Adeno-associated virus vector encoding connexin 26 and its use

Abstract

This invention relates to a recombinant adeno-associated virus (rAAV) vector encoding connexin 26 (CX26). The invention further relates to the use of the AAV vector in the treatment of hereditary hearing loss.

Description

[Technical Field]

[0001] The present invention relates to a recombinant adeno-associated virus (AAV) vector encoding connexin 26 and its therapeutic use. [Background technology]

[0002] The gap junction β2 (GJB2) protein, also known as connexin 26 (CX26), is a member of the connexin protein family, which includes 21 members in humans. Connexin proteins consist of one intracellular loop, two extracellular loops, and four transmembrane domains linked by the cytoplasmic N and C terminals.

[0003] Connexin proteins are responsible for forming intercellular channels called gap junctions. Six connexin proteins assemble to form hexameric hemichannels called connexons, which originate in the cell membrane and bind to the extracellular portion of another connexon on the membrane of an adjacent cell. Gap junctions enable the intercellular diffusion of metabolites, ions, and second messenger molecules. The type of connexin protein that forms the gap junction determines its size and the types of particles that can pass through it. Connexin 26, in particular, is responsible for the transport of potassium ions (K+) and several small molecules.

[0004] In humans, connexin 26 is expressed in many tissues throughout the body. In particular, connexin 26 is expressed in the inner ear, specifically in the non-sensory epithelial supporting cells of the cochlea surrounding sensory hair cells, the fibrous cells lining the inside of the cochlear duct, and the spiral ligament region associated with the stria vascularis. The gap junctions formed between the epithelial supporting cells and fibrous cells provide a pathway for potassium ions (K+) passing through the base of the hair cells to return to the endolymph above the hair cells.

[0005] In 1994, locus 13q12 was first identified as being associated with recessive non-syndromic deafness, i.e., recessive deafness without other clinically recognizable features (Guilford P et al., "A non-syndrome form of neurosensory, recessive deafness maps to the pericentromeric region of chromosome 13q." Nat Genet. 1994 Jan;6(1):24-8). Shortly thereafter, the gene GJB2 encoding CX26 was identified as the causative gene (Kelsell DP et al., "Connexin 26 mutations in hereditary non-syndromic sensorineural deafness." Nature. 1997 May 1;387(6628):80-3). Since then, autosomal recessive mutations in the GJB2 gene have been identified as the most common cause of moderate to severe asymptomatic hereditary hearing impairment (also known as asymptomatic hereditary hearing loss) in most populations (Denoyelle F et al., "Prelingual deafness: high prevalence of a 30delG mutation in the connexin 26 gene." Hum Mol Genet. 1997 Nov;6(12):2173-7; Kemperman MH et al., "Hearing loss and connexin 26." JR Soc Med. 2002 Apr;95(4):171-7). Asymptomatic hearing loss caused by biallelegenic GJB2 variants is known as DFNB1.

[0006] For many years, hearing aids or cochlear implants were the only available treatment options for individuals suffering from hereditary hearing loss. Today, gene therapy is a promising treatment for hereditary hearing loss (also known as hereditary hearing impairment), where the therapeutic gene is typically delivered to the inner ear via a viral vector, such as adeno-associated virus (AAV) vectors. However, a significant limitation of gene therapy is the potential lack of specificity in the type of cells transduced by the viral vector. As mentioned above, CX26 expression in the inner ear is limited to non-sensory supporting cells present in the epithelium and connective tissue. Consequently, the connexin protein is completely absent in the sensory hair cells of the cochlea. In fact, CX26 expression in cochlear inner ear hair cells has been shown to be detrimental to the survival of these cells (Guo J et al., "GJB2 gene therapy and conditional deletion reveal developmental stage-dependent effects on inner ear structure and function." Mol Ther Methods Clin Dev. 2021 Oct 1;23:319-333). Therefore, there is still a need for AAV vectors that enable controlled expression of CX26 in target cells and are thus suitable for gene therapy. In particular, there is still a need for AAV vectors that can prevent or suppress CX26 expression in cells and tissues that may induce some kind of adverse effect.

[0007] The inventors have surprisingly demonstrated that a recombinant AAV-DJ vector containing a polynucleotide comprising a sequence encoding CX26 operably linked to a promoter and a so-called "precursor miR183 target site" enables CX26 expression in non-sensory support cells of the cochlear while preventing CX26 expression in inner ear hair cells. Accordingly, the inventors have demonstrated that administration of the recombinant AAV-DJ vector prevents hearing loss in a mouse model of hereditary hearing loss. Similar cellular patterns of regulatory effects on CX26 expression can be expected when using other "precursor miRNA target sites" of the miR183 family, namely the so-called "precursor miR182 target site" and the so-called "precursor miR96 target site."

[0008] Therefore, these results demonstrate that a recombinant AAV-DJ vector containing a polynucleotide comprising a sequence encoding the connexin 26 protein operably linked to a promoter and a precursor miRNA target site of the miR183 family (such as a precursor miR183 target site) can be effectively used for prophylactic or therapeutic treatment of hereditary hearing loss. [Overview of the project]

[0009] The present invention relates to an adeno-associated virus (AAV) vector comprising an AAV-DJ capsid or a capsid derived from an AAV-DJ capsid, comprising a polynucleotide comprising (i) a nucleic acid sequence encoding a connexin 26 protein (CX26) operably linked to a promoter, and (ii) at least one copy of a miRNA target site of the miR183 family comprising the sequence described in SEQ ID NO: 3, SEQ ID NO: 16, or SEQ ID NO: 31, or a sequence having at least 90% identity with any one of SEQ ID NO: 3, SEQ ID NO: 16, or SEQ ID NO: 31. In some embodiments, the AAV vector comprises 2 to 6 copies, preferably 3 copies, of a miRNA target site of the miR183 family comprising the sequence described in SEQ ID NO: 3, SEQ ID NO: 16, or SEQ ID NO: 31, or a sequence having at least 90% identity with any one of SEQ ID NO: 3, SEQ ID NO: 16, or SEQ ID NO: 31.

[0010] In some embodiments, the promoter is the smCBA promoter. In some embodiments, the connexin 26 protein is human CX26. In some embodiments, the polynucleotide further comprises 5' and 3' inverted terminal repeats (ITRs). In some embodiments, the polynucleotide further comprises at least one copy of another miRNA target site of the miR183 family.

[0011] The present invention also relates to a pharmaceutical composition comprising the AAV vector described herein and at least one pharmaceutically acceptable excipient or carrier.

[0012] The present invention also relates to AAV vectors described herein or pharmaceutical compositions described herein for use as pharmaceuticals.

[0013] The present invention also relates to the AAV vectors or pharmaceutical compositions described herein for use in the treatment of hereditary hearing loss in subjects requiring such treatment. In some embodiments, the hereditary hearing loss is non-symptomatic hearing loss (DFNB1). In some embodiments, the hereditary hearing loss is severe hereditary hearing loss. In some embodiments, the hereditary hearing loss is high-grade hereditary hearing loss. In some embodiments, the hereditary hearing loss is progressive hereditary hearing loss. In some embodiments, the subject is an adult. In some embodiments, the subject is an infant or child.

[0014] definition In this invention, the following terms have the meanings set forth below.

[0015] The human gap junction protein β2 (or GJB2 protein) used herein corresponds to the protein referenced as NP_003995.2 in the NCBI database. The reference human GJB2 protein sequence corresponds to the amino acid sequence described in Sequence ID 1. In the NCBI database (https: / / www.ncbi.nlm.nih.gov), the reference human GJB2 gene sequence corresponds to NCBI gene ID:2706. The human GJB2 gene, also known as the CX26 gene, consists of two exons on chromosome 13q12.11. The human GJB2 transcript contains 2290 nucleotides and encodes 226 amino acid proteins. The human reference GJB2 transcript corresponds to the nucleic acid sequence described in Sequence ID 2. Other names for gap junction protein β2 include, in non-restrictive examples, "NSRD1," "gap junction protein, β2, 26kDa," "gap junction β-2 protein," "connexin 26," "connexin-26," "DFNA3," "DFNB1," "DFNA3A," "DFNB1A," "BAPS," "Cx26," "CX26," "HID," "KID," and "PPK." In this specification, the expressions "gap junction protein β2 or GJB2 protein" and "connexin 26 or CX26" are used interchangeably.

[0016] The terms "a" and "an" refer to one or more (i.e., at least one) grammatical objects of the article. For example, "one element" means one or more elements. Similarly, the terms "at least one" and "one or more" are interchangeable.

[0017] The term "approximately" preceding a number encompasses a range of ±10% or less of the given number. It should be understood that the value referred to by the term "approximately" is also specifically and preferably disclosed.

[0018] The term "code," as in a coding sequence, refers to the inherent properties of specific sequences of nucleotides in nucleic acids, such as genes, complementary DNA (cDNA), or messenger RNA (mRNA), and the biological characteristics arising therefrom, which serve as templates for the synthesis of other polymers and macromolecules in biological processes, whether they are defined sequences of nucleotides (e.g., ribosomal RNA (rRNA), transfer RNA (tRNA), mRNA) or defined sequences of amino acids (e.g., polypeptides or proteins).

[0019] "Expression" refers to the transcription and / or translation of a specific nucleotide sequence, such as a gene.

[0020] A "gene" refers to an RNA molecule, specifically a coding nucleic acid sequence that can be transcribed into either a coding RNA molecule such as mRNA, which can then be translated into a polypeptide or protein, or a non-coding RNA molecule such as rRNA or tRNA. A "transgene" specifically refers to a gene of one species that can be introduced into an organism belonging to a different species. Therefore, it should be noted that a gene may or may not contain a coding sequence (or CDS), i.e., a nucleic acid sequence that actually codes for a protein. A gene, especially one containing a CDS, may also contain untranslated transcription regions (UTRs) such as the 3'UTR and / or 5'UTR, as well as other sequences such as transcribed but untranslated regulatory elements and / or introns.

[0021] As used herein, "hereditary hearing impairment" and "hereditary deafness" can be used interchangeably and refer to a condition where sound cannot be partially or fully heard due to genetic causes. The severity of the hearing impairment (or deafness) described in this specification can vary. The level of severity typically refers to the degree of hearing impairment (or deafness), which can range from mild to severe.

[0022] When used herein in relation to the relationship between sequences of two or more nucleic acids or two or more polypeptides, “identity” refers to the degree of sequence relatedness between nucleic acids or polypeptides (each) determined by the number of matching sequences of two or more nucleotides or two or more amino acid residues, respectively. “Identity” is a measure of the degree of perfect agreement between shorter sequences of two or more sequences that have gap alignments (if any) treated by a particular mathematical model or computer program (i.e., “algorithm”). The identity of related nucleic acid or polypeptide sequences can be readily calculated by known methods. Such methods are not limited to, but include: "Computational Molecular Biology," edited by Lesk, AM, Oxford University Press, New York, 1988; "Biocomputing: Informatics and Genome Projects," edited by Smith, DW, Academic Press, New York, 1993; "Computer Analysis of Sequence Data, Part 1," edited by Griffin, AM and Griffin, HG, Humana Press, New Jersey, 1994; "Sequence Analysis in Molecular Biology," von Heinje, G., Academic Press, 1987; "Sequence Analysis Primer," edited by Gribskov, M. and Develeux, J., M. Stockton Press, New York, 1991; and Carillo et al., SIAM J. Applied Examples include those described in Math. 48, 1073 (1988). The preferred method for determining identity is designed to give the greatest match between the sequences being tested.The method for determining identity is described in publicly available computer programs. Preferred computer program methods for determining identity between two arrays include the GCG program package, including GAP (Devereux et al., Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387-95, Genetics Computer Group, University of Wisconsin, Madison, Wisconsin), BLASTP, BLASTN and FASTA (Altschul et al., J. Mol. Biol. 215, 403-410 (1990)). The BLASTX program is publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul et al., NCB / NLM / NIH, Bethesda, Maryland 20894; Altschul et al., J. Mol. Biol. 215, 403-410 (1990)). It should be noted that the term "polypeptide" as used herein can be used interchangeably with the terms "peptide" or "protein".

[0023] With respect to a cell, "isolated" refers to a cell removed from an organism, i.e., a cell that is no longer part of an organism. Typically, the term "isolated cell" may refer to a cell cultured in vitro. Examples of isolated cells include primary cells, immortalized cell lines and commercially available cell lines. Thus, an entire organism is explicitly excluded from the definition of "isolated cell".

[0024] MicroRNAs, or miRNAs, are small, endogenous non-coding RNA molecules consisting of approximately 18 to 24 nucleotides that play a crucial role in the post-transcriptional regulation of gene expression in eukaryotic cells. A single miRNA can regulate up to several hundred different mRNAs, and it is expected that most mRNAs are targeted by multiple miRNAs. miRNA genes are transcribed by RNA polymerase II or III, and then processed to produce single-stranded mature miRNAs, which are incorporated into the RNA-induced silencing complex (RISC). As the central component of the RISC complex, the miRNA guides RISC to its mRNA target, where it typically binds to the 3' untranslated region (3'UTR) of the mRNA transcript through partial complementary base pairing. Complete base pairing must occur over a short length of 7 or 8 nucleotides complementary to the so-called miRNA "seed" region, usually located at positions 2-8 from the 5' end of the mature miRNA. Gene silencing can be achieved by mRNA cleavage mediated by Argonaut-2 (AG02) or translational repression facilitated by AG01-4, both of which ultimately result in a reduction in the level of the corresponding protein. As used herein, “miRNA,” e.g., “miR183,” refers to a mature miRNA, e.g., mature miR183. In contrast, “precursor miRNA,” or “pre-miRNA,” e.g., “precursor miR183,” refers to the hairpin precursor sequence from which mature miRNA is processed.

[0025] As used herein, the “miR183 family” or “miR183 cluster” refers to a family or cluster consisting of three paralogous miRNAs (miR183 (or miR-183), miR96 (or miR-96), and miR182 (or miR-182)) that exhibit sequence homology. The miR-183 / 182 / 96 cluster is a gene located on the short arm of chromosome 7 (7q32.2) that generates a single polycistronic transcript producing miR183, miR96, and miR182. The miR183 family is particularly essential for the proper development of sensory organs.

[0026] As used herein, “microRNA target site,” “miRNA target site,” or “miR target site” refers to a nucleic acid sequence that can bind to a miRNA (i.e., a mature miRNA). As used herein, the terms “microRNA target site,” “miRNA target site,” or “miR target site” encompass both endogenous target sites that may be present in natural transcripts, and artificial, or engineered, target sites (i.e., target sites that do not occur naturally) that can be inserted as regulatory elements (or regulatory sequences) into vectors, particularly AAV vectors, to control the expression of a target nucleic acid sequence, such as a gene of interest. In particular, in vectors, a miRNA target site can be operably ligated to or inserted into a gene sequence, and in particular into a transcribed sequence of a gene. By definition, a miRNA target site must contain a nucleic acid sequence that is at least partially complementary to the corresponding miRNA, for example, a nucleic acid sequence complementary to the corresponding miRNA over at least 5 nucleotides, usually 6-7 nucleotides. Therefore, the miR183 target site must contain a nucleic acid sequence that is at least partially complementary to miR183, for example, a nucleic acid sequence complementary to miR183 over a length of at least 7-8 nucleotides. Similarly, the miR182 target site must contain a nucleic acid sequence that is at least partially complementary to miR182, for example, a nucleic acid sequence complementary to miR182 over a length of at least 7-8 nucleotides, and the miR96 target site must contain a nucleic acid sequence that is at least partially complementary to miR96, for example, a nucleic acid sequence complementary to miR96 over a length of at least 7-8 nucleotides. In addition, miRNA target sites, especially artificial or manipulated miRNA target sites (i.e., target sites that do not occur naturally), may contain or consist of a nucleic acid sequence complementary to miRNA over the entire length of the miRNA (i.e., over 18-24 nucleotides of the miRNA). When inserted into a vector, the miR target site enables the binding of the corresponding miRNA, and therefore, when this vector is introduced into a host cell expressing the miRNA, it can mediate miRNA-induced silencing of the expression of the target nucleic acid, such as the gene of interest.For example, when inserted into a vector, the miR183 target site enables the binding of miR183, and therefore, introducing this vector into host cells expressing miR183 can mediate miR183-induced silencing of the expression of a target nucleic acid, such as a target gene. Similarly, when inserted into a vector, the miR182 target site enables the binding of miR182, and therefore, introducing this vector into host cells expressing miR182 can mediate miR182-induced silencing of the expression of a target nucleic acid, such as a target gene. When inserted into a vector, the miR96 target site enables the binding of miR96, and therefore, introducing this vector into host cells expressing miR96 can mediate miR96-induced silencing of the expression of a target nucleic acid, such as a target gene. Methods for evaluating whether a nucleic acid sequence can be a suitable miRNA target site (e.g., a miR183 target site, a miR182 target site, or a miR96 target site) are well known in the art. One such method involves inserting the nucleic acid sequence to be evaluated into a vector containing a reporter gene, such as a gene encoding a GFP protein (green fluorescent protein), under the control of a ubiquitous promoter, i.e., a constitutive promoter. The nucleic acid sequence to be evaluated may be operably ligated to the gene at its 3'UTR, or it may be inserted into the gene. The vector containing the nucleic acid sequence to be evaluated and the reporter gene is then introduced into host cells expressing the corresponding miRNA (e.g., miR183, miR182, or miR96), such as HEK293 cells expressing miR183, miR182, and miR96. Inhibition of reporter gene expression in the host cells indicates that the nucleic acid sequence to be evaluated is a suitable miRNA target site, particularly when compared to a control condition in which the vector contains only the reporter gene under the control of a promoter.

[0027] As used herein, “miR183 family microRNA target site,” “miR183 family miRNA target site,” or “miR183 family miR target site” refers to a nucleic acid sequence to which miRNAs (i.e., mature miRNAs) belonging to the miR183 family (sometimes referred to as the miR183 cluster) can be bound. Thus, the term “miR183 family microRNA target site,” “miR183 family miRNA target site,” or “miR183 family miR target site” refers to a nucleic acid sequence to which miR183, miR182, and / or miR96 can be bound. In other words, the term “miR183 family microRNA target site,” “miR183 family miRNA target site,” or “miR183 family miR target site” encompasses the miR183 target site, the miR182 target site, and the miR96 target site.

[0028] "Nucleic acid" refers to a polymer of nucleotides covalently linked by phosphodiester bonds, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), in either single-stranded or double-stranded form (i.e., polynucleotide). Therefore, nucleic acids as used herein may be single-stranded, partially double-stranded, or fully double-stranded. The nucleotides constituting the nucleic acids of this disclosure may be unmodified (natural) nucleotides or non-natural, i.e., modified nucleotides. Examples of unmodified (i.e., natural or naturally occurring) nucleotides include adenosine monophosphate (AMP), deoxyadenosine monophosphate (dAMP), cytidine monophosphate (CMP), deoxycytidine monophosphate (dCMP), guanosine monophosphate (GMP), deoxyguanosine monophosphate (dGMP), thymidine monophosphate (TMP), deoxythymidine monophosphate (dTMP), and uridine monophosphate (UMP). The term "nucleic acid" also includes nucleic acids, including known analogues of natural nucleotides that have similar binding properties to the reference nucleic acid and are metabolized in a similar manner to naturally occurring nucleotides.

[0029] A “nucleic acid sequence” or “nucleotide sequence” refers to a continuous sequence of nucleotides in a single nucleic acid. Unless otherwise indicated, a particular nucleic acid sequence implicitly includes its conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologues, SNPs (single nucleotide polymorphisms), and complementary sequences, as well as sequences explicitly indicated. In particular, certain nucleic acid sequences described herein implicitly include their corresponding complementary sequences. It should be noted that certain nucleic acid sequences described herein implicitly include DNA sequences and their corresponding RNA sequences.

