A dual-carrier system for treating hearing loss and its application

Abstract

The present invention belongs to the field of gene therapy within the medical field, and specifically involves the use of overexpression of normal genes to restore hearing in patients with hereditary deafness caused by gene mutation or deletion. The present invention provides a dual-vector system for expressing the OTOF protein, comprising two nucleotide sequences: a first nucleotide sequence comprising two ITR sequences and a gene expression cassette inserted between the ITR sequences; and a second nucleotide sequence comprising two ITR sequences and a gene expression cassette inserted between the ITR sequences. The gene expression cassette of the first nucleotide sequence comprises a promoter, an OTOF N-terminal coding sequence, an intein N-terminal coding sequence, and polyA; and the gene expression cassette of the second nucleotide sequence comprises a promoter, an intein C-terminal coding sequence, an OTOF C-terminal coding sequence, and polyA. Also provided is an adeno-associated virus packaged by the aforementioned vector. The vector and virus can be used in the field of large-gene dual-vector delivery for gene therapy for deafness to restore hearing in both ears through unilateral administration.

Description

Technical field:

[0001] The present invention belongs to the field of gene therapy in the medical field, and in particular is an application of overexpression of normal genes to restore hereditary hearing loss caused by gene mutation or deletion. Background technology:

[0002] The ear is a vital organ in the human body, consisting of the outer ear, middle ear, and inner ear. Its primary function is to perceive sound and maintain balance. Abnormal ear function can lead to a range of bodily problems, including deafness, tinnitus, and vertigo.

[0003] Deafness is a common hearing disorder that can be categorized as either congenital or acquired, and is often linked to both genetic and environmental factors. Congenital deafness affects 2 in every 1,000 births, and 50-60% of these cases are caused by gene mutations. Deafness caused by gene mutations can be categorized as dominant or recessive. Dominant deafness genes include ACTG1, CCDC50, CD164, CEACAM16, and DIAPH1, while recessive deafness genes include CLDN14, PJVK, GRXCR1, MYO7A, MYO6, MYO3A, MYO15A, OTOF, OTOG, OTOA, STRC, TMC1, SLC22A4, SLC26A4, SLC26A5, TECTA, GJB2, and GJB6. The discovery of these deafness genes provides potential targets for the precise treatment of hereditary deafness. Therefore, when the above genes cause deafness, gene therapy can be used as the preferred strategy for curing deafness.

[0004] Gene therapy refers to a method of correcting, compensating, or inhibiting the DNA or RNA level to restore the disease caused by abnormal nucleic acid sequence or expression in the body and achieve the purpose of treatment. 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 AAV packaging, such as BDP1, CDH23, COL11A2, LOXHD1, MET, MYO15A, MYO3A, MYO7A, OTOG, OTOF, OTOGL, PCDH15, PTPRQ, STRC, TECTA, TARA, etc. Their coding regions are all longer than 4kb, and together with the related regulatory elements, they will exceed the packaging limit of AAV vectors. Although the use of DNA recombinant dual vectors has solved this problem, the in vivo recombination efficiency of DNA recombinant dual vector packaging is low.

[0005] Among the deafness-related genes with coding sequence length greater than 4kb, OTOF plays an important role in hearing. OTOF protein is mainly expressed in the inner ear hair cells of the cochlea, and its main function is to bind calcium ions (Ca 2+ ) and initiates the release of downstream neurotransmitters. Deletion or loss-of-function mutations in the OTOF gene can cause deafness in DFNB9.

[0006] The OTOF gene (NCBI Gene ID: 9381) has different transcripts after transcription, including isoform 1 (NM_194248.3), isoform 2 (NM_004802.4), isoform 3 (NM_194322.3), isoform 4 (NM_194323.3), and isoform 5 (NM_001287489.2). They all originate from different splicing forms of the same RNA, with the splicing form associated with hearing in inner ear hair cells being isoform 1 or isoform 5. Both transcripts have a CDS region length of 5994 base pairs and encode a protein of 1997 amino acids.

[0007] For congenital deafness caused by OTOF gene mutations, although AAV has the characteristics of non-integration in delivery, long expression time, and low immunogenicity, it is the preferred delivery vehicle, but the AAV packaging capacity is less than 4.7kb, and there is a packaging limitation problem, which cannot meet the packaging of the OTOF gene (the total length of the OTOF gene plus the regulatory sequence exceeds 7kb). In order to solve the packaging problem of OTOF, there are currently three main methods. One is overload packaging, which mainly uses this method to package a gene expression element with a length of 7.5kb into an AAV virus and inject it into the cochlea of ​​mice. After a period of action, it can be observed that about 30% of the inner ear hair cells express OTOF protein, and the hearing of the mice is restored to about 58dB. However, overload packaging has the problems of low packaging efficiency, difficult product control and low transfection efficiency, and is not the best solution. The second approach is to shorten the coding sequence required for a functional OTOF. OTOF is a C2 domain protein composed of six C2 domains (A, B, C, D, E, and F) and a TEM domain. Studies have found that mini-OTOFs composed of several of these domains can partially restore OTOF function, but cannot restore hearing in animals. The third approach is to use a dual-vector approach for DNA recombination to produce full-length mature OTOF mRNA and complete protein translation. This can be categorized into overlapping, trans-splicing, or a combination of overlapping and trans-splicing.

[0008] Although DNA recombination strategies can yield a full-length, functional OTOF protein (otoferlin), this strategy suffers from suboptimal recombination efficiency, hindering protein expression and accumulation. To overcome this issue, protein recombination, particularly intein-mediated protein trans-splicing, offers rapid and efficient recombination. Inteins were first discovered in fungi and yeast and have subsequently been found in a variety of microorganisms, including bacteria, viruses, and archaea. Inteins can form both intramolecular and intermolecular protein linkages. Intermolecular linkages can be achieved using naturally separated inteins or artificially separated pre-existing intramolecular inteins. When the N- and C-termini of an intein are linked to the C- and N-termini of the target protein, respectively, the electrophilic group of the second moiety attacks the nucleophilic group of the first moiety, forming a covalent bond and, through conformational modification, forming a complete two-part linked protein. Molecular design requires that the first amino acid of the C-terminus be serine, threonine, or cysteine.

[0009] Given the potential for improved protein recombination efficiency in dual vectors, the present invention proposes a protein-level recombination strategy to reconstruct deafness-related proteins. The method involves using AAV vectors and intein recombination to express OTOF proteins, thereby restoring abnormal gene function and achieving efficient expression. Summary of the invention:

[0010] The current method of using dual-vector AAV delivery and DNA recombination in the OTOF expression system has low recombination efficiency. At the same time, the current dual-vector delivery technology only achieves the expression of OTOF in the unilateral cochlea and the restoration of unilateral hearing. If binaural hearing restoration is required, binaural cochlear injection must be performed. Therefore, the second injection means inconvenience to the patient and increased risk. In order to solve the above technical problems, the present invention will provide a dual-vector system capable of expressing OTOF protein and an adeno-associated virus packaged thereby. This method can restore bilateral hearing by unilateral administration in the field of large-gene dual-vector delivery for deafness gene therapy.

[0011] One of the technical solutions provided by the present invention is a dual-vector system for expressing OTOF protein, wherein the dual-vector system comprises two nucleotide sequences:

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

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

[0014] The expression cassette of the first nucleotide sequence includes: a promoter, an OTOF N-terminal coding sequence, an Intein N-terminal coding sequence and PolyA;

[0015] The expression cassette of the second nucleotide sequence includes: a promoter, an Intein C-terminal coding sequence, an OTOF C-terminal coding sequence and PolyA;

[0016] The amino acid sequence of the OTOF is shown in SEQ ID NO.1 or SEQ ID NO.2 in the sequence listing;

[0017] Wherein, a split point is set on the OTOF amino acid sequence, and the nucleotide coding sequence from the N-terminus of the OTOF amino acid sequence to the split point is the OTOF N-terminal coding sequence, and the nucleotide coding sequence from the next amino acid of the split point to the C-terminus of the OTOF amino acid sequence is the OTOF C-terminal coding sequence;

[0018] Furthermore, the cleavage site of OTOF includes but is not limited to the amino acid preceding serine, threonine or cysteine ​​in the amino acid sequence of the OTOF protein, and the order is from the N-terminus to the C-terminus of OTOF;

[0019] The promoter includes but is not limited to: CAG promoter, CMV promoter, CBA promoter, UbC promoter, SFFV promoter, EF1α promoter, PGK promoter, or promoters of genes encoding Myo7A, Myo15, Atoh1, POU4F3, Lhx3, Myo6, α9AchR, α10AchR, OTOF, etc.;

[0020] The PolyA is an adenine modification at the tail end during the maturation of mRNA, which can make the mRNA more stable. The PolyA sequence of the present invention contains AATAAA and its variants, including but not limited to ATTAAA, AGTAAA, CATAAA, TATAAA, GATAAA, ACTAAA, AATTA, AAGAAA, AATGAA, AATCAA, AACAAA, AATCAA, AATCAA, AATTAGA, AATTAAA or AATAAG;

[0021] Furthermore, the ITR sequence (inverted terminal repeat sequence) is derived from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 or AAV9;

[0022] The expression cassette further comprises other expression elements, including but not limited to expression regulatory elements and tag elements;

[0023] Furthermore, the expression control elements include but are not limited to the following functional control elements: (1) for regulating the expression of target proteins, such as IRES, for initiating the translation of downstream genes; (2) control elements for expressing miRNA and siRNA sequences; (3) introns; (4) localization sequences, for localizing the expression of target proteins to the nucleus, cytoplasm or various organelles, and for secretion outside the cell; (5) sequences for accelerating protein degradation, such as PEST sequences; (6) the control element can also be a partial kozak sequence, the kozak sequence is GNCNCN, such as GCCACC; (7) enhancers, which can be derived from SV40 virus, CMV virus or adenovirus, etc.; (8) the control element can be WPRE.

[0024] Furthermore, the tag element includes but is not limited to FLAG, HA, MYC, fluorescent protein, luciferase, SUMO protein, ubiquitin protein, GST, etc.

[0025] Furthermore, the nucleotide sequence of the OTOF encoding gene is shown in SEQ ID NO.3 or SEQ ID NO.4 in the sequence listing;

[0026] Furthermore, the intein includes but is not limited to intein sequences in proteins such as MxeGyrA, pabPolIII, MjaKlbA, SspDnaB, SceVMA, SspDnaE, ​​NpuDnaE, ​​AvaDnaE, ​​CraDnaE, ​​CspDnaE, ​​CwaDnaE, ​​MchtDnaE, ​​OliDnaE, ​​TerDnaE, ​​gp41-1, gp41-8, IMPDH-1, and RmaDnaB;

[0027] Furthermore, for the amino acid sequence of the OTOF protein shown in SEQ ID NO. 1 or 2, the splitting sites include but are not limited to amino acid residues 827, 930, 954 and 1130;

[0028] Furthermore, for the amino acid sequence of the OTOF protein shown in SEQ ID NO. 1 or 2, the splitting site is: amino acid residue 827, that is, the nucleotide coding sequence of the amino acid sequence from positions 1 to 827 is the OTOF N-terminal coding sequence, and the nucleotide coding sequence of the amino acid sequence from positions 828 to 1997 is the OTOF C-terminal coding sequence;

[0029] Furthermore, for the amino acid sequence of the OTOF protein shown in SEQ ID NO. 1 or 2, the splitting site is: amino acid residue 930, that is, the nucleotide coding sequence of the amino acid sequence from positions 1 to 930 is the OTOF N-terminal coding sequence, and the nucleotide coding sequence of the amino acid sequence from positions 931 to 1997 is the OTOF C-terminal coding sequence;

[0030] Furthermore, for the amino acid sequence of the OTOF protein shown in SEQ ID NO. 1 or 2, the splitting site is: amino acid residue 954, that is, the nucleotide coding sequence of the amino acid sequence from positions 1 to 954 is the OTOF N-terminal coding sequence, and the nucleotide coding sequence of the amino acid sequence from positions 955 to 1997 is the OTOF C-terminal coding sequence;

[0031] Furthermore, for the amino acid sequence of the OTOF protein shown in SEQ ID NO. 1 or 2, the splitting site is: amino acid residue 1130, that is, the nucleotide coding sequence of the amino acid sequence from positions 1 to 1130 is the OTOF N-terminal coding sequence, and the nucleotide coding sequence of the amino acid sequence from positions 1131 to 1997 is the OTOF C-terminal coding sequence;

[0032] Preferably, the amino acid sequence of the OTOF is shown as SEQ ID NO.2.

[0033] Preferably, the first nucleotide sequence is on a plasmid comprising ITRs and an expression cassette in which the first nucleotide sequence is inserted between the ITR sequences; the second nucleotide sequence is on a plasmid comprising ITRs and an expression cassette in which the second nucleotide sequence is inserted between the ITR sequences;

[0034] Furthermore, the plasmid containing ITR includes but is not limited to: pAAV, pAAV-CMV, pX601, pX551, pAAV-MCS plasmid, etc.

[0035] The second technical solution provided by the present invention is an adeno-associated virus packaging vector system, which comprises: the dual-vector system for expressing the OTOF protein described in the first technical solution, a vector carrying the AAV rep and cap genes, and a helper virus vector, wherein the above-mentioned vectors are packaged into an AAV vector;

[0036] Furthermore, the vectors carrying AAV rep and cap genes include but are not limited to: AAV1, AAV2, AAV5, AAV8, AAV9, Anc80, PHP.eB, AAV-DJ or AAVrh.10 vectors, etc.;

[0037] Furthermore, the helper virus vector is an adenovirus or herpes virus helper virus vector, preferably a pHelper plasmid.

[0038] The third technical solution provided by the present invention is a method for packaging an adeno-associated virus, wherein the adeno-associated virus packaging vector system described in the second technical solution is transferred into a host cell for packaging;

[0039] Furthermore, the dual vectors in the packaging vector system described in Technical Solution 2 are respectively transferred into host cells with the vector carrying the AAV rep and cap genes and the helper virus vector for packaging;

[0040] Furthermore, the host cell is a cell line capable of viral replication and stable inheritance, including but not limited to Hela-S3, HEK-293, HEK-293T, HEK-293FT, A549 and Sf9 cells;

[0041] Preferably, the host cell is HEK-293 or HEK-293T cell.

[0042] The fourth technical solution provided by the present invention is an adeno-associated virus obtained by the packaging method described in the third technical solution. The virus is a pair of viruses, which are respectively packaged with the OTOF N-terminal coding sequence, the Intein N-terminal coding sequence, and the OTOF C-terminal coding sequence, the Intein C-terminal coding sequence.

[0043] The fifth technical solution provided by the present invention is the use of the dual vector system described in the first technical solution or the adeno-associated virus described in the fourth technical solution, especially in the preparation of a drug or preparation for treating deafness, hearing loss or hearing dysfunction;

[0044] Furthermore, the deafness disease or hearing impairment or hearing function abnormality is caused by a gene mutation including the OTOF gene, and the mutation includes but is not limited to base substitution, frameshift mutation, deletion mutation, insertion mutation, etc.

[0045] The sixth technical solution provided by the present invention is a preparation, formula or medicine prepared by the dual vector system described in the first technical solution or the adeno-associated virus described in the fourth technical solution;

[0046] Furthermore, the preparation, formulation or drug may be in any dosage form, including but not limited to injection and ointment dosage forms;

[0047] Furthermore, in the preparation, formulation or medicament, the above-mentioned dual vector system or adeno-associated virus is the only active ingredient;

[0048] Furthermore, the preparation or formulation or drug may comprise common solvents, buffers, such as common pharmaceutical carriers and adjuvants, including one or more of neutral salt buffer, acidic salt buffer, alkaline salt buffer, glucose, mannose, mannitol, proteins, polypeptides and amino acids, antibiotics, chelating agents, adjuvants or preservatives;

[0049] Furthermore, the buffer is phosphate buffer, Tris buffer, 0.01% poloxamer PBS buffer solution or HEPES buffer;

[0050] Furthermore, in the preparation, formulation or medicine, the active ingredient may also be contained in other carriers, such as nanoparticles, liposomes and positive lipid particles.

[0051] Furthermore, the preparation, formula or drug is administered into one ear or both ears;

[0052] Preferably, the preparation, formulation or drug is administered to one ear;

[0053] Furthermore, when administering to a unilateral ear, the administration method is cochlear injection, including but not limited to cochlear round window injection, oval window injection, semicircular canal injection, and utricle injection;

[0054] Furthermore, a single or multiple doses for life, with a total dose of 1×10 9 -1×10 13 A viral genome.

[0055] Beneficial effects:

[0056] The present invention applies the AAV dual vector and Intein recombination method to OTOF expression, significantly improving the efficiency of complete OTOF protein expression. At the same time, it achieves the effect of bilateral hearing restoration after unilateral cochlear administration. - / - The hearing of the injected ear of the mice was restored to the level of wild-type mice, while the hearing of the contralateral non-injected ear was also improved to 60 decibels. Description of the drawings:

[0057] Figure 1 pAAV-CMV plasmid map;

[0058] Figure 2 pAAV-CMV-OTOF-N-S1-Npu-N-intein plasmid map;

[0059] Figure 3 pAAV-CMV-Npu-C-intein-OTOF-C-S1 plasmid map;

[0060] Figure 4 pAAV-CMV-OTOF-N-S2-Npu-N-intein plasmid map;

[0061] Figure 5 pAAV-CMV-Npu-C-intein-OTOF-C-S2 plasmid map;

[0062] Figure 6 pAAV-CMV-OTOF-N-S4-Npu-N-intein plasmid map;

[0063] Figure 7 pAAV-CMV-Npu-C-intein-OTOF-C-S4 plasmid map;

[0064] Figure 8 pAAV-CMV-OTOF-N-S1-Rma-N-intein plasmid map;

[0065] Figure 9 pAAV-CMV-Rma-C-intein-OTOF-C-S1 plasmid map;

[0066] Figure 10 pAAV-CMV-OTOF-N-S2-Rma-N-intein plasmid map;

[0067] Figure 11 pAAV-CMV-Rma-C-intein-OTOF-C-S2 plasmid map;

[0068] Figure 12 pAAV-CMV-OTOF-N-S3-Rma-N-intein plasmid map;

[0069] Figure 13 pAAV-CMV-Rma-C-intein-OTOF-C-S3 plasmid map;

[0070] Figure 14 pAAV-CMV-OTOF-N-S4-Rma-N-intein plasmid map;

[0071] Figure 15 pAAV-CMV-Rma-C-intein-OTOF-C-S4 plasmid map;

[0072] Figure 16 pAAV-CMV-OTOF-FL plasmid map;

[0073] Figure 17pAAV-CMV-OTOF-N-AK plasmid map;

[0074] Figure 18 pAAV-AK-OTOF-C-PolyA plasmid map;

[0075] Figure 19 pAAV-CMV-OTOF-N-AP plasmid map;

[0076] Figure 20 pAAV-AP-OTOF-C-PolyA plasmid map;

[0077] Figure 21 pAAV-CMV-OTOF-N-TS plasmid map;

[0078] Figure 22 pAAV-TS-OTOF-C-PolyA plasmid map;

[0079] Figure 23 Example 6 imaging results;

[0080] Figure 24 Example 7 Imaging results;

[0081] Figure 25 Example 8 imaging results;

[0082] Figure 26 Example 9 imaging results;

[0083] Figure 27 Imaging results of Example 10;

[0084] Figure 28 Comparison of OTOF Intein recombination and OTOF DNA recombination results;

[0085] Figure 29 Otof - / - Base sequence comparison between gene mutant mice and wild-type mice;

[0086] Figure 30 Otof - / - Comparison of hearing between mutant mice and wild-type mice;

[0087] Figure 31 Hearing recovery after 1 month in Example 13;

[0088] Figure 32 Hearing recovery after 2 months in Example 13;

[0089] Figure 33 Hearing recovery after 1 month in Example 14;

[0090] Figure 34 Hearing recovery after 2 months in Example 14;

[0091] Figure 35 Hearing recovery effect in mice in the low-dose group;

[0092] Figure 36 OTOF expression in wild-type and gene-deficient mice;

[0093] Figure 37 The expression of OTOF in the treated and contralateral ears of the treated mice;

[0094] Figure 38 Statistics of OTOF expression in mice.

[0095] Figure 39 AAV1 serotype OTOF restored hearing in mice. Specific implementation method:

[0096] In order to make the purpose, technical solutions and advantages of this application more clear, the following is a further detailed description of this application in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain this patent and are not intended to limit the present invention.

