal recessive non-syndromic hearing loss. The Strc gene encodes an extracellular structural protein called stereocilin (also known as "STRC protein"), which is associated with the hair bundles of sensory hair cells in the inner ear. The hair bundles are composed of stiff microvilli called stereocilia, and the role of the Strc gene is to maintain the sticky bundle of microvilli and couple this bundle to the overlying tectorial membrane, which is located in the cochlea of ​​the inner ear. Normal expression of the Strc gene in the inner ear is essential for auditory function. The lack of normal STRC protein causes the sensory hair bundles to separate from the overlying tectorial membrane, but the non-separation of the sensory hair bundles from the overlying tectorial membrane is necessary for proper sound-evoked stimulation.

[0007] Global statistics indicate that DFNB 16 accounts for a significant proportion of inherited hearing loss, particularly among those with moderate hearing loss. As such, it is the second most common form of inherited hearing loss and the most common form affecting the sensory hair cells of the inner ear. Patients with DFNB 16 experience moderate to severe hearing loss and are typically treated with hearing aids or cochlear implants. However, there are currently no clinically available biological treatments for DFNB 16 hearing loss.

[0008] Treating deafness is one of the major challenges facing medicine today. While hearing aids and cochlear implants can provide significant benefits to patients, there remains an unmet medical need for a one-time, precise, and permanent strategy to potentially cure hereditary hearing loss.

[0009] Currently, most methods to address deafness are physical methods such as hearing aids, vibrating sound bridges, and cochlear implants. Although patients can achieve varying degrees of improvement in hearing function, there are large individual differences and significant limitations and weaknesses. For example, treatment effects are limited, frequency sensitivity, speech discrimination, and perception difficulties in noisy environments, and the device needs to be used with caution. Approximately 300,000 patients worldwide have received cochlear implants, but this only accounts for a small fraction of all deaf patients. For most patients, there is still an urgent need for fundamental and effective drug treatments, but there are no approved treatments to date, and this is an area with serious unmet needs.

[0010] For hereditary deafness with a clear cause, gene therapy is considered an ideal treatment method. It uses normal genes to compensate for defective genes, and a single dose can fundamentally restore or improve hearing, achieving lasting restoration of auditory function and closer to natural sounds. In the past decade, with the rapid development of delivery vectors and gene therapy technologies around the world, related drugs have made major breakthroughs in disease treatment, and some gene therapy drugs have been approved for marketing. However, in the field of hereditary deafness, there are no gene therapy drugs on the market.

[0011] Gene therapy refers to a method of correcting, compensating or inhibiting at the DNA or RNA level, so that the subject can recover from diseases caused by abnormal nucleic acid sequences or abnormal expressions in the body, thereby achieving the purpose of treating the disease. Currently, most gene therapies require vector delivery, and adeno-associated virus (AAV) is one of the safe and efficient delivery vectors with a packaging capacity of approximately 4.7Kb. However, in the field of deafness, the length of many gene coding regions is not suitable for adeno-associated virus packaging, such as BDP1, CDH23, COL11A2, LOXHD1, MET, MYO15A, MYO3A, MYO7A, OTOG, OTOF, OTOGL, PCDH15, PTPRQ, STRC, TECTA, TARA, etc. Their coding region lengths are all over 4kb, and together with the related regulatory elements, they will exceed the packaging limit of AAV vectors.

[0012] Using a dual adeno-associated virus system (dual AAV vector system) to deliver large gene sequences (e.g., genes larger than 4kB, such as the STRC gene) can overcome the gene size limitation of a single AAV vector. However, clinical research on the use of AAV gene therapy for the treatment of deafness is currently underway. Summary of the Invention

[0013] The present invention provides a treatment method for hereditary deafness DFNB16. AAV vectors have been proven to be effective transgene delivery tools and are one of the most promising vectors for human gene therapy transfer technology. Their safety and ability to be persistently expressed in inner ear cells have made significant progress in gene therapy for deafness. Therefore, AAV-based gene therapy is a boon for deaf patients. However, the AAV packaging capacity is less than 4.7kb, and there is a packaging limitation problem. The coding sequences of many proteins that are critical to the function of the inner ear exceed the AAV vector packaging limit, including Strc (the total length including regulatory sequences exceeds 9kb). In the dual AAV vector system, larger exogenous genes are divided into two segments and packaged into different AAV particles. By using dual AAV vectors to co-transform the same cell, the exogenous gene fragments in each AAV vector can be reassembled, transcribed and expressed in the target cell. Ideally, the dual AAV vector system can expand the exogenous gene carrying capacity to 9kb.

[0014] However, the efficiency of Strc gene delivery using dual AAV vectors is a challenge. Due to the limited packaging capacity of AAV vectors, the system of the present invention screened a large number of STRC protein cleavage sites and codon-optimized the Strc gene nucleotide sequence to increase the expression of the full-length STRC protein. Finally, the most suitable AAV subtype, expression element, and optimal STRC protein cleavage site were selected, thereby producing normal, functional STRC protein in vivo.

[0015] This invention is a gene therapy based on dual AAV vector technology. Through a single unilateral intracochlear administration, the dual AAV vector system of the invention delivers a transgene encoding Strc to cochlear hair cells, thereby expressing normal, functional Strc protein to treat Strc-mediated hearing loss. This article provides a method and composition for treating autosomal recessive nonsyndromic hearing loss DFNB16 by delivering the Strc gene encoding the Strc protein using a dual vector system.

[0016] In response to the shortcomings of the existing technology, this article provides a composition and method for delivering Strc gene cDNA into human 293T cells and Strc ko mice using a dual-vector system and expressing STRC protein to form a complete and functional STRC protein, which can be used to increase the expression of wild-type STRC protein or provide wild-type STRC protein to subjects to treat autosomal recessive non-syndromic DFNB16 deafness disease associated with STRC gene mutations.

[0017] The dual AAV vector system separates the Strc gene target sequence into two AAV virus particles. The two AAV vectors each carry part of the target gene sequence, and the two gene sequences do not overlap. The dual AAV vector system ultimately produces a complete and functional STRC protein in cells. In the dual AAV vector system, the 3' end of the AAV genome encoding the N-terminal sequence of the target protein in one AAV vector contains an N-terminal intein coding sequence; the 5' end of the AAV genome encoding the C-terminal sequence of the target protein in the other AAV vector contains an C-terminal intein coding sequence; and both AAV genome sequences contain an independent promoter. Ultimately, based on splicing at the polypeptide level in the cell, the two polypeptide segments of the target protein N-terminal sequence and the target protein C-terminal sequence will complete splicing to form an active STRC protein.

[0018] The present invention improves the expression level of STRC protein entering cells by screening the STRC protein / Strc gene cleavage site and codon optimization in the dual AAV vector system, laying a foundation for the preparation of targeted drugs for the treatment of sensorineural hearing loss using the Strc gene.

[0019] Therefore, in a first aspect, the present invention provides a dual vector system for expressing STRC protein, comprising a first nucleic acid vector and a second nucleic acid vector, wherein

[0020] The first nucleic acid vector comprises a first nucleotide sequence; and the second nucleic acid vector comprises a second nucleotide sequence;

[0021] The first nucleotide sequence comprises an expression cassette inserted between two first ITR sequences;

[0022] The second nucleotide sequence comprises an expression cassette inserted between two second ITR sequences;

[0023] The expression cassette of the first nucleotide sequence comprises a promoter, an N-terminal coding sequence of STRC, an N-terminal coding sequence of an intein, and polyA;

[0024] 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 a STRC, and polyA; and

[0025] A STRC cleavage site is provided in the STRC amino acid sequence, for example, the STRC amino acid sequence is as shown in SEQ ID NO: 2 or a functional fragment thereof, for example, the functional fragment is an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 2;

[0026] The N-terminal coding sequence of STRC is the nucleotide coding sequence from the N-terminus of the STRC amino acid sequence to the STRC cleavage site; the C-terminal coding sequence of STRC is the nucleotide coding sequence from the amino acid after the STRC cleavage site to the C-terminus of the STRC amino acid sequence.

[0027] In some embodiments, the STRC cleavage site is located at the amino acid preceding serine, threonine, or cysteine ​​in the STRC amino acid sequence.

[0028] In some embodiments, in the binary vector system for expressing STRC protein of the present invention, the promoter of the expression cassette of the first nucleotide sequence or the second nucleotide sequence is selected from CAG promoter, CMV promoter, CBA promoter, UbC promoter, SFFV promoter, EF1α promoter, PGK promoter, or Myo7A, Myo15, Atoh1, POU4F3, Lhx3, Myo6, α9AchR, α10AchR, OTOF and the promoter of STRC encoding gene.

[0029] In some embodiments, in the binary vector system for expressing STRC protein of the present invention, the poly A of the expression cassette of the first nucleotide sequence or the second nucleotide sequence comprises AATAAA (SEQ ID NO: 20) and a variant of AATAAA; the variant of AATAAA comprises ATTAAA (SEQ ID NO: 21), AGTAAA (SEQ ID NO: 22), CATAAA (SEQ ID NO: 23), TATAAA (SEQ ID NO: 24), GATAAA (SEQ ID NO: 25), ACTAAA (SEQ ID NO: 26), AATATA (SEQ ID NO: 27), AAGAAA (SEQ ID NO: 28), AATAAT (SEQ ID NO: 29), AAAAAA (SEQ ID NO: 30), AATGAA (SEQ ID NO: 31), AATCAA (SEQ ID NO: 32), AACAAA (SEQ ID NO: 33), AATCAA (SEQ ID NO: 34), AATAAC (SEQ ID NO: 35), AATAGA (SEQ ID NO: 36), AATTAA (SEQ ID NO: 37), or AATAAG (SEQ ID NO: 38). ID NO: 38); for example, the polyA is a nucleotide sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the poly A signal sequence shown in SEQ ID NO: 14 or SEQ ID NO: 17; 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.

[0030] In some embodiments, in the dual-vector system for expressing STRC protein of the present invention, 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 posttranscriptional 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 with the nucleotide sequence shown in SEQ ID NO: 13, for example, the nucleotide sequence shown in SEQ ID NO: 16; for example, the tag element is HA.

[0031] In some embodiments, in the binary vector system for expressing STRC protein of the present invention, the intein 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 intein is derived from RmaDnaB, for example, the N-terminus of the intein is the RmaDnaB intein N-terminus as shown in SEQ ID NO:5, and the C-terminus of the intein is the RmaDnaB intein C-terminus as shown in SEQ ID NO:6. In some embodiments, the intein is derived from NpuDnaE, ​​for example, the N-terminus of the intein is the N-terminus of the NpuDnaE intein as shown in SEQ ID NO:39, and the C-terminus of the intein is the C-terminus of the NpuDnaE intein as shown in SEQ ID NO:41.

[0032] In some embodiments, in the dual-vector system for expressing STRC protein of the present invention, 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 pAAV, pAAV-CMV, pX601, pX551 or pAAV-MCS plasmid.

[0033] In some embodiments, in the binary vector system for expressing STRC protein of the present invention, the STRC cleavage site is as shown in Table 1.