[0030] "Operatively linked" or "operably linked" refers to a functional link between a regulatory sequence and a nucleic acid sequence (e.g., a gene), where the former regulates the expression of the latter. For example, when a first nucleic acid sequence is functionally related to a second nucleic acid sequence, the first nucleic acid sequence is operationally linked to the second nucleic acid sequence. In particular, when a promoter affects the transcription or expression of a gene, the promoter is operationally linked to the gene. Similarly, when a regulatory sequence affects the expression of a gene (i.e., induces or inhibits (or represses) it), the regulatory sequence is operationally linked to the gene. Operationally linked sequences may be consecutive.

[0031] A "pharmaceutically acceptable excipient" or "pharmaceutically acceptable carrier" refers to an excipient or carrier that does not cause adverse reactions, allergic reactions, or other adverse reactions when administered to animals such as humans. This includes all solvents, such as dispersions, coatings, antimicrobial agents, antifungal agents, isotonic agents, and absorption retarders. A pharmaceutically acceptable excipient or carrier refers to any type of non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating agent, or formulation aid. Formulations intended for human administration must meet the sterility, pyrogenicity, general safety, and purity standards required by regulatory authorities such as the EMA (European Medicines Agency) or the FDA (U.S. Food and Drug Administration).

[0032] A "vector" refers to a medium that can introduce a nucleic acid sequence (e.g., a DNA or RNA molecule), such as RNA or a nucleic acid encoding a target polypeptide or protein, into a host cell in order to transform, transfect, or transduce the host cell and to promote the expression (e.g., transcription and / or translation) of the introduced nucleic acid sequence.

[0033] An "expression vector" refers to a vector containing regulatory elements (or regulatory sequences) operatively linked to a target nucleic acid sequence to be expressed, such as a gene of interest. Thus, an expression vector contains sufficient cis-regulatory elements to control the expression of the target nucleic acid sequence, and any other elements that may be required to control the expression of the target nucleic acid sequence may be supplied by a host cell or an in vitro expression system (e.g., miRNAs that bind to miR target sites). Examples of cis-regulatory elements include promoters and miR target sites such as the miR183 target site, miR182 target site, and miR96 target site described herein.

[0034] "Subject" refers to warm-blooded animals, more preferably mammals. The term "mammal" here refers to all mammals, including humans. Preferably, mammals are primates, more preferably humans.

[0035] "Treatment" refers to therapeutic (or curative) treatment, prophylactic treatment, or both therapeutic (or curative) and prophylactic treatment aimed at preventing, reducing, slowing (delaying progression), or curing one or more symptoms or manifestations of hereditary hearing loss, such as moderate to severe non-symptomatic hearing loss. In some embodiments, a subject is considered "successfully treated" for hereditary hearing loss if, after administration of a therapeutically effective dose or amount of the recombinant AAV vector described herein, the subject shows improvement in hearing, i.e., a delay in the progression of hearing loss. In some embodiments, a subject is considered "successfully treated" for hearing loss if, after administration of a therapeutically effective dose or amount of the recombinant AAV vector described herein, hearing loss is completely or partially prevented in the subject (i.e., hearing is completely or partially prevented in the subject). Methods for measuring or evaluating hearing loss (or hearing impairment) are well known to those skilled in the art. Examples of such methods include pure-tone audiometry (PTA) (also known as pure-tone audiograms), including pure-tone air conduction audiometry and pure-tone bone conduction audiometry, speech audiometry, auditory behavioral response audiometry, visually enhanced audiometry, conditioned play audiometry, ABR (auditory brainstem response) measurement, DPOAE (distortion component otoacoustic emissions) measurement, TEOAE (transient evoked otoacoustic emissions) measurement, speech audiometry under noise, word comprehension tests, tympanotomy, stapedius reflex tests, and tuning fork tests. Other methods that may be suitable for evaluating the effectiveness of treatment for hereditary hearing loss in subjects include brain functional imaging (such as functional near-infrared spectroscopy or fNIRS) and functional ultrasound imaging (fUS). By evaluating the presence of intracochlear potentials (or EP, also known as intracochlear DC potentials), which are positive voltages in the range of approximately 80 to 100 mV found in the intracochlear lymphatic space and controlled by K+ transport across the lateral wall of the cochle, the effectiveness of treatments for hereditary hearing loss in subjects, preferably non-human subjects, can also be assessed. [Modes for carrying out the invention]

[0036] The present invention relates to an adeno-associated virus (AAV) vector comprising an AAV-DJ capsid or a capsid derived from an AAV-DJ capsid, comprising a polynucleotide comprising (i) a nucleic acid sequence encoding a connexin 26 protein (also known as CX26) operably linked to a promoter, and (ii) at least one copy of a miR183 family miRNA target site comprising, consisting of, or having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with the sequence described in SEQ ID NO: 3, SEQ ID NO: 16, or SEQ ID NO: 31.

[0037] Another object considered herein is an adeno-associated virus (AAV) vector comprising an AAV-DJ capsid or a capsid derived from an AAV-DJ capsid, comprising a polynucleotide comprising (i) a nucleic acid sequence encoding CX26 operably ligated to a promoter, and (ii) at least one copy of a miR183 family miRNA target site comprising, consisting of, or having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with the sequence described in SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15.

[0038] AAV or an AAV vector containing a capsid, which is an AAV-DJ capsid used herein, may be referred to as AAV-DJ or an AAV-DJ vector, respectively. Accordingly, in some embodiments, the present invention relates to an AAV-DJ vector comprising (i) a nucleic acid sequence encoding CX26 operably ligated to a promoter, and (ii) at least one copy of a miR183 family miRNA target site comprising, consisting of, or having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with the sequence described in SEQ ID NO: 3, SEQ ID NO: 16, or SEQ ID NO: 31.

[0039] The polynucleotides contained in the AAV vectors described herein may be double-stranded (ds) nucleic acids or single-stranded (ss) nucleic acids. In some embodiments, the AAV-DJ vectors described herein contain double-stranded (ds) polynucleotides. In some embodiments, the AAV-DJ vectors described herein contain single-stranded (ss) polynucleotides.

[0040] As used herein, the terms “AAV vector” and “recombinant AAV vector (or rAAV)” can be used interchangeably. Accordingly, the present invention also relates to a recombinant AAV vector comprising an AAV-DJ capsid or a capsid derived from an AAV-DJ capsid, comprising a polynucleotide comprising (i) a nucleic acid sequence encoding CX26 operably ligated to a promoter, and (ii) at least one copy of a miR183 family miRNA target site comprising, consisting of, or having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with the sequence described in SEQ ID NO: 3, SEQ ID NO: 16, or SEQ ID NO: 31. In some embodiments, the present invention relates to a recombinant AAV-DJ vector comprising (i) a nucleic acid sequence encoding CX26 operably linked to a promoter, and (ii) at least one copy of a miR183 family miRNA target site comprising, consisting of, or having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with the sequence described in SEQ ID NO: 3, SEQ ID NO: 16, or SEQ ID NO: 31.

[0041] The present invention also relates to AAV particles comprising an AAV-DJ capsid or a capsid derived from an AAV-DJ capsid, comprising a polynucleotide comprising (i) a nucleic acid sequence encoding CX26 operably linked to a promoter, and (ii) at least one copy of a miR183 family miRNA target site comprising, consisting of, or having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with the sequence described in SEQ ID NO: 3, SEQ ID NO: 16, or SEQ ID NO: 31. In some embodiments, the present invention relates to AAV-DJ particles comprising (i) a nucleic acid sequence encoding CX26 operably linked to a promoter, and (ii) at least one copy of a miR183 family miRNA target site comprising, consisting of, or having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with the sequence described in SEQ ID NO: 3, SEQ ID NO: 16, or SEQ ID NO: 31.

[0042] The present invention also relates to AAV virus particles comprising an AAV-DJ capsid or a capsid derived from an AAV-DJ capsid, comprising a polynucleotide comprising (i) a nucleic acid sequence encoding CX26 operably linked to a promoter, and (ii) at least one copy of a miR183 family miRNA target site comprising, consisting of, or having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with the sequence described in SEQ ID NO: 3, SEQ ID NO: 16, or SEQ ID NO: 31. In some embodiments, the present invention relates to an AAV-DJ virus particle comprising a polynucleotide comprising (i) a nucleic acid sequence encoding CX26 operably linked to a promoter, and (ii) at least one copy of a miR183 family miRNA target site comprising, consisting of, or having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with the sequence described in SEQ ID NO: 3, SEQ ID NO: 16, or SEQ ID NO: 31.

[0043] In some embodiments, the polynucleotide contained in the AAV vector described herein is an expression cassette. Thus, in some embodiments, the expression cassette used herein corresponds to a single polynucleotide, i.e., a single nucleic acid. The expression cassette contained in the AAV vector described herein comprises at least (i) a nucleic acid sequence encoding CX26 operably ligated to a promoter, and (ii) at least one copy of a miR183 family miRNA target site containing, consisting of, or having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with the sequence described in SEQ ID NO: 3, SEQ ID NO: 16, or SEQ ID NO: 31.

[0044] The expression cassette may include two ITRs, namely a 5'ITR and a 3'ITR, located at its 5' end and 3' end, respectively. Thus, the expression cassette may be sandwiched between the two ITRs, namely a 5'ITR and a 3'ITR.

[0045] The expression cassette may further include regulatory elements (or control sequences), particularly regulatory elements, that are operably ligated to the nucleic acid sequence encoding CX26. Thus, the expression cassette may include enough cis-regulatory elements to control the expression of the nucleic acid sequence encoding CX26, such as polyadenylation signals (or poly(A) signals), chimerichtrons, and / or WPREs.

[0046] The miR183 family miRNA target sites used herein are selected from the miR183 target sites, miR182 target sites, and miR96 target sites.

[0047] Human microRNA 183 (miR183 or miR-183) belongs to the miR183 family, which consists of three homologous miRNAs: miR183 (or miR-183), miR96 (or miR-96), and miR182 (or miR-182). miRNAs of the miR183 family are particularly necessary for the proper development of sensory organs. In particular, miRNAs of the miR183 family are expressed in hair cells of vertebrates.

[0048] In some embodiments, the miR183 family miRNA target sites include, consist of, or have a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical to the sequence described in SEQ ID NO: 3.

[0049] As used herein, the “miR183 target site containing, consisting of, or having the sequence described in Sequence ID No. 3” corresponds to the so-called “precursor miR183 target site.”

[0050] The human miR-183 / 182 / 96 cluster gene consists of a single exon on chromosome 7q32.2. Mature miR183 is produced by the processing of a hairpin precursor called precursor miR183 or pre-miR183. Human precursor miR183 is 110 nucleotides long and consists of the sequence described in Sequence ID No. 4, which is referenced as NR_029615.1 in the NCBI database or MI0000273 in miRBase (https: / / www.mirbase.org).

[0051] Processing of the hairpin precursor miR183, which is folded into a stem-loop structure, produces mature miR183. Human mature miR183, called miR183-5p (or hsa-miR183-5p or hsa-miR-183-5p), is 22 nucleotides long and consists of the sequence described in SEQ ID NO: 5, referenced as MIMAT0000261 in miRBase. The sequence of miR183-5p corresponds to nucleotides 27-48 of the human precursor miR183 in SEQ ID NO: 4. The miR183 target site complementary to the sequence of hsa-miR183-5p (i.e., SEQ ID NO: 5) consists of the sequence described in SEQ ID NO: 29. Human mature miR183, called miR183-3p (or hsa-miR183-3p or hsa-miR-183-3p), is also 22 nucleotides long and consists of the sequence described in SEQ ID NO: 6, referenced as MIMAT0004560 in miRBase. The sequence of miR183-3p corresponds to nucleotides 66-87 of the human precursor miR183 in SEQ ID NO: 4. The miR183 target site complementary to the sequence of hsa-miR183-3p (i.e., SEQ ID NO: 6) consists of the sequence described in SEQ ID NO: 30. As used herein, the term “mature miR183” (or “mature miR-183”) encompasses both miR183-5p and miR183-3p. In other words, as used herein, both miR183-5p and miR183-3p can be referred to as “mature miR183” (or “mature miR-183”).

[0052] In vivo, mature miR183 can bind to target mRNA containing a short sequence complementary to the seed region of the mature miR183. For example, the seed region of hsa-miR183-5p is AUGGCAC, corresponding to nucleotides 2-8 of hsa-miR183-5p (SEQ ID NO: 5). Similarly, the seed region of hsa-miR183-3p is UGAAUUA, corresponding to nucleotides 2-8 of hsa-miR183-3p (SEQ ID NO: 6).

[0053] As shown in the experimental section below, we have surprisingly demonstrated that a 110-nucleotide miR183 target site having the sequence described in Sequence ID No. 3, which is complementary to the human sequence of precursor miR183 (i.e., Sequence ID No. 4), can be successfully used as a regulatory element to control CX26 expression in inner ear hair cells expressing miR183 when inserted into an AAV vector. As shown above, the miR183 target site consisting of a sequence complementary to the human sequence of precursor miR183 may also be referred to herein as the “precursor miR183 target site” (i.e., Sequence ID No. 3). Upon introduction into cells, the miR183 target site contained in this AAV vector is transcribed along the sequence encoding CX26 and is therefore present in the resulting mRNA. The 110-nucleotide miR183 target site having the sequence described in Sequence ID No. 3 is expected to fold into a stem-loop structure. To our surprise, and contrary to expectations, the inventors demonstrated that the stem-loop structure does not hinder the binding of miR183 to the miR183 target site of Sequence ID No. 3.

[0054] In some embodiments, the polynucleotides in the AAV vector described herein include, consist of, or comprise at least one copy of the miR183 target site having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with the sequence described in SEQ ID NO: 3, or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% or more identity with the sequence in SEQ ID NO: 3.

[0055] In some embodiments, a mirR183 target site containing, comprising, or having a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical to sequence number 3 has a length of at least 100 nucleotides. In some embodiments, a mirR183 target site containing, comprising, or having a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical to sequence number 3 has a length of at least 80, 85, 90, 95, 100, or 105 nucleotides. In some embodiments, a mirR183 target site comprising, consisting of, or having a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical to sequence number 3 has a length of 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, or 110 nucleotides.

[0056] The miR183 target sites used herein are functional miR183 target sites, i.e., they enable the binding of miR183. In some embodiments, a miR183 target site that includes, consists of, or has a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with Sequence ID No. 3 described herein is a functional miR183 target site, i.e., it enables the binding of miR183.

[0057] In some embodiments, the mirR183 target site contains, consists of, or has the sequence described in Sequence ID No. 3, in which up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 nucleotides are substituted with different nucleotides relative to the corresponding nucleotide in Sequence ID No. 3. In some embodiments, such mirR183 target sites that include, consist of, or have the sequence described in Sequence ID No. 3 having a nucleotide substitution are functional mirR183 target sites, i.e., they enable the binding of miR183.

[0058] In some embodiments, the functional mirR183 target site described herein includes at least one sequence complementary to the seed region of a mature miR183, such as miR183-5p or miR183-3p. In some embodiments, the functional mirR183 target site includes a sequence complementary to the seed region of miR183-5p, preferably a sequence complementary to the seed region of hsa-miR183-5p (i.e., a sequence complementary to nucleotides 2-8 of SEQ ID NO: 5). Thus, in some embodiments, the functional mirR183 target site includes a sequence complementary to AUGGCAC. In some embodiments, the functional mirR183 target site includes a sequence complementary to the seed region of miR183-3p, preferably a sequence complementary to the seed region of hsa-miR183-3p (i.e., a sequence complementary to nucleotides 2-8 of SEQ ID NO: 6). Thus, in some embodiments, the functional mirR183 target site includes a sequence complementary to UGAAUUA.

[0059] In some embodiments, the miR183 family miRNA target sites are miR182 target sites that include, consist of, or have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with the sequence described in SEQ ID NO: 16.

[0060] As used herein, the “miR182 target site comprising, consisting of, or having the sequence described in Sequence ID No. 16” corresponds to the so-called “precursor miR182 target site.”

[0061] As mentioned above, the human miR-183 / 182 / 96 cluster gene consists of a single exon on chromosome 7q32.2. Mature miR182 is produced by the processing of a hairpin precursor called precursor miR182 or pre-miR182. Human precursor miR182 is 110 nucleotides long and has the sequence described in Sequence ID No. 32, which is referenced as NR_029614.1 in the NCBI database or MI0000272 in miRBase (https: / / www.mirbase.org).

[0062] Processing of the hairpin precursor miR182, which is folded into a stem-loop structure, results in mature miR182. Human mature miR182, called miR182-5p (or hsa-miR182-5p or hsa-miR-182-5p), is 24 nucleotides long and consists of the sequence described in Sequence ID No. 33, which is referenced as MIMAT0000259 in miRBase. The sequence of miR182-5p corresponds to nucleotides 23-46 of the human precursor miR182 in Sequence ID No. 32. The miR182 target site complementary to the sequence of hsa-miR182-5p (i.e., Sequence ID No. 33) consists of the sequence described in Sequence ID No. 12. The human mature miR182, called miR182-3p (or hsa-miR182-3p or hsa-miR-182-3p), is 21 nucleotides long and consists of the sequence described in Sequence ID No. 34, referenced as MIMAT0000260 in miRBase. The sequence of miR182-3p corresponds to nucleotides 67-87 of the human precursor miR182 in Sequence ID No. 32. The miR182 target site complementary to the sequence of hsa-miR182-3p (i.e., Sequence ID No. 34) consists of the sequence described in Sequence ID No. 13. As used herein, the term “mature miR182” (or “mature miR-182”) encompasses both miR182-5p and miR182-3p. In other words, as used herein, both miR182-5p and miR182-3p can be referred to as “mature miR182” (or “mature miR-182”).

[0063] In vivo, mature miR182 can bind to target mRNA containing a short sequence complementary to the seed region of the mature miR182. For example, the seed region of hsa-miR182-5p is UUGGCAA, corresponding to nucleotides 2-8 of hsa-miR182-5p (sequence number 33). Similarly, the seed region of hsa-miR182-3p is GGUUCUA, corresponding to nucleotides 2-8 of hsa-miR182-3p (sequence number 34).

[0064] In some embodiments, the polynucleotides in the AAV vector described herein include, consist of, or comprise at least one copy of a miR182 target site having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with the sequence described in SEQ ID NO: 16, preferably a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% or more identity with SEQ ID NO: 16.

[0065] In some embodiments, a mirR182 target site containing, comprising, or having a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical to sequence number 16 has a length of at least 100 nucleotides. In some embodiments, a mirR182 target site containing, comprising, or having a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical to sequence number 16 has a length of at least 80, 85, 90, 95, 100, or 105 nucleotides. In some embodiments, the mirR182 target site includes, consists of, or has a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical to sequence number 16, and has a length of 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, or 110 nucleotides.

[0066] The miR182 target sites used herein are functional miR182 target sites, i.e., they enable the binding of miR182. In some embodiments, a miR182 target site that includes, consists of, or has a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with Sequence ID No. 16 described herein is a functional miR182 target site, i.e., it enables the binding of miR182.

[0067] In some embodiments, the mirR182 target site includes, consists of, or has the sequence described in Sequence ID No. 16, in which up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 nucleotides are substituted with different nucleotides relative to the corresponding nucleotide in Sequence ID No. 16. In some embodiments, such mirR182 target sites that include, consist of, or have the sequence described in SEQ ID NO: 16 having a nucleotide substitution are functional mirR182 target sites, i.e., they enable the binding of miR182.