[0097] The present invention achieves efficient expression of OTOF protein in the host through a dual-vector (or AAV dual-vector) delivery combined with Intein recombination, thereby achieving the recovery of deafness, hearing loss, or hearing dysfunction, while achieving the technical effect of restoring hearing in both ears through unilateral ear administration.

[0098] The present invention utilizes an intein recombination method to express the OTOF protein. The OTOF regions involved are: isoform 5 (NM_001287489.2), whose translated amino acid sequence is NP_001274418.1; and isoform 1 (NM_194248.3), whose translated amino acid sequence is NP_919224.1.

[0099] In some embodiments of the present invention, the target protein required for deafness restoration or improvement is OTOF as shown in SEQ ID NO.1 or SEQ ID NO.2. The target protein can also be a sequence with a similarity of 65%-100% to either SEQ ID NO.1 or 2, such as 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75%, 70%, or 65%. At the same time, the protein may also have appropriate truncations, such as proteins with a length of 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, or 60% of the full-length protein of either SEQ ID NO.1 or 2. The protein may also have appropriate insertions, such as proteins with a length of 101%, 102%, 103%, 104%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, or 140% of the full-length protein. The relevant sequences have been organized into Table 1.

[0100] Table 1 OTOF sequences and corresponding relationships

[0101] SEQ ID NO. NCBI ID feature 3 CCDS1725.1 Isoform 1, nucleic acid sequence 1 NP_919224.1 Isoform 1, amino acid sequence 4 CCDS74497.1 Isoform5, nucleic acid sequence 2 NP_001274418.1 Isoform 5, amino acid sequence

[0102] For the amino acid sequence of SEQ ID NO. 1 or 2, in addition to the nucleotide sequence of SEQ ID NO. 3 or 4 corresponding thereto, codon optimization can also be performed, and the corresponding CAI can be 0.65-1.0, for example, 1.0, 0.99, 0.98, 0.97, 0.96, 0.95, 0.90, 0.85, 0.80, 0.75, 0.70 or 0.65.

[0103] In addition to the above-mentioned OTOF protein sequences from humans, OTOF protein sequences can also be selected from other animals, such as mice (protein sequences are NP_001273350.1, NP_001300696.1, NP_001093865.1 or NP_114081.2), rats (protein sequence is NP_001263649.1), pigs (protein sequence is XP_020943388.1) , monkey (protein sequence is XP_014967378.2, XP_014967379.2, XP_028687700.1, XP_014967380.2 or XP_028687701.1) and sequences with a similarity of 65%-100%, such as 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75%, 70%, 65%.

[0104] Inteins can perform protein splicing, acting either after or during protein translation, covalently linking two distinct protein segments. Inteins were first discovered in yeast and molds. Sequence alignment and analysis predict that over 600 intein genes exist in viruses, bacteria, archaea, and eukaryotic microorganisms. Most inteins are complete proteins, while a small fraction have separate N- and C-termini, each attached to a portion of a protein. These separate N- and C-termini then recombine after translation, forming the complete protein through nucleophilic chemical reactions and conformational changes. In this application, inteins with separate N- and C-termini are preferred. Intein can be selected from MxeGyrA, pabPolIII, MjaKlbA, SspDnaB, SceVMA, SspDnaE, ​​NpuDnaE, ​​AvaDnaE, ​​CraDnaE, ​​CspDnaE, ​​CwaDnaE, ​​MchtDnaE, ​​OliDnaE, ​​TerDnaE, ​​gp41-1, gp41-8, IMPDH-1, RmaDnaB, etc. The amino acid sequences of some inteins used in the embodiments of the present invention are shown in Table 2:

[0105] Table 2 Partial Intein Amino Acid Sequences

[0106]

[0107] In this study, the target protein was assembled into two vectors using intein recombination. The OTOF was first divided into two segments, the N-terminus and the C-terminus, based on the split point. The separation criteria were: 1) compatibility with AAV packaging and 2) the first amino acid at the C-terminus was serine, threonine, or cysteine. The N-terminus of the intein was fused to the C-terminus of the OTOF protein, and the C-terminus of the intein was fused to the N-terminus of the OTOF protein.

[0108] Preferably, the OTOF protein is split into two segments with approximately equal lengths at the N-terminus and C-terminus, or the N-terminus / C-terminus length is 0.3-3, for example, the N-terminus length / C-terminus length is 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.5, 1.7, 1.8, 2.0, 2.0, 2.5 or 3.0.

[0109] More preferably, the methods for splitting the N-terminus and C-terminus of the OTOF protein include but are not limited to those listed in Table 3 of Example 1.

[0110] More preferably, for the amino acid sequence of the OTOF protein shown in SEQ ID NO. 1 or 2, the N-terminus and the C-terminus are split into amino acids 1-827 and amino acids 828-1997, amino acids 1-930 and amino acids 931-1997, amino acids 1-954 and amino acids 955-1997, and amino acids 1-1130 and 1131-1997.

[0111] In the present invention, when the target protein is assembled into two vectors using the Intein method, a dual vector expressing the OTOF protein is constructed, and the dual vector includes two nucleotide sequences:

[0112] The first nucleotide sequence includes two ITR sequences and an expression cassette inserted between the ITR sequences. The second nucleotide sequence includes two ITR sequences and an expression cassette inserted between the ITR sequences. ITR is a sequence used for adeno-associated protein recognition and DNA packaging. It is also involved in the recovery and replication of the adeno-associated virus genome. The preferred ITR sequences are from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and ITR sequences with a similarity of 65%-100% to the above sequences, such as 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75%, 70%, and 65%.

[0113] The N-terminus of the aforementioned Intein is fused to the C-terminus of the N-terminus of the OTOF protein, and the obtained sequence is constructed into the expression cassette of the first nucleotide sequence, so that the expression cassette of the first nucleotide sequence includes: a promoter, an OTOF N-terminus coding sequence, an Intein N-terminus coding sequence, and PolyA;

[0114] The C-terminus of the aforementioned Intein is fused to the N-terminus of the C-terminus of the OTOF protein, and the obtained sequence is constructed into the expression cassette of the second nucleotide sequence, so that the expression cassette of the second nucleotide sequence includes: a promoter, the C-terminal coding sequence of the Intein, the C-terminal coding sequence of the OTOF, and PolyA;

[0115] The first nucleotide sequence and the second nucleotide sequence mentioned above can be used in any order to construct the N-terminal or C-terminal sequence of OTOF.

[0116] In the present invention, the aforementioned promoter refers to a sequence capable of initiating transcription of a downstream target protein, typically used to recruit transcription factors and express the target protein in a specific spatial and temporal location. The promoter includes, but is not limited to, the following promoters: The promoter can be an RNA polymerase II promoter or an RNA polymerase III promoter. Promoters can also be classified as broadly expressed promoters, such as the CMV promoter and the CAG promoter. Promoters can also be tissue-specific promoters, preferably highly expressed in the ear, further highly expressed in the cochlea or vestibule, and further highly expressed in the cochlea. Tissue-specific promoters may be derived from partial or complete sequences 1-10,000 bp upstream of the transcription start site of the OTOF gene, as well as sequences with sequence similarity of 65%-100%, such as 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75%, 70%, or 65%. Preferably, promoters suitable for the present invention include but are not limited to CAG promoter, CMV promoter, CBA promoter, UbC promoter, SFFV promoter, EF1α promoter, PGK promoter, or promoters corresponding to genes such as Myo7A, Myo15, Atoh1, POU4F3, Lhx3, Myo6, α9AchR, α10AchR, OTOF, etc.

[0117] PolyA refers to the adenine modification at the tail end of mRNA during maturation, which can make mRNA more stable. The PolyA sequence of the present invention includes AATAAA and its variants, including ATTAAA, AGTAAA, CATAAA, TATAAA, GATAAA, ACTAAA, AATATA, AAGAAA, AATGAA, AATCAA, AACAAA, AATCAA, AATCAA, AATTAGA, AATTAAA or AATAAG, etc.

[0118] The first or second nucleotide sequence may also contain one or more regulatory elements, such as two, three, four, five, etc. The regulatory elements in the first and second segments may be the same or different. These include but are not limited to the following regulatory elements: (1) for regulating the expression of the target protein, such as IRES, for initiating the translation of downstream genes. IRES is a sequence that can initiate protein translation within mRNA. For example, IRES can be derived from viruses such as FMDV, EMCV, HRV, HIV, HAV, HCV, and PV; (2) regulatory elements for expressing miRNA and siRNA sequences; (3) introns; (4) localization sequences, for localizing the expression of the target protein to the nucleus, cytoplasm, or various organelles, and for secretion outside the cell; (5) sequences that accelerate protein degradation, such as PEST sequences; (6) the regulatory element may also be a partial kozak sequence. The kozak sequence is GNCNCN, such as GCCACC. (7) Enhancers refer to sequences that can enhance gene expression, such as CMV enhancers, SV40 virus enhancers, or adenovirus enhancers. They may be upstream, downstream, or within the target gene, may or may not be tissue-specific, and may or may not transcribe eRNA. (8) Regulatory elements may be WPREs.

[0119] The first or second nucleotide sequence may also contain one or more tags, such as two, three, four, or five tags. The tags in the first and second segments may be the same or different. Examples include FLAG, HA, MYC, fluorescent protein, luciferase, etc., and may also be used to improve protein properties, such as SUMO protein, ubiquitin protein, GST, etc.

[0120] The first or second nucleotide sequence constructed above can be a plasmid, but can also be other forms of linear or circular nucleic acids, which can be selected through appropriate expression and regulatory elements to co-express the N-terminus and C-terminus of the target protein in cells, tissues, organs and individuals. The preferred scope of this patent is expression in the cochlea. Preferably, the present invention uses a plasmid containing ITRs, including pAAV, pAAV-CMV, pX601, pX551, pAAV-MCS, etc., to load the target gene expression cassette and then perform AAV packaging.

[0121] When the first and second nucleotide sequences constructed above are viral vectors, they can be used for any form of AAV packaging. The first and second nucleotide sequences are respectively combined with a vector carrying the AAV rep and cap genes and a helper virus vector and then packaged into a host cell to obtain an adeno-associated virus; the host cell is a cell line capable of viral replication and stable inheritance, including but not limited to Hela-S3, HEK-293, HEK-293T, HEK-293FT, A549 or Sf9 cells, preferably HEK-293 or HEK-293T cells. The AAV virus sample obtained by packaging may contain empty shell viruses, and the empty shell virus content may be 0%-99%, for example, 0%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, preferably 0%-50%.

[0122] The viruses obtained by the above packaging method are a pair of adeno-associated viruses, which are respectively packaged with the N-terminal coding sequence of OTOF and the N-terminal coding sequence of Intein, as well as the C-terminal coding sequence of Intein and the C-terminal coding sequence of OTOF. The above pair of adeno-associated viruses can co-express the N-terminus and C-terminus of the target protein into cells, tissues, organs and individuals, and perform protein splicing through the Intein protein, which can play a role after or during protein translation, and covalently link the two different protein segments to obtain a full-length functional OTOF protein. It is suitable for use in various mammalian cells, including human, mouse, dog, pig, rabbit, hamster, sheep, cat, horse or non-human primate. It can be used in the ear, inner ear, cochlea, inner ear hair cells, or cells unrelated to the ear. The cells can be in vitro, in vivo, or ex vivo.

[0123] Therefore, the above-mentioned dual vector or adeno-associated virus can be used to prepare drugs for deafness diseases, hearing damage or hearing dysfunction, where the deafness diseases, hearing damage or hearing dysfunction are caused by gene mutations including the OTOF gene, and the mutations include but are not limited to base substitutions, frameshift mutations, deletion mutations, insertion mutations, etc.

[0124] The preparation or formulation prepared from the aforementioned dual vector or adeno-associated virus can be a powder or solution. The preparation or formulation can contain common solvents and buffers, such as common pharmaceutical carriers and adjuvants, including neutral salt buffer, acidic salt buffer, alkaline salt buffer, glucose, mannose, mannitol, proteins, peptides, amino acids, antibiotics, chelating agents, adjuvants, and preservatives. Buffers include phosphate buffer, Tris buffer, and HEPES buffer. The formulation can also be contained in other carriers, such as nanoparticles, liposomes, and positive lipid particles. The solution can simulate the components of perilymph fluid, with a NaCl concentration of 20-200 mM, a KCl concentration of 1-5 mM, a CaCl2 concentration of 0.1-10 mM, a glucose concentration of 1-10 mM, a HEPES concentration of 2-50 mM, and a pH of 6-9. The solution is ultimately sterile and can be dissolved in water, glycerol, ethanol, polyols, and oils. The solution can be used directly or diluted.

[0125] The administration method adopted by the present invention is: In the present invention, the dual vector or the final packaged adeno-associated virus can be delivered to the ear by various methods, and can be delivered to the ear from the round window, oval window, semicircular canal, utricle and other areas with or without auxiliary tools. The administration can be a single administration or multiple administrations based on the protein expression and hearing recovery. The delivery dose can be 1-200 μL, and the amount of AAV contained is 1×10 9 -1×10 13 For example, 1-2 μL containing 1 × 10 9 , 1×10 10 or 1×10 11 AAV virus, delivered in humans in 10-100 μL, contains 1 × 10 9 -1×10 13 The virus can be delivered to one ear or both ears. The present invention preferably delivers the virus to one ear, which can ultimately achieve the effect of bilateral hearing restoration.

[0126] The present invention will be further explained below with reference to specific embodiments.

[0127] Example 1 OTOF (SEQ ID NO. 1 or 2) Intein segmentation site selection

[0128] A split point is set on the OTOF amino acid sequence. The nucleotide coding sequence from the N-terminus of the OTOF amino acid sequence to the split point is the OTOF N-terminal coding sequence, and the nucleotide coding sequence from the position immediately following the split point to the C-terminus of the OTOF amino acid sequence is the OTOF C-terminal coding sequence. The N-terminus of OTOF is fused to the N-terminus of the intein, and the C-terminus of the intein is fused to the C-terminus of OTOF. There are multiple options for the split point of OTOF, some of which are listed in Table 3.

[0129] Table 3 Partial segmentation sites applicable to OTOF shown in SEQ ID NO. 1 or 2

[0130]

[0131]

[0132]

[0133]

[0134]

[0135] Example 2: Construction of the first / second nucleotide sequence using the pAAV-CMV plasmid containing the ITR sequence

[0136] The amino acid sequence of the OTOF protein shown in SEQ ID NO.2 was divided into the 827th amino acid residue (S1), the 930th amino acid residue (S2), and the 1130th amino acid residue (S4); NpuDnaE was used as the intein, and the pAAV-CMV plasmid ( Figure 1 , the sequence is shown in SEQ ID NO.9) as the first segment / second segment nucleotide sequence for constructing the vector, that is, constructing a dual vector expressing the OTOF protein, specifically as follows:

[0137] The pAAV-CMV plasmid was digested with BstBI and HindIII, and the plasmid fragments were recovered from a gel. Gene fragment 1 (encoding amino acids 1-827 of OTOF and the N-terminus of NpuDnaE Intein), gene fragment 2 (encoding the C-terminus of NpuDnaE Intein and amino acids 828-1997 of OTOF), gene fragment 3 (encoding amino acids 1-930 of OTOF and the N-terminus of NpuDnaE Intein), gene fragment 4 (encoding the C-terminus of NpuDnaE Intein and amino acids 931-1997 of OTOF), gene fragment 5 (encoding amino acids 1-1130 of OTOF and the N-terminus of NpuDnaE Intein), and gene fragment 6 (encoding the C-terminus of NpuDnaE Intein and amino acids 1131-1997 of OTOF) were synthesized and digested with BstBI and HindIII. The digested pAAV-CMV plasmid was then ligated with the digested gene fragments 1-6 using the T4 ligase system. After ligation, 2 μL of the ligation product was added to 50 μL of DH5α competent cells, ice-bathed for 30 minutes, heat-shocked for 1 minute, and immediately placed on ice for 1 minute. 200 μL of LB liquid medium was added and incubated at 37°C for 30 minutes. 200 μL of the bacterial solution was evenly applied to an ampicillin-resistant solid LB medium and cultured at 37°C overnight. Five single clones were picked and sequenced correctly. The following six plasmids were obtained for subsequent examples:

[0138] pAAV-CMV-OTOF-N-S1-Npu-N-intein plasmid (plasmid map see Figure 2 );

[0139] pAAV-CMV-Npu-C-intein-OTOF-C-S1 plasmid (plasmid map see Figure 3 );

[0140] pAAV-CMV-OTOF-N-S2-Npu-N-intein plasmid (plasmid map see Figure 4 );

[0141] pAAV-CMV-Npu-C-intein-OTOF-C-S2 plasmid (plasmid map see Figure 5 );

[0142] pAAV-CMV-OTOF-N-S4-Npu-N-intein plasmid (plasmid map see Figure 6 );

[0143] pAAV-CMV-Npu-C-intein-OTOF-C-S4 plasmid (plasmid map see Figure 7 ).

[0144] pAAV-CMV-OTOF-N-S1-Npu-N-intein, pAAV-CMV-Npu-C-intein-OTOF-C-S1; pAAV-CMV-OTOF-N-S2-Npu-N-intein, pAAV-CMV-Npu-C-intein-OTOF-C-S2; pAAV-CMV-OTOF-N-S4-Npu-N-intein, pAAV-CMV-Npu-C-intein-OTOF-C-S4; the transcription products of the above plasmids all contain covalently linked OTOF (partial) and NpuDnaE intein (partial). The transcription products are listed in Table 4.

