Method for performing gene therapy and regeneration of cochlear supporting cells

Abstract

A method for performing gene therapy and regeneration of cochlear supporting cells. Specifically, provided is a cochlear-supporting-cell-specific promoter as shown in SEQ ID NO: 1, comprising: (1) an enhancer sequence as shown in SEQ ID NO: 2 or a sequence having at least 90% sequence identity thereto and retaining the function of SEQ ID NO: 2; and (2) a core promoter element SEQ ID NO: 3. The promoter specifically drives the expression of a transgene in the cochlear supporting cells.

Description

Methods for gene therapy and regeneration of cochlear supporting cells Technical Field

[0001] The present invention relates to gene engineering technology, in particular to a method for gene therapy and regeneration of cochlear supporting cells. Background Art

[0002] Hearing loss is the fourth leading cause of life expectancy for people with disabilities worldwide, affecting 6% to 8% of the global population and significantly reducing quality of life. In recent years, efforts to treat hearing loss have increasingly focused on gene therapy as a potential solution. Some deafness genes are expressed in cochlear supporting cells (SCs), making gene therapy targeting SCs in the inner ear highly desirable. For example, epidemiological studies have shown that a large proportion of hereditary deafness cases (30–50% across ethnic groups) are caused by loss-of-function mutations in a single gene, GJB2. This gene encodes connexin 26 (Cx26), a major cochlear gap junction (GJ) protein, and is primarily expressed in SCs. Therefore, if this protein could be specifically re-expressed in SCs, it is expected to restore hearing loss caused by GJB2 deficiency, providing a theoretical basis for clinical treatment of this hereditary deafness. However, specific gene therapy approaches targeting SCs remain scarce. There is a need to identify novel SC-targeted gene therapy approaches to treat hereditary sensorineural hearing loss, such as that caused by GJB2 deficiency.

[0003] However, current vectors commonly used in inner ear gene therapy to promote the expression of specific transgenes in cochlear cells are generally broad and cannot target specific cell types.

[0004] Summary of the Invention

[0005] In response to the deficiencies of the prior art, the present invention provides a polynucleotide containing a cochlear supporting cell SC promoter region, and a vector containing the region, which can be used to promote the expression of a specific transgene in cochlear supporting cells SC.

[0006] The present invention provides a cochlear supporting cell-specific enhancer comprising the sequence shown in SEQ ID NO: 2 or a homologous sequence having at least 90% sequence identity therewith and retaining the function of SEQ ID NO: 2. The homologous sequence is a sequence of mammals, preferably primates, more preferably hominids.

[0007] The present invention also provides a cochlear supporting cell-specific promoter, comprising:

[0008] (1) the sequence shown in SEQ ID NO: 2 or a sequence having at least 90% sequence identity thereto and retaining the function of SEQ ID NO: 2, and

[0009] (2) Core promoter elements.

[0010] In one or more embodiments, (2) the core promoter element is a promoter suitable for promoting gene expression in mammalian cells.

[0011] In one or more embodiments, (2) the core promoter element is the promoter of the SHH gene.

[0012] In one or more embodiments, (2) the sequence of the core promoter element is shown in SEQ ID NO: 3 or a homologous sequence having at least 90% sequence identity therewith. The homologous sequence is a SHH gene promoter sequence of mammals, preferably primates, more preferably humans.

[0013] The present invention also provides a nucleic acid construct comprising the cochlear supporting cell-specific promoter described in any embodiment herein.

[0014] In one or more embodiments, the nucleic acid construct further comprises a gene of interest operably linked to the cochlear supporting cell-specific promoter.

[0015] In one or more embodiments, the gene is selected from the group consisting of: a deafness gene, a transcription factor gene, a signaling pathway gene, and a tool gene for manipulating genes in cochlear supporting cells or expressed in cochlear supporting cells.

[0016] In one or more embodiments, the nucleic acid construct further comprises a multiple cloning site and / or a terminator. Preferably, the terminator is a polyA terminator.

[0017] In one or more embodiments, the nucleic acid construct is a vector, such as a cloning vector, an integration vector, an expression vector. In one or more embodiments, the vector is an AAV vector.

[0018] The present invention also provides an rAAV vector system comprising an AAV vector and a nucleic acid construct encoding genes required by the AAV virus, wherein the AAV vector comprises the cochlear supporting cell-specific promoter described in any embodiment of the present invention.

[0019] In one or more embodiments, the AAV vector further comprises a gene of interest operably linked to the cochlear supporting cell-specific promoter.

[0020] In one or more embodiments, the desired genes of the AAV virus include one or more selected from the group consisting of Rep, Cap, E2A, E4, and VA genes.

[0021] The present invention also provides an rAAV virus particle comprising the cochlear supporting cell-specific promoter described in any embodiment of the present invention.

[0022] The present invention also provides a host cell comprising or having integrated into its genome: the cochlear supporting cell-specific promoter, nucleic acid construct or AAV vector system described in any embodiment of the present invention.

[0023] The present invention also provides a pharmaceutical composition comprising pharmaceutically acceptable excipients and one or more selected from the following: the cochlear supporting cell-specific promoter, nucleic acid construct, rAAV vector system, rAAV virus particles, and host cells described in any embodiment of the present invention.

[0024] The present invention also provides an in vitro method for expressing a gene in cochlear supporting cells, comprising the step of introducing a nucleic acid construct comprising the gene into the cochlear supporting cells, wherein the gene is operably linked to the cochlear supporting cell-specific promoter described in any embodiment of the present invention.

[0025] In one or more embodiments, the genes are selected from the group consisting of: deafness genes, transcription factors, signaling pathway genes, and tool genes for manipulating genes in cochlear supporting cells or expressed in cochlear supporting cells. Deafness genes are deafness genes expressed in cochlear supporting cells, including GJB2, GJB3, GJB6, MYH14, WFS1, EYA4, TMPRSS3, DCDC2, GPSM2, and KARS. Transcription factor genes are transcription factor genes associated with hair cell regeneration, including ATOH1, POU4F3, GFI1, IKZF2, INSM1, and SIX1. Signaling pathway genes are genes in the Wnt, Notch, Hippo, and TGF-beta signaling pathways associated with hair cell regeneration.

[0026] The present invention also provides the use of the sequence shown in SEQ ID NO: 2 or a sequence having at least 90% sequence identity thereto and retaining the function of SEQ ID NO: 2 in a chimeric promoter.

[0027] The present invention also provides a method for preparing a chimeric promoter, comprising: operably linking (1) a sequence shown in SEQ ID NO: 2 or a sequence having at least 90% sequence identity therewith and retaining the function of SEQ ID NO: 2 with (2) a core promoter element.

[0028] The present invention also provides use of the cochlear supporting cell-specific promoter described in any embodiment herein in the preparation of a drug for treating a disease caused by a gene defect in cochlear supporting cells, wherein the drug is used to express the gene in cochlear supporting cells.

[0029] The genes are selected from the group consisting of: deafness genes, transcription factors, signaling pathway genes, and tool genes for manipulating genes in cochlear supporting cells or expressed in cochlear supporting cells. Deafness genes are deafness genes expressed in cochlear supporting cells, including GJB2, GJB3, GJB6, MYH14, WFS1, EYA4, TMPRSS3, DCDC2, GPSM2, and KARS. Transcription factor genes are transcription factor genes associated with hair cell regeneration, including ATOH1, POU4F3, GFI1, IKZF2, INSM1, and SIX1. Signaling pathway genes are genes in the Wnt, Notch, Hippo, and TGF-beta signaling pathways.

[0030] In one or more embodiments, the disease is hereditary deafness caused by a gene defect in cochlear supporting cells. Preferably, the disease is hereditary deafness caused by a functional loss of GJB2.

[0031] The present invention also provides a method for treating a disease caused by a gene defect in cochlear supporting cells, comprising the step of expressing the gene in cochlear supporting cells, wherein the gene is operably linked to the cochlear supporting cell-specific promoter described in any embodiment of the present invention.

