Composition and method for expression of OTOF gene by dual vector system

Abstract

The present invention relates to a composition and a method for expressing an OTOF gene by a dual-vector system. Specifically provided are a fusion polypeptide, comprising an otoferlin protein portion, and an intein system portion. The gene encoding OTOF is transferred into the cell by means of a recombinant AAV dual-vector system.

Description

Compositions and methods for expressing Otof gene using a dual vector system Technical Field

[0001] 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-vector system. Background Art

[0002] Hearing loss is one of the most common sensory impairments in humans and can be caused by both environmental and genetic factors. Some people may be born with hearing loss, while others may gradually lose their hearing over time. Hearing loss affects over 1.5 billion people worldwide, approximately one-fifth of the global population. Of these, 470 million people suffer from disabling hearing loss (moderate or greater hearing impairment), and 34 million children are deaf or have hearing loss. It is estimated that by 2050, over 700 million people may suffer from disabling hearing loss. In my country, approximately 16% of the population has hearing impairment, and approximately 5% of the population has disabling hearing loss. The prevalence of deafness among newborns in my country ranges from 1% to 3.47%, meaning that 1-3 deaf children are born in every 1,000 births, or approximately 30,000 deaf children each year. Environmental causes of hearing loss are primarily related to various environmental factors or complications, such as the use of ototoxic drugs, infections during pregnancy, neonatal hypoxia, and radiation exposure. Genetic factors are primarily due to individual genetic defects in hearing loss, leading to varying degrees of hearing loss. The disease-causing genes are passed down through various genetic pathways and can occur at any age. Genetic factors account for approximately 60% of hearing loss cases. Currently, over 200 genes associated with hearing loss have been identified, involving over 1,500 pathogenic variants. To date, no medications are available clinically to treat hereditary hearing loss. Hereditary hearing loss is divided into syndromic hearing loss (SHL) and non-syndromic hearing loss (NSHI) based on whether or not there are abnormalities or lesions in extra-auricular tissue. Syndromic hearing loss accounts for approximately 30% of hereditary hearing loss and, in addition to hearing impairment, is accompanied by many other manifestations. Non-syndromic hearing loss accounts for approximately 70% to 80% of hereditary hearing loss and only presents with hearing loss, with no hereditary damage to other organs.

[0003] Hereditary nonsyndromic deafness is primarily a monogenic disorder, which can be categorized by inheritance pattern, including autosomal dominant deafness (DFNA), autosomal recessive deafness (DFNB), X-linked deafness (DFNX), Y-linked deafness (DFNY), mitochondrial inheritance, and epigenetic inheritance. DFNA accounts for approximately 18% of hereditary deafness, DFNB for approximately 80%, and DFNX for approximately 1%. Mitochondrial inheritance accounts for less than 1%, and DFNY and epigenetic inheritance have only been reported in isolated cases. A total of 110 genes associated with nonsyndromic deafness have been identified, including 45 genes associated with DFNA and 70 genes associated with DFNB. Ten genes are associated with both DFNA and DFNB (CLOL11A2, GJB2, GJB6, MYO3A, MYO6, MYO7A, PTPRQ, TCB1D24, TECTA, and TMC1), and five genes are associated with DFNX. Each type is described according to the naming convention of the deafness gene, for example, DFNA1 is the first autosomal dominant deafness type discovered.

[0004] DFNB9 hearing impairment is a common form of congenital hearing loss. Patients with DFNB9 hearing loss typically suffer from severe sensorineural hearing loss, caused by mutations in the Otof gene, which encodes otoferlin. The Otof gene encodes otoferlin, a transmembrane protein belonging to the Ferlin family of proteins. Otoferlin contains six calcium-binding domains and two Fer domains, and plays important roles in cell membrane ion exchange, signal transduction, and neurotransmitter release. The Otof gene was the first gene discovered to be associated with non-syndromic auditory neurophy spectrum disorder (ANDS), also known as auditory neuropathy. Otoferlin plays a key role in the transmission of sound information at inner hair cell synapses. Over 56% of non-syndromic ANSD cases are caused by mutations in the Otof gene, affecting approximately 200,000 people worldwide. Currently, patients with Otof gene mutations can only benefit from traditional hearing aids, which are ineffective in clinical treatment, and there are no approved drug treatments.

[0005] Mutations in the Otof gene can cause congenital, severe, prelingual hereditary hearing loss, which is inherited in an autosomal recessive manner. Patients with Otof gene mutations often suffer from severe bilateral hearing loss from birth, and their auditory brainstem response (ABR) is absent or highly abnormal. This is currently the most common cause of hearing loss. Currently, most methods for addressing hearing loss use physical methods such as hearing aids, vibrating sound bridges, and cochlear implants. Although patients can achieve varying degrees of improvement in hearing function, there are large individual differences and significant limitations and weaknesses. For example, treatment effects are limited, frequency sensitivity, speech discrimination, and perception difficulties in noisy environments, and the device needs to be used with caution. Approximately 300,000 patients worldwide have received cochlear implants, but this only accounts for a small fraction of all deaf patients. For most patients, there is still an urgent need for fundamental and effective drug treatments, and there are no approved treatments to date, making this an area of ​​serious unmet need.

[0006] Summary of the Invention

[0007] 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.

[0008] 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.

[0009] In one or more embodiments, the otoferlin protein is set forth in SEQ ID NO: 1 or a sequence at least 90% identical thereto.

[0010] In one or more embodiments, the fusion polypeptide is a first fusion polypeptide or a second fusion polypeptide.

[0011] 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.

[0012] 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), 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.

[0013] 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.

[0014] 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.

[0015] In one or more embodiments, the C-terminal fragment of the intein (C-intein) is a split intein-C, as shown in SEQ ID NO:3.

[0016] In one or more embodiments, the first fusion polypeptide has the sequence shown in any one of SEQ ID NOs: 5-16, or a variant thereof having at least 80% sequence identity thereto.

[0017] In one or more embodiments, the second fusion polypeptide has the sequence set forth in any one of SEQ ID NOs: 17-28, or a variant thereof having at least 80% sequence identity thereto.

[0018] 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.

[0019] The present invention also provides a polynucleotide comprising a sequence selected from the group consisting of:

[0020] (1) a nucleic acid sequence encoding a fusion polypeptide or polypeptide composition according to any embodiment of the present invention,

[0021] (2) a variant having at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity with (1),

[0022] (3) The complementary sequence of (1) or (2).

[0023] In one or more embodiments, the polynucleotide encoding the first fusion polypeptide has a sequence as set forth 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 set forth in any one of SEQ ID NOs: 41-52, or a variant thereof having at least 80% sequence identity.

[0024] The present invention also provides a nucleic acid construct, wherein the nucleic acid construct:

[0025] (1) expressing a fusion polypeptide or polypeptide composition as described in any embodiment of the present invention, and / or

[0026] (2) A sequence comprising a polynucleotide described herein.

[0027] 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.

[0028] In one or more embodiments, the nucleic acid construct comprises two expression cassettes, each comprising a polynucleotide encoding a first fusion polypeptide and a second fusion polypeptide.

[0029] 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.

[0030] In one or more embodiments, 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 inverted terminal repeat (ITR) sequences flanking the polynucleotide. The polynucleotide 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.

