Viral vector for delivering GJB2 and use thereof

Abstract

Provided is an expression cassette for expressing GJB2 protein, which comprises a cochlear supporting cell and cochlear lateral wall-specific promoter and a nucleic acid encoding GJB2 protein. Also provided is a recombinant adeno-associated viral vector, the genome of which comprises an expression cassette for expressing GJB2 protein inserted between two ITR sequences; and a combination of at least two recombinant adeno-associated viral vectors. The recombinant adeno-associated viral vector or combination thereof can be used for treating or preventing hearing loss associated with defects in the GJB2 gene.

Description

Viral vectors for delivering GJB2 and their uses Technical Field

[0001] This invention relates to AAV viral vectors that deliver gap-joined β-2 (GJB2) coding sequences and their application in gene therapy, particularly in the treatment of hearing loss. Background Technology

[0002] Hearing loss is the most common disabling disease in clinical practice, severely impacting normal life and placing a huge burden on society. According to the WHO, nearly 1.5 billion people worldwide have varying degrees of hearing loss, with 466 million suffering from disabling hearing loss, accounting for 5% of the total population, including 34 million children. It is projected that by 2050, 2.5 billion people globally will have varying degrees of hearing loss, with 700 million suffering from disabling hearing loss.

[0003] Heredity and environment are the two major factors causing deafness. Environmental causes are mainly related to various environmental factors or certain complications, such as the use of ototoxic drugs, infections during pregnancy, neonatal hypoxia, and radiation exposure. Hereditary factors are mainly due to individual defects in deafness genes, leading to varying degrees of hearing loss. These pathogenic genes are passed on to the next generation through different inheritance pathways and can occur at any age, with permanent effects. Heredity is a major cause of deafness, accounting for approximately 60% of cases. Currently, more than 120 genes have been identified as related to deafness, involving more than 1500 pathogenic variants. To date, there are no drugs available clinically to treat hereditary deafness.

[0004] Based on the presence of clinical symptoms in organs other than the auditory system, hereditary hearing loss can be divided into syndromic hearing loss (SHL) and non-syndromic hearing loss (NSHL). Syndromic hearing loss (SHL) is often accompanied by clinical manifestations in other systems, including the eyes, heart, kidneys, nervous system, skin, and bones, accounting for 30% of hereditary hearing loss. Pendred syndrome, Usher syndrome (USH), and Waardenburg syndrome (WS) are the most well-known SHL types; while USH, Pendred syndrome, and Jervell and Lange-Nielsen syndrome (JLNS) have successfully achieved inner ear gene therapy in preclinical animal model studies. Non-syndromic hearing loss (NSHL) has four types: autosomal dominant (DFNA), autosomal recessive (DFNB), X-linked (DFNX), and mitochondrial non-syndromic hearing loss. Approximately 70% of hereditary hearing loss patients have non-syndromic hearing loss. The most common mode of inheritance for non-homocytic leukemia (NSHL) is autosomal recessive (75%-80%), followed by autosomal dominant (20%), X-linked inheritance (<2%), and mitochondrial inheritance (<1%). To date, over 120 genes have been reported to be associated with NSHL, including 51 DFNA-related deafness genes, 78 DFNB-related deafness genes, and 10 deafness genes associated with both DFNA and DFNB: CLOL11A2, GJB2, GJB6, MYO3A, MYO6, MYO7A, PTPRQ, TCB1D24, TECTA, and TMC1. Five DFNX-related deafness genes are also identified. The GJB2 gene is the most common cause of NSHL, accounting for approximately 50% of autosomal recessive deafness cases. The GJB2 gene is a first-line diagnostic gene for clinical deafness.

[0005] The GJB2 gene, located on human chromosome 13q12, encodes connexin 26 (also known as "Cx26 protein" or "GJB2 protein"), which is highly expressed in the cochlear gap junction (GJ). In normal individuals, the GJB2 protein encoded by the GJB2 gene, together with gap junction proteins from adjacent cells, forms a complete gap junction channel. This channel plays a crucial role in signal transduction and substance exchange, and is also an important channel for the intercellular transfer of electrolytes, second messengers, and metabolites. The potassium ion cycle in cochlear hair cells and cochlear lymph is regulated by the gap junction protein channel. Potassium ions enter the stria vascularis through the gap junction, are released by intermediate cells into the stria vascularis space, and return to the endolymph. When mutations occur in the coding region of the GJB2 gene, leading to alterations in the function of the GJB2 protein, the structure of the gap junction protein changes, thereby affecting the normal opening and closing of the gap junction protein channel. Abnormalities in the structure of the gap junction protein can impair the circulation of potassium ions back into the endolymph. When the concentration of potassium ions changes, and reaches a certain level, it can lead to potassium poisoning, damage to the cochlear hair cells, and sensorineural hearing loss.

