CN116925239B - Composition and method for expressing Otof gene by dual vector system - Google Patents

Abstract

The present invention relates to compositions and methods for dual vector system expression Otof genes, and in particular provides fusion polypeptides comprising a portion of an otoferlin protein and a portion of an intein system. The present invention delivers the gene encoding Otof into cells via a recombinant AAV dual vector system.

Description

Composition and method for expressing Otof gene by dual vector system

Technical Field

The present invention relates to the field of biomolecules, and in particular to a splicing method and application of a fusion polypeptide in a dual-carrier system.

Background Art

Hearing loss is one of the most common sensory impairment diseases in humans and can be caused by environmental and genetic factors. Some people may be born with hearing loss, while others may slowly lose their hearing over time. Hearing loss affects more than 1.5 billion people worldwide, about one-fifth of the world's population, of which 470 million suffer from disabling hearing loss (moderate and above hearing impairment), and 34 million children suffer from deafness or hearing loss. It is estimated that by 2050, more than 700 million people may suffer from disabling hearing loss. The number of people with hearing impairment in my country accounts for about 16% of the total population, and patients with disabling hearing impairment account for about 5% of the total population. The incidence of deafness in newborns in my country is 1% to 3.47%, that is, there are 1-3 deaf children for every 1,000 newborns, and about 30,000 deaf children are born each year. The environmental causes of hearing loss are mainly related to the use of ototoxic drugs, pregnancy infections, neonatal hypoxia and radiation exposure and other environmental factors or certain complications. Genetic factors are mainly due to individual deafness gene defects, which lead to different degrees of hearing loss. The pathogenic genes will be passed on to the next generation through different genetic methods and can occur at any age. Deafness caused by genetic factors accounts for about 60%. More than 200 genes have been found to be related to deafness, involving more than 1,500 pathogenic variants. So far, there is no drug that can be used to treat hereditary deafness in clinical practice. Hereditary deafness is divided into syndromic deafness (SHL) and non-syndromic deafness (NSHI) according to whether it is accompanied by abnormalities or lesions of extra-auricular tissues. Syndromic deafness accounts for about 30% of hereditary deafness. In addition to hearing impairment, it is also accompanied by many other manifestations. Non-syndromic deafness accounts for about 70% to 80% of hereditary deafness. There are only symptoms of hearing loss, and there is no hereditary damage to other organs.

Hereditary non-syndromic deafness is mainly a monogenic disease, which can be divided into autosomal dominant deafness (DFNA), autosomal recessive deafness (DFNB), X-linked deafness (DFNX), Y-linked deafness (DFNY), mitochondrial inheritance and epigenetic inheritance according to its inheritance mode. DFNA accounts for about 18% of hereditary deafness, DFNB accounts for about 80%, DFNX accounts for about 1%, mitochondrial inheritance <1%, and DFNY and epigenetic inheritance are only reported in individual cases. It is reported that there are 110 genes related to non-syndromic deafness that have been identified, including 45 DFNA-related genes, 70 DFNB-related genes, 10 genes related to both DFNA and DFNB (CLOL11A2, GJB2, GJB6, MYO3A, MYO6, MYO7A, PTPRQ, TCB1D24, TECTA, TMC1), and 5 DFNX-related genes. Each type is described by the naming convention for the deafness gene, for example DFNA1 is the first autosomal dominant deafness type discovered.

DFNB9 hearing impairment is a common form of congenital hereditary deafness. Patients with DFNB9 hearing impairment usually have severe sensorineural hearing loss, which is caused by a gene mutation in the Otof gene encoding the otoferlin protein. The otoferlin protein encoded by the Otof gene is a transmembrane protein belonging to the Ferlin protein family. The Otoerlin protein contains six calcium ion binding domains and two Fer domains, and plays an important role in cell membrane ion exchange, signal transduction, and neurotransmitter release. The Otof gene is the first gene discovered to be associated with non-syndromic auditory neurophy spectrum disorder (ANDS). Auditory neurophy spectrum disorder is also called auditory neuropathy. Otoferlin protein is a protein that plays a key role in the transmission of sound information at the inner hair cell synapse. More than 56% of non-syndromic ANSD is caused by mutations in the Otof gene, and approximately 200,000 people worldwide are affected. At present, patients with Otof gene mutations can only benefit from traditional hearing aids and the clinical treatment effect is poor. There is currently no approved drug treatment plan.

Mutations in the Otof gene can cause congenital prelingual severe hereditary deafness, which is inherited in an autosomal recessive manner. Patients with Otof gene mutations often suffer from severe bilateral deafness from birth, and the auditory brainstem response (ABR) is absent or highly abnormal, which is currently the most common cause of hearing loss. At present, most of the methods to solve deafness are physical methods such as hearing aids, vibrating sound bridges and cochlear implants. Although patients can achieve different degrees of improvement in hearing function, there are large individual differences and great limitations and weaknesses. For example, the treatment effect is limited, frequency sensitivity, speech discrimination and perception difficulties in noisy environments, and the device needs to be used with caution. About 300,000 patients worldwide have received cochlear implants, but this only accounts for a small part of all deaf patients. For most patients, there is still an urgent need for fundamental and effective drug treatments, and there is no approved treatment so far. This is an area with serious unmet needs.

Summary of the invention

The present invention provides a dual-vector system, the purpose of which is to transfer the Otof gene into the inner hair cells of the cochlea through the dual vectors to express normal functional otoferlin protein to treat Otof-mediated hearing loss.

In a first aspect, the present invention provides a fusion polypeptide comprising a portion of an otoferlin protein and a portion of an intein system.

In one or more embodiments, the otoferlin protein is set forth in SEQ ID NO: 1, or a sequence having at least 90% identity thereto.

In one or more embodiments, the fusion polypeptide is a first fusion polypeptide or a second fusion polypeptide.

In one or more embodiments, the first fusion polypeptide comprises or consists of amino acids 1 to n of the otoferlin protein shown in SEQ ID NO:1 and the N-terminal fragment (N-intein) of the intein, wherein n is any integer from 750 to 1250, preferably n is any integer from 790 to 1200, and more preferably n is any integer from 797 to 1169.

In one or more embodiments, the second fusion polypeptide comprises or consists of amino acids (n+1) to 1997 of the otoferlin protein shown in SEQ ID NO:1 and the C-terminal fragment (C-intein) of the intein, wherein n is any integer from 750 to 1250, preferably n is any integer from 790 to 1200, and more preferably n is any integer from 797 to 1169.

In one or more embodiments, n is any one or more of 797, 819, 844, 854, 866, 872, 878, 882, 887, 900, 904, 918, 926, 950, 970, 976, 977, 980, 992, 994, 995, 1001, 1003, 1005, 1040, 1050, 1064, 1083, 1161, 1169.

In one or more embodiments, the N-terminal fragment of the intein (N-intein) is split intein-N, as shown in the sequence of SEQ ID NO:2.

In one or more embodiments, the C-terminal fragment of the intein (C-intein) is a split intein-C, as shown in the sequence of SEQ ID NO:3.

In one or more embodiments, the first fusion polypeptide has a sequence as shown in any one of SEQ ID NOs: 5-16, or a variant thereof having at least 80% sequence identity thereto.

In one or more embodiments, the second fusion polypeptide has the sequence shown in any one of SEQ ID NOs: 17-28, or a variant thereof having at least 80% sequence identity thereto.

The present invention also provides a polypeptide composition, comprising the first fusion polypeptide and the second fusion polypeptide described in the first aspect of the present invention.

The present invention also provides a polynucleotide comprising a sequence selected from the following:

(1) a nucleic acid sequence encoding a fusion polypeptide or polypeptide composition as described in any embodiment of the present invention,

(2) a variant having at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity with (1),

(3) A complementary sequence of (1) or (2).

In one or more embodiments, the polynucleotide encoding the first fusion polypeptide has a sequence as shown in any one of SEQ ID NOs: 29-40, or a variant thereof having at least 80% sequence identity. In one or more embodiments, the polynucleotide encoding the second fusion polypeptide has a sequence as shown in any one of SEQ ID NOs: 41-52, or a variant thereof having at least 80% sequence identity.

The present invention also provides a nucleic acid construct, wherein:

(1) expressing a fusion polypeptide or polypeptide composition as described in any embodiment of the present invention, and/or

(2) A sequence comprising a polynucleotide described herein.

In one or more embodiments, the nucleic acid construct is an expression cassette, and the polynucleotides encoding the first fusion polypeptide and the second fusion polypeptide are both located in the expression cassette.

In one or more embodiments, the nucleic acid construct is two expression cassettes, respectively comprising polynucleotides encoding the first fusion polypeptide and the second fusion polypeptide.

In one or more embodiments, the nucleic acid construct is a vector, such as a cloning vector, an integration vector, or an expression vector, preferably an AAV vector.

In one or more embodiments, the nucleic acid construct is two AAV vectors, respectively comprising a polynucleotide encoding a first fusion polypeptide and a second fusion polypeptide, and one or more inverted terminal repeat (ITR) sequences flanking the polynucleotides. The polynucleotides can be in the form of a single-stranded (ss) or self-complementary (sc) AAV nucleic acid vector, for example, in the form of a single-stranded or self-complementary recombinant viral genome.

In one or more embodiments, the nucleic acid construct further comprises an element for expressing the polynucleotide contained therein. In one or more embodiments, the polynucleotide is operably linked to a constitutive promoter.

In one or more embodiments, the nucleic acid construct has the sequence shown in SEQ ID NO:4.

The present invention also provides an rAAV vector system, comprising two AAV vectors and a nucleic acid construct encoding genes required by the AAV virus, wherein the two AAV vectors respectively contain polynucleotides encoding a first fusion polypeptide and a second fusion polypeptide, and the first fusion polypeptide and the second fusion polypeptide are as described in the first aspect of the present invention.