[0032] In one or more embodiments, the method includes the step of introducing into cochlear supporting cells a nucleic acid construct or vector comprising the coding sequence of the gene operably linked to the cochlear supporting cell-specific promoter.

[0033] In one or more embodiments, the gene is a deafness gene expressed by cochlear supporting cells, including GJB2, GJB3, GJB6, MYH14, WFS1, EYA4, TMPRSS3, DCDC2, GPSM2, and KARS.

[0034] In one or more embodiments, the disease is hereditary deafness caused by loss of function of GJB2.

[0035] The present invention also provides a method for inducing cochlear supporting cells to differentiate into hair cells, comprising the step of expressing transcription factor genes and / or signaling pathway genes associated with hair cell regeneration in cochlear supporting cells, wherein the genes are operably linked to the cochlear supporting cell-specific promoter described in any embodiment herein. The method is an in vitro method.

[0036] In one or more embodiments, the method includes the step of introducing a nucleic acid construct or vector comprising the coding sequence of the transcription factor and / or signaling pathway gene into cochlear supporting cells, wherein the coding sequence is operably linked to the cochlear supporting cell-specific promoter.

[0037] In one or more embodiments, the transcription factor genes include ATOH1, POU4F3, GFI1, IKZF2, INSM1, SIX1.

[0038] In one or more embodiments, the signaling pathway genes are genes in the Wnt, Notch, Hippo, or TGF-beta signaling pathways associated with hair cell regeneration.

[0039] Beneficial effects of the present invention:

[0040] AAV-ie-CAG-nls-EGFP and AAV-ie-CAG-nls-EGFP-mGJB2 were injected into the cochlea of ​​newborn mice, and ABR audiometry was performed on day 30. The experimental group, in which CAG-driven GJB2 expression was expressed in the SC and hair cells (HCs), showed hearing loss. This suggests that broad-spectrum promoters such as CAG-driven gene expression are not suitable for gene therapy of deafness caused by GJB2 mutations.

[0041] Immunofluorescence staining of the cochlea of ​​newborn mice after injection of the virus AAV-Sox47-mNeonGreen (Sox47 promoter sequence: SEQ ID NO: 1) revealed that the green fluorescent protein mNeonGreen is specifically expressed in the SCs. Furthermore, the SC-specific promoter of the present invention has therapeutic effects on hereditary deafness. Furthermore, the introduction of exogenous genes is safe, preventing overexpression in hair cells and potential damage to these cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 shows ABR audiometry after injection of control AAV-ie-CAG-nls-EGFP and AAV-ie-CAG-nls-EGFP-mouse GJB2 into the cochlea of ​​newborn mice. In the experimental group, CAG-driven GJB2 expression in the SC and HC regions resulted in hearing loss. This suggests that broad-spectrum promoters such as CAG-driven gene expression are not suitable for gene therapy of deafness caused by GJB2 mutations.

[0043] Figure 2 shows immunofluorescence staining of the cochlea taken from newborn mice on the 14th day after the virus AAV-ie-CAG-mNeonGreen was injected into the cochlea. The green fluorescent protein mNeonGreen was expressed in both HC and SC, and there was also fluorescence expression in other areas, indicating that the CAG promoter is widely expressed in various cells in the cochlea and has no specificity.

[0044] Figure 3 shows immunofluorescence staining results for the Sox47 promoter. Neonatal mice were injected with AAV-ie-Sox47-mNeonGreen (Sox47 sequence: SEQ ID NO: 1) and then harvested for immunofluorescence staining on day 14. The results showed that the Sox47 promoter drives the expression of the mNeonGreen reporter gene specifically in cochlear supporting cells.

[0045] Figure 4 shows immunofluorescence staining of the SHH gene promoter. Immunofluorescence staining of the cochlea of ​​newborn mice, obtained 14 days after injection of the AAV-ie-SHH-mNeonGreen (SHH sequence: SEQ ID NO: 3), revealed that the mNeonGreen reporter gene was not expressed in either cochlear hair cells or cochlear supporting cells. This suggests that SHH alone cannot drive transgene expression specifically in cochlear supporting cells.

[0046] Figure 5 shows the ABR results of hearing tests performed after EGFP or GJB2 driven by the AAV-Sox47 promoter was injected into the mouse cochlea. The results show that, unlike the hearing impairment caused by GJB2 driven by the CAG promoter in Figure 1, GJB2 driven by the Sox47 promoter did not affect the mouse hearing.

[0047] Figure 6 shows the results of restoring GJB2 expression in the SC region of GJB2 knockout mice driven by the Sox47 promoter. Compared with KO mice, GJB2 expression was restored after Sox47-GJB2 injection.

[0048] Figure 7 shows the ABR results of hearing in mice after Sox47 promoter-driven re-expression of GJB2. Compared with KO mice, the hearing of KO mice recovered to a certain extent after Sox47-GJB2 injection. DETAILED DESCRIPTION

[0049] Unless otherwise defined, the practice of the present invention will employ conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. These techniques are fully explained in the literature, such as Molecular Cloning: A Laboratory Manual, Second Edition (Sambrook et al., 1989); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Animal Cell Culture (RI Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987 edition and periodic updates thereof); PCR: The Polymerase Chain Reaction (Mullis et al., eds., 1994); A Practical Guide to Molecular Cloning (Perbal Bernard V., 1988); Phage Display: A Laboratory Manual (Barbas et al., 2001).

[0050] Specific promoter

[0051] The inventors have discovered an enhancer that can specifically enhance the expression-initiating function of a core promoter element in cochlear supporting cells, which has a sequence shown in SEQ ID NO: 2 or a homologous sequence with at least 90% identity thereto. This enhancer can be used to construct a chimeric promoter. Based on this, compared with the prior art, the present invention provides a cochlear supporting cell-specific promoter and a treatment method that specifically targets cochlear SCs: by using a promoter that can specifically target cochlear SCs, a viral vector containing the promoter targets a specific gene to SCs, promoting the expression of transgenes in SCs, thereby: 1. performing gene therapy for hearing loss caused by deafness gene mutations; 2. achieving the regeneration of SCs into hair cells by introducing exogenous genes to treat hereditary deafness, and being safe and avoiding overexpression in hair cells that damages hair cells.

[0052] A promoter is a DNA sequence that RNA polymerase recognizes, binds to, and initiates transcription. It is located upstream of the start codon of a structural gene (usually within approximately 100 to 1000 bp) that controls transcription of the structural gene. It contains conserved sequences required for specific RNA polymerase binding and transcription initiation, but the promoter itself is not transcribed. Its general structure comprises a core promoter element and upstream regulatory elements. The core promoter element, in turn, includes the transcription start site and the TATA box, primarily serving as sites for RNA polymerase binding and transcription initiation. Upstream regulatory elements can alter the efficiency of transcription by binding to corresponding trans-acting factors.

[0053] In some embodiments, the cochlear supporting cell-specific promoter of the present invention comprises: (1) a sequence as set forth in SEQ ID NO: 2 or a homologous sequence having at least 90% sequence identity thereto and retaining the function of SEQ ID NO: 2, and (2) a core promoter element as set forth in SEQ ID NO: 3. The present invention also provides a method for preparing a chimeric promoter, comprising: operably linking (1) and (2). In one or more embodiments, (1) is located 5' to (2).

[0054] Herein, the core promoter element contained in the specific promoter is any promoter suitable for promoting gene expression in mammalian cells. In one or more embodiments, (2) the core promoter element is a promoter that is not expressed in cochlear hair cells. The sequences of these promoters can be easily obtained by those skilled in the art. In an exemplary embodiment, the core promoter element is a promoter of a mammalian, preferably human, SHH gene. The promoter of the human SHH gene is shown in SEQ ID NO: 3.

[0055] In other embodiments, the cochlear supporting cell-specific promoter of the present invention is SOX47 (SEQ ID NO: 1) or a sequence having at least 90% sequence identity thereto.

[0056] The present invention also relates to homologous sequences having at least 50%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the above-mentioned nucleic acid sequences (e.g., sequences of the components of a specific promoter). As used herein, "homologous sequence" refers to a sequence from a species of the same order, family, genus, or species as the species of the source sequence and having the sequence identity.