[0068] In some embodiments, the functional mirR182 target site includes at least one sequence complementary to the seed region of a mature miR182, such as miR182-5p or miR182-3p. In some embodiments, the functional mirR182 target site includes a sequence complementary to the seed region of miR182-5p, preferably a sequence complementary to the seed region of hsa-miR182-5p (i.e., a sequence complementary to nucleotides 2-8 of SEQ ID NO: 33). Thus, in some embodiments, the functional mirR182 target site includes a sequence complementary to UUGGCAA. In some embodiments, the functional mirR182 target site includes a sequence complementary to the seed region of miR182-3p, preferably a sequence complementary to the seed region of hsa-miR182-3p (i.e., a sequence complementary to nucleotides 2-8 of SEQ ID NO: 34). Thus, in some embodiments, the functional mirR182 target site includes a sequence complementary to GGUUCUA.

[0069] In some embodiments, the miR183 family miRNA target site is a miR96 target site that includes, consists of, or has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with the sequence described in SEQ ID NO: 31.

[0070] As used herein, the "miR96 target site comprising, consisting of, or having the sequence described in Sequence ID No. 31" corresponds to the so-called "precursor miR96 target site."

[0071] As mentioned above, the human miR-183 / 182 / 96 cluster gene consists of a single exon on chromosome 7q32.2. Mature miR96 is produced by the processing of a hairpin precursor called precursor miR96 or pre-miR96. Human precursor miR96 is 78 nucleotides long and has the sequence described in Sequence ID No. 17, which is referenced as NR_029512.1 in the NCBI database or MI0000098 in miRBase (https: / / www.mirbase.org).

[0072] Mature miR96 is produced by the processing of the hairpin precursor miR96, which is folded into a stem-loop structure. Human mature miR96, called miR96-5p (or hsa-miR96-5p or hsa-miR-96-5p), is 23 nucleotides long and has the sequence described in SEQ ID NO: 35, which is referenced as MIMAT0000095 in miRBase. The sequence of miR96-5p corresponds to nucleotides 9-31 of the human precursor miR96 in SEQ ID NO: 17. Human mature miR96, called miR96-3p (or hsa-miR96-3p or hsa-miR-96-3p), is 22 nucleotides long and has the sequence described in SEQ ID NO: 36, which is referenced as MIMAT0004510 in miRBase. The sequence of miR96-3p corresponds to nucleotides 52-73 of the human precursor miR96 in SEQ ID NO: 17. As used herein, the term “mature miR96” (or “mature miR-96”) encompasses both miR96-5p and miR96-3p. In other words, as used herein, both miR96-5p and miR96-3p can be referred to as “mature miR96” (or “mature miR-96”).

[0073] In vivo, mature miR96 can bind to target mRNA containing a short sequence complementary to the seed region of the mature miR96. For example, the seed region of hsa-miR96-5p is UUGGCAC, corresponding to nucleotides 2-8 of hsa-miR96-5p (SEQ ID NO: 35). Similarly, the seed region of hsa-miR96-3p is AUCAUGU, corresponding to nucleotides 2-8 of hsa-miR96-3p (SEQ ID NO: 36).

[0074] In some embodiments, the polynucleotides in the AAV vector described herein include, consist of, or comprise at least one copy of a miR96 target site having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with the sequence described in SEQ ID NO: 31, preferably a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% or more identity with the sequence in SEQ ID NO: 31.

[0075] In some embodiments, a mirR96 target site containing, comprising, or having a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 31 has a length of at least 70 nucleotides. In some embodiments, a mirR96 target site containing, comprising, or having a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 31 has a length of at least 55, 60, or 65 nucleotides. In some embodiments, mirR96 target sites that include, consist of, or have a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with SEQ ID NO: 31 have a length of 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, or 78 nucleotides.

[0076] The miR96 target sites used herein are functional mirR96 target sites, i.e., they enable the binding of miR96. In some embodiments, a mirR96 target site that includes, consists of, or has a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with SEQ ID NO: 31 described herein is a functional mirR96 target site, i.e., it enables the binding of miR96.

[0077] In some embodiments, the mirR96 target site contains, consists of, or has the sequence described in Sequence ID No. 31, in which up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleotides are substituted with different nucleotides relative to the corresponding nucleotide in Sequence ID No. 31. In some embodiments, such a mirR96 target site containing, consisting of, or having the sequence described in Sequence ID No. 31 with nucleotide substitutions is a functional mirR96 target site, i.e., it enables the binding of miR96.

[0078] In some embodiments, the functional mirR96 target site includes at least one sequence complementary to the seed region of a mature miR96, such as miR96-5p or miR96-3p. In some embodiments, the functional mirR96 target site includes a sequence complementary to the seed region of miR96-5p, preferably a sequence complementary to the seed region of hsa-miR96-5p (i.e., a sequence complementary to nucleotides 2-8 of SEQ ID NO: 35). Thus, in some embodiments, the functional mirR96 target site includes a sequence complementary to UUGGCAC. In some embodiments, the functional mirR96 target site includes a sequence complementary to the seed region of miR96-3p, preferably a sequence complementary to the seed region of hsa-miR96-3p (i.e., a sequence complementary to nucleotides 2-8 of SEQ ID NO: 36). Thus, in some embodiments, the functional mirR96 target site includes a sequence complementary to AUCAUGU.

[0079] As used herein, the expression "at least one copy" preferably includes one, two, three, four, five, six, seven, eight, nine, ten, or more copies of a miRNA target site of the miR183 family that includes, consists of, or has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with the sequence described herein as Sequence ID No. 3, Sequence ID No. 16, or Sequence ID No. 31.

[0080] As used herein, the term “copy” has its usual meaning, meaning that several copies of a miRNA target site of the miR183 family correspond to the same sequence. In other words, as used herein, the expression “at least one copy” preferably includes one, two, three, four, five, six, seven, eight, nine, ten, or more identical copies of a miRNA target site of the miR183 family that includes, consists of, or has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with the sequence described herein as SEQ ID NO: 3, SEQ ID NO: 16, or SEQ ID NO: 31.

[0081] In some embodiments, the polynucleotides in the AAV vector described herein include, consist of, or comprise 2 to 10 copies, preferably 2 to 6 copies, of miR183 family miRNA target sites that include, or comprise a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with the sequence described in SEQ ID NO: 3, SEQ ID NO: 16, or SEQ ID NO: 31, or a sequence having such identity.

[0082] In some embodiments, the polynucleotides in the AAV vector described herein include, consist of, or comprise 2 to 10 copies, preferably 2 to 6 copies, of a miR183 target site having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with the sequence described in SEQ ID NO: 3 or the sequence described herein.

[0083] In some embodiments, the polynucleotides in the AAV vector described herein include, consist of, or comprise 2 to 10 copies, preferably 2 to 6 copies, of a miR182 target site having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with the sequence described in SEQ ID NO: 16 or the sequence described herein.

[0084] In some embodiments, the polynucleotides in the AAV vector described herein include, consist of, or comprise 2 to 10 copies, preferably 2 to 6 copies, of a miR96 target site having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with the sequence described in SEQ ID NO: 31 or the sequence described herein.

[0085] In the polynucleotides in the AAV vectors described herein, copies of the miRNA target sites of the miR183 family described herein may be contiguous. In other words, in some embodiments, copies of the miRNA target sites of the miR183 family described herein are not separated from each other, in particular by spacers.

[0086] In some embodiments, the polynucleotides in the AAV vectors described herein include, consist of, or comprise three copies, preferably three consecutive copies, of a miR183 target site having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with the sequence described in SEQ ID NO: 3. Accordingly, the polynucleotides in the AAV vectors described herein may include a control sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with the control sequence described in SEQ ID NO: 7. The control sequence of SEQ ID NO: 7 consists of or comprises three copies of a miR183 target site having the sequence described in SEQ ID NO: 3.

[0087] In some embodiments, the polynucleotides in the AAV vector described herein include, consist of, or comprise three copies, preferably three consecutive copies, of a miR182 target site having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with the sequence described in SEQ ID NO: 16 or SEQ ID NO: 16.

[0088] In some embodiments, the polynucleotides in the AAV vector described herein include, consist of, or comprise three copies, preferably three consecutive copies, of a miR96 target site having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with the sequence described in SEQ ID NO: 31 or SEQ ID NO: 31.

[0089] In the polynucleotides of the AAV vectors described herein, copies of the miR183 family miRNA target sites described herein may be separated from each other. In particular, in the polynucleotides of the AAV vectors described herein, copies of the miR183 family miRNA target sites described herein may be separated from each other by spacers. In other words, spacers may optionally be inserted between two copies of the miR183 family miRNA target sites described herein.

[0090] As used herein, “spacer” refers to a non-coding sequence. Such spacers may be characterized by a length of approximately 5 to 25 nucleotides, preferably approximately 10 to 20 nucleotides, and more preferably approximately 20 nucleotides. Spacers are commonly used in the art and are well known to those skilled in the art. For example, spacers are described in Hammarsten et al., “Herpes simplex virus: selection of origins of DNA replication,” Nucleic Acids Res. 1997 May 1;25(9):1753-60. Examples of spacers include spacers containing, consisting of, or having the sequence described in SEQ ID NO: 8 (ATAACTAAAAGATTCGGA), SEQ ID NO: 9 (AATATATATATATTATTA), SEQ ID NO: 10 (AAAAACATATAAAATAAT), or SEQ ID NO: 11 (CTTTCTTTTCCCAATTTT). For example, such spacer may contain, consist of, or have the sequence described in SEQ ID NO: 8.

[0091] In the polynucleotides of the AAV vectors described herein, one or more copies of the miR183 family miRNA target sites described herein may be operationally ligated to the nucleic acid sequence encoding CX26. "Operationally ligated" (or "operationally ligated") means that one or more copies of the miR183 family miRNA target sites described herein can affect the expression of the nucleic acid sequence encoding CX26.

[0092] In some embodiments, one or more copies of the miRNA target sites of the miR183 family described herein are contiguous with the nucleic acid sequence encoding CX26. In some embodiments, one or more copies of the miRNA target sites of the miR183 family described herein are adjacent to the nucleic acid sequence encoding CX26 at 5' or 3', preferably 3'. In some embodiments, one or more copies of the miRNA target sites described herein are inserted into an untranslated region (UTR) adjacent to the nucleic acid sequence encoding CX26. Examples of untranslated regions include the 5'UTR, 3'UTR, and introns. In some embodiments, one or more copies of the miRNA target sites of the miR183 family described herein are inserted into the 5'UTR adjacent to the nucleic acid sequence encoding CX26. In some embodiments, one or more copies of the miRNA target sites of the miR183 family described herein are inserted into the 3'UTR adjacent to the nucleic acid sequence encoding CX26. In some embodiments, one or more copies of the miRNA target sites of the miR183 family described herein are inserted into a nucleic acid sequence encoding CX26, preferably an intron.

[0093] In some embodiments, the polynucleotides in the AAV vector described herein include at least one miRNA target site of the miR183 family that includes, consists of, or has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with the sequence described herein as SEQ ID NO: 3, SEQ ID NO: 16, or SEQ ID NO: 31, and at least one other miR183 target site, such as a miR183 target site, that includes, consists of, or has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with the sequence described herein as SEQ ID NO: 29, or SEQ ID NO: 30, or either SEQ ID NO: 29, or SEQ ID NO: 30. For example, the polynucleotides in the AAV vector described herein may include, consist of, or have at least one miR183 target site that includes, consists of, or has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with the sequence described in SEQ ID NO: 3 as described herein, or SEQ ID NO: 3, and at least one other miR183 target site, such as a miR183 target site that includes, consists of, or has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with the sequence described in SEQ ID NO: 29 or SEQ ID NO: 30, or either SEQ ID NO: 29 or SEQ ID NO: 30.

[0094] In some embodiments, the polynucleotide in the AAV vector described herein further comprises at least one other miR target site (i.e., a miR target site other than the miR183 family of miR targets described herein). In some embodiments, the polynucleotide in the AAV vector described herein further comprises two, three, four, five, six, seven, eight, nine, ten or more other miR target sites, i.e., copies of either the same other miR target site or different miR target sites.

[0095] In some embodiments, the polynucleotide in the AAV vector described herein further comprises 2 to 10, preferably 2 to 6 copies of another miR target site (i.e., a miR target site other than the miRNA target site of the miR183 family described herein). In some embodiments, the polynucleotide in the AAV vector described herein further comprises 3 copies of another miR target site (i.e., a miR target site other than the miRNA target site of the miR183 family described herein).

[0096] In particular, other miR target sites may be miR target sites recognized by miRNAs expressed in sensory neurons and / or hair cells. Other miR target sites may be other miRNA targets of the miR183 family, namely the miR194 target site, miR140 target site, miR18a target site, miR99a target site, miR30b target site, miR15a target site, miR210 target site, miR124 target site and / or miR376 target site.

[0097] In some embodiments, other miR target sites are other miRNA targets of the miR183 family. For example, the polynucleotide in the AAV vector described herein may include, consist of, or have at least one miR183 target site containing, a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with the sequence described in Sequence ID No. 3 herein, and a miR182 target site and / or a miR96 target site. The polynucleotide in the AAV vector described herein may include, consist of, or have at least one miR182 target site, a miR183 target site and / or a miR96 target site containing, a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with the sequence described in Sequence ID No. 16 herein, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with the sequence described in Sequence ID No. 16 herein, and / or a miR96 target site. Alternatively, the polynucleotides in the AAV vectors described herein may include, consist of, or have at least one miR96 target site, miR183 target site and / or miR182 target site having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with the sequence described in Sequence ID No. 31 described herein.

[0098] Other miR target sites may be so-called "mature miR target sites," i.e., miR target sites having a sequence complementary to the corresponding mature miR. For example, the miR182 target site may be a so-called "mature miR182 target site," i.e., a miR182 target site having a sequence complementary to mature miR182 (such as hsa-miR182-5p or hsa-miR182-3p). Accordingly, in some embodiments, the miR182 target site (or mature miR182 target site) contains, consists of, or has a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with the sequence described in SEQ ID NO: 12 or SEQ ID NO: 13, or either SEQ ID NO: 12 or SEQ ID NO: 13. Similarly, in some embodiments, the miR96 target site (or mature miR96 target site) includes, consists of, or has a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with the sequence described in SEQ ID NO: 14 or SEQ ID NO: 15, or either SEQ ID NO: 14 or SEQ ID NO: 15.

[0099] Other miR target sites may be so-called "precursor miR target sites," that is, miR target sites having a sequence complementary to the corresponding precursor miR. For example, the miR182 target site may be a so-called "precursor miR182 target site," that is, a miR182 target site having a sequence complementary to precursor miR182. Accordingly, in some embodiments, the miR182 target site (or precursor miR182 target site) contains, consists of, or has a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with the sequence described in Sequence ID No. 16 or Sequence ID No. 16. Similarly, in some embodiments, the miR96 target site (or precursor miR96 target site) includes, consists of, or has a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical to the sequence described in SEQ ID NO: 31 or SEQ ID NO: 31.

[0100] In some embodiments, the polynucleotides in the AAV vectors described herein contain 2 to 10, preferably 2 to 6, miR target sites that include, consist of, or have at least one miR target site that is identical to, the sequence described in SEQ ID NO: 3, SEQ ID NO: 16, or SEQ ID NO: 31, or a sequence that is identical to, or has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more of, the sequence described in SEQ ID NO: 3, SEQ ID NO: 16, or SEQ ID NO: 31, or a sequence that is identical to, or has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more of, the sequence described in SEQ ID NO: 3, or a sequence that is identical to, or has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more of, the sequence that is identical to, or has at least 2 to 10, preferably 2 to 6, miR target sites that are miR target sites of the miR183 family.

[0101] In some embodiments, the polynucleotides in the AAV vectors described herein include three miR target sites that are miR target sites of the miR183 family, or consist of at least one miR target site having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with the sequence described in SEQ ID NO: 3, SEQ ID NO: 16, or SEQ ID NO: 31. For example, the polynucleotides in the AAV vectors described herein may include three miR target sites that are miR target sites of the miR183 family, or consist of at least one miR target site having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with the sequence described in SEQ ID NO: 3, or a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with the sequence described in SEQ ID NO: 3.

[0102] In the polynucleotides in the AAV vectors described herein, the miR target sites or copies of miR target sites described herein may be contiguous. Alternatively, in the polynucleotides in the AAV vectors described herein, the miR target sites or copies of miR target sites described herein may be separated from each other. In particular, in the polynucleotides in the AAV vectors described herein, the miR target sites or copies of miR target sites described herein may be separated from each other by spacers described herein. In other words, spacers may optionally be inserted between the miR target sites or copies of miR target sites described herein.

[0103] In some embodiments, the capsid of the AAV vector described herein is an AAV-DJ capsid. Therefore, in some embodiments, the AAV vector described herein is an AAV-DJ vector.

[0104] AAV-DJ capsids are chimeric hybrid capsids derived from eight serotypes, mainly AAV-2, AAV-8, and AAV-9 (Grimm D et al., "In vitro and in vivo gene therapy vector evolution via multispecies interbreeding and retargeting of adeno-associated viruses." J Virol. 2008 Jun;82(12):5887-911).

[0105] In some embodiments, the AAV-DJ capsid contains, comprises, or has the amino acid sequence described in SEQ ID NO: 18, which corresponds to the protein referenced as 3J1Q_A in the NCBI database. For example, the AAV-DJ capsid may be encoded by the nucleic acid sequence described in SEQ ID NO: 19.

[0106] In some embodiments, the AAV-DJ capsid is up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 5 The AAV-DJ capsid contains, consists of, or has the amino acid sequence described in Sequence ID No. 18, in which 0, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or 75 amino acids are substituted with different amino acids based on the corresponding amino acids in Sequence ID No. 18. In some embodiments, such an AAV-DJ capsid containing, consisting of, or having the amino acid sequence described in Sequence ID No. 18 with amino acid substitutions is a functional AAV-DJ capsid, i.e., it enables transduction of cells with the same efficiency and / or specificity as an AAV-DJ capsid containing, consisting of, or having the amino acid sequence described in Sequence ID No. 18.

[0107] Methods for evaluating the efficiency and / or specificity of transduction of an AAV vector by a given capsid are well known to those skilled in the art. Such methods include, for example, detecting or measuring the expression of a target gene (e.g., GJB2) contained in the AAV vector containing a given capsid in cells and / or tissues targeted by the AAV vector containing a given capsid by immunostaining or RT-qPCR (real-time quantitative polymerase chain reaction). Such methods also include, for example, detecting or measuring the expression of a target tagged gene (e.g., tagged GJB2 as described herein) contained in the AAV vector containing a given capsid in cells and / or tissues targeted by the AAV vector containing a given capsid by immunostaining. Such methods also include, for example, detecting or measuring the expression of a reporter gene contained in the AAV vector containing a given capsid in cells and / or tissues targeted by the AAV vector containing a given capsid by fluorescence microscopy.

[0108] In some embodiments, the AAV vector capsid described herein is the AAV-DJ capsid-derived capsid described herein. In some embodiments, the AAV-DJ capsid-derived capsid is a capsid comprising, consisting of, or having an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with SEQ ID NO: 18, preferably an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 18. In some embodiments, an AAV-DJ capsid containing, comprising, or having an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with SEQ ID NO: 18 is a functional AAV-DJ derived capsid, i.e., it enables cell transduction with the same efficiency and / or specificity as an AAV-DJ capsid containing, comprising, or having the amino acid sequence described in SEQ ID NO: 18. Accordingly, in some embodiments, the AAV vector described herein comprises an AAV-DJ capsid or a functional AAV-DJ derived capsid described herein.

[0109] In some embodiments, the AAV-DJ capsid-derived capsid contains, comprises, or has the amino acid sequence described in SEQ ID NO: 20, which is more than 99% identical to the amino acid sequence described in SEQ ID NO: 18.

[0110] In some embodiments, the polynucleotides in the AAV vectors described herein include nucleic acid sequences encoding human connexin 26 protein (i.e., human CX26 or hCX26) or a functional fragment or variant thereof.

[0111] In some embodiments, the nucleic acid encodes human CX26 comprising, consisting of, or having a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with the amino acid sequence described in SEQ ID NO: 1. In some embodiments, such human connexin 26 (hCX26) protein having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with SEQ ID NO: 1 is an hCX26 functional fragment or variant, i.e., it enables the formation of gap junctions, in particular the transport of potassium ions (K+) and several small molecules.