[0145] Table 4 Transcriptional and translation products of Npu fused to OTOF at different N-termini and C-termini

[0146] Serial number Plasmid name Transcriptional translation products 1 pAAV-CMV-OTOF-N-S1-Npu-N-intein OTOF 1-827-NpuDnaE Intein N-terminus 2 pAAV-CMV-Npu-C-intein-OTOF-C-S1 NpuDnaE Intein C-terminus-OTOF828-1997 3 pAAV-CMV-OTOF-N-S2-Npu-N-intein OTOF 1-930-NpuDnaE Intein N-terminus 4 pAAV-CMV-Npu-C-intein-OTOF-C-S2 NpuDnaE Intein C-terminus-OTOF931-1997 5 pAAV-CMV-OTOF-N-S4-Npu-N-intein OTOF 1-1130-NpuDnaE Intein N-terminus 6 pAAV-CMV-Npu-C-intein-OTOF-C-S4 NpuDnaE Intein C-terminus-OTOF 1131-1997

[0147] Example 3: Construction of the first / second nucleotide sequence using the pAAV-CMV plasmid containing the ITR sequence

[0148] The amino acid sequence of the OTOF protein shown in SEQ ID NO.2 was divided into the 827th amino acid residue (S1), the 930th amino acid residue (S2), the 954th amino acid residue (S3), and the 1130th amino acid residue (S4); and the RmaDnaB Intein was used to express the OTOF protein in the pAAV-CMV plasmid ( Figure 1 , the sequence is shown in SEQ ID NO.9) as the first segment / second segment nucleotide sequence for constructing the vector, that is, constructing a dual vector expressing the OTOF protein, specifically as follows:

[0149] The pAAV-CMV plasmid was digested with BstBI and HindIII, and the plasmid fragment was recovered from the gel. Gene fragment 7 (encoding gene of amino acids 1-827 of OTOF and N-terminal of RmaDnaB Intein, as shown in SEQ ID NO.5 (including the partial sequence connected to the plasmid)), gene fragment 8 (encoding gene of C-terminal of RmaDnaB Intein and amino acids 828-1997 of OTOF, as shown in SEQ ID NO.6 (including the partial sequence connected to the plasmid)), gene fragment 9 (encoding gene of amino acids 1-930 of OTOF and N-terminal of RmaDnaB Intein, as shown in SEQ ID NO.7 (including the partial sequence connected to the plasmid)), gene fragment 10 (encoding gene of C-terminal of RmaDnaB Intein and amino acids 931-1997 of OTOF, as shown in SEQ ID NO.8 (including the partial sequence connected to the plasmid)), gene fragment 11 (encoding gene of amino acids 1-954 of OTOF and N-terminal of RmaDnaB Intein), gene fragment 12 (encoding gene of amino acids 1-954 of OTOF and N-terminal of RmaDnaB Intein), gene fragment 13 (encoding gene of amino acids 1-954 of OTOF and N-terminal of RmaDnaB Intein), gene fragment 14 (encoding gene of amino acids 1-954 of OTOF and N-terminal of RmaDnaB Intein), gene fragment 15 (encoding gene of amino acids 1-954 of OTOF and N-terminal of RmaDnaB Intein), gene fragment 16 (encoding gene of amino acids 1-954 of OTOF and N-terminal of RmaDnaB Intein), gene fragment 17 (encoding gene of amino acids 1-954 of OTOF and N-terminal of RmaDnaB Intein), gene fragment 18 (encoding gene of amino acids C-terminus and OTOF amino acid 955-1997 encoding gene), gene fragment 13 (OTOF amino acid 1-1130 and RmaDnaB Intein N-terminus encoding gene), gene fragment 14 (RmaDnaB Intein C-terminus and OTOF amino acid 1131-1997 encoding gene), and digested by BstBI and HindIII. The T4 ligase system was then used to connect the digested pAAV-CMV plasmid to the digested gene fragments 7-14 respectively. After connection, 2 μL of the ligation product was added to 50 μL DH5α competent cells, ice bathed for 30 minutes, heat shocked for 1 minute, and immediately placed on ice for 1 minute. 200 μL LB liquid culture medium was added and incubated at 37 ° C for 30 minutes. After that, 200 μL of bacterial solution was evenly applied to the solid LB culture medium of ampicillin resistance and cultured at 37 ° C overnight. Pick 5 monoclonal shake bacteria and sequence correctly. The following 8 plasmids were obtained for subsequent examples:

[0150] pAAV-CMV-OTOF-N-S1-Rma-N-intein plasmid (plasmid map see Figure 8 );

[0151] pAAV-CMV-Rma-C-intein-OTOF-C-S1 plasmid (plasmid map see Figure 9 );

[0152] pAAV-CMV-OTOF-N-S2-Rma-N-intein plasmid (plasmid map see Figure 10 );

[0153] pAAV-CMV-Rma-C-intein-OTOF-C-S2 plasmid (plasmid map see Figure 11 );

[0154] pAAV-CMV-OTOF-N-S3-Rma-N-intein plasmid (plasmid map see Figure 12 );

[0155] pAAV-CMV-Rma-C-intein-OTOF-C-S3 plasmid (plasmid map see Figure 13 );

[0156] pAAV-CMV-OTOF-N-S4-Rma-N-intein plasmid (plasmid map see Figure 14 );

[0157] pAAV-CMV-Rma-C-intein-OTOF-C-S4 plasmid (plasmid map see Figure 15 );

[0158] pAAV-CMV-OTOF-N-S1-Rma-N-intein, pAAV-CMV-Rma-C-intein-OTOF-C-S1; pAAV-CMV-OTOF-N-S2-Rma-N-intein, pAAV-CMV-Rma-C-intein-OTOF-C-S2; pAAV-CMV-OTOF-N-S3-Rma-N-intein, pAAV-CMV-Rma-C-intein-OTOF-C-S3; pAAV-CMV-OTOF-N-S4-Rma-N-intein, pAAV-CMV-Rma-C-intein-OTOF-C-S4; the transcription products of the above plasmids all contain covalently linked OTOF (part) and RmaDnaB Intein (part). The transcription products are listed in Table 5.

[0159] Table 5 Transcriptional and translation products of Rma fused to different N-termini and C-termini of OTOF

[0160]

[0161]

[0162] Example 4: Constructing OTOF shown in SEQ ID NO.2 into different recombinant plasmids

[0163] According to the method similar to Example 2 and Example 3, a full-length plasmid expressing OTOF protein was constructed: pAAV-CMV-OTOF-FL plasmid (containing CMV promoter, OTOF isoform 5 (SEQ ID NO. 4), bGH PolyA, map see Figure 16 ).

[0164] According to the method similar to Example 2 and Example 3, the plasmid for DNA recombination was constructed:

[0165] pAAV-CMV-OTOF-N-AK plasmid (containing CMV promoter, OTOF 1-930 amino acid coding sequence, SD sequence, AK sequence, map see Figure 17 ), pAAV-AK-OTOF-C-PloyA plasmid (containing AK sequence, SA sequence, OTOF 931-1997 amino acid coding sequence, bGH polyA sequence, map see Figure 18 );

[0166] pAAV-CMV-OTOF-N-AP plasmid (containing CMV promoter, OTOF 1-930 amino acid coding sequence, SD sequence, AP sequence, map see Figure 19 ), pAAV-AP-OTOF-C-PloyA plasmid (containing AP sequence, SA sequence, OTOF 931-1997 amino acid coding sequence, bGHpolyA sequence, map see Figure 20 );

[0167] pAAV-CMV-OTOF-N-TS plasmid (containing CMV promoter, OTOF 1-930 amino acid coding sequence, SD sequence, map see Figure 21 ), pAAV-TS-OTOF-C-PloyA plasmid (containing SA sequence, OTOF 931-1997 amino acid coding sequence, bGH polyA sequence, map see Figure 22 ).

[0168] AK and AP are sequences that the adeno-associated virus (AAV) uses to undergo DNA recombination after entering cells. After DNA recombination, it undergoes trans-cleavage through the SA-SD sequence to form complete mRNA, expressing the full-length, functional OTOF protein. pAAV-CMV-OTOF-N-TS and pAAV-TS-OTOF-C-PolyA AAVs undergo ITR recombination and SA-SD trans-cleavage to form complete, mature mRNA, expressing the full-length, functional OTOF protein.

[0169] Example 5 Preparation of Adeno-Associated Virus

[0170] The eight plasmids constructed by the method of the present invention in Example 3 and the six plasmids constructed in Example 4 were co-transfected with the pHelper plasmid and the pRC plasmid of PHP.eB (at a molar ratio of 1:1:1 using PEI transfection reagent) into HEK-293T cells (approximately 1 μg of plasmid was added per million cells). After culturing for 3 days in a DMEM medium containing 10% fetal bovine serum at 37°C and a 5% carbon dioxide incubator, the cells were washed once with PBS buffer, collected, and repeatedly frozen and thawed five times. Solid NaCl was added to a final concentration of 500 mM, and the supernatant was centrifuged at 10,000 g for half an hour. The supernatant was filtered through a 0.45 μm filter membrane and purified using the iodixanol method. A portion of the sample was concentrated to obtain a viral titer of 1±0.2×10 13 The number of viral genomes / ml, the solvent is 0.01% poloxamer PBS buffer solution, and the empty shell ratio of the adeno-associated virus is approximately 50%. The specific experimental system is shown in Table 6 below. The viruses will be named according to the plasmid name. For example, the adenovirus No. 1 packaged with pAAV-CMV-OTOF-N-S1-Rma-N-intein and pHelper plasmids and PHP.eB is named pAAV-CMV-OTOF-N-S1-Rma-N-intein adeno-associated virus.

[0171] Preparation method of 0.01% poloxamer PBS buffer solution:

[0172] (1) Prepare PBS buffer: 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, and the remainder water;

[0173] (2) Poloxamer F68 was added to the PBS buffer prepared in step (1) at a mass-to-volume ratio to a final concentration of 0.01%.

[0174] The 0.01% poloxamer PBS buffer solution involved in the present invention is prepared by this method.

[0175] Table 6 Transfection system

[0176]

[0177] Example 6 The plasmids constructed in Examples 2 and 3 were recombined in cells (plasmid ratio 1:1)

[0178] The seven pairs of plasmids constructed in Examples 2 and 3 and the pAAV-CMV-OTOF-FL constructed in Example 4 were transfected into HEK-293T cells cultured in six-well plates (the number of cells per well was about 1×10 62 μg of plasmid was added to 100 μL Opti-MEM to prepare a plasmid premix. 4 μL of PEI was then added to 100 μL Opti-MEM to prepare a PEI premix. The plasmid and PEI premixes were then mixed, allowed to stand for 10 minutes, and then dripped onto cultured HEK-293T cells. After 48 hours of culture, the cell culture medium was aspirated and added with 200 μL of cell lysis buffer (1% Triton-X 100, 50 mM Tris-HCl, 1 mM PMSF, pH = 7.4). The cells were incubated on ice for 10 minutes. The cell lysate was harvested and centrifuged at 12,000 g for 10 minutes at 4°C. The supernatant was retained. One-quarter volume of 5× loading buffer (0.25 M Tris-HCl, 10% SDS, 0.05% bromophenol blue, 50% glycerol, 0.25 M DTT) was added to the supernatant and heated at 90°C for 10 minutes to obtain a protein sample. Take 50 μL of protein sample for polyacrylamide gel electrophoresis, transfer to PVDF gel and incubate with OTOF N-terminal antibody (Cat. No. A20266, abclonal) or C-terminal antibody (Cat. No. PA5-52935, Invitrogen) at room temperature for 2 hours, and incubate with corresponding HRP-conjugated secondary antibody for 1 hour. Add ECL reagent for imaging, the results are as follows Figure 23 As shown, NpuS1, NpuS2, RmaS1, RmaS2 and RmaS4 all have high recombination efficiency, among which NpuS2 and RmaS2 have the highest efficiency.

[0179] The amount of plasmid used for transfection is summarized in Table 7.

[0180] Table 7 Plasmid transfection dosage

[0181]

[0182]

[0183] Example 7 S2 plasmid was recombined in cells at a ratio of 1:2

[0184] The pAAV-CMV-OTOF-N-S2-Rma-N-intein plasmid and the pAAV-CMV-Rma-C-intein-OTOF-C-S2 plasmid were prepared at a mass ratio of 1:1 (1 μg, 1 μg), 1:2 (0.65 μg, 1.35 μg), 1:3 (0.5 μg, 1.5 μg), and 1:4 (0.4 μg, 1.6 μg), respectively. 2 μg of pAAV-CMV-OTOF-FL was transfected into HEK-293T cells cultured in six-well plates (approximately 1 × 10 cells per well). 62 μg of plasmid was added to 100 μL Opti-MEM to prepare a plasmid premix. 4 μL of PEI was then added to 100 μL Opti-MEM to prepare a PEI premix. The plasmid premix and PEI premix were then mixed, allowed to stand for 10 minutes, and then dripped onto cultured HEK-293T cells. After 48 hours of culture, the cell culture medium was aspirated and 200 μL of cell lysis buffer (1% Triton-X100, 50 mM Tris-HCl, 1 mM PMSF, pH 7.4) was added. The cells were incubated on ice for 10 minutes. The cell lysate was harvested and centrifuged at 12,000 g for 10 minutes at 4°C. The supernatant was retained. One-quarter volume of 5× loading buffer (0.25 M Tris-HCl, 10% SDS, 0.05% bromophenol blue, 50% glycerol, 0.25 M DTT) was added to the supernatant and heated at 90°C for 10 minutes to obtain a protein sample. 50 μL of protein sample was subjected to polyacrylamide gel electrophoresis and transferred to a PVDF gel membrane. The sample was then incubated with an antibody against the N-terminus of OTOF (A20266, abclonal) or the C-terminus of OTOF (PA5-52935, Invitrogen) at room temperature for 2 hours and then incubated with the corresponding HRP-conjugated secondary antibody for 1 hour. ECL reagent was added for imaging. The results were as follows: Figure 24 , it can be seen that for RmaS2, the transfection efficiency is highest when N:C=1:2.

[0185] Example 8 S2 plasmid was recombined in cells at different ratios such as 1:2.5

[0186] The pAAV-CMV-OTOF-N-S2-Rma-N-intein plasmid and the pAAV-CMV-Rma-C-intein-OTOF-C-S2 plasmid were prepared at a mass ratio of 1:2 (0.65 μg, 1.35 μg), 1:2.5 (0.55 μg, 1.45 μg), 1:3 (0.5 μg, 1.5 μg), and 1:3.5 (0.4 μg, 1.6 μg). 2 μg of pAAV-CMV-OTOF-FL was transfected into HEK-293T cells cultured in six-well plates (approximately 1 × 10 cells per well). 62 μg of plasmid was added to 100 μL Opti-MEM to prepare a plasmid premix. 4 μL of PEI was then added to 100 μL Opti-MEM to prepare a PEI premix. The plasmid and PEI premixes were then mixed, allowed to stand for 10 minutes, and then dripped onto cultured HEK-293T cells. After 48 hours of culture, the cell culture medium was aspirated and 200 μL of cell lysis buffer (1% Triton-X 100, 50 mM Tris-HCl, 1 mM PMSF, pH 7.4) was added. The cells were incubated on ice for 10 minutes. The cell lysate was harvested and centrifuged at 12,000 g for 10 minutes at 4°C. The supernatant was retained. One-quarter volume of 5× loading buffer (0.25 M Tris-HCl, 10% SDS, 0.05% bromophenol blue, 50% glycerol, 0.25 M DTT) was added to the supernatant and heated at 90°C for 10 minutes to obtain a protein sample. 50 μL of protein sample was subjected to polyacrylamide gel electrophoresis and transferred to a PVDF gel membrane. Antibodies against the N-terminus (A20266, abclonal) or C-terminus (PA5-52935, Invitrogen) of OTOF were incubated at room temperature for 2 hours and then incubated with the corresponding HRP-conjugated secondary antibody for 1 hour. ECL reagent was added for imaging. The results were as follows: Figure 25 As shown, it can be seen that when the ratio of RmaS2 is further adjusted to N:C = 1:2.5, the transfection efficiency is the highest.

[0187] Example 9 S1 plasmid was recombined in cells at a ratio of 1:2

[0188] The pAAV-CMV-OTOF-N-S1-Rma-N-intein plasmid and the pAAV-CMV-Rma-C-intein-OTOF-C-S1 plasmid were prepared at a mass ratio of 1:1 (1 μg, 1 μg), 1:2 (0.65 μg, 1.35 μg), 1:3 (0.5 μg, 1.5 μg), and 1:4 (0.4 μg, 1.6 μg), respectively. 2 μg of pAAV-CMV-OTOF-FL was transfected into HEK-293T cells cultured in six-well plates (approximately 1 × 10 cells per well). 6The N-terminal plasmid and the C-terminal plasmid were added to 100 μL Opti-MEM in the appropriate proportions to obtain a plasmid premix. 4 μL PEI was then added to 100 μL Opti-MEM to obtain a PEI premix. The plasmid premix and PEI premix were then mixed, allowed to stand for 10 minutes, and then dripped onto cultured HEK-293T cells. The cells were cultured for 48 hours. The cell culture medium was aspirated, and 200 μL of cell lysis buffer (1% Triton-X 100, 50 mM Tris-HCl, 1 mM PMSF, pH = 7.4) was added. The cells were incubated on ice for 10 minutes. The cell lysate was harvested and centrifuged at 12,000 g for 10 minutes at 4°C. The supernatant was retained. One-quarter volume of 5× loading buffer (0.25 M Tris-HCl, 10% SDS, 0.05% bromophenol blue, 50% glycerol, 0.25 M DTT) was added to the supernatant and heated at 90°C for 10 minutes to obtain a protein sample. 50 μL of protein sample was subjected to polyacrylamide gel electrophoresis and transferred to a PVDF gel membrane. Antibodies against the N-terminus (A20266, abclonal) or C-terminus (PA5-52935, Invitrogen) of OTOF were incubated at room temperature for 2 hours and then incubated with the corresponding HRP-conjugated secondary antibody for 1 hour. ECL reagent was added for imaging. The results were as follows: Figure 26 As shown, for RmaS1, by optimizing the transfection ratio of the N-terminus and the C-terminus, the transfection efficiency is highest when N:C=1:2.

[0189] Example 10 Transfection efficiency at different culture times

[0190] The pAAV-CMV-OTOF-N-S2-Rma-N-intein plasmid and the pAAV-CMV-Rma-C-intein-OTOF-C-S2 plasmid were transfected into HEK-293T cells cultured in six-well plates (about 1×10 cells per well) at a mass ratio of 1:2.5 (0.55 μg, 1.45 μg). 62 μg of plasmid was added to 100 μL Opti-MEM to obtain a plasmid premix. 4 μL of PEI was added to 100 μL Opti-MEM to obtain a PEI premix. The plasmid premix and PEI premix were then mixed, allowed to stand for 10 minutes, and then dripped onto cultured HEK-293T cells. The cells were cultured for 2, 4, 6, 8, 12, 24, 48, and 72 hours, respectively. The cell culture medium was aspirated and 200 μL of cell lysis buffer (1% Triton-X 100, 50 mM Tris-HCl, 1 mM PMSF, pH = 7.4) was added. The cells were incubated on ice for 10 minutes, and the cell lysates were collected and centrifuged at 12,000 g for 10 minutes at 4°C. The supernatant was retained. Add 1 / 4 volume of 5× loading buffer (0.25MTris-HCl, 10% SDS, 0.05% bromophenol blue, 50% glycerol, 0.25M DTT) to the supernatant and heat at 90°C for 10 minutes to obtain the protein sample. Take 50μL of protein sample for polyacrylamide gel electrophoresis, transfer to PVDF gel, and incubate with antibodies against the N-terminus (A20266, abclonal) or C-terminus of OTOF (PA5-52935, Invitrogen) at room temperature for 2 hours, and incubate with the corresponding HRP-conjugated secondary antibody for 1 hour. Add ECL reagent for imaging, and the results are as follows Figure 27 As shown, it can be seen that the recombination speed of OTOF using Intein is very fast, and the ratio of fragments and full-length OTOF at different time points is basically the same.

[0191] Example 11 Comparison of OTOF Intein Recombination and OTOF DNA Recombination

[0192] The viruses packaged in Example 5 were added in pairs to 400 μL of serum-free DMEM medium (as shown in Table 8), mixed evenly, and then added to HEK-293T cells (about 1×10 6Cells were cultured in a 4-well plate (the culture medium was aspirated before virus addition) and incubated for 4 hours. Then, 1.6 mL of DMEM medium containing 10% fetal bovine serum was added. After two days of culture, the cell culture medium was aspirated and 200 μL of cell lysis buffer (1% triton-X 100, 50 mM Tris-HCl, 1 mM PMSF, pH = 7.4) was added. The cells were placed on ice for 10 minutes, and the cell lysate was collected. The cells were centrifuged at 12,000 g for 10 minutes at 4°C, and the supernatant was retained. 1 / 4 volume of 5× loading buffer (0.25 M Tris-HCl, 10% SDS, 0.05% bromophenol blue, 50% glycerol, 0.25 M DTT) was added to the supernatant and heated at 90°C for 10 minutes to obtain a protein sample. 50 μL of protein sample was subjected to polyacrylamide gel electrophoresis. After transfer to PVDF gel, the OTOF antibody (Cat. No. A20266, abclonal) was diluted 1:3000 and incubated with the corresponding HRP-conjugated secondary antibody for 1 hour. ECL reagent was added for imaging. The experiment was repeated three times, and the grayscale of the full-length band was counted. The results are as follows: Figure 28 It can be seen that the recombination efficiency of the protein recombined by the OTOF Intein used in the present invention is significantly higher than that of the OTOF DNA recombination result.

[0193] Table 8 Protein and nucleic acid recombination systems

[0194]

[0195]

[0196] Example 12 Construction of Otof gene-deficient mice

[0197] Otof - / - Genetic mutant mice were constructed based on the 129S2 / SvPasCrl mouse strain using the CRISPR / Cas9 method. - / - The model mice carried a homozygous mutation in the Otof gene NM_001100395.1:c.2503_2504insA, which caused a frameshift in the Otof gene. Figure 29 As shown, Otof - / - The mutant mice have a single base insertion, which causes a frame shift in the reading frame. - / - Mutant mice and wild-type mice, hearing comparison Figure 30 As shown, the mutant mice had complete hearing loss in both ears.

[0198] Example 13: Administration of S1 Intein to the unilateral ear of infant mice and its effects

[0199] HEK-293T cells were co-transfected with plasmids pAAV-CMV-OTOF-N-S1-Rma-N-intein and pAAV-CMV-Rma-C-intein-OTOF-C-S1, pHelper plasmid, and PHP.eB pRC plasmid at a molar ratio of 1:1:1 using PEI transfection reagent (approximately 1 μg of plasmid was added per million cells). The cells were cultured in DMEM medium containing 10% fetal bovine serum in an incubator containing 5% carbon dioxide at 37°C for 3 days, washed once with PBS buffer, collected, and repeatedly frozen and thawed five times. Solid NaCl was added to a final concentration of 500 mM, and the cells were centrifuged at 10,000 g for half an hour. The supernatant was filtered through a 0.45 μm filter membrane, and a gradient of iodixanol solution was prepared and added to a centrifuge tube (5 mL of 60% iodixanol, 5 mL of 40% iodixanol, 6 ml of 25% iodixanol, 8 ml of 15% iodixanol) was added to the top layer and centrifuged at 350,000 g for 1 hour. The virus layer at the 40% and 60% interface was aspirated. The virus was centrifuged at 10,000 g using a 50KDa ultrafiltration tube and replaced with 0.01% poloxamer PBS buffer solution five times. The virus titer was determined by qPCR and the titer was adjusted to obtain a final virus titer of 1 ± 0.2 × 10 13 The viruses containing 100 viral genomes / ml, namely pAAV-CMV-OTOF-N-S1-Rma-N-intein PHP.eB AAV and pAAV-CMV-Rma-C-intein-OTOF-C-S1 PHP.eB AAV, were prepared using a 0.01% poloxamer PBS buffer solution as the solvent.