[0057] The present invention also relates to polynucleotides that hybridize to the sequence of the specific promoter described above and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that hybridize to the polynucleotide of the present invention under stringent conditions. In the present invention, "stringent conditions" refer to: (1) hybridization and elution at relatively low ionic strength and relatively high temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) the addition of a denaturing agent during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C; or (3) hybridization occurs only when the identity between the two sequences is at least 90%, more preferably at least 95%.

[0058] Nucleic acid constructs

[0059] The present invention also provides nucleic acid constructs comprising the above-mentioned specific promoters for cloning or expressing genetic functions such as genes. The nucleic acid constructs can be expression cassettes or vectors. Therefore, in addition to the specific promoter, the nucleic acid constructs can also include other elements required for cloning or expression, such as multiple cloning sites and / or terminators (e.g., polyA signal sequences). For example, the nucleic acid constructs can be RNA constructs or DNA vectors that can be transfected into cells and expressed. Herein, "nucleic acid molecule," "polynucleotide," and "nucleic acid" are used interchangeably.

[0060] To express a polypeptide or protein, the nucleic acid construct further comprises a gene of interest, operably linked to the specific promoter. Herein, a gene can be a coding sequence that encodes a polypeptide or protein. In some embodiments of the present invention, the gene is selected from the group consisting of: deafness genes, transcription factor genes, signaling pathway genes, and various tool genes that manipulate genes in cochlear supporting cells or are expressed in cochlear supporting cells.

[0061] Herein, "deafness genes" are deafness genes expressed by cochlear supporting cells, including but not limited to GJB2, GJB3, GJB6, MYH14, WFS1, EYA4, TMPRSS3, DCDC2, GPSM2, and KARS. Herein, "transcription factor genes" are transcription factor genes associated with hair cell regeneration, including but not limited to ATOH1, POU4F3, GFI1, IKZF2, INSM1, and SIX1. Herein, "signaling pathway genes" are genes in the Wnt, Notch, Hippo, and TGF-beta signaling pathways associated with hair cell regeneration.

[0062] When a gene or its sequence is known, the coding nucleic acid of each gene can be prepared using conventional methods in the art, and corresponding vectors can be constructed. Recombinant vectors can be constructed using methods well known to those skilled in the art, such as Sambrook (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory), Ausubel (1989, Short Protocols in Molecular Biology, Wiley) or other standard textbooks. Alternatively, the nucleic acid construct can be reconstructed into a liposome for delivery to a target cell. Containing the nucleic acid construct of the present invention can be transferred to a host cell by a well-known method, which varies according to the type of cell host. For example, calcium chloride transfection is commonly used for prokaryotic cells, while calcium phosphate treatment or electroporation can be used for other cell hosts, as shown in Sambrook (see above).

[0063] The vectors described herein contain nucleic acid molecules or expression cassettes as described herein. The vector can be a plasmid, a cosmid, a virus, or a viral vector. The vector includes a cloning vector, an integration vector, or an expression vector. The expression vector typically contains sequences for plasmid maintenance and for cloning and expressing exogenous nucleotide sequences. The sequences (collectively referred to as "flanking sequences" in certain embodiments) typically include one or more of the following nucleotide sequences: a promoter (e.g., a specific promoter as described herein), one or more enhancer sequences, an origin of replication, a transcription termination sequence, a complete intron sequence containing donor and acceptor splice sites, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a multiple cloning site for inserting a nucleic acid encoding a protein to be expressed, and a selectable marker element. Typically, the promoter is located at the 5' end of the multiple cloning site or the polypeptide coding sequence. In one or more embodiments, the expression vector uses an AAV series vector as a backbone.

[0064] The vector may optionally contain a "tag" coding sequence, i.e., an oligonucleotide molecule located at the 5' or 3' end of the polypeptide coding sequence; the oligonucleotide sequence encodes polyhistidine (such as 6His) or another "tag" such as FLAG, HA or myc. This tag is typically fused to the polypeptide when the polypeptide is expressed and can serve as a means for affinity purification or detection of the protein from the host cell. Affinity purification can be accomplished, for example, by column chromatography using antibodies against the tag as an affinity matrix. The tag can optionally be subsequently removed from the purified protein by various means, such as using certain peptidases for cleavage.

[0065] The flanking sequences may be homologous (i.e., from the same species and / or strain as the host cell), heterologous (i.e., from a species other than the host cell species or strain), hybrid (i.e., a combination of flanking sequences from more than one source), synthetic, or natural. Likewise, the source of the flanking sequences may be any prokaryotic or eukaryotic organism, any vertebrate or invertebrate organism, or any plant, provided that the flanking sequences function in and can be activated by the host cell machinery.

[0066] Selectable marker genes encode a protein that is essential for the survival and growth of host cells grown in selective culture media. Typical selectable marker genes encode (a) resistance to antibiotics or other toxins (e.g., ampicillin, tetracycline, or kanamycin for prokaryotic host cells); (b) supplementing the auxotrophic deficiency of the cell; or (c) providing a protein that is not available from a complex or defined culture medium. Specific selectable markers are kanamycin resistance genes, ampicillin resistance genes, and tetracycline resistance genes. Advantageously, the neomycin resistance gene can also be used for selection in prokaryotic and eukaryotic host cells.

[0067] AAV vectors and AAV particles

[0068] As used herein, the term "adeno-associated virus" or "AAV" includes members of the class of viruses associated with that name, which belongs to the genus Dependoparvovirus, family Parvoviridae. Adeno-associated virus is a single-stranded DNA virus that grows exclusively in cells, where certain functions are provided by a co-infecting helper virus. General information and a review of AAV can be found, for example, in Carter, 1989, Handbook of Parvoviruses, Volume 1, pages 169-228. Various serotypes of this virus are known to be suitable for gene delivery, and the various serotypes are closely related structurally and functionally. All AAV serotypes apparently exhibit very similar replication characteristics mediated by homologous rep genes; and all carry three related capsid proteins. At least 13 sequentially numbered AAV serotypes are known in the art. Non-limiting exemplary serotypes that can be used in the methods disclosed herein include any of the 13 serotypes, such as AAV2, AAV8, and AAV9. AAV particles comprise, consist essentially of, or consist of the following three major viral proteins: VP1, VP2, and VP3. In one or more embodiments, AAV refers to serotype AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVPHP.B, or AAVrh74.

[0069] In some embodiments, the nucleic acid construct of the present invention is an AAV vector that expresses the gene of interest via a recombinant AAV (rAAV) vector system. As used herein, "AAV vector" refers to a vector comprising, consisting essentially of, or consisting of a specific promoter and one or more AAV inverted terminal repeats (ITRs) as described herein. When present in a host cell that provides functionality of the rep and cap gene products, such an AAV vector can be replicated and packaged into infectious viral particles; for example, by transfecting the host cell. In one or more embodiments, the AAV vector contains a specific promoter as described herein, at least one nucleic acid encoding at least one protein or RNA, and / or an enhancer and / or terminator packaged into the flanking ITRs of infectious AAV particles. The plasmid containing the AAV vector may also contain elements for preparation purposes, such as antibiotic resistance genes and the like.

[0070] The present invention also provides an rAAV vector system comprising the AAV vector and other nucleic acid constructs encoding genes required by the AAV virus, wherein the AAV vector comprises the cochlear supporting cell-specific promoter described in any embodiment herein. In one or more embodiments, the AAV vector further comprises a gene of interest as described herein, which is operably linked to the cochlear supporting cell-specific promoter. In one or more embodiments, the AAV vector system comprises an Anc80L65 plasmid, a helper plasmid, and the AAV vector.

[0071] As used herein, the term "viral capsid" or "capsid" refers to the protein shell or capsid of the virus particle. The function of the capsid is to encapsidate, protect, transport the viral genome, and release it into the host cell. The capsid is generally composed of oligomeric structural subunits of proteins ("capsid proteins"). As used herein, the term "encapsid" refers to being contained within the viral capsid. The viral capsid of AAV is composed of a mixture of three viral capsid proteins: VP1, VP2, and VP3.