[0034] In some specific embodiments, in the dual-vector system for expressing STRC protein of the present invention, amino acid 656 of the STRC amino acid sequence shown in SEQ ID NO: 2 is used as the STRC cleavage site, and the RmaDnaB intein is used; the N-terminal coding sequence of STRC and the N-terminal coding sequence of the RmaDnaB intein are connected and fused to construct a first nucleotide sequence, and the pAAV-CMV plasmid is used as a vector; the C-terminal coding sequence of the RmaDnaB intein and the C-terminal coding sequence of STRC are connected and fused to construct a second nucleotide sequence, and the pAAV-CMV plasmid is used as a vector;

[0035] The 708th amino acid of the STRC amino acid sequence shown in SEQ ID NO: 2 is used as the STRC cleavage site, and the RmaDnaB intein is used; the N-terminal coding sequence of STRC is connected and fused with the N-terminal coding sequence of the RmaDnaB intein to construct a first nucleotide sequence, and the pAAV-CMV plasmid is used as a vector; the C-terminal coding sequence of the RmaDnaB intein is connected and fused with the C-terminal coding sequence of STRC to construct a second nucleotide sequence, and the pAAV-CMV plasmid is used as a vector;

[0036] Using amino acid 722 of the STRC amino acid sequence shown in SEQ ID NO: 2 as the STRC cleavage site and using the RmaDnaB intein; connecting and fusing the N-terminal coding sequence of STRC with the N-terminal coding sequence of the RmaDnaB intein to construct a first nucleotide sequence, using the pAAV-CMV plasmid as a vector; connecting and fusing the C-terminal coding sequence of the RmaDnaB intein with the C-terminal coding sequence of STRC to construct a second nucleotide sequence, using the pAAV-CMV plasmid as a vector; or

[0037] The 917th amino acid of the STRC amino acid sequence shown in SEQ ID NO: 2 is used as the STRC cleavage site, and the RmaDnaB intein is used; the N-terminal coding sequence of STRC is connected and fused with the N-terminal coding sequence of the RmaDnaB intein to construct a first nucleotide sequence, and the pAAV-CMV plasmid is used as a vector; the C-terminal coding sequence of the RmaDnaB intein is connected and fused with the C-terminal coding sequence of STRC to construct a second nucleotide sequence, and the pAAV-CMV plasmid is used as a vector;

[0038] For example, the N-terminal coding sequence of the RmaDnaB intein encodes the N-terminal portion of RmaDnaB shown in SEQ ID NO:5, and the C-terminal coding sequence of the RmaDnaB intein encodes the C-terminal portion of RmaDnaB shown in SEQ ID NO:6.

[0039] In some embodiments, in the binary vector system for expressing the STRC protein of the present invention, the expression cassette of the first nucleotide sequence and the expression cassette of the second nucleotide sequence each contain a signal sequence operably linked to a promoter sequence and under the control of the promoter; preferably, the signal sequence is a nucleotide sequence encoding SEQ ID NO: 3.

[0040] In some embodiments, in the binary vector system for expressing the STRC protein of the present invention, the expression cassette of the first nucleotide sequence and the expression cassette of the second nucleotide sequence each comprise a combination of a WPRE nucleotide sequence and an SV40 polyadenylation sequence at the N-terminus of the 3' ITR sequence, for example, a nucleotide sequence as set forth in SEQ ID NO: 12, or a nucleotide sequence at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 12; or a combination of a WPRE3 nucleotide sequence and an SV40 late polyadenylation sequence, for example, a nucleotide sequence as set forth in SEQ ID NO: 15, or a nucleotide sequence at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 15.

[0041] In a second aspect, the present invention provides an adeno-associated virus packaging vector system, which comprises the dual-vector system for expressing STRC protein described in the first aspect of the present invention, a vector carrying AAV rep and cap genes, and a helper virus vector, which are packaged into an AAV vector. Preferably, the amino acid sequence of the STRC protein is as shown in SEQ ID NO: 2.

[0042] In some embodiments, in the adeno-associated virus packaging vector system of the present invention, 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.

[0043] In a third aspect, the present invention provides a method for packaging an adeno-associated virus, wherein the adeno-associated virus packaging vector system described in the second aspect of the present invention is transferred into a host cell for packaging.

[0044] In some embodiments, the host cell is selected from Hela-S3 cells, HEK-293 cells, HEK-293T cells, HEK-293FT cells, A549 cells, and Sf9 cells.

[0045] In a fourth aspect, the present invention provides a dual adeno-associated virus vector, which is obtained by the packaging method according to the second aspect of the present invention.

[0046] In the fifth aspect, the present invention provides the use of the dual vector system for expressing STRC protein described in the first aspect of the present invention or the dual adeno-associated virus vector described in the fourth aspect of the present invention for preparing a drug or preparation for treating deafness diseases or hearing loss or hearing dysfunction.

[0047] In the sixth aspect, the present invention provides a medicine or preparation for treating deafness, hearing loss or hearing dysfunction, which is prepared by the dual-vector system for expressing STRC protein described in the first aspect of the present invention or the adeno-associated virus described in the fourth aspect of the present invention, wherein the adeno-associated virus is obtained by transferring the packaging vector system of the adeno-associated virus into a host cell for packaging, and the packaging vector system of the adeno-associated virus includes a dual-vector system for expressing STRC protein, a vector carrying AAVrep and cap genes, and a helper virus vector.

[0048] In some embodiments, the drug or formulation of the present invention further comprises a neutral salt buffer, an acidic salt buffer, an alkaline salt buffer, glucose, mannose, mannitol, proteins, polypeptides, amino acids, antibiotics, chelating agents, adjuvants, preservatives, nanoparticles, liposomes and positive lipid particles.

[0049] In some embodiments, the drug or preparation of the present invention is administered by injection through the round window, oval window, semicircular canal, or common canal of the cochlea; and a single or multiple administration throughout life, with a total dose of 1×10 9 -1×10 13 Viral genome. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] FIG1 is a schematic diagram of intein-mediated STRC protein expression in a dual-vector system.

[0051] Figure 2 illustrates the vector backbone elements in the binary vector system, for example, its sequence can be as shown in SEQ ID NO: 7, wherein the 5'ITR sequence is located at 1 bp-141 bp, the CMV promoter sequence is located at 169 bp-752 bp, the Kozak sequence is located at 792 bp-797 bp, the EGFP sequence is located at 801 bp-1517 bp, the WPRE sequence is located at 1536 bp-2124 bp, the SV40 PolyA sequence is located at 2131 bp-2252 bp, the 3'ITR sequence is located at 2290 bp-2430 bp, the f1 Ori sequence is located at 2505 bp-2960 bp, the kana resistance sequence is located at 3242 bp-4156 bp, and the Ori sequence is located at 4327 bp-4515 bp.

[0052] Figure 3 is a schematic diagram of constructing a dual vector system based on the wild-type human Strc nucleotide sequence (taking the cleavage site at 650Leu as an example), wherein both the first and second nucleic acid vectors have a signal peptide coding sequence located at the C-terminus of the promoter sequence.

[0053] Figure 4 is a schematic diagram of constructing a dual-vector system based on the codon-optimized human Strc nucleotide sequence (taking the cleavage site at 656Asn as an example), wherein both the first and second nucleic acid vectors have a signal peptide coding sequence located at the C-terminus of the promoter sequence.

[0054] Figure 5 is a schematic diagram of constructing a dual-vector system based on the codon-optimized human Strc nucleotide sequence (taking the cleavage site at 656Asn as an example), wherein the first nucleic acid vector has a signal peptide coding sequence located at the C-terminus of the promoter sequence, and the second nucleic acid vector has the signal peptide coding sequence located at the C-terminus of the promoter sequence removed.

[0055] FIG6 shows the expression of full-length STRC protein in co-transfected 293T cells using a dual vector system based on the wild-type human Strc nucleotide sequence and having different cleavage sites shown in Table 1.

[0056] Figure 7 shows the expression of full-length STRC protein in co-transfected 293T cells by a dual-vector system of human Strc nucleotide sequences after codon optimization with different cleavage sites, which shows that the codon-optimized cleavage point sequence leads to increased expression of full-length STRC protein. Panel A in Figure 7 shows the expression of full-length STRC protein in co-transfected 293T cells by a dual-vector system of human Strc nucleotide sequences after codon optimization with different cleavage sites, wherein both the first and second nucleic acid vectors have a signal peptide coding sequence located at the C-terminus of the promoter sequence; Panel B in Figure 7 shows the expression of full-length STRC protein in co-transfected 293T cells by a dual-vector system of human Strc nucleotide sequences after codon optimization with different cleavage sites, wherein the first nucleic acid vector has a signal peptide coding sequence located at the C-terminus of the promoter sequence, and the second nucleic acid vector has the signal peptide coding sequence located at the C-terminus of the promoter sequence removed.

[0057] Figure 8 shows the auditory brainstem response (ABR) results in the Strc knockout mouse model treated with a dual-vector system, which shows that the ABR threshold is lowered in the treated Strc knockout mouse model, and some hearing frequency bands are significantly restored, which is close to the hearing threshold of WT mice.

[0058] Figure 9 shows immunofluorescence results of STRC protein expression in the cochlea of ​​Strc knockout mice treated with the dual-vector system. After audiometry, the mice were sacrificed and the cochlea was stained for basilar membrane flat-mount staining. Immunofluorescence results showed that the dual-AAV vector system of the present invention achieved STRC protein expression in the mouse cochlea and restored hearing. DETAILED DESCRIPTION

[0059] Unless otherwise defined hereinafter, all technical and scientific terms used in this specification have the same meaning as those of ordinary skill in the art to which the present invention pertains. All publications, patent applications, patents and other references mentioned herein are incorporated herein by reference in their entirety. In addition, the materials, methods and examples described herein are merely illustrative and are not intended to be restrictive. Other features, objects and advantages of the present invention will become apparent from this specification and the accompanying drawings and from the appended claims.

[0060] I. Definition

[0061] As used herein, the term "about" when used in conjunction with a numerical value is intended to encompass numerical values ​​within a range having a lower limit that is 5% less than the specified numerical value and an upper limit that is 5% greater than the specified numerical value. The term is also intended to encompass values ​​within ±1%, ±0.5%, or ±0.1% of the specified number.

[0062] As used herein, the term "comprising" or "including" means including the recited elements, integers, steps, or groups of elements, integers, or steps, but does not exclude any other elements, integers, or steps, or other groups of elements, integers, or steps. As used herein, unless otherwise indicated, the term "comprising" or "including" also encompasses the situation consisting of the recited elements, integers, or steps. For example, when referring to a polynucleotide "comprising" a particular sequence, it is intended to encompass a polynucleotide consisting of that particular sequence.

[0063] Herein, the expression "and / or," when used in conjunction with two or more items, is intended to mean any one of the associated listed items, or any multiple or all possible combinations of the associated listed items.

