Compositions and methods for expressing Otoferlin

Abstract

To provide methods and compositions for expressing Otoferlin, e.g., utilizing adeno-associated viral (AAV) particles.SOLUTION: Such methods and compositions may be useful for treatment of diseases such as deafness, autosomal recessive 9 (DFNB9).

Description

[Technical Field]

[0001] Cross-references to related applications This application claims priority to U.S. Provisional Patent Application No. 62 / 502,462, filed on 5 May 2017, the full disclosure of which is incorporated herein by reference. Research funded by the federal government This invention was made with government support under authorization numbers EY000331, EY021721, and DC012118, granted by the National Institutes of Health. The government has certain rights to the invention. [Background technology]

[0002] Background of the Invention Non-symptomatic hearing loss is a type of hearing loss generally caused by defects or damage to the inner and / or middle ear. Mutations in the OTOF gene, which encodes the protein otoferrin, are thought to cause a type of non-symptomatic hearing loss called "hearing loss, autosomal recessive 9 (DFNB9)." Treatment for DFNB9 and other similar forms of hearing loss currently involves cochlear transplantation for severe or profound hearing loss and the use of hearing aids for milder forms of hearing loss. There remains a need for alternative forms of treatment that do not rely, or rely less, on electronic devices to restore hearing. [Overview of the project]

[0003] Provided herein are compositions and methods for expressing otoferlin, for example, in cells or subjects. As described herein, delivery of OTOF cDNA to OTOF knockout mice via a dual adeno-associated virus (AAV) system containing different portions of OTOF cDNA has been found to be able to rescue the mice's hearing to near wild-type levels.

[0004] In some respects, the present disclosure relates to a method for increasing the expression of otoferlin in cells, the method comprising: contacting cells with a first AAV particle comprising a first polynucleotide; and contacting cells with a second AAV particle comprising a second polynucleotide, wherein the first polynucleotide comprises a reverse-terminal repeat sequence adjacent to an expression cassette containing, 5' to 3': (a) a promoter, (b) a partial coding sequence encoding the N-terminal portion of the otoferlin polypeptide, (c) a splice donor site, and (d) a first homologous region containing a sequence homologous to the sequence in the second polynucleotide; and the second polynucleotide comprises a reverse-terminal repeat sequence adjacent to an expression cassette containing, 5' to 3': (a) a second homologous region containing a sequence homologous to the sequence in the first polynucleotide, (b) a splice acceptor site, (c) a partial coding sequence encoding the C-terminal portion of the otoferlin polypeptide, and (d) a polyadenylation (pA) signal sequence.

[0005] In some embodiments, the homologous regions in the first and second polynucleotides are between 50 and 500 nucleotides. In some embodiments, the homologous regions in the first and second polynucleotides are between 50 and 300 nucleotides. In some embodiments, the homologous regions include the nucleotide sequence of SEQ ID NO: 3. In some embodiments, the promoter is a chimeric CMVβ-actin (smcBA) promoter. In some embodiments, the promoter includes the sequence of SEQ ID NO: 4. In some embodiments, the otoferlin polypeptide includes the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6. In some embodiments, the splice donor site includes the sequence of SEQ ID NO: 7. In some embodiments, the splice acceptor site includes the sequence of SEQ ID NO: 8. In some embodiments, the reverse terminal repeat sequence is an AAV2 reverse terminal repeat sequence. In some embodiments, the first and second AAV particles are AAV2 serotype particles. In some embodiments, the cells are ex vivo. In some embodiments, the cells are in vivo. In some embodiments, the cells are in a mammalian subject. In some embodiments, the subjects have hearing loss and autosomal recessive 9 (DFNB9).

[0006] In other aspects, the Disclosure provides a composition comprising a first AAV particle comprising a first polynucleotide; and a second AAV particle comprising a second polynucleotide, wherein the first polynucleotide comprises a reverse terminal repeat sequence adjacent to an expression cassette containing, from 5' to 3': (a) a promoter, (b) a partial coding sequence encoding the N-terminal portion of an otoferlin polypeptide, (c) a splice donor site, and (d) a first homologous region containing a sequence homologous to the sequence in the second polynucleotide; and the second polynucleotide comprises a reverse terminal repeat sequence adjacent to an expression cassette containing, from 5' to 3': (a) a second homologous region containing a sequence homologous to the sequence in the first polynucleotide, (b) a splice acceptor site, (c) a partial coding sequence encoding the C-terminal portion of an otoferlin polypeptide, and (d) a polyadenylation (pA) signal sequence.

[0007] In some embodiments, the homologous regions in the first and second polynucleotides are between 50 and 500 nucleotides. In some embodiments, the homologous regions in the first and second polynucleotides are between 50 and 300 nucleotides. In some embodiments, the homologous region comprises the nucleotide sequence of SEQ ID NO: 3. In some embodiments, the promoter is the chimeric CMVβ-actin (smcBA) promoter. In some embodiments, the promoter comprises the sequence of SEQ ID NO: 4. In some embodiments, the otoferlin polypeptide comprises the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6. In some embodiments, the splice donor site comprises the sequence of SEQ ID NO: 7. In some embodiments, the splice acceptor site comprises the sequence of SEQ ID NO: 8. In some embodiments, the inverted terminal repeat is the AAV2 inverted terminal repeat. In some embodiments, the first and second AAV particles are AAV2 serotype particles. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier.

[0008] In yet another aspect, the disclosure provides a kit comprising a composition as described herein or a first AAV particle as described herein and a second AAV particle as described herein. These and other aspects are described in more detail herein.

[0009] Brief Description of the Drawings The following drawings form a part of this specification and are included to further demonstrate certain aspects of the present disclosure, which can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

Brief Description of the Drawings

[0011] [Figure 3A-1] Figure 3A shows the annotated sequence of the expression cassette containing the inverted terminal repeat (TR) for the plasmid in Figure 1A. [Figure 3A-2] Figure 3A shows the annotated sequence of the expression cassette containing the inverted terminal repeat (TR) for the plasmid in Figure 1A. [Figure 3A-3] Figure 3A shows the annotated sequence of the expression cassette containing the inverted terminal repeat (TR) for the plasmid in Figure 1A. [Figure 3B-1] Figure 3B shows the annotated sequence of the expression cassette containing the inverted terminal repeat (TR) for the plasmid in Figure 1B. [Figure 3B-2] Figure 3B shows the annotated sequence of the expression cassette containing the inverted terminal repeat (TR) for the plasmid in Figure 1B. [Figure 3B-3] Figure 3B shows the annotated sequence of the expression cassette containing the inverted terminal repeat (TR) for the plasmid in Figure 1B.

[0012] [Figure 4] Figure 4 is a series of photographs showing the expression of the OTOF protein in HEK 293 cells treated with AAV2-OTOF-NT (AAV-NT), or AAV2-OTOF-NT and AAV2-OTOF-CT (AAV2-NT+CT). [Figure 5] Figure 5 is a series of photographs showing GFP expression in surface preparations of the organ of Corti in the cochlea from wild-type mice treated with AAV2-GFP. [Figure 6A-6B] Figures 6A–D are a series of photographs and graphs showing OTOF expression in the cochlea of ​​P1–P3 mice. Figure 6A shows OTOF protein expression during mid-turn. Figure 6B shows OTOF protein expression at the apex. [Figure 6C-6D] Figure 6C shows the differences in basal, middle-turn, and apical OTOF expression between wild-type mice (WT, n=6) and OTOF knockout mice treated with AAV2-OTOF-NT and AAV2-OTOF-CT (Res. KO NT+CT, n=6). In each pair of bars, the left bar represents WT, and in each pair of bars, the right bar represents Res. KO NT+CT. Figure 6D shows RT-PCR results of OTOF mRNA in wild-type (WT), OTOF knockout mice (KO), and OTOF knockout mice treated with AAV2-OTOF-NT and AAV2-OTOF-CT (Res. KO).

[0013] [Figure 7A] Figures 7A-D show auditory evaluations in mice. Figure 7A is a trace of auditory brainstem response (ABR) patterns induced by auditory stimulation in OTOF knockout mice, which are either wild-type mice (WT), untreated mice (KO / KO NT), or mice treated with AAV2-OTOF-NT and AAV2-OTOF-CT (rescue KO). [Figure 7B]Figure 7B shows the auditory brainstem response (ABR) thresholds in wild-type mice (WT), untreated otoferlin knockout mice (KO), otoferlin knockout mice treated with AAV2-OTOF-NT and AAV2-OTOF-CT (Res KO NT+CT), and otoferlin knockout mice treated with AAV2-OTOF-NT (KO +NT). [Figure 7C] Figure 7C shows the time course of hearing recovery in wild-type mice (WT), untreated OTOF knockout mice (KO), otoferlin knockout mice treated with AAV2-OTOF-NT and AAV2-OTOF-CT (rescue KO NT+CT), and otoferlin knockout mice treated with AAV2-OTOF-NT (KONT). [Figure 7D] Figure 7D shows the click ABR thresholds in wild-type mice (WT), untreated otoferlin knockout mice (KO), otoferlin knockout mice treated with AAV2-OTOF-NT and AAV2-OTOF-CT (Res. KO(NT+CT)), and otoferlin knockout mice treated with AAV2-OTOF-NT (KO+NT).