[0072] "AAV virus particle" or "AAV particle" refers to a virus particle composed of at least one AAV capsid protein and an encapsidated polynucleotide AAV vector. Therefore, the production of AAV particles necessarily includes the production of AAV vectors, because such vectors are contained in AAV particles. The present invention provides a rAAV virus particle comprising a specific promoter as described herein, and optionally further comprising a gene of interest operably linked to the specific promoter. In one or more embodiments, the rAAV virus particle is selected from one or more of rAAV1, rAAV2, rAAV4, rAAV5, rAAV6, rAAV7, rAAV8, rAAV9, rAAV10, rAAV11, rAAV12, rAAV13, rAAVPHP.B or rAAVrh74.

[0073] As used herein, the term "helper" with respect to a virus or plasmid refers to a virus or plasmid that provides additional components necessary for replication and packaging of any of the AAV vectors disclosed herein. The components encoded by the helper virus can include any genes required for virion assembly, encapsidation, genome replication and / or packaging. For example, a helper virus or plasmid can encode enzymes necessary for viral genome replication. Non-limiting examples of helper viruses and plasmids suitable for use with AAV constructs include pHELPer (plasmid), adenovirus (virus), or herpes virus (virus).

[0074] Methods for producing and using rAAV vectors are known in the art. Methods for producing rAAV particles and heterologous nucleic acids are also known in the art and are commercially available (see, for example, US 2007 / 0015238 and US2012 / 0322861, which are incorporated herein by reference in their entirety). For example, a plasmid comprising a heterologous nucleic acid sequence can be combined with one or more helper plasmids (e.g., comprising rep genes (e.g., encoding Rep78, Rep68, Rep52, and Rep40) and cap genes (encoding VP1, VP2, and / or VP3) and transfected or permanently integrated into a production cell line so that the rAAV particles can be packaged and subsequently purified.

[0075] In some embodiments, the one or more helper plasmids include a first helper plasmid comprising a rep gene and a cap gene and a second helper plasmid comprising an E1a gene, an E1b gene, an E4 gene, an E2a gene, and a VA gene. In some embodiments, the rep gene is a rep gene derived from AAV2, and the cap gene is derived from AAV44.9. Helper plasmids and methods for preparing such plasmids are known in the art and are commercially available.

[0076] cell

[0077] The present invention also includes host cells that contain or have integrated into their genome the specific promoters or nucleic acid constructs described herein. Host cells can be used as recipients for vectors. Host cells can be "transfected" or "transformed," which refers to the process by which exogenous nucleic acids are transfected or transduced into host cells. Transformed cells include the primary subject cell and its progeny. As used herein, the terms "engineered" and "recombinant" cells or host cells often refer to cells into which exogenous nucleic acid sequences, such as vectors, have been introduced. Thus, recombinant cells can be distinguished from naturally occurring cells that do not contain the introduced recombinant nucleic acids.

[0078] Suitable host cells are as described above, including but not limited to Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (eg, HepG2), cochlear supporting cells, etc.

[0079] As used herein, packaging cells (or helper cells) are cells for producing viral vectors. Production of recombinant AAV viral vectors requires trans-provided Rep and Cap proteins and the gene sequence of the helper AAV replication from adenovirus. In some aspects, packaging / helper cells contain a plasmid stably integrated into the cell genome. In other aspects, packaging cells can be transiently transfected. Typically, packaging cells are eukaryotic cells, such as mammalian cells or insect cells. In one or more embodiments, the cell for producing viral vectors is a HEK293 cell, including HEK293T, HEK293H, HEK293F, HEK293S, HEK293T / 17, HEK293T / 17SF, HEK293FT, HEK293SG, HEK293E, HEK293-6E, HEK293FTM, HEK293SGGD cells.

[0080] The nucleic acid construct / recombinant expression vector of the present invention can be transferred into the cell of interest. The method of transfer is conventional in the art, including but not limited to: viral transduction, microinjection, particle bombardment, gene gun transformation and electroporation. In certain embodiments, electroporation is used to transfer the nucleic acid construct or recombinant expression vector. When the host cells are cultured under appropriate conditions, they synthesize the fusion protein, which can then be collected from the culture medium (if the host cells secrete it into the culture medium) or directly from the host cells that produce it (if it is not secreted).

[0081] Methods for preparing AAV vectors and viruses

[0082] A variety of methods can be used to prepare AAV viral vectors. In one or more embodiments, packaging is achieved by using a helper virus or helper plasmid and a cell line. The helper virus or helper plasmid contains elements and sequences that promote the production of the viral vector. In some embodiments, the helper plasmid is stably integrated into the genome of the packaging cell line, so that the packaging cell line does not require additional transfection with the helper plasmid.

[0083] In one or more embodiments, the cell is a packaging cell line or a helper cell line. In one or more embodiments, the helper cell line is a eukaryotic cell; for example, HEK 293 cells or 293T cells. In one or more embodiments, the helper cell is a yeast cell or an insect cell.

[0084] The helper plasmid can comprise, for example, at least one viral helper DNA sequence derived from a replication-incompetent viral genome encoding trans-acting full virion proteins required for packaging replication-incompetent AAV, and virion proteins for producing virions capable of packaging replication-incompetent AAV at high titers without producing replication-incompetent AAV.

[0085] Helper plasmids for packaging AAV are known in the art, see, for example, U.S. Patent Publication No. 2004 / 0235174A1, which is incorporated herein by reference. As described therein, as a non-limiting example, an AAV helper plasmid may contain the Ad5 genes E2A, E4, and VA as helper viral DNA sequences, controlled by their respective native promoters or heterologous promoters. The AAV helper plasmid may additionally contain an expression cassette for expressing a marker protein, such as a fluorescent protein, to allow easy detection of transfection of desired target cells.

[0086] Provided herein are methods for producing AAV particles, comprising transfecting a packaging cell line with any of the AAV helper plasmids disclosed herein; and any of the AAV vectors disclosed herein. In some embodiments, the AAV helper plasmid and the AAV vector are co-transfected into the packaging cell line. In some embodiments, the cell line is a mammalian cell line, such as a human embryonic kidney (HEK) 293 cell line. Provided herein are cells comprising any of the AAV vectors and / or AAV particles disclosed herein.

[0087] Pharmaceutical composition

[0088] The present invention also provides a pharmaceutical composition comprising a pharmaceutically acceptable excipient and one or more selected from the following: a specific promoter, a nucleic acid construct, a rAAV vector system, a rAAV virus particle, and a host cell as described herein. In one or more embodiments, the pharmaceutical composition comprises an AAV vector and a pharmaceutically acceptable excipient, wherein the AAV vector comprises a specific promoter as described herein operably linked to a gene of interest. In one or more embodiments, the pharmaceutical composition comprises rAAV virus particles and a pharmaceutically acceptable excipient, wherein the rAAV virus particles comprise a polynucleotide comprising a specific promoter as described herein operably linked to a gene of interest.

[0089] As used herein, the term "pharmaceutically acceptable excipient" encompasses any standard pharmaceutical carrier, such as phosphate-buffered saline solution, water, and emulsions, such as oil / water or water / oil emulsions, and various types of wetting agents. The composition may also include stabilizers and preservatives. For examples of carriers, stabilizers, and adjuvants, see Martin (1975) Remington's Pharm. Sci. 15th Edition (Mack Publ. Co., Easton).

[0090] As described herein, pharmaceutical compositions can be formulated by any method known or developed in the art of pharmacology, including but not limited to contacting the active ingredient (e.g., viral particles or recombinant vectors) with excipients or other auxiliary ingredients, dividing or packaging the product into dosage units. The viral particles herein can be formulated to have desired characteristics, such as increased stability, increased cell transfection, sustained or delayed release, biodistribution or tropism, modulation or enhanced translation of the encoded protein in vivo, and release profile of the encoded protein in vivo.