[0064] The intein or protein intein (also known as intein) described herein is a polypeptide chain within an immature precursor protein. Through a series of self-catalytic reactions, such as rearrangement, transesterification, and cyclization, it can be excised from the precursor protein and its two end polypeptide segments (exteins) connected by a natural peptide bond. This is to say, protein self-splicing achieves a rearrangement of the protein structure. A split intein is a structural type of intein. Structurally, its N-terminal and C-terminal regions are separated from each other. When the two fragments containing the N-terminal and C-terminal regions of the intein are connected, the exteins at both ends can be spliced ​​together according to the standard intein splicing pathway.

[0065] Most inteins are composed of terminal splicing regions at both ends and a homing endonuclease domain or linker domain in the middle. Inteins can be divided into three types: canonical inteins, mini inteins, and split inteins. Both canonical inteins and mini inteins contain splicing domains at both ends and a middle region. The difference between them is that the middle region of canonical inteins is an endonuclease domain, while the middle region of mini inteins is a linker domain. The lengths of the linker domains of different mini inteins are not the same. The middle region of the split intein is disconnected at a specific site to form an N-terminal fragment and a C-terminal fragment, and are respectively located on two genes far apart on the genome. During the translation and maturation of the precursor protein, these two intein fragments recognize each other and restore endonuclease activity, mediating protein trans splicing. In this context, a dual AAV vector system can be used to deliver a nucleic acid encoding a split intein. For example, in the present invention, the N-terminal fragment of the intein is shown in SEQ ID NO: 5, and the C-terminal fragment of the intein is shown in SEQ ID NO: 6.

[0066] Typically, inteins are composed of 10 modules, starting from the N-terminus: A, N2, B, N4, C, D, E, H, F, and G. A, N2, B, and N4 are the N-terminal splicing regions, F and G are the C-terminal splicing regions, and C, D, E, and H are the homing endonuclease active regions or linker domains. The motifs involved in intein splicing within the A, B, F, and G modules contain highly conserved amino acid residues at the splice sites, essential for the affinity displacement reaction during intein splicing. The motifs within the A module of the intein typically contain amino acids with hydroxyl or sulfhydryl groups, such as Ser and Cys. The motif within the B module contains the highly conserved amino acid sequence Thr-XX-His, which is also found in serine proteases. The conserved amino acid residues within the motif involved in the splicing reaction within the G module are Asn, Ser, Cys, Thr, and His. In addition, the conserved sites in the motif of the A module (such as Ser, Cys) can be replaced by Ala, Gln or Pro in some inteins, and the same is true for the motif of the G module.

[0067] In this article, "hearing loss" refers to hearing below the normal hearing threshold level determined by audiometry, including mild, moderate, severe and profound hearing loss, and deafness. Hearing loss can be described by a percentage of hearing loss, for example, 30%, 60%, 80% or even 100% hearing loss, or by a grading description of hearing loss. The hearing loss can be hearing loss caused by or associated with a gene defect, such as congenital deafness and pre-lingual deafness caused by genetic factors, or hearing loss associated with genetic factors, induced by environmental factors (for example, aging, noise, drugs or infection-induced). Hearing loss can be asymptomatic (that is, there is no associated visible outer ear or other organ abnormality) or symptomatic. In some embodiments, hearing loss is sensorineural hearing loss.

[0068] In this article, "hearing loss-associated genes" refer to genes whose mutations can cause hearing loss or susceptibility to hearing loss by changing the ability of the inner ear to function normally. In this article, such genes are also referred to as "hearing loss genes". More than 100 genes have been identified as being associated with hearing loss (see, Hereditary Hearing Loss Homepage, https: / / hereditaryhearingloss.org / , which lists the gene locations and identification data of currently known single-gene asymptomatic hearing loss). In the case of causing susceptibility to hearing loss, individuals carrying the hearing loss gene mutations may show greater susceptibility to hearing loss due to environmental factors, such as aging, noise, drugs or infections, relative to healthy individuals.

[0069] As used herein, "cochlear inner hair cells" refer to isolated or in vitro cochlear inner hair cells, cell lines, or cell populations derived from a mammal, or inner hair cells in the cochlea of ​​a mammal.

[0070] As used herein, "cochlear outer hair cells" refer to cochlear outer hair cells, cell lines, or cell populations isolated from or in vitro of a mammal, or outer hair cells in the cochlea of ​​a mammal.

[0071] As used herein, an "isolated" nucleic acid refers to a nucleic acid molecule that has been artificially synthesized or separated from at least some components of its natural environment. For example, an isolated nucleic acid can be part of a larger nucleic acid, or part of a vector or composition of matter, or can be contained within a cell and still be "isolated" provided that the larger nucleic acid, vector, composition of matter, or specific cell is not the natural environment of the nucleic acid.

[0072] As used herein, the term "operably linked," also referred to as "effectively linked" or "functionally linked," means that two or more polynucleotide (e.g., DNA) segments are in a relationship that allows them to function in the intended manner. For example, a promoter sequence is operably linked to a coding sequence if it stimulates or regulates the transcription of the coding sequence in a suitable host cell or other expression system. Generally, promoters that are operably linked to a transcribable sequence are contiguous with the transcribable sequence, i.e., they are cis-acting. However, some transcriptional regulatory sequences (e.g., enhancers) do not need to be physically adjacent to or in close proximity to the coding sequence whose transcription they enhance.

[0073] "Promoter" refers to a polynucleotide sufficient to direct transcription of downstream polynucleotides. In some embodiments, the nucleic acid vectors described herein may include one or more regulatory elements. Those of ordinary skill in the art can select regulatory elements suitable for use in mammalian cells or human host cells. Non-limiting examples of regulatory elements include promoters, transcription termination sequences, translation termination sequences, enhancers, and polyadenylation elements. The nucleic acid vectors described herein may include a promoter sequence operably linked to a nucleotide sequence encoding a polypeptide of interest (e.g., STRC protein). The promoters used in the present invention include, but are not limited to, cytomegalovirus (CMV) promoters, SV40 promoters, Rous sarcoma virus (RSV) promoters, chimeric CMV / chicken beta actin promoters (CBA), and truncated forms of CBA (smCBA). In some embodiments, the promoter is a CMV promoter.

[0074] "Signal peptide" is an amino acid sequence attached to the N-terminus of a foreign protein sequence that promotes the secretion of the foreign protein outside the cell. The mature form of the extracellular protein does not have a signal sequence and is removed during the secretion process.

[0075] The term "full-length STRC protein" refers to a STRC protein produced by effectively linking the N-terminal portion and the C-terminal portion of the STRC protein expressed in the dual-vector system of the present invention, which can be the full-length STRC protein shown in SEQ ID NO.2 or its functional derivative or functional fragment.

[0076] As used herein, 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 has a single-stranded DNA genome.

[0077] AAV is a dependent virus that requires other viruses such as adenovirus, herpes simplex virus, human papillomavirus, or auxiliary factors to provide auxiliary functional proteins for replication.

[0078] The first AAV virus isolated was serotype 2 (AAV2). The AAV2 genome is approximately 4.7 kb long, flanked by 145-bp inverted terminal repeats (ITRs) at either end, forming a palindromic hairpin structure. The genome also contains two large open reading frames (ORFs), encoding the rep and cap genes, respectively.

[0079] ITRs are cis-acting elements of the AAV vector genome, playing a crucial role in AAV virus integration, rescue, replication, and genome packaging. The ITR sequence contains the Rep protein binding site (RBS) and the terminal resolution site (TRs), which are recognized by the Rep protein and produce a nick at the TRs. The ITR sequence also forms a unique "T"-shaped secondary structure, playing a crucial role in the AAV virus life cycle.

[0080] The rest of the AAV2 genome can be divided into two functional regions, the rep gene region and the cap gene region.

[0081] The rep gene region encodes four Rep proteins: Rep78, Rep68, Rep52, and Rep40. Rep proteins play an important role in the replication, integration, rescue, and packaging of AAV viruses. Rep78 and Rep68 specifically bind to the terminal melting sites trs and GAGY repeat motifs in the ITR, initiating the replication of the AAV genome from single-stranded to double-stranded. The trs and GAGC repeat motifs and / or GAGY repeat motifs in the ITR are the center of AAV genome replication. Therefore, although the ITR sequences are different in various serotypes of AAV viruses, they can all form a hairpin structure and contain Rep binding sites. There is a p19 promoter at position 19 on the AAV2 genome map, which initiates the expression of Rep52 and Rep40, respectively. Rep52 and Rep40 have ATP-dependent DNA helicase activity but do not have the function of binding to DNA.

[0082] The cap gene encodes the AAV capsid proteins VP1, VP2, and VP3. 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 facilitates VP3 entry into the cell nucleus; and VP3 is the primary protein in AAV particles.

[0083] As used herein, the term "AAV vector" refers to an efficient exogenous gene transfer tool, i.e., an AAV vector, that has been transformed from wild-type AAV virus as people gain a better understanding of the AAV virus life cycle and its related molecular biological mechanisms. The modified AAV vector genome only contains the ITR sequence of the AAV virus and the exogenous sequence to be transferred. The Rep and Cap proteins required for AAV virus packaging are provided in trans by other exogenous plasmids, thereby reducing the possible harm caused by packaging the rep and cap genes into the AAV vector. Furthermore, the AAV virus itself is not pathogenic, which makes the AAV vector recognized as one of the safest viral vectors.

[0084] There are many AAV virus serotypes, and different serotypes have different tissue infection tropisms. Therefore, the use of AAV vectors can transport exogenous genes to specific organs and tissues.

[0085] The existing technology has a relatively mature packaging system for AAV vectors, which facilitates the large-scale production of AAV vectors.

[0086] The term "vector genome (vg)" refers to the nucleic acid sequence that is packaged within the rAAV capsid to form the rAAV vector.

[0087] As used herein, "individual" and "subject" are used interchangeably to 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. As used herein, mammals include individuals at all stages of development, including embryonic and fetal stages.

[0088] As used herein, the term "treatment" refers to clinical intervention intended to alter the natural course of a disease in the individual being treated. Desired therapeutic effects include, but are not limited to, preventing the onset or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or palliating the disease state, and alleviating or improving prognosis. The term "treatment" also encompasses modification or improvement of at least one physical parameter, including physical parameters that may not be discernible by the patient.

[0089] As used herein, the term "prevention" refers to preventing or delaying the onset or development or progression of a disease or condition. As used herein, "prevention" generally refers to hospital intervention performed before at least one symptom of a disease occurs.

[0090] Various aspects of the present invention are described below.

[0091] II. Dual Vector System

[0092] The present invention utilizes protein trans-splicing, that is, the CDS of the N-terminus and C-terminus of STRC are constructed into two different plasmids for expression, and the complete full-length protein is assembled through intein splicing. The N-terminal plasmid adds the N-terminal coding sequence of the intein at the 3' end of the STRC N-terminal CDS sequence, such as the nucleic acid sequence encoding the Rm-N-intein sequence (SEQ ID NO: 5), and the first amino acid residue of the Rm-N-intein sequence contains Cys. The C-terminal plasmid adds the C-terminal coding sequence of the intein at the 5' end of the STRC C-terminal CDS sequence, such as the nucleic acid sequence encoding the Rm-C-intein (SEQ ID NO: 6), and the terminal sequence of the Rm-C-intein contains His and Asn, and the first amino acid residue of the STRC C-terminal CDS sequence is Cys, Ser, and Thr for intein splicing.