[0031] 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.

[0032] In one or more embodiments, the nucleic acid construct has the sequence shown in SEQ ID NO:4.

[0033] The present invention also provides an rAAV vector system, comprising two AAV vectors and a nucleic acid construct encoding the 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.

[0034] 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.

[0035] In one or more embodiments, the AAV vector system comprises an Anc80L65 plasmid, a helper plasmid, and the AAV vector.

[0036] The present invention also provides an rAAV virus particle comprising the polynucleotide according to any embodiment of the present invention.

[0037] In one or more embodiments, the rAAV viral particle is selected from one or more of rAAV1, rAAV2, rAAV4, rAAV5, rAAV6, rAAV7, rAAV8, rAAV9, rAAV10, rAAV11, rAAV12, rAAV13, rAAVPHP.B, or rAAVrh74.

[0038] The present invention also provides a host cell, wherein:

[0039] (1) contains, expresses and / or secretes a fusion polypeptide or polypeptide composition as described herein,

[0040] (2) comprising a polynucleotide, nucleic acid construct or AAV vector system as described in any embodiment herein,

[0041] (3) The polynucleotide described in any embodiment of the present invention is integrated into the chromosome.

[0042] In one or more embodiments, the host cell is a HEK293 cell.

[0043] 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.

[0044] 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.

[0045] In one or more embodiments, the method comprises the steps of 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.

[0046] In one or more embodiments, the cell is an animal cell, preferably a HEK293 cell.

[0047] In one or more embodiments, the method further comprises the step of intein-mediated trans-splicing of the protein.

[0048] The present invention also provides the use of the fusion polypeptide, polypeptide composition, polynucleotide, nucleic acid construct, and host cell described in any embodiment herein in the preparation of a drug for treating a disease caused by an Otof gene defect.

[0049] In one or more embodiments, the disease is DFNB9 hearing impairment.

[0050] The present invention also provides a pharmaceutical composition comprising pharmaceutically acceptable excipients and one or more selected from the following: the fusion polypeptide, polypeptide composition, polynucleotide, nucleic acid construct, rAAV vector system, rAAV virus particles, and host cells described in any embodiment of the present invention.

[0051] 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 described in any embodiment of the present invention. Preferably, the hereditary deafness is DFNB9 hereditary deafness.

[0052] Beneficial effects of the present invention:

[0053] The present invention constructs the 5′ and 3′ ends of the otoferlin gene into two different rAAV vectors. Through a recombinant AAV dual-vector system, the transgene encoding Otof is delivered to the inner hair cells of the cochlea, ultimately achieving expression of the 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

[0054] FIG1 shows a schematic diagram of double AAV intein-mediated trans-splicing of the OTOF protein using inverted terminal repeats, promoter, and polyadenylation sequences.

[0055] Figure 2 shows the vector backbone elements in the binary vector system, where 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.

[0056] FIG3 shows a schematic diagram of the cleavage sites of the full-length OTOF protein divided into two AAV vectors, and Western blotting was used for in vitro screening verification.

[0057] Figure 4 shows that HEK-293T cells co-transfected with both the N-terminal portion (N-Otof) and the C-terminal portion (C-Otof) encoding otoferlin protein resulted in the expression of full-length OTOF. AD, Expression of full-length otoferlin protein at different cleavage sites.

[0058] Figure 5 shows 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.

[0059] Figure 6 shows the immunofluorescence results of an adult Otof- / - knockout mouse model that received treatment. After audiometry, the mice were sacrificed 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

[0060] 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 (MJ Gait, ed., 1984); Animal Cell Culture (RI Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987 edition and periodic updates thereof); PCR: The Polymerase Chain Reaction (Mullis et al., eds., 1994); A Practical Guide to Molecular Cloning (Perbal Bernard V., 1988); Phage Display: A Laboratory Manual (Barbas et al., 2001).

[0061] The term "disease and / or condition" refers to a physical state of a subject that is associated with the diseases and / or conditions described herein.

[0062] 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.

[0063] Fusion peptide

[0064] The present invention first provides a fusion polypeptide comprising a portion comprising an otoferlin protein and a portion comprising an intein system.

[0065] As used herein, the term "intein" refers to an insertion sequence within a host protein. The intein gene is not an independent gene and must be inserted into an extein gene for replication and transcription. It can be excised from the precursor protein and the exteins on both sides can be linked to form a mature protein. Its corresponding nucleotide sequence is embedded within the nucleic acid sequence corresponding to the host protein, existing within the same open reading frame (ORF) as the host protein gene. It undergoes synchronous transcription and translation with the host protein gene. After translation to form the protein precursor, the intein is excised from the host protein, thereby forming a mature, active protein. The amino acid sequence encoding the splice donor (e.g., 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 splice acceptor (e.g., the C-terminal fragment of the intein (C-intein), also known as the split intein-C) is shown in SEQ ID NO: 3.

[0066] 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 of the otoferlin protein as shown in SEQ ID NO: 1 and the N-terminal fragment of the intein (N-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. The second fusion polypeptide comprises or consists of amino acids (n+1) to 1997 of the otoferlin protein as shown in SEQ ID NO: 1 and the C-terminal fragment of the intein (C-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 exemplary embodiments, 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.

[0067] The present invention also provides a polypeptide composition comprising a first fusion polypeptide and 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 as set forth in SEQ ID NO: 1 and the N-terminal fragment of an intein (N-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. The second fusion polypeptide comprises or consists of amino acids (n+1) to 1997 of the otoferlin protein as set forth in SEQ ID NO: 1 and the C-terminal fragment of an intein (C-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 exemplary embodiments, 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 the sequence set forth in SEQ ID NO:5, or a variant thereof having at least 80% sequence identity, and the second fusion polypeptide has the sequence set forth 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 the sequence set forth in SEQ ID NO:6, or a variant thereof having at least 80% sequence identity, and the second fusion polypeptide has the sequence set forth 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 the sequence set forth in SEQ ID NO:7, or a variant thereof having at least 80% sequence identity, and the second fusion polypeptide has the sequence set forth 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 the sequence set forth in SEQ ID NO:8, or a variant thereof having at least 80% sequence identity, and the second fusion polypeptide has the sequence set forth 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 the sequence set forth in SEQ ID NO: 9, or a variant thereof having at least 80% sequence identity, and the second fusion polypeptide has the sequence set forth 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 the sequence set forth in SEQ ID NO: 10, or a variant thereof having at least 80% sequence identity, and the second fusion polypeptide has the sequence set forth 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 the sequence set forth in SEQ ID NO: 11, or a variant thereof having at least 80% sequence identity, and the second fusion polypeptide has the sequence set forth 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 the sequence set forth in SEQ ID NO: 12, or a variant thereof having at least 80% sequence identity, and the second fusion polypeptide has the sequence set forth in SEQ ID NO: 24, or a variant thereof having at least 80% sequence identity. The first fusion polypeptide has the sequence set forth in SEQ ID NO: 13, or a variant thereof having at least 80% sequence identity, and the second fusion polypeptide has the sequence set forth 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 the sequence set forth in SEQ ID NO: 14, or a variant thereof having at least 80% sequence identity, and the second fusion polypeptide has the sequence set forth in SEQ ID NO: 26, or a variant thereof having at least 80% sequence identity. The first fusion polypeptide has the sequence set forth in SEQ ID NO: 15, or a variant thereof having at least 80% sequence identity, and the second fusion polypeptide has the sequence set forth 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 the sequence set forth in SEQ ID NO: 16, or a variant thereof having at least 80% sequence identity, and the second fusion polypeptide has the sequence set forth in SEQ ID NO: 28, or a variant thereof having at least 80% sequence identity.