In one or more embodiments, the desired genes of the AAV virus include one or more selected from the group consisting of: Rep, Cap, E2A, E4, and VA genes.

In one or more embodiments, the AAV vector system comprises an Anc80L65 plasmid, a helper plasmid, and the AAV vector.

The present invention also provides an rAAV virus particle comprising the polynucleotide described in any embodiment of the present invention.

In one or more embodiments, the rAAV virus particles are selected from one or more of rAAV1, rAAV2, rAAV4, rAAV5, rAAV6, rAAV7, rAAV8, rAAV9, rAAV10, rAAV11, rAAV12, rAAV13, rAAVPHP.B or rAAVrh74.

The present invention also provides a host cell, wherein:

(1) comprising, expressing and/or secreting a fusion polypeptide or polypeptide composition as described herein,

(2) comprising a polynucleotide, a nucleic acid construct or an AAV vector system as described in any embodiment herein,

(3) The polynucleotide described in any embodiment of the present invention is integrated into the chromosome.

In one or more embodiments, the host cell is a HEK293 cell.

The third aspect of the present invention provides a method for preparing otoferlin protein, comprising contacting a first fusion polypeptide and a second fusion polypeptide under conditions suitable for intein splicing, wherein the first fusion polypeptide and the second fusion polypeptide are as described in the first aspect of the present invention.

In one or more embodiments, the method comprises the step of incubating the host cell described in any embodiment herein under conditions suitable for intein splicing.

In one or more embodiments, the method comprises the following steps: introducing the AAV vector system described in any embodiment herein into a cell, and incubating the host cell under conditions suitable for the expression of the first fusion polypeptide and the second fusion polypeptide.

In one or more embodiments, the cell is an animal cell, preferably a HEK293 cell.

In one or more embodiments, the method further comprises the step of intein-mediated trans-splicing of the protein.

The present invention also provides the use of the fusion polypeptide, polypeptide composition, polynucleotide, nucleic acid construct, and host cell described in any embodiment of the present invention in the preparation of a drug for treating a disease caused by an Otof gene defect.

In one or more embodiments, the disease is DFNB9 hearing impairment.

The present invention also provides a pharmaceutical composition comprising a pharmaceutically acceptable excipient and one or more selected from the following: the fusion polypeptide, polypeptide composition, polynucleotide, nucleic acid construct, rAAV vector system, rAAV virus particle, host cell described in any embodiment of the present invention.

The present invention also provides a method for treating hereditary deafness, comprising administering to a patient in need thereof a therapeutically effective amount of the fusion polypeptide, polypeptide composition, polynucleotide, nucleic acid construct, host cell or pharmaceutical composition of any embodiment of the present invention. Preferably, the hereditary deafness is DFNB9 hereditary deafness.

Beneficial effects of the present invention:

The present invention constructs the 5'-end and 3'-end of the Otoferlin gene into two different rAAV vectors, and delivers the transgene encoding Otof to the inner hair cells of the cochlea through the recombinant AAV dual-vector system, and finally achieves the expression of Otoferlin protein in the hair cells to treat Otof-mediated hearing loss. This means that gene replacement therapy can play a role in preventing or treating hereditary deafness caused by Otoferlin mutations.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 shows a schematic diagram of double AAV intein-mediated trans-splicing of OTOF protein using inverted terminal repeats, promoter and polyadenylation sequences.

Figure 2 shows the vector backbone elements in the dual vector system, wherein the ITR sequence is 1-141, the CMV Promoter sequence is 169-752, the Kozak sequence is 792-797, the EGFP sequence is 801-1517, the WPRE sequence is 1536-2124, the SV40 PolyA sequence is 2131-2252, the ITR sequence is 2290-2430, the f1 Ori sequence is 2505-2960, the kana resistance sequence is 3242-4156, and the Ori sequence is 4327-4515.

FIG3 shows a schematic diagram of the cleavage sites of the OTOF full-length protein split into two AAV vectors, and Western blotting was used for in vitro screening verification.

Figure 4 shows that HEK-293T cells co-transfected with both the N-terminal part (N-Otof) and the C-terminal part (C-Otof) encoding otoferlin protein resulted in the expression of full-length OTOF. A-D, expression of full-length otoferlin protein at different cleavage sites.

Figure 5 is the auditory brainstem response (ABR) results of the treated adult Otof-/- knockout mouse model, showing that the ABR threshold of the treated adult Otof-/- knockout mouse model was lowered, and some hearing frequency bands were significantly restored, which was close to the hearing threshold of WT mice.

Figure 6 is the immunofluorescence result of the treated adult Otof-/- knockout mouse model. After the audiometry, the mice were killed and the cochlea was taken for basilar membrane staining. The immunofluorescence results showed that the dual AAV system of the present application achieved the expression of otoferlin protein in the mouse cochlea to varying degrees. The dual AAV system of the present application achieved the expression of otoferlin protein in the mouse cochlea, restored the release of synaptic vesicles in the inner ear hair cells, and restored hearing in both ears.

DETAILED DESCRIPTION

Unless otherwise defined, the practice of the present invention will employ conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. These techniques are fully explained in the literature, such as Molecular Cloning: A Laboratory Manual, Second Edition (Sambrook et al., 1989); Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Animal Cell Culture (R. I. Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Current Protocols in Molecular Biology (F. M. Ausubel et al., ed., 1987 edition and periodic updates thereof); PCR: The Polymerase Chain Reaction (Mullis et al., ed., 1994); A Practical Guide to Molecular Cloning (Perbal Bernard V., 1988); Phage Display: A Laboratory Manual (Barbas et al., 2001).

The term "disease and/or condition" refers to a physical state of a subject associated with the diseases and/or conditions described herein.

The term "subject" or "patient" may refer to patients or other animals, especially mammals, such as humans, dogs, monkeys, cows, horses, etc., who receive the pharmaceutical composition of the present invention to treat, prevent, improve and/or alleviate the diseases or conditions of the present invention.

Fusion peptide

The present invention first provides a fusion polypeptide, comprising a portion of otoferlin protein and a portion of an intein system.

Herein, the term intein is an insertion sequence located in the host protein. The intein gene is not an independent gene and must be inserted into the extein gene to replicate and transcribe. It can be cut from the precursor protein and the exteins on both sides are connected to form a mature protein. Its corresponding nucleotide sequence is embedded in the nucleic acid sequence corresponding to the host protein, exists in the same open reading frame (ORF) as the host protein gene, and is synchronously transcribed and translated with the host protein gene. After the protein precursor is formed by translation, the intein is cut from the host protein to form a mature active protein. The amino acid sequence encoding the splicing donor (for example, the N-terminal fragment of the intein (N-intein), also known as the split intein-N) is shown in SEQ ID NO: 2. The amino acid sequence encoding the splicing acceptor (for example, the C-terminal fragment of the intein (C-intein), also known as the split intein-C) is shown in SEQ ID NO: 3.

The host protein herein is otoferlin protein, as shown in SEQ ID NO: 1. Therefore, the fusion polypeptide includes a first fusion polypeptide or a second fusion polypeptide. The first fusion polypeptide comprises or consists of amino acids 1 to n and an N-terminal fragment (N-intein) of the otoferlin protein shown in SEQ ID NO: 1, wherein n is any integer from 750 to 1250, preferably n is any integer from 790 to 1200, and more preferably n is any integer from 797 to 1169. The second fusion polypeptide comprises or consists of amino acids (n+1) to 1997 and an C-terminal fragment (C-intein) of the otoferlin protein shown in SEQ ID NO: 1, wherein n is any integer from 750 to 1250, preferably n is any integer from 790 to 1200, and more preferably n is any integer from 797 to 1169. In an exemplary embodiment, in one or more embodiments, n is any one or more of 797, 819, 844, 854, 866, 872, 878, 882, 887, 900, 904, 918, 926, 950, 970, 976, 977, 980, 992, 994, 995, 1001, 1003, 1005, 1040, 1050, 1064, 1083, 1161, 1169. In one or more embodiments, n is any one or more of 797, 844, 854, 866, 872, 882, 900, 926, 950, 970, 977, 980, 992, 994, 1003, 1064, 1161, 1169.