[0093] The present invention provides a dual vector system for expressing STRC protein, which comprises a first nucleic acid vector and a second nucleic acid vector, wherein

[0094] The first nucleic acid vector comprises a first nucleotide sequence; and the second nucleic acid vector comprises a second nucleotide sequence;

[0095] The first nucleotide sequence comprises an expression cassette inserted between two first ITR sequences;

[0096] The second nucleotide sequence comprises an expression cassette inserted between two second ITR sequences;

[0097] The expression cassette of the first nucleotide sequence comprises a promoter, an N-terminal coding sequence of STRC, an N-terminal coding sequence of an intein, and polyA;

[0098] 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 a STRC and polyA.

[0099] Inteins

[0100] Inteins can splice proteins and exert their effects by covalently linking two different proteins after or during protein translation. The earliest inteins were discovered in fungi. Comparison and analysis of intein sequences predicts that there are over 600 intein genes present in viruses, bacteria, archaea, and eukaryotic microorganisms. Most inteins are complete proteins, but a small number of inteins have separate N- and C-termini. Inteins are linked to a portion of a protein at each end, and then reassemble after translation to produce the complete protein through nucleophilic chemical reactions and conformational changes.

[0101] In the present invention, preferably, the intein is separated from the N-terminus and the C-terminus. The intein can 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.

[0102] In some embodiments, the intein is an RmaDnaB intein, e.g., having an N-terminal portion of the RmaDnaB intein set forth in SEQ ID NO: 5 and a C-terminal portion of the RmaDnaB intein set forth in SEQ ID NO: 6. In some embodiments, the intein is an NpuDnaE intein, e.g., having an N-terminal portion of the NpuDnaE intein set forth in SEQ ID NO: 39 and a C-terminal portion of the NpuDnaE intein set forth in SEQ ID NO: 41.

[0103] STRC proteins

[0104] In some embodiments, the STRC protein comprises or consists of the amino acid sequence of SEQ ID NO:2.

[0105] In some embodiments, a cleavage site is provided on the amino acid sequence of the STRC protein to cleave the STRC protein into the N-terminal portion of the STRC protein (also referred to herein as the "N-terminus of the STRC") and the C-terminal portion of the STRC protein (also referred to herein as the "C-terminus of the STRC"). The N-terminus of the STRC is the sequence from the N-terminus of the STRC amino acid sequence to the cleavage site, and the C-terminus of the STRC is the sequence from the amino acid residue immediately adjacent to the cleavage site to the C-terminus of the STRC amino acid sequence. The N-terminus of the STRC is connected and fused to the N-terminus of the intein, and the C-terminus of the intein is connected and fused to the C-terminus of the STRC. There are multiple options for the cleavage site of the STRC. Table 1 below lists the locations of some of these cleavage sites on the STRC protein and the corresponding N-terminal portion of the STRC and the C-terminal portion of the STRC.

[0106] Table 1. Location of cleavage sites on STRC proteins and the corresponding N-terminal and C-terminal parts of STRC

[0107] In some embodiments, the STRC protein is cleaved at one or more of the following amino acid residues to form the N-terminal portion of the STRC protein and the C-terminal portion of the STRC protein: 650Leu, 947Leu, 651Ser, 967Leu, 657Cys, 982Arg, 669Arg, 983Ser, 688Pro, 989Leu, 697Pro, 1001Phe, 698Ser, 1012Leu, 701Ile, 1027Thr, 710Phe, 1047Leu, 722Lys, 1053Cys, 747L 19Val, 942Gln, 1086Leu, 802Leu, 1089Leu, 811Val, 656Asn, 819Pro, 708Ala, 825Asp, 933Ala, 834Tyr, 960Glu, 885Leu, 1052Leu, 895Asp, 1078Ala, 912Arg, 1080Ser, 917Gln, 1091Ala, 936Leu, 119Val, or 942Gln, wherein the amino acid position is relative to SEQ ID NO: 2.

[0108] In some embodiments, the STRC protein is cleaved at one or more of the following groups of amino acid residues to form a STRC protein N-terminal portion and a STRC protein C-terminal portion: 656, 722, 917, or 708, wherein the amino acid positions are relative to SEQ ID NO: 2.

[0109] In some embodiments, the STRC protein is cleaved into a STRC protein N-terminal portion and a STRC protein C-terminal portion selected from any one of the following groups:

[0110] Table 2 Exemplary cleavage sites of the STRC protein shown in SEQ ID NO: 2

[0111] Optionally, wherein the amino acid position is relative to SEQ ID NO:2.

[0112] In some embodiments, the STRC protein is cleaved into an N-terminal portion of the STRC protein and a C-terminal portion of the STRC protein selected from any one of the following groups:

[0113] 1-656aa+657-1775aa;

[0114] 1-722aa+723-1775aa;

[0115] 1-917aa+918-1775aa; or

[0116] 1-708aa+709-1775aa,

[0117] Optionally, wherein the amino acid position is relative to SEQ ID NO:2.

[0118] In some embodiments, the STRC protein is cleaved into the amino acid sequence of the N-terminal portion of the STRC protein of 1-656 aa and the amino acid sequence of the C-terminal portion of the STRC protein of 657-1775 aa.

[0119] In some embodiments, the STRC protein is cleaved into the amino acid sequence of the N-terminal portion of the STRC protein of 1-722 aa and the amino acid sequence of the C-terminal portion of the STRC protein of 723-1775 aa.

[0120] In some embodiments, the STRC protein is cleaved into the amino acid sequence of the N-terminal portion of the STRC protein of 1-917 aa and the amino acid sequence of the C-terminal portion of the STRC protein of 918-1775 aa.

[0121] In some embodiments, the STRC protein is cleaved into the amino acid sequence of the N-terminal portion of the STRC protein of 1-708 aa and the amino acid sequence of the C-terminal portion of the STRC protein of 709-1775 aa.

[0122] vector plasmid

[0123] The vector plasmid of the present invention can be any plasmid that can replicate in a host cell and express a corresponding polypeptide.

[0124] In some embodiments, the vector plasmid comprises two ITR sequences, namely a 5' inverted terminal repeat (5'ITR) sequence and a 3' inverted terminal repeat (3'ITR) sequence.

[0125] In some embodiments, in the dual-vector system for expressing STRC protein of the present invention, 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 pAAV, pAAV-CMV, pX601, pX551 or pAAV-MCS plasmid.

[0126] Dual vector system

[0127] The present invention provides a dual vector system comprising a first nucleic acid vector and a second nucleic acid vector, wherein:

[0128] The first nucleic acid vector comprises, in a 5'-3' direction: a 5' inverted terminal repeat (5'ITR) sequence, a nucleic acid sequence encoding the N-terminal portion of the STRC protein, a nucleic acid sequence encoding the N-terminal portion of the intein, and a 3' inverted terminal repeat (3'ITR) sequence;

[0129] The second nucleic acid vector comprises, in 5'-3' direction: a 5'ITR sequence, a nucleic acid sequence encoding the C-terminal portion of an intein, a nucleic acid sequence encoding the C-terminal portion of a STRC protein, and a 3'ITR sequence, and

[0130] Optionally, after the first nucleic acid vector and the second nucleic acid vector are introduced into a host cell, the N-terminal portion of the STRC protein and the C-terminal portion of the STRC protein are operably linked to produce a STRC protein.

[0131] In some embodiments, the present invention provides a two-vector system comprising a first nucleic acid vector and a second nucleic acid vector, wherein:

[0132] The first nucleic acid vector comprises, in a 5'-3' direction: a 5' inverted terminal repeat (5'ITR) sequence, a nucleic acid sequence encoding the N-terminal portion of the STRC protein, a nucleic acid sequence encoding the N-terminal portion of the intein, and a 3' inverted terminal repeat (3'ITR) sequence;

[0133] The second nucleic acid vector comprises, in 5'-3' direction: a 5'ITR sequence, a nucleic acid sequence encoding the C-terminal portion of an intein, a nucleic acid sequence encoding the C-terminal portion of a STRC protein, and a 3'ITR sequence, and

[0134] The amino acid sequence of the STRC protein has a STRC cleavage site, for example, the amino acid sequence of the STRC protein is as shown in SEQ ID NO: 2 or a functional fragment thereof, for example, an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 2;

[0135] The N-terminal portion of the STRC protein is the sequence from the N-terminus of the STRC amino acid sequence to the STRC cleavage site;

[0136] The C-terminal portion of the STRC protein is a sequence from the amino acid following the STRC cleavage site to the C-terminus of the STRC amino acid sequence;

[0137] Optionally, after the first nucleic acid vector and the second nucleic acid vector are introduced into a cell, the N-terminal portion of the STRC protein and the C-terminal portion of the STRC protein are operably linked to produce a full-length STRC protein.

[0138] 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.

[0139] In some embodiments, a tissue-specific promoter is used in a two-vector system, for example, a promoter that mediates expression in the ear, such as the synapsin promoter or the GFAP promoter.

[0140] In some embodiments, any one of the following promoters is used in the binary vector system: cytomegalovirus (CMV) promoter, SV40 promoter, Rous sarcoma virus (RSV) promoter, CAG promoter, chimeric CMV / chicken beta actin (CBA) promoter, truncated CBA (smCBA) promoter, UbC promoter, SFFV promoter, EF1α promoter, PGK promoter, or promoters of Myo7A, Myo15, Atoh1, POU4F3, Lhx3, Myo6, α9AchR, α10AchR, OTOF and STRC encoding genes. In some embodiments, the promoter is a CMV promoter.

[0141] The dual vector system of the present invention may also include one or more additional regulatory sequences that can function before or after transcription. The regulatory sequences may be part of the native transgenic locus or may be heterologous regulatory sequences. A portion of the 5'UTR or 3'UTR of the native transgenic transcript may be included in the dual vector system of the present invention.

[0142] The regulatory sequence may be any sequence that promotes transgene expression, i.e., serves 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.

[0143] Enhancers are cis-regulatory elements that affect the transcription of genes on the same molecule of DNA. Enhancers can be located upstream, downstream, within introns, or even relatively far from the genes they regulate.

[0144] The preferred post-transcriptional regulatory element used in the dual vector system of the present invention is the woodchuck hepatitis post-transcriptional regulatory element (WPRE) or a variant thereof. Compared with an AAV vector without WPRE or a variant thereof, an AAV vector containing WPRE or a variant thereof increases the expression of STRC protein.

[0145] In one embodiment, the dual vector system of the present invention comprises a WPRE nucleotide sequence as set forth in SEQ ID NO: 13. In another embodiment, the dual vector system of the present invention comprises a post-transcriptional regulatory element having a nucleotide sequence that is at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the WPRE nucleotide sequence as set forth in SEQ ID NO: 13, wherein the nucleotide sequence substantially retains the functional activity of the post-transcriptional regulatory element as set forth in SEQ ID NO: 13, for example, a truncated variant of WPRE. Reducing the size of the AAV genome enables increased flexibility in introducing other regulatory elements into the vector in addition to transgenes. In one embodiment, the truncated variant of WPRE has the WPRE3 nucleotide sequence as set forth in SEQ ID NO: 16.