[0014] [Figures 8A-8B] Figures 8A and 8B show otoferlin protein expression in endohair cells of OTOF-rescue KO mice. Figure 8A shows OTOF protein expression in P12 and older mice treated with AAV2-OTOF-NT and AAV2-OTOF-CT. Figure 8B shows the percentage of endohair cells expressing OTOF in wild-type mice (WT, n=5) and OTOF knockout mice treated with AAV2-OTOF-NT and AAV2-OTOF-CT (rescue KO, n=5). In each pair of bars, the left bar represents WT, and in each pair of bars, the right bar represents rescue KO. [Figure 9A-9B]Figures 9A and 9B are a series of graphs showing hearing assessments. Figure 9A shows the ABR threshold values ​​in wild-type mice (WT), OTOF knockout mice (KO), and OTOF knockout mice treated with AAV2-OTOF-NT and AAV2-OTOF-CT (rescue KO). Figure 9B shows the hearing lifespan in WT, KO, and rescue KO mice. [Figure 10] Figure 10 is a plasmid map containing the CMV enhancer, chicken beta-actin promoter, 5' section of human otoferrin cDNA (otoferrin NT), splice donor sequence (APSD), homologous sequence for recombination (APhead), and AAV2 reverse terminal repeat (TR) adjacent to it. [Figure 11] Figure 11 is a plasmid map containing the homologous sequence for recombination (APhead), the splice acceptor sequence (APSA), the 3' section of human otoferlin cDNA encoding an isoform of otoferlin 1 (otoferlin CT), the bovine growth hormone polyadenylation signal (bGH polyA), and the AAV2 reverse terminal repeat (TR) adjacent to it.

[0015] [Figure 12] Figure 12 is a plasmid map containing the homologous sequence for recombination (APhead), the splice acceptor sequence (APSA), the 3' section of mouse otoferrin cDNA encoding an isoform of otoferrin 5 (otoferrin CT), the bovine growth hormone polyadenylation signal (bGH polyA), and the AAV2 reverse terminal repeat (TR) adjacent to it. [Figure 13-1] Figure 13 shows the annotated sequence of the human OTOF N-terminal expression cassette, including the reverse terminal repeat (TR), for the plasmid in Figure 10. [Figure 13-2] Figure 13 shows the annotated sequence of the human OTOF N-terminal expression cassette, including the reverse terminal repeat (TR), for the plasmid in Figure 10. [Figure 13-3]Figure 13 shows the annotated sequence of the human OTOF N-terminal expression cassette, including the reverse terminal repeat (TR), for the plasmid in Figure 10.

[0016] [Figure 14-1] Figure 14 shows the annotated sequence of the human OTOF C-terminal expression cassette for isoform 1, including the reverse terminal repeat (TR), for the plasmid in Figure 11. [Figure 14-2] Figure 14 shows the annotated sequence of the human OTOF C-terminal expression cassette for isoform 1, including the reverse terminal repeat (TR), for the plasmid in Figure 11. [Figure 14-3] Figure 14 shows the annotated sequence of the human OTOF C-terminal expression cassette for isoform 1, including the reverse terminal repeat (TR), for the plasmid in Figure 11. [Figure 15-1] Figure 15 shows the annotated sequence of the human OTOF C-terminal expression cassette for isoform 5, including the reverse terminal repeat (TR), for the plasmid in Figure 12. [Figure 15-2] Figure 15 shows the annotated sequence of the human OTOF C-terminal expression cassette for isoform 5, including the reverse terminal repeat (TR), for the plasmid in Figure 12. [Figure 15-3] Figure 15 shows the annotated sequence of the human OTOF C-terminal expression cassette for isoform 5, including the reverse terminal repeat (TR), for the plasmid in Figure 12. [Modes for carrying out the invention]

[0017] Detailed description of the invention As described herein, it has been found that hearing in otoferlin knockout mice can be restored by treating the mice with two separate AAV particles, one containing the 5' portion of OTOF cDNA and the other containing the 3' portion of OTOF cDNA, each containing a homologous region to promote homologous recombination between the 5' and 3' portions in vivo. This homologous region is flanked by a splice donor sequence on the 5' side of the 5' portion of OTOF cDNA and a splice acceptor sequence on the 3' side of the 3' portion of OTOF cDNA. Thus, compositions and methods are provided to increase otoferlin expression.

[0018] Definition of an example Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which this invention pertains. Any methods and materials similar to or equivalent to those described herein may be used for carrying out or testing the present invention, but preferred methods and materials are described herein. For the purposes of this invention, the following terms are defined below:

[0019] As used herein, the terms “nucleic acid” and “polynucleotide sequence” refer to deoxyribonucleotides or ribonucleotide polymers, either single-stranded or double-stranded, and encompass known analogs of naturally occurring nucleotides that can function in the same way as naturally occurring nucleotides, unless otherwise specified.

[0020] The terms “substantially corresponding,” “substantially homologous,” or “substantially identical,” as used herein, refer to a characteristic of a nucleic acid or amino acid sequence in which a selected nucleic acid or amino acid sequence has at least about 70 or about 75 percent sequence identity with respect to a selected reference nucleic acid or reference amino acid sequence. More typically, the selected sequence and the reference sequence have at least about 76, 77, 78, 79, 80, 81, 82, 83, 84, or 85 percent sequence identity, and more preferably at least about 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95 percent sequence identity. Even more preferably, highly homologous sequences often share greater sequence identity than at least about 96, 97, 98, or 99 percent between the selected sequence and the reference sequence with which it is compared.

[0021] The degree of sequence identity may be calculated over the entire length of the sequences being compared, or by excluding small deletions or absences that total less than about 25 percent of the selected reference sequence. The reference sequence may be a subset of a larger sequence, such as a portion of a gene or adjacent sequence, or a repeating portion of a chromosome. However, in the case of sequence homology of two or more polynucleotide sequences, the reference sequence typically contains at least about 18–25 nucleotides, more typically 26–35 nucleotides, and even more typically at least about 40, 50, 60, 70, 80, 90, or 100 nucleotides.

[0022] When highly homologous fragments are desired, the degree of percentage identity between two sequences can be at least about 80%, preferably at least about 85%, and more preferably about 90% or 95%, or higher, as can be easily determined by one or more sequence comparison algorithms well known to those skilled in the art, such as the FASTA program analysis described, for example, by Pearson and Lipman.

[0023] Polynucleotides In some aspects, polynucleotides are provided to deliver a portion of the coding sequence of the OTOF gene encoding the otoferrin protein to cells. In some embodiments, the coding sequence is derived from the human OTOF gene (see, for example, NCBI gene ID: 9381 and cDNA sequences NM_001287489.1, NM_004802.3, NM_194248.2, NM_194322.2, and NM_194323.2). In some embodiments, the coding sequence is derived from the mouse OTOF gene (see, for example, NCBI gene ID 83762 and cDNA sequences NM_001100395.1, NM_001286421.1, NM_001313767.1, and NM_031875.2). In some embodiments, first and second polynucleotides are provided. It should be understood that terms such as "first," "second," "third," etc., do not imply any particular order or importance unless explicitly stated otherwise.

[0024] In some embodiments, the first polynucleotide includes a reverse-terminal repeat sequence adjacent to an expression cassette, containing, from 5' to 3', one or more of the following: (a) a promoter, (b) a partial coding sequence encoding the N-terminal portion of the otoferlin polypeptide, (c) a splice donor site, and (d) a homologous region of the first polynucleotide containing a sequence homologous to the sequence in the second polynucleotide. In some embodiments, the first polynucleotide includes at least two, at least three, or all four of (a), (b), (c), and (d).

[0025] In some embodiments, the second polynucleotide includes a reverse-terminal repeat sequence adjacent to the expression cassette, comprising, from 5' to 3', one or more of the following: (a) a second homologous region containing a sequence homologous to the sequence in the first polynucleotide, (b) a splice acceptor site, (c) a partial coding sequence encoding the C-terminal portion of the otoferlin polypeptide, and (d) a polyadenylation (pA) signal sequence. In some embodiments, the second polynucleotide comprises at least two, at least three, or all four of (a), (b), (c), and (d).

[0026] The partial coding sequences contained within the polynucleotides described herein may be designed such that, upon delivery of the polynucleotide, the partial coding sequences are joined together, for example, through homologous recombination, to form a complete coding sequence encoding an otoferlin polypeptide.

[0027] In some embodiments, the polynucleotide is a plasmid (a circular nucleic acid comprising, for example, one or more origins of replication, selectable markers, and reporter genes). In some embodiments, the polynucleotide described herein, such as a plasmid, may also contain a marker or reporter gene, for example, LacZ or a fluorescent protein, and an origin of replication. In some embodiments, the plasmid is transfected into producer cells that produce AAV particles containing an expression cassette contained within the plasmid.

[0028] In some embodiments, polynucleotides are nucleic acid vectors, such as recombinant adeno-associated virus (AAV) vectors. Useful exemplary AAV nucleic acid vectors provided herein include single-stranded (ss) or self-complementary (sc) AAV nucleic acid vectors.

[0029] In some embodiments, recombinant AAV particles comprise a polynucleotide, such as a single-stranded (ss) or self-complementary (sc) AAV nucleic acid vector. In some embodiments, the polynucleotide comprises an expression construct as described herein, and a reverse-terminal repeat (ITR) sequence adjacent to the expression construct (e.g., a wild-type ITR sequence or an artificially created ITR sequence). In some embodiments, the polynucleotide is capsidated by a viral capsid.