[0112] In some embodiments, the nucleic acid sequence encoding human CX26 includes, consists of, or comprises the sequence described in SEQ ID NO: 21 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with SEQ ID NO: 21.

[0113] In some embodiments, CX26, in particular human CX26, is tagged. "Tagging" means that CX26, in particular human CX26, is fused to a peptide (a so-called "peptide tag" or "tag").

[0114] Examples of tags include human influenza hemagglutinin tags (also known as HA tags), polyarginine tags, polyhistidine tags, myc tags, strep tags, GST tags, maltose-binding protein tags, or fluorescent protein tags. In particular, the tag may be an HA tag.

[0115] Methods for fusing peptide tags to proteins are well-known and routinely used. Simply put, such methods involve inserting a nucleic acid sequence encoding a peptide tag into a polynucleotide containing a nucleic acid sequence encoding a protein. The nucleic acid sequence encoding the peptide tag can be inserted at the 3' or 5' position of the protein-encoding nucleic acid sequence so that the peptide tag is located at the N-terminus or C-terminus, respectively. Furthermore, a short nucleic acid sequence encoding a linker or spacer may be present between the nucleic acid sequence encoding the peptide tag and the nucleic acid sequence encoding the protein in the polynucleotide.

[0116] Accordingly, in some embodiments, the polynucleotide in the AAV vector described herein comprises a nucleic acid sequence encoding a peptide tag, wherein the nucleic acid sequence is preferably continuous with a nucleic acid sequence encoding CX26.

[0117] In some embodiments, the promoter operably ligated to the nucleic acid sequence encoding CX26 is a constitutive promoter. In other words, in some embodiments, the polynucleotide in the AAV vector described herein contains a constitutive promoter. As used herein, a constitutive promoter may be defined as a promoter that enables uncontrolled and / or continuous transcription of the coding sequence operably ligated thereto.

[0118] Examples of constitutive promoters include the hybrid cytomegalovirus (CMV) initial / chicken β-actin (CBA) promoter, the shortened hybrid CMV-CBA promoter (which has shortened hybrid chicken β-actin / rabbit β-globin introns to create a smaller promoter called smCBA), the cytomegalovirus (CMV) promoter (optionally containing a CMV enhancer), the chicken β-actin (CBA) promoter, the CAG promoter, the retrovirus Roussarcoma virus (RSV) LTR promoter (optionally containing an RSV enhancer), the SV40 promoter, the dihydrofolate reductase promoter, the β-actin promoter, the phosphoglycerate kinase (PGK) promoter, and the EF1α promoter.

[0119] In some embodiments, the constitutive promoter includes an enhancer. In some embodiments, the constitutive promoter does not include an enhancer.

[0120] In some embodiments, the constitutive promoter is either the smCBA promoter or the CMV promoter.

[0121] In some embodiments, the constitutive promoter is an smCBA promoter. The smCBA promoter may contain, consist of, or have the sequence described in SEQ ID NO: 22 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with SEQ ID NO: 22.

[0122] In some embodiments, the polynucleotide in the AAV vector described herein further comprises one or more, preferably two, inverted terminal repeats (ITRs). In particular, the polynucleotide in the AAV vector described herein may comprise a 5'ITR and a 3'ITR. The polynucleotide in the AAV vector described herein may be flanked by the 5'ITR and the 3'ITR.

[0123] Examples of ITRs include ITRs of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12 serotypes.

[0124] In some embodiments, the polynucleotide in the AAV vector described herein comprises two ITRs of the AAV2 serotype, preferably a 5'ITR and a 3'ITR. The 5'ITR may contain, consist of, or have the sequence described in SEQ ID NO: 23 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with SEQ ID NO: 23. The 3'ITR may contain, consist of, or have the sequence described in SEQ ID NO: 37 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with SEQ ID NO: 37. In some embodiments, the polynucleotides in the AAV vector described herein each contain, consist of, or include two ITRs, preferably a 5'ITR and a 3'ITR, each containing the sequence described in SEQ ID NO: 23 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with SEQ ID NO: 23. In some embodiments, the polynucleotides in the AAV vector described herein include a 5'ITR containing, comprising, or having a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical to the sequence described in SEQ ID NO: 23, and a 3'ITR containing, comprising, or having a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical to the sequence described in SEQ ID NO: 37, or SEQ ID NO: 37.

[0125] In some embodiments, the AAV vectors described herein include a polynucleotide comprising (i) a nucleic acid sequence encoding CX26 operably ligated to a promoter; (ii) at least one copy of a miRNA target site of the miR183 family comprising, consisting of, or having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with the sequence described in SEQ ID NO: 3, SEQ ID NO: 16, or SEQ ID NO: 31; and (iii) an ITR. In some embodiments, the AAV vectors described herein are 5' to 3' · 5'ITR as described herein, • Promoters as described herein, • The nucleic acid sequence encoding CX26 as described herein, · At least one copy of a miRNA target site of the miR183 family that includes, consists of, or has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with the sequence described herein as sequence 3, sequence 16, or sequence 31, and · 3'ITR as described herein It contains polynucleotides.

[0126] In some embodiments, the polynucleotides in the AAV vectors described herein further comprise a polyadenylation signal (or poly(A) signal).

[0127] Examples of poly(A) signals include bovine growth hormone (bGH) poly(A), mouse β-globin poly(A), mouse α-globin poly(A), human collagen poly(A), polyomavirus poly(A), herpes simplex virus thymidine kinase gene (HSV TK) poly(A), IgG heavy chain gene poly(A), human growth hormone poly(A), SV40 late and early poly(A), and poly(A) signals selected from the group including or consisting of AATAAA, ATTAAA, TATAAA, AGTAAA, and CATAAA.

[0128] In some embodiments, the poly(A) signal is a bovine growth hormone (bGH) poly(A) signal. The bGH poly(A) signal may include, consist of, or have the sequence described in SEQ ID NO: 24 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with SEQ ID NO: 24.

[0129] In some embodiments, the AAV vectors described herein include (i) a nucleic acid sequence encoding CX26 operably linked to a promoter; (ii) at least one copy of a miR183 family miRNA target site comprising, consisting of, or having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with the sequence described in SEQ ID NO: 3, SEQ ID NO: 16, or SEQ ID NO: 31; (iii) a poly(A) signal; and optionally (iv) one or more, preferably two, ITRs described herein.

[0130] In some embodiments, the AAV vector described herein is 5' to 3'. • Promoters as described herein, • The nucleic acid sequence encoding CX26 as described herein, · At least one copy of a miRNA target site of the miR183 family that includes, consists of, or has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with the sequence described herein as sequence 3, sequence 16, or sequence 31, and • Poly(A) Signal as described herein It contains polynucleotides.

[0131] In some embodiments, the AAV vector described herein is 5' to 3'. · 5'ITR as described herein, • Promoters as described herein, • The nucleic acid sequence encoding CX26 as described herein, · At least one copy of a miR183 family miRNA target site containing, consisting of, or having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with the sequence described herein as sequence 3, sequence 16, or sequence 31, • Poly(A) signals as described herein, and · 3'ITR as described herein It contains polynucleotides.

[0132] In some embodiments, the polynucleotides in the AAV vectors described herein further comprise chimeric introns.

[0133] Examples of chimeric introns include human hemoglobin subunit β(HBB2) introns, SV40-derived introns, chicken β-actin gene-derived introns, introns containing enhancers such as RSV enhancers or CMV enhancers, synthetic introns based on GJB2 introns, and synthetic introns containing repressors.

[0134] In some embodiments, the chimeric intron is a human hemoglobin subunit β(HBB2) intron. Therefore, the chimeric intron may contain, consist of, or have the sequence described in Sequence ID No. 25 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with Sequence ID No. 25.

[0135] In some embodiments, the AAV vectors described herein include (i) a nucleic acid sequence encoding CX26 operably linked to a promoter; (ii) at least one copy of a miR183 family miRNA target site comprising, consisting of, or having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with the sequence described in SEQ ID NO: 3, SEQ ID NO: 16, or SEQ ID NO: 31; and (iii) a poly(A) signal, (iv) a chimeric intron, and optionally (v) one or more, preferably two, ITRs described herein.

[0136] Therefore, the AAV vectors described herein are from 5' to 3'. • Promoters as described herein, • Chimeraintron as described herein, • The nucleic acid sequence encoding CX26 as described herein, · At least one copy of a miRNA target site of the miR183 family that includes, consists of, or has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with the sequence described herein as sequence 3, sequence 16, or sequence 31, and • Poly(A) Signal as described herein It may also contain polynucleotides that include [the specified element].

[0137] Alternatively, the AAV vectors described herein are 5' to 3'. • Promoters as described herein, • The nucleic acid sequence encoding CX26 as described herein, · At least one copy of a miR183 family miRNA target site containing, consisting of, or having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with the sequence described herein as sequence 3, sequence 16, or sequence 31, • Chimeraintron as described herein, and • Poly(A) Signal as described herein It may also contain polynucleotides that include [the specified element].

[0138] In some embodiments, the AAV vector described herein is 5' to 3'. · 5'ITR as described herein, • Promoters as described herein, • Chimeraintron as described herein, • The nucleic acid sequence encoding CX26 as described herein, · At least one copy of a miR183 family miRNA target site containing, consisting of, or having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with the sequence described herein as sequence 3, sequence 16, or sequence 31, • Poly(A) signals as described herein, and · 3'ITR as described herein It contains polynucleotides.

[0139] In some embodiments, the polynucleotides in the AAV vectors described herein further comprise woodchuck hepatitis virus post-transcriptional regulatory elements (WPREs). WPREs are DNA sequences that enhance protein expression by forming tertiary structures that stabilize mRNA obtained from the transcription of a protein-coding nucleic acid sequence.

[0140] WPRE may contain, consist of, or have the sequence described in Sequence ID No. 26, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with Sequence ID No. 26.

[0141] In some embodiments, the AAV vectors described herein include (i) a nucleic acid sequence encoding CX26 operably linked to a promoter; (ii) at least one copy of a miR183 family miRNA target site comprising, consisting of, or having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with the sequence described herein as SEQ ID NO: 3, SEQ ID NO: 16, or SEQ ID NO: 31; (iii) a WPRE; (iv) a poly(A) signal; optionally (v) a chimeric intron; and optionally (vi) one or more, preferably two, ITRs described herein.

[0142] Therefore, the AAV vectors described herein are from 5' to 3'. • Promoters as described herein, • Chimera Intron as specified herein, • The nucleic acid sequence encoding CX26 as described herein, · At least one copy of a miRNA target site of the miR183 family that includes, consists of, or has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with the sequence described herein as sequence 3, sequence 16, or sequence 31, and • WPRE as described in this specification, • Poly(A) Signal as described herein It may also contain polynucleotides that include [the specified element].

[0143] Alternatively, the AAV vectors described herein are 5' to 3'. • Promoters as described herein, • The nucleic acid sequence encoding CX26 as described herein, · At least one copy of a miR183 family miRNA target site containing, consisting of, or having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with the sequence described herein as sequence 3, sequence 16, or sequence 31, • Chimera Intron as specified herein, ·WPRE as described herein, and • Poly(A) Signal as described herein It may also contain polynucleotides that include [the specified element].

[0144] In some embodiments, the AAV vector described herein is 5' to 3'. · 5'ITR as described herein, • Promoters as described herein, • Chimera Intron as specified herein, • The nucleic acid sequence encoding CX26 as described herein, · At least one copy of a miR183 family miRNA target site containing, consisting of, or having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with the sequence described herein as sequence 3, sequence 16, or sequence 31, • WPRE as described in this specification, • Poly(A) signals as described herein, and · 3'ITR as described herein It contains polynucleotides.

[0145] In some embodiments, the AAV-DJ vector described herein is 5' to 3'. • AAV2 serotype 5'ITR, preferably comprising or consisting of the sequence described in SEQ ID NO: 23 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with SEQ ID NO: 23. smCBA promoter, preferably comprising or consisting of the sequence described in SEQ ID NO: 22 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with SEQ ID NO: 22. • Human CX26, preferably a nucleic acid sequence encoding human CX26 comprising or consisting of the amino acid sequence described in SEQ ID NO: 1 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with SEQ ID NO: 1. - miRNA target sites of the miR183 family comprising or consisting of a sequence described in SEQ ID NO: 3, SEQ ID NO: 16, or SEQ ID NO: 31, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with SEQ ID NO: 3, SEQ ID NO: 16, or SEQ ID NO: 31, preferably at least one copy, preferably three copies, particularly three consecutive copies, of a miR183 target site comprising or consisting of a sequence described in SEQ ID NO: 3, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with SEQ ID NO: 3, preferably three copies, particularly three consecutive copies. • A bovine growth hormone (bGH) poly(A) signal comprising or consisting of the sequence described in SEQ ID NO: 24 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with SEQ ID NO: 24, preferably a bGH poly(A) signal, and A 3'ITR of an AAV2 serotype, preferably comprising or consisting of the sequence described in SEQ ID NO: 23 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with SEQ ID NO: 23, or a 3'ITR of an AAV2 serotype comprising or consisting of the sequence described in SEQ ID NO: 37 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity with SEQ ID NO: 37 It contains polynucleotides.

[0146] In some embodiments, the AAV-DJ vector described herein is 5' to 3'. • The 5'ITR of the AAV2 serotype consisting of the sequence described in Sequence ID No. 23, • The smCBA promoter consisting of the sequence described in Sequence ID No. 22, • Human CX26, preferably a nucleic acid sequence encoding human CX26 comprising or consisting of the amino acid sequence described in SEQ ID NO: 1 or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% or more identity with SEQ ID NO: 1. • At least one copy, preferably three copies, particularly three consecutive copies, of the miR183 target site consisting of the sequence described in Sequence ID No. 3. • A bGH poly(A) signal consisting of the sequence described in Sequence ID No. 24, and • 3'ITR of the AAV2 serotype consisting of the sequence described in Sequence ID No. 37 It contains polynucleotides.

[0147] Accordingly, in some embodiments, the AAV-DJ vector described herein contains a polynucleotide, also known as an expression cassette, which contains or comprises the sequence described in SEQ ID NO: 38. SEQ ID NO: 38 is 5' to 3' • The 5'ITR of the AAV2 serotype consisting of the sequence described in Sequence ID No. 23, • The smCBA promoter consisting of the sequence described in Sequence ID No. 22, • The nucleic acid sequence described in Sequence ID No. 21, which encodes human CX26 having the amino acid sequence described in Sequence ID No. 1, • Three consecutive copies of the miR183 target site, each consisting of the sequence described in Sequence ID No. 3 (corresponding to the sequence described in Sequence ID No. 7), • A bGH poly(A) signal consisting of the sequence described in Sequence ID No. 24, and • 3'ITR of the AAV2 serotype consisting of the sequence described in Sequence ID No. 37 Includes.

[0148] Another object of the present invention is a method for producing a recombinant AAV vector as described herein, comprising transfecting one or more cells (also known as packaging cells) with a construct or vector comprising a polynucleotide or expression cassette as described herein, a construct or vector comprising a functional AAV Cap gene encoding an AAV-DJ capsid, and a construct or vector comprising a functional AAV Rep gene.

[0149] In some embodiments, one or more cells or packaging cells are isolated cells.

[0150] As used herein, the term “gene” encompasses any nucleic acid that codes for a functional polypeptide or protein. For example, the term “Cap gene coding for AAV-DJ capsid” encompasses any nucleic acid sequence that codes for AAV-DJ capsid.

[0151] In some embodiments, the functional AAV Cap gene and functional AAV Rep gene encoding the AAV-DJ capsid may be included in the same construct or vector. Accordingly, in some embodiments, the method for producing the recombinant AAV vector described herein includes transfecting one or more cells (packaging cells) with a construct or vector comprising the polynucleotide or expression cassette described herein, as well as a construct or vector comprising the functional AAV Cap gene and functional AAV Rep gene encoding the AAV-DJ capsid.

[0152] Examples of packaging cells that can be used in the methods described herein include, but are not limited to, insect cells (such as Sf9 cells), mammalian cells, and especially human cells (such as HEK cells). Examples of constructs that can be used in the methods described herein include nucleic acids such as plasmids. Examples of vectors that can be used in the methods described herein include viruses such as baculoviruses, HSV-1 (herpes simplex virus), Adv5 (adenovirus 5), and HCMV (human cytomegalovirus).

[0153] In some embodiments, one or more packaging cells are insect cells such as Sf9 cells. In some embodiments, the method for producing the recombinant AAV vector described herein includes transfecting insect cells with a baculovirus vector (i.e., a baculovirus). Thus, in some embodiments, the method for producing the recombinant AAV vector described herein includes transfecting insect cells with a baculovirus vector containing a polynucleotide or expression cassette described herein, a baculovirus vector containing a functional AAV Cap gene encoding the AAV-DJ capsid, and a baculovirus vector containing a functional AAV Rep gene. In some embodiments, the method for producing the recombinant AAV vector described herein includes transfecting insect cells with a baculovirus vector containing a polynucleotide or expression cassette described herein, and a baculovirus vector containing a functional AAV Cap gene encoding the AAV-DJ capsid and a functional AAV Rep gene.

[0154] In some embodiments, one or more packaging cells are mammalian cells, particularly human cells such as HEK (human embryonic kidney) cells. In some embodiments, the method for producing the recombinant AAV vector described herein includes transfecting mammalian cells, particularly human cells, with constructs such as plasmids. Thus, in some embodiments, the method for producing the recombinant AAV vector described herein includes transfecting mammalian cells, particularly human cells, with constructs comprising polynucleotides or expression cassettes described herein, constructs comprising a functional AAV Cap gene encoding the AAV-DJ capsid, and constructs comprising a functional AAV Rep gene.

[0155] The method may optionally include transfecting packaging cells with a helper construct or helper vector to facilitate the packaging of the polynucleotides or expression cassettes described herein into the AAV-DJ capsid.

[0156] Methods for transfecting packaging cells (sometimes called host cells) with (i) a construct or vector containing at least the coding nucleic acid sequence of interest to be packaged in a recombinant AAV vector, (ii) a construct or vector containing a functional AAV Cap gene, and (ii) a construct or vector containing a functional AAV Rep gene, and optionally (iii) a helper construct are well known in the art. Alternatively, as shown above, the functional AAV Cap gene and the functional AAV Rep gene may be included in the same construct or vector.

[0157] For example, a construct used to transfect packaging cells (mammalian cells, particularly human cells, etc.) may be a plasmid, i.e., a plasmid containing the polynucleotide or expression cassette described herein, a plasmid containing the functional AAV Cap gene and functional AAV Rep gene encoding the AAV-DJ capsid, and optionally a helper plasmid. Alternatively, as shown above, the functional AAV Cap gene and functional AAV Rep gene may each be contained in different plasmids.

[0158] Examples of such plasmids are shown in the following experimental section. Plasmids containing polynucleotides or expression cassettes described herein may also contain the polynucleotide or expression cassette of SEQ ID NO: 38. An example of a plasmid containing the expression cassette of SEQ ID NO: 38 is plasmid pCA027, which has the sequence described in SEQ ID NO: 42. Plasmids containing the functional AAV Cap gene and functional AAV Rep gene encoding the AAV-DJ capsid may also contain plasmid pCK003, which is mentioned in the following experimental section. The helper plasmid may also contain plasmid pALD-X80, which is mentioned in the following experimental section.

[0159] In some embodiments, the method for preparing recombinant AAV vectors described herein is • Transfecting packaging cells with (i) a construct or vector comprising a polynucleotide or expression cassette as described herein, and (ii) a construct or vector comprising a functional AAV Cap gene and a functional AAV Rep gene encoding an AAV-DJ capsid (alternatively, the functional AAV Cap gene and the functional AAV Rep gene may each be contained in different constructs or vectors), and optionally (iii) a helper construct, • Culturing packaging cells Includes.