The present invention also provides a polypeptide composition, comprising the first fusion polypeptide and the second fusion polypeptide. In one or more embodiments, the first fusion polypeptide comprises or consists of the amino acids 1 to n and the N-terminal fragment (N-intein) of the otoferlin protein shown in SEQ ID NO: 1, wherein n is any integer from 750 to 1250, preferably n is any integer from 790 to 1200, and more preferably n is any integer from 797 to 1169. The second fusion polypeptide comprises or consists of the amino acids (n+1) to 1997 and the C-terminal fragment (C-intein) of the otoferlin protein shown in SEQ ID NO: 1, wherein n is any integer from 750 to 1250, preferably n is any integer from 790 to 1200, and more preferably n is any integer from 797 to 1169. In an exemplary embodiment, in one or more embodiments, n is any one or more of 797, 819, 844, 854, 866, 872, 878, 882, 887, 900, 904, 918, 926, 950, 970, 976, 977, 980, 992, 994, 995, 1001, 1003, 1005, 1040, 1050, 1064, 1083, 1161, 1169. In one or more embodiments, n is any one or more of 797, 844, 854, 866, 872, 882, 900, 926, 950, 970, 977, 980, 992, 994, 1003, 1064, 1161, 1169. In one or more embodiments, the first fusion polypeptide has a sequence as shown in SEQ ID NO:5 or a variant thereof having at least 80% sequence identity, and the second fusion polypeptide has a sequence as shown in SEQ ID NO:17 or a variant thereof having at least 80% sequence identity. In one or more embodiments, the first fusion polypeptide has a sequence as shown in SEQ ID NO:6 or a variant thereof having at least 80% sequence identity, and the second fusion polypeptide has a sequence as shown in SEQ ID NO:18 or a variant thereof having at least 80% sequence identity. In one or more embodiments, the first fusion polypeptide has a sequence as shown in SEQ ID NO:7 or a variant thereof having at least 80% sequence identity, and the second fusion polypeptide has a sequence as shown in SEQ ID NO:19 or a variant thereof having at least 80% sequence identity. In one or more embodiments, the first fusion polypeptide has a sequence as shown in SEQ ID NO:8 or a variant thereof having at least 80% sequence identity, and the second fusion polypeptide has a sequence as shown in SEQ ID NO:20 or a variant thereof having at least 80% sequence identity. In one or more embodiments, the first fusion polypeptide has a sequence as shown in SEQ ID NO:9 or a variant thereof having at least 80% sequence identity, and the second fusion polypeptide has a sequence as shown in SEQ ID NO:21 or a variant thereof having at least 80% sequence identity. In one or more embodiments, the first fusion polypeptide has a sequence as shown in SEQ ID NO:10 or a variant thereof having at least 80% sequence identity, and the second fusion polypeptide has a sequence as shown in SEQ ID NO:22 or a variant thereof having at least 80% sequence identity. In one or more embodiments, the first fusion polypeptide has a sequence as shown in SEQ ID NO:11 or a variant thereof having at least 80% sequence identity, and the second fusion polypeptide has a sequence as shown in SEQ ID NO:23 or a variant thereof having at least 80% sequence identity. In one or more embodiments, the first fusion polypeptide has a sequence as shown in SEQ ID NO:12 or a variant thereof having at least 80% sequence identity, and the second fusion polypeptide has a sequence as shown in SEQ ID NO:24 or a variant thereof having at least 80% sequence identity. The first fusion polypeptide has a sequence as shown in SEQ ID NO: 13 or a variant thereof having at least 80% sequence identity, and the second fusion polypeptide has a sequence as shown in SEQ ID NO: 25 or a variant thereof having at least 80% sequence identity. In one or more embodiments, the first fusion polypeptide has a sequence as shown in SEQ ID NO: 14 or a variant thereof having at least 80% sequence identity, and the second fusion polypeptide has a sequence as shown in SEQ ID NO: 26 or a variant thereof having at least 80% sequence identity. The first fusion polypeptide has a sequence as shown in SEQ ID NO: 15 or a variant thereof having at least 80% sequence identity, and the second fusion polypeptide has a sequence as shown in SEQ ID NO: 27 or a variant thereof having at least 80% sequence identity. In one or more embodiments, the first fusion polypeptide has a sequence as shown in SEQ ID NO: 16 or a variant thereof having at least 80% sequence identity, and the second fusion polypeptide has a sequence as shown in SEQ ID NO: 28 or a variant thereof having at least 80% sequence identity.

In some embodiments, the sequence of the variant of the present invention may have at least 95%, 96%, 97%, 98% or 99% identity to its source sequence. The sequence identity of the present invention can be measured using sequence analysis software. For example, the computer program BLAST, especially BLASTP or TBLASTN, using default parameters is used. The present invention also includes molecules having antibody heavy chain variable regions with CDRs, as long as their CDRs have more than 90% (preferably more than 95%, and most preferably more than 98%) homology with the CDRs identified herein.

Nucleotide sequence

The present invention provides a polynucleotide comprising: a nucleic acid sequence encoding the fusion polypeptide or polypeptide composition described herein or a complementary sequence thereof.

In one or more embodiments, the nucleic acid sequence encoding the first fusion polypeptide is a nucleotide sequence encoding the N-terminal portion of the target protein and a splicing donor sequence N-intein sequence (e.g., N-Otof-N-intein). Preferably, its nucleic acid sequence is a sequence as shown in any one of SEQ ID NOs: 29-40 or a variant thereof having at least 80% sequence identity.

In one or more embodiments, the nucleic acid sequence encoding the second fusion polypeptide is a nucleotide sequence encoding the C-terminal portion of the target protein and a splicing donor sequence C-intein sequence (e.g., C-intein-C-Otof). Preferably, its nucleic acid sequence is a sequence as shown in any one of SEQ ID NOs: 41-52 or a variant thereof having at least 80% sequence identity.

In some embodiments, the nucleic acid sequence encoding the first fusion polypeptide has a sequence as shown in SEQ ID NO:29 or a variant having at least 80% sequence identity therewith, and the nucleic acid sequence encoding the second fusion polypeptide has a sequence as shown in SEQ ID NO:41 or a variant having at least 80% sequence identity therewith. In some embodiments, the nucleic acid sequence encoding the first fusion polypeptide has a sequence as shown in SEQ ID NO:30 or a variant having at least 80% sequence identity therewith, and the nucleic acid sequence encoding the second fusion polypeptide has a sequence as shown in SEQ ID NO:42 or a variant having at least 80% sequence identity therewith. In some embodiments, the nucleic acid sequence encoding the first fusion polypeptide has a sequence as shown in SEQ ID NO:31 or a variant having at least 80% sequence identity therewith, and the nucleic acid sequence encoding the second fusion polypeptide has a sequence as shown in SEQ ID NO:43 or a variant having at least 80% sequence identity therewith. In some embodiments, the nucleic acid sequence encoding the first fusion polypeptide has a sequence as shown in SEQ ID NO:6832 or a variant having at least 80% sequence identity therewith, and the nucleic acid sequence encoding the second fusion polypeptide has a sequence as shown in SEQ ID NO:44 or a variant having at least 80% sequence identity therewith. In some embodiments, the nucleic acid sequence encoding the first fusion polypeptide has a sequence as shown in SEQ ID NO:33 or a variant having at least 80% sequence identity therewith, and the nucleic acid sequence encoding the second fusion polypeptide has a sequence as shown in SEQ ID NO:45 or a variant having at least 80% sequence identity therewith. In some embodiments, the nucleic acid sequence encoding the first fusion polypeptide has a sequence as shown in SEQ ID NO:34 or a variant having at least 80% sequence identity therewith, and the nucleic acid sequence encoding the second fusion polypeptide has a sequence as shown in SEQ ID NO:46 or a variant having at least 80% sequence identity therewith. In some embodiments, the nucleic acid sequence encoding the first fusion polypeptide has a sequence as shown in SEQ ID NO:35 or a variant having at least 80% sequence identity therewith, and the nucleic acid sequence encoding the second fusion polypeptide has a sequence as shown in SEQ ID NO:47 or a variant having at least 80% sequence identity therewith. In some embodiments, the nucleic acid sequence encoding the first fusion polypeptide has a sequence as shown in SEQ ID NO:36 or a variant having at least 80% sequence identity therewith, and the nucleic acid sequence encoding the second fusion polypeptide has a sequence as shown in SEQ ID NO:48 or a variant having at least 80% sequence identity therewith. In some embodiments, the nucleic acid sequence encoding the first fusion polypeptide has a sequence as shown in SEQ ID NO:37 or a variant having at least 80% sequence identity therewith, and the nucleic acid sequence encoding the second fusion polypeptide has a sequence as shown in SEQ ID NO:49 or a variant having at least 80% sequence identity therewith. In some embodiments, the nucleic acid sequence encoding the first fusion polypeptide has a sequence as shown in SEQ ID NO:38 or a variant having at least 80% sequence identity therewith, and the nucleic acid sequence encoding the second fusion polypeptide has a sequence as shown in SEQ ID NO:50 or a variant having at least 80% sequence identity therewith. In some embodiments, the nucleic acid sequence encoding the first fusion polypeptide has a sequence as shown in SEQ ID NO:39 or a variant having at least 80% sequence identity therewith, and the nucleic acid sequence encoding the second fusion polypeptide has a sequence as shown in SEQ ID NO:51 or a variant having at least 80% sequence identity therewith. In some embodiments, the nucleic acid sequence encoding the first fusion polypeptide has a sequence as shown in SEQ ID NO:40 or a variant having at least 80% sequence identity therewith, and the nucleic acid sequence encoding the second fusion polypeptide has a sequence as shown in SEQ ID NO:52 or a variant having at least 80% sequence identity therewith.

As will be appreciated by those skilled in the art, due to the degeneracy of the genetic code, a very large number of nucleic acids can be produced, all of which encode the fusion polypeptides of the present invention. Thus, once a specific amino acid sequence has been identified, one skilled in the art can produce any number of different nucleic acids by simply modifying the sequence of one or more codons in a manner that does not alter the amino acid sequence of the encoded protein. Therefore, the present invention also relates to polynucleotides that hybridize with the above-mentioned polynucleotide sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that can hybridize with the polynucleotides of the present invention under stringent conditions. In the present invention, "stringent conditions" refers to: (1) hybridization and elution at relatively low ionic strength and relatively high temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) the addition of a denaturant during hybridization, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42°C, etc.; or (3) hybridization occurs only when the identity between the two sequences is at least 90%, preferably at least 95%. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.

The full-length nucleic acid sequence of the present invention or its fragments can usually be obtained by PCR amplification, recombination or artificial synthesis. A feasible method is to synthesize the relevant sequence by artificial synthesis, especially when the fragment length is short. Usually, by synthesizing multiple small fragments first and then connecting them, a very long sequence fragment can be obtained. In addition, the coding sequence of the heavy chain and the expression tag (such as 6His) can be fused together to form a fusion protein.

Once the relevant sequence is obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the host cells after proliferation by conventional methods. The biomolecules (nucleic acids, proteins, etc.) involved in the present invention include biomolecules in isolated form. At present, the DNA sequence encoding the protein of the present invention (or its fragment, or its derivative) can be obtained completely by chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequence of the present invention by chemical synthesis.

The present invention also provides a nucleic acid construct (such as an expression cassette or vector) comprising at least one of the above nucleotides. The nucleic acid construct contains a promoter, a sequence encoding a fusion polypeptide, and may also contain other elements required for expression, including but not limited to a poly A signal sequence.