[0030] Accordingly, in some embodiments, the AAV particles comprise a viral capsid and polynucleotides as described herein capsidated by the viral capsid. In some embodiments, the viral capsid comprises 60 capsid protein subunits, including VP1, VP2, and VP3. In some embodiments, the VP1, VP2, and VP3 subunits are present in the capsid in a ratio of approximately 1:1:10, respectively.

[0031] In some embodiments, the polynucleotides described herein (e.g., the first and second polynucleotides) contain homologous regions to facilitate homologous recombination between the polynucleotides, for example, once delivered to a cell (see, for example, Ghosh et al. Efficient transgene reconstitution with hybrid dual AAV vectors carrying the minimized bridging sequences. Hum Gene Ther. 2011 Jan;22(1):77-83). In some embodiments, the first homologous region and the second homologous region have a threshold level of sequence identity with each other to facilitate homologous recombination. In some embodiments, the first homologous region has at least 75%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the second homologous region.

[0032] Unless otherwise specified, the percent sequence identity and / or similarity of two sequences used herein can be determined using the algorithm of Karlin and Altschul (1990), modified as in Karlin and Altschul (1993). Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al. (1990). A BLAST search can be performed using the NBLAST program, score=100, wordlength=12, to obtain sequences with the desired percent sequence identity. To obtain gap-accommodated alignment for comparison purposes, Gapped BLAST can be used as described (Altschul et al., 1997). When using BLAST and Gapped BLAST, the default parameters of the respective programs (NBLAST and XBLAST) can be used according to the published methods. In some embodiments, each homologous region is independently between 50-500, 50-400, 50-300, 100-500, 100-400, 100-300, 200-500, 200-400, or 200-300 nucleotides. In some embodiments, the homologous regions are identical, and each homologous region is between 50-500, 50-400, 50-300, 100-500, 100-400, 100-300, 200-500, 200-400, or 200-300 nucleotides.

[0033] In some embodiments, a region homology is a nucleotide sequence [ka] and include sequences that are at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical.

[0034] In some embodiments, the polynucleotides described herein may comprise one or more regulatory elements. Those skilled in the art can select regulatory elements for use in suitable host cells, e.g., mammalian or human host cells. Regulatory elements include, for example, promoters, transcription termination sequences, translation termination sequences, enhancers, and polyadenylation elements. The polynucleotides described herein may comprise promoter sequences operably linked to a nucleotide sequence encoding a desired polypeptide, such as otoferlin. Promoters intended for use in the subject invention include, but are not limited to, cytomegalovirus (CMV) promoters, SV40 promoters, Roussarcoma virus (RSV) promoters, chimeric CMV / chicken β-actin promoters (CBAs), and truncated CBAs (smCBAs) (see, for example, Hayre et al. 2006 and U.S. Patent No. 8,298,818, which are specifically incorporated herein by explicit reference). In some embodiments, the promoter is a truncated chimeric CMV β-actin (smcBA) promoter.

[0035] In some embodiments, the promoter controls the nucleotide sequence. [ka] and include sequences that are at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical.

[0036] In some embodiments, the polynucleotide described herein comprises a subcoding sequence that encodes the N-terminal or C-terminal portion of an otoferlin polypeptide, where the subcoding sequence can be spliced ​​in vivo to encode the otoferlin polypeptide or otherwise combined together. In some embodiments, the otoferlin polypeptide is a human otoferlin polypeptide. In some embodiments, the otoferlin polypeptide is a long isoform of a human otoferlin polypeptide (see, for example, Yasunaga et al. OTOF Encodes Multiple Long and Short Isoforms: Genetic Evidence That the Long Ones Underlie Recessive Deafness DFNB9. Am. J. Hum. Genet. 67:591-600, 2000).

[0037] In some embodiments, the otoferlin polypeptide comprises a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to one or both of the following amino acid sequences:

[0038] Human OTOF isoform 1 - Genbank number AF183185.1 [ka] [ka]

[0039] Human OTOF isoform 5 - Genbank number NP_001274418 [ka] [ka]

[0040] In some embodiments, the otoferlin polypeptide is a mouse otoferlin polypeptide. In some embodiments, the otoferlin polypeptide comprises a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the following amino acid sequence:

[0041] Mouse OTOF Isoform 1 - Genbank number NP_001093865.1 [ka] [ka]

[0042] In some embodiments, the polynucleotides described herein include splice donor or splice acceptor sites. In some embodiments, the splice donor and / or splice acceptor sites contain a splice consensus sequence. In some embodiments, the splice donor and / or splice acceptor sites contain a splice consensus sequence derived from alkaline phosphatase.

[0043] In some embodiments, the splice donor site is a nucleotide sequence [ka] and include sequences that are at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical.

[0044] In some embodiments, the splice acceptor site is a nucleotide sequence [ka] and include sequences that are at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical.

[0045] In some embodiments, the oligonucleotides described herein include an ITR sequence. The ITR sequence of the polynucleotides described herein may be derived from any AAV serotype (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) or from more than one serotype. In some embodiments of the polynucleotides provided herein, the ITR sequence is derived from AAV2. ITR sequences and plasmids containing ITR sequences are known in the art and commercially available (for example, products and services available from Vector Biolabs, Philadelphia, PA; Cellbiolabs, San Diego, CA; Agilent Technologies, Santa Clara, CA; and Addgene, Cambridge, MA; as well as "Gene delivery to skeletal muscle results in sustained expression and systemic delivery of a therapeutic protein." Kessler PD, Podsakoff GM, Chen X, McQuiston SA, Colosi PC, Matelis LA, Kurtzman GJ, Byrne BJ. Proc Natl Acad Sci US A. 1996 Nov 26;93(24):14082-7; and Curtis A. Machida. "Methods in Molecular Medicine" TM"Viral Vectors for Gene Therapy Methods and Protocols." 10.1385 / 1-59259-304-6:201 (C) Humana Press Inc. 2003. Chapter 10, "Targeted Integration by Adeno-Associated Virus." Matthew D. Weitzman, Samuel M. Young Jr., Toni Cathomen and Richard Jude Samulski; refer to U.S. Patents 5,139,941 and 5,962,313, all of which are incorporated herein by reference.

[0046] An example AAV2 ITR sequence for the one adjacent to the 5' end of the expression construct is: [ka] Includes. An example AAV2 ITR sequence for the one adjacent to the 3' end of the expression construct is: [ka] Includes.

[0047] In some embodiments, the polynucleotides described herein may further optionally include one or more transcription termination sequences, one or more translation termination sequences, one or more signal peptide sequences, one or more internal ribosome entry sites (IRESs), and / or one or more enhancer elements, or any combination thereof. Transcription termination regions can typically be obtained from the 3' untranslated region of a eukaryotic or viral gene sequence. Transcription termination sequences may be located downstream of the coding sequence to enable efficient termination. Signal peptide sequences are amino-terminal peptide sequences that encode information responsible for the placement of a polypeptide operably linked to one or more post-translational cellular destinations (e.g., specific organelle compartments, or sites of protein synthesis and / or activity, and even the extracellular environment). In some embodiments, the polynucleotides described herein include bovine growth hormone polyadenylation signals.

[0048] In some embodiments, the expression construct contained within the polynucleotide described herein has a size of 5 kilobases or less, 4 kilobases or less, or 3 kilobases or less. In some embodiments, the expression construct has a size between 4 and 5 kilobases.

[0049] In some embodiments, the polynucleotides described herein are contained within one or more recombinant AAV particles (e.g., first and second AAV particles). The rAAV particles may be of any AAV serotype (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10), including any derivative (including non-spontaneous variants of serotypes) or pseudotypes. Non-restrictive examples of derivatives and pseudotypes include AAV2-AAV3 hybrids, AAVrh.10, AAVhu.14, AAV3a / 3b, AAVrh32.33, AAV-HSC15, AAV-HSC17, AAVhu.37, AAVrh.8, CHt-P6, AAV2.5, AAV6.2, AAV2i8, AAV-HSC15 / 17, AAVM41, AAV9.45, AAV6(Y445F / Y731F), AAV2.5T, AAV-HAE1 / 2, AAV clone 32 / 83, AAVShH10, AAV2 (Y->F), AAV8 (Y733F), AAV2.15, AAV2.4, AAVM41, and AAVr3.45. Such AAV serotypes and derivatives / pseudotypes, and methods for producing such derivatives / pseudotypes, are known in the art (see, for example, Mol Ther. 2012 Apr;20(4):699-708. doi: 10.1038 / mt.2011.287. Epub 2012 Jan 24. "The AAV vector toolkit: poised at the clinical crossroads." Asokan A1, Schaffer DV, Samulski RJ.). In some embodiments, the first and second AAV particles are AAV2 serotype particles.

[0050] Methods for producing AAV particles and polynucleotides are known in the art and commercially available (see, for example, Zolotukhin et al. Production and purification of serotype 1, 2, and 5 recombinant adeno-associated viral vectors. Methods 28 (2002) 158-167; and U.S. Patent Publication Nos. US20070015238 and US20120322861 incorporated herein by reference; and plasmids and kits available from ATCC and Cell Biolabs, Inc.). For example, polynucleotides (for example, as plasmids) may be combined with one or more helper plasmids containing, for example, rep genes (for example, encoding Rep78, Rep68, Rep52, and Rep40) and cap genes (for example, encoding VP1, VP2, and VP3) and transfected into producer cell lines so that AAV particles can be packaged and subsequently purified.