[0160] In some embodiments, the method further includes isolating and optionally purifying the recombinant AAV vector prepared from packaging cells.

[0161] Another object of the present invention is a set of constructs or vectors suitable for transfecting packaging cells and producing the AAV vectors described herein. Therefore, another object of the present invention is • Constructs or vectors comprising polynucleotides or expression cassettes as described herein, • Constructs or vectors containing functional AAV Cap genes and functional AAV Rep genes encoding AAV-DJ capsids, and Optionally, helper structures or helper vectors It is a set of constructs or vectors that include [this element].

[0162] In some embodiments, the construct or set of vectors is • Constructs or vectors containing a polynucleotide or expression cassette that includes or consists of the sequence described in Sequence ID No. 38, • Constructs or vectors containing functional AAV Cap genes encoding the AAV-DJ capsid and functional AAV Rep genes such as the AAV2 Rep gene, and Optionally, helper structures or helper vectors Includes.

[0163] In some embodiments, the functional AAV Cap gene and the functional AAV Rep gene are contained in different constructs or vectors, and this set of constructs or vectors is • Constructs or vectors comprising polynucleotides or expression cassettes as described herein, • Constructs or vectors containing the functional AAV Cap gene encoding the AAV-DJ capsid, • Constructs or vectors containing the functional AAV Rep gene, and Optionally, helper structures or helper vectors Includes.

[0164] In some embodiments, the set of constructs is a set of plasmids, and therefore • Plasmids containing polynucleotides or expression cassettes as described herein, • Plasmids containing the AAV Cap gene encoding the AAV-DJ capsid and the functional AAV Rep gene, and Optionally, a helper plasmid Includes.

[0165] In some embodiments, this set of plasmids is • Plasmids containing a polynucleotide or expression cassette that includes or consists of the sequence described in Sequence ID No. 38, • Plasmids containing functional AAV Cap genes encoding the AAV-DJ capsid and functional AAV Rep genes such as the AAV2 Rep gene, and Optionally, a helper plasmid Includes.

[0166] In some embodiments, the functional AAV Cap gene and the functional AAV Rep gene are contained in different plasmids, and this set of plasmids is • Plasmids containing polynucleotides or expression cassettes as described herein, • Plasmid containing the functional AAV Cap gene encoding the AAV-DJ capsid, • Plasmids containing the functional AAV Rep gene, and Optionally, a helper plasmid Includes.

[0167] Another object of the present invention is a cell or packaging cell containing a construct (e.g., plasmid) or vector and / or a set of AAV vectors described herein. In some embodiments, the cell or packaging cell is an isolated cell. In particular, the cell or packaging cell may be used for the preparation of recombinant AAV vectors described herein.

[0168] These cells or packaging cells may be animal cells, such as insect cells or mammalian cells. In particular, these cells or packaging cells may be human cells, such as immortalized human cells, such as HEK (human embryonic kidney) cells.

[0169] It should be noted that, with respect to animal, mammalian, and human cells, the terms “cell” and “packaging cell” preferably refer to cells of a cultured cell line. Animals and humans into which the AAV vectors described herein have been introduced are expressly excluded from the definition of “cell” or “packaging cell.”

[0170] Another object of the present invention is a composition comprising, or essentially comprising, at least one of the AAV vectors described herein.

[0171] As used herein with respect to compositions, pharmaceutical compositions, or drugs, “essentially consisting of” means that at least one AAV vector described herein is the only active agent, therapeutic agent, or biologically active agent in the composition.

[0172] Another object of the present invention is a pharmaceutical composition comprising, or essentially comprising, at least one AAV vector described herein and at least one pharmaceutically acceptable excipient or carrier.

[0173] Pharmaceutically acceptable excipients or carriers that can be used in this pharmaceutical composition include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffering substances such as phosphates (e.g., phosphate-buffered saline), lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts or electrolytes, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulosic substances (e.g., sodium carboxymethylcellulose), polyethylene glycol, polyacrylate, wax, polyethylene-polyoxypropylene block polymer, polyethylene glycol, lanolin, and water.

[0174] The buffer or buffering agent may refer to an agent capable of maintaining physiological pH, such as HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer, Dulbecco's phosphate-buffered saline (DPBS) buffer, or phosphate-buffered saline (PBS) buffer. Such buffers may contain disodium hydrogen phosphate and sodium chloride, or potassium dihydrogen phosphate and potassium chloride. The buffer may also be based on a physiologically suitable solution containing synthetic perilymphatic solution (generally 20-200 mM NaCl, 1-5 mM KCl, 0.1-10 mM CaCl2, 1-10 mM glucose, and 2-50 mM HEPES with a pH in the range of about 6-9) or pluronic acid F68 as a surfactant.

[0175] pharmaceutically acceptable excipients or carriers may also include surfactants that can reduce surface tension and function as cleaning agents, wetting agents, emulsifiers, foaming agents, and dispersants. Suitable surfactants include nonionic agents such as polyoxyethylene sorbitan (e.g., Tween 20, 40, 60, 80, or 85) and other sorbitans (e.g., Span 20, 40, 60, 80, or 85), Pluronic F-68, or other pluronic acids (usually 0.01% to 0.001%).

[0176] pharmaceutically acceptable excipients or carriers may also include preservatives or chemical stabilizers. Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, parabens, ethyl vanillin, glycerin, phenol, and parachlorophenol. Suitable chemical stabilizers include gelatin and albumin.

[0177] Another object of the present invention is a drug comprising, comprising, or essentially comprising, at least one AAV vector described herein and optionally at least one pharmaceutically acceptable excipient or carrier described herein.

[0178] In some embodiments, the AAV vectors, compositions, pharmaceutical compositions, or drugs described herein are formulated for administration to a subject.

[0179] In some embodiments, the AAV vectors, compositions, pharmaceutical compositions, or drugs described herein are for single-dose use or are formulated for single-dose use. In some embodiments, the AAV vectors, compositions, pharmaceutical compositions, or drugs described herein are for repeated use or are formulated for repeated use.

[0180] The AAV vectors, compositions, pharmaceutical compositions, or drugs described herein may be formulated for systemic or topical administration. The AAV vectors, compositions, pharmaceutical compositions, or drugs described herein may be for systemic administration, or may be formulated for systemic administration. The AAV vectors, compositions, pharmaceutical compositions, or drugs described herein may be for topical administration, or may be formulated for topical administration.

[0181] In some embodiments, the AAV vectors, compositions, pharmaceutical compositions, or drugs described herein are formulated for administration by injection, for example, local injection into the ear, particularly the inner ear.

[0182] Examples of forms suitable for injection include solutions such as sterile aqueous solutions, gels, dispersions, emulsions, suspensions, and active pharmaceutical ingredient forms such as powders that are suitable for preparing a solution or suspension by adding liquid before use.

[0183] In some embodiments, the AAV vectors, compositions, pharmaceutical compositions, or drugs described herein are formulated as vesicles such as biocompatible gels, liposomes, nanoparticles, or cell-derived exosomes.

[0184] In some embodiments, the regimens or dosages used for administering the AAV vectors, compositions, pharmaceutical compositions, or drugs described herein can be varied depending on various parameters, particularly the mode of administration used, the relevant medical condition, or the desired duration of treatment. For example, it is well within the skill of those skilled in the art to start with a dose lower than the amount required to achieve the desired therapeutic effect and gradually increase the dose of the AAV vector described herein until the desired effect is achieved.

[0185] For example, the dose of recombinant AAV vector (i.e., AAV vector) described herein administered to a subject is approximately 10 per ear. 8 ~about 10 13This may be within the range of rAAV genome copies. Therefore, the AAV vectors, compositions, pharmaceutical compositions or drugs described herein may be approximately 10 per ear. 8 ~about 10 13 It may be formulated for administration in doses of a recombinant AAV vector (i.e., an AAV vector) within the range of rAAV genome copies.

[0186] In some embodiments, the subjects may be “patients” who are awaiting or receiving medical care, or who have been, are currently, or will be subject to medical treatment in the past, or who are being observed for the onset of a target disease or condition, particularly hereditary hearing loss or hereditary hearing impairment. In some embodiments, the subjects are male. In some embodiments, the subjects are female.

[0187] In some embodiments, the target audience is adults (e.g., those aged 18, 19, 20, 21, 22, 23, 24, or 25 or older). In some embodiments, the target audience is adults aged 25 to 70.

[0188] In some embodiments, the subjects are children (e.g., under 21, 20, 19, or 18 years of age). In some embodiments, the subjects are infants (i.e., children under 5, 4, 3, 2, or 1 year of age). In some embodiments, the subjects are children 6 months to 18 years of age, preferably 6 months or 1 year to 10 years of age. In some embodiments, the subjects are children 6 months to 5 years of age, preferably 6 months or 1 year to 4 years of age. In some embodiments, the subjects are children 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years of age.

[0189] In some embodiments, the subject suffers from hereditary hearing loss or hereditary hearing impairment.

[0190] In some embodiments, the subject suffers from non-symptomatic hearing impairment (also known as non-symptomatic hearing loss). In some embodiments, the subject suffers from autosomal recessive non-symptomatic hearing impairment (also known as autosomal recessive non-symptomatic hearing loss). In some embodiments, the subject suffers from autosomal dominant non-symptomatic hearing impairment (also known as autosomal dominant non-symptomatic hearing loss).

[0191] In some embodiments, the subject suffers from asymptomatic hearing loss DFNB1. In some embodiments, the subject suffers from asymptomatic hearing loss DFNA3.

[0192] In some embodiments, the subject has biallelic loss-of-function (LOF) variants in the GJB2 gene and / or the GJB6 gene. Examples of LOF variants in the GJB2 gene include nonsense variants, splice site variants, insertion variants (particularly frameshift insertion variants), and deletion variants (particularly frameshift deletion variants). Examples of LOF variants in the GJB6 gene include LOF variants resulting from insertions and / or deletions in the GJB6 gene. In some embodiments, the subject has biallelic LOF variants in the GJB2 gene and / or insertions and / or deletions in the GJB6 gene. In some embodiments, the subject has biallelic LOF variants resulting from insertions and / or deletions in the GJB6 gene. As used herein, “having biallelic loss-of-function (LOF) variants” means that the subject has LOF variants on both alleles of the gene (the LOF variants on each allele are either identical or different).

[0193] In some embodiments, the subject has biallelelic pathogenic variants of the GJB2 gene and / or the GJB6 gene. Examples of pathogenic variants of the GJB2 gene include missense variants and intraframe deletion and / or insertion variants. As used herein, “having biallelelic pathogenic variants of a gene” means that the subject has pathogenic variants on both alleles of the gene (the pathogenic variants on each allele are either identical or different).

[0194] In some embodiments, the subject has biallele deletions of the GJB2 gene and / or the GJB6 gene, particularly biallele macrodeletions of the GJB2 gene and / or the GJB6 gene. In some embodiments, the subject has biallele deletions of the GJB2 gene, particularly biallele macrodeletions of the GJB2 gene. In some embodiments, the subject has biallele deletions of the GJB6 gene, particularly biallele macrodeletions of the GJB6 gene.

[0195] In some embodiments, the subject has a monoallelic loss-of-function (LOF) variant in the GJB2 gene and / or the GJB6 gene. In some embodiments, the subject has a monoallelic LOF variant in the GJB2 gene and / or an insertion and / or deletion in the GJB6 gene. In some embodiments, the subject has a monoallelic LOF variant resulting from an insertion and / or deletion in the GJB6 gene. As used herein, “having a monoallelic LOF variant” means that the subject has an LOF variant on one allele of the gene.

[0196] In some embodiments, the subject has a monoallelic pathogenic variant of the GJB2 gene, such as a missense variant or an intraframe deletion and / or insertion variant of the GJB2 gene, and / or a monoallelic pathogenic variant of the GJB6 gene. As used herein, “having a monoallelic pathogenic variant of a gene” means that the subject has a pathogenic variant on one allele of the gene.

[0197] In some embodiments, the subject has a single-alele deletion of the GJB2 gene and / or the GJB6 gene, particularly a single-alele macrodeletion of the GJB2 gene and / or the GJB6 gene. In some embodiments, the subject has a single-alele deletion of the GJB2 gene, particularly a single-alele macrodeletion of the GJB2 gene. In some embodiments, the subject has a single-alele deletion of the GJB6 gene, particularly a single-alele macrodeletion of the GJB6 gene.

[0198] To date, identified variants of the GJB2 gene, including pathogenic and highly pathogenic mutations, are listed in the Deafness Variation database (available at https: / / deafnessvariationdatabase.org / gene / GJB2). Furthermore, eight major deletions in the GJB2 promoter region are described in Safka Brozkova D et al., "The Cause of Hereditary Hearing Loss in GJB2 Heterozygotes—A Comprehensive Study of the GJB2 / DFNB1 Region." Genes (Basel). 2021 May 1;12(5):684.

[0199] Another object of the present invention is a kit comprising at least one AAV vector, composition, pharmaceutical composition or drug described herein and optionally instructions for use.

[0200] "Kit" refers to any product (e.g., packaging or container) containing at least one of the AAV vectors, compositions, pharmaceutical compositions or drugs described herein. The Kit may be promoted, distributed or sold as a unit for the medical use described herein, or as a unit for the therapeutic use or treatment described herein.

[0201] Another object of the present invention is the AAV vector, composition, or pharmaceutical composition described herein for use as a medicine.

[0202] Another object of the present invention is the AAV vectors, compositions, pharmaceutical compositions, or drugs described herein for use in the treatment (i.e., preventive treatment and / or therapeutic or curative treatment) of hereditary hearing impairment (or hereditary hearing loss) in subjects requiring such treatment. In some embodiments, the AAV vectors, compositions, pharmaceutical compositions, or drugs described herein are for use in the prevention of hereditary hearing impairment (or hereditary hearing loss) in subjects requiring such treatment. In some embodiments, the AAV vectors, compositions, pharmaceutical compositions, or drugs described herein are for use in the therapeutic or curative treatment of hereditary hearing impairment (or hereditary hearing loss) in subjects requiring such treatment.

[0203] The terms “hereditary hearing impairment” and “hereditary hearing loss” as used herein are interchangeable and refer to a condition in which a person is partially or completely unable to hear sounds due to a genetic cause. The severity of hearing impairment (or hearing loss) as described herein may vary. The severity level usually refers to the degree of hearing impairment (or hearing loss), which can range from mild to severe.

[0204] In some embodiments, hereditary hearing loss is asymptomatic hearing loss (or asymptomatic hearing impairment). In some embodiments, hereditary hearing loss is autosomal recessive asymptomatic hearing loss (or autosomal recessive asymptomatic hearing impairment). In some embodiments, hereditary hearing loss is autosomal dominant asymptomatic hearing loss (or autosomal dominant asymptomatic hearing impairment).

[0205] In some embodiments, hereditary hearing loss is non-symptomatic hearing loss DFNB1. DFNB1 is a hereditary disorder in which subjects suffer from or develop mild to severe hearing impairment, which manifests at birth or later in life. Thus, DFNB1 includes later-onset forms (age-related hearing loss (ARHL), i.e., presbycusis). In particular, DFNB1 can be caused by pathogenic variants (sometimes called pathogenic mutations) and / or deletions of the GJB2 gene (which encodes the connexin 26 protein). A deletion of the GJB2 gene means any deletion that includes any part of the GJB2 gene, including the promoter, regulatory sequences, and / or GJB2 non-coding and coding transcription sequences. A deletion of the GJB2 gene may also extend to the GJB6 gene (which encodes the connexin 30 protein), which is adjacent to GJB2. Cx30 knockout mice (Cx30 - / -In a mouse study—Teubner B et al., "Connexin 30 (Gjb6) deficiency causes severe hearing impairment and lack of endocochlear potential." Hum Mol Genet. 2003 Jan 1;12(1):13-21—overexpression of Cx26 completely restored auditory sensitivity and prevented hair cell death (Ahmad et al., "Restoration of connexin 26 protein level in the cochlea completely rescues hearing in a mouse model of human connexin 30-linked deafness." Proc Natl Acad Sci US A. 2007 Jan 23;104(4):1337-41) has been demonstrated. Therefore, in some embodiments, hereditary hearing loss is associated with DFNB1 mutations in the GJB2 and / or GJB6 genes, or deletions affecting the GJB2 and / or GJB6 genes (including deletions affecting the expression of the GJB2 and / or GJB6 genes). Some DFNB1 subjects exhibit progressive hearing loss (particularly childhood-onset). Some DFNB1 subjects exhibit premature presbycusis, i.e., premature age-related hearing loss (Boucher S et al., "Ultrarare heterozygous pathogenic variants of genes causing dominant forms of early-onset deafness underlie severe presbycusis." Proc Natl Acad Sci US A. 2020 Dec 8;117(49):31278-31289).For example, some individuals in the DFNB1 group may develop severe age-related hearing loss early in adulthood.

[0206] In some embodiments, hereditary hearing loss is non-symptomatic hearing loss DFNA3. DFNA3 is a hereditary disorder in which individuals suffer from or develop mild to severe hearing impairment. DFNA3 is inherited in an autosomal dominant manner. In particular, DFNA3 can be caused by a dominant pathogenic mutation (sometimes called a dominant pathogenic variant) in the GJB2 gene.

[0207] In some embodiments, hereditary hearing loss is caused by de novo pathogenic mutations in the GJB2 gene, particularly de novo-dominant pathogenic mutations in the GJB2 gene.

[0208] In some embodiments, hereditary hearing loss (or hearing impairment) is severe hereditary hearing loss (or hearing impairment). A person with severe hereditary hearing loss can be defined as a person who cannot hear sounds with an intensity of less than approximately 91 dB HL (hearing level) but can perceive sounds with a higher intensity.

[0209] In some embodiments, hereditary hearing loss (or hearing impairment) is severe hereditary hearing loss (or hearing impairment). A person with severe hereditary hearing loss can be defined as a person who cannot hear sounds with an intensity of less than approximately 71 dB HL but can perceive sounds with a higher intensity.

[0210] In some embodiments, hereditary hearing loss (or hearing impairment) is moderate hereditary hearing loss (or hearing impairment). A person with moderate hereditary hearing loss can be defined as a person who cannot hear sounds with an intensity of less than approximately 41 dB HL but can perceive sounds with a higher intensity.

[0211] In some embodiments, hereditary hearing loss (or hearing impairment) is progressive hereditary hearing loss (or hearing impairment). Individuals with progressive hereditary hearing loss can be defined as those whose hearing progresses from normal to severe or profound hearing loss over several years.

[0212] In some embodiments, hereditary hearing loss (or hearing impairment) is age-related hearing loss (ARHL), or presbycusis. In some embodiments, hereditary hearing loss (or hearing impairment) is presbycusis. In some embodiments, hereditary hearing loss (or hearing impairment) is severe presbycusis.

[0213] In some embodiments, hereditary hearing loss (or hearing impairment) is childhood-onset hereditary hearing loss (or hearing impairment). Subjects with childhood-onset hereditary hearing loss can be defined as subjects who develop hearing loss in childhood and whose hearing loss worsens over time.

[0214] In some embodiments, hereditary hearing loss (or hearing impairment) is congenital hereditary hearing loss (or hearing impairment). Subjects with congenital hereditary hearing loss can be defined as subjects with hearing loss at birth, particularly severe or profound hearing loss.

[0215] Another object of the present invention is a method for treating (i.e., preventive treatment and / or therapeutic or curative treatment) hereditary hearing loss (or hereditary hearing impairment) described herein in a subject requiring such treatment, comprising administering to the subject at least one AAV vector, composition, pharmaceutical composition, or drug described herein. In some embodiments, the method is for the prevention of hereditary hearing loss (or hereditary hearing impairment) described herein in a subject requiring such treatment. In some embodiments, the method is for the therapeutic or curative treatment of hereditary hearing loss (or hereditary hearing impairment) described herein in a subject requiring such treatment.