In the case of known nucleotide sequence, each nucleotide molecule can be prepared by the method routine in this area, and the corresponding vector is constructed. Recombinant vectors can be constructed using methods well known to those skilled in the art, referring to, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory), Ausubel et al. (1989, Short Protocols in Molecular Biology, Wiley) or the technology described in other standard textbooks. Alternatively, the polynucleotide and the vector can be reconstructed into a liposome for delivery to a target cell. The vector containing the polynucleotide of the present invention can be transferred to a host cell by a well-known method, and it varies according to the type of the cell host. For example, calcium chloride transfection is generally used for prokaryotic cells, and calcium phosphate treatment or electroporation can be used for other cell hosts, referring to Sambrook et al. (see above).

The vectors described herein contain the polynucleotides or expression cassettes described herein. The vectors may be plasmids, cosmids, viruses, or viral vectors. The vectors include cloning vectors, integration vectors, and may also be expression vectors. In addition to the nucleotide molecules described herein, the expression vectors generally contain other elements commonly contained in vectors, such as multiple cloning sites, resistance genes, replication initiation sites, and the like. In one or more embodiments, the recombinant expression vector uses an AAV series vector as a backbone.

Expression vectors typically contain sequences for plasmid maintenance and for cloning and expressing exogenous nucleotide sequences. The sequences (collectively referred to as "flanking sequences" in certain embodiments) typically include one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a complete intron sequence containing donor and acceptor splice sites, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a multilinker region for inserting a nucleic acid encoding a protein to be expressed, and a selectable marker element.

The vector may optionally contain a "tag" encoding sequence, i.e., an oligonucleotide molecule located at the 5' or 3' end of the fusion polypeptide; the oligonucleotide sequence encodes polyhistidine (such as 6His) or another "tag", such as FLAG, HA or myc. This tag is typically fused to the polypeptide when the polypeptide is expressed, and can serve as a means for affinity purification or detection of the protein from the host cell. Affinity purification can be accomplished, for example, by column chromatography using antibodies against this tag as an affinity matrix. The tag can optionally be subsequently removed from the purified protein by various means, such as using certain peptidases for cleavage.

The flanking sequences may be homologous (i.e., from the same species and/or strain as the host cell), heterologous (i.e., from a species other than the host cell species or strain), hybrid (i.e., a combination of flanking sequences from more than one source), synthetic, or natural. Likewise, the source of the flanking sequences may be any prokaryotic or eukaryotic organism, any vertebrate or invertebrate organism, or any plant, provided that the flanking sequences function in and can be activated by the host cell machinery.

Selectable marker genes encode a protein that is necessary for the survival and growth of host cells grown in selective media. Typical selectable marker genes encode (a) resistance to antibiotics or other toxins (e.g., ampicillin, tetracycline, or kanamycin for prokaryotic host cells); (b) complement the auxotrophic deficiency of the cell; or (c) provide proteins that are not available from complexes or defined culture media for important nutrients. Specific selectable markers are kanamycin resistance genes, ampicillin resistance genes, and tetracycline resistance genes. Advantageously, the neomycin resistance gene can also be used for selection in prokaryotic and eukaryotic host cells.

The expression and cloning vectors of the present invention will typically contain a promoter that is recognized by the host organism and operably linked to a molecule encoding a protein or polypeptide. A promoter is a non-transcribed sequence located upstream of the start codon of a structural gene (usually within about 100 to 1000 bp) that controls the transcription of the structural gene. Suitable promoters for use in various hosts are also well known in the art. For example, suitable promoters for use with mammalian host cells include, but are not limited to, promoters obtained from viral genomes such as polyoma virus, fowlpox virus, adenovirus (such as adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus, and most preferably simian virus 40 (SV40). Other suitable mammalian promoters include heterologous mammalian promoters, such as heat shock promoters and actin promoters.

In some embodiments, the fusion polypeptide of the present invention is expressed by a recombinant AAV (rAAV) vector system. The term "adeno-associated virus" or "AAV" as used herein includes members of the virus class associated with the name, which belongs to the Dependoparvovirus genus, Parvoviridae family. Adeno-associated virus is a single-stranded DNA virus that grows only in cells, where certain functions are provided by co-infected helper viruses. General information and reviews of AAV can be found, for example, in Carter, 1989, Handbook of Parvoviruses, Volume 1, pages 169-228. It is known that various serotypes of the virus are suitable for gene delivery, and the various serotypes are closely related in structure and function. All AAV serotypes apparently exhibit very similar replication characteristics mediated by homologous rep genes; and all carry three related capsid proteins. At least 13 sequentially numbered AAV serotypes are known in the art. Non-limiting exemplary serotypes that can be used for the methods disclosed herein include any of the 13 serotypes, such as AAV2, AAV8, and AAV9. AAV particles comprise, consist essentially of, or consist of the following three major viral proteins: VP1, VP2, and VP3. In one or more embodiments, AAV refers to serotype AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVPHP.B, or AAVrh74.

As used herein, "AAV vector" refers to a vector comprising, consisting essentially of, or consisting of one or more coding sequences for the fusion polypeptides herein and one or more AAV inverted terminal repeats (ITRs). Such AAV vectors can be replicated and packaged into infectious viral particles when present in a host cell that provides functionality of the rep and cap gene products; for example, by transfecting the host cell. In one or more embodiments, the AAV vector contains a promoter, at least one nucleic acid encoding at least one protein or RNA, and/or an enhancer and/or terminator packaged into the flanking ITRs of infectious AAV particles. The plasmid containing the AAV vector may also contain elements for preparation purposes, such as antibiotic resistance genes, etc.

As used herein, the term "viral capsid" or "capsid" refers to the protein shell or capsid of the virus particle. The function of the capsid is to encapsidate, protect, transport the viral genome, and release it into the host cell. The capsid is usually composed of oligomeric structural subunits of proteins ("capsid proteins"). As used herein, the term "encapsid" refers to being contained within the viral capsid. The viral capsid of AAV is composed of a mixture of three viral capsid proteins: VP1, VP2 and VP3.

"AAV virus particle" or "AAV particle" refers to a virus particle composed of at least one AAV capsid protein and an encapsidated polynucleotide AAV vector. Therefore, the production of AAV particles necessarily includes the production of AAV vectors, because such vectors are contained in AAV particles.

As used herein, the term "helper" with respect to a virus or plasmid refers to a virus or plasmid that is used to provide additional components necessary for replication and packaging of any of the AAV vectors disclosed herein. The components encoded by the helper virus may include any genes required for virion assembly, encapsidation, genome replication and/or packaging. For example, a helper virus or plasmid may encode an enzyme necessary for viral genome replication. Non-limiting examples of helper viruses and plasmids suitable for use with AAV constructs include pHELPer (plasmid), adenovirus (virus) or herpes virus (virus).

Methods for producing and using rAAV vectors are known in the art. Methods for producing rAAV particles and heterologous nucleic acids are also known in the art and are commercially available (see, e.g., US 2007/0015238 and US2012/0322861, which are incorporated herein by reference in their entirety). For example, a plasmid comprising a heterologous nucleic acid sequence can be combined with one or more auxiliary plasmids (e.g., comprising a rep gene (e.g., encoding Rep78, Rep68, Rep52, and Rep40) and a cap gene (encoding VP1, VP2, and/or VP3) and transfected or permanently integrated into a production cell line so that the rAAV particles can be packaged and subsequently purified.

In some embodiments, one or more helper plasmids include a first helper plasmid comprising a rep gene and a cap gene and a second helper plasmid comprising an E1a gene, an E1b gene, an E4 gene, an E2a gene, and a VA gene. In some embodiments, the rep gene is a rep gene derived from AAV2, and the cap gene is derived from AAV44.9. Helper plasmids and methods for preparing such plasmids are known in the art and are commercially available.

Host cells

The present invention also includes host cells expressing the fusion polypeptides described herein, or comprising the nucleotides or nucleic acid constructs described herein. Host cells can be used as recipients of vectors. Host cells can be "transfected" or "transformed," which refers to the process of transfection or transduction of exogenous nucleic acids into host cells. Transformed cells include primary subject cells and their progeny. The terms "engineered" and "recombinant" cells or host cells used herein often refer to cells into which exogenous nucleic acid sequences, such as vectors, have been introduced. Therefore, recombinant cells can be distinguished from naturally occurring cells that do not contain introduced recombinant nucleic acids.

Suitable host cells are as described above, including but not limited to Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (eg, HepG2), and the like.

As used herein, packaging cells (or helper cells) are cells for producing viral vectors. Production of recombinant AAV viral vectors requires trans-provided Rep and Cap proteins and gene sequences from adenovirus-assisted AAV replication. In some aspects, packaging/helper cells contain plasmids that are stably integrated into the cell genome. In other aspects, packaging cells can be transiently transfected. Typically, packaging cells are eukaryotic cells, such as mammalian cells or insect cells. In one or more embodiments, the cell for producing viral vectors is a HEK293 cell, including HEK293T, HEK293H, HEK293F, HEK293S, HEK293T/17, HEK293T/17SF, HEK293FT, HEK293SG, HEK293E, HEK293-6E, HEK293FTM, HEK293SGGD cells.

The nucleic acid construct/recombinant expression vector of the present invention can be transferred into the cell of interest. The method of transfer is a conventional method in the art, including but not limited to: viral transduction, microinjection, particle bombardment, gene gun transformation and electrotransformation, etc. In certain embodiments, electrotransformation is used to transfer the nucleic acid construct or recombinant expression vector. When the host cell is cultured under appropriate conditions, it synthesizes the fusion protein, which can then be collected from the culture medium (if the host cell secretes it into the culture medium) or directly collected from the host cell that produces it (if it is not secreted).