[0068] In some embodiments, the sequence of the variants of the present invention may be at least 95%, 96%, 97%, 98% or 99% identical to the sequence from which it is derived. The sequence identity of the present invention can be measured using sequence analysis software. For example, the computer program BLAST, particularly BLASTP or TBLASTN, is used with default parameters. The present invention also includes molecules having antibody heavy chain variable regions with CDRs, as long as their CDRs have greater than 90% (preferably greater than 95%, and most preferably greater than 98%) homology to the CDRs identified herein.

[0069] Nucleotide sequence

[0070] The present invention provides polynucleotides comprising: a nucleic acid sequence encoding the fusion polypeptide or polypeptide composition described herein or a complementary sequence thereof.

[0071] 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 protein of interest and a splice donor sequence N-intein sequence (e.g., N-Otof-N-intein). Preferably, the nucleic acid sequence thereof is as set forth in any one of SEQ ID NOs: 29-40, or a variant thereof having at least 80% sequence identity thereto.

[0072] 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 protein of interest and a splice donor sequence, C-intein sequence (e.g., C-intein-C-Otof). Preferably, the nucleic acid sequence thereof is as shown in any one of SEQ ID NOs: 41-52, or a variant thereof having at least 80% sequence identity thereto.

[0073] In some embodiments, the nucleic acid sequence encoding the first fusion polypeptide has the sequence set forth in SEQ ID NO:29, or a variant thereof having at least 80% sequence identity, and the nucleic acid sequence encoding the second fusion polypeptide has the sequence set forth in SEQ ID NO:41, or a variant thereof having at least 80% sequence identity. In some embodiments, the nucleic acid sequence encoding the first fusion polypeptide has the sequence set forth in SEQ ID NO:30, or a variant thereof having at least 80% sequence identity, and the nucleic acid sequence encoding the second fusion polypeptide has the sequence set forth in SEQ ID NO:42, or a variant thereof having at least 80% sequence identity. In some embodiments, the nucleic acid sequence encoding the first fusion polypeptide has the sequence set forth in SEQ ID NO:31, or a variant thereof having at least 80% sequence identity, and the nucleic acid sequence encoding the second fusion polypeptide has the sequence set forth in SEQ ID NO:43, or a variant thereof having at least 80% sequence identity. In some embodiments, the nucleic acid sequence encoding the first fusion polypeptide has the sequence set forth in SEQ ID NO:6832, or a variant thereof having at least 80% sequence identity, and the nucleic acid sequence encoding the second fusion polypeptide has the sequence set forth in SEQ ID NO:44, or a variant thereof having at least 80% sequence identity. In some embodiments, the nucleic acid sequence encoding the first fusion polypeptide has the sequence set forth in SEQ ID NO:33, or a variant thereof having at least 80% sequence identity, and the nucleic acid sequence encoding the second fusion polypeptide has the sequence set forth in SEQ ID NO:45, or a variant thereof having at least 80% sequence identity. In some embodiments, the nucleic acid sequence encoding the first fusion polypeptide has the sequence set forth in SEQ ID NO:34, or a variant thereof having at least 80% sequence identity, and the nucleic acid sequence encoding the second fusion polypeptide has the sequence set forth in SEQ ID NO:46, or a variant thereof having at least 80% sequence identity. In some embodiments, the nucleic acid sequence encoding the first fusion polypeptide has the sequence set forth in SEQ ID NO:35, or a variant thereof having at least 80% sequence identity, and the nucleic acid sequence encoding the second fusion polypeptide has the sequence set forth in SEQ ID NO:47, or a variant thereof having at least 80% sequence identity. In some embodiments, the nucleic acid sequence encoding the first fusion polypeptide has the sequence set forth in SEQ ID NO:36, or a variant thereof having at least 80% sequence identity, and the nucleic acid sequence encoding the second fusion polypeptide has the sequence set forth in SEQ ID NO:48, or a variant thereof having at least 80% sequence identity. In some embodiments, the nucleic acid sequence encoding the first fusion polypeptide has the sequence set forth in SEQ ID NO:37, or a variant thereof having at least 80% sequence identity thereto, and the nucleic acid sequence encoding the second fusion polypeptide has the sequence set forth in SEQ ID NO:49, or a variant thereof having at least 80% sequence identity thereto.In some embodiments, the nucleic acid sequence encoding the first fusion polypeptide has the sequence set forth in SEQ ID NO:38, or a variant thereof having at least 80% sequence identity, and the nucleic acid sequence encoding the second fusion polypeptide has the sequence set forth in SEQ ID NO:50, or a variant thereof having at least 80% sequence identity. In some embodiments, the nucleic acid sequence encoding the first fusion polypeptide has the sequence set forth in SEQ ID NO:39, or a variant thereof having at least 80% sequence identity, and the nucleic acid sequence encoding the second fusion polypeptide has the sequence set forth in SEQ ID NO:51, or a variant thereof having at least 80% sequence identity. In some embodiments, the nucleic acid sequence encoding the first fusion polypeptide has the sequence set forth in SEQ ID NO:40, or a variant thereof having at least 80% sequence identity, and the nucleic acid sequence encoding the second fusion polypeptide has the sequence set forth in SEQ ID NO:52, or a variant thereof having at least 80% sequence identity.

[0074] As will be appreciated by those skilled in the art, due to the degeneracy of the genetic code, a vast 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 to the polynucleotide sequences described above 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 hybridize to the polynucleotides of the present invention under stringent conditions. In the present invention, "stringent conditions" refer 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 denaturing agent 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%, more preferably at least 95%. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.

[0075] The full-length nucleic acid sequences of the present invention or fragments thereof can generally be obtained by PCR amplification, recombination, or synthetic methods. One feasible method is to synthesize the relevant sequences by synthetic methods, especially when the fragment length is relatively short. Generally, very long fragments can be obtained by first synthesizing multiple small fragments and then ligating them. In addition, the coding sequence of the heavy chain can be fused with an expression tag (such as 6His) to form a fusion protein.

[0076] Once the relevant sequence is obtained, it can be obtained in large quantities using 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 propagated host cells by conventional methods. The biomolecules (nucleic acids, proteins, etc.) involved in the present invention include biomolecules in isolated form. Currently, DNA sequences encoding proteins of the present invention (or fragments thereof, or derivatives thereof) can be obtained entirely through chemical synthesis. This 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 sequences of the present invention through chemical synthesis.

[0077] The present invention also provides a nucleic acid construct (e.g., an expression cassette or vector) comprising at least one of the above nucleotide sequences. 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.

[0078] When the nucleotide sequence is known, each nucleotide molecule can be prepared using a method conventional in the art, and a corresponding vector can be constructed. Recombinant vectors can be constructed using methods well known to those skilled in the art, see, 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 vector can be reconstructed into liposomes for delivery to target cells. The vector containing the polynucleotide of the present invention can be transferred to a host cell by a well-known method, which varies according to the type of cell host. For example, calcium chloride transfection is commonly used for prokaryotic cells, while calcium phosphate treatment or electroporation can be used for other cell hosts, see Sambrook et al. (see above).