[0200] Then the adeno-associated virus constructed above was injected into the P0 / P1 Otof construct in Example 12. - / - The drug was administered to the mutant mice, specifically through round window injection into the right cochlea, with a single dose of 2×10 10 The number of viral genomes (1×10 10 The ABR index of mice was tested to confirm the hearing recovery of mice. The results after 1 month were as follows Figure 31 As shown in the figure, the circle marks the non-drug group (n=8), the square marks the contralateral ear of the drug (n=10), the triangle marks the drug ear (n=10), and the rhombus marks the wild-type animal group (n=20). The results after 2 months are shown in Figure 32 As shown in the figure, the circle marks the non-drug group (n=8), the square marks the contralateral ear (n=3), the triangle marks the drug-treated ear (n=3), and the rhombus marks the wild-type animal group (n=11). - / -After unilateral cochlear administration of the drug to the model mice, the hearing of the injected ear was restored, while the hearing of the contralateral non-injected ear was also restored.

[0201] Example 14: S2 Intein administration to one side of the ear of infant mice and its effect

[0202] HEK-293T cells were co-transfected with plasmids pAAV-CMV-OTOF-N-S2-Rma-N-intein and pAAV-CMV-Rma-C-intein-OTOF-C-S2, pHelper plasmid, and PHP.eB pRC plasmid at a molar ratio of 1:1:1 using PEI transfection reagent (approximately 1 μg of plasmid per million cells). The cells were cultured in DMEM medium containing 10% fetal bovine serum in an incubator containing 5% carbon dioxide at 37°C for 3 days, washed once with PBS buffer, collected, and repeatedly frozen and thawed five times. Solid NaCl was added to a final concentration of 500 mM, and the cells were centrifuged at 10,000 g for half an hour. The supernatant was filtered through a 0.45 μm filter membrane, and a gradient of iodixanol solution was prepared and added to a centrifuge tube (5 mL of 60% iodixanol, 5 mL of 40% iodixanol, 6 ml of 25% iodixanol, 8 ml of 15% iodixanol) was added to the top layer and centrifuged at 350,000 g for 1 hour. The virus layer at the 40% and 60% interface was aspirated. The virus was centrifuged at 10,000 g using a 50KDa ultrafiltration tube and replaced with 0.01% poloxamer PBS buffer solution five times. The virus titer was determined by qPCR and the titer was adjusted to obtain a final virus titer of 1 ± 0.2 × 10 13 The virus has a viral genome count / ml and an empty shell rate of about 50%, namely, pAAV-CMV-OTOF-N-S2-Rma-N-intein PHP.eB AAV and pAAV-CMV-Rma-C-intein-OTOF-C-S2 PHP.eBAAV, and the solvent is 0.01% poloxamer PBS buffer solution.

[0203] Then the adeno-associated virus constructed above was injected into the P0 / P1 Otof construct in Example 12. - / - The drug was administered to the mutant mice, specifically by injection into the right cochlea through the round window, with each mouse receiving 2×10 10 The number of viral genomes (1×10 10 The ABR index of mice was tested to confirm the hearing recovery of mice. The results after 1 month were as follows Figure 33As shown in the figure, the circle marks the non-drug group (n=10), the square marks the contralateral ear of the drug (n=33), the triangle marks the drug ear (n=33), and the rhombus marks the wild-type animal group (n=20). The results after 2 months are shown in Figure 34 In the figure, the circle marks the non-administered group (n=8), the square marks the contralateral ear (n=27), the triangle marks the administered ear (n=27), and the rhombus marks the wild-type animal group (n=11). - / - After unilateral cochlear administration of the drug to the mutant mice, the hearing of the injected ear was restored (triangle mark) as well as the hearing of the contralateral uninjected ear (square mark).

[0204] Example 15 Safety Observation of the Dosage Group

[0205] After injection of the AAVs constructed in Examples 13 and 14 of the present invention into the cochlea of ​​6-8 week-old wild-type CD-1 mice through the round window, the mice were observed for daily activities, hair smoothness, and food intake for three months. No significant differences were found between the mice and the control group that did not receive the AAV injection. There was no difference in hearing between the treated and control groups. The cochleae of the mice were dissected on days 28 and 91, and immunofluorescence staining of inner ear hair cells was performed. No significant difference in hair cell number was found between the treated and control groups.

[0206] Example 16: Low-dose drug administration to one ear and its effect

[0207] HEK-293T cells were co-transfected with plasmids pAAV-CMV-OTOF-N-S2-Rma-N-intein and pAAV-CMV-Rma-C-intein-OTOF-C-S2, pHelper plasmid, and PHP.eB pRC plasmid at a molar ratio of 1:1:1 using PEI transfection reagent (approximately 1 μg of plasmid per million cells). The cells were cultured in DMEM medium containing 10% fetal bovine serum in an incubator containing 5% carbon dioxide at 37°C for 3 days, washed once with PBS buffer, collected, and repeatedly frozen and thawed five times. Solid NaCl was added to a final concentration of 500 mM, and the cells were centrifuged at 10,000 g for half an hour. The supernatant was filtered through a 0.45 μm filter membrane, and a gradient of iodixanol solution was prepared and added to a centrifuge tube (5 mL of 60% iodixanol, 5 mL of 40% iodixanol, 6 ml of 25% iodixanol, 8 ml of 15% iodixanol) was added to the top layer and centrifuged at 350,000 g for 1 hour. The virus layer at the 40% and 60% interface was aspirated. The virus was centrifuged at 10,000 g using a 50KDa ultrafiltration tube and replaced with 0.01% poloxamer PBS buffer solution five times. The virus titer was determined by qPCR and the titer was adjusted to obtain a final virus titer of 1 ± 0.2 × 1013 Viruses containing 100 viral genomes / ml, namely pAAV-CMV-OTOF-N-S2-Rma-N-intein PHP.eB AAV and pAAV-CMV-Rma-C-intein-OTOF-C-S2 PHP.eB AAV, were prepared using a 0.01% poloxamer PBS buffer solution.

[0208] Then the adeno-associated virus constructed above was injected into the Otof construct in Example 12. - / - The drug was administered to the mutant mice, specifically through round window injection into the right cochlea, with a single dose of 5×10 9 The number of viral genomes (2.5×10 9 The ABR index of mice was tested to confirm the hearing recovery of mice. The results after 1 month are as follows Figure 35 As shown, it can be seen that the AAV constructed by the present invention is effective in the regulation of Otof - / - After unilateral cochlear administration of the drug to the model mice, the hearing of the injected ear was restored, while the hearing of the contralateral non-injected ear was also restored.

[0209] Example 17 In vivo protein immunofluorescence detection after administration

[0210] The mice administered with the drug in Example 14 were sacrificed one month later, and the cochlear tissue was peeled off and soaked in 4% paraformaldehyde at 4°C overnight, and decalcified with 10% EDTA solution for three days. Before staining, the cells were incubated with 0.3% Triton X-100 PBS buffer for 10 minutes and blocked with 10% donkey blood at room temperature for one hour. The cells were incubated with anti-OTOF antibodies PA5-52935 (C-terminus) or ab53233 (N-terminus) at 4°C overnight, and washed three times with 0.1% Triton X-100 PBS for 10 minutes each. After incubation with the corresponding fluorescent secondary antibody and DAPI for 1 hour, the cells were washed three times with 0.1% Triton X-100 PBS for 10 minutes each. Finally, confocal fluorescence microscopy was used for imaging, and the results are as follows:

[0211] Figure 36 Is OTOF in wild-type mice and Otof - / - The expression of OTOF in the cochlea of ​​gene mutant mice is shown in the figure. The cells stained with DAPI in a single row are inner ear hair cells. It can be seen that OTOF is clearly expressed in inner ear hair cells in wild-type mice, while Otof - / - OTOF is completely absent in mutant mice.

[0212] Figure 37 Figure A shows the expression of Otof in the injected ear and the contralateral ear. - / -The expression of OTOF protein in the ear of the mouse. The left picture is a flat section of the entire cochlea. The right picture is a magnified view of the part in the left frame. - / - This image shows OTOF protein expression in the contralateral ear of a mouse treated with the drug. Apex, Middle, and Base refer to different regions of the cochlear hair cells. The image shows that OTOF is expressed in the majority of inner ear hair cells in the treated ear, and OTOF is also significantly expressed in the contralateral ear.

[0213] Figure 38 Is OTOF after administration of Otof - / - Expression statistics in mice. Panel A shows OTOF expression in inner ear hair cells of the treated ear, with over 60% expressing the protein. Panel B shows OTOF expression in the contralateral ear, with approximately 40% expressing the protein. The apical turn, middle turn, and basal turn in the figure refer to different parts of the cochlea. The vertical axis shows the percentage of inner ear hair cells infected.

[0214] Example 18: Administration to one side of the ear of four-week-old mice and its effects

[0215] Plasmids pAAV-CMV-OTOF-N-S2-Rma-N-intein and pAAV-CMV-Rma-C-intein-OTOF-C-S2 were co-transfected with pHelper plasmid and AAV1 pRC plasmid at a molar ratio of 1:1:1 using PEI transfection reagent (approximately 1 μg of plasmid was added per million cells). The cells were cultured in DMEM medium containing 10% fetal bovine serum in an incubator containing 5% carbon dioxide at 37°C for 3 days, washed once with PBS buffer, collected and frozen and thawed five times, solid NaCl was added to a final concentration of 500 mM, and the supernatant was centrifuged at 10,000 g for half an hour. The supernatant was filtered through a 0.45 μm filter membrane, and a gradient of iodixanol solution was prepared and added to a centrifuge tube (5 mL 60% iodixanol, 5 mL 40% iodixanol, 6 ml 25% iodixanol, 8 ml 15% iodixanol) was added to the top layer and centrifuged at 350,000 g for 1 hour. The virus layer at the 40% and 60% interface was aspirated. The virus was centrifuged at 10,000 g using a 50KDa ultrafiltration tube and replaced with 0.01% poloxamer PBS buffer solution five times. The virus titer was determined by qPCR and the titer was adjusted to obtain a final virus titer of 1 ± 0.2 × 10 13Adeno-associated viruses with a virus genome count of 100 viral genomes / ml and an empty shell rate of approximately 50%, namely pAAV-CMV-OTOF-N-S2-Rma-N-intein AAV1 and pAAV-CMV-Rma-C-intein-OTOF-C-S2 AAV1, were prepared using a 0.01% poloxamer PBS buffer solution as the solvent.

[0216] Then the adeno-associated virus constructed above was injected into the Otof construct in Example 12. - / - Gene mutant mice (four weeks old) were given the drug, specifically, the drug was injected into the right cochlea through the posterior semicircular canal, and each mouse was given 2×10 10 The number of viral genomes (1×10 10 The ABR index of mice was tested to confirm the hearing recovery of mice. The results after 1 month were as follows Figure 39 As shown in the figure, the circle marks the non-drug group (n=8), the square marks the contralateral ear of the drug (n=6), the triangle marks the drug ear (n=6), and the rhombus marks the wild-type animal group (n=8). The results show that the adeno-associated virus packaged by the present invention also has a good effect on hearing restoration in adult mice, and it can also restore hearing in both ears after unilateral ear injection.