Protein preparation method

The present invention also provides a method for preparing otoferlin protein, comprising contacting a first fusion polypeptide and a second fusion polypeptide under conditions suitable for intein splicing. In one or more embodiments, the method comprises the following steps: introducing the AAV vector system described in any embodiment of the present invention into a cell, and incubating the host cell under conditions suitable for the expression of the first fusion polypeptide and the second fusion polypeptide. The duration of the intein splicing or incubation is not limited and can be determined by a person skilled in the art based on experience and needs, for example, at least 0.5h, preferably 0.5h-15 days. The temperature of the intein splicing or incubation is not limited, as long as the temperature is suitable for the survival of immune effector cells, for example, 25-37°C. The intein splicing or incubation can also be carried out under an ambient _CO2 of 0-20% (v/v).

Method for preparing AAV viral vector

A variety of methods can be used to prepare AAV viral vectors. In one or more embodiments, packaging is achieved by using a helper virus or a helper plasmid and a cell line. The helper virus or helper plasmid contains elements and sequences that promote the production of viral vectors. In some embodiments, the helper plasmid is stably integrated into the genome of the packaging cell line so that the packaging cell line does not need to be additionally transfected with the helper plasmid.

In one or more embodiments, the cell is a packaging cell line or a helper cell line. In one or more embodiments, the helper cell line is a eukaryotic cell; for example, a HEK 293 cell or a 293T cell. In one or more embodiments, the helper cell is a yeast cell or an insect cell.

The helper plasmid can comprise, for example, at least one viral helper DNA sequence derived from a replication-incompetent viral genome encoding trans-acting full virion proteins required for packaging of replication-incompetent AAV, and virion proteins for producing virions capable of packaging replication-incompetent AAV at high titers without producing replication-incompetent AAV.

Auxiliary plasmids for packaging AAV are known in the art, see, for example, U.S. Patent Publication No. 2004/0235174A1, which is incorporated herein by reference. As described herein, as a non-limiting example, the AAV auxiliary plasmid may contain the Ad5 genes E2A, E4 and VA controlled by respective native promoters or heterologous promoters as auxiliary viral DNA sequences. The AAV auxiliary plasmid may additionally contain an expression cassette for expressing a marker protein such as a fluorescent protein to allow easy detection of transfection of desired target cells.

Provided herein is a method for producing AAV particles, comprising transfecting a packaging cell line with any of the AAV helper plasmids disclosed herein; and any of the AAV vectors disclosed herein. In some embodiments, the AAV helper plasmid and the AAV vector are co-transfected into the packaging cell line. In some embodiments, the cell line is a mammalian cell line, such as a human embryonic kidney (HEK) 293 cell line. Provided herein is a cell comprising any of the AAV vectors and/or AAV particles disclosed herein.

The present invention also provides a method for introducing a target gene into a subject's cell, comprising contacting the cell with an effective amount of any AAV virus particle described herein, wherein the particle contains any AAV vector described herein comprising a fusion polypeptide encoding sequence.

Pharmaceutical composition

Also provided herein are pharmaceutical compositions comprising the AAV vectors and/or AAV particles described herein. Typically, the AAV particles are administered for therapy.

As described herein, pharmaceutical compositions can be formulated by any method known or developed in the field of pharmacology, including but not limited to contacting an active ingredient (e.g., a viral particle or a recombinant vector) with an excipient or other auxiliary ingredient, dividing or packaging the product into dosage units. The viral particles herein can be formulated to have desired characteristics, such as increased stability, increased cell transfection, sustained or delayed release, biodistribution or tropism, regulation of the encoded protein in vivo or enhanced translation, and release profiles of the encoded protein in vivo.

Thus, the pharmaceutical composition can further comprise saline, lipidoids, liposomes, lipid nanoparticles, polymers, lipid complexes, core-shell nanoparticles, peptides, proteins, cells transfected with viral vectors (e.g., for transplantation into a subject), nanoparticle mimics, or a combination thereof. In some embodiments, the pharmaceutical composition is formulated as nanoparticles. In some embodiments, the nanoparticles are self-assembled nucleic acid nanoparticles.

According to the pharmaceutical composition herein, it can be prepared, packaged and/or sold in bulk with a single unit dose and/or with multiple single unit doses. The amount of the active ingredient is generally equal to the dosage of the active ingredient to be administered to the subject and/or the appropriate ratio of such dosage, such as half or one-third of such dosage. The preparation of the present invention may include one or more excipients, and the amount of each excipient together increases the stability of the viral vector, increases cell transfection or viral vector transduction, increases the expression of the protein encoded by the viral vector, and/or changes the release profile of the protein encoded by the viral vector. In some embodiments, the pharmaceutical composition includes an excipient. Non-limiting examples of excipients include solvents, dispersion media, diluents or other liquid vehicles, dispersion or suspension aids, surfactants, isotonic agents, thickeners or emulsifiers, preservatives or combinations thereof.

In some embodiments, the pharmaceutical composition comprises a cryoprotectant. The term "cryoprotectant" refers to an agent that can reduce or eliminate damage to a substance during freezing. Non-limiting examples of cryoprotectants include sucrose, trehalose, lactose, glycerol, dextrose, raffinose and/or mannitol.

Treatment

The present disclosure provides a method for preventing or treating a condition, comprising administering to a subject a therapeutically effective amount of a pharmaceutical composition described herein. In some embodiments, the condition is a hereditary deafness disease. Preferably, the hereditary deafness disease is DFNB9 hereditary deafness.

As used herein, "treatment" includes any beneficial or desired effect on the symptoms or pathology of a disease or pathological condition, and may include even a small reduction in one or more measurable markers of the disease or condition being treated (e.g., a hereditary deafness disease). Treatment may optionally include a reduction or alleviation of the symptoms of the disease or condition, or a delay in the progression of the disease or condition. "Treatment" does not necessarily mean complete eradication or cure of the disease or condition or its associated symptoms. As used herein, "treatment" of a disease in a subject refers to (1) preventing the occurrence of symptoms or disease in a subject who is susceptible to or does not yet show symptoms of the disease; (2) inhibiting the disease or preventing its development; or (3) ameliorating or causing regression of the disease or disease symptoms. As understood in the art, "treatment" is a method for obtaining beneficial or desired results, including clinical results. Beneficial or desired results may include one or more, but are not limited to, alleviation or amelioration of one or more symptoms, whether detectable or undetectable, reduction in the extent of a condition (including a disease), a stable (i.e., not worsening) state of a condition (including a disease), a delay or slowing of a condition (including a disease), progression, improvement or palliation of a condition (including a disease), state, and remission (whether partial or total).

As used herein, the term "pharmaceutically acceptable carrier" encompasses any standard pharmaceutical carrier, such as phosphate buffered saline solution, water and emulsions, such as oil/water or water/oil emulsions, and various types of wetting agents. The composition may also include stabilizers and preservatives. For examples of carriers, stabilizers and adjuvants, see Martin (1975) Remington's Pharm. Sci. 15th Edition (Mack Publ. Co., Easton).

The "subject" of diagnosis or treatment is a cell or an animal, such as a mammal or a human. The subject is not limited to a particular species, including non-human animals receiving diagnosis or treatment and those animals receiving infection or animal models, including but not limited to simian, mouse, rat, dog or rabbit species, and other livestock, sports animals or pets. In some embodiments, the subject is a human.

The term "effective amount" as used herein is intended to mean an amount sufficient to achieve the desired effect. In the case of therapeutic or preventive applications, the effective amount will depend on the type and severity of the disorder in question and the characteristics of the individual subject, such as general health, age, sex, body weight and tolerance to the pharmaceutical composition. In the context of gene therapy, in some embodiments, the effective amount is an amount sufficient to restore some or all of the function of a defective gene in a subject. In some other embodiments, the effective amount of AAV viral particles is an amount sufficient to cause the gene to be expressed in a subject. In some embodiments, the effective amount is the amount required to increase galactose metabolism in a subject in need. A skilled technician will be able to determine the appropriate amount based on these and other factors.

In some embodiments, the effective amount will depend on the size and nature of the application in question. It also depends on the nature and sensitivity of the target subject and the method used. Those skilled in the art will be able to determine the effective amount based on these and other considerations. According to an embodiment, the effective amount may include, consist essentially of, or consist of one or more administrations of the composition.

As used herein, the term "administering" is intended to mean delivering a substance to a subject, such as an animal or a human. Administration can be performed in one dose, continuously or intermittently throughout the course of treatment. Methods for determining the most effective mode of administration and dosage are known to those skilled in the art and will vary with the composition used for treatment, the purpose of treatment, and the age, health or sex of the subject being treated. Single or multiple administrations can be performed, with dosage levels and patterns selected by the treating physician, or in the case of pets and other animals, by the treating veterinarian.

Dosage and Administration

Methods for determining the most effective mode of administration and dosage are known to those skilled in the art and will vary with the composition used for treatment, the purpose of the treatment, and the subject being treated. Single or multiple administrations may be performed, with the dosage level and pattern being selected by the treating physician. The dosage may be affected by the route of administration. Suitable dosage formulations and methods of administering the agents are known in the art. Non-limiting examples of such suitable dosages may be as low as 10E9 vector genomes to as high as 10E17 vector genomes per administration.

In embodiments of the methods described herein, the number of viral particles (e.g., AAV) administered to a subject ranges from about 10E9 to about 10E17. In certain embodiments, about 10E10 to about 10E12, about 10E11 to about 10E13, about 10E11 to about 10E12, about 10E11 to about 10E14, about 5×10E11 to about 5×10E12, or about 10E12 to about 10E13 viral particles are administered to a subject.