[0051] In some embodiments, one or more helper plasmids include a first helper plasmid containing the rep gene and the cap gene, as well as a second helper plasmid containing other genes that assist in AAV production, such as the E1a gene, E1b gene, E4 gene, E2a gene, and VA gene. In some embodiments, the rep gene is a rep gene derived from AAV2. Helper plasmids and methods for producing such plasmids are known in the art and commercially available (for example, pDM, pDG, pDP1rs, pDP2rs, pDP3rs, pDP4rs, pDP5rs, pDP6rs, pDG(R484E / R585E), and pDP8.ape plasmids (from PlasmidFactory, Bielefeld, Germany); other products and services available from Vector Biolabs, Philadelphia, PA; Cellbiolabs, San Diego, CA; Agilent Technologies, Santa Clara, CA; and Addgene, Cambridge, MA; pxx6; Grimm et al. (1998), Novel Tools for Production and Purification of Recombinant Adenoassociated Virus Vectors, Human Gene Therapy, Vol. 9, 2745-2760; Kern, A. et al. (2003), Identification of a Heparin-Binding Motif on Adeno-Associated Virus Type 2 Capsids, Journal of Virology, Vol. 77, 11072-11081.;Grimm et al. (2003), Helper Virus-Free, Optically Controllable, and Two-Plasmid-Based Production of Adeno-associated Virus Vectors of Serotypes 1 to 6, Molecular Therapy,Vol.See also 7, 839-850; Kronenberg et al. (2005), A Conformational Change in the Adeno-Associated Virus Type 2 Capsid Leads to the Exposure of Hidden VP1 N Termini, Journal of Virology, Vol. 79, 5296-5303; and Moullier, P. and Snyder, RO (2008), International efforts for recombinant adenoassociated viral vector reference standards, Molecular Therapy, Vol. 16, 1185-1188).

[0052] A non-limiting, exemplary method for producing AAV particles is described below. ORFs of rep and cap for a desired AAV serotype, and one or more helper plasmids containing adenovirus VA, E2A(DBP) and E4 genes under the transcriptional control of the innate promoter, are produced or obtained. HEK293 cells (available from ATCC®) are transfected with the helper plasmid(s) and the polynucleotide-containing plasmids described herein via CaPO4-mediated transfection, lipids, or polymer molecules such as polyethyleneimine (PEI). Alternatively, in another non-limiting example, an Sf9 lineage producer stable cell line is infected with a single recombinant baculovirus containing polynucleotides. As a further non-limiting alternative, in another example, a HEK293 or BHK cell line is infected with a polynucleotide-containing HSV, and optionally with one or more helper HSVs containing the rep and cap ORFs as described herein, and the adenovirus VA, E2A(DBP) and E4 genes under the transcriptional control of the innate promoter. The HEK293, BHK, or Sf9 cells are then incubated for at least 60 hours to enable AAV particle production. The AAV particles can then be purified using any method known in the art or described herein, for example, by an iodixanol stepwise gradient, a CsCl gradient, chromatography, or polyethylene glycol (PEG) precipitation.

[0053] This disclosure also intends to describe host cells containing at least one of the disclosed AAV particles or polynucleotides. Such host cells include mammalian host cells, preferably human host cells, and may be isolated or in cell or tissue culture. In the case of genetically modified animal models (e.g., mice), the transformed host cells may be in the body of the non-human animal itself.

[0054] Methods and Subjects Methods are provided for increasing the expression of otoferlin in cells in several aspects. In some embodiments, the method comprises contacting cells with a first AAV particle as described herein, comprising a first polynucleotide as described herein; and contacting cells with a second AAV particle as described herein, comprising a second polynucleotide as described herein. In some embodiments, the cells are mammalian cells, such as mouse or human cells. In some embodiments, the cells are ex vivo. In some embodiments, the cells are in vivo. In some embodiments, the cells are ear cells (e.g., human ear cells). In some embodiments, the cells are inner ear cells (e.g., human inner ear cells). In some embodiments, the cells are in a subject (e.g., a mammalian subject such as a human subject).

[0055] Other aspects of this disclosure relate to the treatment of diseases or conditions caused by reduced or absent expression or activity of otoferlin. In some embodiments, the method comprises administering to a subject a therapeutically effective dose of a first AAV particle containing the first polynucleotide as described herein and a second AAV particle containing the second polynucleotide as described herein. In some embodiments, the subject is a human subject and has hearing loss, autosomal recessive 9 (DFNB9). In some embodiments, the subject is a human subject having impaired vestibular function or vestibular disorder (see, for example, Dulon et al. Otoferlin is Critical for a Highly Sensitive and Linear Calcium Dependent Exocytosis at Vestibular Hair Cell Ribbon Synapses. J Neurosci. 2009; 29(34): 10474-10487).

[0056] "Treating" a disease, as the term is used herein, means reducing the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject. The compositions described above or elsewhere herein are typically administered to a subject in an effective amount, i.e., an amount capable of producing a desired result. The desired result depends on the active agent being administered. For example, an effective amount of AAV particles can be the amount of particles capable of transferring an expression construct to a host organ, tissue, or cell. A therapeutically acceptable amount can be an amount capable of treating a disease, for example DFNB9. As is well known in the fields of medicine and veterinary medicine, the dosage for a particular subject depends on many factors including the size, body surface area, age of the subject, the particular composition being administered, the active ingredient(s) in the composition, the time and route of administration, general health status, and other drugs being administered concurrently.

[0057] AAV particles or polynucleotides may be delivered in the form of a composition, such as a composition comprising an active ingredient such as the AAV particles described herein and a pharmaceutically acceptable carrier as described herein. AAV particles or polynucleotides can be prepared in various compositions and can also be formulated in a suitable pharmaceutical vehicle for administration to a human or animal subject. In some embodiments, when first and second AAV particles are utilized, the first and second AAV particles may be contained within the same composition or within different compositions and may be administered together or separately.

[0058] In some embodiments, the AAV particles administered to a subject are in a composition having a concentration on the order ranging from 10 6 ~10 14 particles / ml or 10 3 ~10 15 particles / ml, or, for example, about 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11, 10 12 , 10 13 , or 10 14 It may be provided in any value between any range, such as particles / ml. In one embodiment, 10 13 AAV particles exceeding particles / ml are administered. In some embodiments, the number of AAV particles administered to the subject is 10 6 ~10 14 Vector genome (vgs) / ml or 10 3 ~10 15 The order may be in the range of vgs / ml, or, for example, about 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , or 10 14 It may be any value within any range, such as vgs / ml. 13 AAV particles exceeding vgs / ml are administered. AAV particles can be administered as a single dose or divided into two or more doses, as required to achieve treatment of the specific disease or disorder being treated. In some embodiments, 0.0001 ml to 10 mls are delivered to the subject.

[0059] In some embodiments, the number of AAV particles administered to the subject is 10 6 ~10 14 The order may be in the range of vg / kg, or, for example, about 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , or 10 14The value may be any value within any range, such as vgs / kg. In some embodiments, when a first AAV particle containing the first polynucleotide as described herein and a second AAV particle containing the second polynucleotide as described herein are administered, the amount administered is the same for both particles. In some embodiments, when a first AAV particle containing the first polynucleotide as described herein and a second AAV particle containing the second polynucleotide as described herein are administered, the amount administered differs for each particle.

[0060] Where desired, AAV particles may also be administered in combination with other agents or treatments (including systemic or topical administration of one or more therapeutic polypeptides, bioactive fragments, or variants thereof), such as proteins or polypeptides or various pharmaceutically active agents. In fact, the other components that may be included are virtually limited, provided that the additional agents do not cause significant adverse effects upon contact with target cells or host tissues. Thus, AAV particles may be delivered together with a variety of other agents or treatments as required in particular cases. In some embodiments, the treatment of AAV particles may be accompanied by the use of hearing aids.

[0061] In certain situations, it is preferable to deliver AAV particles to one or more cells, tissues, or organs by subcutaneous, parenteral, intravenous, intramuscular, intraperitoneal, oral or nasal inhalation, or direct injection, using a suitably formulated pharmaceutical composition as described herein. In some embodiments, the administration is a suitable route for systemic delivery, such as by intravenous injection or infusion. In some embodiments, the administration is to the ear, and for example, via intracochlear administration. Pharmaceutical forms of AAV particle compositions suitable for injectable use include sterile aqueous solutions or dispersions. In some embodiments, the form is sterile and fluid enough to allow for easy injection. In some embodiments, the form is stable under manufacturing and storage conditions, as well as protected from microbial contamination, such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, saline solution, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and / or vegetable oils. Proper fluidity can be maintained by the use of coatings such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants.

[0062] For the administration of injectable aqueous solutions, for example, the solution may be suitably buffered if necessary, and the liquid diluent may first be isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, intravitreous, subretinal, subcutaneous, and nasoperitoneal administration. In this regard, sterile aqueous media that can be employed in light of this disclosure are known to those skilled in the art. For example, one dose may be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of fluid for subcutaneous injection, or injected into the proposed injection site (see, e.g., Remington's Pharmaceutical Sciences, 15th Edition, pp. 1035-1038 and 1570-1580). Some variation in dosage will inevitably occur depending on the condition of the subject being treated. In any case, the person responsible for administration will determine the appropriate dose for each individual subject. Furthermore, for administration to humans, the preparation must meet sterility, pyrogenicity, and general safety and purity standards, as required, for example, by the standards of the FDA's Biomedical Review Division.