[0146] In one embodiment, the binary vector system of the present invention comprises a polyadenylation sequence, e.g., a bovine growth hormone polyadenylation sequence, an SV40 polyadenylation sequence, and / or an SV40 late polyadenylation sequence. In one embodiment, the binary vector system of the present invention comprises an SV40 polyadenylation sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO: 14. In one embodiment, the binary vector system of the present invention comprises an SV40 late polyadenylation sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO: 17.

[0147] In some embodiments, the two-vector system of the present invention comprises a combination of a WPRE nucleotide sequence and an SV40 polyadenylation sequence, for example, the two-vector system of the present invention has the nucleotide sequence set forth in SEQ ID NO: 12, or a nucleotide sequence that is at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 12. The combination of the WPRE nucleotide sequence and the SV40 polyadenylation sequence allows for high-level expression of the transgene.

[0148] In some embodiments, the dual vector system of the present invention comprises a combination of a WPRE3 nucleotide sequence and an SV40 late polyadenylation sequence, for example, the dual vector system of the present invention has the nucleotide sequence set forth in SEQ ID NO: 15, or a nucleotide sequence that is at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 15. The combination of the WPRE3 nucleotide sequence and the SV40 late polyadenylation sequence (also referred to as "W3SL") can efficiently express larger exogenous genes while occupying less AAV packaging capacity.

[0149] In some embodiments, a signal peptide sequence is provided in the two-vector system that post-translationally targets the operably linked polypeptide to one or more cellular destinations (including, for example, specific organelle compartments) or to sites of protein synthesis and / or activity, and even to the extracellular environment.

[0150] The present invention uses the dual-vector system to deliver the STRC protein gene in two parts to inner ear cells, inner hair cells, or outer hair cells, where the N-terminal and C-terminal parts of the STRC protein expressed undergo trans-splicing to form the full-length STRC protein. The present invention demonstrates that the dual-vector system for expressing the STRC protein can effectively transduce the targeted inner ear cells, inner hair cells, or outer hair cells, producing the STRC protein in these cells and durably restoring hearing loss caused by STRC gene knockout.

[0151] In a preferred embodiment, the dual vector system of the present invention allows for the expression of homologous polypeptides having an amino acid sequence that is at least 70% identical and / or similar to SEQ ID NO: 2. More preferably, the homologous sequence is 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%, at least 99% identical and / or similar to SEQ ID NO: 2. When the homologous polypeptide is much shorter than SEQ ID NO: 2, local alignment may be considered.

[0152] In another embodiment, the dual vector system of the present invention can allow for the expression of functional fragments of STRC protein polypeptides. The term "functional fragment" herein refers to any fragment that retains at least one biological function of the STRC protein polypeptide of interest.

[0153] The full-length STRC protein can be obtained by transforming host cells using the dual vector system of the present 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.

[0154] III. Uses of the Dual Vector System

[0155] The dual vector system of the present invention is used to administer to patients suffering from DFNB16 deafness. "Patient suffering from DFNB16 deafness" refers to a patient, particularly a human patient, who is believed to have (or has been diagnosed with) a mutation in the gene for a constitutive STRC protein, which triggers abnormal expression, abnormal function, or both of the STRC protein.

[0156] In some embodiments, the dual vector system of the present invention is a dual AAV vector system. In some embodiments, the first AAV vector and the second AAV vector in the dual AAV vector system are vectors each having a capsid of the same or different AAV origin, for example, the first AAV vector and the second AAV vector in the dual AAV vector system are vectors each having an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-Anc80 capsid or an AAV vector with a chimeric capsid, in particular an AAV vector with an AAV-Anc80 capsid. Preferably, the synthetic adeno-associated virus vector Anc80L65 is used, which has been shown to have the highest transduction efficiency of inner ear hair cells reported to date (Suzuki et al., Sci. Rep. 7: 45524 (2017)).

[0157] The dual AAV vector system, after administration, treats STRC mutation-related autosomal recessive non-syndromic DFNB16 deafness disease by increasing the expression of wild-type STRC protein or providing wild-type STRC protein to the subject.

[0158] After administration, the dual-vector system of the present invention can trigger the expression of the full-length STRC protein polypeptide, or a functional fragment thereof, in inner ear cells, inner hair cells, or outer hair cells.

[0159] The patients to whom the dual vector system of the present invention is administered are preferably newborn human infants, usually less than 6 months old, or even less than 3 months old (if they were diagnosed with DFNB16 deafness in childhood). These human infants are more preferably between 3 months and 1 year old.

[0160] The two-vector system of the present invention can also be administered to, for example, infants (2-6 years), children (6-12 years), adolescents (12-18 years), or adults (18 years and older).

[0161] As used herein, the term "treating" is intended to mean administering a therapeutically effective amount of the dual-vector system of the present invention to a patient suffering from DFNB16 deafness to partially or completely restore the patient's hearing. Such restoration can be assessed by testing auditory brainstem responses (ABRs) using electrophysiological equipment. "Treatment of STRC mutation-induced hearing loss" specifically refers to complete restoration of hearing function. The term "preventing" refers to reducing or delaying hearing loss within the auditory frequency range.

[0162] Example

[0163] The technical solutions in the embodiments of the present invention are clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. Unless otherwise specified, the various reaction reagents involved in the embodiments can be purchased through commercial channels.

[0164] Example 1. Design of intein cleavage sites in the amino acid sequence of the full-length STRC protein

[0165] Intein cleavage sites as shown in Table 2 were designed in the human STRC protein shown in SEQ ID NO: 2, with a total of 47 cleavage sites.

[0166] Each STRC protein N-terminal fragment (also referred to herein as "N-Strc", "5'Strc") and each STRC protein C-terminal fragment (also referred to herein as "C-Strc", "3'Strc") in Table 2 is encoded by the corresponding nucleotide sequence shown in SEQ ID NO: 1.

[0167] Example 2. Construction of a dual-vector system for expressing STRC protein using a plasmid containing ITR sequences

[0168] The schematic diagram of the dual-vector system used in this example is shown in Figure 1. The pAAV-CMV-EGFP-WPRE-SV40 plasmid (synthesized by Nanjing GenScript, Figure 2) was used as the plasmid backbone to construct a first nucleic acid vector expressing the N-terminus of the STRC protein and a second nucleic acid vector expressing the C-terminus of the STRC protein. Figure 3 illustrates a dual-vector system constructed for cleavage site number 1 (i.e., with Leu 650aa of the STRC protein as the cleavage site), wherein the second nucleic acid vector is connected to the end of the coding sequence (CDS) expressing the C-terminus of the STRC protein (the HA tag sequence YPYDVPDYA (SEQ ID NO: 8) is used to verify in vitro expression), thereby obtaining a dual-vector system.

[0169] Specifically, the first nucleic acid vector and the second nucleic acid vector in the dual-vector system are obtained by transformation on the pAAV-CMV-EGFP-WPRE-SV40 plasmid backbone shown in Figure 2. Specifically, the pAAV-CMV-EGFP-WPRE-SV40 plasmid backbone (Figure 2) is used for transformation, and the EGFP reporter gene sequence and other sequences in the pAAV-CMV-EGFP-WPRE-SV40 plasmid backbone are replaced with a target sequence comprising a 5'Strc NT portion and an intein N-terminal sequence by double digestion with EcoRI and EcoRV to obtain the first nucleic acid vector; the EGFP reporter gene sequence in the pAAV-CMV-EGFP-WPRE-SV40 plasmid backbone is replaced with a target sequence comprising an intein C-terminal sequence and an intein C-terminal sequence and encoding a 3'Strc CT by double digestion with EcoRI and EcoRV to obtain the second nucleic acid vector. The obtained first nucleic acid vector comprises an inverted terminal repeat sequence (ITR), a CMV promoter sequence, a Kozak sequence, a cDNA sequence encoding the N-terminal portion (5'Strc NT) of the STRC protein, an N-terminal intein sequence ("N-intein") (the amino acid sequence encoded is shown in SEQ ID NO: 5), a WPRE sequence, an SV40 polyA sequence, an Ori sequence, and a resistance sequence; the second nucleic acid vector comprises an inverted terminal repeat sequence (ITR), a CMV promoter sequence, an C-terminal intein sequence ("C-intein") (the amino acid sequence encoded is shown in SEQ ID NO: 6), a Kozak sequence, a cDNA sequence encoding the C-terminal portion (3'Strc CT) of the STRC protein, a WPRE sequence, an SV40 polyA sequence, an Ori sequence, and a resistance sequence. After the first and second nucleic acid vectors are expressed in cells, expression of the full-length STRC protein is achieved through protein trans-splicing.

[0170] The synthesis of the sequences and the construction of the vectors were entrusted to Nanjing GenScript Biotechnology Co., Ltd.

[0171] 47 pairs of plasmids corresponding to the 47 cleavage sites in Table 2 were obtained and used in subsequent examples.

[0172] Example 3. Recombination of constructed plasmids in cells

[0173] The 47 pairs of plasmids obtained in Example 2 were transfected into HEK-293T cells (purchased from ATCC) in pairs, and the expression of full-length STRC protein was analyzed by Western blotting 48 hours after transfection. The specific experimental method is as follows.

[0174] Cell transfection: Inoculate HEK-293T cells (Human Embryonic Kidney 293T cells, hereinafter referred to as "293T cells") to a density of 70-90% and prepare for transfection. Prepare tube A: 125 μL serum-free DMEM medium + 8 μL Lipofectamine 3000 reagent (Invitrogen, catalog number: L3000015), and mix thoroughly. Prepare tube B: 125 μL serum-free DMEM medium + 2 μg first nucleic acid vector + 2 μg second nucleic acid vector + 8 μL P3000 reagent (Invitrogen, catalog number: L3000015), and mix thoroughly. Add the mixture in tube B to tube A, mix gently and thoroughly, and let it stand at room temperature for 10-15 minutes. Tube A contains a mixture of culture medium and Lipofectamine 3000 transfection reagent, and tube B contains a mixture of culture medium, vector plasmid DNA, and transfection enhancer P3000. The obtained DNA-liposome complex was added to 293T cells for transfection, and the cells were incubated at 37° C. in 95% air and 5% CO 2 . 48 hours after transfection, the cells were harvested by centrifugation.

[0175] After transfection, the cell pellet, harvested by centrifugation, was thoroughly resuspended in an appropriate amount of RIPA lysis buffer (Thermo Fisher Scientific, Catalog No. 89900) supplemented with 1% protease inhibitor cocktail (Thermo Fisher Scientific, Catalog No. 87786) and 1% PMSF. Lyse on ice for 30 minutes, vortexing every 10 minutes to thoroughly resuspend the cell pellet in the lysis buffer. Centrifuge at 12,000 rpm for 15 minutes, collect the supernatant (do not aspirate the pellet), add 5X loading buffer to the sample in proportion, boil at 75°C for 15 minutes, cool on ice, and after centrifugation, collect the supernatant for Western blot analysis.