In some embodiments, the AAV particles repair the genetic defects of the subject. In some embodiments, the ratio of the target polynucleotide (e.g., DFNB9) or polypeptide repaired in the successfully treated cells, tissues, organs, or subjects to the unrepaired target polynucleotide or polypeptide is at least about 1.5: 1, about 2: 1, about 3: 1, about 4: 1, about 5: 1, about 6: 1, about 7: 1, about 8: 1, about 9: 1, about 10: 1, about 20: 1, about 50: 1, about 100: 1, about 1000: 1, about 10,000: 1, about 100,000: 1 or about 1,000,000: 1. The amount or ratio of the repaired target polynucleotide or polypeptide can be determined by any method known in the art, including but not limited to Western blotting, Northern blotting, Southern blotting, PCR, sequencing, mass spectrometry, flow cytometry, immunohistochemistry, immunofluorescence, fluorescence in situ hybridization, sequencing, immunoblotting, and ELISA.

In some embodiments, the viral particles are introduced into the subject intravenously, intrathecally, intracerebrally, intraventricularly, intranasally, intratracheally, intraauricularly, intraocularly or periocularly, orally, rectally, transmucosally, by inhalation, transdermally, parenterally, subcutaneously, intradermally, intramuscularly, intrapleurally, topically, intralymphatically, intracisternally; such introduction may also be intraarterially, intracardially, subventricularly, epidurally, intracerebrally, intraventricularly, subretinally, intravitreally, intraarticularly, intraperitoneally, intrauterinely, or any combination thereof. In some embodiments, the delivery of the viral particles is systemic.

For intraocular treatment of otological diseases, there are a variety of administration modes known to those skilled in the art, including but not limited to: round window approach, cochlear fenestration, vestibular and endolymphatic sac approach, semicircular canal approach, etc., such as bilateral and unilateral round window injections.

Administration of the AAV vectors, AAV particles, or compositions of the present disclosure can be achieved in one dose, continuously, or intermittently throughout the course of treatment. In some embodiments, the AAV vectors, AAV particles, or compositions of the present disclosure are administered parenterally by injection, infusion, or implantation. The AAV particles and compositions of the present disclosure can be administered in combination with other known treatments for the condition being treated.

Reagent test kit

In some embodiments, the reagents, vectors or compositions described herein can be assembled into pharmaceutical or diagnostic or research kits to facilitate their use in therapeutic, diagnostic or research applications. In some embodiments, the kits of the present disclosure include any of the AAV vectors, AAV particles, host cells, polypeptide compositions or pharmaceutical compositions described herein.

In some embodiments, the kit also includes instructions for use. Specifically, such a kit can include one or more reagents as described herein, and instructions for describing the intended application and correct use of these reagents. In some embodiments, the kit can include instructions for one or more components of the mixed kit and/or separation and mixed samples and applied to the experimenter. In some embodiments, the reagent in the kit is a pharmaceutical preparation and dosage suitable for the method of administration of a specific application and reagent. Kits for research purposes can contain components of appropriate concentration or amount for carrying out various experiments.

Kits can be designed to facilitate the use of the methods described herein, and can take many forms. Where applicable, each composition of the kit can be provided in liquid form (e.g., in solution) or in solid form (e.g., dry powder). In some cases, some compositions can be constructible or otherwise processable (e.g., processed into active forms), for example, by adding suitable solvents or other substances (e.g., water or cell culture media), which may or may not be provided with the kit. In some embodiments, the composition can be provided in a preservation solution (e.g., a cryopreservation solution). Non-limiting examples of preservation solutions include DMSO, paraformaldehyde. In some embodiments, the preservation solution contains a certain amount of metalloproteinase inhibitors.

In some embodiments, the test kit contains any one or more components described herein in one or more containers. Therefore, in some embodiments, the test kit can include a container for holding a reagent as described herein. The reagent can be in the form of a liquid, gel or solid (powder). The reagent can be aseptically prepared, packaged in a syringe and transported frozen. Alternatively, they can be contained in a vial or other container for storage. The second container can have other reagents prepared aseptically. Alternatively, the test kit can include a premixed active agent and transported in a syringe, vial, tube or other container. The test kit can have one or more or all components required for the reagent to be applied to the subject, such as a syringe, a local application device or an IV needle tube and a bag.

The present invention will be described below in the form of specific examples. It should be understood that these examples are merely illustrative and are not intended to limit the scope of the present invention. The methods and materials used in the examples are, unless otherwise stated, materials and methods conventional in the art.

Example

Example 1: Screening of cleavage sites of full-length Otoferlin protein in HEK-293T cells

Using a double trans-splicing vector system, the first vector is connected to the splicing donor sequence (C-intein sequence) after encoding the N-Otof sequence (i.e., the N-terminal part of Otof), and the second vector is connected to the C-Otof coding sequence (i.e., the C-terminal part of Otof) after its corresponding splicing acceptor sequence (N-intein sequence), and has a C-terminal HA tag. Its double vector sequence was transformed on the pAAV-CMV-EGFP-WPRE-SV40 plasmid backbone (Figure 2), and the EGFP reporter gene sequence was replaced with the required target sequence using EcoRI and EcoRV double enzyme digestion. The target sequence synthesis and vector construction were entrusted to Nanjing GenScript Biotechnology Co., Ltd. to obtain N-Otof and C-Otof-HA vectors. The selected groups of cleavage sites were co-transfected into HEK-293T cells at the same molar ratio. In order to confirm the trans-splicing and processing efficiency mediated by intein, the expression of full-length OTOF was analyzed by Western blotting 48 hours after transfection.

1.1 Cell transfection

1) Seed HEK-293T cells to 70-90% density and prepare for transfection;

2) Tube A: 125 μL serum-free medium + 8 μL Lipofectamine 3000 reagent, mix thoroughly;

3) Tube B: 125 μL serum-free medium + DNA + 8 μL P3000 reagent, mix thoroughly;

Each group of cutting points corresponds to the following (corresponding to the wb lane number), and the cells were transfected with the same molar number 2.72517E-10.

Among them, the sequences of the first fusion polypeptides containing splice donor sequences of groups 1, 6, 10, 13, 17, 18, 19, 20, 23, 27, 29, and 30 are shown in SEQ ID NOs: 5-16; the sequences of the second fusion polypeptides containing splice acceptor sequences of these groups are shown in SEQ ID NOs: 17-28;

4) Add the mixture in tube B to tube A and mix gently and thoroughly;

5) Leave at room temperature for 10-15 minutes;

6) Adding DNA-liposome complexes to cells;

7) Incubate at 37°C, 95% air and 5% CO ₂ , and collect samples 48 hours after transfection.

1.2 Protein extraction

1) Remove the culture medium and add 1 mL PBS to wash 2-3 times;

2) Gently disperse the cells with 1 mL of PBS, collect the cell suspension into a centrifuge tube, centrifuge at 700 g for 5 min, and remove the supernatant;

3) Add 120 μL (single well of six-well plate) of RIPA lysis buffer (add Cocktail (100×) before use) and lyse on ice for 30 min, vortex every 10 min to fully resuspend the cell pellet in the lysis buffer;

4) Finally, centrifuge at 12000 rpm for 15 min to collect the supernatant (do not aspirate the precipitate);

5) Add 5× Sample Loading Buffer (GenScript) to the supernatant, mix well, place in a 75°C metal bath, and incubate for 15 minutes. Centrifuge at 12000 rpm for 2 minutes before loading (15 μL per loading, 15-well gel).

1.3Western Blot

1.3.1 Electrophoresis

1)1x running buffer preparation: 950ml ddH ₂ O+50ml 20x running buffer;

2) Install the electrophoresis device, pour in 1x running buffer, and check for leaks;

3) Carefully remove the comb, apply constant voltage of 100V for 1 hour (Biyuntian 4-12%, HEPES system).

1.3.2 Transfer

1) Prepare 1x transfer buffer in advance and precool it;

1x transfer buffer preparation: 700ml water + 200ml ethanol + 100ml 10x transfer buffer

2) Remove the glue, separate the two pieces of glass with a shovel, and cut off the upper layer of glue;

3) Place the lower layer of gel in transfer buffer for equilibrium;

4) Soak the PVDF membrane in methanol (activation for 3 minutes) and place it in transfer buffer;

5) Place the transfer clamp with the black side facing down, and arrange in the order of: sponge - 1 layer of filter paper - glue - PVDF membrane - 1 layer of filter paper - sponge. Use a shovel to expel the air and clamp it tightly (no bubbles);

6) After installing the transfer device, pour the transfer buffer into the electrophoresis tank;

7) Transfer the membrane at a constant current of 300 mA on ice for 90 min.

1.3.3 Membrane washing

1) Disassemble the device and take out the PVDF membrane; cut the membrane according to the size of the target protein and mark it;

2) Place the cut PVDF membrane in an incubation box, add 5% skim milk (prepared using TBST), and block at room temperature for 1 hour on a shaker;

1.3.4 Antibody incubation

1) Add primary antibodies, anti-otoferlin (sc-271092) mouse IgG1 antibody (Santa Cruz, 1:500); β-Actin (8H10D10) Mouse mAb antibody (CST, 1:20000), and incubate overnight on a shaker at 4°C;

2) Recover the antibody and wash with TBST 3 times, 10-15 min each time

3) Add secondary antibody HRP-conjugated Affinipure Goat Anti-Mouse IgG (H+L) (1:5000) and incubate at room temperature on a shaker for 1-2 hours

4) Recover the antibody and wash with TBST three times, each time for 10-15 min.

1.3.5 Develop with Novozyme developer, observe the results on a Western imaging system and take photos.

The results are shown in Figure 4: the control was untransfected HEK-293T cells; Western blotting showed that full-length otoferlin protein was expressed in lanes 1, 3, 4, 5, 6, 8, 10, 13, 14, 15, 17, 18, 19, 20, 23, 27, 29, and 30.

Example 2: Analysis of auditory function and cochlear morphology in Otof knockout mice

2.1 Detection of auditory function in Otof knockout mice

The auditory brainstem response (ABR) and distortion product otoacoustic emission (DPOAE) were used to detect the overall auditory function differences between Otof knockout mice (ordered by Saiye Biotechnology Co., Ltd.) and their wild-type mice.