[0217] The above-described embodiments merely represent several implementation methods of the present invention. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that, without departing from the concept of this patent, a person skilled in the art would be able to make various variations, combinations, and improvements to the above-described embodiments, all of which fall within the scope of protection of this patent. Therefore, the scope of protection of this patent shall be determined by the claims. Sequence Listing <110> Fudan University Eye, Ear, Nose and Throat Hospital Shanghai Dingxin Gene Technology Co., Ltd. <120> A dual-carrier system for treating hearing loss and its application <130> 1 <141> 2022-05-17 <160> 9 <170> SIPOSequenceListing 1.0 <210> 1 <211> 1997 <212> PRT <213> Homo sapiens <400> 1 Met Ala Leu Leu Ile His Leu Lys Thr Val Ser Glu Leu Arg Gly Arg 1 5 10 15 Gly Asp Arg Ile Ala Lys Val Thr Phe Arg Gly Gln Ser Phe Tyr Ser 20 25 30 Arg Val Leu Glu Asn Cys Glu Asp Val Ala Asp Phe Asp Glu Thr Phe 35 40 45 Arg Trp Pro Val Ala Ser Ser Ile Asp Arg Asn Glu Met Leu Glu Ile 50 55 60 Gln Val Phe Asn Tyr Ser Lys Val Phe Ser Asn Lys Leu Ile Gly Thr 65 70 75 80 Phe Arg Met Val Leu Gln Lys Val Val Glu Glu Ser His Val Glu Val 85 90 95 Thr Asp Thr Leu Ile Asp Asp Asn Asn Ala Ile Ile Lys Thr Ser Leu 100 105 110 Cys Val Glu Val Arg Tyr Gln Ala Thr Asp Gly Thr Val Gly Ser Trp 115 120 125 Asp Asp Gly Asp Phe Leu Gly Asp Glu Ser Leu Gln Glu Glu Glu Lys 130 135 140 Asp Ser Gln Glu Thr Asp Gly Leu Leu Pro Gly Ser Arg Pro Ser Ser 145 150 155 160 Arg Pro Pro Gly Glu Lys Ser Phe Arg Arg Ala Gly Arg Ser Val Phe 165 170 175 Ser Ala Met Lys Leu Gly Lys Asn Arg Ser His Lys Glu Glu Pro Gln 180 185 190 Arg Pro Asp Glu Pro Ala Val Leu Glu Met Glu Asp Leu Asp His Leu 195 200 205 Ala Ile Arg Leu Gly Asp Gly Leu Asp Pro Asp Ser Val Ser Leu Ala 210 215 220 Ser Val Thr Ala Leu Thr Thr Asn Val Ser Asn Lys Arg Ser Lys Pro 225 230 235 240 Asp Ile Lys Met Glu Pro Ser Ala Gly Arg Pro Met Asp Tyr Gln Val 245 250 255 Ser Ile Thr Val Ile Glu Ala Arg Gln Leu Val Gly Leu Asn Met Asp 260 265 270 Pro Val Val Cys Val Glu Val Gly Asp Asp Lys Lys Tyr Thr Ser Met 275 280 285 Lys Glu Ser Thr Asn Cys Pro Tyr Tyr Asn Glu Tyr Phe Val Phe Asp 290 295 300 Phe His Val Ser Pro Asp Val Met Phe Asp Lys Ile Ile Lys Ile Ser 305 310 315 320 Val Ile His Ser Lys Asn Leu Leu Arg Ser Gly Thr Leu Val Gly Ser 325 330 335 Phe Lys Met Asp Val Gly Thr Val Tyr Ser Gln Pro Glu His Gln Phe 340 345 350 His His Lys Trp Ala Ile Leu Ser Asp Pro Asp Asp Ile Ser Ser Gly 355 360 365 Leu Lys Gly Tyr Val Lys Cys Asp Val Ala Val Val Gly Lys Gly Asp 370 375 380 Asn Ile Lys Thr Pro His Lys Ala Asn Glu Thr Asp Glu Asp Asp Ile 385 390 395 400 Glu Gly Asn Leu Leu Leu Pro Glu Gly Val Pro Pro Glu Arg Gln Trp 405 410 415 Ala Arg Phe Tyr Val Lys Ile Tyr Arg Ala Glu Gly Leu Pro Arg Met 420 425 430 Asn Thr Ser Leu Met Ala Asn Val Lys Lys Ala Phe Ile Gly Glu Asn 435 440 445 Lys Asp Leu Val Asp Pro Tyr Val Gln Val Phe Phe Ala Gly Gln Lys 450 455 460 Gly Lys Thr Ser Val Gln Lys Ser Ser Tyr Glu Pro Leu Trp Asn Glu 465 470 475 480 Gln Val Val Phe Thr Asp Leu Phe Pro Pro Leu Cys Lys Arg Met Lys 485 490 495 Val Gln Ile Arg Asp Ser Asp Lys Val Asn Asp Val Ala Ile Gly Thr 500 505 510 His Phe Ile Asp Leu Arg Lys Ile Ser Asn Asp Gly Asp Lys Gly Phe 515 520 525 Leu Pro Thr Leu Gly Pro Ala Trp Val Asn Met Tyr Gly Ser Thr Arg 530 535 540 Asn Tyr Thr Leu Leu Asp Glu His Gln Asp Leu Asn Glu Gly Leu Gly 545 550 555 560 Glu Gly Val Ser Phe Arg Ala Arg Leu Leu Leu Gly Leu Ala Val Glu 565 570 575 Ile Val Asp Thr Ser Asn Pro Glu Leu Thr Ser Ser Thr Glu Val Gln 580 585 590 Val Glu Gln Ala Thr Pro Ile Ser Glu Ser Cys Ala Gly Lys Met Glu 595 600 605 Glu Phe Phe Leu Phe Gly Ala Phe Leu Glu Ala Ser Met Ile Asp Arg 610 615 620 Arg Asn Gly Asp Lys Pro Ile Thr Phe Glu Val Thr Ile Gly Asn Tyr 625 630 635 640 Gly Asn Glu Val Asp Gly Leu Ser Arg Pro Gln Arg Pro Arg Pro Arg 645 650 655 Lys Glu Pro Gly Asp Glu Glu Glu Val Asp Leu Ile Gln Asn Ala Ser 660 665 670 Asp Asp Glu Ala Gly Asp Ala Gly Asp Leu Ala Ser Val Ser Ser Thr 675 680 685 Pro Pro Met Arg Pro Gln Val Thr Asp Arg Asn Tyr Phe His Leu Pro 690 695 700 Tyr Leu Glu Arg Lys Pro Cys Ile Tyr Ile Lys Ser Trp Trp Pro Asp 705 710 715 720 Gln Arg Arg Arg Leu Tyr Asn Ala Asn Ile Met Asp His Ile Ala Asp 725 730 735 Lys Leu Glu Glu Gly Leu Asn Asp Ile Gln Glu Met Ile Lys Thr Glu 740 745 750 Lys Ser Tyr Pro Glu Arg Arg Leu Arg Gly Val Leu Glu Glu Leu Ser 755 760 765 Cys Gly Cys Cys Arg Phe Leu Ser Leu Ala Asp Lys Asp Gln Gly His 770 775 780 Ser Ser Arg Thr Arg Leu Asp Arg Glu Arg Leu Lys Ser Cys Met Arg 785 790 795 800 Glu Leu Glu Asn Met Gly Gln Gln Ala Arg Met Leu Arg Ala Gln Val 805 810 815 Lys Arg His Thr Val Arg Asp Lys Leu Arg Leu Cys Gln Asn Phe Leu 820 825 830 Gln Lys Leu Arg Phe Leu Ala Asp Glu Pro Gln His Ser Ile Pro Asp 835 840 845 Ile Phe Ile Trp Met Met Ser Asn Asn Lys Arg Val Ala Tyr Ala Arg 850 855 860 Val Pro Ser Lys Asp Leu Leu Phe Ser Ile Val Glu Glu Glu Thr Gly 865 870 875 880 Lys Asp Cys Ala Lys Val Lys Thr Leu Phe Leu Lys Leu Pro Gly Lys 885 890 895 Arg Gly Phe Gly Ser Ala Gly Trp Thr Val Gln Ala Lys Val Glu Leu 900 905 910 Tyr Leu Trp Leu Gly Leu Ser Lys Gln Arg Lys Glu Phe Leu Cys Gly 915 920 925 Leu Pro Cys Gly Phe Gln Glu Val Lys Ala Ala Gln Gly Leu Gly Leu 930 935 940 His Ala Phe Pro Pro Val Ser Leu Val Tyr Thr Lys Lys Gln Ala Phe 945 950 955 960 Gln Leu Arg Ala His Met Tyr Gln Ala Arg Ser Leu Phe Ala Ala Asp 965 970 975 Ser Ser Gly Leu Ser Asp Pro Phe Ala Arg Val Phe Phe Ile Asn Gln 980 985 990 Ser Gln Cys Thr Glu Val Leu Asn Glu Thr Leu Cys Pro Thr Trp Asp 995 1000 1005 Gln Met Leu Val Phe Asp Asn Leu Glu Leu Tyr Gly Glu Ala His Glu 1010 1015 1020 Leu Arg Asp Asp Pro Pro Ile Ile Val Ile Glu Ile Tyr Asp Gln Asp 1025 1030 1035 1040 Ser Met Gly Lys Ala Asp Phe Met Gly Arg Thr Phe Ala Lys Pro Leu 1045 1050 1055 Val Lys Met Ala Asp Glu Ala Tyr Cys Pro Pro Arg Phe Pro Pro Gln 1060 1065 1070 Leu Glu Tyr Tyr Gln Ile Tyr Arg Gly Asn Ala Thr Ala Gly Asp Leu 1075 1080 1085 Leu Ala Ala Phe Glu Leu Leu Gln Ile Gly Pro Ala Gly Lys Ala Asp 1090 1095 1100 Leu Pro Pro Ile Asn Gly Pro Val Asp Val Asp Arg Gly Pro Ile Met 1105 1110 1115 1120 Pro Val Pro Met Gly Ile Arg Pro Val Leu Ser Lys Tyr Arg Val Glu 1125 1130 1135 Val Leu Phe Trp Gly Leu Arg Asp Leu Lys Arg Val Asn Leu Ala Gln 1140 1145 1150 Val Asp Arg Pro Arg Val Asp Ile Glu Cys Ala Gly Lys Gly Val Gln 1155 1160 1165 Ser Ser Leu Ile His Asn Tyr Lys Lys Asn Pro Asn Phe Asn Thr Leu 1170 1175 1180 Val Lys Trp Phe Glu Val Asp Leu Pro Glu Asn Glu Leu Leu His Pro 1185 1190 1195 1200 Pro Leu Asn Ile Arg Val Val Asp Cys Arg Ala Phe Gly Arg Tyr Thr 1205 1210 1215 Leu Val Gly Ser His Ala Val Ser Ser Leu Arg Arg Phe Ile Tyr Arg 1220 1225 1230 Pro Pro Asp Arg Ser Ala Pro Ser Trp Asn Thr Thr Val Arg Leu Leu 1235 1240 1245 Arg Arg Cys Arg Val Leu Cys Asn Gly Gly Ser Ser Ser His Ser Thr 1250 1255 1260 Gly Glu Val Val Val Thr Met Glu Pro Glu Val Pro Ile Lys Lys Leu 1265 1270 1275 1280 Glu Thr Met Val Lys Leu Asp Ala Thr Ser Glu Ala Val Val Lys Val 1285 1290 1295 Asp Val Ala Glu Glu Glu Lys Glu Lys Lys Lys Lys Lys Lys Gly Thr 1300 1305 1310 Ala Glu Glu Pro Glu Glu Glu Glu Pro Asp Glu Ser Met Leu Asp Trp 1315 1320 1325 Trp Ser Lys Tyr Phe Ala Ser Ile Asp Thr Met Lys Glu Gln Leu Arg 1330 1335 1340 Gln Gln Glu Pro Ser Gly Ile Asp Leu Glu Glu Lys Glu Glu Val Asp 1345 1350 1355 1360 Asn Thr Glu Gly Leu Lys Gly Ser Met Lys Gly Lys Glu Lys Ala Arg 1365 1370 1375 Ala Ala Lys Glu Glu Lys Lys Lys Lys Thr Gln Ser Ser Gly Ser Gly 1380 1385 1390 Gln Gly Ser Glu Ala Pro Glu Lys Lys Lys Pro Lys Ile Asp Glu Leu 1395 1400 1405 Lys Val Tyr Pro Lys Glu Leu Glu Ser Glu Phe Asp Asn Phe Glu Asp 1410 1415 1420 Trp Leu His Thr Phe Asn Leu Leu Arg Gly Lys Thr Gly Asp Asp Glu 1425 1430 1435 1440 Asp Gly Ser Thr Glu Glu Glu Arg Ile Val Gly Arg Phe Lys Gly Ser 1445 1450 1455 Leu Cys Val Tyr Lys Val Pro Leu Pro Glu Asp Val Ser Arg Glu Ala 1460 1465 1470 Gly Tyr Asp Ser Thr Tyr Gly Met Phe Gln Gly Ile Pro Ser Asn Asp 1475 1480 1485 Pro Ile Asn Val Leu Val Arg Val Tyr Val Val Arg Ala Thr Asp Leu 1490 1495 1500 His Pro Ala Asp Ile Asn Gly Lys Ala Asp Pro Tyr Ile Ala Ile Arg 1505 1510 1515 1520 Leu Gly Lys Thr Asp Ile Arg Asp Lys Glu Asn Tyr Ile Ser Lys Gln 1525 1530 1535 Leu Asn Pro Val Phe Gly Lys Ser Phe Asp Ile Glu Ala Ser Phe Pro 1540 1545 1550 Met Glu Ser Met Leu Thr Val Ala Val Tyr Asp Trp Asp Leu Val Gly 1555 1560 1565 Thr Asp Asp Leu Ile Gly Glu Thr Lys Ile Asp Leu Glu Asn Arg Phe 1570 1575 1580 Tyr Ser Lys His Arg Ala Thr Cys Gly Ile Ala Gln Thr Tyr Ser Thr 1585 1590 1595 1600 His Gly Tyr Asn Ile Trp Arg Asp Pro Met Lys Pro Ser Gln Ile Leu 1605 1610 1615 Thr Arg Leu Cys Lys Asp Gly Lys Val Asp Gly Pro His Phe Gly Pro 1620 1625 1630 Pro Gly Arg Val Lys Val Ala Asn Arg Val Phe Thr Gly Pro Ser Glu 1635 1640 1645 Ile Glu Asp Glu Asn Gly Gln Arg Lys Pro Thr Asp Glu His Val Ala 1650 1655 1660 Leu Leu Ala Leu Arg His Trp Glu Asp Ile Pro Arg Ala Gly Cys Arg 1665 1670 1675 1680 Leu Val Pro Glu His Val Glu Thr Arg Pro Leu Leu Asn Pro Asp Lys 1685 1690 1695 Pro Gly Ile Glu Gln Gly Arg Leu Glu Leu Trp Val Asp Met Phe Pro 1700 1705 1710 Met Asp Met Pro Ala Pro Gly Thr Pro Leu Asp Ile Ser Pro Arg Lys 1715 1720 1725 Pro Lys Lys Tyr Glu Leu Arg Val Ile Ile Trp Asn Thr Asp Glu Val 1730 1735 1740 Val Leu Glu Asp Asp Asp Phe Phe Thr Gly Glu Lys Ser Ser Asp Ile 1745 1750 1755 1760 Phe Val Arg Gly Trp Leu Lys Gly Gln Gln Glu Asp Lys Gln Asp Thr 1765 1770 1775 Asp Val His Tyr His Ser Leu Thr Gly Glu Gly Asn Phe Asn Trp Arg 1780 1785 1790 Tyr Leu Phe Pro Phe Asp Tyr Leu Ala Ala Glu Glu Lys Ile Val Ile 1795 1800 1805 Ser Lys Lys Glu Ser Met Phe Ser Trp Asp Glu Thr Glu Tyr Lys Ile 1810 1815 1820 Pro Ala Arg Leu Thr Leu Gln Ile Trp Asp Ala Asp His Phe Ser Ala 1825 1830 1835 1840 Asp Asp Phe Leu Gly Ala Ile Glu Leu Asp Leu Asn Arg Phe Pro Arg 1845 1850 1855 Gly Ala Lys Thr Ala Lys Gln Cys Thr Met Glu Met Ala Thr Gly Glu 1860 1865 1870 Val Asp Val Pro Leu Val Ser Ile Phe Lys Gln Lys Arg Val Lys Gly 1875 1880 1885 Trp Trp Pro Leu Leu Ala Arg Asn Glu Asn Asp Glu Phe Glu Leu Thr 1890 1895 1900 Gly Lys Val Glu Ala Glu Leu His Leu Leu Thr Ala Glu Glu Ala Glu 1905 1910 1915 1920 Lys Asn Pro Val Gly Leu Ala Arg Asn Glu Pro Asp Pro Leu Glu Lys 1925 1930 1935 Pro Asn Arg Pro Asp Thr Ser Phe Ile Trp Phe Leu Asn Pro Leu Lys 1940 1945 1950 Ser Ala Arg Tyr Phe Leu Trp His Thr Tyr Arg Trp Leu Leu Leu Lys 1955 1960 1965 Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Ala Leu Phe Leu Tyr 1970 1975 1980 Ser Val Pro Gly Tyr Leu Val Lys Lys Ile Leu Gly Ala 1985 1990 1995 <210> 2 <211> 1997 <212> PRT <213> Homo sapiens <400> 2 Met Ala Leu Leu Ile His Leu Lys Thr Val Ser Glu Leu Arg Gly Arg 1 5 10 15 Gly Asp Arg Ile Ala Lys Val Thr Phe Arg Gly Gln Ser Phe Tyr Ser 20 25 30 Arg Val Leu Glu Asn Cys Glu Asp Val Ala Asp Phe Asp Glu Thr Phe 35 40 45 Arg Trp Pro Val Ala Ser Ser Ile Asp Arg Asn Glu Met Leu Glu Ile 50 55 60 Gln Val Phe Asn Tyr Ser Lys Val Phe Ser Asn Lys Leu Ile Gly Thr 65 70 75 80 Phe Arg Met Val Leu Gln Lys Val Val Glu Glu Ser His Val Glu Val 85 90 95 Thr Asp Thr Leu Ile Asp Asp Asn Asn Ala Ile Ile Lys Thr Ser Leu 100 105 110 Cys Val Glu Val Arg Tyr Gln Ala Thr Asp Gly Thr Val Gly Ser Trp 115 120 125 Asp Asp Gly Asp Phe Leu Gly Asp Glu Ser Leu Gln Glu Glu Glu Lys 130 135 140 Asp Ser Gln Glu Thr Asp Gly Leu Leu Pro Gly Ser Arg Pro Ser Ser 145 150 155 160 Arg Pro Pro Gly Glu Lys Ser Phe Arg Arg Ala Gly Arg Ser Val Phe 165 170 175 Ser Ala Met Lys Leu Gly Lys Asn Arg Ser His Lys Glu Glu Pro Gln 180 185 190 Arg Pro Asp Glu Pro Ala Val Leu Glu Met Glu Asp Leu Asp His Leu 195 200 205 Ala Ile Arg Leu Gly Asp Gly Leu Asp Pro Asp Ser Val Ser Leu Ala 210 215 220 Ser Val Thr Ala Leu Thr Thr Asn Val Ser Asn Lys Arg Ser Lys Pro 225 230 235 240 Asp Ile Lys Met Glu Pro Ser Ala Gly Arg Pro Met Asp Tyr Gln Val 245 250 255 Ser Ile Thr Val Ile Glu Ala Arg Gln Leu Val Gly Leu Asn Met Asp 260 265 270 Pro Val Val Cys Val Glu Val Gly Asp Asp Lys Lys Tyr Thr Ser Met 275 280 285 Lys Glu Ser Thr Asn Cys Pro Tyr Tyr Asn Glu Tyr Phe Val Phe Asp 290 295 300 Phe His Val Ser Pro Asp Val Met Phe Asp Lys Ile Ile Lys Ile Ser 305 310 315 320 Val Ile His Ser Lys Asn Leu Leu Arg Ser Gly Thr Leu Val Gly Ser 325 330 335 Phe Lys Met Asp Val Gly Thr Val Tyr Ser Gln Pro Glu His Gln Phe 340 345 350 His His Lys Trp Ala Ile Leu Ser Asp Pro Asp Asp Ile Ser Ser Gly 355 360 365 Leu Lys Gly Tyr Val Lys Cys Asp Val Ala Val Val Gly Lys Gly Asp 370 375 380 Asn Ile Lys Thr Pro His Lys Ala Asn Glu Thr Asp Glu Asp Asp Ile 385 390 395 400 Glu Gly Asn Leu Leu Leu Pro Glu Gly Val Pro Pro Glu Arg Gln Trp 405 410 415 Ala Arg Phe Tyr Val Lys Ile Tyr Arg Ala Glu Gly Leu Pro Arg Met 420 425 430 Asn Thr Ser Leu Met Ala Asn Val Lys Lys Ala Phe Ile Gly Glu Asn 435 440 445 Lys Asp Leu Val Asp Pro Tyr Val Gln Val Phe Phe Ala Gly Gln Lys 450 455 460 Gly Lys Thr Ser Val Gln Lys Ser Ser Tyr Glu Pro Leu Trp Asn Glu 465 470 475 480 Gln Val Val Phe Thr Asp Leu Phe Pro Pro Leu Cys Lys Arg Met Lys 485 490 495 Val Gln Ile Arg Asp Ser Asp Lys Val Asn Asp Val Ala Ile Gly Thr 500 505 510 His Phe Ile Asp Leu Arg Lys Ile Ser Asn Asp Gly Asp Lys Gly Phe 515 520 525 Leu Pro Thr Leu Gly Pro Ala Trp Val Asn Met Tyr Gly Ser Thr Arg 530 535 540 Asn Tyr Thr Leu Leu Asp Glu His Gln Asp Leu Asn Glu Gly Leu Gly 545 550 555 560 Glu Gly Val Ser Phe Arg Ala Arg Leu Leu Leu Gly Leu Ala Val Glu 565 570 575 Ile Val Asp Thr Ser Asn Pro Glu Leu Thr Ser Ser Thr Glu Val Gln 580 585 590 Val Glu Gln Ala Thr Pro Ile Ser Glu Ser Cys Ala Gly Lys Met Glu 595 600 605 Glu Phe Phe Leu Phe Gly Ala Phe Leu Glu Ala Ser Met Ile Asp Arg 610 615 620 Arg Asn Gly Asp Lys Pro Ile Thr Phe Glu Val Thr Ile Gly Asn Tyr 625 630 635 640 Gly Asn Glu Val Asp Gly Leu Ser Arg Pro Gln Arg Pro Arg Pro Arg 645 650 655 Lys Glu Pro Gly Asp Glu Glu Glu Val Asp Leu Ile Gln Asn Ala Ser 660 665 670 Asp Asp Glu Ala Gly Asp Ala Gly Asp Leu Ala Ser Val Ser Ser Thr 675 680 685 Pro Pro Met Arg Pro Gln Val Thr Asp Arg Asn Tyr Phe His Leu Pro 690 695 700 Tyr Leu Glu Arg Lys Pro Cys Ile Tyr Ile Lys Ser Trp Trp Pro Asp 705 710 715 720 Gln Arg Arg Arg Leu Tyr Asn Ala Asn Ile Met Asp His Ile Ala Asp 725 730 735 Lys Leu Glu Glu Gly Leu Asn Asp Ile Gln Glu Met Ile Lys Thr Glu 740 745 750 Lys Ser Tyr Pro Glu Arg Arg Leu Arg Gly Val Leu Glu Glu Leu Ser 755 760 765 Cys Gly Cys Cys Arg Phe Leu Ser Leu Ala Asp Lys Asp Gln Gly His 770 775 780 Ser Ser Arg Thr Arg Leu Asp Arg Glu Arg Leu Lys Ser Cys Met Arg 785 790 795 800 Glu Leu Glu Asn Met Gly Gln Gln Ala Arg Met Leu Arg Ala Gln Val 805 810 815 Lys Arg His Thr Val Arg Asp Lys Leu Arg Leu Cys Gln Asn Phe Leu 820 825 830 Gln Lys Leu Arg Phe Leu Ala Asp Glu Pro Gln His Ser Ile Pro Asp 835 840 845 Ile Phe Ile Trp Met Met Ser Asn Asn Lys Arg Val Ala Tyr Ala Arg 850 855 860 Val Pro Ser Lys Asp Leu Leu Phe Ser Ile Val Glu Glu Glu Thr Gly 865 870 875 880 Lys Asp Cys Ala Lys Val Lys Thr Leu Phe Leu Lys Leu Pro Gly Lys 885 890 895 Arg Gly Phe Gly Ser Ala Gly Trp Thr Val Gln Ala Lys Val Glu Leu 900 905 910 Tyr Leu Trp Leu Gly Leu Ser Lys Gln Arg Lys Glu Phe Leu Cys Gly 915 920 925 Leu Pro Cys Gly Phe Gln Glu Val Lys Ala Ala Gln Gly Leu Gly Leu 930 935 940 His Ala Phe Pro Pro Val Ser Leu Val Tyr Thr Lys Lys Gln Ala Phe 945 950 955 960 Gln Leu Arg Ala His Met Tyr Gln Ala Arg Ser Leu Phe Ala Ala Asp 965 970 975 Ser Ser Gly Leu Ser Asp Pro Phe Ala Arg Val Phe Phe Ile Asn Gln 980 985 990 Ser Gln Cys Thr Glu Val Leu Asn Glu Thr Leu Cys Pro Thr Trp Asp 995 1000 1005 Gln Met Leu Val Phe Asp Asn Leu Glu Leu Tyr Gly Glu Ala His Glu 1010 1015 1020 Leu Arg Asp Asp Pro Pro Ile Ile Val Ile Glu Ile Tyr Asp Gln Asp 1025 1030 1035 1040 Ser Met Gly Lys Ala Asp Phe Met Gly Arg Thr Phe Ala Lys Pro Leu 1045 1050 1055 Val Lys Met Ala Asp Glu Ala Tyr Cys Pro Pro Arg Phe Pro Pro Gln 1060 1065 1070 Leu Glu Tyr Tyr Gln Ile Tyr Arg Gly Asn Ala Thr Ala Gly Asp Leu 1075 1080 1085 Leu Ala Ala Phe Glu Leu Leu Gln Ile Gly Pro Ala Gly Lys Ala Asp 1090 1095 1100 Leu Pro Pro Ile Asn Gly Pro Val Asp Val Asp Arg Gly Pro Ile Met 1105 1110 1115 1120 Pro Val Pro Met Gly Ile Arg Pro Val Leu Ser Lys Tyr Arg Val Glu 1125 1130 1135 Val Leu Phe Trp Gly Leu Arg Asp Leu Lys Arg Val Asn Leu Ala Gln 1140 1145 1150 Val Asp Arg Pro Arg Val Asp Ile Glu Cys Ala Gly Lys Gly Val Gln 1155 1160 1165 Ser Ser Leu Ile His Asn Tyr Lys Lys Asn Pro Asn Phe Asn Thr Leu 1170 1175 1180 Val Lys Trp Phe Glu Val Asp Leu Pro Glu Asn Glu Leu Leu His Pro 1185 1190 1195 1200 Pro Leu Asn Ile Arg Val Val Asp Cys Arg Ala Phe Gly Arg Tyr Thr 1205 1210 1215 Leu Val Gly Ser His Ala Val Ser Ser Leu Arg Arg Phe Ile Tyr Arg 1220 1225 1230 Pro Pro Asp Arg Ser Ala Pro Ser Trp Asn Thr Thr Val Arg Leu Leu 1235 1240 1245 Arg Arg Cys Arg Val Leu Cys Asn Gly Gly Ser Ser Ser His Ser Thr 1250 1255 1260 Gly Glu Val Val Val Thr Met Glu Pro Glu Val Pro Ile Lys Lys Leu 1265 1270 1275 1280 Glu Thr Met Val Lys Leu Asp Ala Thr Ser Glu Ala Val Val Lys Val 1285 1290 1295 Asp Val Ala Glu Glu Glu Lys Glu Lys Lys Lys Lys Lys Lys Gly Thr 1300 1305 1310 Ala Glu Glu Pro Glu Glu Glu Glu Pro Asp Glu Ser Met Leu Asp Trp 1315 1320 1325 Trp Ser Lys Tyr Phe Ala Ser Ile Asp Thr Met Lys Glu Gln Leu Arg 1330 1335 1340 Gln Gln Glu Pro Ser Gly Ile Asp Leu Glu Glu Lys Glu Glu Val Asp 1345 1350 1355 1360 Asn Thr Glu Gly Leu Lys Gly Ser Met Lys Gly Lys Glu Lys Ala Arg 1365 1370 1375 Ala Ala Lys Glu Glu Lys Lys Lys Lys Thr Gln Ser Ser Gly Ser Gly 1380 1385 1390 Gln Gly Ser Glu Ala Pro Glu Lys Lys Lys Pro Lys Ile Asp Glu Leu 1395 1400 1405 Lys Val Tyr Pro Lys Glu Leu Glu Ser Glu Phe Asp Asn Phe Glu Asp 1410 1415 1420 Trp Leu His Thr Phe Asn Leu Leu Arg Gly Lys Thr Gly Asp Asp Glu 1425 1430 1435 1440 Asp Gly Ser Thr Glu Glu Glu Arg Ile Val Gly Arg Phe Lys Gly Ser 1445 1450 1455 Leu Cys Val Tyr Lys Val Pro Leu Pro Glu Asp Val Ser Arg Glu Ala 1460 1465 1470 Gly Tyr Asp Ser Thr Tyr Gly Met Phe Gln Gly Ile Pro Ser Asn Asp 1475 1480 1485 Pro Ile Asn Val Leu Val Arg Val Tyr Val Val Arg Ala Thr Asp Leu 1490 1495 1500 His