[0176] Western blotting to detect STRC protein expression: Wash the glass plates and secure them flat on a rack, clamping them with the concave surface facing inward. Position the plates symmetrically, front and back. Prepare separating gel and seal with isopropanol. After 0.5 hours, discard the isopropanol and place on its side with a pump to dry. Prepare stacking gel, adding until overflowing, and insert a comb. After 45 minutes, remove the gel plates and attach them to the clamps in the electrophoresis tank. Add running buffer from the center of the tank until it reaches 1 / 2 the tank volume. Carefully remove the comb. Load the sample, conduct electrophoresis at a constant voltage of 100V for 1 hour, and transfer the membrane at a constant current of 300mA on ice for 90 minutes.

[0177] After transfer, remove the PVDF membrane and incubate it with blocking solution (5% skim milk powder in TBST buffer) at room temperature for 1 hour. Add the primary antibodies (HA-Tag Mouse mAb, Cell Signaling Technology, catalog number: 6E2; β-Actin Mouse mAb, Cell Signaling Technology, catalog number: 8H10D10) to the blocked PVDF membrane and incubate overnight at 4°C. Use the HA antibody as the primary antibody to detect the expression of the full-length STRC protein, and the β-actin antibody as the primary antibody to detect the level of the internal control protein β-actin in Western blotting. β-actin protein levels generally do not change, so it can be used to check whether the sample loading amount is consistent during Western blotting.

[0178] The next day, the incubated PVDF membrane was removed, rinsed three times with 1X TBST for 5 minutes each time, incubated with the secondary antibody (HRP-conjugated Affinipure Goat Anti-Mouse IgG (H+L), Proteintech, catalog number: SA00001-1) at room temperature for 1 hour, rinsed three times with 1X TBST for 5 minutes each time; then, chemiluminescent reagent (ECL) was added for development in a dark room.

[0179] The expression results of the target proteins after the 47 pairs of plasmids obtained in Example 2 were transfected into 293T cells in pairs are shown in Figure 6. In Figure 6, lane "Ctrl" represents the cell protein control without transfection of plasmids; lane "FL" represents the full-length protein STRC; lanes "1" to "47" respectively represent the protein expression levels of STRC after the 47 pairs of plasmids obtained in Example 2 were co-transfected into 293T cells.

[0180] As shown in Figure 6, for the cleavage site where the first amino acid residue of the C-terminus of STRC is Ser (cleavage sites numbered 1-38 of the STRC protein), the paired plasmids corresponding to cleavage sites 10 and 22 of the STRC protein, etc., were co-transfected into 293T cells, resulting in significantly higher expression levels of full-length STRC protein. For the cleavage site where the first amino acid residue of the C-terminus of STRC is Cys (cleavage sites numbered 39-47 of the STRC protein), the paired plasmids corresponding to cleavage sites 39 and 40 of the STRC protein, etc., were co-transfected into 293T cells, resulting in significantly higher expression levels of full-length STRC protein.

[0181] Example 4. Codon optimization, vector construction and recombinant expression in cells

[0182] The codon optimization of the Strc gene and the construction of the cleavage point plasmid were commissioned to Nanjing GenScript Biotechnology Co., Ltd. Specifically, the cDNA sequences corresponding to the cleavage sites 10 and 22 of the STRC protein in Example 3 were codon optimized; and the cDNA sequences corresponding to the cleavage sites 39 and 40 of the STRC protein in Example 3 were codon optimized. The codon-optimized nucleotide sequence of the Strc gene is shown in SEQ ID NO: 4, and the encoded signal peptide sequence is shown in SEQ ID NO: 3.

[0183] Using the codon-optimized Strc gene nucleotide sequence, a binary vector system 48-55 (Table 3) expressing the STRC protein was constructed similarly to that described in Example 2. Sequence synthesis and vector construction were commissioned to Nanjing GenScript Biotechnology Co., Ltd.

[0184] Table 3 Coding sequence information of STRC proteins in paired plasmids after codon optimization

[0185] Note: In Table 3, "GS-1-656" indicates that the codon-optimized nucleotide sequence encoding positions 1-656 of the N-terminal portion of STRC; "Signal Peptide-GS-657-1775" indicates that the codon-optimized nucleotide sequence encoding positions 657-1775 of the C-terminal portion of STRC is linked to the N-terminus with the nucleotide sequence encoding the signal peptide shown in SEQ ID NO: 3. These sequences correspond to the corresponding sequences in the codon-optimized Strc gene nucleotide sequence in SEQ ID NO: 4.

[0186] The eight plasmid pairs obtained corresponding to Table 3 were transfected into HEK-293T cells in pairs using the method described in Example 3, and the expression of the full-length STRC protein was detected by Western blotting. The Western blotting results are shown in Figure 7.

[0187] In Figure 7, lane "Ctrl" represents a cell protein control without transfection of the plasmid; lanes "48" to "51" respectively represent the protein expression levels of STRC after co-transfection of paired plasmids 48-51 in Table 3 (i.e., the C-terminal portion (CT) of STRC encoded by the second plasmid vector also has a signal peptide coding sequence) (Figure 4) into 293T cells; lanes "52" to "55" respectively represent the protein expression levels of STRC after co-transfection of paired plasmids 52-55 in Table 3 (i.e., the C-terminal portion (CT) of STRC encoded by the second plasmid vector does not have a signal peptide coding sequence) (Figure 5) into 293T cells.

[0188] The results in Figure 7 show that even when the C-terminal portion (CT) of the STRC encoded by the second plasmid vector lacks a signal peptide coding sequence, the full-length STRC protein can be generated, omitting the need for a signal peptide sequence. Codon optimization significantly increased the expression of the full-length STRC protein in cells.

[0189] Example 5. Analysis of auditory function and cochlear morphology in Strc gene knockout mice

[0190] 5.1 Construction of Strc gene knockout mouse model

[0191] Suzhou Saiye Biotechnology Co., Ltd. was commissioned to use the Crispr-Cas9 system to construct a mouse model with complete Strc gene knockout; the expression of the Strc gene in the knockout mice was then detected by sequencing, RT-PCR and immunofluorescence.

[0192] PCR (crude genomic DNA) method: Place a 2-5 mm section of mouse tail in a microcentrifuge tube, add 200 μL of lysis buffer (vazyme, PD101-01), vortex, and incubate in a 55°C water bath for 20 minutes. After incubation, heat the sample at 95°C or in a boiling water bath for 5 minutes to inactivate Proteinase K (vazyme, PD101-01). Vortex the lysate thoroughly, centrifuge at 12,000 rpm for 5 minutes, and remove the supernatant for PCR. PCR amplification reaction system (50 μL): 2× Taq Plus Master Mix (Dye Plus) 25 μL, lysate 2 μL, primer 1 (5'-CTGCTAGGCATCTAACTGGTCTG-3' (SEQ ID NO: 9), 10 μM) 2 μL, primer 2 (5'-CATGGGACCATCCACCTTACATT-3' (SEQ ID NO: 10), 10 μM) 2 μL, primer 3 (5'-ACGAGGAAATCATGAAGTCGAAGTG-3' (SEQ ID NO: 11), 10 μM) 2 μL, ddH2O 17 μL; the amplified products were directly detected by agarose gel electrophoresis.

[0193] Immunofluorescence staining method: Place the temporal bone of a newborn mouse on ice and dissect it quickly. Gently rinse the temporal bone once with 1XPBS. Coat a 10mm coverslip with 0.5μL of Cell Tak per coverslip and allow to dry (freshly coated). Place the coated coverslip in a culture dish containing the cochlea and adhere the cochlea to the coverslip with the front of the cochlea facing up. Place the adhered coverslip in a 4-well culture dish (previously added with 3ml of 1X PBS and placed on ice). Once all coverslips are adhered, remove the PBS and add 4% paraformaldehyde (PFA) (freshly prepared) to the four-well culture dish. Fix for 1 hour at room temperature and wash three times with 1X PBST. If the mouse is older than 7 days old, remove the entire ear tissue and place it in 1× PBS. Dissect the temporal bone under a microscope and place it in 4% PFA. Use forceps to gently perforate the top of the temporal bone and repeatedly inject PFA with a syringe. Fix the temporal bone in 4% PFA overnight at 4°C. Rinse three times in 1× PBS for 5 minutes each, then decalcify the bone in 0.5M EDTA (pH 8.0): decalcify for 3-4 hours from P0 to P7 (P0-P7 is postnatal day 0-7 of the mouse); decalcify for 1 day from P8 to P15; and decalcify for 2 days from P15 to P30. Wash the temporal bone three times with 1× PBST, place it in 1× PBS, and dissect the cochlea under a microscope. The coverslips coated with Cell-tak were placed in the culture dish containing the cochlea. The cochlea was adhered to the coverslips (front side up). The adhered coverslips were placed in a 4-well dish (previously added with 3 mL of PBS). After all the adhered coverslips were placed in the 4-well dish, they were blocked with blocking solution (PBS containing 1% Triton X-100 and 10% donkey serum) for 1-2 hours (85 μL / well). The primary antibody solution (STRC antibody, rabbit source, obtained from Huaan Bio) was prepared in PBT (PBS containing 1% Triton X-100 and 1% donkey serum). The primary antibody solution was added to the 4-well dish at 80 μL / well for incubation. The 4-well dish was placed in a large dish and lined with a wet napkin to prevent the primary antibody from evaporating. The dish was incubated at 4°C overnight. The cells were washed 3 times with 1× PBST (during which the secondary antibody could be prepared). The secondary antibody (Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor Alexa Fluor 488 (Invitrogen, A-11008) and Alexa Fluor Plus 555 phalloidin (Invitrogen, A30106) were added to PBT in the appropriate proportions and incubated at room temperature for 1 hour (80 μL / well) in the dark. The samples were washed three times with 1× PBST. 6 μL of DAKO was added to each sample, coverslips were placed, and the slides were sealed with nail polish and stored at 4°C.

[0194] Results: The cochlear basilar membrane sections of Strc knockout mice at different time points were prepared as described above, and immunofluorescence staining was performed using the stereocilia marker actin fluorescent dye Phalloidin, the kinocilia marker acetyl α-tubulin and Strc antibody. The results showed that Strc was not expressed in the inner ear of the knockout mice.

[0195] 5.2 Auditory function testing of Strc gene knockout mice

[0196] P30 (30 days after birth) wild-type C57BL / 6J mice of the same litter whose Strc gene was not knocked out were used as controls. Auditory brainstem response (ABR), distortion product otoacoustic examination (DPOAE) and CP analysis were performed on the Strc gene knockout mice 30 days after birth, thereby comprehensively evaluating the overall hearing status and inner ear hair cell function of the Strc gene knockout mice.

[0197] A. ABR Threshold Analysis

[0198] ABR tests, including auditory response threshold, latency, and inter-wave duration, use clicks, particularly at different frequencies (4 kHz, 8 kHz, 12 kHz, 16 kHz, 24 kHz, and 32 kHz), as stimuli. The researchers then tested the mice's hearing thresholds to analyze their hearing sensitivity and assess whether they have normal hearing function from hair cells to the cerebral cortex. Higher ABR thresholds indicate more severe hearing loss in Strc knockout mice.