[0091] Thus, the pharmaceutical composition can further comprise saline, lipidoids, liposomes, lipid nanoparticles, polymers, lipid complexes, core-shell nanoparticles, peptides, proteins, cells transfected with viral vectors (e.g., for transplantation into a subject), nanoparticle mimics, or combinations thereof. In some embodiments, the pharmaceutical composition is formulated as nanoparticles. In some embodiments, the nanoparticles are self-assembled nucleic acid nanoparticles.

[0092] According to the pharmaceutical composition herein, it can be prepared, packaged and / or sold in bulk with a single unit dose and / or with multiple single unit doses. The amount of the active ingredient is generally equal to the dosage of the active ingredient to be administered to the subject and / or the appropriate ratio of such dosage, for example, half or one-third of such dosage. The preparation of the present invention may include one or more excipients, and the amount of each excipient together increases the stability of the viral vector, increases cell transfection or viral vector transduction, increases the expression of the protein encoded by the viral vector, and / or changes the release profile of the protein encoded by the viral vector. In some embodiments, the pharmaceutical composition includes an excipient. Non-limiting examples of an excipient include a solvent, a dispersion medium, a diluent or other liquid vehicle, a dispersion or suspension aid, a surfactant, an isotonic agent, a thickener or an emulsifier, a preservative or a combination thereof.

[0093] In some embodiments, the pharmaceutical composition comprises a cryoprotectant. The term "cryoprotectant" refers to an agent that can reduce or eliminate damage to a substance during freezing. Non-limiting examples of cryoprotectants include sucrose, trehalose, lactose, glycerol, dextrose, raffinose, and / or mannitol.

[0094] Uses and methods

[0095] The present invention provides the use of the sequence represented by SEQ ID NO: 2, or a homologous sequence having at least 90% sequence identity thereto and retaining the function of SEQ ID NO: 2, in a chimeric promoter. Furthermore, the present invention provides the use of the specific promoter in the preparation of a medicament for treating a disease caused by a gene defect in cochlear supporting cells, wherein the medicament is used to express the gene in cochlear supporting cells.

[0096] The present invention provides a method for introducing a target gene into a subject's cells, comprising contacting the cells with an effective amount of any of the AAV viral particles described herein, wherein the particle comprises a polynucleotide comprising a specific promoter described herein operably linked to the gene of interest.

[0097] The present invention provides a method for expressing a gene of interest in cochlear supporting cells, comprising the step of introducing a nucleic acid construct comprising the gene into the cochlear supporting cells, wherein the gene is operably linked to a specific promoter described herein. Typically, the promoter is in frame with the gene and located upstream of the gene. Examples of genes of interest are described elsewhere herein.

[0098] The specific promoter described herein is operably linked to a therapeutic gene, providing a theoretical basis for clinical targeted treatment of hereditary hearing loss. The present invention provides a method for treating a disease caused by a gene defect in cochlear supporting cells, comprising the step of expressing the gene in cochlear supporting cells, wherein the gene is operably linked to the cochlear supporting cell-specific promoter described herein. The method comprises the step of introducing a nucleic acid construct or vector comprising the coding sequence of the gene into the cochlear supporting cells, wherein the coding sequence of the gene is operably linked to the cochlear supporting cell-specific promoter. Exemplarily, the gene is a deafness gene expressed by cochlear supporting cells, including GJB2, GJB3, GJB6, MYH14, WFS1, EYA4, TMPRSS3, DCDC2, GPSM2, and KARS. In some embodiments, the gene is GJB2. In one or more embodiments, the disease is hereditary deafness caused by a functional loss of GJB2.

[0099] The present invention also provides a method for inducing cochlear supporting cells to differentiate into hair cells, comprising the step of expressing transcription factors and / or signaling pathway genes associated with hair cell regeneration in cochlear supporting cells, wherein the genes are operably linked to the specific promoter described herein. Specifically, the method comprises the step of introducing a nucleic acid construct or vector comprising a coding sequence for the transcription factor and / or signaling pathway gene into the cochlear supporting cells, wherein the coding sequence is operably linked to the cochlear supporting cell-specific promoter. Exemplarily, the transcription factor genes include ATOH1, POU4F3, GFI1, IKZF2, INSM1, and SIX1, and the signaling pathway genes are genes in the Wnt, Notch, Hippo, and TGF-beta signaling pathways associated with hair cell regeneration.

[0100] The term "disease and / or condition" refers to the physical state of a subject associated with a disease and / or condition described herein. As used herein, "treatment" includes any beneficial or desired effect on the symptoms or changes of a disease or pathological condition, and may include even a small reduction in one or more measurable markers of the disease or condition being treated (e.g., a hereditary deafness disease). Treatment may optionally include a reduction or alleviation of the symptoms of the disease or condition, or a delay in the progression of the disease or condition. "Treatment" does not necessarily mean complete eradication or cure of the disease or condition or its associated symptoms. As used herein, "treatment" of a disease in a subject means (1) preventing the occurrence of symptoms or disease in a subject who is susceptible to or does not yet exhibit symptoms of the disease; (2) inhibiting the disease or arresting its development; or (3) ameliorating or causing regression of the disease or disease symptoms. As understood in the art, "treatment" is an approach used to obtain beneficial or desired results, including clinical results. Beneficial or desired results may include one or more of, but are not limited to, alleviation or amelioration of one or more symptoms, whether detectable or undetectable, lessening of the extent of a condition (including a disease), a stable (i.e., not worsening) state of a condition (including a disease), a delay or slowing of a condition (including a disease), progression, improvement or palliation of a condition (including a disease), state, and remission (whether partial or total).

[0101] The term "subject" or "patient" may refer to a cell or animal, such as a mammal or human, that receives a pharmaceutical composition of the present invention to treat, prevent, ameliorate, and / or alleviate a disease or condition of the present invention. The subject is not limited to a particular species and includes non-human animals receiving diagnosis or treatment and those receiving infection or animal models, including but not limited to dog, monkey, cow, horse, ape, mouse, rat, canine, or lagomorphic species, as well as other livestock, sports animals, or pets. In some embodiments, the subject is human.

[0102] As used herein, the term "effective amount" is intended to mean an amount sufficient to achieve the desired effect. In the case of therapeutic or preventive applications, the effective amount will depend on the type and severity of the disorder in question and the characteristics of the individual subject, such as general health, age, sex, weight, and tolerance to the pharmaceutical composition. In the context of gene therapy, in some embodiments, the effective amount is an amount sufficient to restore some or all of the function of a defective gene in a subject. In other embodiments, the effective amount of AAV viral particles is an amount sufficient to cause gene expression in a subject. In some embodiments, the effective amount is the amount required to increase galactose metabolism in a subject in need. A skilled technician will be able to determine the appropriate amount based on these and other factors.

[0103] In some embodiments, the effective amount will depend on the size and nature of the application in question. It also depends on the nature and sensitivity of the target subject and the method of use. Those skilled in the art will be able to determine the effective amount based on these and other considerations. According to embodiments, the effective amount can include, consist essentially of, or consist of one or more administrations of the composition.

[0104] As used herein, the term "administering" is intended to mean delivering a substance to a subject, such as an animal or a human. Administration can be performed in one dose, continuously, or intermittently throughout the course of treatment. Methods for determining the most effective mode of administration and dosage are known to those skilled in the art and will vary with the composition used for treatment, the therapeutic purpose, and the age, health, or sex of the subject being treated. Single or multiple administrations can be performed, with the dosage level and pattern being selected by the treating physician, or in the case of pets and other animals, by the treating veterinarian.

[0105] Dosage and administration

[0106] Methods for determining the most effective mode of administration and dosage are known to those skilled in the art and will vary with the composition used for treatment, the purpose of the treatment, and the subject being treated. Single or multiple administrations may be performed, with the dosage level and pattern being selected by the treating physician. Dosage may be affected by the route of administration. Suitable dosage formulations and methods of administering the agents are known in the art. Non-limiting examples of such suitable dosages may range from as little as 10E9 vector genomes to as much as 10E17 vector genomes per administration.