[0216] Another object of the present invention is a method for improving or restoring the hearing of a subject suffering from hereditary hearing loss (or hereditary hearing impairment) as described herein, comprising administering to the subject at least one AAV vector, composition, pharmaceutical composition, or drug described herein. Another object of the present invention is a method for slowing, reducing, or minimizing the progression of hearing loss in a subject suffering from hereditary hearing loss (or hereditary hearing impairment) as described herein, comprising administering to the subject at least one AAV vector, composition, pharmaceutical composition, or drug described herein.

[0217] Another object of the present invention is a method for partially or completely preventing hearing loss in a subject who has or is susceptible to hereditary hearing loss (or hereditary hearing impairment) as described herein, comprising administering to the subject at least one AAV vector, composition, pharmaceutical composition or drug described herein. Another object of the present invention is a method for partially or completely maintaining hearing in a subject who has or is susceptible to hereditary hearing loss (or hereditary hearing impairment) as described herein, comprising administering to the subject at least one AAV vector, composition, pharmaceutical composition or drug described herein.

[0218] In some embodiments, the method includes administering a therapeutically effective dose of the AAV vector described herein.

[0219] The therapeutically effective dose is, for example, about 10 per ear. 8 ~about 10 13 The dosage may correspond to a range of individual rAAV genome copies. Naturally, the dosages shown herein are illustrative and can be adjusted to an optimal dose by taking into account parameters such as the affinity and tolerability of the AAV vector in the composition, pharmaceutical composition, or drug.

[0220] The specific therapeutically effective dose for any particular subject may be determined by a variety of factors, including the hereditary hearing loss during treatment, the severity of the hearing loss, the activity of the AAV vector, composition, pharmaceutical composition, or drug used, the subject's age, weight, overall health status, sex, and diet, the time of administration, the route of administration, the excretion rate of the specific AAV vector, composition, pharmaceutical composition, or drug used, the duration of treatment, drugs used in combination with or concurrently with the specific AAV vector, composition, pharmaceutical composition, or drug used, and similar factors well known in the medical field. The total dose required for each treatment may be administered in multiple doses or single doses.

[0221] Another object of the present invention is a pharmaceutical composition for treating hereditary hearing loss (or hereditary hearing impairment) as described herein in a subject requiring such treatment, or a pharmaceutical composition for use in such treatment (i.e., prophylactic treatment and / or therapeutic or curative treatment), comprising at least one AAV vector as described herein and optionally at least one pharmaceutically acceptable excipient or carrier.

[0222] Another object of the present invention is the use of at least one AAV vector, composition or pharmaceutical composition described herein in the manufacture of a drug for the treatment (i.e., prophylactic treatment and / or therapeutic or curative treatment) of hereditary hearing loss (or hereditary hearing impairment) described herein in subjects requiring such treatment.

[0223] In some embodiments, the pharmaceutical composition or drug is for the prevention of hereditary hearing loss (or hereditary hearing impairment) as described herein in subjects requiring it. In some embodiments, the pharmaceutical composition or drug is for the therapeutic or curative treatment of hereditary hearing loss (or hereditary hearing impairment) as described herein in subjects requiring it.

[0224] Another object of the present invention is a method for specifically expressing CX26 in non-sensory cells of the cochlea of ​​a subject who is suffering from or susceptible to hereditary hearing loss (or hereditary hearing impairment) as described herein, the method comprising administering to the subject at least one AAV vector, composition, pharmaceutical composition or drug as described herein.

[0225] In some embodiments, this method enables CX26 expression in non-sensory cells of the cochlear while reducing off-target expression of CX26. In particular, in some embodiments, this method enables CX26 expression in non-sensory cells of the cochlear while preventing CX26 expression in sensory cells of the cochlear, especially inner ear hair cells.

[0226] In some embodiments, the non-sensory cells of the cochlea include supporting cells (such as supporting cells of the non-sensory epithelium of the cochlear), fibrous cells (such as fibrous cells lining the inside of the cochlear duct), and stria vascularis cells.

[0227] As shown in the experimental section below, the inventors demonstrated that a recombinant AAV-DJ vector containing an expression cassette (such as the expression cassette for SEQ ID NO: 38) comprising a CX26 coding sequence operably linked to a promoter and a so-called SEQ ID NO: 3 precursor miR183 target site allows for the maximization of CX26 expression in non-sensory cells of the cochlear while preventing CX26 expression in inner ear hair cells. Furthermore, the inventors demonstrated that in vivo administration of such a recombinant AAV-DJ vector can effectively treat (i.e., preventive and / or therapeutic) hereditary hearing loss in a mouse model of hereditary hearing loss. In particular, therapeutic effects were observed in three different mouse models of hereditary hearing loss induced by Gjb2 deletion: the OtogL-cre;Gjb2-Flox mouse model (Examples 3-4 & 6-8), the ROSA26-creERT2;Gjb2-Flox mouse model (Example 5), and the FoxG1-cre;Gjb2-Flox mouse model (Example 9).

[0228] Furthermore, in vitro transfection experiments revealed that the precursor miR183 target site appears to be particularly well-suited to mediating miR-induced silencing in cells expressing miR183. [Table 1] JPEG2026514375000003.jpg211159JPEG2026514375000004.jpg211159JPEG2026514375000005.jpg210159JPEG2026514375000006.jpg21315 9JPEG2026514375000007.jpg210159JPEG2026514375000008.jpg209159JPEG2026514375000009.jpg212159JPEG2026514375000010.jpg21115 9JPEG2026514375000011.jpg209159JPEG2026514375000012.jpg207159JPEG2026514375000013.jpg215159JPEG2026514375000014.jpg21515 9JPEG2026514375000015.jpg212159JPEG2026514375000016.jpg212159JPEG2026514375000017.jpg210159JPEG2026514375000018.jpg16159 [Brief explanation of the drawing]

[0229] [Figure 1]Figures 1A–1D are combinations of confocal micrographs showing immunofluorescence staining for GFP, MyoVIIa (control staining for hair cells), Sox2 (control staining for supporting cells), and DAPI (DNA staining) in cochlear sections of mice injected with either the AAVDJ-CMV-eGFP vector (Figures 1A–1B) or the AAVDJ-smCBA-eGFP-miRT vector (Figures 1C–1D). Figures 1B and 1D show GFP expression only, while Figures 1A and 1C show signals combined into one of GFP, MyoVIIa, Sox2, and DAPI. Arrows indicate mouse cochlear cells expressing GFP. [Figure 2] Figures 2A–2F are combinations of confocal micrographs showing immunofluorescence staining for GFP, MyoVIIa (control staining for hair cells), Sox2 (control staining for supporting cells), and DAPI (DNA staining) in cochlear sections of non-human primates injected with either no vector (Figures 2A–2B), the AAVDJ-CMV-eGFP vector (Figures 2C–2D), or the AAVDJ-smCBA-eGFP-miRT vector (Figures 2E–2F). Figures 2B, 2D, and 2F show GFP expression only, while Figures 2A, 2C, and 2E show signals combined into one of GFP, MyoVIIa, Sox2, and DAPI. Arrows indicate non-human primate cochlear cells expressing GFP. [Figure 3] Figures 3A–3F are combinations of confocal micrographs showing immunofluorescence staining for GFP, MyoVIIa (control staining for hair cells), Sox2 (control staining for supporting cells), and DAPI (DNA staining) in the apex (Figures 3A–3B), middle (Figures 3C–3D), and base (Figures 3E–3F) of non-human primate cochlea injected with the AAVDJ-smCBA-eGFP-miRT vector. Figures 3B, 3D, and 3F show GFP expression only, while Figures 3A, 3C, and 3E show signals combined into one of GFP, MyoVIIa, Sox2, and DAPI. Arrows indicate non-human primate cochlear cells expressing GFP. [Figure 4]This histogram shows the percentage of GFP-expressing cells in boundary cells (BC), internal drug cell cells (IPh), fiber cells, Hensens cells, and Claudius cells (HC, CC) and inner ear hair cells (IHC) of non-human primates injected with the AAVDJ-CMV-eGFP vector or the AAVDJ-smCBA-eGFP-miRT vector as directed. **P-value < 0.01 [Figure 5] This graph shows the mean auditory brainstem response (ABR) threshold (expressed as decibel sound pressure level (dB SPL)) measured in the left ear (black square) and right ear (white circle - negative control) of mice with severe congenital hearing impairment 8 weeks after treatment. OtogLcre / +,Gjb2flox / flox mice were simultaneously injected with AAVDJ-smCBA-mGjb2-miRT vector and AAVDJ-CMV-eGFP vector through the round window membrane of the left ear at P0, while the right ear was left untreated (negative control). Hearing evaluation was performed 8 weeks after AAV injection. n=11 mice. [Figure 6] Figures 6A and 6B are a combination of graphs showing auditory brainstem response (ABR) thresholds (expressed in dB SPL) measured in the left ear (black square) and right ear (white circle - negative control) of one mouse with severe congenital hearing impairment 3 weeks (Figure 6A) and 8 weeks (Figure 6B) after treatment. OtogLcre / +,Gjb2flox / flox mice were simultaneously injected with AAVDJ-smCBA-mGjb2-miRT vector and AAVDJ-CMV-eGFP vector through the round window membrane of the left ear at P0, while the right ear was left untreated (negative control). Hearing evaluations were performed 3 weeks and 8 weeks after AAV injection. n=1 mouse. [Figure 7]Figures 7A-7C are combinations of graphs showing auditory brainstem response (ABR) thresholds (expressed in dB SPL) measured in the left ear (black square) and right ear (white circle - negative control) of one progressive hearing-impaired mouse 3 weeks (Figure 7A), 8 weeks (Figure 7B), and 4 months (Figure 7C) after treatment. OtogLcre / +,Gjb2flox / flox mice were simultaneously injected with AAVDJ-smCBA-mGjb2-miRT vector and AAVDJ-CMV-eGFP vector through the round window membrane of the left ear at P0, while the right ear was left untreated (negative control). Hearing evaluations were performed 3 weeks, 8 weeks, and 4 months after AAV injection. n=1 mouse. [Figure 8] Figures 8A and 8B are a combination of graphs showing auditory brainstem response (ABR) thresholds (expressed in dB SPL) measured four weeks after treatment in the left ear (black square) and right ear (white circle - negative control) of two mice with severe congenital hearing impairment. The AAVDJ-smCBA-mGjb2-FLAGtag-miRT vector was injected into the posterior semicircular canal of the vestibular region of the left ear of OtogLcre / +,Gjb2flox / flox mice at P2, while the right ear was left untreated (negative control). Hearing evaluation was performed four weeks after AAV injection. Figure 8A shows the ABR threshold measured from mouse #1, and Figure 8B shows the ABR threshold measured from mouse #2. [Figure 9] This graph shows the auditory brainstem response (ABR) threshold (expressed in dB SPL) measured in the left ear (black square) and right ear (white circle - negative control) of one inducible ROSA26CreERT2 / +,Gjb2flox / flox mouse 5 weeks after treatment. In phase 2, Gjb2 gene inactivation was induced by intraperitoneal injection of hydroxytamoxifen (OHT) into ROSA26CreERT2 / +,Gjb2flox / flox mice. The AAVDJ-smCBA-mGjb2-miRT vector was also injected into the posterior semicircular canal of the vestibular region of the left ear of these mice, while the right ear was left untreated (negative control). Hearing evaluation was performed 5 weeks after AAV injection. n=1 mouse. [Figure 10]Figures 10A-10C are combinations of graphs showing auditory brainstem response (ABR) thresholds (expressed in dB SPL) measured in the left ear (black square) and right ear (white circle - negative control) of one progressive hearing-impaired mouse 3 weeks (Figure 10A), 6 weeks (Figure 10B), and 3 months (Figure 10C) after treatment. The AAVDJ-smCBA-mGjb2-miRT vector was injected into the posterior semicircular canal of the vestibular region of the left ear of OtogLcre / +,Gjb2flox / flox mice at P16, while the right ear was left untreated (negative control). Hearing evaluations were performed 3 weeks, 6 weeks, and 3 months after AAV injection. n=1 mouse. [Figure 11] Figures 11A–11H are combinations of confocal micrographs showing immunofluorescence staining for CX26, acetylated tubulin (AcTub-control staining for the organ of Corti, particularly columnar cells, base of border cells, and internal drug ganglion cells), and DAPI (DNA staining) in cochlear sections of the right ear (Figures 11A–11B and 11E–11F) and left ear (Figures 11C–11D and 11G–11H) of one progressive hearing-impaired mouse 3 months after treatment. The AAVDJ-smCBA-mGjb2-miRT vector was injected at P16 into the posterior semicircular canal of the vestibular region of the left ear of an OtogLcre / +,Gjb2flox / flox mouse, while the right ear was left untreated (negative control). Figures 11B, 11D, 11F, and 11H show CX26 expression only, while Figures 11A, 11C, 11E, and 11G show signals combined into one of CX26, AcTub, and DAPI. Figures 11E through 11H are 2x magnified views of the white squares shown in Figures 11A through 11D. [Figure 12]Figures 12A to 12C are combinations of graphs showing brainstem response (ABR) thresholds (expressed in dB SPL) measured in the left ear (black square) and right ear (white circle - negative control) of three progressive hearing-impaired mice four weeks after treatment. The AAVDJ-smCBA-mGjb2-miRT vector was injected into the posterior semicircular canal of the vestibular region of the left ear of OtogLcre / +,Gjb2flox / flox mice at P16, while the right ear was left untreated (negative control). Hearing evaluation was performed four weeks after AAV injection. Figure 12A shows the ABR threshold measured from mouse #1, Figure 12B shows the ABR threshold measured from mouse #2, and Figure 12C shows the ABR threshold measured from mouse #3. [Figure 13] Figures 13A–13F are combinations of confocal micrographs showing immunofluorescence staining for GFP and MyoVIIa (control staining for hair cells) in cochlear sections of mice injected with either the AAVDJ-smCBA-eGFP vector (Figures 13A–13C) or the AAVDJ-smCBA-eGFP-miRT vector (Figures 13D–13F). Figures 13A and 13D show MyoVIIa expression only, Figures 13B and 13E show GFP expression only, and Figures 13C and 13F show a combined signal of GFP and MyoVIIa. The thick arrows indicate outer hair cells of the mouse cochlea expressing MyoVIIa, and the thin arrows indicate inner ear hair cells of the mouse cochlea expressing MyoVIIa. These show the colocalization of MyoVIIa and GFP signals, which was detected only in the cochlea of ​​mice injected with the AAVDJ-smCBA-eGFP vector. [Figure 14]Figures 14A–14C are a set of graphs and histograms showing the mean auditory brainstem response (ABR) threshold (Figure 14A) expressed in decibel sound pressure level (dB SPL), ABR threshold variability (Figure 14B) expressed in dB SPL, ABR wave I amplitude (Figure 14C) expressed in nV, and ABR wave I latency (Figure 14D) expressed in ms, measured for the left ear of mice 3 and 7 weeks after treatment. The AAVDJ-smCBA-hGjb2-miRT vector was injected into the left ear of OtogLcre / +,Gjb2flox / flox mice and control mice OtogL+ / +,Gjb2flox / flox via a round window membrane at P0–P3, or not injected (uninjected). Hearing evaluations were performed 3 and 7 weeks after AAV injection. n=28 uninjected OtogLcre / +,Gjb2flox / flox mice, n=15 AAV-injected OtogLcre / +,Gjb2flox / flox mice, and n=14 AAV-injected OtogL+ / +,Gjb2flox / flox mice. ** and ## = p < 0.05, *** and ### = p < 0.001. [Figure 15]Figures 15A–15D are a set of graphs illustrating the characterization of further FoxG1-cre;Gjb2-Flox (severe hearing loss) models (Figure 15A) and the efficacy of the AAVDJ-smCBA-hGjb2-miRT vector in FoxG1Cre / + Gjb2flox / flox mice (Figures 15B–15D). Figure 15A shows the mean auditory brainstem response (ABR) threshold (left ear), expressed as decibel sound pressure level (dB SPL) as mean ± standard deviation (SD) for control FoxG1+ / + Gjb2flox / flox mice (n=6, gray line, gray squares), FoxG1Cre / + Gjb2flox / + mice (n=6, black line, black diamonds), and FoxG1Cre / + Gjb2flox / flox mice (n=4, black line, black squares). Figures 15B-15D: FoxG1Cre / + Gjb2flox / flox mice were injected with the AAVDJ-smCBA-hGjb2-miRT vector at P1 via the round window membrane of the left ear, while the right ear was left untreated (negative control). Hearing evaluation was performed 3 weeks after AAV injection. The dots in Figures 15B and 15C represent the ABR threshold fluctuations of each responding FoxG1Cre / + Gjb2flox / flox mouse (3 out of 4 mice). Black dots highlight the mouse with the best response. Figure 15D shows the evaluation of ABR wave I amplitude. The dots represent the wave I amplitude of each responding FoxG1Cre / + Gjb2flox / flox mouse (3 out of 4 mice, for all frequencies tested). The black dots highlight the most responsive mice, and the 3μV dotted line indicates the minimum threshold amplitude in the control FoxG1+ / + Gjb2flox / flox mice. [Figure 16]Figures 16A–16C compare the inhibitory effects obtained using three copies of the target site complementary to the precursor hsa-miR-183 (i.e., 3*premiR183-TS), three copies of the target site complementary to mature hsa-miR-183-5p+mature hsa-miR-183-3p (i.e., 3*miR183-5P3P-TS), and three copies of the target site complementary to mature hsa-miR-183-5p (i.e., 3*miR183-5P-TS). Where “No miRTS” is indicated, it indicates the absence of the miR183 target site. Figure 16A is a graph showing GFP fluorescence measured in protein lysates of HeLa cells transfected with a construct encoding GFP operably linked to the indicated miR183 target site. The optical density at 528 nm is expressed as the Log2 factor change of GFP fluorescence in “indicated cells” versus “control conditions (HeLa cells transfected with a GFP construct that does not contain the miR target site)”. Error bars represent the standard deviation (n=2). Figure 16B is a representative image of Western blot analysis of CX26 protein levels in HeLa cells transfected with a construct encoding CX26 operably linked to the indicated miR183 target site. HSP90 is used as a loading control. Figure 16C is a graph showing the quantification of CX26 protein levels detected by the Western blot analysis shown in Figure 16B (compared to a control without the miR183 target sequence - “no miRTS”) by concentration metric analysis. Error bars represent the standard deviation (n=2). [Examples]

[0230] The present invention is further illustrated by the following embodiments.

[0231] Example 1: AAV-DJ containing an expression cassette with a precursor miR183 target site efficiently and specifically transduces supporting cells of the organ of Corti in mice and non-human primates. material and method AAV Vector AAV-DJ vectors were constructed in HEK293T cells transfected with three different plasmids. The first plasmid contains two AAV2-ITRs (inverted terminal repeats) that define the range of the expression cassette, including the F1 origin of replication, the ampicillin resistance gene, and the smCBA (shortened CMV-chicken-β-actin) promoter, an eGFP (highly sensitive green fluorescent protein) reporter gene, three repeat sequences of the human precursor miR183 target site inserted into the 3'UTR of the gene, and a bovine growth hormone (bGH) polyadenylation signal sequence. The second plasmid (pCK003) contains the AAV2-Rep gene and a sequence encoding Cap AAV-DJ (i.e., the AAV-DJ capsid consisting of the amino acid sequence described in Sequence ID No. 18). The third plasmid (i.e., helper plasmid-pALD-X80 (Alvedron)) contains genes (VA, E2A, E4) that encode proteins that assist in the replication of the AAV vector.