[0079] The vectors described herein contain the polynucleotides or expression cassettes described herein. The vectors can be plasmids, cosmids, viruses, or viral vectors. Vectors include cloning vectors, integrating vectors, and expression vectors. In addition to the nucleotide molecules described herein, expression vectors typically contain other elements typically found in vectors, such as multiple cloning sites, resistance genes, and replication origins. In one or more embodiments, the recombinant expression vector utilizes an AAV vector family as a backbone.

[0080] Expression vectors typically contain sequences for plasmid maintenance and for cloning and expressing exogenous nucleotide sequences. Such 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.

[0081] 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 the 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.

[0082] 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.

[0083] Selectable marker genes encode a protein that is essential for the survival and growth of host cells grown in selective culture media. Typical selectable marker genes encode (a) resistance to antibiotics or other toxins (e.g., ampicillin, tetracycline, or kanamycin for prokaryotic host cells); (b) supplement the auxotrophic deficiency of the cell; or (c) provide a protein that is not available from a complex or defined culture medium for an important nutrient. 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.

[0084] 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 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.

[0085] 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 genus Dependoparvovirus, family Parvoviridae. Adeno-associated virus is a single-stranded DNA virus that grows only in cells, where certain functions are provided by a co-infecting helper virus. 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 this 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 in 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.

[0086] As used herein, "AAV vector" refers to a vector comprising, consisting essentially of, or consisting of the coding sequence for one or more fusion polypeptides herein and one or more AAV inverted terminal repeats (ITRs). Such an AAV vector 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 the infectious AAV particles. The plasmid containing the AAV vector may also contain elements for manufacturing purposes, such as antibiotic resistance genes, etc.

[0087] 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 generally 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.

[0088] "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. Thus, the production of AAV particles necessarily includes the production of AAV vectors, as such vectors are contained within AAV particles.

[0089] As used herein, the term "helper" with respect to a virus or plasmid refers to a virus or plasmid that provides additional components necessary for replication and packaging of any of the AAV vectors disclosed herein. The components encoded by the helper virus can include any genes required for virion assembly, encapsidation, genome replication and / or packaging. For example, a helper virus or plasmid can encode enzymes 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).

[0090] 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, for example, US2007 / 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 helper plasmids (e.g., comprising rep genes (e.g., encoding Rep78, Rep68, Rep52, and Rep40) and cap genes (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.

[0091] In some embodiments, the 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.

[0092] host cells

[0093] The present invention also includes host cells that express the fusion polypeptides described herein, or contain the nucleotide or nucleic acid constructs described herein. Host cells can be used as recipients for vectors. Host cells can be "transfected" or "transformed," which refers to the process by which exogenous nucleic acids are transfected or transduced into the host cell. Transformed cells include the primary subject cell and its progeny. As used herein, the terms "engineered" and "recombinant" cells or host cells often refer to cells into which an exogenous nucleic acid sequence, such as a vector, has been introduced. Thus, recombinant cells can be distinguished from naturally occurring cells that do not contain the introduced recombinant nucleic acid.

[0094] 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.

[0095] 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 the gene sequence of the helper AAV replication from adenovirus. In some aspects, packaging / helper cells contain a plasmid 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.

[0096] The nucleic acid construct / recombinant expression vector of the present invention can be transferred into the cell of interest. The method of transfer is conventional in the art, including but not limited to: viral transduction, microinjection, particle bombardment, gene gun transformation and electroporation. In certain embodiments, electroporation is used to transfer the nucleic acid construct or recombinant expression vector. When the host cells are cultured under appropriate conditions, they synthesize the fusion protein, which can then be collected from the culture medium (if the host cells secrete it into the culture medium) or directly from the host cells that produce it (if it is not secreted).

[0097] Protein preparation method

[0098] 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 it is a temperature 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).

[0099] Method for preparing AAV viral vector

[0100] 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 helper plasmid and a cell line. The helper virus or helper plasmid contains elements and sequences that promote the production of the viral vector. 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 require additional transfection with the helper plasmid.

[0101] 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 HEK293 cell or a 293T cell. In one or more embodiments, the helper cell is a yeast cell or an insect cell.

[0102] 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 replication-incompetent AAV, and virion proteins for producing virions capable of packaging replication-incompetent AAV at high titers without producing replication-incompetent AAV.

[0103] Helper 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 therein, as a non-limiting example, an AAV helper plasmid may contain the Ad5 genes E2A, E4, and VA as helper viral DNA sequences, controlled by their respective native promoters or heterologous promoters. The AAV helper 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.

[0104] Provided herein are methods 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 are cells comprising any of the AAV vectors and / or AAV particles disclosed herein.

[0105] The present invention also provides a method for introducing a target gene into a subject's cells, comprising contacting the cells 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.

[0106] Pharmaceutical composition

[0107] Also provided herein are pharmaceutical compositions comprising the AAV vectors and / or AAV particles described herein. Typically, AAV particles are administered for therapy.

[0108] As described herein, pharmaceutical compositions can be formulated by any method known or developed in the art of pharmacology, including but not limited to contacting the active ingredient (e.g., viral particles or recombinant vectors) with excipients or other auxiliary ingredients, 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, modulation or enhanced translation of the encoded protein in vivo, and release profile of the encoded protein in vivo.

[0109] 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 combinations thereof. In some embodiments, the pharmaceutical composition is formulated as nanoparticles. In some embodiments, the nanoparticles are self-assembled nucleic acid nanoparticles.

[0110] 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, for example, 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 an excipient include a solvent, a dispersion medium, a diluent or other liquid vehicle, a dispersion or suspension aid, a surfactant, an isotonic agent, a thickener or an emulsifier, a preservative or a combination thereof.

[0111] 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.

[0112] Treatment

[0113] 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.

[0114] 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 disorder). 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 exhibit symptoms of the disease; (2) inhibiting the disease or arresting its development; or (3) ameliorating or causing regression of the disease or disease symptoms. As understood in the art, "treatment" is an approach used to obtain beneficial or desired results, including clinical results. Beneficial or desired results may include one or more of, but are not limited to, alleviation or amelioration of one or more symptoms, whether detectable or undetectable, lessening of 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).

[0115] 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 ed. (Mack Publ. Co., Easton).

[0116] A "subject" for 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 and includes non-human animals undergoing diagnosis or treatment and those undergoing infection or animal models, including but not limited to simian, murine, rat, canine, or lagomorphic species, as well as other livestock, sports animals, or pets. In some embodiments, the subject is a human.

[0117] As used herein, the term "effective amount" 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, 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 other embodiments, the effective amount of AAV viral particles is an amount sufficient to cause gene expression 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.

[0118] 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 of use. Those skilled in the art will be able to determine the effective amount based on these and other considerations. According to embodiments, the effective amount can include, consist essentially of, or consist of one or more administrations of the composition.

[0119] 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 therapeutic purpose, and the age, health, or sex of the subject being treated. Single or multiple administrations can be performed, with the dosage level and pattern being selected by the treating physician, or in the case of pets and other animals, by the treating veterinarian.