1) Weigh the mice and inject 1% sodium pentobarbital intraperitoneally according to the appropriate mass to volume ratio.

2) After the mouse is anesthetized, place it in the soundproof box of the TDT workstation, insert the positive electrode of the electrode into the subcutaneous part of the mouse's brain midline, insert the negative electrode into the subcutaneous part behind the mouse's ear, and insert the ground wire into the mouse's thigh muscle. Check the electrode indicator to confirm that the connection is normal.

3) Open the test file on the computer, select the test frequency of 4-32kHz, and record the critical threshold above the noise.

By testing the hearing threshold of mice to 4KHz, 8KHz, 12KHz, 16KHz, 24KHz, and 32KHz tone bursts, the duration of which is 10ms. This allows the hearing sensitivity of mice to be analyzed, and overall, to determine whether the mice have normal hearing function from hair cells to the cerebral cortex. ABR and DPOAE can well reflect the integrity of cochlear hair cells and cochlear function.

2.2 Electrophysiological function detection of hair cells in Otof gene knockout mice

The electrochemical changes of hair cells in the cochlea of Otof knockout mice were recorded by whole-cell extracellular recording method using patch clamp technology, so as to detect the changes of electrophysiological function of hair cells in Otof knockout mice.

2.3 Detection of hair cell number in Otof knockout mice

Otoferlin is highly expressed in the inner hair cells of the mouse cochlea, but is lowly expressed in the outer hair cells. Immunofluorescence staining was used to detect the number of surviving inner ear hair cells in Otof knockout mice at different time points, including P0, P7, P14, and P30.

1) Cut the entire ear tissue of the mouse and put it in 1×HBSS. Dissect out the temporal bone under a microscope and put it in 4% PFA. Use forceps to gently make a hole at the top of the temporal bone and repeatedly blow PFA into it with a syringe. Shake on a shaker for 1-2 hours.

2) Decalcify the temporal bones in 0.5 M EDTA: P0-P7, decalcify for 3-4 hours; P8-P15, decalcify for 1 day; P15-P30, decalcify for 2 days.

3) Rinse with 1×PBST for 3 times.

4) Place the temporal bone in 1× HBSS and dissect out the cochlea under a microscope.

5) Place the coverslip coated with Cell-tak into the dish containing the cochlea and adhere the cochlea to the slide (front side facing up).

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

7) Fix at room temperature for 1 hour and wash three times with PBST.

8) Add 100 μL blocking medium to each well and block for 1 hour.

9) Use PBT-1 to dilute the primary antibody in proportion, 80-100 μL per well, and incubate at 4°C overnight.

10) Wash three times with 1×PBST, 5 min each time.

11) Use PBT-2 to dilute the secondary antibody in proportion, 80-100 μL per well, and incubate at room temperature in the dark for 1 hour.

12) Wash three times with 1×PBST, 5 min each time.

13) Remove the slides from the clip and place them on a glass slide. Add 6 μL of DAKO to each slide, cover with a new slide, seal the slide with nail polish, observe and take photos.

Example 3: Effect Verification

The verified cleavage sites with higher expression levels, such as cleavage sites 1, 6, 10, 13, 17, 18, 19, 20, 23, 27, 29, and 30 ( FIG. 4 , D), were selected to construct the double AAV virus.

1) Prepare HEK-293T cells: subculture at a ratio of 1:3 and culture in 150 mm sterile dishes until 8 dishes are grown, with a cell density of 80%-90%.

2) Preparation: DMEM, PEI, N-Otof and C-Otof-HA plasmids in Example 1, Anc80L65 plasmid (GenBank: KT235804.1), and helper plasmid (GenBank: AF369965.1).

3) Prepare three sterile 15 ml EP tubes. Mark the first one as tube A. Add 3.444 ml of DMEM and 0.756 ml of PEI (1 μg/μl) into tube A and let it stand for 5 minutes.

4) The remaining two tubes are labeled B1 and B2, and 7 μg of N-Otof or C-Otof-HA plasmid, 9 μg of Anc80L65 plasmid, and 14 μg of helper plasmid are added thereto respectively, and DMEM is added to 2.1 ml.

5) Add the liquid in tube A to tubes B1 and B2 respectively, drop by drop, 2.1 ml per tube, let stand at room temperature for 20-25 minutes, and then add the mixture to the prepared HEK-293T cells.

6) After 12 hours, discard the culture medium and add 20 ml of 293T cell transfection medium to each plate. After 48 hours, collect the supernatant into a sterile bottle and store at 4°C. Add 20 ml of 293T transfection medium to each plate in a large dish.

7) After another 48 hours, the cells and supernatant were collected into the aforementioned sterile bottles for virus purification and titer detection. The titer was determined by virus reverse terminal repeat-specific qPCR.

The virus purification steps are as follows:

1) Collect the supernatant and cells in a 50mL centrifuge tube (chloroform-resistant) and centrifuge for 7 minutes at 11,000r at 4°C. Aspirate the supernatant and keep part of the resuspended cells. Add 1/10 volume of chloroform and shake at 37°C at 220r for 3 hours.

2) 11000r, 4°C, 20min, collect the supernatant and mix it with the supernatant collected in the previous step.

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

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

5) Add 700 μl of enzymatic solution (containing DNase I and RNase) to the precipitate and incubate in a 37°C water bath for 1 hour. Mix by pipetting several times during this time.

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

7) Add an equal volume of virus dissolution solution (20% PEG8000+2M Nacl+0.002% F68+PBS) and incubate at 4°C overnight.

8) Centrifuge at 12000r/min for 7min, discard the supernatant, and centrifuge again for 3min, discard the supernatant.

9) Dissolve the precipitate with a solution containing DNaseⅠ and RNase, add an appropriate amount of enzymatic solution to dissolve it, and then pass it through chloroform to obtain the supernatant, which is the final virus.

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

The titer detection steps are as follows:

1) Digest genomic DNA and plasmid DNA, incubate at 37°C for 20 min; 95°C for 10 min, the system is as follows:

(37℃ digestion of genomic DNA and plasmid DNA; 95℃ heat treatment to inactivate DNaseI)

2) Add an equal volume of lysis buffer (10 μl) and 2 μl 20 mg/ml proteinase K, incubate at 55°C for 30 min; 95°C for 10 min. (lysis buffer: 10 mM Tris-HCl pH 8.0 + 5 mM EDTA + 100 ug/ml proteinase K) (leave at room temperature)

Take 4 μl of the PCR product, dilute it 100 times to 400 μl with ddH2O, and use 2 μl as the template for RT-QPCR. The system is as follows:

The program was as follows: 95°C, 5 min; 95°C, 10 s; 58°C, 30 s (40X); 95°C, 15 s; 60°C, 1 min; 95°C, 15 s;

Titer calculation formula: y = 38.71-3.54x; y = CT; tittering = 1000000*power(10,x)

AAV-Otof-NT and AAV-Otof-CT recombinant vector viruses (8.5e ¹² +8.5e ¹² vg/mL (ratio 1:1, total virus volume 3.4x10 ¹⁰ vg)) were injected into the left cochlea of adult Otof-/- mice around P30 through the posterior semicircular canal of the cochlea. The adult mice were anesthetized by injection of 1% sodium pentobarbital. After anesthesia, hair removal was performed around the ear, an incision was made behind the ear to expose the semicircular canal, and the semicircular canal was punctured and manually injected with a glass micropipette. After injection, the skin incision was sealed with tissue glue. The injected mice were then placed on a 37°C heating pad for recovery. After surgery, the adult mice were fully recovered in about 30 minutes and returned to the mouse cage. Standard postoperative care was used after surgery. Two weeks later, the mice were tested for ABR to evaluate the impact of the injection on the overall hearing of the mice.

In order to determine whether the dual vector system using AAV vectors, which encodes N-Otof with N-Intein and C-Otof with C-Intein, can restore hearing loss and the extent of the restoration of hearing loss, auditory brainstem response (ABR) was used to detect the recovery of hearing function in mice of different ages after gene therapy. The sensory epithelium of the cochlea on the Otof-/- virus-injected side after audiometry was microdissected and immunolabeled for otoferlin (inner hair cells) and Myosin7a (hair cells) to study the recombinant expression of the exogenous Otof gene in the cochlear cells of ko mice;

1) Cut the entire ear tissue of the mouse and put it in 1×HBSS. Dissect out the temporal bone under a microscope and put it in 4% PFA. Use forceps to gently make a hole at the top of the temporal bone and repeatedly blow PFA into it with a syringe. Fix it at room temperature for 1-2 hours.

2) The temporal bones were immersed in 0.5 M EDTA for decalcification: P0-P7, decalcification for 3-4 hours; P8-P15, decalcification for 1 day; P15-P60, decalcification for 2 days.

3) Rinse with 1×PBST three times.

4) Place the temporal bone in 1× HBSS and dissect out the cochlea under a microscope. Divide the entire sensory epithelium into three segments of equal length, labeled as the top, middle, and bottom of the cochlea, and store them in HBSS until staining.

5) Place the coverslip coated with Cell-tak into the dish containing the cochlea and adhere the cochlea to the slide (front side facing up).

6) Place the adhered slides in a 4-well dish (previously added with 3 mL PBS). After all the adhered slides are placed in the 4-well dish, permeabilize and block with PBS containing 1% Triton X-100 and 10% donkey serum at room temperature for 1 hour.

7) Use PBT-1 to dilute the primary antibody in proportion, otoferlin antibody (ab53233, 1:200, Abcam), Myosin7a antibody (256790, 1:1000, Proteus), 80-100 μl per well, 4°C overnight.

8) Wash three times with 1×PBST, 5 min each time.

9) Use PBT-2 to dilute the corresponding secondary antibody in proportion, 80-100 μl per well, and incubate at room temperature in the dark for 1 hour.