Pro Ala Asp Ile Asn Gly Lys Ala Asp Pro Tyr Ile Ala Ile Arg 1505 1510 1515 1520 Leu Gly Lys Thr Asp Ile Arg Asp Lys Glu Asn Tyr Ile Ser Lys Gln 1525 1530 1535 Leu Asn Pro Val Phe Gly Lys Ser Phe Asp Ile Glu Ala Ser Phe Pro 1540 1545 1550 Met Glu Ser Met Leu Thr Val Ala Val Tyr Asp Trp Asp Leu Val Gly 1555 1560 1565 Thr Asp Asp Leu Ile Gly Glu Thr Lys Ile Asp Leu Glu Asn Arg Phe 1570 1575 1580 Tyr Ser Lys His Arg Ala Thr Cys Gly Ile Ala Gln Thr Tyr Ser Thr 1585 1590 1595 1600 His Gly Tyr Asn Ile Trp Arg Asp Pro Met Lys Pro Ser Gln Ile Leu 1605 1610 1615 Thr Arg Leu Cys Lys Asp Gly Lys Val Asp Gly Pro His Phe Gly Pro 1620 1625 1630 Pro Gly Arg Val Lys Val Ala Asn Arg Val Phe Thr Gly Pro Ser Glu 1635 1640 1645 Ile Glu Asp Glu Asn Gly Gln Arg Lys Pro Thr Asp Glu His Val Ala 1650 1655 1660 Leu Leu Ala Leu Arg His Trp Glu Asp Ile Pro Arg Ala Gly Cys Arg 1665 1670 1675 1680 Leu Val Pro Glu His Val Glu Thr Arg Pro Leu Leu Asn Pro Asp Lys 1685 1690 1695 Pro Gly Ile Glu Gln Gly Arg Leu Glu Leu Trp Val Asp Met Phe Pro 1700 1705 1710 Met Asp Met Pro Ala Pro Gly Thr Pro Leu Asp Ile Ser Pro Arg Lys 1715 1720 1725 Pro Lys Lys Tyr Glu Leu Arg Val Ile Ile Trp Asn Thr Asp Glu Val 1730 1735 1740 Val Leu Glu Asp Asp Asp Phe Phe Thr Gly Glu Lys Ser Ser Asp Ile 1745 1750 1755 1760 Phe Val Arg Gly Trp Leu Lys Gly Gln Gln Glu Asp Lys Gln Asp Thr 1765 1770 1775 Asp Val His Tyr His Ser Leu Thr Gly Glu Gly Asn Phe Asn Trp Arg 1780 1785 1790 Tyr Leu Phe Pro Phe Asp Tyr Leu Ala Ala Glu Glu Lys Ile Val Ile 1795 1800 1805 Ser Lys Lys Glu Ser Met Phe Ser Trp Asp Glu Thr Glu Tyr Lys Ile 1810 1815 1820 Pro Ala Arg Leu Thr Leu Gln Ile Trp Asp Ala Asp His Phe Ser Ala 1825 1830 1835 1840 Asp Asp Phe Leu Gly Ala Ile Glu Leu Asp Leu Asn Arg Phe Pro Arg 1845 1850 1855 Gly Ala Lys Thr Ala Lys Gln Cys Thr Met Glu Met Ala Thr Gly Glu 1860 1865 1870 Val Asp Val Pro Leu Val Ser Ile Phe Lys Gln Lys Arg Val Lys Gly 1875 1880 1885 Trp Trp Pro Leu Leu Ala Arg Asn Glu Asn Asp Glu Phe Glu Leu Thr 1890 1895 1900 Gly Lys Val Glu Ala Glu Leu His Leu Leu Thr Ala Glu Glu Ala Glu 1905 1910 1915 1920 Lys Asn Pro Val Gly Leu Ala Arg Asn Glu Pro Asp Pro Leu Glu Lys 1925 1930 1935 Pro Asn Arg Pro Asp Thr Ala Phe Val Trp Phe Leu Asn Pro Leu Lys 1940 1945 1950 Ser Ile Lys Tyr Leu Ile Cys Thr Arg Tyr Lys Trp Leu Ile Ile Lys 1955 1960 1965 Ile Val Leu Ala Leu Leu Gly Leu Leu Met Leu Gly Leu Phe Leu Tyr 1970 1975 1980 Ser Leu Pro Gly Tyr Met Val Lys Lys Leu Leu Gly Ala 1985 1990 1995 <210> 3 <211> 5994 <212> DNA <213> Homo sapiens <400> 3 atggccttgc tcatcacct spacecagtc sparrow ggggcagggg sparrow 60 gccaaagtga ctttccgagg gcaatccttc tactctcggg tcctggagaa ctgtgaggat 120 gtggctgact ttgatgagac atttcggtgg ccggtggcca gcagcatcga cagaatgag 180 atgctggaga ttcaggtttt caactacagc aaagtctca gcaacaagct catcgggacc 240 ttccgcatgg tgctgcagaa ggtggtagag gagagccatg tggaggtgac tgacacgctg 300 attgatgaca acaatgctat catcaagacc agcctgtgcg tggaggtccg gtatcaggcc 360 actgacggca cagtgggctc ctgggacgat ggggacttcc tgggagatga gtctcttcaa 420 gaggaagaga aggacagcca agagacggat ggactgctcc caggctcccg gcccagctcc 480 cggcccccag gagagagag cttccggaga gccgggagga gcgtgttctc cgccatgaag 540 ctcggcaaa accggtctca caggaggag cccaagac cagatgaaccc ggcggtgctg 600 gagatggaag accttgacca tctggccatt cggctaggag atggactgga tcccgactcg 660 gtgtctctag cctcagtcac agctctcacc actaatgtct ccaacaagcg atctaagcca 720 gacattaaga tggagccaag tgctgggcgg cccatggatt accaggtcag catcacggtg 780 atcgaggccc ggcagctggt gggcttgaac atggaccctg tggtgtgcgt ggaggtgggt 840 gacgacaaga agtacacatc catgaaggag tccactaact gcccctatta caacgagtac 900 ttcgtcttcg acttccatgt ctctccggat gtcatgtttg acaagatcat caagatttcg 960 gtgattcact ccaagaacct gctgcgcagt ggcaccctgg tgggctcctt caaaatggac 1020 gtgggaaccg tgtactcgca gccagagcac cagttccatc acaagtgggc catcctgtct 1080 gaccccgatg acatctcctc ggggctgaag ggctacgtga agtgtgacgt tgccgtggtg 1140 ggcaaagggg acaacatcaa gacgccccac aaggccaatg agaccgacga agatgacatt 1200 gaggggaact tgctgctccc cgagggggtg ccccccgaac gccagtgggc ccggttctat 1260 gtgaaaattt accgagcaga ggggctgccc cgtatgaaca caagcctcat ggccaatgta 1320 aagaaggctt tcatcggtga aaacaaggac ctcgtggacc cctacgtgca agtcttcttt 1380 gctggccaga agggcaagac ttcagtgcag aagagcagct atgagcccct gtggaatgag 1440 caggtcgtct ttacagacct cttcccccca ctctgcaaac gcatgaaggt gcagatccga 1500 gactcggaca aggtcaacga cgtggccatc ggcacccact tcattgacct gcgcaagatt 1560 tctaatgacg gagacaaagg cttcctgccc acactgggcc cagcctgggt gaacatgtac 1620 ggctccacac gtaactacac gctgctggat gagcatcagg acctgaacga gggcctgggg 1680 gagggtgtgt ccttccgggc ccggctcctg ctgggcctgg ctgtggagat cgtagacacc 1740 tccaaccctg agctcaccag ctccacagag gtgcaggtgg agcaggccac gcccatctcg 1800 gagagctgtg caggtaaaat ggaagaattc tttctctttg gagccttcct ggaggcctca 1860 atgatcgacc ggagaaacgg agacaagccc atcacctttg aggtcaccat aggcaactat 1920 gggaacgaag ttgatggcct gtcccggccc cagcggcctc ggccccggaa ggagccgggg 1980 gatgaggaag aagtagacct gattcagaac gcaagtgatg acgaggccgg tgatgccggg 2040 gacctggcct cagtctcctc cactccacca atgcggcccc aggtcaccga caggaactac 2100 ttccatctgc cctacctgga gcgaaagccc tgcatctaca tcaagagctg gtggccggac 2160 cagcgccgcc gcctctacaa tgccaacatc atggaccaca ttgccgacaa gctggaagaa 2220 ggcctgaacg acatacagga gatgatcaaa acggagaagt cctaccctga gcgtcgcctg 2280 cggggcgtcc tggaggagct gagctgtggc tgctgccgct tcctctccct cgctgacaag 2340 gaccagggcc actcatcccg caccaggctt gaccgggagc gcctcaagtc ctgcatgagg 2400 gagctggaaa acatggggca gcaggccagg atgctgcggg cccaggtgaa gcggcacacg 2460 gtgcgggaca agctgaggct gtgccagaac ttcctgcaga agctgcgctt cctggcggac 2520 gagccccagc acagcattcc cgacatcttc atctggatga tgagcaacaa caagcgtgtc 2580 gcctatgccc gtgtgccctc caaggacctg ctcttctcca tcgtggagga ggagactggc 2640 aaggactgcg ccaaggtcaa gacgctcttc cttaagctgc cagggaagcg gggcttcggc 2700 tcggcaggct ggacagtgca ggccaaggtg gagctgtacc tgtggctggg cctcagcaaa 2760 cagcgcaagg agttcctgtg cggcctgccc tgtggcttcc aggaggtcaa ggcagcccag 2820 ggcctgggcc tgcatgcctt cccacccgtc agcctggtct acaccaagaa gcaggcgttc 2880 cagctccgag cgcacatgta ccaggcccgc agcctctttg ccgccgacag cagcggactc 2940 tcagacccct ttgcccgcgt cttcttcatc aatcagagtc agtgcacaga ggtgctgaat gagaccctgt gtcccacctg ggaccagatg ctggtgttcg acaacctgga gctctatggt 3060 gaagctcatg agctgaggga cgatccgccc atcattgtca ttgaatcta tgaccaggat 3120. tccatgggca aagctgactt catgggccgg accttcgcca aacccctggt gaagatggca gacgaggcgt actgcccacc ccgcttccca cctcagctcg stagnate gatctaccgt 3240 ggcaacgcca cagctggaga cctgctggcg gccttcgagc tgctgcagat tggaccagca 3300 gggaaggctg acctgcccc catcaatggc ccggtggacg tggaccgagg tcccatcatg cccgtgccca tgggcatccg gcccgtgctc agcaagtacc gagtggaggt gctgttctgg 3420. ggcctacggg acctaaagcg ggtgaacctg gcccaggtgg accggccacg ggtggacatc 3480 gagtgtgcag ggaagggggt gcagtcgtcc ctgatccaca attataagaa gaaccccaac 3540 ttcacaccc tcgtcaagtg gtttgaagtg gacctcccag agaacgagct gctgcacccg 3600 cccttgaaca tccgtgtggt ggactgccgg gccttcggtc gctacacact ggtgggctcc 3660 catgccgtca gctccctgcg acgcttcatc taccggcccc cagaccgctc ggccccagc 3720 tggaacacca cggtcaggct tctccggcgc tgccgtgtgc tgtgcaatgg gggctcctcc 3780 tctcactcca caggggaggt tgtggtgact atggagccag aggtaccat caggaactg 3840 gagaccatgg tgaagctgga cgcgacttct gaagctgttg tcaggtgga tgtggctgag 3900 gagagagggagagagagagggagggagggagggagggagggagg 3960 ccagacgaga gcatgctgga ctggtggtcc aagtactttg cctccattga caccatgaag 4020 gagcaacttc gacaacaga gccctctgga attgacttgg aggagaagga ggaagtggac 4080 ataccgagg gcctgaaggg gtcaatgaag ggcaaggaga aggcaagggc tgccaaggg 4140 gagaagaga agaaactca gagctctggc tctggccagg ggtccgaggc ccccgagaag 4200 aagaaaccca agattgatga gcttaaggta taccccaaag agctggagtc cgagtttgat 4260 aactttgagg actggctgca cactttcaac ttgcttcggg gcaagaccgg ggatgatgag 4320 gatggctcca ccgaggagga gcgcattgtg ggacgcttca agggctccct ctgcgtgtac 4380 aaagtgccac tcccagagga cgtgtcccgg gaagccggct acgactccac ctacggcatg 4440 ttccagggca tcccgagcaa tgaccccatc aatgtgctgg tccgagtcta tgtggtccgg 4500 gccacggacc tgcaccctgc tgacatcaac ggcaaagctg acccctacat cgccatccgg 4560 ctaggcaaga ctgacatccg cgacaaggag aactacatct ccaagcagct caaccctgtc 4620 tttgggaagt cctttgacat cgaggcctcc ttccccatgg aatccatgct gacggtggct 4680 gtgtatgact gggacctggt gggcactgat gacctcattg gggaaaccaa gatcgacctg 4740 gagaaccgct tctacagcaa gcaccgcgcc acctgcggca tcgcccagac ctactccaca 4800 catggctaca atatctggcg ggaccccatg aagcccagcc agatcctgac ccgcctctgc 4860 aaagacggca aagtggacgg cccccacttt gggccccctg ggagagtgaa ggtggccaac 4920 cgcgtcttca ctgggccctc tgagattgag gacgagaacg gtcagaggaa gcccacagac 4980 gagcatgtgg cgctgttggc cctgaggcac tgggaggaca tcccccgcgc aggctgccgc 5040 ctggtgccag agcatgtgga gacgaggccg ctgctcaacc ccgacaagcc gggcatcgag 5100 cagggccgcc tggagctgtg ggtggacatg ttccccatgg acatgccagc ccctgggacg 5160 cctctggaca tctcacctcg gaagcccaag aagtacgagc tgcgggtcat catctggaac 5220 acagatgagg tggtcttgga ggaggacgac ttcttcacag gggagaagtc cagtgacatc 5280 5340 cactccctca ctggcgaggg caacttcaac tggcgctacc tgttcccctt cgactacctg 5400 gcggcggagg agaagatcgt catctccaag aaggagtcca tgttctcctg ggacgagacc 5460 ggatacaaga tccccgcgcg gctcaccctg cagatctggg atgcggacca cttctccgct 5520 gacgacttcc tggggccat cgagctggac ctgaaccggt tccgcgggg cgcaaagaca 5580 gccaagcagt gcaccatgga gatggccacc ggggaggtgg acgtgcccct cgtgtccatc 5640 ttcaagcaaa agcgcgtcaa aggctggtgg cccctcctgg cccgcaatga gaacgatgag 5700 tttgagctca cgggcaaggt ggaggctgag ctgcatttac tgacagcaga ggaggcagag 5760 aagaacccag tgggcctggc ccgcaatgaa cctgaccccc tagagaaacc caaccggccc 5820 gacacgagct tcatctggtt cctgaaccct ctcaagtcgg ctcgctactt cttgtggcac 5880<q acgtatcgct ggctgctcct caaactgttg ctgctcctgc tgctgctcct cctcctcgcc 5940 ctgttcctct actctgtgcc tggctacctg gtcaagaaaa tcctcggggc ctga 5994 <210> 4 <211> 5994 [[ID= / / ID=16]]<212> DNA <213> Homo sapiens <400> 4 atggccttgc tcatccacct caagacagtc tcggagctgc ggggcagggg cgaccggatc 60 gccaaagtga ctttccgagg gcaatccttc tactctcggg tcctggagaa ctgtgaggat 120 gtggctgact ttgatgagac atttcggtgg ccggtggcca gcagcatcga cagaaatgag 180 atgctggaga ttcaggtttt caactacagc aaagtcttca gcaacaagct catcgggacc 240 ttccgcatgg tgctgcagaa ggtggtagag gagagccatg tggaggtgac tgacacgctg 300 attgatgaca acaatgctat catcaagacc agcctgtgcg tggaggtccg gtatcaggcc 360 actgacggca cagtgggctc ctgggacgat ggggacttcc tgggagatga gtctcttcaa 420 gaggaagaga aggacagcca agagacggat ggactgctcc caggctcccg gcccagctcc 480 cggcccccag gagagaagag cttccggaga gccgggagga gcgtgttctc cgccatgaag 540 ctcggcaaaa accggtctca caaggaggag ccccaaagac cagatgaacc ggcggtgctg 600 gagatggaag accttgacca tctggccatt cggctaggag atggactgga tcccgactcg 660 gtgtctctag cctcagtcac agctctcacc actaatgtct ccaacaagcg atctaagcca 720 gacattaaga tggagccaag tgctgggcgg cccatggatt accaggtcag catcacggtg 780 atcgaggccc ggcagctggt gggcttgaac atggaccctg tggtgtgcgt ggaggtgggt 840 gacgacaaga agtacacatc catgaaggag tccactaact gcccctatta caacgagtac 900 ttcgtcttcg acttccatgt ctctccggat gtcatgtttg acaagatcat caagatttcg 960 gtgattcact ccaagaacct gctgcgcagt ggcaccctgg tgggctcctt caaaatggac 1020 gtgggaaccg tgtactcgca gccagagcac cagttccatc acaagtgggc catcctgtct 1080 gaccccgatg acatctcctc ggggctgaag ggctacgtga agtgtgacgt tgccgtggtg 1140 ggcaaagggg acaacatcaa gacgccccac aaggccaatg agaccgacga agatgacatt 1200 gaggggaact tgctgctccc cgagggggtg ccccccgaac gccagtgggc ccggttctat 1260 gtgaaaattt accgagcaga ggggctgccc cgtatgaaca caagcctcat ggccaatgta 1320 aagaaggctt tcatcggtga aaacaaggac ctcgtggacc cctacgtgca agtcttcttt 1380 gctggccaga agggcaagac ttcagtgcag aagagcagct atgagcccct gtggaatgag 1440 caggtcgtct ttacagacct cttcccccca ctctgcaaac gcatgaaggt gcagatccga 1500 gactcggaca aggtcaacga cgtggccatc ggcacccact tcattgacct gcgcaagatt 1560 tctaatgacg gagacaaagg cttcctgccc acactgggcc cagcctgggt gaacatgtac 1620 ggctccacac gtaactacac gctgctggat gagcatcagg acctgaacga gggcctgggg 1680 gagggtgtgt ccttccggggc ccggctcctg ctggggcctgg ctgtggagat cgtagacacc 1740. tccaaccctg agctcaccag ctccacagag gtgcaggtgg agcaggccac gcccatctcg gagagctgtg caggtaaaat ggaagaattc tttctctttg gagccttcct ggaggcctca atgatcgacc ggagaaacgg agacaagccc atcacctttg aggtcaccat aggcaactat gggacgaag ttgatggcct gtcccggccc cagcggcctc ggccccgga ggagccgggg gatgaggaag aagtagacct gattcagaac gcaagtgatg acgaggccgg tgatgccggg gacctggcct cagtctcctc cactccacca atgcggcccc aggtcaccga caggaactac ttccatctgc cctacctgga gcgaaagccc tgcatctaca tcaagagctg gtggccggac 2160 cagcgccgcc gcctctacaa tgccaacatc atggaccaca ttgccgacaa gctggaagaa ggcctgaacg acatacagga gatgatcaaa acggagaagt cctaccctga gcgtcgcctg cggggcgtcc tggaggagct gagctgtggc tgctgccgct tcctctccct cgctgacaag 2340 gaccagggcc actcatcccg caccaggctt gaccggggagc gcctcaagtc ctgcatgagg gagctgga acatggggca gcaggccagg atgctgcggg cccaggtga gcggcacacg gtgcgggaca agctgaggct gtgccagaac ttcctgcaga agctgcgctt cctggcggac 2520 gagccccagc acagcattcc cgacatcttc atctggatga tgagcaacaa caagcgtgtc gcctatgccc gtgtgccctc caaggacctg ctcttctcca tcgtggagga ggagactggc 2640 aaggactgcg ccaaggtcaa gacgctcttc cttaagctgc caggagcg gggcttcggc tcggcaggct ggacagtgca ggccaaggtg gagctgtacc tgtggctggg cctcagcaaa cagcgcaagg agttcctgtg cggcctgccc tgtggcttcc aggaggtcaa ggcagcccag 2820 ggcctgggcc tgcatgcctt cccacccgtc agcctggtct acaccaagaa gcaggcgttc 2880 cagctccgag cgcacatgta ccaggcccgc agcctctttg ccgccgacag cagcggactc 2940 tcagacccct ttgcccgcgt cttcttcatc aatcagagtc agtgcacaga ggtgctgaat gagaccctgt gtcccacctg ggaccagatg ctggtgttcg acaacctgga gctctatggt 3060 gaagctcatg agctgaggga cgatccgccc atcattgtca ttgaatcta tgaccaggat 3120. tccatgggca aagctgactt catgggccgg accttcgcca aacccctggt gaagatggca 3180 gacgaggcgt actgcccacc ccgcttccca cctcagctcg agtactacca gatctaccgt 3240 ggcaacgcca cagctggaga cctgctggcg gccttcgagc tgctgcagat tggaccagca 3300 gggaaggctg acctgccccc catcaatggc ccggtggacg tggaccgagg tcccatcatg 3360 cccgtgccca tgggcatccg gcccgtgctc agcaagtacc gagtggaggt gctgttctgg 3420 ggcctacggg acctaaagcg ggtgaacctg gcccaggtgg accggccacg ggtggacatc 3480 gagtgtgcag ggaagggggt gcagtcgtcc ctgatccaca attataagaa gaaccccaac 3540 ttcaacaccc tcgtcaagtg gtttgaagtg gacctcccag agaacgagct gctgcacccg 3600 cccttgaaca tccgtgtggt ggactgccgg gccttcggtc gctacacact ggtgggctcc 3660 catgccgtca gctccctgcg acgcttcatc taccggcccc cagaccgctc ggcccccagc 3720 tggaacacca cggtcaggct tctccggcgc tgccgtgtgc tgtgcaatgg gggctcctcc 3780 tctcactcca caggggaggt tgtggtgact atggagccag aggtacccat caagaaactg 3840 gagaccatgg tgaagctgga cgcgacttct gaagctgttg tcaggtgga tgtggctgag 3900 gagagagggagagagagagggagggagggagggagggagggagg 3960 ccagacgaga gcatgctgga ctggtggtcc aagtactttg cctccattga caccatgaag 4020 gagcaacttc gacaacaga gccctctgga attgacttgg aggagaagga ggaagtggac 4080 ataccgagg gcctgaaggg gtcaatgaag ggcaaggaga aggcaagggc tgccaaggg 4140 gagaagaga agaaactca gagctctggc tctggccagg ggtccgaggc ccccgagaag 4200 aagaaaccca agattgatga gcttaggta taccccaag agctggagtc cgagttttgat 4260 aactttgagg actggctgca cacttcaac tgcttcggg gcaagaccgg ggatgatgag 4320 gatggctcca ccgaggagga gcgcattgtg ggacgcttca agggctccct ctgcgtgtac 4380 aaagtgccac tcccagagga cgtgtcccgg gaagccggct acgactccac ctacggcatg 4440 4500 gccacggacc tgcaccctgc tgacaccac gccaagctg accctacat cgccatccgg 4560 ctaggcaaga ctgacatccg cgacaaggag aactacatct ccaagcagct caaccctgtc 4620 tttgggaagt cctttgacat cgaggcctcc ttccccatgg aatccatgct gacggtggct 4680 gtgtatgact gggacctggt gggcactgat gacctcattg gggaaaccaa gatcgacctg 4740 gagaaccgct tctacagcaa gcaccgcgcc acctgcggca tcgcccagac ctactccaca 4800 catggctaca atatctggcg ggaccccatg aagcccagcc agatcctgac ccgcctctgc 4860 aaagacggca aagtggacgg cccccacttt gggccccctg ggagagtgaa ggtggccaac 4920 cgcgtcttca ctgggccctc tgagattgag gacgagaacg gtcagaggaa gcccacagac 4980 gagcatgtgg cgctgttggc cctgaggcac tgggaggaca tcccccgcgc aggctgccgc 5040 ctggtgccag agcatgtgga gacgaggccg ctgctcaacc ccgacaagcc gggcatcgag 5100 cagggccgcc tggagctgtg ggtggacatg ttccccatgg acatgccagc ccctgggacg 5160 cctctggaca tctcacctcg gaagcccaag aagtacgagc tgcgggtcat catctggaac 5220 acagatgagg tggtcttgga ggacgacgac ttcttcacag gggagaagtc cagtgacatc 5280 ttcgtgaggg ggtggctgaa gggccagcag gaggacaagc aggacacaga cgtccactac 5340 cactccctca ctggcgaggg caacttcaac tggcgctacc tgttcccctt cgactacctg 5400 gcggcggagg agaagatcgt catctccaag aaggagtcca tgttctcctg ggacgagacc 5460 gagtacaaga tcccgcg gctcaccctg cagatctggg atgcggacca cttctccgct 5520 gacgacttcc tgggggccat cgagctggac ctgaaccggt tcccgcgggg cgcaaagaca 5580 gccaagcagt gcaccatgga gatggccacc ggggaggtgg acgtgcccct cgtgtccatc 5640 ttcaagcaaa agcgcgtcaa aggctggtgg cccctcctgg cccgcaatga gaacgatgag 5700 tttgagctca cgggcaaggt ggaggctgag ctgcatttac tgacagcaga ggaggcagag 5760 aagaacccag tgggcctggc ccgcaatgaa cctgacccc tagagaaacc caaccggcc 5820 gacacggcct tcgtctggtt cctcaaccct ctcaagtcca tcaagtacct catctgcacc 5880 cggtacaagt ggctcatcat caagatcgtg ctggcgctgt tggggctgct catgttgggg 5940 ctcttcctct acagcctccc tgctacatg gtcaaaaagc tccttggggc atga 5994 <210> 5 <211> 2844 <212> DNA <213> Artificial sequence () <400> 5 ggattcgaac atcgattgaa ttccccgggg atcctctagg ccaccatggc cttgctcatc 60 cacctcaaga cagtctcgga gctgcggggc aggggcgacc ggatcgccaa agtgactttc 120 cgagggcaat ccttctactc tcgggtcctg gagaactgtg aggatgtggc tgactttgat 180 gagacatttc ggtggccggt ggccagcagc atcgacagaa atgagatgct ggagattcag 240 gttttcaact acagcaaagt cttcagcaac aagctcatcg ggaccttccg catggtgctg 300 cagaaggtgg tagaggagag ccatgtggag gtgactgaca cgctgattga tgacaacaat 360 gctatcatca agaccagcct gtgcgtggag gtccggtatc aggccactga cggcacagtg 420 ggctcctggg acgatgggga cttcctggga gatgagtctc ttcaagagga agagaaggac 480 agccaagaga cggatggact gctcccaggc tcccggccca gctcccggcc cccaggagag 540 aagagcttcc ggagagccgg gaggagcgtg ttctccgcca tgaagctcgg caaaaaccgg 600 tctcacaagg aggagcccca aagaccagat gaaccggcgg tgctggagat ggaagacctt 660 gaccatctgg ccattcggct aggagatgga ctggatcccg actcggtgtc tctagcctca 720 gtcacagctc tcaccactaa tgtctccaac aagcgatcta agccagacat taagatggag 780 ccaagtgctg ggcggcccat ggattaccag gtcagcatca cggtgatcga ggcccggcag 840 ctggtgggct tgaacatgga ccctgtggtg tgcgtggagg tgggtgacga caagaagtac 900 acatccatga aggagtccac taactgcccc tattacaacg agtacttcgt cttcgacttc 960 catgtctctc cggatgtcat gtttgacaag atcatcaaga tttcggtgat tcactccaag 1020 aacctgctgc gcagtggcac cctggtgggc tccttcaaaa tggacgtggg aaccgtgtac 1080 tcgcagccag agcaccagtt ccatcacaag tgggccatcc tgtctgaccc cgatgacatc 1140 tcctcggggc tgaagggcta cgtgaagtgt gacgttgccg tggtgggcaa aggggacaac 1200 atcaagacgc cccacaaggc caatgagacc gacgaagatg