[0063] Sterile injectable solutions are prepared by incorporating the required amount of AAV in a suitable solvent, along with some of the other components listed above as needed, followed by filtration sterilization or other sterilization techniques. Generally, dispersions are prepared by incorporating various sterile active ingredients into a sterile medium containing a basic dispersion medium and other necessary components from those listed above. For sterile powders for the preparation of sterile injectable solutions, preferred preparation methods are vacuum drying and freeze-drying techniques, which yield powders of the active ingredient and any further desired components from a pre-filtered solution.

[0064] The amount of AAV particles or polynucleotide composition and the timing of administration of such composition are within the scope of the knowledge of those skilled in the art who benefit from this teaching. However, a therapeutically effective dose of the disclosed composition may be achieved by a single dose, such as a single injection of a sufficient number of infectious particles, in order to provide a therapeutic benefit to a patient receiving such treatment. Alternatively, in some circumstances, it may be desirable to provide multiple or consecutive doses of the AAV particle composition over a relatively short or relatively long period, as may be determined by the physician supervising the administration of such composition. The composition may include AAV particles alone or in combination with one or more additional active ingredients that may be obtained from natural or recombinant sources or chemically synthesized.

[0065] The toxicity and efficacy of the compositions used in the methods disclosed herein can be determined by standard pharmaceutical procedures, which involve determining the LD50 (the dose that is lethal to 50% of the population) using either cultured cells or experimental animals. The dose-to-toxicity ratio is the therapeutic index, which can be expressed as the ratio LD50 / ED50. Compositions exhibiting a large therapeutic index are preferred. Compositions exhibiting toxic side effects may also be used, but care must be taken to design a delivery system that minimizes the potential damage caused by such side effects. The dosages of the compositions described herein generally fall within a range that includes an ED50 that is little to no toxicity. The dosage may vary within this range depending on the form of administration adopted and the route of administration used.

[0066] Aspects of this disclosure relate to methods for use with subjects such as human or non-human primate subjects. Non-limiting examples of non-human primate subjects include macaques (e.g., crab-eating macaques or rhesus macaques), marmosets, tamarins, spider monkeys, night monkeys, velvet monkeys, squirrel monkeys, baboons, gorillas, chimpanzees, and orangutans. In some embodiments, subjects are human subjects. Other exemplary subjects include domesticated animals such as dogs and cats; livestock such as horses, cattle, pigs, sheep, goats, and chickens; and other animals such as mice, rats, guinea pigs, and hamsters.

[0067] In some embodiments, subjects have or are suspected of having a disease that can be treated with gene therapy. In some embodiments, subjects have or are suspected of having hearing loss, autosomal recessive 9 (DFNB9). DFNB9 is an autosomal recessive form of hearing loss thought to be caused by mutations in the OTOF gene resulting in reduced expression, function, or both of the otoferlin protein. The otoferlin protein has been shown to be important for exocytosis at the auditory ribbon synapse (see, for example, Roux et al. Otoferlin, defective in a human deafness form, is essential for exocytosis at the auditory ribbon synapse. (2006) Cell 127(2):277-89). Subjects with DFNB9 can be identified by a skilled physician using, for example, electrophysiological testing of the auditory brainstem response (ABR) and a combination of genetic testing to identify mutations in the OTOF gene (see, for example, OMIM entries 603681 and 601071).

[0068] In some embodiments, subjects are human subjects having one or more of the following nonsense or missense mutations in the OTOF gene: TYR730TER, GLN829TER, PRO1825ALA, PRO50ARG, LEU1011PRO, ILE515THR, ARG1939GLN, or GLY541SER. In some embodiments, subjects are human subjects having an A-to-G transition at the intron 8 / exon 9 junction (IVS8-2A-G), or a G-to-A transition at position +1, which is the first intron nucleotide at the splice donor site in exon 5, or a G-to-C transversion at the donor splice site in intron 39. In some embodiments, subjects are human subjects having a single base pair deletion at exon 16 (1778G), which leads to a stop codon, and a 6141G-A change, which results in an ARG-to-GLN substitution at exon 48.

[0069] composition Other aspects of this disclosure relate to compositions comprising AAV particles or polynucleotides as described herein. In some embodiments, the AAV particles described herein are added to compositions, for example, pharmaceutical compositions.

[0070] In some embodiments, the composition comprises a pharmaceutically acceptable carrier. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which AAV is administered. Such a pharmaceutical carrier may be a sterile liquid, such as water or oil, which includes petroleum such as mineral oil, vegetable oils such as peanut oil, soybean oil, and sesame oil, animal oil, or oils of synthetic origin. Saline solutions and aqueous dextrose and glycerol solutions may also be used as liquid carriers. Non-limiting examples of pharmaceutically acceptable carriers include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, saline, syrup, methylcellulose, ethylcellulose, hydroxypropyl methylcellulose, polyacrylic acid, lubricants (such as talc, magnesium stearate, and mineral oil), wetting agents, emulsifiers, suspending agents, preservatives (such as methyl, ethyl, and propyl hydroxybenzoates), and pH adjusters (such as inorganic and organic acids and bases).

[0071] Other examples of carriers include phosphate-buffered saline, HEPES-buffered saline, and water for injection, any of which may be optionally combined with one or more of the following: calcium chloride dihydrate, anhydrous disodium phosphate, magnesium chloride hexahydrate, potassium chloride, potassium dihydrogen phosphate, sodium chloride, or sucrose. Other examples of carriers that may be used include saline (e.g., sterile, pyrogen-free saline), saline buffers (e.g., citrate buffer, phosphate buffer, acetate buffer, and bicarbonate buffer), amino acids, urea, alcohol, ascorbic acid, phospholipids, proteins (e.g., serum albumin), EDTA, sodium chloride, liposomes, mannitol, sorbitol, and glycerol. USP-grade carriers and excipients are particularly useful for the delivery of AAV particles to human subjects. Such compositions may further optionally include liposomes, lipids, lipid complexes, microspheres, fine particles, nanospheres, or nanoparticles, or may be otherwise formulated for administration to cells, tissues, organs, or other subjects of the body requiring them. Methods for producing such compositions are well known and, for example, Remington: The Science and Practice of Pharmacy, 22 nd This can be found in edition, Pharmaceutical Press, 2012.

[0072] Typically, such compositions may contain at least about 0.1% of the therapeutic agent (e.g., AAV particles) or more, although the proportion of the active ingredient(s) may, of course, vary and may conveniently be between about 1 or 2% and about 70 or 80% or more of the total weight or volume of the formulation. Naturally, the amount of the therapeutic agent(s) (e.g., AAV particles) in each therapeutically useful composition may be prepared in such a way that a suitable dose is obtained at some given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, shelf life of the product, and other pharmacological considerations will be contemplated by those skilled in the art to prepare such pharmaceutical formulations, and so that a variety of doses and treatment regimens may be desirable.

[0073] In some embodiments, the compositions described herein may be administered to subjects in need, such as subjects having DFNB9. In some embodiments, the methods described herein may include administering one or more compositions containing AAV particles as described herein to subjects in need. In some embodiments, the subjects are human subjects. In some embodiments, the subjects have or are suspected of having a disease that can be treated with gene therapy, such as DFNB9. In some embodiments, the subjects have been diagnosed with DFNB9.

[0074] kit Other aspects of this disclosure relate to kits comprising AAV particles or polynucleotides as described herein in one or more containers. The kits may optionally include pharmaceutically acceptable carriers and / or diluents. In some embodiments, the kits include instructions or packaging materials describing how to administer the AAV particles or polynucleotides contained within the kit to selected cells or recipients. The kit containers may be of any suitable material (e.g., glass, plastic, metal, etc.) and may be of any suitable size, shape, or configuration. In some embodiments, the kits may include one or more ampoules or syringes containing the AAV particles or polynucleotides in a suitable liquid or solution form. [Examples]

[0075] example Hearing rescue in OTOF knockout mice using an adeno-associated virus gene therapy approach. Introduction Otoferlin is an important calcium sensor for neurotransmitter release in the ear (see Roux 2006, for example). Otoferlin is expressed primarily in the inner hair cells of the cochlea and in a very small number of other cells in the central nervous system (see Yasunaga et al. 1999 & 2000, for example). It is a member of the ferlin family of transmembrane proteins, sharing a C2 domain with synaptotagmin, PKC, and PLC.

[0076] Mutations in the human OTOF gene, which encodes human otoferlin, cause a type of non-symptomatic hearing loss called "deafness, autosomal recessive 9 (DFNB9)." OTOF knockout mice have also been shown to have severe hearing loss despite normal inner hair cell development and auditory ribbon synapse formation (see, for example, Roux et al. (2006) Otoferlin, defective in a human deafness form, is essential for exocytosis at the auditory ribbon synapse. Cell. 127:277-289). However, Otof - / - Mice lose auditory brainstem responses across all sound frequencies due to the complete abolition of synaptic exocytosis and, consequently, the abolition of neurotransmitter release from synaptic vesicles.

[0077] DFNB9 manifests in humans as two phenotypes: as asymptomatic bilateral hearing loss prior to language acquisition, and less frequently as a temperature-sensitive, asymptomatic auditory neuropathy. It was first discovered in an affected Lebanese family (Chaib et al. 1996) and has since been found in numerous parts of the world (see, for example, Adato et al. 2000, Rodriguez-Ballesteros et al. 2003, Choi et al. 2009, and Matsunaga et al. 2012).