[0120] Dosage and administration

[0121] 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. 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 range from as little as 10E9 vector genomes to as much as 10E17 vector genomes per administration.

[0122] 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.

[0123] In some embodiments, the AAV particles repair a gene defect in a subject. In some embodiments, the ratio of repaired target polynucleotides (e.g., DFNB9) or polypeptides to unrepaired target polynucleotides or polypeptides in successfully treated cells, tissues, organs, or subjects 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 repaired target polynucleotides or polypeptides 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.

[0124] In some embodiments, the viral particles are introduced into the subject intravenously, intrathecally, intracerebrally, intraventricularly, intranasally, intratracheally, intraaurally, intraocularly or periocularly, orally, rectally, transmucosally, by inhalation, transdermally, parenterally, subcutaneously, intradermally, intramuscularly, intrapleurally, topically, intralymphatically, intracisternally; such introduction can 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.

[0125] For intraocular treatment of otological diseases, there are various modes of administration 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 injection.

[0126] 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.

[0127] Reagent test kit

[0128] 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.

[0129] In some embodiments, the test kit also includes instructions for use. Specifically, such a test kit can include one or more reagents as described herein, and instructions for the intended application and correct use of these reagents. In some embodiments, the test kit can include instructions for mixing one or more components of the test kit and / or separating and mixing samples and applying to a subject. In some embodiments, the reagent in the test kit is a pharmaceutical formulation and dosage suitable for a specific application and the method of administration of the reagent. The test kit can contain the components of the appropriate concentration or amount for carrying out various experiments for research purposes.

[0130] Kit can be designed to facilitate the use of the methods described herein, and can take many forms. Where applicable, every kind of composition of 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 processable in addition (e.g., processed into active form), for example, by adding suitable solvent or other substances (e.g., water or cell culture medium), which may or may not be provided together with kit. In some embodiments, composition can be provided in a preservation solution (e.g., a cryopreservation solution). The non-limiting examples of a preservation solution include DMSO, paraformaldehyde. In some embodiments, the preservation solution contains a certain amount of metalloproteinase inhibitors.

[0131] In some embodiments, the test kit contains any one or more components as 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 bottle or other container for storage. A second container can have other reagents of aseptic preparation. Alternatively, the test kit can include a premixed active agent and transported in a syringe, bottle, tube, or other container. The test kit can have one or more or all components required for administering the reagent to the subject, such as a syringe, a local application device, or an IV needle and bag.

[0132] Some specific implementation plan projects:

[0133] 1. A fusion polypeptide comprising a portion of an otoferlin protein and a portion of an intein system,

[0134] Preferably, the otoferlin protein is represented by SEQ ID NO: 1 or a sequence having at least 90% identity thereto.

[0135] 2. The fusion protein according to item 1, wherein the fusion polypeptide is the first fusion polypeptide or the second fusion polypeptide,

[0136] 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 of the intein (N-intein), wherein n is any integer from 750 to 1250.

[0137] 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 of the intein (C-intein), wherein n is any integer from 750 to 1250.

[0138] Preferably, the N-terminal fragment of the intein is shown as SEQ ID NO: 2 or a sequence having at least 90% identity thereto, and / or the C-terminal fragment of the intein is shown as SEQ ID NO: 3 or a sequence having at least 90% identity thereto.

[0139] 3. The fusion protein of item 1 or 2, wherein 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, and / or the second fusion polypeptide has a sequence as shown in any one of SEQ ID NOs: 17-28 or a variant thereof having at least 80% sequence identity.

[0140] 4. A polypeptide composition comprising the first fusion polypeptide and the second fusion polypeptide according to item 2 or 3.

[0141] 5. A polynucleotide comprising a sequence selected from the group consisting of:

[0142] (1) a nucleic acid sequence encoding the fusion polypeptide described in any one of items 1 to 3 or the polypeptide composition described in item 4,

[0143] (2) a variant having at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity with (1),

[0144] (3) The complementary sequence of (1) or (2).

[0145] 6. The polynucleotide of item 5, wherein 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, and / or 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.

[0146] 7. A nucleic acid construct, wherein:

[0147] (1) expressing the fusion polypeptide described in any one of items 1 to 3 or the polypeptide composition described in item 4, and / or

[0148] (2) comprising a sequence of the polynucleotide according to item 5 or 6,

[0149] Preferably, the nucleic acid construct is a vector, more preferably an AAV vector.

[0150] 8. The nucleic acid construct according to item 7, characterized in that

[0151] The nucleic acid construct comprises an expression cassette, and the polynucleotides encoding the first fusion polypeptide and the second fusion polypeptide are both located in the expression cassette, or

[0152] The nucleic acid construct comprises two expression cassettes, each comprising a polynucleotide encoding a first fusion polypeptide and a second fusion polypeptide.

[0153] Preferably, the nucleic acid construct is two AAV vectors, comprising polynucleotides encoding the first fusion polypeptide and the second fusion polypeptide, respectively, and one or more terminal inverted repeat sequences flanking the polynucleotides.

[0154] 9. The nucleic acid construct according to item 7 or 8, characterized in that the nucleic acid construct further comprises an element for expressing the polynucleotide contained therein,

[0155] Preferably, the nucleic acid construct has the sequence shown in SEQ ID NO:4.

[0156] 10. 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 comprise polynucleotides encoding a first fusion polypeptide and a second fusion polypeptide, wherein the first fusion polypeptide and the second fusion polypeptide are as described in item 2 or 3.

[0157] Preferably, the genes required for AAV virus include one or more selected from the following: Rep, Cap, E2A, E4 and VA genes,

[0158] Preferably, the AAV vector system comprises Anc80L65 plasmid, helper plasmid and the AAV vector.

[0159] 11. An rAAV virus particle comprising the polynucleotide according to item 5 or 6,

[0160] Preferably, 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.

[0161] 12. A host cell, wherein:

[0162] (1) comprising, expressing and / or secreting the fusion polypeptide described in any one of items 1 to 3 or the polypeptide composition described in item 4,

[0163] (2) comprising the polynucleotide according to item 5 or 6, the nucleic acid construct according to any one of items 7 to 9, or the AAV vector system according to item 10, or

[0164] (3) The polynucleotide according to item 5 or 6 is integrated into the chromosome,

[0165] Preferably, the host cell is a HEK293 cell.

[0166] 13. A method for preparing an 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 item 2 or 3.

[0167] Preferably, the method comprises the step of incubating the host cell according to item 12 under conditions suitable for intein splicing.

[0168] 14. Use of the fusion polypeptide described in any one of items 1 to 3, the polypeptide composition described in item 4, the polynucleotide described in item 5 or 6, the nucleic acid construct described in any one of items 7 to 9, or the host cell described in item 12 in the preparation of a medicament for treating a disease caused by an Otof gene defect.

[0169] Preferably, the disease is DFNB9 hearing impairment.

[0170] 15. A pharmaceutical composition comprising a pharmaceutically acceptable excipient and one or more selected from the following: the fusion polypeptide described in any one of items 1-3, the polypeptide composition described in item 4, the polynucleotide described in item 5 or 6, the nucleic acid construct described in any one of items 7-9, the rAAV vector system described in item 10, the rAAV virus particle described in item 11, and the host cell described in item 12.