acattgaggg gaacttgctg 1260 ctccccgagg gggtgccccc cgaacgccag tgggcccggt tctatgtgaa aatttaccga 1320 gcagaggggc tgccccgtat gaacacaagc ctcatggcca atgtaaagaa ggctttcatc 1380 ggtgaaaaca aggacctcgt ggacccctac gtgcaagtct tctttgctgg ccagaagggc 1440 aagacttcag tgcagaagag cagctatgag cccctgtgga atgagcaggt cgtctttaca 1500 gacctcttcc ccccactctg caaacgcatg aaggtgcaga tccgagactc ggacaaggtc 1560 aacgacgtgg ccatcggcac ccacttcatt gacctgcgca agatttctaa tgacggagac 1620 aaaggcttcc tgcccacact gggcccagcc tgggtgaaca tgtacggctc cacacgtaac 1680 tacacgctgc tggatgagca tcaggacctg aacgagggcc tgggggaggg tgtgtccttc 1740 cgggcccggc tcctgctggg cctggctgtg gagatcgtag acacctccaa ccctgagctc 1800 accagctcca cagaggtgca ggtggagcag gccacgccca tctcggagag ctgtgcaggt 1860 aaaatggaag aattctttct ctttggagcc ttcctggagg cctcaatgat cgaccggaga 1920 aacggagaca agcccatcac ctttgaggtc accataggca actatgggaa cgaagttgat 1980 ggcctgtccc ggccccagcg gcctcggccc cggaaggagc cgggggatga ggaagaagta 2040 gacctgattc agaacgcaag tgatgacgag gccggtgatg ccggggacct ggcctcagtc 2100 tcctccactc caccaatgcg gccccaggtc accgacagga actacttcca tctgccctac 2160 ctggagcgaa agccctgcat ctacatcaag agctggtggc cggaccagcg ccgccgcctc 2220 tacaatgcca acatcatgga ccacattgcc gacaagctgg aagaaggcct gaacgacata 2280 caggagatga tcaaaacgga gaagtcctac cctgagcgtc gcctgcgggg cgtcctggag 2340 gagctgagct gtggctgctg ccgcttcctc tccctcgctg acaaggacca gggccactca 2400 tcccgcacca ggcttgaccg ggagcgcctc aagtcctgca tgagggagct ggaaaacatg 2460 gggcagcagg ccaggatgct gcgggcccag gtgaagcggc acacggtgcg ggacaagctg 2520 aggctgtgtc tggctggcga tactctcatt acctggccg atggacgacg agtgcctatt 2580 agagaactgg tgtcacagca gaatttttcc gtgtgggctc tgaatcctca gacttaccgc 2640 ctggagaggg ctagagtgag tagagctttc tgtaccggca tcaaacctgt gtaccgcctc 2700 accactagac tggggagatc cattagggcc actgccaacc accgatttct cacacctcag 2760 ggctggaaac gagtcgatga actccagcct ggagattacc tggctctgcc taggagaatc 2820 cctactgcct cctaaagct tgcc2844 <210> 6 <211> 3723 <212> DNA <213> Container() <400> 6 ggattcgaac atcgattgaa ttccccgggg atcctctagg ccaccatggc ggcggcgtgc ccggaactgc gtcagctggc gcagagcgat gtgtattggg atccgattgt gagcattgaa ccggatggcg tggaagaagt gtttgatctg accgtgccgg gcccgcataa ctttgtggcg 180 aacgatatta ttgcgcataa ctgccagaac ttcctgcaga agctgcgctt cctggcggac 240 gagccccagc acagcattcc cgacatcttc atctggatga tgagcaacaa caagcgtgtc gcctatgccc gtgtgccctc caaggacctg ctcttctcca tcgtggagga ggagactggc 360 aaggactgcg ccaaggtcaa gacgctcttc cttaagctgc caggggagcg gggcttcggc tcggcaggct ggacagtgca ggccaaggtg gagctgtacc tgtggctggg cctcagcaaa cagcgcaagg agttcctgtg cggcctgccc tgtggcttcc aggaggtcaa ggcagcccag 540 ggcctgggcc tgcatgcctt cccacccgtc agcctggtct acaccaagaa gcaggcgttc 600 cagctccgag cgcacatgta ccaggcccgc agcctctttg ccgccgacag cagcggactc 660 tcagacccct ttgcccgcgt cttcttcatc aatcagagtc agtgcacaga ggtgctgaat 720 gagaccctgt gtcccacctg ggaccagatg ctggtgttcg acaacctgga gctctatggt 780 gaagctcatg agctgaggga cgatccgccc atcattgtca ttgaaatcta tgaccaggat 840 tccatgggca aagctgactt catgggccgg accttcgcca aacccctggt gaagatggca 900 gacgaggcgt actgcccacc ccgcttccca cctcagctcg agtactacca gatctaccgt 960 ggcaacgcca cagctggaga cctgctggcg gccttcgagc tgctgcagat tggaccagca 1020 gggaaggctg acctgccccc catcaatggc ccggtggacg tggaccgagg tcccatcatg 1080 cccgtgccca tgggcatccg gcccgtgctc agcaagtacc gagtggaggt gctgttctgg 1140 ggcctacggg acctaaagcg ggtgaacctg gcccaggtgg accggccacg ggtggacatc 1200 gagtgtgcag ggaagggggt gcagtcgtcc ctgatccaca attataagaa gaaccccaac 1260 ttcaacaccc tcgtcaagtg gtttgaagtg gacctcccag agaacgagct gctgcacccg 1320 cccttgaaca tccgtgtggt ggactgccgg gccttcggtc gctacacact ggtgggctcc 1380 catgccgtca gctccctgcg acgctcatc taccggcccc cagaccgctc gcccccagc 1440 tggacacca cggtcaggct tctccggcgc tgccgtgtgc tgtgcaatgg gggctcctcc 1500 tctcactcca caggggaggt tgtggtgact atggagccag aggtaccat cagggaactg 1560 gagaccatgg tgaagctgga cgcgacttct gaagctgttg tcaggtgga tgtggctgag 1620 gagagagggagagagagggagggagggagggagggagggagg 1680 ccagacgaga gcatgctgga ctggtggtcc aagtactttg cctccattga caccatgaag 1740 gagcaacttc gacacaga gccctctgga attgacttgg aggaagga ggaagtggac 1800 ataccgagg gcctgaaggg gtcaatgag ggcaaggag aggcaagggc tgccaaggg 1860 gagaagaga agaaactca gagctctggc tctggccagg ggtccgaggc ccccgagaag 1920 aagaaaccca agattgatga gcttaggta taccccaag agctggagtc cgagttttgat 1980 aactttgagg actggctgca cacttcaac tgcttcggg gcaagaccgg ggatgatgag 2040 gatggctcca ccgaggagga gcgcattgtg ggacgcttca agggctccct ctgcgtgtac 2100 aaagtgccac tcccagagga cgtgtcccgg gaagccggct acgactccac ctacggcatg 2160 ttccagggca tcccgagcaa tgaccccatc aatgtgctgg tccgagtcta tgtggtccgg 2220 gccacggacc tgcaccctgc tgacatcaac ggcaaagctg acccctacat cgccatccgg 2280 ctaggcaaga ctgacatccg cgacaaggag aactacatct ccaagcagct caaccctgtc 2340 tttgggaagt cctttgacat cgaggcctcc ttccccatgg aatccatgct gacggtggct 2400 gtgtatgact gggacctggt gggcactgat gacctcattg gggaaaccaa gatcgacctg 2460 gagaaccgct tctacagcaa gcaccgcgcc acctgcggca tcgcccagac ctactccaca 2520 catggctaca atatctggcg ggaccccatg aagcccagcc agatcctgac ccgcctctgc 2580 aaagacggca aagtggacgg cccccacttt gggccccctg ggagagtgaa ggtggccaac 2640 cgcgtcttca ctgggccctc tgagattgag gacgagaacg gtcagaggaa gcccacagac 2700 gagcatgtgg cgctgttggc cctgaggcac tgggaggaca tcccccgcgc aggctgccgc 2760 ctggtgccag agcatgtgga gacgaggccg ctgctcaacc ccgacaagcc gggcatcgag 2820 cagggccgcc tggagctgtg ggtggacatg ttccccatgg acatgccagc ccctgggacg 2880 cctctggaca tctcacctcg gaagcccaag aagtacgagc tgcgggtcat catctggaac 2940 Acagatgagg tggtcttgga ggaggacgac ttcttcacag gggagaagtc cagtgacatc 3000 3060 cactccctca ctggcgaggg caacttcaac tggcgctacc tgttcccctt cgactacctg 3120 gcggcggagg agaagatcgt catctccaag aaggagtcca tgttctcctg ggacgagacc 3180 ggatacaaga tccccgcgcg gctcaccctg cagatctggg atgcggacca cttctccgct 3240 gacgacttcc tggggccat cgagctggac ctgaaccggt tcccggggg cgcaaagaca 3300 gccaagcagt gcaccatgga gatggccacc ggggaggtgg acgtgcccct cgtgtccatc 3360 ttcaagcaaa agcgcgtcaa aggctggtgg cccctcctgg cccgcaatga gaacgatgag 3420 tttgagctca cgggcaaggt ggaggctgag ctgcatttac tgacagcaga ggaggcagag 3480 aagaacccag tgggcctggc ccgcaatgaa cctgaccccc tagagaacc caaccggccc gacacggcct tcgtctggtt cctcaaccct ctcaagtcca tcaagtacct catctgcacc 3600 cggtacaagt ggctcatcat caagatcgtg ctggcgctgt tggggctgct catgttgggg ctcttcctct acagcctccc tggctacatg gtcaaaagc tccttggggc atgaaagctt 3720 gcc 3723 <210> 7 <211> 3153 <212> DNA <213> Container() <400> 7 ggattcgaac atcgattgaa ttccccgggg atcctctagg ccaccatggc cttgctcatc cacctcaaga cagtctcgga gctgcggggc aggggcgacc ggatcgccaa agtgactttc cgagggcaat ccttctactc tcgggtcctg gagaactgtg aggatgtggc tgactttgat 180 gagacatttc ggtggccggt ggccagcagc atcgacagaa atgagatgct ggagattcag gttttcaact acagcaaagt cttcagcaac aagctcatcg ggaccttccg catggtgctg cagaaggtgg tagaggagag ccatgtggag gtgactgaca cgctgattga tgacaacaat gctatcatca agaccagcct gtgcgtggag gtccggtatc aggccactga cggcacagtg 420 ggctcctggg acgatgggga cttcctggga gatgagtctc ttcagagga agagaggac 480 agccaagaga cggatggact gctcccaggc tcccggcccca gctcccggcc cccaggagag 540 aagagcttcc ggagagccgg gaggagcgtg ttctccgcca tgaagctcgg caaaaccgg 600 tctcacaagg aggagcccca aagaccagat gaaccggcgg tgctggagat ggaagacctt 660 gaccatctgg ccattcggct aggagatgga ctggatcccg actcggtgtc tctagcctca 720 gtcacagctc tcaccactaa tgtctccaac aagcgatcta agccagacat taagatggag 780 ccaagtgctg ggcggccat ggattaccag gtcagcatca cggtgatcga ggcccggcag 840 ctggtgggct tgaacatgga ccctgtggtg tgcgtggagg tggtgacga caagaagtac 900 acatccatga aggagtccac taactgcccc tattacaacg agtactcgt cttcgacttc 960 catgtctctc cggatgtcat gtttgacaag atcatcaaga ttcggtgat tcactcaag 1020 aacctgctgc gcagtggcac cctggtgggc tccttcaaa tggacgtggg aaccgtgtac 1080 tcgcagccag agcaccagtt ccatcacaag tgggccatcc tgtctgaccc cgatgacatc 1140 tcctcggggc tgaagggcta cgtgaagtgt gacgttgccg tggtgggcaa aggggacaac 1200 atcaagacgc cccacaaggc caatgagacc gacgaagatg acattgaggg gaacttgctg 1260 ctccccgagg gggtgccccc cgaacgccag tgggcccggt tctatgtgaa aatttaccga 1320 gcagaggggc tgccccgtat gaacacaagc ctcatggcca atgtaaagaa ggctttcatc 1380 ggtgaaaaca aggacctcgt ggacccctac gtgcaagtct tctttgctgg ccagaagggc 1440 aagacttcag tgcagaagag cagctatgag cccctgtgga atgagcaggt cgtctttaca 1500 gacctcttcc ccccactctg caaacgcatg aaggtgcaga tccgagactc ggacaaggtc 1560 aacgacgtgg ccatcggcac ccacttcatt gacctgcgca agatttctaa tgacggagac 1620 aaaggcttcc tgcccacact gggcccagcc tgggtgaaca tgtacggctc cacacgtaac 1680 tacacgctgc tggatgagca tcaggacctg aacgagggcc tgggggaggg tgtgtccttc 1740 cgggcccggc tcctgctggg cctggctgtg gagatcgtag acacctccaa ccctgagctc 1800 accagctcca cagaggtgca ggtggagcag gccacgccca tctcggagag ctgtgcaggt 1860 aaaatggaag aattctttct ctttggagcc ttcctggagg cctcaatgat cgaccggaga 1920 aacggagaca agcccatcac ctttgaggtc accataggca actatgggaa cgaagttgat 1980 ggcctgtccc ggccccagcg gcctcggccc cggaaggagc cgggggatga ggaagaagta 2040 gacctgattc agaacgcaag tgatgacgag gccggtgatg ccggggacct ggcctcagtc 2100 tcctccactc caccaatgcg gccccaggtc accgacagga actacttcca tctgccctac 2160 ctggagcgaa agccctgcat ctacatcaag agctggtggc cggaccagcg ccgccgcctc 2220 tacaatgcca acatcatgga ccacattgcc gacaagctgg aagaaggcct gaacgacata 2280 caggagatga tcaaaacgga gaagtcctac cctgagcgtc gcctgcgggg cgtcctggag 2340 gagctgagct gtggctgctg ccgcttcctc tccctcgctg acaaggacca gggccactca 2400 tcccgcacca ggcttgaccg ggagcgcctc aagtcctgca tgagggagct ggaaaacatg 2460 gggcagcagg ccaggatgct gcgggcccag gtgaagcggc acacggtgcg ggacaagctg 2520 aggctgtgcc agaacttcct gcagaagctg cgcttcctgg cggacgagcc ccagcacagc 2580 attcccgaca tcttcatctg gatgatgagc aacaacaagc gtgtcgccta tgcccgtgtg ccctccaagg acctgctctt ctccatcgtg gaggaga ctggcaagga ctgcgccaag gtcaagacgc tcttccttaa gctgccaggg aagcggggct tcggctcggc aggctggaca 2760 gtgcaggcca aggtggagct gtacctgtgg ctgggcctca gcaaacagcg caaggagttc ctgtgcggcc tgccctgtct ggctggcgat actctcatta ccctggccga tggacgacga 2880 gtgcctatta gagaactggt gtcacagcag aatttttccg tgtgggctct gaatcctcag acttaccgcc tggagagggc taggtgagt agagctttct gtaccggcat caaacctgtg 3060. taccgcctca ccctagact ggggagatcc attagggcca ctgccaacca ccgatttctc acacctcagg gctggaaacg agtcgatgaa ctccagcctg gagattacct ggctctgcct aggagatcc ctactgcctc ctaaagctt gcc 3153 <210> 8 <211> 3414 <212> DNA <213> Container() <400> 8 ggattcgaac atcgattgaa ttccccgggg atcctctagg ccaccatggc ggcggcgtgc 60 ccggaactgc gtcagctggc gcagagcgat gtgtattggg atccgattgt gagcattgaa 120 ccggatggcg tggagaagt gtttgatctg accgtgccgg gcccgcataa ctttgtggcg 180 aacgatatta ttgcgcataa ctgtggcttc caggaggtca aggcagccca gggcctgggc 240 ctgcatgcct tcccacccgt cagcctggtc tacaccaaga agcaggcgtt ccagctccga 300 gcgcacatgt accaggcccg cagcctcttt gccgccgaca gcagcggact ctcagacccc 360 tttgcccgcg tcttcttcat caatcagagt cagtgcacag aggtgctgaa tgagaccctg 420 tgtcccacct gggaccagat gctggtgttc gacaacctgg agctctatgg tgaagctcat 480 gagctgagg acgatccgcc catcattgtc attgaaatct atgaccagga ttccatgggc 540 aaagctgact tcatgggccg gaccttcgcc aaacccctgg tgaagatggc agacgaggcg 600 tactgcccac cccgcttccc acctcagctc gagtactacc agatctaccg tggcaacgcc 660 acagctggag acctgctggc ggccttcgag ctgctccaga ttggaccagc agggaaggct 720 gacctgcccc ccatcaatgg cccggtggac gtggaccgag gtccatcat gccgtgccc 780 atgggcatcc gggccgtgct cagcaagtac cgagtggagg tgctgttctg gggcctacgg 840 gacctaaagc gggtgaacct ggcccaggtg gaccggccac gggtggacat cgagtgtgca 900 gggaaggggg tgcagtcgtc cctgatccac aattataaga agaacccca cttcacacc 960 ctcgtcaagt ggttgaagt ggaccccca gagaacgagc tgctgcaccc gcccttgaac 1020 atccgtgtgg tggactgccg ggccttcggt cgctacacac tggtgggctc ccatgccgtc 1080 agctccctgc gacgctcat ctaccggccc ccagaccgct cggcccccg ctggacacc 1140 acggtcaggc ttctccggcg ctgccgtgtg ctgtgcaatg ggggctcctc ctctcactcc 1200 acaggggagg ttgtggtgac tatggagcca gaggtaccca tcaagaact ggagaccatg 1260 gtgaagctgg acgcgacttc tgaagctgtt gtcaggtgg atgtggctga ggaggagaag 1320 gagagagagagagaa gggcactgcg gagggag gaggagg gccagacgag 1380 agcatgctgg actgtggtc caagtacttt gcctccattg acaccatgaa ggagcaactt 1440 cgacaacaag agccctctgg aattgacttg gaggaagagg aggaagtgga caataccgag 1500 ggcctgaagg ggtcaatgaa gggcaaggag aaggcaaggg ctgccaaaga ggagaagaag 1560 aagaaaactc agagctctgg ctctggccag gggtccgagg cccccgagaa gaagaaccc 1620 aagattgatg agcttaaggt ataccccaaa gagctggagat ccgagtttga taactttgag 1680 gactggctgc acactttcaa cttgcttcgg ggcaagaccg gggatgatga ggatggctcc 1740 accgagagg agcgcattgt gggacgcttc aagggctccc tctgcgtgta caaagtgcca 1800 ctcccagagg acgtgtcccg ggaagccggc tacgactcca cctacggcat gttccagggc 1860 atccccgagca atgaccccat caatgtgctg gtccgagtct atgtggtccg ggccacggac 1920 ctgcaccctg ctgacatcaa cggcaaagct gacccctaca tcgccatccg gctaggcaag 1980 actgacatcc gcgacaagga gaactacatc tccaagcagc tcaaccctgt ctttgggaag 2040 tcctttgaca tcgaggcctc cttccccatg gaatccatgc tgacggtggc tgtgtatgac 2100 tgggacctgg tgggcactga tgacctcatt ggggaaacca agatcgacct ggagaaccgc 2160 ttctacagca agcaccgcgc cacctgcggc atcgcccaga cctactccac acatggctac 2220 aatatctggc gggaccccat gaagcccagc cagatcctga cccgcctctg caaagacggc 2280 aaagtggacg gcccccactt tgggccccct gggagagtga aggtggccaa ccgcgtcttc 2340 actgggccct ctgagattga ggacgagaac ggtcagagga agcccacaga cgagcatgtg 2400 gcgctgttgg ccctgaggca ctgggaggac atcccccgcg caggctgccg cctggtgcca 2460 gagcatgtgg agacgaggcc gctgctcaac cccgacaagc cgggcatcga gcagggccgc 2520 ctggagctgt gggtggacat gttccccatg gacatgccag cccctgggac gcctctggac 2580 atctcacctc ggaagcccaa gaagtacgag ctgcgggtca tcatctggaa cacagatgag 2640 gtggtcttgg aggacgacga cttcttcaca ggggagaagt ccagtgacat cttcgtgagg 2700 gggtggctga agggccagca ggaggacaag caggacacag acgtccacta ccactccctc 2760 actggcgagg gcaacttcaa ctggcgctac ctgttcccct tcgactacct ggcggcggag 2820 gagaagatcg tcatctccaa gaaggagtcc atgttctcct gggacgagac cgagtacaag 2880 atccccgcgc ggctcaccct gcagatctgg gatgcggacc acttctccgc tgacgacttc 2940 ctgggggcca tcgagctgga cctgaaccgg ttcccgcggg gcgcaaagac agccaagcag tgcaccatgg agatggccac cggggaggtg gacgtgcccc tcgtgtccat cttcaagcaa aagcgcgtca aaggctggtg gcccctcctg gcccgcaatg agaacgatga gtttgagctc acgggcaagg tggaggctga gctgcattta ctgacagcag aggaggcaga gaagaaccca gtgggcctgg cccgcaatga acctgacccc ctagagaaac ccaaccggcc cgacacggcc ttcgtctggt tcctcaaccc tctcaagtcc atcaagtacc tcatctgcac ccggtacaag tggctcatca tcaagatcgt gctggcgctg ttggggctgc tcatgttggg gctcttcctc 3360 3414. ttcctcc ctggctacat ggtcaaaaag ctccttgggg catgaaagct tgcc <210> 9 <211> 3822 <212> DNA <213> Container() <400> 9 60. cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccggggcgtc gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca 120 actccatcac tagggttcc tgcggccgca cgcgtggagc tagcccatat atggagttcc 180 ggacattgat tattgactag ttattaatag yours cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc 300 aacgacccc gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat 420 caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa atggccccgcc tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag 600 cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt 660 tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc atcaacgcca atgggcggta ggcgtgtacg gtgggaggtc your father gagctttggg attcgaacat 780. cgattgaatt ccccggggat cctctaggcc accaagcttg cctcgagccg actgtgcctt 840 ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg 900 ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt 960 gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca 1020 atagcaggca tgctggggat gcggtgggct ctatggctga ttttgtaggt cgacctgctc 1080 gagcaggaac ccctagtgat ggagttggcc actccctctc tgcgcgctcg ctcgctcact 1140 gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc 1200 gagcgagcgc gcagctgcct gcaggggcgc ctgatgcggt attttctcct tacgcatctg 1260 tgcggtattt cacaccgcat acgtcaaagc aaccatagta cgcgccctgt agcggcgcat 1320 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag 1380 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc 1440 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc 1500 ccaaaaaact tgatttgggt gatggttcac gtagtgggcc atcgccctga tagacggttt 1560 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa 1620 caacactcaa ccctatctcg ggctattctt ttgatttata agggattttg ccgatttcgg 1680 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aaaaaatat 1740 taacgtttac aattttatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa 1800 gccagccccg acacccgcca acaccgctg acgcgccctg acgggcttgt ctgctcccgg 1860 catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac 1920 cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta 1980 atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg 2040 gaacccctat ttgtttatttt ttctaaatac attcaaatat gtatccgctc atgagacaat 2100 aaccctgata aatgcttcaa tatattgaa aaaggaagag tatgagtatt caacatttcc 2160 gtgcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa 2220 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac 2280 tggatctcaa cagcggtaag atccttgaga gttttcgcc cgaagaacgt tttccaatga 2340 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag 2400 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca 2460 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca 2520 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa 2580 ccgctttttt gcaacacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc 2640 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa 2700 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa aattaatag 2760 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct 2820 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac 2880 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa 2940 ctatggatga agaataga cagatcgctg agataggtgc ctcactgatt aagcattggt 3000 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat 3060 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg 3120 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc 3180 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg 3240 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag 3300 cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact 3360 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg 3420 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc 3480 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg 3540 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg 3600 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag 3660 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc 3720 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct 3780 ttttacggtt cctggccttt tgctggcctt ttgctcacat gt 3822