[0078] Current treatments for humans with DFNB9 involve cochlear transplantation and hearing aids. In addition, for temperature-sensitive DFNB9, prevention of fever and other conditions causing elevated body temperature is crucial. As a proof-of-concept for using AAV to deliver OTOF as a treatment for DFNB9, the applicant aimed to use adeno-associated virus (AAV) as a means of restoring OTOF expression in knockout mice. Mouse OTOF cDNA is 5979 base pairs long, while most AAVs cannot package genomes exceeding approximately 4.8 kilobases. Consequently, a dual-vector system was used to deliver the 5' and 3' portions of the cDNA separately as separate AAV constructs so that the full-length cDNA could be reassembled in vivo once delivered.

[0079] method Dual AAV vector construct Mouse OTOF cDNA was divided into two segments, the 5' and 3' segments, and inserted into two AAV ITR-containing plasmids. The sequences of each of the two cassettes in the plasmids are shown below, and maps of each construct are shown in Figures 1 and 2. Annotated versions of the cassettes are shown in Figures 3A and 3B. Each cassette contains homologous regions to facilitate homologous recombination between the 5' and 3' ends of the cDNA in vivo (see Ghosh et al., 2011). Once recombined in vivo, the full-length cDNA contains splice donor / splice acceptor pairs that cause splicing excision from the homologous regions. The vectors were packaged into AAV2 serotype particles using the standard plasmid transfection method previously described (see Zolotukhin et al. Production and purification of serotype 1, 2, and 5 recombinant adeno-associated viral vectors. Methods 28 (2002) 158-167). The viral particles were purified using the standard method previously described (see Zolotukhin et al. Production and purification of serotype 1, 2, and 5 recombinant adeno-associated viral vectors. Methods 28 (2002) 158-167). Viral particles containing the 5' portion of the OTOF cDNA are also referred to herein as "AAV2-OTOF-NT". Viral particles containing the 3' portion of the OTOF cDNA are also referred to herein as "AAV2-OTOF-CT".

[0080] pTR22-smCBA-Otoferlin NT-APSD-APhead [ka] [ka] [ka] [ka] [ka]

[0081] pTR22-APhead-APSA-OtoferlinCT [ka] [ka] [ka] [ka] [ka]

[0082] HEK 293 cell transfection HEK 293 cells were grown on polylysine-coated coverslips in culture medium. 1 μl of each virus was used in each well as follows: virus-free control cells, cells with the n-terminal portion of AAV2-OTOF (AAV2-OTOF-NT), cells with the c-terminal portion of AAV2-OTOF (AAV2-OTOF-CT), and cells with both viruses (AAV2-OTOF-NT and AAV2-OTOF-CT). The cells were stained with anti-OTOF antibody and prepared on glass slides.

[0083] OTOF knockout mice The OTOF knockout mice used were those created in a previous study (see Roux et al. (2006) Otoferlin, defective in a human deafness form, is essential for exocytosis at the auditory ribbon synapse. Cell. 127:277-289). Briefly, two fragments containing the 5' and 3' portions of the Otof genome sequence up to exons 14 and 15 were amplified by PCR. The 5kb BamHI-XhoI-BssHII and 6kb BssHII-SfiI-BamHI-NaeI ​​129 / SvPas fragments were inserted into pUC19 (New England BioLabs), which had been pre-modified by inserting the BamHI-XhoI-BssHII-SfiI-NaeI-HindIII polylinker. The loxP-hygro-loxP (a gene that confers resistance to hygromycin under the control of the phosphoglycerate kinase gene [Pgk-1] promoter) cassette was inserted into the BssHII site. All constructs were sequenced, and the resulting sequences were compared with the 129 / SvPas genome sequence. Hygromycin-resistant 282 CK35 ES cells were screened for homologous recombination and monoinsertion events by Southern blot analysis.

[0084] To create chimeric animals, two clones were injected into C57BL / 6N blastocysts. The transmission of the mutant Otof allele was detected by PCR in agouti offspring. Positive offspring from the F1 generation were crossed with Pgk-1-cre mice in a mixed C57BL / 6-129 / SvPas background. F2 animals carrying the allele lacking the hygromycin cassette (Otof tm1Ugds allele) were selected by PCR using primers 5'-CACTTGCTTTGTCT CATCTCC-3' (SEQ ID NO: 12) and 5'-GTCACTTCTTCTGGGTATTTC-3' (SEQ ID NO: 13), which produced 507 base pair PCR products. Heterozygous animals were selected for Otof - / - Otof + / - , and Otof + / + Intrabred mice were created to produce knockout mice. Knockout mice were produced in the C57BL / 6-129 / SvPas background as described above. Since this background strain is known to have some age-related hearing loss, the mice were backcrossed with the FVB mouse strain up to the 10th generation to obtain an FVB allogeneic background without the known age-related hearing loss.

[0085] Delivery of AAV to mice OTOF knockout mice (newborn and older than P10) were injected with 1 microliter of viral particles from each of the two AAV constructs using round window membrane (RWM) injection as previously described (see Akil et al. (2012) Restoration of Hearing in the VGLUT3 Knockout Mouse Using Virally-Mediated Gene Therapy. Neuron. 75(2): 283-293 and Akil et al. (2015) Surgical Method for Virally Mediated Gene Delivery to the Mouse Inner Ear through the Round Window Membrane. J. Vis. Exp. (97), e52187). AAV2-OTOF-NT (6.32 × 10⁻¹⁰) 12 (vg / ml) and AAV2-OTOF-CT (4.5 × 10 12 AAV2-OTOF-NT (1.43 × 10⁻¹⁰) was delivered to P1-3 mice via RWM. 13 (vg / ml) and AAV2-OTOF-CT (3.12 × 10) 13 AAV2-GFP (vg / ml) was delivered to mice with P≧12 via RWM. An ABR test was performed 7 days after injection. OTOF protein expression in mice was measured using an anti-OTOF antibody to label cells in whole-cochlear specimens. Reverse transcriptase (RT)-PCR was used to screen for the presence of OTOF mRNA within mouse cochlear tissue. Wild-type mice were also injected with AAV2-GFP using the same technique to evaluate viral delivery to the cochlea via AAV2. The cochlea were prepared as whole-cochlear specimens and stained with anti-GFP antibody.

[0086] Auditory Brainstem Response (ABR) Test Auditory testing was performed on otoferlin knockout (OTOF KO) mice, rescued OTOF KO mice, and wild-type (WT) littermates, as previously described (Akil et al. (2006) Progressive deafness and altered cochlear innervation in knockout mice lacking prosaposin. J. Neurosci. 26:13076-13088 and Akil et al. (2016) Mouse Auditory Brainstem Response Testing. Bio Protoc. 6(6)). In short, all auditory tests were performed in a soundproof room. Prior to auditory testing, mice were anesthetized by intraperitoneal injection of a mixture of ketamine hydrochloride (Ketaset, 100 mg / ml) and xylazine hydrochloride (xyla-ject, 10 mg / ml), and boosted with one-fifth of the initial dose as needed. Throughout the recording period, body temperature was maintained with a heating pad and monitored with a rectal probe.

[0087] Evoked auditory brainstem response (ABR) thresholds were recorded separately from the scalp of mice. Responses were recorded using subcutaneous needle electrodes at the parietal region, below the left auricle (reference), and below the opposite ear (ground). The auditory stimuli used included clicks (5 ms duration, 31 Hz) and tone pips at 8, 16, and 32 kHz (10 ms duration, cos² shaped, 21 Hz). Measurements were recorded using the TDT BioSig III system (Tucker Davis Technologies). For each stimulus, electroencephalogram (EEG) activity was recorded for 20 ms (at a sampling rate of 25 kHz) and filtered (0.3–3 kHz). For click responses, waveforms from 512 stimuli were averaged. To identify frequency-specific tone burst stimuli (8, 16, and 32 kHz), waveforms from 1000 stimuli were examined. ABR waveforms were recorded at intervals of 5 dB sound pressure level (SPL) from the maximum amplitude. The threshold was defined as the lowest stimulus level at which a clear and recurring response peak for wave IV was observed visually. These threshold determinations were confirmed by analysis of the stored waveforms. Comparisons between the animal groups were performed using one-way ANOVA and Bonferroni's post-hoc test.

[0088] result Two different AAV plasmid constructs were created to deliver the 5' and 3' halves of mouse OTOF cDNA to the inner ear of OTOF knockout mice (OTOF N-terminal virus and OTOF C-terminal virus). The two constructs were packaged separately into AAV2 particles. The AAV2 particles were then pooled together and used to treat HEK 293 cells or injected into the inner ear of OTOF knockout mice. HEK 293 cells expressed the otoferlin protein only when transfected with both viruses (Figure 4). Otoferlin protein expression was not observed in untreated cells or in cells transfected with only the OTOF N-terminal (Figure 4) or OTOF C-terminal virus.

[0089] Next, the ability of AAV2 to transduce mouse cochlea was evaluated using the AAV2-GFP reporter virus. It was shown that AAV2 transfects a number of cell types, including inner hair cells (IHC), outer hair cells (OHC), columnar cells (P), and other indicator cells (SC) (Figure 5). Therefore, AAV2 can effectively transduce mouse cochlea.

[0090] Mice were then treated with pooled OTOF N-terminal and C-terminal viruses and compared to various controls. Otoferlin protein was found to be expressed after treatment with both viruses (Figure 6). The largest number of transfected inner hair cells (IHCs) was observed at the basal level, with fewer at the middle turn and apex (Figure 6). IHC counting demonstrated that approximately 11% of IHCs were labeled overall, with significant differences observed between the basal (approximately 29%), middle turn (approximately 8%), and apex (approximately 2%) (Figure 6). Using RT-PCR, it was shown that OTOF mRNA was present in the whole cochlear extract and was the same size in both wild-type and OTOF knockout mice treated with both viruses (Figure 6). In contrast, OTOF mRNA expression was not demonstrated in the cochlea of ​​untreated OTOF knockout mice. No product was detected when RT-PCR was performed in the absence of reverse transcriptase.