[0171] The present invention will be described below by way 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, conventional materials and methods in the art.

[0172] Example

[0173] Example 1: Screening of cleavage sites of full-length otoferlin protein in HEK-293T cells

[0174] 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 dual 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 KingScript 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 efficiency of trans-splicing and processing mediated by the intein, the expression of full-length OTOF was analyzed by Western blotting 48 hours after transfection.

[0175] 1.1 Cell transfection

[0176] 1) Seed HEK-293T cells to 70-90% density and prepare for transfection;

[0177] 2) Tube A: 125 μL serum-free medium + 8 μL Lipofectamine 3000 reagent, mix thoroughly;

[0178] 3) Tube B: 125 μL serum-free medium + DNA + 8 μL P3000 reagent, mix thoroughly;

[0179] The cut points of each group correspond to the following (corresponding to the WB lane number), and the cells were transfected with the same molar number of 2.72517E-10.

[0180] Among them, the sequences of the first fusion polypeptides containing splice donor sequences in 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 in these groups are shown in SEQ ID NOs: 17-28;

[0181] 4) Add the mixture in tube B to tube A and mix gently and thoroughly;

[0182] 5) Let stand at room temperature for 10-15 minutes;

[0183] 6) Adding DNA-liposome complexes to cells;

[0184] 7) Incubate at 37°C, 95% air and 5% CO2, and collect samples 48 hours after transfection.

[0185] 1.2 Protein extraction

[0186] 1) Remove the culture medium and wash 2-3 times with 1 mL of PBS.

[0187] 2) Gently disperse the cells with 1 mL of PBS, collect the cell suspension into a centrifuge tube, centrifuge at 700 g for 5 minutes, and discard the supernatant;

[0188] 3) Add 120 μL (per well of a six-well plate) of RIPA lysis buffer (add Cocktail (100×) before use) and lyse the cells on ice for 30 min. Vortex every 10 min to fully resuspend the cell pellet in the lysis buffer.

[0189] 4) Finally, centrifuge at 12,000 rpm for 15 minutes and collect the supernatant (do not aspirate the precipitate);

[0190] 5) Add 5× Sample Loading Buffer (GenScript) to the supernatant, mix thoroughly, and incubate in a 75°C metal bath for 15 minutes. Centrifuge at 12,000 rpm for 2 minutes before loading the sample (15 μL per well of a 15-well gel).

[0191] 1.3 Western Blot

[0192] 1.3.1 Electrophoresis

[0193] 1)1x running buffer preparation: 950ml ddH2O+50ml 20x running buffer;

[0194] 2) Install the electrophoresis apparatus, add 1x running buffer, and check for leaks.

[0195] 3) Carefully remove the comb, apply a constant voltage of 100V for 1 hour (Biyuntian 4-12%, HEPES system).

[0196] 1.3.2 Transfer

[0197] 1) Prepare 1x transfer buffer in advance and pre-cool.

[0198] 1x transfer buffer preparation: 700ml water + 200ml ethanol + 100ml 10x transfer buffer

[0199] 2) Remove the glue, separate the two pieces of glass with a spatula, and cut off the upper layer of glue;

[0200] 3) Place the lower layer of gel in transfer buffer for equilibrium;

[0201] 4) Soak the PVDF membrane in methanol (activation for 3 minutes) and place it in transfer buffer;

[0202] 5) Place the transfer clamp with the black side facing down and arrange the membrane in the following order: sponge - 1 layer of filter paper - glue - PVDF membrane - 1 layer of filter paper - sponge. Use a spatula to remove air and clamp tightly (avoid bubbles).

[0203] 6) After installing the transfer device, pour the transfer buffer into the electrophoresis tank;

[0204] 7) Transfer the membrane on ice at a constant current of 300 mA for 90 min.

[0205] 1.3.3 Membrane washing

[0206] 1) Disassemble the device and take out the PVDF membrane; cut the membrane according to the size of the target protein and mark it;

[0207] 2) Place the cut PVDF membrane in an incubation box, add 5% skim milk (prepared with TBST), and block on a shaker at room temperature for 1 hour;

[0208] 1.3.4 Antibody incubation

[0209] 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 at 4°C on a shaker;

[0210] 2) Recover the antibody and wash with TBST 3 times, 10-15 minutes each time

[0211] 3) Add secondary antibody HRP-conjugated Affinipure Goat Anti-Mouse IgG (H+L) (1:5000) and incubate on a shaker at room temperature for 1-2 hours

[0212] 4) Recover the antibody and wash with TBST three times, 10-15 min each time.

[0213] 1.3.5 Develop the sample using Novozyme developer, observe the results using a Western imaging system, and take photos.

[0214] The results are shown in Figure 4: the control is untransfected HEK-293T cells; Western blotting shows expression of full-length otoferlin protein in lanes 1, 3, 4, 5, 6, 8, 10, 13, 14, 15, 17, 18, 19, 20, 23, 27, 29, and 30.

[0215] Example 2: Auditory function analysis and cochlear morphology analysis of Otof gene knockout mice

[0216] 2.1 Auditory function testing of Otof knockout mice

[0217] 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.

[0218] 1) Weigh the mice and inject 1% sodium pentobarbital intraperitoneally according to the appropriate mass-to-volume ratio.

[0219] 2) After the mouse is anesthetized, place it in a soundproof box at the TDT workstation. Insert the positive electrode of the electrode subcutaneously at the midline of the mouse's brain, the negative electrode subcutaneously behind the ear, and the ground wire into the thigh muscle. Check the electrode indicators to confirm proper connection.

[0220] 3) Open the test file on the computer, select the test frequency of 4-32kHz, and record the critical threshold above the noise.

[0221] The hearing thresholds of mice were tested for 10ms tone bursts of 4, 8, 12, 16, 24, and 32 kHz. This allowed us to analyze the mice's hearing sensitivity and assess whether their hearing function, from hair cells to the cerebral cortex, was normal. ABR and DPOAE are excellent indicators of cochlear hair cell integrity and cochlear function.

[0222] 2.2 Electrophysiological function analysis of hair cells in Otof knockout mice

[0223] The patch clamp technique and the whole-cell extracellular recording method were used to record the electrochemical changes of the hair cells in the cochlea of ​​Otof gene knockout mice, so as to detect the changes in the electrophysiological function of the hair cells in Otof gene knockout mice.

[0224] 2.3 Detection of hair cell number in Otof knockout mice

[0225] Otoferlin is highly expressed in the inner hair cells of the mouse cochlea, but is expressed at a low level in the outer hair cells. Immunofluorescence staining was used to determine the number of surviving inner hair cells in Otof knockout mice at different time points, including P0, P7, P14, and P30.

[0226] 1) Excise the entire ear tissue of the mouse and place it in 1× HBSS. Dissect the temporal bone under a microscope and place it in 4% PFA. Use forceps to gently punch a hole in the top of the temporal bone and repeatedly blow PFA into it with a syringe. Incubate on a shaker for 1-2 hours.

[0227] 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.

[0228] 3) Rinse with 1×PBST three times.

[0229] 4) Place the temporal bone in 1× HBSS and dissect out the cochlea under a microscope.