[0232] By adding the miR183 precursor target site (corresponding to the nucleic acid sequence described in SEQ ID NO: 3) to the expression cassette of this AAV vector, long-term detargeting of hair cells becomes possible. The selection of miR183 was based on qPCR expression profiling of microRNAs in the inner ear of elderly people (Sekine et al., "Expression Profiling of MicroRNAs in the Inner Ear of Elderly People by Real-Time PCR Quantification." Audio Neurootol. 2017;22(3):135-145). The precursor miR183 target site (i.e., SEQ ID NO: 3, hereafter referred to as miRT) was selected to obtain optimal efficacy compared to shorter, mature miR183 target sites (e.g., miR183-5p and / or miR183-3p).

[0233] The expression cassette was encapsulated using the AAV-DJ serotype with tropism for Sox2-positive supporting cells in the cochlea.

[0234] The resulting AAV-DJ vector was designated AAVDJ-smCBA-eGFP-miRT and corresponds to an AAV-DJ vector (i.e., an AAV vector containing a capsid that is an AAV-DJ capsid) containing a polynucleotide (i.e., an expression cassette) that, from 5' to 3', comprises a 5' AAV2-ITR, a smCBA promoter operably linked to a gene encoding eGFP, three copies of a precursor miR183 target site (i.e., three copies of SEQ ID NO: 3), a bGH polyadenylation signal, and a 3' AAV2-ITR.

[0235] A control AAV-DJ vector was similarly prepared using an expression cassette that, from 5' to 3', comprises a 5' AAV2-ITR, a CMV promoter operably linked to a gene encoding eGFP, a bGH polyadenylation signal, and a 3' AAV2-ITR. The control vector was designated AAVDJ-CMV-eGFP.

[0236] A second control AAV-DJ vector, i.e., the AAVDJ-smCBA-eGFP vector, was prepared for in vivo administration to mice using an expression cassette that, from 5' to 3', comprises a 5' AAV2-ITR, a smCBA promoter operably linked to a gene encoding eGFP, a bGH polyadenylation signal, and a 3' AAV2-ITR.

[0237] Mouse At P0 or P15, mice (C57B6 / N, Janvier Labs) were injected via the round window membrane with 1 μL of either the AAVDJ-CMV-eGFP vector, the AAVDJ-smCBA-eGFP vector, or the AAVDJ-smCBA-eGFP-miRT vector at a total dose level corresponding to 5.0×10 10 vg corresponding to 5.0×10 13 vg / mL (viral genomes / ml).

[0238] Non-human primates In non-human primates (cynomolgus monkeys, Charles River Laboratories) at approximately 22 months of age, either 40 μL of the AAVDJ-CMV-eGFP vector or the AAVDJ-smCBA-eGFP-miRT vector was injected via the round window membrane at a total dose level of 4.0×10 11 vg corresponding to a concentration of 1.0×10 13 vg / mL. Ventilation of the oval window was performed to maximize the local distribution of the vector along the cochlear length.

[0239] Cochlear treatment In the case of mice, the cochleae were harvested 15 days after injection, fixed overnight at 4°C with 4% PFA, and then decalcified with 0.5 mM EDTA at 4°C for 4 hours.

[0240] In the case of non-human primates, the animals were euthanized 21 days after injection, 1.5 L of PBS was injected via the intracardiac route for approximately 10 minutes, and then 2.2 L of 4% paraformaldehyde (pH 7.4) was injected for approximately 15 minutes. The excised cochleae were perfused with the same fixative for 2 hours, then transferred to a 4% PFA solution, and finally transferred to a PBS (1×) solution at +4°C. Subsequently, the cochleae were decalcified with 0.5 mM EDTA for 5 days.

[0241] Immunofluorescence staining Cochleae from mice and non-human primates were blocked and permeabilized in PBS (1×) + 20% NGS (normal goat serum) + 0.5% Triton at room temperature for 1 hour, and then incubated overnight at 4°C with antibodies against GFP (1:250 dilution), MyoVIIa (1:300), or Sox2 (1:200) in PBS (1×) + 3% NGS + 0.1% Triton. After washing three times with PBS (1×) + 0.1% Triton, secondary antibodies (all at 1:700 dilution of Alexa fluor 647, Alexa fluor 568, Alexa fluor 488 or 1:250 dilution of Phalloidine Alexa fluor 488) and DAPI (dilution 1:4000) were added at room temperature for 1 hour. Negative control: no addition of secondary antibody.

[0242] Imaging Stained cochlea were imaged using an Andor Dragonfly spinning disk confocal microscope. Maximum intensity projection images were generated using ImageJ software (NIH).

[0243] Quantification The proportion of GFP-expressing cells in different cell populations of the organ of Corti (border cells, medial phalangeal cells, fiber cells, Hensens cells, Claudius cells, and inner ear hair cells) was quantified using a cell counter plugin (ImageJ / NIH). The ratio of GFP-expressing to non-expressing cells was calculated and plotted using Graphpad Prism. Statistical significance was calculated using Student's t-test.

[0244] result In both mice (Figures 1A-1B) and non-human primates (Figures 2C-2D and 4), the AAVDJ-CMV-eGFP control vector primarily transduced supporting cells of the outer helical groove, interdental cells of the organ of Corti, columnar cells, and fibrous cells. The AAVDJ-CMV-eGFP vector also resulted in significant GFP expression (25% in non-human primates) in inner ear hair cells (IHC) of both mice (Figures 1A-1B) and non-human primates (Figures 2C-2D and 4).

[0245] A vector of the same DJ serotype containing the precursor miR183 target site, namely the AAVDJ-smCBA-eGFP-miRT vector, showed efficient transduction of major supporting cell types in both mice (Figures 1C-1D) and non-human primates (Figures 2E-2F, 3A-3F, and 4). These supporting cells correspond to target cells where CX26 expression can be restored to enable therapeutic effects. As shown in Figures 3A-3F, the AAVDJ-smCBA-eGFP-miRT vector enables GFP expression along the frequency axis of the cochlea. However, GFP expression was not detected in inner ear hair cells (IHC), indicating that the AVDJ-smCBA-eGFP-miRT vector did not enable GFP expression in IHC.

[0246] To confirm the effect of the precursor miR183 target site on transgene expression in vivo, two-week-old C57Bl6n mice were injected intracochlea with AAV-DJ vectors containing / without the precursor miR183 target site (AAVDJ-smCBA-eGFP-miRT or AAVDJ-smCBA-eGFP, respectively). Two weeks after injection, the animals were euthanized, and the GFP expression profile was analyzed using whole-mount imaging of the cochlea. As shown in Figures 13A-13F, injection of AAVDJ-smCBA-eGFP resulted in widespread GFP expression in the inner ear hair cell compartment of the cochlear (see Figures 13A-13C), while injection of AAVDJ-smCBA-eGFP-miRT resulted in selective GFP expression in supporting cells, with only slight or no detection of GFP in inner ear hair cells (see Figures 13D-13F). These data further support the fact that the precursor miR183 target site is efficient in inhibiting transgene expression in miR183-expressing cells such as cochlear hair cells. Furthermore, compared to the GFP signal observed in these cells after injection of AAVDJ-smCBA-eGFP (see Figure 13B), injection of AAVDJ-smCBA-eGFP resulted in a particularly strong GFP signal in supporting cells (see Figure 13F).

[0247] As shown in Figure 4, no significant difference in the proportion of GFP-expressing cells was observed between cells transduced with AAVDJ-CMV-eGFP and cells transduced with AAVDJ-smCBA-eGFP-miRT in border cells (BC), internal drug cell (IPh), fibrillary cells, Hensens cells, and Claudius cells (HC, CC). Notably, transduction with the AAVDJ-smCBA-miRT vector resulted in a significant decrease in the proportion of GFP-positive cells in inner ear hair cells (IHC) compared to transduction with the same serotype vector that does not contain the precursor miR183 target site, i.e., the AAV-DJ-CMV-eGFP vector (Figure 4).

[0248] Therefore, in addition to primarily transducing supporting cells that normally express CX26, another advantage of the AAVDJ-smCBA-eGFP-miRT vector is that it prevents CX26 expression in hair cells where CX26 expression can be detrimental to hair cell survival and thus to hearing.

[0249] Example 2: Preparation of the AAVDJ-GJB2 vector As described in Example 1, the AAVDJ-GJB2 vector was constructed in HEK293T cells transfected with three different plasmids. The first plasmid contained an F1 origin of replication, an ampicillin resistance gene, an expression cassette flanked by two AAV2-ITRs (a 5'ITR consisting of the nucleic acid described in SEQ ID NO: 23 and a 3'ITR consisting of the nucleic acid described in SEQ ID NO: 37) and an smCBA promoter (SEQ ID NO: 22), a GJB2c cDNA sequence (a mouse cDNA sequence encoding a mouse protein described in SEQ ID NO: 27 or a human cDNA sequence encoding a human protein described in SEQ ID NO: 1), three copies of the human precursor miR183 target site (three copies of SEQ ID NO: 3), and a bovine growth hormone (bGH) polyadenylation signal (described in SEQ ID NO: 24). The expression cassette containing the 5'-AAV2-ITR of SEQ ID NO: 23, the smCBA promoter of SEQ ID NO: 22, the human cDNA sequence of SEQ ID NO: 21 encoding human CX26 as described in SEQ ID NO: 1, three copies of the human precursor miR183 target site of SEQ ID NO: 3 (corresponding to SEQ ID NO: 7), the bGH polyadenylation signal of SEQ ID NO: 24, and the 3'-AAV2-ITR of SEQ ID NO: 37 corresponds to the expression cassette of SEQ ID NO: 38. The first plasmid containing the F1 origin of replication, the ampicillin resistance gene, and the expression cassette of SEQ ID NO: 38 is called pCA027, which has the sequence described in SEQ ID NO: 42. The second plasmid (pCK003) contains the AAV2-Rep gene and the sequence encoding Cap AAV-DJ (i.e., the AAV-DJ capsid consisting of the amino acid sequence described in SEQ ID NO: 18). The third plasmid (i.e., helper plasmid - pALD-X80 (Alvedron)) contains genes (VA, E2A, E4) encoding proteins that assist in the replication of the AAV vector.

[0250] The resulting AAV-DJ vector, called the AAVDJ-smCBA-mGjb2-miRT vector, corresponds to an AAV-DJ vector containing a polynucleotide (i.e., an expression cassette) including a 5'AAV2-ITR (SEQ ID NO: 23) from 5' to 3', an smCBA promoter (SEQ ID NO: 22) operably ligated to the mouse cDNA sequence encoding the mouse Cx26 protein described in SEQ ID NO: 27, three copies of the precursor miR183 target site (i.e., three copies of SEQ ID NO: 3), a bGH polyadenylation signal (SEQ ID NO: 24), and a 3'AAV2-ITR (SEQ ID NO: 23). In a variant of the AAVDJ-smCBA-mGjb2-miRT vector, called the AAVDJ-smCBA-mGjb2-FLAGtag-miRT vector, the C-terminus of the mouse cDNA sequence encoding the Cx26 protein was tagged with a nucleic acid sequence encoding a FLAG tag (DYKDDDDK, corresponding to SEQ ID NO: 28).

[0251] Example 3: Hearing loss relief by vector injection at postnatal day 0 (P0) (OtogL-cre;Gjb2-Flox mouse model) material and method Mouse model To demonstrate the therapeutic effect of the AAVDJ-smCBA-mGjb2-miRT vector described in Example 2, a first mouse model of Gjb2-induced hearing loss was developed. Since complete knockout of the Gjb2 gene is lethal in mice due to placental abnormalities (either fetal or early postnatal), conditional knockout using the Cre / loxP system was employed. This model was named the OtogL-cre;Gjb2-Flox mouse model.

[0252] In this mouse model, Cre recombinase expression is driven by the promoter of the OtogL gene, thus specifically expressed in cochlear cells that express Cx26. Cre recombinase (referred to as OtogL-cre) recognizes the loxP site adjacent to the Gjb2 gene and induces Gjb2 inactivation, which occurs mainly in cochlear support cells.

[0253] OtogLcre / +,Gjb2flox / flox mice may exhibit one of three distinct hearing impairment (HI) phenotypes: (i) severe congenital hearing impairment (i.e., severe hearing impairment from birth), (ii) high congenital hearing impairment (i.e., high hearing impairment from birth), or (iii) progressive hearing impairment after normal hearing at birth.

[0254] Treatment for mice The AAVDJ-smCBA-mGjb2-miRT vector (6.5 × 10¹⁶) described in Example 2 was injected into the left ear of 20 OtogLcre / +,Gjb2flox / flox neonatal mice and 12 control OtogL+ / +,Gjb2flox / flox littermates via a round window membrane. 9 vg)+AAVDJ-CMV-eGFP vector (3.7×10 9 vg) was administered simultaneously at P0 at a total of 1 μL per cochlea. The right ear was not surgically treated (negative control). Hearing was evaluated 3 and 8 weeks after surgery.

[0255] Auditory Brainstem Response Measurement Auditory brainstem responses (ABR) were recorded using a dedicated workstation to evaluate the auditory function of mice. Mice were anesthetized by intraperitoneal injection of ketamine and xylazine. Body temperature was maintained at 37°C using a heating pad under feedback control. Animals were placed in a soundproof box. Acoustic stimuli (i.e., tone bursts at 5, 10, 15, 20, 32, and 40 kHz) were delivered at close range using a speaker. Needle electrodes were inserted subcutaneously at the top of the head (active), ventrolateral to the left ear (reference), and on the tail (ground) to collect bioelectric potentials. Responses were amplified and averaged over 300 identical stimuli. The hearing threshold level was determined as the SPL (sound pressure level) at which the peak of wave I could be visually distinguished above the noise floor, and the ABR threshold was determined. Higher ABR thresholds were associated with hearing loss. In other words, the higher the ABR threshold for a given tested frequency, the more impaired hearing was at that frequency.

[0256] result As shown in Fig. 5, mice with severe congenital hearing impairment (11 OtogLcre / +, Gjb2flox / flox mice) showed lower average auditory brainstem response (ABR) thresholds at all tested frequencies in the injected ear (i.e., the left ear) compared to the non-injected ear (i.e., the right ear) 8 weeks after injection of the AAVDJ-smCBA-mGjb2-miRT vector.

[0257] Figs. 6A - 6B show the ABR thresholds evaluated in one of the mice with severe congenital HI 3 weeks (Fig. 6A) and 8 weeks (Fig. 6B) after surgery. This mouse showed significantly lower ABR thresholds at all tested frequencies in the left ear compared to the right ear both 3 weeks (Fig. 6A) and 8 weeks (Fig. 6B) after injection of the AAVDJ-smCBA-mGjb2-miRT vector.

[0258] These results demonstrate that injection of the AAVDJ-smCBA-mGjb2-miRT vector was able to prevent severe congenital deafness of genetic origin for at least 8 weeks from the injection of this vector at P0.

[0259] Figs. 7A - 7C show the ABR thresholds evaluated in one mouse with progressive hearing impairment (HI) 3 weeks (Fig. 7A), 8 weeks (Fig. 7B) and 4 months (Fig. 7C) after surgery. As shown above, the progressive HI mouse showed normal hearing at birth and gradually became deaf over time. As shown in Figs. 7A - 7B, this individual showed low ABR thresholds at all tested frequencies in both the treated and untreated ears 3 weeks (Fig. 7A) and 8 weeks (Fig. 7B) after surgery. However, 4 months after surgery (Fig. 7C), the ABR thresholds of the right untreated ear increased at all tested frequencies, indicating hearing loss. In contrast, the ABR thresholds in the left treated ear remained lower (compared to the untreated ear) at all tested frequencies.

[0260] These data indicate that injection of the AAVDJ-smCBA-mGjb2-miRT vector in P0 can delay the onset of genetically-derived progressive hearing loss.

[0261] Example 4: Hearing loss relief by vector injection in P2 (OtogL-cre;Gjb2-Flox mouse model) material and method Treatment for mice The posterior semicircular canal of the left ear vestibule of 7 OtogLcre / +,Gjb2flox / flox neonatal mice and 7 control OtogL+ / +,Gjb2flox / flox littermates was injected with the AAVDJ-smCBA-mGjb2-FLAGtag-miRT vector (2.7 × 10⁻¹⁰) described in Example 2. 9 The vg) vector was injected at P2 at a dose of 1 μL per cochlea. No surgery was performed on the right ear. Hearing evaluation was performed 4 weeks after surgery. Note that the AAVDJ-smCBA-mGjb2-FLAGtag-miRT vector was prepared independently of the AAVDJ-smCBA-mGjb2-miRT vector in Example 3 (i.e., a different production batch).

[0262] Auditory Brainstem Response Measurement The auditory brainstem response (ABR) threshold was measured using the protocol described in Example 3 above.

[0263] result As shown in Figures 8A and 8B, two mice with severe congenital sensory impairment showed lower ABR thresholds in the injected side (i.e., left ear) compared to the non-injected side (i.e., right ear) at all frequencies except one tested, four weeks after injection of the AAVDJ-smCBA-mGjb2-miRT vector.

[0264] These results, obtained using different batches of vectors, demonstrate that injection of the AAVDJ-smCBA-mGjb2-miRT vector in P2 can reliably prevent severe congenital hearing loss of genetic origin.

[0265] Example 5: Hearing loss relief by vector injection in P2 (ROSA26-creERT2;Gjb2-Flox mouse model) material and method Mouse model We developed a second conditional knockout mouse model for Gjb2-induced hearing loss (dependent on an inducible Cre recombinase that targets all cells upon activation). This inducible model was named the ROSA26-creERT2;Gjb2-Flox mouse model.

[0266] In this mouse model, fusion creERT2 recombinase expression is driven by the constitutively active and ubiquitous locus ROSA26. Upon administration of hydroxytamoxifen (OHT), fusion creERT2 recombinase dissociates from its cytoplasmic anchor, enters the nucleus, and can induce Gjb2 gene inactivation. Therefore, time-specific control of Gjb2 gene inactivation can be achieved.

[0267] ROSA26CreERT2 / +,Gjb2flox / flox mice injected with hydroxytamoxifen (OHT) in Phase 2 exhibited severe to profound hearing impairment (HI).

[0268] Treatment for mice In Phase 2, three ROSA26CreERT2 / +,Gjb2flox / flox neonatal mice were intraperitoneally injected with hydroxytamoxifen (OHT), and the AAVDJ-smCBA-mGjb2-miRT vector described in Example 2 was injected into the posterior semicircular canal of the vestibule of the left ear (2.7 × 10⁻¹⁰). 9 ~1.3 × 10 10 (vg). No surgery was performed on the right ear. Hearing evaluation was performed 5 weeks after surgery.

[0269] Auditory Brainstem Response Measurement The auditory brainstem response (ABR) threshold was measured using the protocol described in Example 3 above.

[0270] result Of the three ROSA26CreERT2 / +,Gjb2flox / flox mice, two showed severe HI in the right ear, suggesting efficient Gjb2 deletion in the cochlea (not shown). As shown in Figure 9, one of the two mice showed a decrease in ABR threshold in the injected side (i.e., left ear) compared to the non-injected side (i.e., right ear) 5 weeks after injection of the AAVDJ-smCBA-mGjb2-miRT vector.

[0271] These results demonstrate that injection of the AAVDJ-smCBA-mGjb2-miRT vector in P2 could also prevent genetically caused neonatal-onset hearing loss.

[0272] Example 6: Hearing loss relief by vector injection in P16 (OtogL-cre;Gjb2-Flox mouse model) material and method Treatment for mice AAVDJ-smCBA-mGjb2-miRT vector (1.3 × 10¹⁴) was injected into the posterior semicircular canal of the left ear vestibule of OtogLcre / +,Gjb2flox / flox mice and control OtogL+ / +,Gjb2flox / flox littermates. 10 vg) was injected at P16 at a dose of 1 μL per cochlea. No surgery was performed on the right ear. Hearing evaluations were performed 3 weeks, 6 weeks, and 3 months after surgery.