Claims

1. A dual vector system for expressing OTOF protein, characterized in that: The dual vector system includes two nucleotide sequences: The first nucleotide sequence includes two ITR sequences and an expression cassette inserted between the ITR sequences; The second nucleotide sequence includes two ITR sequences and an expression cassette inserted between the ITR sequences; The expression cassette of the first nucleotide sequence includes: a promoter, an OTOF N-terminal coding sequence, an Intein N-terminal coding sequence and PolyA; The expression cassette of the second nucleotide sequence includes: a promoter, an Intein C-terminal coding sequence, an OTOF C-terminal coding sequence and PolyA; A split point is set on the OTOF amino acid sequence, and the nucleotide coding sequence from the N-terminus of the OTOF amino acid sequence to the split point is the OTOF N-terminal coding sequence, and the nucleotide coding sequence from the next amino acid of the split point to the C-terminus of the OTOF amino acid sequence is the OTOF C-terminal coding sequence; The amino acid sequence of OTOF is shown in SEQ ID NO. 1 or SEQ ID NO. 2 in the sequence listing; The cleavage site of OTOF is the amino acid preceding serine or cysteine ​​in the amino acid sequence of the OTOF protein, and the cleavage site is the 827th, 930th or 1130th amino acid residue of SEQ ID NO.1 or SEQ ID NO.

2.

2. A dual vector system for expressing an OTOF protein according to claim 1, characterized in that: The promoter includes but is not limited to: CAG promoter, CMV promoter, CBA promoter, UbC promoter, SFFV promoter, EF1α promoter, PGK promoter, or Myo7A, Myo15, Atoh1, POU4F3, Lhx3, Myo6, α9AchR, α10AchR, OTOF Promoters of other coding genes; The PolyA comprises AATAAA and variants thereof, including but not limited to ATTAAA, AGTAAA, CATAAA, TATAAA, GATAAA, ACTAAA, AATATA, AAGAAA, AATAAT, AAAAAA, AATGAA, AATCAA, AACAAA, AATCAA, AATAAC, AATAGA, AATTAA or AATAAG; The ITR sequence is derived from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 or AAV9.

3. A dual vector system for expressing an OTOF protein according to claim 1, characterized in that: The expression cassette further comprises an expression control element or a tag element.

4. The dual vector system for expressing an OTOF protein according to claim 1, wherein: The intein is selected from the intein sequence of MxeGyrA, pabPolIII, MjaKlbA, SspDnaB, SceVMA, SspDnaE, ​​NpuDnaE, ​​AvaDnaE, ​​CraDnaE, ​​CspDnaE, ​​CwaDnaE, ​​MchtDnaE, ​​OliDnaE, ​​TerDnaE, ​​gp41-1, gp41-8, IMPDH-1, or RmaDnaB.

5. A dual vector system for expressing an OTOF protein according to claim 1, characterized in that: The first nucleotide sequence is an expression cassette on a plasmid containing ITRs and inserted between the ITR sequences; the second nucleotide sequence is an expression cassette on a plasmid containing ITRs and inserted between the ITR sequences.

6. A dual vector system for expressing an OTOF protein according to claim 5, characterized in that: The plasmid containing ITR is pAAV, pAAV-CMV, pX601, pX551 or pAAV-MCS plasmid.

7. The dual vector system for expressing an OTOF protein according to claim 1, wherein: The OTOF shown in SEQ ID NO. 2 is divided at amino acid position 827 and NpuDnaE Intein is used; the N-terminal coding sequence of OTOF is ligated and fused with the N-terminal coding sequence of NpuDnaE Intein, and the pAAV-CMV plasmid is used as a vector to construct a first nucleotide sequence; the C-terminal coding sequence of NpuDnaE Intein is ligated and fused with the C-terminal coding sequence of OTOF, and the pAAV-CMV plasmid is used as a vector to construct a second nucleotide sequence; or The OTOF shown in SEQ ID NO. 2 is divided at amino acid position 930 and NpuDnaE Intein is used; the N-terminal coding sequence of OTOF is ligated and fused with the N-terminal coding sequence of NpuDnaE Intein, and the first nucleotide sequence is constructed using the pAAV-CMV plasmid as a vector; the C-terminal coding sequence of NpuDnaE Intein is ligated and fused with the C-terminal coding sequence of OTOF, and the second nucleotide sequence is constructed using the pAAV-CMV plasmid as a vector; or The OTOF shown in SEQ ID NO. 2 is divided at amino acid position 1130 and NpuDnaE Intein is used; the N-terminal coding sequence of OTOF is ligated and fused with the N-terminal coding sequence of NpuDnaE Intein, and the pAAV-CMV plasmid is used as a vector to construct a first nucleotide sequence; the C-terminal coding sequence of NpuDnaE Intein is ligated and fused with the C-terminal coding sequence of OTOF, and the pAAV-CMV plasmid is used as a vector to construct a second nucleotide sequence; or The OTOF shown in SEQ ID NO. 2 is divided at amino acid position 827 and RmaDnaB Intein is used; the N-terminal coding sequence of OTOF is ligated and fused with the N-terminal coding sequence of RmaDnaB Intein, and the pAAV-CMV plasmid is used as a vector to construct a first nucleotide sequence; the C-terminal coding sequence of RmaDnaB Intein is ligated and fused with the C-terminal coding sequence of OTOF, and the pAAV-CMV plasmid is used as a vector to construct a second nucleotide sequence; or The OTOF shown in SEQ ID NO. 2 is divided at amino acid position 930 and RmaDnaB Intein is used; the N-terminal coding sequence of OTOF is ligated and fused with the N-terminal coding sequence of RmaDnaB Intein, and the pAAV-CMV plasmid is used as a vector to construct a first nucleotide sequence; the C-terminal coding sequence of RmaDnaB Intein is ligated and fused with the C-terminal coding sequence of OTOF, and the pAAV-CMV plasmid is used as a vector to construct a second nucleotide sequence; or The OTOF shown in SEQ ID NO.2 was divided at amino acid position 1130 and RmaDnaB Intein was used; the N-terminal coding sequence of OTOF was connected and fused with the N-terminal coding sequence of RmaDnaB Intein, and the first nucleotide sequence was constructed using the pAAV-CMV plasmid as a vector; the C-terminal coding sequence of RmaDnaB Intein was connected and fused with the C-terminal coding sequence of OTOF, and the second nucleotide sequence was constructed using the pAAV-CMV plasmid as a vector.

8. An adeno-associated virus packaging vector system, characterized in that: The packaging vector system comprises: a dual vector system for expressing OTOF protein according to any one of claims 1 to 7, carrying AAV rep 、 cap The gene vector and the helper virus vector are packaged into AAV vectors.

9. The adeno-associated virus packaging vector system according to claim 8, wherein: The AAV rep 、 cap The gene vector is selected from AAV1, AAV2, AAV5, AAV8, AAV9, Anc80, PHP.eB, AAV-DJ or AAVrh.10 vector; the helper virus vector is pHelper plasmid.

10. An in vitro packaging method for adeno-associated virus, characterized in that: The adeno-associated virus packaging vector system according to claim 8 is transferred into host cells for packaging.

11. The in vitro packaging method of an adeno-associated virus according to claim 10, characterized in that: The host cell is selected from Hela-S3, HEK-293, HEK-293T, HEK-293FT, A549 or Sf9 cells.

12. The adeno-associated virus obtained by packaging according to the method of claim 10.

13. Use of the dual-vector system for expressing the OTOF protein according to any one of claims 1 to 7 or the adeno-associated virus according to claim 12 in the preparation of a preparation for treating deafness, hearing loss, or hearing dysfunction caused by OTOF gene mutation or loss of function or dysfunction of its encoded protein.

14. A preparation for treating deafness, hearing loss, or hearing dysfunction caused by OTOF gene mutation or loss of function or dysfunction of its encoded protein, prepared by the dual vector system expressing OTOF protein according to any one of claims 1 to 7 or the adeno-associated virus according to claim 12.

15. The preparation according to claim 14, wherein The preparation further comprises one or more of neutral salt buffer, acidic salt buffer, alkaline salt buffer, glucose, mannose, mannitol, protein, polypeptide and amino acid, antibiotic, chelating agent, preservative, nanoparticle, liposome or positive lipid particle.

16. The preparation according to claim 14, wherein The preparation also contains an adjuvant.

17. The preparation according to claim 14, wherein Administration is by injection, including injection into the round window of the cochlea, oval window, semicircular canal, and utricle; Single or multiple administrations for life, with a total dose of 1×10 9 -1×10 13 A viral genome.

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