[0107] In embodiments of the methods described herein, the number of viral particles (e.g., AAV) administered to a subject ranges from about 10E9 to about 10E17. In certain embodiments, about 10E10 to about 10E12, about 10E11 to about 10E13, about 10E11 to about 10E12, about 10E11 to about 10E14, about 5×10E11 to about 5×10E12, or about 10E12 to about 10E13 viral particles are administered to a subject.

[0108] In some embodiments, the AAV particles repair a gene defect in a subject. In some embodiments, the ratio of repaired target polynucleotides (e.g., GJB2) or polypeptides to unrepaired target polynucleotides or polypeptides in successfully treated cells, tissues, organs, or subjects is at least about 1.5:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 20:1, about 50:1, about 100:1, about 1000:1, about 10,000:1, about 100,000:1, or about 1,000,000:1. The amount or ratio of repaired target polynucleotides or polypeptides can be determined by any method known in the art, including but not limited to Western blotting, Northern blotting, Southern blotting, PCR, sequencing, mass spectrometry, flow cytometry, immunohistochemistry, immunofluorescence, fluorescence in situ hybridization, sequencing, immunoblotting, and ELISA.

[0109] In some embodiments, the viral particles are introduced into the subject intravenously, intrathecally, intracerebrally, intraventricularly, intranasally, intratracheally, intraaurally, intraocularly or periocularly, orally, rectally, transmucosally, by inhalation, transdermally, parenterally, subcutaneously, intradermally, intramuscularly, intrapleurally, topically, intralymphatically, intracisternally; such introduction can also be intraarterially, intracardially, subventricularly, epidurally, intracerebrally, intraventricularly, subretinally, intravitreally, intraarticularly, intraperitoneally, intrauterinely, or any combination thereof. In some embodiments, the delivery of the viral particles is systemic.

[0110] Administration of the AAV vectors, AAV particles, or compositions of the present disclosure can be achieved in one dose, continuously, or intermittently throughout the course of treatment. In some embodiments, the AAV vectors, AAV particles, or compositions of the present disclosure are administered parenterally by injection, infusion, or implantation. The AAV particles and compositions of the present disclosure can be administered in combination with other known treatments for the condition being treated.

[0111] Reagent test kit

[0112] In some embodiments, the specific promoters, nucleic acid constructs, cells, viral particles, or pharmaceutical compositions described herein can be assembled into pharmaceutical or diagnostic or research kits to facilitate their use in therapeutic, diagnostic, or research applications. In some embodiments, the kits of the present disclosure include any of the AAV vectors, AAV particles, cells, or pharmaceutical compositions described herein.

[0113] In some embodiments, the test kit also includes instructions for use. Specifically, such a test kit can include one or more reagents as described herein, and instructions for the intended application and correct use of these reagents. In some embodiments, the test kit can include instructions for mixing one or more components of the test kit and / or separating and mixing samples and applying to a subject. In some embodiments, the reagent in the test kit is a pharmaceutical formulation and dosage suitable for a specific application and the method of administration of the reagent. The test kit can contain the components of the appropriate concentration or amount for carrying out various experiments for research purposes.

[0114] Kit can be designed to facilitate the use of the methods described herein, and can take many forms. Where applicable, every kind of composition of kit can be provided in liquid form (e.g., in solution) or in solid form (e.g., dry powder). In some cases, some compositions can be constructible or processable in addition (e.g., processed into active form), for example, by adding suitable solvent or other substances (e.g., water or cell culture medium), which may or may not be provided together with kit. In some embodiments, composition can be provided in a preservation solution (e.g., a cryopreservation solution). The non-limiting examples of a preservation solution include DMSO, paraformaldehyde. In some embodiments, the preservation solution contains a certain amount of metalloproteinase inhibitors.

[0115] In some embodiments, the test kit contains any one or more components as described herein in one or more containers. Therefore, in some embodiments, the test kit can include a container for holding a reagent as described herein. The reagent can be in the form of a liquid, gel, or solid (powder). The reagent can be aseptically prepared, packaged in a syringe, and transported frozen. Alternatively, they can be contained in a bottle or other container for storage. A second container can have other reagents of aseptic preparation. Alternatively, the test kit can include a premixed active agent and transported in a syringe, bottle, tube, or other container. The test kit can have one or more or all components required for administering the reagent to the subject, such as a syringe, a local application device, or an IV needle and bag.

[0116] Some specific implementation plan projects:

[0117] 1. A cochlear supporting cell-specific enhancer comprising the sequence shown in SEQ ID NO: 2 or a homologous sequence having at least 90% sequence identity thereto and retaining the function of SEQ ID NO: 2,

[0118] Preferably, the homologous sequence is a sequence of a mammal, preferably of the order Primates, more preferably of the family Hominidae.

[0119] 2. A cochlear supporting cell-specific promoter comprising:

[0120] (1) the sequence shown in SEQ ID NO: 2 or a sequence having at least 90% sequence identity thereto and retaining the function of SEQ ID NO: 2, and

[0121] (2) core promoter elements,

[0122] Preferably, (2) the core promoter element is the promoter of the SHH gene,

[0123] Preferably, (2) the sequence of the core promoter element is as shown in SEQ ID NO: 3 or a homologous sequence thereof having at least 90% sequence identity,

[0124] More preferably, the homologous sequence is a SHH gene promoter sequence of a mammal, preferably a primate, more preferably a hominid.

[0125] 3. A nucleic acid construct comprising the cochlear supporting cell-specific promoter described in item 2,

[0126] Preferably, the nucleic acid construct further comprises a gene of interest, wherein the gene is operably linked to the cochlear supporting cell-specific promoter; and / or, the nucleic acid construct is a vector,

[0127] More preferably, the gene is selected from: a deafness gene, a transcription factor gene, a signal pathway gene, a tool gene for manipulating genes in cochlear supporting cells or expressed in cochlear supporting cells.

[0128] 4. A rAAV vector system comprising an AAV vector and a nucleic acid construct encoding a gene required by the AAV virus, wherein the AAV vector comprises the cochlear supporting cell-specific promoter described in item 2,

[0129] Preferably, the AAV vector further comprises a gene of interest, which is operably linked to the cochlear supporting cell-specific promoter.

[0130] 5. A rAAV virus particle comprising the cochlear supporting cell-specific promoter described in item 2.

[0131] 6. A host cell comprising or having integrated into its genome: the cochlear supporting cell-specific promoter according to item 2, the nucleic acid construct according to item 3, or the AAV vector system according to item 4.

[0132] 7. A pharmaceutical composition comprising a pharmaceutically acceptable excipient and one or more selected from the following: the cochlear supporting cell-specific promoter described in Item 2, the nucleic acid construct described in Item 3 or the AAV vector system described in Item 4, the rAAV virus particle described in Item 5, and the host cell described in Item 6.

[0133] 8. A method for expressing a gene in cochlear supporting cells, comprising the step of introducing a nucleic acid construct comprising the gene into the cochlear supporting cells, wherein the gene is operably linked to the cochlear supporting cell-specific promoter according to item 2.

[0134] 9. Use of the sequence shown in SEQ ID NO: 2 or a homologous sequence having at least 90% sequence identity thereto and retaining the function of SEQ ID NO: 2 in a chimeric promoter.

[0135] 10. A method for preparing a chimeric promoter comprising: operably linking (1) a sequence as set forth in SEQ ID NO: 2 or a sequence having at least 90% sequence identity thereto and retaining the function of SEQ ID NO: 2 to (2) a core promoter element.

[0136] 11. Use of the cochlear supporting cell-specific promoter described in item 2 in the preparation of a drug for treating a disease caused by a gene defect in cochlear supporting cells, wherein the drug is used to express the gene in the cochlear supporting cells.

[0137] Preferably, the disease is hereditary deafness caused by a gene defect in the cochlear supporting cells,

[0138] More preferably, the disease is hereditary deafness caused by loss of function of GJB2.