[0273] Auditory Brainstem Response Measurement The auditory brainstem response (ABR) threshold was measured using the protocol described in Example 3 above.

[0274] Confocal microscopy Three months after injection of the AAVDJ-smCBA-mGjb2-miRT vector into the left ear, the right and left cochleas were collected from one progressively deaf OtogLcre / +,Gjb2flox / flox mouse. Immediately after euthanasia of the mouse, the cochlea were removed and the samples were fixed in phosphate-buffered saline (PBS) with 4% paraformaldehyde (PFA) at room temperature for 1 hour. Next, for decalcification, the cochlea were incubated in 0.35 M ethylenediaminetetraacetic acid (EDTA) for 3 days. The samples were then re-fixed in PBS with 4% paraformaldehyde at room temperature for 1 hour, incubated in sucrose (20%) for 12 hours, and then embedded in OCT compound (optimal cutting temperature compound). Longitudinal sections were prepared to a thickness of 10 μm using a cryostat. The tissue was permeabilized by incubation of the sections in a PBS solution containing 0.1% TritonX-100 and 20% normal goat serum (NGS). These sections were then incubated overnight at 4°C in PBS with 10% bovine serum albumin (BSA) with the following primary antibodies: anti-CX26 polyclonal antibody (catalog #51-2800, Invitrogen), anti-acetylated tubulin monoclonal antibody (catalog #T6793, Sigma), phalloidin, and DAPI (4',6-diamidino-2-phenylindole). Incubation with secondary antibodies and phalloidin was performed in PBS with 10% BSA for 1 hour and 30 minutes. Staining was observed using a confocal microscope.

[0275] result One of the OtogLcre / +,Gjb2flox / flox mice developed progressive hearing impairment (HI). Figures 10A–10C show the ABR thresholds evaluated in the progressive HI mouse 3 weeks (Figure 10A), 6 weeks (Figure 10B), and 4 months (Figure 10C) after surgery. As shown in Figures 10A–10B, this individual showed low ABR thresholds at all tested frequencies in both the treated and untreated ears 3 weeks (Figure 10A) and 6 weeks (Figure 10B) after surgery. However, 3 months after surgery (Figure 10C), the ABR thresholds increased at all frequencies in the right untreated ear, indicating hearing loss. In contrast, the ABR threshold remained lower in the left treated ear (compared to the untreated ear).

[0276] These data indicate that injection of the AAVDJ-smCBA-mGjb2-miRT vector in P16 can delay the onset of genetically-derived progressive hearing loss.

[0277] Connexin 26 expression was measured by confocal microscopy of cochlear sections from progressive HI mice three months after injection of the AAVDJ-smCBA-mGjb2-miRT vector in P16. Figures 11A-11B and 11E-11F show images obtained from the cochlea of ​​the untreated right ear, where CX26 expression cannot be detected. In contrast, Figures 11C-11D and 11G-11H show images obtained from the cochlea of ​​the treated left ear, where CX26 expression is detected.

[0278] These images confirm that injection of the AAVDJ-smCBA-mGjb2-miRT vector effectively restored CX26 protein expression in the injected ears of progressive HI mice.

[0279] Example 7: Hearing loss relief by vector injection in P16 (OtogL-cre;Gjb2-Flox mouse model) material and method In mice with progressive hearing impairment (HI) clearly identified by behavioral testing (response to sudden noises) and control OtogL+ / +,Gjb2flox / flox littermates, the posterior semicircular canal of the left ear vestibular region was injected with the AAVDJ-smCBA-mGjb2-miRT vector (1.3 × 10⁻¹⁰). 10 vg) was injected at P16 at a dose of 1 μL per cochlea. No surgery was performed on the right ear. Hearing was evaluated 4 weeks after surgery.

[0280] Auditory Brainstem Response Measurement The auditory brainstem response (ABR) threshold was measured using the protocol described in Example 3 above.

[0281] result Figures 12A–12C show the ABR thresholds evaluated in three of four mice with early progressive HI four weeks after surgery. As shown in Figures 12A–12C, these three individuals showed lower ABR thresholds in the left treated ear compared to the right treated ear at all or nearly all tested frequencies.

[0282] These data confirm that injection of the AAVDJ-smCBA-mGjb2-miRT vector in P16 could prevent or delay the onset of early progressive hearing loss due to genetic causes.

[0283] Example 8: Hearing loss relief by vector injection in P0-P3 (OtogL-cre;Gjb2-Flox mouse model) Treatment for mice AAVDJ-smCBA-hGjb2-miRT vector (1.5 × 10¹⁶) was injected into the inner ear of the left ear of control OtogL+ / +,Gjb2flox / flox and OtogLcre / +,Gjb2flox / flox littermates via the round window membrane. 10The mice were injected with 1 μL of the vg) vector per cochlea at P0-P3. The right ear was not surgically treated. Hearing was evaluated 3 and 7 weeks after surgery. These mice were also injected with AAVDJ-smCBA-hGjb2-miRT, which encodes human CX26. As detailed in Example 2, the AAVDJ-smCBA-hGjb2-miRT vector contains the expression cassette of SEQ ID NO: 38.

[0284] Auditory brainstem response measurement and ABR wave I amplitude and latency analysis Auditory brainstem response (ABR) thresholds and ABR threshold variability were measured using the protocol described in Example 3 above. ABR threshold variability represents the change in hearing threshold between injected animals and uninjected animals. Statistical analysis was performed using two-way ANOVA followed by all-pairs multiple comparisons (Holm-Sidak method).

[0285] The first peak of the ABR wave is called wave I and reflects the synchronous output generated in the auditory nerve. The interpretation of the ABR can take into account wave amplitude, which indicates the number of neuronal firings, and wave latency, which indicates the transmission velocity. Wave I amplitude of the ABR was measured in nV, and wave I latency of the ABR was measured in ms. Statistical analysis was performed using one-way analysis of variance.

[0286] result Figures 14A and 14B show the ABR threshold and ABR threshold variability evaluated in 28 uninjected OtogLcre / +,Gjb2flox / flox mice, 15 AAV-injected OtogLcre / +,Gjb2flox / flox mice, and 14 AAV-injected OtogL+ / +,Gjb2flox / flox mice 3 and 7 weeks after surgery. As shown in Figure 14A, on average, the AAV-injected OtogLcre / +,Gjb2flox / flox group showed lower ABR thresholds in the left treated ear at all or nearly all tested frequencies compared to the uninjected OtogLcre / +,Gjb2flox / flox group at both 3 and 7 weeks after injection. The ABR threshold changes in Figure 14B indicate that AAV-injected OtogLcre / +,Gjb2flox / flox mice had a significantly lower ABR threshold, and that hearing recovery in the OtogLcre / +,Gjb2flox / flox group improved between 3 weeks and 7 weeks after injection.

[0287] As shown in Figures 14C to 14D, Gjb2 deletion in the OtogL-cre;Gjb2-Flox mouse model results in a decrease in wave I amplitude and an increase in wave I latency. Injection of OtogLcre / +,Gjb2flox / flox mice with AAVDJ-smCBA-hGjb2-miRT resulted in a significant recovery of both wave I amplitude and latency.

[0288] These data confirm that injection of the AAVDJ-smCBA-hGjb2-miRT vector in P0-P3 could restore the ABR threshold in severe hearing loss of genetic origin.

[0289] Example 9: Hearing loss relief by vector injection in P1 (FoxG1-cre;Gjb2-Flox mouse model) material and method Mouse model To confirm the efficacy of in vivo treatment with this AAV vector, a third mouse model of Gjb2-induced hearing loss was developed. Using the Cre / loxP system, a further Gjb2 conditional knockout mouse model (FoxG1-cre; Gjb2-Flox mouse model) was developed. Mice in which the coding sequence of the Gjb2 gene is flanked by two loxP sequences were crossed with mice in which the Cre gene is expressed under the FoxG1 promoter. Cre recombinase expression occurs in supporting cells, stria vascularis cells, and fibrous cells (not shown).

[0290] Two-week-old control FoxG1+ / + Gjb2flox / flox mice (in which Gjb2 deletion does not occur) show normal auditory brainstem responses (ABR), while FoxG1Cre / + Gjb2flox / flox littermates show an elevated ABR threshold of over 60 dB at all frequencies tested (Figure 15A), reflecting severe hearing loss. FoxG1Cre / + Gjb2flox / + mice show ABR waveforms comparable to those of control mice.

[0291] FoxG1-Cre-mediated Gjb2 deletion is likely to induce severe hearing loss in FoxG1Cre / + Gjb2flox / flox mice. Therefore, the FoxG1-cre;Gjb2-Flox mouse model is a further experimental model of hearing loss for evaluating Gjb2 gene therapy.

[0292] Treatment for mice Four FoxG1Cre / + Gjb2flox / flox neonatal mice (P1) and three control FoxG1+ / + Gjb2flox / flox littermates were injected with 1 μL of AAVDJ-smCBA-hGjb2-miRT vector (1.5 × 10⁻¹⁰) via the round window membrane into the left cochlea. 13The mice were injected with a batch of vg / mL titer, and the right ear (the opposite side) was not surgically treated. Hearing was evaluated 3 weeks after surgery. These mice were also injected with AAVDJ-smCBA-hGjb2-miRT, which encodes human CX26. As detailed in Example 2, the AAVDJ-smCBA-hGjb2-miRT vector contains the expression cassette of SEQ ID NO: 38.

[0293] Auditory Brainstem Response Measurement The auditory brainstem response (ABR) threshold was measured using the protocol described in Example 3 above.

[0294] The I-wave amplitude of the ABR was measured as described in Example 8 above.

[0295] result Three of the four FoxG1Cre / + Gjb2flox / flox mice showed improved ABR threshold (Figures 15B-15C) and improved I-wave amplitude (Figure 15D) for all tested frequencies in the injected ear compared to the opposite ear. The fourth FoxG1Cre / + Gjb2flox / flox mouse showed similar ABR thresholds in both ears (unresponsive mouse, not shown).

[0296] The responsive mice that showed the greatest decrease in ABR threshold also showed improvement in the best I-wave amplitude (Figure 15D).

[0297] Therefore, these data indicate that injection of the AAVDJ-smCBA-hGjb2-miRT vector improved the severe hearing loss phenotype in 3 out of 4 FoxG1Cre / + Gjb2flox / flox mice (the 4th mouse may have suffered trauma from the injection).

[0298] Example 10: Comparison of inhibitory effects on miR183 target sites material and method cell culture HEK293 cells (human fetal kidney 293 cells), HEK293T cells (human fetal kidney 293 cells expressing SV40 large T antigen), and HeLa cells (Henrietta Lachs cervical tumor cells) were maintained in DMEM (Dulbeccoo's modified Eagle medium) containing 10% fetal bovine serum (FBS) under standard conditions.

[0299] Transfection The pAAV-smCBA-eGFP plasmid (Genscript) was modified to enable expression of either GFP or human CX26. The coding sequence (either the eGFP coding sequence encoding the human CX26 protein described in SEQ ID NO: 1 or the human GJB2 cDNA sequence) was modified. • Three consecutive repeat sequences of the human precursor miR183 target site, i.e., three consecutive copies of SEQ ID NO: 3 (corresponding to SEQ ID NO: 7, 3*premiR183-TS), · Three repeat sequences of both the human miR183-5p mature target site (corresponding to SEQ ID NO: 29) and the human miR183-3p mature target site (corresponding to SEQ ID NO: 30), i.e., three copies of SEQ ID NO: 39 corresponding to SEQ ID NO: 30 and SEQ ID NO: 29 separated by spacer TCAC (3*miR183-5P3P-TS corresponding to SEQ ID NO: 40), or • Three repeat sequences of the mature target site of human miR183-5p, namely three copies of SEQ ID NO: 29 (corresponding to SEQ ID NO: 41, 3*miR183-5P-TS) It was operably connected to one of the following.

[0300] smCBA-GJB2 or smCBA-eGFP plasmids, which do not contain miR target sites, were used as controls. Cells were placed in 5 × 10⁶ well plates. 4 cells / cm 2 Cells were seeded at a density and transfected with polyethyleneimine (PEI) using 2 μg of plasmid per well. Cells were harvested 60 hours after transfection for downstream analysis.

[0301] Protein extraction Cells were homogenized in RIPA lysis buffer (Merck) supplemented with a protease inhibitor cocktail. The cell lysates were clarified by centrifugation. The protein concentration of the clarified lysates was determined using the bicinchoninic acid method (BCA, ThermoFisher).

[0302] Western blot The clarified protein lysate was denatured by adding Laemmli buffer (Biorad) containing 25 mM dithiothreitol (ThermoFisher), also known as DTT, and boiled at 95°C for 5 minutes. 25 μg of protein per sample was separated by SDS-PAGE electrophoresis and blotted onto polyvinylidene fluoride (PVDF) membranes. Proteins were detected using anti-GFP (Abcam), anti-GJB2 (Invitrogen), or anti-HSP90 (Abcam) specific antibodies, and the membranes were scanned using a LI-COR DLx imaging system.

[0303] GFP fluorescence measurement 50 μL of total protein lysate was placed in a 96-well plate, and the optical density at 568 nm was acquired using a BioTek Synergy microplate reader (Agilent). GFP fluorescence values ​​were normalized to the total protein concentration of each sample.

[0304] result To evaluate the efficiency of different miR183 target sequences in mediating miR inhibition of transgene expression, either hsa-miR-183-5p alone or in combination with hsa-miR183-3p, or sequences complementary to the precursor hsa-miR-183 (also called premiR183) (hereinafter referred to as miR183-5P-TS (corresponding to SEQ ID NO: 29)), miR183-5P3P-TS (corresponding to SEQ ID NO: 29 + SEQ ID NO: 30) and premiR183-TS (corresponding to SEQ ID NO: 3)), were cloned downstream of an AAV expression cassette encoding GFP. HeLa cells expressing hsa-miR-183-5p were transfected with different constructs, and transgene expression was evaluated after 60 hours. GFP fluorescence in cell lysates was used as a readout for transgene expression, as it reflects GFP protein levels.

[0305] As shown in Figure 16A, GFP fluorescence is significantly reduced in HeLa cells transfected with GFP-premiR183-TS compared to HeLa cells transfected with a GFP cassette that does not contain the miR183 target sequence. Note that the use of a mature miR183 target site (either miR183-5P-TS or miR183-5P3P-TS) does not prevent GFP expression in HeLa cells.

[0306] To determine the efficiency of miR183 target sequences in inhibiting human CX26 expression, miR183-5P-TS (SEQ ID NO: 29), miR183-5P3P-TS (SEQ ID NO: 29 + SEQ ID NO: 30), or premiR183-TS (SEQ ID NO: 3) were cloned downstream of the GJB2 coding sequence, and the constructs were transfected into HeLa cells. Protein lysates were analyzed by Western blotting 60 hours after transfection.

[0307] Notably, the results of CX26 Western blot analysis were remarkably similar to those obtained by GFP fluorescence analysis, and only the precursor miR183 target site showed a clear inhibitory effect on CX26 expression in HeLa cells (see Figures 16B-16C). Overall, these results indicate that the precursor miR183 target site is more efficient than the mature miR183 target site in silencing transgene expression in HeLa cells.

[0308] conclusion The results provided herein demonstrate that an AAV-DJ vector containing an expression cassette comprising a coding sequence operably linked to a promoter and at least one copy of the precursor miR183 target site can specifically transduce supporting cells while preventing gene expression in cochlear hair cells (Example 1). Furthermore, in vitro transfection experiments performed in HeLa cells show that the precursor miR183 target site is more efficient than the mature miR183 target site (e.g., the miR183-5p target site) in silencing transgene expression (Example 10). In addition, the results provided herein provide proof of concept that hereditary hearing loss can be effectively treated (i.e., prophylactic and / or therapeutic – Examples 3-9) by injection of an AAV-DJ vector containing an expression cassette (e.g., the expression cassette for SEQ ID NO: 38) comprising a cDNA encoding CX26 operably linked to a promoter and at least one copy of the precursor miR183 target site. The therapeutic effect of an AAV-DJ vector comprising an expression cassette containing cDNA encoding CX26 operably linked to a promoter and at least one copy of the precursor miR183 target site was observed in three different mouse models mimicking hereditary hearing loss induced by loss of function of Gjb2: OtogL-cre;Gjb2-Flox mouse model (Examples 3-4 and 6-8), ROSA26-creERT2;Gjb2-Flox mouse model (Example 5), and FoxG1-cre;Gjb2-Flox mouse model (Example 9).

[0309] The data provided herein demonstrate that injection of the AAVDJ-smCBA-mGjb2-miRT vector (in P0 or P2) in neonatal mice enabled (i) effective treatment of severe congenital hearing loss of genetic origin at least 8 weeks after surgery, and (ii) effective treatment of progressive hearing loss of genetic origin at least 4 months after surgery. Preliminary results also demonstrate that injection of the AAVDJ-smCBA-mGjb2-miRT vector in neonatal mice enabled effective treatment of neonatal-onset hearing loss of genetic origin, particularly severe hearing loss of genetic origin. Finally, these data demonstrate that injection of the AAVDJ-smCBA-mGjb2-miRT vector in P16 mice enabled effective treatment of progressive hearing loss of genetic origin, including early progressive hearing loss of genetic origin, at least 3 months after surgery.

Claims

1. An adeno-associated virus (AAV) vector comprising an AAV-DJ capsid or a capsid derived from an AAV-DJ capsid, comprising a polynucleotide comprising (i) a nucleic acid sequence encoding a connexin 26 protein (CX26) operably linked to a promoter, and (ii) at least one copy of a miRNA target site of the miR183 family comprising the sequence described in SEQ ID NO: 3, SEQ ID NO: 16, or SEQ ID NO: 31, or a sequence having at least 90% identity with any one of SEQ ID NO: 3, SEQ ID NO: 16, or SEQ ID NO:

31.

2. The AAV vector according to claim 1, comprising 2 to 6 copies, preferably 3 copies, of the miRNA target site of the miR183 family, which includes the sequence described in SEQ ID NO: 3, SEQ ID NO: 16, or SEQ ID NO: 31, or a sequence having at least 90% identity with any one of SEQ ID NO: 3, SEQ ID NO: 16, or SEQ ID NO:

31.

3. The AAV vector according to claim 1 or 2, wherein the promoter is an smCBA promoter.

4. The AAV vector according to any one of claims 1 to 3, wherein the connexin 26 protein is human CX26.

5. The AAV vector according to any one of claims 1 to 4, wherein the polynucleotide further comprises 5' and 3' inverted terminal repeat sequences (ITRs).

6. The AAV vector according to any one of claims 1 to 5, wherein the polynucleotide further comprises at least one copy of another miRNA target site selected from the miR183 family.

7. A pharmaceutical composition comprising an AAV vector according to any one of claims 1 to 6 and at least one pharmaceutically acceptable excipient or carrier.

8. An AAV vector according to any one of claims 1 to 6 or a pharmaceutical composition according to claim 7 for use as a medicine.

9. An AAV vector according to any one of claims 1 to 6 or a pharmaceutical composition according to claim 7 for use in treating hereditary hearing loss in a subject requiring it.

10. The hereditary hearing loss is non-symptomatic hearing loss (DFNB1), the AAV vector or pharmaceutical composition for use according to claim 9.

11. The hereditary hearing loss is severe hereditary hearing loss, the AAV vector or pharmaceutical composition for use according to claim 9 or 10.

12. The AAV vector or pharmaceutical composition for use according to claim 9 or 10, wherein the hereditary hearing loss is severe hereditary hearing loss.

13. The AAV vector or pharmaceutical composition for use according to claim 9 or 10, wherein the hereditary hearing loss is progressive hereditary hearing loss.

14. The subject is an adult, an AAV vector or pharmaceutical composition for use according to any one of claims 9 to 13.

15. The subject is an infant or a child, as described in any one of claims 9 to 13, an AAV vector or pharmaceutical composition for use.

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