[0199] Auditory Brainstem Response (ABR): Mice were anesthetized with sodium pentobarbital (10 mg / mL) and placed in a double-walled acoustic chamber (IAC, Bronx, NY, USA) for recording and measurement using the Smart EP evoked potential system. Subcutaneous needle electrodes (F-E2, Astro-Med Inc., Rhode Island) were used as electrodes. The detection electrode was implanted at the top of the mouse's brain, while the reference and ground electrodes were implanted outside the left and right ears, respectively. Biosignals were bandpass filtered for frequencies below 100 Hz and above 3000 Hz, amplified 200,000 times, and acquired at a 25 kHz A / D sampling rate. The noise suppression level was set to 31.00 μV during data acquisition. The data were averaged and displayed directly by a computer, with a computational time of approximately 10 ms. The threshold was determined by increasing the sound pressure level (SPL) in 10 dB increments. The animal's body temperature was maintained constant throughout the experiment.

[0200] B. Outer Hair Cell Function Analysis

[0201] The amplitude and threshold of distortion product otoacoustic emissions (DPOAE) in mice were measured by DPOAE. Two long-duration pure tones (2f1-f2) of different frequencies with a certain frequency ratio were used to induce DPOAE, and the function of mouse cochlear outer hair cells was preliminarily analyzed.

[0202] Distortion Product Otoacoustic Emission (DPOAE) Detection: Mice were anesthetized with sodium pentobarbital (10 mg / mL) and placed in a double-walled acoustic chamber (IAC, Bronx, NY, USA) for recording and measurement using an IL092 otoacoustic emission instrument (Otodynamic, UK). The probe used was a laboratory-prepared probe, ensuring a good seal between the probe and the mouse's external auditory canal during the experiment. The probe was connected to the IL092 otoacoustic emission instrument via a wire. The relevant parameters were set to / = 1.207. The threshold was set at a DPOAE intensity just 3 dB above the local noise level. To measure the DPOAE input and output curves, the original sound intensity was attenuated in 15 dB increments from 80 dB SPL to below the threshold. DPOAE thresholds were recorded at 5, 6, 7, and 8 kHz. The intensities of the 2DPOAEs elicited at 70 and 60 dB SPL were also recorded. DPOAE latencies were measured at a 70dB initial tone, and DPOAE latencies at 5, 6, and 7 kHz were recorded. Data were processed directly using the appropriate software. The animal's body temperature was maintained constant throughout the experimental procedure.

[0203] C. Electrocochleography (ECochleography) is an electrophysiological technique that records the electrical activity of the cochlea and primary cochlear nerve fibers after acoustic stimulation (CP analysis).

[0204] CP analysis includes analysis of cochlear microphonics (CM) and compound action potential (CAP or AP, including three negative potentials N1, N2, and N3). A short sound is delivered through a stimulator, and parameters such as the latency, threshold intensity, and amplitude of CM and AP are recorded, and CM and AP graphs are drawn. The output intensity and delay of the stimulator are adjusted to observe the phase changes of the cochlear microphonics potential and the auditory nerve action potential.

[0205] 5.3 Morphological Analysis of the Cochlea in Strc Knockout Mice

[0206] A. Detection of changes in hair cell number in Strc knockout mice by immunostaining

[0207] Cochlear basilar membrane sections of Strc knockout mice were prepared at P14, P21, P30, P60 and P90 time points (i.e., 14 days, 21 days, 30 days, 60 days and 90 days after birth), and the basilar membranes of Strc knockout mice were finely dissected and immunofluorescently stained using hair cell marker myo7a antibody (Proteus-bioscienes, catalog number: 25-6790) and STRC antibody (Signalway Antibody, catalog number: CB17). The number of inner ear hair cells in Strc gene knockout mice was changed (relative to wild-type mice).

[0208] B. Study the morphology and structural changes of cilia in Strc knockout mice by SEM and TEM

[0209] Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to examine the changes in the morphology, number, structure of inner ear hair cell cilia and actin filaments in stereocilia of Strc gene knockout mice at different time points: P3, P14, P30, P60 and P90.

[0210] Scanning electron microscopy (SEM) methods: Tissues were fixed overnight in 1 / 2 Karnovsky's fixative (2% PFA, 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4) and post-fixed in 1% osmium hydroxide for 1 hour. All samples were dehydrated through a series of graded ethanol solutions, washed three times with hexamethyldisilazane, and air-dried at room temperature. Tissues were optimally positioned and mounted on carbon tape, which was then placed on an aluminum base. The treated tissue samples were then coated with a 10 nm gold / palladium mixture and observed using a FEI XL30 SEM operating at 10 kV.

[0211] Example 6. Preparation of adeno-associated virus

[0212] In this example, a dual-vector system was constructed that can efficiently express larger exogenous genes while occupying less AAV packaging capacity, and paired adeno-associated viruses were prepared for infecting mice.

[0213] 6.1 Preparation of a binary vector system containing a truncated WPRE

[0214] According to the results of Examples 3 and 4, the cleavage site was selected as Asn at position 656 of the STRC protein; and the cleavage site was selected as Lys at position 722 of the STRC protein, and two-vector systems containing truncated WPRE corresponding to numbers 52 and 53 in Table 3 were constructed, wherein the WPRE+SV40 poly (A) (717 bp) nucleotide sequence shown in SEQ ID NO: 12 in the pAAV-CMV-EGFP-WPRE-SV40 plasmid backbone shown in Figure 2 was replaced with the WPRE3-SV40 late poly (A) (432 bp) nucleotide sequence shown in SEQ ID NO: 15, and two sets of two-vector systems 52 and 53 containing truncated WPRE were constructed.

[0215] 6.2 Preparation of dual AAV viruses

[0216] Four adeno-associated viruses (AAVs) were prepared in HEK-293T cells using the four plasmids corresponding to the two binary vector systems 52 and 53 containing the truncated WPRE. The two pairs of dual AAV viruses were then paired. The specific method is as follows. For viral packaging: Prepare 10 150mm dishes of HEK-293T cells at a cell density of 80%-90% confluency. Prepare tube A with 4880μL of serum-free DMEM medium and 120μL of PEI transfection reagent, mixing thoroughly. Prepare Tube B: 4958 μL serum-free DMEM medium + 15 μL pAnc80L65 plasmid (GenBank: KT235804.1, providing the capsid protein of Anc80L65) (Addgene plasmid #68837) + 15 μL pHelper plasmid (GenBank: AF369965.1) + 12 μL of the first or second nucleic acid vector in the dual-vector system constructed in Example 6.1, and mix thoroughly. Add the liquid in Tube A to Tube B, adding dropwise and gently mixing. Let it stand at room temperature for 20-25 minutes. Add the mixture to the prepared HEK-293T cells, adding 1 ml of DNA-liposome complex per plate (can be prepared in batches). After 12 hours, replace the medium. After 48 hours, collect the supernatant into a sterile bottle and store at 4°C. Add fresh medium and continue culturing for 48 hours. After that, collect the cells and supernatant into the aforementioned sterile bottle.

[0217] AAV purification: After collecting the supernatant and cells, cells were lysed using a hot-cold-hot cycle. Cells were alternately placed in dry ice-cold ethanol and a 37°C water bath three times, followed by centrifugation at 1167g for 15 minutes at 4°C. Genomic and plasmid DNA was then removed, and the supernatant was transferred to a 50mL centrifuge tube. DNase and RNase were added to final concentrations of 10 U / mL and 10 mg / mL, respectively, and incubated at 37°C for 30 minutes. The supernatant was centrifuged at 13490 rpm for 20 minutes at 4°C. The supernatant from the previous centrifugation was filtered using a sterile 50mL syringe and a 0.22μm filter. A gradient of iodixanol was then prepared: 8mL of 15% (v / v) iodixanol, 5.5mL of 25% (v / v) iodixanol, 5mL of 40% (v / v) iodixanol, and 4.5mL of 60% (v / v) iodixanol. During this step, iodixanol is irritating to the eyes, skin, and respiratory tract; personal protective equipment is required and the procedure must be performed in a fume hood. The supernatant was carefully dripped onto a 15% iodixanol surface and ultracentrifuged using a fixed-angle titanium rotor at 301,580 g for 1 hour 40 minutes at 12°C, using maximum acceleration and deceleration. Purified AAV virus was obtained between the 40% and 60% iodixanol interfaces and was carefully aspirated using a blunt stainless steel needle. The resulting AAV viruses were paired and designated double AAV virus 52 and double AAV virus 53.

[0218] AAV titer determination: AAV titer was determined using qPCR. The titer was expressed as GC / mL, where GC represents the genomic particle concentration. The purified virus titer was verified to be above 10e+12 GC / mL. qPCR primers were designed to target the ITR sequence. The primer sequences were: upstream primer fwd ITR primer, 5'-GGAACCCCTAGTGATGGAGTT (SEQ ID NO: 18); downstream primer rev ITR primer, 5'-CGGCCTCAGTGAGCGA (SEQ ID NO: 19).

[0219] Titer calculation formula: Titer = 1000000*power(10 x )

[0220] The formula for calculating x is: x = (38.71-y) / 3.54

[0221] 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.

[0222] For example, the CT value of the qPCR result is 13.925=y. Substituting it into the above formula, x=(38.71-13.925) / 3.54=7, thus, the virus titer value is =1000000*power(10 7 ), that is, 1.00E+13.

[0223] Example 7. Restoration of deafness in Strc gene knockout mice by dual-vector AAV delivery of exogenous Strc gene

[0224] 7.1 Administration of AAV Virus in Mice

[0225] Neonatal mice were anesthetized using hypothermia-induced anesthesia. Strc knockout mice from P2 to P3 (i.e., 2 to 3 days after birth) were placed in an ice bath for 2-3 minutes and then removed and placed on an ice pad for subsequent surgical procedures. The surgery was performed only on the left ear of each mouse, with the right ear serving as a negative control. During the surgery, an incision was made behind the left ear to expose the round window. Care was taken to avoid damaging the facial nerve during the surgery. Then, a microinjection system (Nanoliter 2000, WPI) was used to inject the dual AAV virus 52 and dual AAV virus 53 prepared in Example 6.2 into the cochlea through a capillary glass electrode (10 mm in diameter) through the round window. Since the cochlea of ​​young mice can accommodate 2 μL of AAV virus solution, the volume of the injected virus was selected to be 1-2 μL (N-terminal virus corresponding to each group of cleavage sites: C-terminal virus = 1:1, to obtain a dual AAV virus mixture Anc80L65-Tr). After surgery, the wound was sealed, analgesics and anti-inflammatory medications were applied, and the mice were placed on a 37°C hot plate to recover. The mice fully recovered within approximately 10 minutes and were returned to their mothers. Standard postoperative care was followed.