[0091] Next, hearing tests were performed to determine whether otoferrin expressed by delivery of both the N and C-terminal viruses had the ability to rescue hearing function. ABR waveforms from wild-type and OTOF knockout mice treated with both viruses were similar, demonstrating hearing recovery in rescued KO mice, while untreated OTOF knockout mouse controls and OTOF knockout mice transfected with only the OTOF N-terminal virus did not show hearing recovery (Figure 7). At P70, partial hearing recovery (improved ABR threshold) was observed in OTOF knockout mice treated with both viruses for clicks and at specific frequencies of 8, 16, and 32 kHz, although the ABR threshold appeared to be slightly elevated at 8 and 16 kHz, it was still significantly better than in untreated OTOF knockout mice (Figure 7). Notably, despite some variability in the ABR threshold, hearing was maintained for more than 4 months in OTOF knockout mice treated with both viruses (KO NT+CT) (Figure 7). Untransfected KO controls and KOs transfected with OTOF NT remained hearing impaired (Figure 7).

[0092] Next, OTOF knockout mice older than P12 were treated with both viruses. The double-transfected IHCs expressed OTOF, showing uniform transfection rates at both the basal (not shown) and apical (Figure 8) levels. IHC counts demonstrated that approximately 41% of IHCs were labeled overall, with only slight differences observed between the basal (approximately 38%), intermediate (approximately 42%), and apical (approximately 47%) levels (Figure 8).

[0093] At P60, all OTOF knockout mice treated with both viruses demonstrated normal ABR thresholds to click stimuli, although at certain frequencies of 8, 16, and 32 kHz, the ABR thresholds appeared slightly elevated, but were still significantly better than untreated OTOF knockout mice (Figure 9). Time-course changes in hearing recovery following injection of both viruses into OTOF knockout mice older than P12 showed that hearing was maintained for more than 30 weeks in treated mice, and the ABR thresholds were restored to WT levels (Figure 9).

[0094] These results demonstrate that using more than one AAV construct to deliver different portions of OTOF cDNA can result in functional cDNA in vivo. These results also demonstrate that hearing loss can be treated by delivering OTOF cDNA using an AAV delivery system.

[0095] Example 2: Human OTOF dual vector construct Provided below are examples of dual vector sequences for expressing human otoferlin protein isoforms 1 and 5. The cDNAs encoding both isoforms 1 and 5 contain the same N-terminal sequence so that the same N-terminal vector can be used to express both isoforms. The vector maps and annotated sequences corresponding to the sequences below are shown in Figures 10–15.

[0096] pTR22-smCBA-Otoferlin NT Hs var 1+5-APSD-APhead [ka] [ka] [ka]

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[0097] pTR22-APhead-APSA-オトフェルリンCT Hs var 1

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[0098] pTR22-APhead-APSA-オトフェルリンCT Hs var 5

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[0099] References 1) A dato A1, Raskin L, Petit C, Bonne-Tamir B Deafness heterogeneity in a Druze isolate from the Middle East: novel OTOF and PDS mutations, low prevalence of GJB2 35delG mutation and indication for a new DFNB locus.Eur J Hum Genet. 2000 Jun;8(6):437-42. 2) Allocca M, Doria M, Petrillo M, Colella P, Garcia-Hoyos M, Gibbs D, Kim SR, Maguire A, Rex TS, Di Vicino U, Cutillo L, Sparrow JR, Williams DS, Bennett J, Auricchio A. Serotype-dependent packaging of large genes in adeno-associated viral vectors results in effective gene delivery in mice. J Clin Invest. 2008 May;118(5):1955-64. 3) Chaib, H., Place, C., Salem, N., Chardenoux, S., Vincent, C., Weissenbach, J., El-Zir, E., Loiselet, J., Petit, C. A gene responsible for a sensorineural nonsyndromic recessive deafness maps to chromosome 2p22-23. Hum. Molec. Genet. 1996 5: 155-158. 4) Choi, B. Y., Ahmed, Z. M., Riazuddin, S., Bhinder, M. A., Shahzad, M., Husnain, T., Riazuddin, S., Griffith, A. J., Friedman, T. B. Identities and frequencies of mutations of the otoferlin gene (OTOF) causing DFNB9 deafness in Pakistan. Clin. Genet. 2009 75: 237-243. 5) Dong B, Nakai H, Xiao W. Characterization of genome integrity for oversized recombinant AAV vector. Mol Ther. 2010 Jan;18(1):87-92. 6) Ghosh A, Yue Y, Duan D. Efficient transgene reconstitution with hybrid dual AAV vectors carrying the minimized bridging sequences. Hum Gene Ther. 2011 Jan;22(1):77-83.

[0100] 7) Hirsch ML, Agbandje-McKenna M, Samulski RJ. Little vector, big gene transduction: fragmented genome reassembly of adeno-associated virus. Mol Ther. 2010 Jan;18(1):6-8. 8) Lai Y, Yue Y, Duan D. Evidence for the failure of adeno-associated virus serotype 5 to package a viral genome > or = 8.2 kb. Mol Ther. 2010 Jan;18(1):75-9. 9) Matsunaga T1, Mutai H, Kunishima S, Namba K, Morimoto N, Shinjo Y, Arimoto Y, Kataoka Y, Shintani T, Morita N, Sugiuchi T, Masuda S, Nakano A, Taiji H, Kaga K. A prevalent founder mutation and genotype-phenotype correlations of OTOF in Japanese patients with auditory neuropathy. Clin Genet. 2012 Nov;82(5):425-32. doi: 10.1111 / j.1399-0004.2012.01897.x. Epub 2012 Jun 1 . 10) Rodriguez-Ballesteros M, del Castillo FJ, Martin Y, Moreno-Pelayo MA, Morera C, Prieto F, Marco J, Morant A, Gallo-Teran J, Morales-Angulo C, Navas C, Trinidad G, Tapia MC, Moreno F, del Castillo I. Auditory neuropathy in patients carrying mutations in the otoferlin gene (OTOF). Hum Mutat. 2003 Dec;22(6):451-6. 11) Roux I, Safieddine S, Nouvian R, Grati M, Simmler MC, Bahloul A, Perfettini I, Le Gall M, Rostaing P, Hamard G, Triller A, Avan P, Moser T, Petit C. Otoferlin, defective in a human deafness form, is essential for exocytosis at the auditory ribbon synapse. Cell. 2006 Oct 20;127(2):277-89. 12) Wu Z, Yang H, Colosi P. Effect of genome size on AAV vector packaging. Mol Ther. 2010 Jan;18(1):80-6.

[0101] 13) Yasunaga S, Grati M, Chardenoux S, Smith TN, Friedman TB, Lalwani AK, Wilcox ER, Petit C. Am J Hum Genet. OTOF encodes multiple long and short isoforms: genetic evidence that the long ones underlie recessive deafness DFNB9. 2000 Sep;67(3):591-600. Epub 2000 Jul 19. 14) Yasunaga S, Grati M, Cohen-Salmon M, El-Amraoui A, Mustapha M, Salem N, El-Zir E, Loiselet J, Petit C. A mutation in OTOF, encoding otoferlin, a FER-1-like protein, causes DFNB9, a nonsyndromic form of deafness. Nat Genet. 1999 Apr;21(4):363-9. 15) Didier Dulon, Saaid Safieddine, Sherri M. Jones, Christine Petit. Otoferlin is Critical for a Highly Sensitive and Linear Calcium Dependent Exocytosis at Vestibular Hair Cell Ribbon Synapses. J Neurosci. 2009 August. 16) Zippora Brownstein, Yoni Bhonker and Karen B Avraham. High-throughput sequencing to decipher the genetic heterogeneity of deafness. Brownstein et al. Genome Biology 2012, 13:245 17) Rodriguez-Ballesteros et al. (2003) "Auditory neuropathy in patients carrying mutations in the otoferlin gene (OTOF)" Hum Mutat.; 22 (6):451-456. 18) Petersen MB, Willems PJ: Non-syndromic, autosomal-recessive deafness. Clin Genet. 2006; 69 (5): 371-92.

[0102] 19) Smith R, Gurrola J, Kelley P. OTOF-Related Deafness. In: Pagon R, Bird T, Dolan C, Stephens K, eds. Gene Reviews. Seattle: Internet; 2008 20) Roux I, Safieddine S, Nouvian R et al. Otoferlin, defective in a human deafness form, is essential for exocytosis at the auditory ribbon synapse. Cell 2006;127:277-89 21) Kral A, O'Donoghue GM: Profound deafness in childhood. N Engl J Med. 2010; 363(15):1438-50. doi: 10.1056 / NEJMra0911225. 22) Dyka FM, Boye SL, Chiodo VA, Hauswirth WW, Boye SE., Dual Adeno-Associated Virus Vectors Result in Efficient In Vitro and In Vivo Expression of an Oversized Gene MY07A, Hum Gene Ther Methods. 2014 ; 25 (2):166-77. doi: 10.1089 / hgtb.2013.212. 23) Akil O, Seal RP, Burke K, Wang C, Alemi A, During M, Edwards RH, Lustig LR: Restoration of hearing in the VGLUT3 knockout mouse using virally mediated gene therapy. Neuron. 2012; 75 (2):283-93. doi: 10.1016 / j.neuron.2012.05.019.