[0139] 12. A method for inducing cochlear supporting cells to differentiate into hair cells, comprising the step of expressing transcription factors and / or signaling pathway genes associated with hair cell regeneration in cochlear supporting cells, wherein the genes are operably linked to the cochlear supporting cell-specific promoter of item 2.

[0140] Preferably, the method comprises the step of introducing a nucleic acid construct or vector comprising the coding sequence of the transcription factor and / or signaling pathway gene into cochlear supporting cells, wherein the coding sequence is operably linked to the cochlear supporting cell-specific promoter.

[0141] The present invention will be described below by way of specific examples. It should be understood that these examples are merely illustrative and are not intended to limit the scope of the present invention. The methods and materials used in the examples are, unless otherwise stated, conventional materials and methods in the art.

[0142] Example

[0143] Example 1: Effects of CAG promoter-driven GJB2 expression in SCs and HCs on hearing

[0144] 1.1 AAV-CAG-nls-EGFP (SEQ ID: NO. 6) and AAV-CAG-nls-EGFP-mouse GJB2 (SEQ ID: NO. 7) plasmid construction and viral packaging

[0145] AAV-CAG-nls-EGFP and AAV-CAG-nls-EGFP-mouse GJB2 plasmids (mouse GJB2 gene NCBI accession number: 14619) were constructed and co-transfected with appropriate amounts of AAV-ie capsid protein (reference DOI: 10.1038 / s41467-019-11687-8) and pHelper plasmid (NCBI: AF369965.1) into HEK-293T cells. AAV virus was purified by iodixanol gradient centrifugation, and the viral titer was measured to be 1E12-1E13 GC / mL.

[0146] 1.2 CAG promoter-driven GJB2 expression in the cochlea causes hearing loss

[0147] On day 2 of life, FVB newborn mice were injected through the round window with the viruses AAV-ie-CAG-nls-EGFP and AAV-ie-CAG-nls-EGFP-mouse GJB2. One month later, auditory brainstem response (ABR) testing revealed hearing loss in the AAV-ie-CAG-nls-EGFP-mouse GJB2 group (Figure 1). This suggests that nonspecific cellular expression of the CAG broad-spectrum promoter is detrimental to targeted gene therapy, as GJB2 is primarily expressed in supporting cells. Overexpression in hair cells may cause hearing loss.

[0148] 1.3 CAG promoter drives mNeonGreen expression in SCs and HCs

[0149] On the second day after birth, FVB newborn mice were injected with AAV-ie-CAG-mNeonGreen through the round window. Seven days later, the cochlea was harvested and immunofluorescence staining was performed. The hair cell marker myosin7a was used for staining, and the cell nuclei were stained with DAPI. It was observed that mNeonGreen was expressed in both SCs and HCs (Figure 2).

[0150] Example 2: Sox47 promoter promotes transgene expression in SCs

[0151] 2.1 AAV-Sox47-mNeonGreen plasmid (SEQ ID: NO.8) construction and viral packaging

[0152] The specific promoter Sox47 (SEQ ID NO. 1) was incorporated into the pAAV-mNeonGreen plasmid. The vector was obtained by enzymatic digestion of the pAAV-mNeonGreen plasmid, followed by gene synthesis of the target fragment containing the specific promoter Sox47 (SEQ ID NO. 1), and recombinantly constructed into the complete target plasmid. mNeonGreen (DNA sequence: SEQ ID NO. 4, amino acid sequence: SEQ ID NO. 5) is a green fluorescent protein used to indicate infected and expressing cells, similar to the green fluorescent protein (EGFP). The constructed AAV-Sox47-mNeonGreen viral packaging plasmid was co-transfected with the AAV-ie capsid protein and pHelper plasmids in appropriate amounts into HEK-293T cells. The AAV virus was purified by iodixanol gradient centrifugation, with a viral titer of 1E12-1E13 GC / mL.

[0153] 2.2 Sox47 promoter-driven SCs-targeted NeoGreen

[0154] On the second day after birth, FVB newborn mice were injected with the virus AAV-ie-Sox47-mNeonGreen through the round window. The cochlea was harvested on the 15th day and immunofluorescence staining of the mouse cochlea was performed using the hair cell marker myosin7a and the SCs marker Sox2. It was observed that mNeonGreen was specifically expressed in the SCs region, as shown in Figure 3 (Scale bars: 200 μm; 50 μm).

[0155] The two AAV-ie-Sox47-mNeonGreen viruses containing specific promoters were injected into the inner ears of postnatal day 2-3 (P2-P3) mice via the round window. Mice were anesthetized on ice for 1-2 minutes. After anesthesia, a retroauricular incision was made to expose the round window, and the cochlea was visually inspected and injected manually with a glass micropipette. After injection, the skin incision was sealed with 3M Vethod tissue glue. The injected mice were then placed on a 37°C heating pad for recovery. Newborn mice fully recovered within approximately 10 minutes and were returned to their mothers. Standard postoperative care was followed. Two weeks later, the infection efficiency of cochlear hair cells was assessed and statistically analyzed. Immunofluorescence staining of the mouse cochlea was performed using antibodies against the hair cell marker Myo7a, the supporting cell marker Sox2, and the nuclear marker DAPI. Before cochlear mounting, the cochlea was fixed and decalcified. After dissection and removal of the tectorial membrane, the cochlea was washed three times with PBS, blocked with blocking solution for 1 hour, and then incubated with the primary antibody (1:1000 Myo7a) at 4°C overnight. The tissue was washed three times with PBS and counterstained with secondary antibodies (1:4000 Donkey anti-rabbit Alexa Fluor 555 and 1:1000 DAPI) at room temperature for 1 hour. The tissue was washed three times with PBS. The slides were removed, labeled with mouse information and cochlear processing procedures, and antifade reagent was added to the slides. The slides were gently covered with a coverslip, avoiding air bubbles during the process, and stored in a cool, dark place. Confocal images were acquired on a Zeiss LSM700 laser confocal microscope. Images of the entire cochlea were processed using ImageJ software. The acquired images were projected along the z-axis using ImageJ. The number of cells double-positive for Myo7a-labeled HCs and mNeonGreen was counted. Immunofluorescence staining results showed that the mNeonGreen reporter gene was specifically expressed only in cochlear supporting cells (Figure 3).

[0156] Example 3: The SHH promoter sequence alone (SEQ ID NO: 3) does not drive gene expression in cochlear cells

[0157] 3.1 Plasmid construction and viral packaging

[0158] The SHH promoter (SEQ ID NO: 3) was incorporated into the pAAV-mNeonGreen plasmid to construct the AAV-SHH-mNeonGreen viral packaging plasmid. This plasmid, along with the AAV-ie capsid protein plasmid and pHelper plasmid, was co-transfected into HEK-293T cells at appropriate concentrations. AAV virus was purified by iodide gradient ultracentrifugation, with a viral titer of 1e12-1e13 GC / mL considered optimal.

[0159] 3.2 SHH (SEQ ID NO: 3) alone cannot drive mNeonGreen expression in cochlear cells

[0160] The AAV-ie-SHH-mNeonGreen virus containing the specific promoter was injected into the inner ear of mice at P2-P3 via the round window. Mice were anesthetized on ice for 1-2 minutes. After anesthesia, a retroauricular incision was made to expose the round window, and the cochlea was observed and injected manually with a glass micropipette. After injection, the skin incision was sealed with 3M Vethod tissue glue. Two weeks later, the infection efficiency of cochlear hair cells was assessed and statistically analyzed. Immunofluorescence staining of the mouse cochlea with the hair cell marker myosin7a and the nuclear marker DAPI was performed. Before cochlear mounting, the cochlea was fixed and decalcified. After dissection and removal of the tectorial membrane, the cochlea was washed three times with PBS, blocked with blocking solution for 1 hour, and incubated with the primary antibody (1:1000 Myo7a) overnight at 4°C. The tissue was washed three times with PBS and counterstained with secondary antibodies (1:4000 Donkey anti-rabbit Alexa Fluor 555 and 1:1000 DAPI) at room temperature for 1 hour. Myosin7a-labeled HCs and mNeonGreen double-positive cells were counted. Immunofluorescence staining results showed that the mNeonGreen reporter gene was not expressed in cochlear hair cells or supporting cells (Figure 4). Since the Sox47 promoter (SEQ ID NO: 1) is composed of two parts: an enhancer sequence (SEQ ID NO: 2) and a SHH core promoter sequence (SEQ ID NO: 3), the results of Example 1 and Example 3 indicate that the specificity of the Sox47 promoter is determined by the enhancer sequence (SEQ ID NO: 2).