[0230] 5) Place the coverslip coated with Cell-tak into the dish containing the cochlea and adhere the cochlea to the slide (front side up).

[0231] 6) Place the adhered slides into a 4-well dish (previously filled with 3 mL of PBS). After all adhered slides are placed into the 4-well dish, add 1 mL of 16% PFA.

[0232] 7) Fix at room temperature for 1 hour and wash three times with PBST.

[0233] 8) Add 100 μL blocking medium to each well and block for 1 hour.

[0234] 9) Dilute the primary antibody in PBT-1 in proportion, add 80-100 μL per well, and incubate at 4°C overnight.

[0235] 10) Wash three times with 1×PBST, 5 min each time.

[0236] 11) Dilute the secondary antibody in PBT-2 in proportion, add 80-100 μL per well, and incubate at room temperature in the dark for 1 hour.

[0237] 12) Wash three times with 1× PBST, 5 min each time.

[0238] 13) Remove the slides by clipping them onto a glass slide. Add 6 μL of DAKO to each slide, cover with a new slide, seal the slide with nail polish, and observe and photograph.

[0239] Example 3: Effect Verification

[0240] 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 ( Figure 4 , D), were selected to construct double AAV viruses.

[0241] 1) Prepare HEK-293T cells: subculture at a ratio of 1:3 and culture in 150 mm sterile dishes until the cell density reaches 80%-90%.

[0242] 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).

[0243] 3) Prepare three sterile 15 ml EP tubes, label the first one as tube A. Add 3.444 ml of DMEM and 0.756 ml of PEI (1 μg / μl) to tube A and let it stand for 5 minutes.

[0244] 4) Label the remaining two tubes B1 and B2, add 7 μg of N-Otof or C-Otof-HA plasmid, 9 μg of Anc80L65 plasmid, and 14 μg of helper plasmid, respectively, and add DMEM to 2.1 ml.

[0245] 5) Add the liquid in tube A to tubes B1 and B2 dropwise, 2.1 ml per tube. Let stand at room temperature for 20-25 minutes. Then add the mixture to the prepared HEK-293T cells.

[0246] 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.

[0247] 7) After another 48 hours, the cells and supernatant were collected into the aforementioned sterile bottles for virus purification and titer testing. The titer was determined by viral inverted terminal repeat-specific qPCR.

[0248] Virus purification steps are as follows:

[0249] 1) Place the collected supernatant and cells in a 50 mL centrifuge tube (chloroform-resistant) and centrifuge for 7 minutes at 11,000 rpm and 4°C. Aspirate the supernatant and reserve a portion to resuspend the cell pellet. Add 1 / 10 volume of chloroform and shake at 37°C and 220 rpm for 3 hours.

[0250] 2) Incubate at 11000 r.p.m., 4°C, for 20 min. Collect the supernatant and mix it with the supernatant collected in the previous step.

[0251] 3) Add solid NaCl to a concentration of 1 mol / L and add PEG8000 to a final concentration of 10% (w / v), dissolve completely, and let stand at 4°C overnight.

[0252] 4) Centrifuge at 11,000 rpm, 4°C for 20 min. Discard most of the supernatant, leaving 1-2 mL to resuspend all the precipitate. Transfer the remaining precipitate to several new 2 mL EP tubes. Centrifuge the 2 mL EP tubes containing the precipitate at 12,000 rpm for 7 min.

[0253] 5) Add 700 μl of enzymatic solution (containing DNase I and RNase) to the pellet and incubate in a 37°C water bath for 1 hour. Mix by pipetting several times during this time.

[0254] 6) Add an equal volume of chloroform, shake vigorously, centrifuge at 12000 rpm for 7 min, and collect the supernatant.

[0255] 7) Add an equal volume of virus dissolution solution (20% PEG8000+2M Nacl+0.002% F68+PBS) and incubate at 4°C overnight.

[0256] 8) Centrifuge at 12000 rpm for 7 min, discard the supernatant, and centrifuge again for 3 min, discard the supernatant.

[0257] 9) Dissolve the precipitate with a solution containing DNase I 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.

[0258] 10) Place the open 1.5ml EP tube containing the virus at room temperature for 30 minutes, then temporarily place it in a 4°C refrigerator for subsequent titer testing.

[0259] The titer detection steps are as follows:

[0260] 1) Digest genomic DNA and plasmid DNA by incubating at 37°C for 20 min and 95°C for 10 min using the following system:

[0261] (37°C digestion of genomic DNA and plasmid DNA; 95°C heat treatment to inactivate DNase I)

[0262] 2) Add an equal volume of lysis buffer (10 μl) and 2 μl of 20 mg / ml proteinase K, and incubate at 55°C for 30 min and 95°C for 10 min. (Lysis buffer: 10 mM Tris-HCl pH 8.0 + 5 mM EDTA + 100 μg / ml proteinase K) (Stand at room temperature)

[0263] Take 4 μl of the PCR product, dilute it 100-fold with ddH2O to 400 μl, and use 2 μl as the template for RT-QPCR. The system is as follows:

[0264] 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;

[0265] Titer calculation formula: y = 38.71-3.54x; y = CT; titering = 1000000*power(10, x)

[0266] AAV-Otof-NT and AAV-Otof-CT recombinant vector viruses (8.5 e 12 +8.5e 12 vg / mL (ratio 1:1, total virus volume 3.4x10 10 vg)) was injected into the left cochlea of ​​adult Otof- / - mice around P30. The mice were anesthetized with an injection of 1% sodium pentobarbital. After anesthesia, the periauricular hair was removed, an incision was made behind the ear to expose the semicircular canals, and the semicircular canals were punctured and the injection was performed manually with a glass micropipette. After the injection, the skin incision was sealed with tissue glue. The injected mice were then placed on a 37°C heating pad to recover. After the surgery, the adult mice fully recovered in about 30 minutes and were returned to their cages. Standard postoperative care was provided. Two weeks later, the mice were tested for ABR to assess the impact of the injection on their overall hearing.

[0267] To determine whether and to what extent a dual-vector system using AAV vectors encoding N-Otof with N-Intein and C-Otof with C-Intein could restore hearing loss, auditory brainstem response (ABR) was used to assess auditory function recovery in mice of different ages after gene therapy. The sensory epithelium of the cochlea of ​​Otof- / - virus-injected mice after audiometry was microdissected and immunolabeled for otoferlin (inner hair cells) and Myosin7a (hair cells) to investigate the recombinant expression of the exogenous Otof gene in the cochlear cells of KO mice.

[0268] 1) Excise the entire ear tissue of the mouse and place it in 1× HBSS. Dissect the temporal bone under a microscope and place it in 4% PFA. Use forceps to gently punch a hole in the top of the temporal bone and repeatedly blow PFA into it with a syringe. Fix at room temperature for 1-2 hours.

[0269] 2) Decalcify the temporal bones by immersion in 0.5 M EDTA: P0-P7, decalcify for 3-4 hours; P8-P15, decalcify for 1 day; P15-P60, decalcify for 2 days.

[0270] 3) Rinse with 1×PBST three times.

[0271] 4) Place the temporal bone in 1× HBSS and dissect the cochlea under a microscope. Divide the entire sensory epithelium into three segments of equal length, labeled as the apex, middle, and base of the cochlea, and store in HBSS until staining.