[0103] Other aspects All features disclosed herein can be combined in any combination. Each feature disclosed herein may be replaced by an alternative feature that serves the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each disclosed feature is merely an example of a comprehensive set of equivalent or similar features. From the above description, those skilled in the art will readily identify the essential features of this disclosure and can make various changes and modifications to the disclosure to suit various uses and conditions without departing from its spirit and scope. Therefore, other aspects are also within the scope of the claims.

[0104] Equal parts While several embodiments of the invention are described and illustrated herein, those skilled in the art will readily conceive of various other means and / or structures and / or advantages described herein for performing the function and / or obtaining the result. Each such variation and / or modification is considered to fall within the scope of the embodiments of the invention described herein. More generally, those skilled in the art will readily understand that all parameters, dimensions, materials, and configurations described herein are illustrative, and that actual parameters, dimensions, materials, and / or configurations will depend on the specific application(s) in which the teachings of the invention are used. Those skilled in the art will recognize many equivalents to the particular embodiments of the invention described herein, or can verify them by routine experimentation alone. Therefore, it should be understood that the embodiments described herein are presented only as examples, and within the scope of the appended claims and their equivalents, embodiments of the invention may be carried out in ways other than those specifically described and described herein. The embodiments of the invention of this disclosure relate to each of the individual features, systems, articles, materials, kits, and / or methods described herein. Furthermore, any combination of two or more such features, systems, articles, materials, kits, and / or methods is also included within the scope of the invention of this disclosure, provided that such features, systems, articles, materials, kits, and / or methods are not contradictory to each other.

[0105] All definitions defined and used herein should be understood to govern dictionary definitions, definitions in documents incorporated by reference, and / or the ordinary meaning of the defined terms. All references, patents, and patent applications disclosed herein are incorporated by reference with respect to the subject matter from which they are cited, and in some cases may encompass the entire document. As used herein and in the claims, the indefinite articles "a" and "an" should be understood to mean "at least one" unless explicitly stated otherwise.

[0106] As used herein and in the claims, the phrase “and / or” should be understood to mean “either or both” of the elements thus combined, i.e., elements that exist sometimes associatively and otherwise separately. Any elements listed in “and / or” should be interpreted similarly as “one or more” of the elements thus combined. There may be other elements besides those specifically identified by the “and / or” clause, whether or not they relate to the elements specifically identified. Thus, as a non-restrictive example, a reference to “A and / or B” when used with open-ended language such as “including” may, in one aspect, refer to A only (optionally including elements other than B), in another aspect, refer to B only (optionally including elements other than A), in yet another aspect, refer to both A and B (optionally including other elements), and so on.

[0107] As used herein and in the claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” shall be interpreted as inclusive, that is, including at least one but also including multiple numbers or elements of the list, and optionally additional items not included in the list. Only terms explicitly indicated in the opposite, such as “one of” or “exactly one of,” or, as used in the claims, “consisting of,” would refer to the inclusion of exactly one number of elements or a list of elements. In general, as used herein, the term “or” shall be interpreted only when preceded by terms of exclusivity such as “either,” “one of,” “only one of,” or “exactly one of,” to indicate an exclusive choice (i.e., “one or the other, not both”). “Consisting essentially of” shall, when used in the claims, have its usual meaning as used in the field of patent law.

[0108] As used in the specification and claims, the phrase “at least one” relating to a list of one or more elements should be understood to mean at least one element selected from any one or more elements in the list of elements, but not necessarily including at least one and all of each element specifically enumerated in the list of elements, nor excluding any combination of elements in the list of elements. This definition also allows for the presence of elements other than those specifically identified in the list of elements to which the phrase “at least one” refers, whether related to or unrelated to those specifically identified elements. Therefore, as an unrestricted example, “at least one of A and B” (or equivalently “at least one of A or B” or equivalently “at least one of A and / or B”) may, in one aspect, refer to at least one (including any more than one) A (B does not exist (and optionally includes elements other than B)), in another aspect, refer to at least one (including any more than one) B (A does not exist (and optionally includes elements other than A)), and in yet another aspect, refer to at least one (including any more than one) A and at least one (including any more than one) B (and optionally include other elements), etc.

[0109] Conversely, unless explicitly stated otherwise, in any method of a claim involving more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are listed. In the claims and the above specification, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” and “composed of” should be understood to be open-ended, meaning they include but are not limiting. Only the transitional phrases “consisting of” and “consisting essentially of” are closed or semi-closed transitional phrases, respectively, as described in Section 2111.03 of the U.S. Patent and Trademark Examination Procedure.

Claims

1. A composition for use in treating hearing loss or vestibular disorders in human subjects having mutations in the OTOF gene, The subjects here are the following mutations in the OTOF gene: TYR730TER; GLN829TER; PRO1825ALA; PRO50ARG; LEU1011PRO; ILE515THR; ARG1939GLN; GLY541SER; A-to-G transition at the intron 8 / exon 9 junction (IVS8-2A-G); G-to-A transition at position +1, the first intron nucleotide in the splice donor site of exon 5; G-to-C transversion at the donor splice site of intron 39; a single base pair deletion in exon 16 leading to a stop codon (1778G); and a 6141G-A change resulting in an ARG-to-GLN substitution in exon 48. Having one or more of the following, composition, A first AAV particle containing a first polynucleotide; and Second AAV particle containing a second polynucleotide Including, here, (i) The first polynucleotide is arranged from 5' to 3': (a) promoter, (b) A partial coding sequence that encodes the N-terminal portion of the otoferlin polypeptide, (c) Splice donor site, and (d) A first homologous region containing a sequence homologous to the sequence in the second polynucleotide, It contains an expression cassette adjacent to a reverse terminal repeat sequence, and (ii) The second polynucleotide is positioned from 5' to 3': (a) A second homologous region containing a sequence homologous to the sequence in the first polynucleotide, (b) Splice acceptor site, (c) A partial coding sequence that encodes the C-terminal portion of the otoferlin polypeptide, and (d) Polyadenylated (pA) signal sequence, It contains an expression cassette adjacent to a reverse terminal repeat sequence, The otoferlin polypeptide is a human otoferlin isoform 5 polypeptide having the sequence of SEQ ID NO: 6, and The first homologous region and the second homologous region are between 50 and 300 nucleotides. The aforementioned composition.

2. A composition for use in treating hearing loss or vestibular disorders in subjects having mutations in the OTOF gene, A first AAV particle containing a first polynucleotide; and Second AAV particle containing a second polynucleotide Including, here, AAV particles are 10 ６ ~10 １４ This is the amount of vector genome / ml. (i) The first polynucleotide is arranged from 5' to 3': (a) promoter, (b) A partial coding sequence that encodes the N-terminal portion of the otoferlin polypeptide, (c) Splice donor site, and (d) A first homologous region containing a sequence homologous to the sequence in the second polynucleotide, It contains an expression cassette adjacent to a reverse terminal repeat sequence, and (ii) The second polynucleotide is positioned from 5' to 3': (a) A second homologous region containing a sequence homologous to the sequence in the first polynucleotide, (b) Splice acceptor site, (c) A partial coding sequence that encodes the C-terminal portion of the otoferlin polypeptide, and (d) Polyadenylated (pA) signal sequence, It contains an expression cassette adjacent to a reverse terminal repeat sequence, The otoferlin polypeptide is a human otoferlin isoform 5 polypeptide having the sequence of SEQ ID NO: 6, and The first homologous region and the second homologous region are between 50 and 300 nucleotides. The aforementioned composition.

3. The composition according to claim 2, wherein the target is a mammal.

4. The composition according to claim 3, wherein the subject is a human.

5. The composition according to any one of claims 1 to 4, wherein the first homologous region and the second homologous region comprise the nucleotide sequence of SEQ ID NO:

3.

6. The composition according to any one of claims 1 to 5, wherein the promoter is a chimeric CMV / chicken β-actin promoter or a cleaved chimeric CMV / chicken β-actin promoter.

7. The composition according to claim 6, wherein the promoter contains a sequence having at least 90%, at least 92.5%, at least 95%, at least 98%, or at least 99% identity with SEQ ID NO: 4, or the promoter contains the sequence of SEQ ID NO:

4.

8. The composition according to any one of claims 1 to 7, wherein the splice donor site comprises the sequence of Sequence ID No.

7.

9. The composition according to any one of claims 1 to 8, wherein the splice acceptor region includes the sequence of sequence number 8.

10. The composition according to any one of claims 1 to 9, wherein the pA signal sequence is a bovine growth hormone (bGH) pA signal sequence.

11. The composition according to any one of claims 1 to 10, wherein the reverse terminal repeat sequence is an AAV2 reverse terminal repeat sequence.

12. The composition according to any one of claims 1 to 11, wherein the first and second AAV particles are AAV2 serotype particles.

13. The composition according to any one of claims 1 to 12, further comprising a pharmaceutically acceptable carrier.

14. The composition according to any one of claims 1 to 13, wherein hearing loss or vestibular disorder is DFNB9.

15. A composition according to any one of claims 1 to 14 for use in increasing the expression of otoferlin in cells.

16. The composition according to claim 15, wherein the cells are present in a mammalian subject.

17. Use of the composition according to any one of claims 1 to 16 for manufacturing a medicine for the treatment of hearing loss.

18. The use according to claim 17, wherein the pharmaceutical is for the treatment of DFNB9.

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