[0161] Example 4: Effects of Sox47 promoter-driven GJB2 expression in SCs on hearing

[0162] 4.1 AAV-Sox47-EGFP and AAV-Sox47-human GJB2 (SEQ ID: NO. 9) plasmid construction and viral packaging

[0163] The specific promoter Sox47 (SEQ ID NO. 1) was incorporated into pAAV-EGFP and pAAV-human GJB2 to construct AAV-Sox47-EGFP and AAV-Sox47-human GJB2 (SEQ ID NO. 9) viral packaging plasmids. These plasmids were co-transfected with appropriate amounts of AAV-ie capsid protein and pHelper plasmids into HEK-293T cells. AAV virus was purified by iodixanol gradient centrifugation, with a viral titer of 1E12-1E13 GC / mL. The sequence of human GJB2 is NCBI: 2706.

[0164] 4.2 Sox47 promoter-driven GJB2 expression in SCs has no effect on hearing

[0165] On the second day after birth, C57 / BL6 newborn mice were injected with the viruses AAV-Sox47-EGFP and AAV-Sox47-human GJB2 through the round window. ABR (auditory brainstem response) audiometry was performed one month later, and no difference in hearing was observed between the AAV-Sox47-human GJB2 group and the control group ( Figure 5 ). This indicates that SCs-specific expression of the Sox47 promoter has no effect on hearing and is a safe promoter compared to the CAG promoter in Example 1.

[0166] Example 5, Sox47 promoter drives GJB2 expression in GJB2 conditional knockout mice (Sox9 Cre ;GJB2 Loxp ) The effect of regional re-expression of SCs on hearing

[0167] 5.1 AAV-Sox47-EGFP and AAV-Sox47-human GJB2 plasmid construction and viral packaging

[0168] Same as Example 4.1

[0169] 5.2 Sox47 promoter drives GJB2 expression in Sox9 Cre ;GJB2 Loxp Re-expression in mice

[0170] Sox9 on day 3 after birth Cre ;GJB2 Loxp Newborn mice were injected with AAV-Sox47-EGFP and AAV-Sox47-human GJB2 viruses through the round window. Immunofluorescence staining was performed 14 days later, and it was observed that GJB2 was not expressed in the SCs region in the AAV-Sox47-EGFP group, while GJB2 was re-expressed in the AAV-Sox47-human GJB2 group ( Figure 6 ).

[0171] 5.3 Sox47 promoter drives GJB2 expression in Sox9 Cre ;GJB2 Loxp Re-expression in mice partially restored hearing

[0172] Sox9 Cre ;GJB2 Loxp The mice were induced to have GJB2 knockout in Sox9-marked supporting cells by using the Cre-Loxp system. Cre ;GJB2 Loxp Newborn mice were injected with AAV-Sox47-EGFP and AAV-Sox47-human GJB2 through the round window. Auditory brainstem response (ABR) testing was performed on day 30, revealing that re-expression of GJB2 driven by the Sox47 promoter in the experimental group partially restored hearing (Figure 7).

[0173] sequence of this article

[0174] SEQ ID NO: 1: Sox47 promoter

[0175] SEQ ID NO: 2: Enhancer

[0176] SEQ ID NO: 3: SHH core promoter element

[0177] SEQ ID NO: 4: mNeonGreen DNA sequence

[0178] SEQ ID NO: 5: mNeonGreen amino acid sequence

[0179] SEQ ID NO: 6: AAV-CAG-nls-EGFP plasmid sequence

[0180] SEQ ID NO: 7: AAV-CAG-nls-EGFP-mouse GJB2 plasmid sequence

[0181] SEQ ID NO: 8: AAV-Sox47-mNeonGreen plasmid sequence

[0182] SEQ ID NO: 9: AAV-Sox47-human GJB2 plasmid sequence

Claims

1. A cochlear supporting cell-specific enhancer comprising the sequence shown in SEQ ID NO: 2 or a homologous sequence having at least 90% sequence identity thereto and retaining the function of SEQ ID NO: 2, Preferably, the homologous sequence is a sequence of a mammal, preferably of the order Primates, more preferably of the family Hominidae.

2. A cochlear supporting cell-specific promoter, comprising: (1) the sequence shown in SEQ ID NO: 2 or a sequence having at least 90% sequence identity thereto and retaining the function of SEQ ID NO: 2, and (2) core promoter elements, Preferably, (2) the core promoter element is the promoter of the SHH gene, Preferably, (2) the sequence of the core promoter element is as shown in SEQ ID NO: 3 or a homologous sequence having at least 90% sequence identity thereto, More preferably, the homologous sequence is a SHH gene promoter sequence of a mammal, preferably a primate, more preferably a hominid.

3. A nucleic acid construct comprising the cochlear supporting cell-specific promoter according to claim 2, Preferably, the nucleic acid construct further comprises a gene of interest, wherein the gene is operably linked to the cochlear supporting cell-specific promoter; and / or, the nucleic acid construct is a vector, More preferably, the gene is selected from: a deafness gene, a transcription factor gene, a signal pathway gene, a tool gene for manipulating genes of cochlear supporting cells or expressed in cochlear supporting cells.

4. A rAAV vector system, comprising an AAV vector and a nucleic acid construct encoding a gene required by the AAV virus, wherein the AAV vector comprises the cochlear supporting cell-specific promoter according to claim 2, Preferably, the AAV vector further comprises a gene of interest, which is operably linked to the cochlear supporting cell-specific promoter.

5. A rAAV virus particle comprising the cochlear supporting cell-specific promoter according to claim 2.

6. A host cell comprising or having integrated into its genome: the cochlear supporting cell-specific promoter according to claim 2, the nucleic acid construct according to claim 3, or the AAV vector system according to claim 4.

7. A pharmaceutical composition comprising a pharmaceutically acceptable excipient and one or more selected from the following: the cochlear supporting cell-specific promoter according to claim 2, the nucleic acid construct according to claim 3 or the AAV vector system according to claim 4, the rAAV virus particle according to claim 5, and the host cell according to claim 6.

8. A method for expressing a gene in cochlear supporting cells, comprising the step of introducing a nucleic acid construct comprising the gene into cochlear supporting cells, wherein the gene is operably linked to the cochlear supporting cell-specific promoter of claim 2.

9. Use of the sequence shown in SEQ ID NO: 2 or a homologous sequence having at least 90% sequence identity thereto and retaining the function of SEQ ID NO: 2 in a chimeric promoter.

10. A method for preparing a chimeric promoter, comprising: (1) the sequence shown in SEQ ID NO: 2 or a sequence having at least 90% sequence identity thereto and retaining the function of SEQ ID NO: 2 is operably linked to (2) a core promoter element.

11. Use of the cochlear supporting cell-specific promoter according to claim 2 in the preparation of a drug for treating a disease caused by a gene defect in cochlear supporting cells, wherein the drug is used to express the gene in cochlear supporting cells. Preferably, the disease is hereditary deafness caused by a gene defect in the cochlear supporting cells, More preferably, the disease is hereditary deafness caused by a functional loss of GJB2.

12. A method for inducing cochlear supporting cells to differentiate into hair cells, comprising the step of expressing transcription factors and / or signal pathway genes associated with hair cell regeneration in cochlear supporting cells, wherein the genes are operably linked to the cochlear supporting cell-specific promoter of claim 2, Preferably, the method comprises the step of introducing a nucleic acid construct or vector comprising a coding sequence of the transcription factor and / or signaling pathway gene into cochlear supporting cells, wherein the coding sequence is operably linked to the cochlear supporting cell-specific promoter.

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