[0272] 5) Place the coverslip coated with Cell-tak into the dish containing the cochlea and adhere the cochlea to the slide (front side up).

[0273] 6) Place the adhered slides in a 4-well dish (previously filled with 3 mL of PBS). After all adhered slides are placed in the 4-well dish, permeabilize and block the wells with PBS containing 1% Triton X-100 and 10% donkey serum for 1 hour at room temperature.

[0274] 7) Use PBT-1 to dilute the primary antibodies in proportion, otoferlin antibody (ab53233, 1:200, Abcam), Myosin7a antibody (256790, 1:1000, Proteus), 80-100 μl per well, and incubate at 4°C overnight.

[0275] 8) Wash three times with 1× PBST, 5 min each time.

[0276] 9) Dilute the corresponding secondary antibody in PBT-2 in proportion, add 80-100 μl per well, and incubate at room temperature in the dark for 1 hour.

[0277] 10) Wash three times with 1×PBST, 5 min each time.

[0278] 11) Remove the slides from the holder 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.

[0279] To explore the role of the Otof gene in mouse inner ear development, we first needed to examine Otof expression in the inner ear. Otof is abundantly expressed in the inner ear. To further examine the specific expression site and temporal profile of Otof in the basilar membrane of the inner ear, we performed immunofluorescence staining on wild-type mice at different time points. We also used immunofluorescence staining of the basilar membrane of the cochlea to localize Otof expression in the inner ear. Myosin7a was used as a marker for hair cells. We selected three developmental time points: P0, P7, P14, and P30. At the hair cell level, OTOF colocalized with Myosin7a in basilar membrane flat-mounts at all three developmental time points, indicating that OTOF is expressed in the cytoplasm of hair cells. These experiments demonstrate that OTOF is abundantly expressed in the inner ear, primarily in hair cells.

[0280] We ordered Otof knockout mice from Saiye Biotechnology Co., Ltd. for breeding. To ensure that the Otof protein in the inner ear of the Otof knockout mice was indeed knocked out, we first screened homozygous, heterozygous, and wild-type mice through genotyping. We first tested this goal by immunofluorescence staining. We used WT and Otof- / - mice at different time points, including P0, P7, P14, and P30. The immunofluorescence staining results showed that in wild-type mice, Otof was abundantly expressed in the hair cell cuticle layer, but OTOF expression was no longer detectable in the hair cell cuticle layer of the inner ear of Otof- / - mice. This indicates that the OTOF protein in the inner ear of the Otof- / - mice we used later was completely knocked out.

[0281] Then we tested the hearing status of the Otof knockout mice. First, we used the ABR audiological test 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 some hearing frequency bands were significantly restored; the ABR results showed that after the Otof gene was knocked out, the hearing threshold of the mice increased significantly at low, medium and high frequencies, indicating that Otof plays an important role in the hearing of mice. The dual AAV system of the present 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.

[0282] Two weeks after treatment, the cochlea of ​​the treated adult Otof- / - knockout mouse model was harvested and basal membrane staining was performed. The results are shown in Figure 6. Immunofluorescence results showed that both AAV systems achieved varying degrees of otoferlin protein expression in the mouse cochlea.

Claims

1. A fusion polypeptide comprising a portion of an otoferlin protein and a portion of an intein system, Preferably, the otoferlin protein is represented by SEQ ID NO: 1 or a sequence having at least 90% identity thereto.

2. The fusion protein according to claim 1, wherein The fusion polypeptide is the first fusion polypeptide or the second fusion polypeptide, 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 of the intein, wherein n is any integer from 750 to 1250, 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, the N-terminal fragment of the intein is shown as SEQ ID NO: 2 or a sequence having at least 90% identity thereto, and / or the C-terminal fragment of the intein is shown as SEQ ID NO: 3 or a sequence having at least 90% identity thereto.

3. The fusion protein according to claim 1 or 2, wherein 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, and / or the second fusion polypeptide has a sequence as shown in any one of SEQ ID NOs: 17-28 or a variant thereof having at least 80% sequence identity. 4 . A polypeptide composition comprising the first fusion polypeptide and the second fusion polypeptide according to claim 2 .

5. A polynucleotide comprising a sequence selected from the group consisting of: (1) a nucleic acid sequence encoding the fusion polypeptide according to any one of claims 1 to 3 or the polypeptide composition according to claim 4, (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).

6. The polynucleotide according to claim 5, wherein 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, and / or 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.

7. A nucleic acid construct, wherein: (1) expressing the fusion polypeptide according to any one of claims 1 to 3 or the polypeptide composition according to claim 4, and / or (2) comprising the sequence of the polynucleotide according to claim 5 or 6, Preferably, the nucleic acid construct is a vector, more preferably an AAV vector.

8. The nucleic acid construct according to claim 7, 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, each comprising a polynucleotide encoding a first fusion polypeptide and a second fusion polypeptide, Preferably, the nucleic acid construct is two AAV vectors, respectively comprising polynucleotides encoding the first fusion polypeptide and the second fusion polypeptide, and one or more terminal inverted repeat sequences flanking the polynucleotides.

9. The nucleic acid construct according to claim 7 or 8, wherein The nucleic acid construct also includes elements for expressing the polynucleotide contained therein, Preferably, the nucleic acid construct has the sequence shown in SEQ ID NO:

4.

10. A rAAV vector system, comprising two AAV vectors and a nucleic acid construct encoding a gene required by the AAV virus, wherein the two AAV vectors respectively contain polynucleotides encoding a first fusion polypeptide and a second fusion polypeptide, wherein the first fusion polypeptide and the second fusion polypeptide are as described in claim 2 or 3, Preferably, the genes required for the AAV virus include one or more selected from the following: Rep, Cap, E2A, E4 and VA genes, Preferably, the AAV vector system comprises Anc80L65 plasmid, helper plasmid and the AAV vector.

11. A rAAV virus particle comprising the polynucleotide according to claim 5 or 6, Preferably, 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.

12. A host cell, wherein: (1) comprising, expressing and / or secreting the fusion polypeptide according to any one of claims 1 to 3 or the polypeptide composition according to claim 4, (2) comprising the polynucleotide of claim 5 or 6, the nucleic acid construct of any one of claims 7 to 9, or the AAV vector system of claim 10, or (3) the polynucleotide according to claim 5 or 6 is integrated into the chromosome, Preferably, the host cell is a HEK293 cell.

13. 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 claim 2 or 3, Preferably, the method comprises the step of incubating the host cell of claim 12 under conditions suitable for intein splicing.

14. Use of the fusion polypeptide according to any one of claims 1 to 3, the polypeptide composition according to claim 4, the polynucleotide according to claim 5 or 6, the nucleic acid construct according to any one of claims 7 to 9, and the host cell according to claim 12 in the preparation of a drug for treating a disease caused by Otof gene defect, Preferably, the disease is DFNB9 hearing impairment.

15. A pharmaceutical composition comprising a pharmaceutically acceptable excipient and one or more selected from the following: the fusion polypeptide of any one of claims 1-3, the polypeptide composition of claim 4, the polynucleotide of claim 5 or 6, the nucleic acid construct of any one of claims 7-9, the rAAV vector system of claim 10, the rAAV virus particle of claim 11, and the host cell of claim 12.

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