10) Wash three times with 1×PBST, 5 min each time.

11) Remove the slides from the clip and place them on a glass slide. Add 6 μl of DAKO to each slide, cover with a new slide, seal the slide with nail polish, observe and take photos.

In order to explore the role of the Otof gene in the development of the mouse inner ear, we first need to detect the expression of Otof in the inner ear. Otof is abundantly expressed in the inner ear. In order to further detect the specific expression site of Otof in the inner ear basilar membrane and the time spectrum of expression, we performed immunofluorescence staining on wild-type mice at different time points. And immunofluorescence staining was performed on the cochlear basilar membrane spread to localize the expression of Otof in the inner ear. Myosin7a was used as a marker for hair cells. We selected three time points at different developmental periods: P0, P7, P14, and P30. At the hair cell level, at these three different developmental time points, OTOF was co-localized with Myosin7a in the basilar membrane spread, indicating that OTOF is expressed in the cytoplasm of hair cells. The above experiments show that OTOF is abundantly expressed in the inner ear and is mainly expressed in hair cells.

We ordered Otof knockout mice from Saiye Biotechnology Co., Ltd. for breeding. In order to ensure that the Otof protein in the inner ear of Otof knockout mice has indeed been knocked out, we first screened homozygous, heterozygous and wild-type mice by genotyping. We first tested this purpose by immunofluorescence staining. We used WT and Otof-/- mice at different time points such as P0, P7, P14, and P30. The results of immunofluorescence staining showed that in wild-type mice, Otof was abundantly expressed in the epidermal plate layer of hair cells, but the expression of OTOF could not be detected in the epidermal plate layer of hair cells in the inner ear of Otof-/- mice. This shows that the OTOF protein in the inner ear of Otof-/- mice we used later has been completely knocked out.

Then, the hearing condition of Otof knockout mice was detected. First, we used the ABR audiological detection method to test the homozygous knockout mice 30 days after birth and the wild-type control mice in the same litter. ABR is mainly used to detect whether the functions of all parts in the sound transmission process are normal. As shown in Figure 5, the ABR threshold of the treated adult Otof-/- knockout mouse model decreased two weeks after injection, and the hearing of some frequency bands was significantly restored; the ABR results showed that after knocking out the Otof gene, the hearing threshold of the mouse was significantly increased in low frequency, medium frequency and high frequency, indicating that Otof plays an important role in the hearing of mice. The dual AAV system of this application realizes the expression of otoferlin protein in the cochlea of mice, restores the release of synaptic vesicles in the inner ear hair cells, and restores binaural hearing. The above experimental results show that the Otof gene has an important regulatory effect on the hearing of mice, especially on the function of inner hair cells.

For the treated adult Otof-/- knockout mouse model, the cochlea was taken out for basement membrane staining 2 weeks later, and the results are shown in Figure 6. The immunofluorescence results showed that both AAV systems achieved the expression of otoferlin protein in the mouse cochlea to varying degrees.

Claims (23)

1. A fusion polypeptide consisting of a portion of the otoferlin protein and a portion of the intein system, wherein the fusion polypeptide is the first fusion polypeptide or the second fusion polypeptide, The first fusion polypeptide consists of amino acids 1 to n of the otoferlin protein shown in SEQ ID NO: 1 and the N-terminal fragment of the intein from the N-terminus to the C-terminus, The second fusion polypeptide consists of the C-terminal fragment of the intein and the amino acids (n+1) to 1997 of the otoferlin protein shown in SEQ ID NO: 1 from the N-terminus to the C-terminus, Where n is selected from any integer below: 797, 872, 900, 926, 977, 980, 992, 994, 1003, 1064, 1161, 1169, The N-terminal fragment of the intein is shown in SEQ ID NO:2, and the C-terminal fragment of the intein is shown in SEQ ID NO:3. 2. The fusion polypeptide according to claim 1, wherein The first fusion polypeptide is shown in SEQ ID NO: 5, the second fusion polypeptide is shown in SEQ ID NO: 17, or The first fusion polypeptide is shown in SEQ ID NO:6, the second fusion polypeptide is shown in SEQ ID NO:18, or The first fusion polypeptide is shown in SEQ ID NO:7, the second fusion polypeptide is shown in SEQ ID NO:19, or The first fusion polypeptide is shown in SEQ ID NO: 8, the second fusion polypeptide is shown in SEQ ID NO: 20, or The first fusion polypeptide is shown in SEQ ID NO: 9, the second fusion polypeptide is shown in SEQ ID NO: 21, or The first fusion polypeptide is shown as SEQ ID NO: 10, the second fusion polypeptide is shown as SEQ ID NO: 22, or The first fusion polypeptide is shown in SEQ ID NO: 11, the second fusion polypeptide is shown in SEQ ID NO: 23, or The first fusion polypeptide is shown in SEQ ID NO: 12, the second fusion polypeptide is shown in SEQ ID NO: 24, or The first fusion polypeptide is shown in SEQ ID NO: 13, the second fusion polypeptide is shown in SEQ ID NO: 25, or The first fusion polypeptide is shown in SEQ ID NO: 14, the second fusion polypeptide is shown in SEQ ID NO: 26, or The first fusion polypeptide is shown in SEQ ID NO: 15, the second fusion polypeptide is shown in SEQ ID NO: 27, or The first fusion polypeptide is shown as SEQ ID NO:16, and the second fusion polypeptide is shown as SEQ ID NO:28. 3. A polypeptide composition, consisting of the first fusion polypeptide and the second fusion polypeptide according to any one of claims 1 to 2. 4. A polynucleotide comprising the following sequence: a nucleic acid sequence encoding the fusion polypeptide according to any one of claims 1 to 2 or the polypeptide composition according to claim 3. 5. The polynucleotide according to claim 4, wherein the polynucleotide encoding the first fusion polypeptide is shown in any one of SEQ ID NOs: 29-40, and/or the polynucleotide encoding the second fusion polypeptide is shown in any one of SEQ ID NOs: 41-52. 6. A nucleic acid construct, wherein: (1) expressing the fusion polypeptide according to any one of claims 1 to 2 or the polypeptide composition according to claim 3, and/or (2) A sequence comprising the polynucleotide according to claim 4 or 5. 7. The nucleic acid construct of claim 6, wherein the nucleic acid construct is a vector. 8. The nucleic acid construct of claim 7, wherein the nucleic acid construct is an AAV vector. 9. The nucleic acid construct according to any one of claims 6 to 8, wherein The nucleic acid construct comprises an expression frame, and the polynucleotides encoding the first fusion polypeptide and the second fusion polypeptide are both located in the expression frame, or The nucleic acid construct comprises two expression frames, which respectively comprise polynucleotides encoding a first fusion polypeptide and a second fusion polypeptide. 10. The nucleic acid construct of claim 9, wherein the nucleic acid construct is two AAV vectors, each comprising a polynucleotide encoding a first fusion polypeptide and a second fusion polypeptide, and one or more terminal inverted repeat sequences flanking the polynucleotide. 11. The nucleic acid construct of claim 10, wherein the nucleic acid construct further comprises an element for expressing the polynucleotide contained therein. 12. The nucleic acid construct of claim 11, wherein the nucleic acid construct has the sequence shown in SEQ ID NO:4. 13. A rAAV vector system, comprising two AAV vectors and a nucleic acid construct encoding genes required by the AAV virus, wherein the two AAV vectors respectively contain polynucleotides encoding a first fusion polypeptide and a second fusion polypeptide, and the first fusion polypeptide and the second fusion polypeptide are as described in claim 1 or 2. 14. The rAAV vector system of claim 13, wherein the genes required for the AAV virus are selected from one or more of the following: Rep, Cap, E2A, E4 and VA genes. 15. The rAAV vector system of claim 14, wherein the AAV vector system comprises an Anc80L65 plasmid, a helper plasmid and the AAV vector. 16. An rAAV virus particle comprising the polynucleotide of claim 4 or 5. 17. The rAAV virus particle as described in claim 16 is characterized in that the rAAV virus particle is selected from one or more of rAAV1, rAAV2, rAAV4, rAAV5, rAAV6, rAAV7, rAAV8, rAAV9, rAAV10, rAAV11, rAAV12, rAAV13, rAAVPHP.B or rAAVrh74. 18. A host cell, wherein: (1) comprising, expressing and/or secreting the fusion polypeptide according to any one of claims 1 to 2 or the polypeptide composition according to claim 3, (2) comprising the polynucleotide of claim 4 or 5, the nucleic acid construct of any one of claims 6 to 12, or the AAV vector system of claims 13 to 15, or (3) The polynucleotide according to claim 4 or 5 is integrated into a chromosome. 19. The host cell of claim 18, wherein the host cell is a HEK293 cell. 20. A method for preparing otoferlin protein, comprising contacting a first fusion polypeptide and a second fusion polypeptide under conditions suitable for intein splicing, wherein the first fusion polypeptide and the second fusion polypeptide are as described in any one of claims 1-2. 21. The method of claim 20, comprising the step of incubating the host cell of claim 18 or 19 under conditions suitable for intein splicing. 22. Use of the fusion polypeptide according to any one of claims 1-2, the polypeptide composition according to claim 3, the polynucleotide according to claim 4 or 5, the nucleic acid construct according to any one of claims 6-12, or the host cell according to claim 18 or 19 in the preparation of a medicament for treating a disease caused by Otof gene defect, wherein the disease is DFNB9 hearing impairment. 23. A pharmaceutical composition comprising a pharmaceutically acceptable excipient and one or more selected from the following: the fusion polypeptide of any one of claims 1-2, the polypeptide composition of claim 3, the polynucleotide of claim 4 or 5, the nucleic acid construct of any one of claims 6-12, the rAAV vector system of claims 13-15, the rAAV virus particle of claim 16 or 17, and the host cell of claim 18 or 19.