[0226] Four weeks later, auditory brainstem responses (ABRs) and distortion product otoacoustic emissions (DPOAEs) were performed on the injected mice to evaluate the effects of the dual AAV virus mixture Anc80L65-Tr injection on the mice's overall hearing.

[0227] 7.2 Analysis of the therapeutic effect of mice after injection of dual AAV virus mixture

[0228] Detection of exogenous Strc gene expression:

[0229] The basilar membrane of the cochlea on the gene-treated side was spread and immunofluorescence staining was performed using the stereocilia marker actin fluorescent dye Phalloidin; Strc antibody was used as the primary antibody (rabbit source, obtained from Huaan Biotechnology) and goat anti-rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488, Invitrogen, A-11008 and Alexa Fluor Plus 555 phalloidin, Invitrogen, A30106 as the secondary antibodies to study the expression of exogenous Strc gene in hair cells.

[0230] Detect changes in the number of cochlear hair cells:

[0231] Immunofluorescence staining was used to detect changes in the number of hair cells in the cochlea after gene therapy. Immunofluorescence staining was performed using antibodies against the hair cell markers Myo7a and Strc to observe and count changes in the number of hair cells in the cochlea after virus injection. The primary antibody was Strc antibody (rabbit source, obtained from Huaan Biotechnology); Myo7a antibody, Proteus, catalog number: 256790; and the secondary antibodies were goat anti-rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488, Invitrogen, A-11008, and Alexa Fluor Plus 555 Phalloidin, Invitrogen, A30106.

[0232] Detecting changes in the morphology and structure of stereocilia in the cochlea:

[0233] Immunofluorescence staining and electron microscopy were used to examine the morphological and structural changes of stereocilia in the gene-treated cochlea. Immunofluorescence staining and scanning electron microscopy (SEM) were used to examine the changes in the morphology, number, and structure of stereocilia in the virus-injected cochlea. Transmission electron microscopy (TEM) was used to examine the changes in actin filaments in stereocilia in the virus-injected cochlea.

[0234] As shown in Figure 8, in the treated Strc knockout mouse model, double AAV virus 52 and double AAV virus 53 lowered the ABR threshold and significantly restored some hearing frequency bands, which was close to the hearing threshold of wild-type (WT) mice.

[0235] The immunofluorescence results in Figure 9 show that the dual AAV virus 52 and the dual AAV virus 53 achieved the expression of STRC protein in the mouse cochlea and restored hearing.

[0236] While the exemplary embodiments of the present invention have been described above, it should be understood by those skilled in the art that these disclosures are merely exemplary and that various other substitutions, adaptations, and modifications may be made within the scope of the present invention. Therefore, the present invention is not limited to the specific embodiments listed herein.

[0237] Exemplary sequences

Claims

1. A dual-vector system for expressing STRC protein, which comprises a first nucleic acid vector and a second nucleic acid vector, wherein the first nucleic acid vector contains a first nucleotide sequence; and the second nucleic acid vector contains a second nucleotide sequence; the first nucleotide sequence contains an expression cassette inserted between two first ITR sequences; the second nucleotide sequence contains an expression cassette inserted between two second ITR sequences; the expression cassette of the first nucleotide sequence contains a promoter, an N-terminal coding sequence of STRC, an N-terminal coding sequence of an intein, and polyA; the expression cassette of the second nucleotide sequence contains a promoter, a C-terminal coding sequence of an intein, a C-terminal coding sequence of STRC, and polyA; and a STRC cleavage site is set in the STRC amino acid sequence. For example, the STRC amino acid sequence is as shown in SEQ ID NO:2 or a functional fragment thereof, for example, an amino acid sequence having at least 80% sequence identity with SEQ ID NO:2; the N-terminal coding sequence of STRC is the nucleotide coding sequence from the N-terminus of the STRC amino acid sequence to the STRC cleavage site; the C-terminal coding sequence of STRC is the nucleotide coding sequence from the amino acid at the position immediately after the STRC cleavage site to the C-terminus of the STRC amino acid sequence.

2. The dual-vector system for expressing STRC protein according to claim 1, wherein, The STRC cleavage site is located at the amino acid immediately preceding serine, threonine, or cysteine in the STRC amino acid sequence.

3. The dual-vector system for expressing the STRC 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 the CAG promoter, CMV promoter, CBA promoter, UbC promoter, SFFV promoter, EF1α promoter, PGK promoter, or the promoters of Myo7A, Myo15, Atoh1, POU4F3, Lhx3, Myo6, α9AchR, α10AchR, OTOF, and the STRC coding gene; The polyA of the expression cassette of the first nucleotide sequence or the second nucleotide sequence comprises AATAAA (SEQ ID NO: 20) and variants of AATAAA; the variants of AATAAA comprise ATTAAA (SEQ ID NO: 21), AGTAAA (SEQ ID NO: 22), CATAAA (SEQ ID NO: 23), TATAAA (SEQ ID NO: 24), GATAAA (SEQ ID NO: 25), ACTAAA (SEQ ID NO: 26), AATATA (SEQ ID NO: 27), AAGAAA (SEQ ID NO: 28), AATAAT (SEQ ID NO: 29), AAAAAA (SEQ ID NO: 30), AATGAA (SEQ ID NO: 31), AATCAA (SEQ ID NO: 32), AACAAA (SEQ ID NO: 33), AATCAA (SEQ ID NO: 34), AATAAC (SEQ ID NO: 35), AATAGA (SEQ ID NO: 36), AATTAA (SEQ ID NO: 37) or AATAAG (SEQ ID NO: 38); 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: 14 or SEQ ID NO: 17; 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 STRC 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 the woodchuck 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: 13, for example, the nucleotide sequence shown in SEQ ID NO: 16; for example, the tag element is HA.

5. The dual-vector system for expressing STRC protein according to claim 1, wherein, The intein 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 intein is derived from RmaDnaB. For example, the N-terminus of the intein is the N-terminus of the RmaDnaB intein shown in SEQ ID NO:5, and the C-terminus of the intein is the C-terminus of the RmaDnaB intein shown in SEQ ID NO:6; or the intein is derived from NpuDnaE. For example, the N-terminus of the intein is the N-terminus of the NpuDnaE intein shown in SEQ ID NO:39, and the C-terminus of the intein is the C-terminus of the NpuDnaE intein shown in SEQ ID NO:

41.

6. The dual-vector system for expressing STRC protein according to claim 1, wherein, Insert the first nucleotide sequence into a plasmid containing two first ITR sequences, and insert the second nucleotide sequence 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 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 STRC protein according to any one of claims 1-6, wherein, The STRC cleavage site is shown in Table 1; preferably, the 656th amino acid of the STRC amino acid sequence shown in SEQ ID NO:2 is used as the STRC cleavage site, and the RmaDnaB intein is used; after ligating and fusing the N-terminal coding sequence of STRC with the N-terminal coding sequence of the RmaDnaB intein, a first nucleotide sequence is constructed using the pAAV-CMV plasmid as a vector; After ligating and fusing the C-terminal coding sequence of the RmaDnaB intein with the C-terminal coding sequence of STRC, a second nucleotide sequence is constructed using the pAAV-CMV plasmid as a vector; Use the 708th amino acid of the STRC amino acid sequence shown in SEQ ID NO:2 as the STRC cleavage site, and use the RmaDnaB intein; after ligating and fusing the N-terminal coding sequence of STRC with the N-terminal coding sequence of the RmaDnaB intein, a first nucleotide sequence is constructed using the pAAV-CMV plasmid as a vector; After ligating and fusing the C-terminal coding sequence of the RmaDnaB intein with the C-terminal coding sequence of STRC, a second nucleotide sequence is constructed using the pAAV-CMV plasmid as a vector; Use the 722nd amino acid of the STRC amino acid sequence shown in SEQ ID NO:2 as the STRC cleavage site, and use the RmaDnaB intein; after ligating and fusing the N-terminal coding sequence of STRC with the N-terminal coding sequence of the RmaDnaB intein, a first nucleotide sequence is constructed using the pAAV-CMV plasmid as a vector; After ligating and fusing the C-terminal coding sequence of the RmaDnaB intein with the C-terminal coding sequence of STRC, a second nucleotide sequence is constructed, using the pAAV-CMV plasmid as a vector; or Using the 917th amino acid of the STRC amino acid sequence shown in SEQ ID NO: 2 as the STRC cleavage site and using the RmaDnaB intein; after ligating and fusing the N-terminal coding sequence of STRC with the N-terminal coding sequence of the RmaDnaB intein, a first nucleotide sequence is constructed, using the pAAV-CMV plasmid as a vector; After ligating and fusing the C-terminal coding sequence of the RmaDnaB intein with the C-terminal coding sequence of STRC, a second nucleotide sequence is constructed, using the pAAV-CMV plasmid as a vector; For example, the N-terminal coding sequence of the RmaDnaB intein encodes the N-terminal portion of RmaDnaB shown in SEQ ID NO: 5, and the C-terminal coding sequence of the RmaDnaB intein encodes the C-terminal portion of RmaDnaB shown in SEQ ID NO:

6.

8. The dual-vector system for expressing the STRC protein according to any one of claims 1-7, wherein the expression cassette of the first nucleotide sequence and the expression cassette of the second nucleotide sequence each comprise a signal sequence operably linked to a promoter sequence and under the control of the promoter; preferably, the signal sequence is the nucleotide sequence encoding SEQ ID NO:

3.

9. The dual-vector system for expressing the STRC 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 comprise a combination of a WPRE nucleotide sequence and an SV40 polyadenylation sequence at the N-terminus of the 3' ITR sequence, for example, having the nucleotide sequence shown in SEQ ID NO: 12 or a nucleotide sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 12; or comprise a combination of a WPRE3 nucleotide sequence and an SV40 late polyadenylation sequence, for example, having the nucleotide sequence shown in SEQ ID NO: 15 or a nucleotide sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:

15.

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

2.

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

12. A method for packaging adeno-associated virus, wherein, The packaging vector system of adeno-associated virus according to claim 10 or 11 is transferred into a host cell for packaging.

13. The packaging method of adeno-associated virus according to 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 dual-vector system for expressing STRC protein according to any one of claims 1-9 or the adeno-associated virus according to claim 14 for the preparation of a drug or preparation for treating deafness, hearing impairment, or hearing dysfunction.

16. A drug or preparation for treating deafness diseases, hearing impairments or hearing dysfunctions, which is prepared from the dual-vector system for expressing STRC 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. The packaging vector system of the adeno-associated virus includes a dual-vector system for expressing STRC protein, a vector carrying AAVrep and cap genes, and a helper virus vector.

17. The drug or preparation according to claim 16, wherein, The drug or preparation further comprises a neutral salt buffer, an acidic salt buffer, a basic salt buffer, glucose, mannose, mannitol, protein, polypeptide, amino acid, antibiotic, chelating agent, adjuvant, preservative, nanoparticle, liposome, and positive lipid particle.

18. The medicament or preparation according to claim 16 or 17, wherein, Administration by injection through the round window, oval window, semicircular canals, and common canal of the cochlea; and single or multiple administrations throughout life, with a total dose of 1×10 9 -1×10 13 viral genomes.

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