[0006] The human GJB2 gene is expressed in the supporting cells of the organ of Corti in the cochlea and on the lateral wall of the cochlea. For GJB2 deficiency, there remains a need in the art for gene therapies that can effectively deliver the functional GJB2 coding sequence to its specific locations in the inner ear to efficiently express the functional GJB2 protein in the supporting cells of the organ of Corti and on the lateral wall of the cochlea. Summary of the Invention

[0007] To meet the need for developing gene therapies targeting GJB2 defects, this invention provides an AAV viral vector for high-level expression of GJB2 in mammalian cochlear supporting cells and the lateral wall of the cochlea, thereby providing a useful tool for clinical targeted treatment of GJB2-related hearing loss.

[0008] In a first aspect, the present invention provides isolated promoter sequences comprising the nucleotide sequences shown in SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, or variants thereof having at least about 90% identity (e.g., at least 95%, 96%, 97%, 98%, 99%, or higher). The isolated promoter sequences are capable of driving the specific expression of exogenous target nucleic acids effectively linked thereto in the inner ear supporting cells and the lateral wall of the cochlea.

[0009] In a second aspect, the present invention provides an expression box that includes effective connections in a 5'-3' direction:

[0010] (a) A promoter sequence element comprising the promoter sequences shown in SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or a variant promoter sequence having at least about 90% identity with them (e.g., having at least 95%, 96%, 97%, 98%, 99% or higher identity); and

[0011] (b) A nucleic acid encoding a functional GJB2 protein of a mammal, such as a human, a non-human primate, a mouse, a pig, or a rat, wherein the nucleic acid encoding the GJB2 protein has at least 85%, for example, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the sequence of SEQ ID NO:2;

[0012] Optionally, the expression box is inserted between two ITR sequences.

[0013] In some embodiments, the expression cassette of the present invention further includes additional expression regulatory elements, such as the marmot hepatitis post-transcriptional regulatory element (WPRE) or a variant thereof.

[0014] In a third aspect, the present invention provides a nucleic acid vector, for example, a nucleic acid expression vector, which includes the expression frame of the present invention.

[0015] In some embodiments, the expression box of the present invention is inserted between two ITR sequences, which may be the same or different, for example, the two ITR sequences are independently derived from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9. In some embodiments, the two ITR sequences are the same, being the ITR sequence of AAV2.

[0016] This invention also provides the use of the expression cassette or nucleic acid vector of this invention for preparing recombinant adeno-associated virus (rAAV) vectors. In some embodiments, it is used to prepare recombinant AAV-ie vectors with infection tropism for inner ear supporting cells. In some embodiments, it is used to prepare recombinant AAV1 vectors with infection tropism for the lateral wall of the cochlea.

[0017] In a fourth aspect, the present invention provides a recombinant adeno-associated virus (rAAV) vector, preferably a recombinant AAV-ie vector or a recombinant AAV1 vector, the genome of which contains the expression cassette of the present invention inserted between two ITR sequences. In some embodiments, the expression cassette of the present invention is inserted between two ITR sequences, and the two ITR sequences may be identical or different, for example, the two ITR sequences are independently derived from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9. In some embodiments, the two ITR sequences are identical, being the ITR sequence of AAV2. In some embodiments, the genome of the recombinant adeno-associated virus vector of the present invention can self-complement to form a double-stranded DNA molecule.

[0018] In a fifth aspect, the present invention provides a combination of recombinant adeno-associated virus (rAAV) vectors, which is a combination of at least two recombinant adeno-associated virus (rAAV) vectors of the present invention, preferably a combination of two recombinant adeno-associated virus (rAAV) vectors of the present invention, for example, a combination of recombinant AAV-ie vector and recombinant AAV1 vector.

[0019] In a sixth aspect, the present invention provides a pharmaceutical composition comprising the nucleic acid vector of the present invention, the recombinant AAV viral vector of the present invention, or a combination of the recombinant AAV viral vectors of the present invention, and a pharmaceutically acceptable excipient.

[0020] In a seventh aspect, the present invention provides the use of the nucleic acid vector of the present invention, the recombinant AAV viral vector of the present invention, or a combination of the recombinant AAV viral vectors of the present invention for the preparation of a medicament for the treatment or prevention of hearing loss associated with defects in the GJB2 gene.

[0021] In an eighth aspect, the present invention provides a medicament or preparation for treating or preventing hearing loss associated with defects in the GJB2 gene, which is prepared from the recombinant AAV viral vector of the present invention or a combination of recombinant AAV viral vectors of the present invention, wherein the adeno-associated virus is obtained by packaging an adeno-associated virus packaging vector system into a host cell, the adeno-associated virus packaging vector system comprising the nucleic acid vector of the present invention expressing the GJB2 protein, a vector carrying the AAV rep and cap genes, and an auxiliary viral vector.

[0022] In some embodiments, the medicament or formulation of the present invention further comprises a neutral salt buffer, an acidic salt buffer, an alkaline salt buffer, glucose, mannose, mannitol, protein, polypeptide, amino acid, antibiotic, chelating agent, adjuvant, preservative, nanoparticles, liposomes, and positively charged lipid particles.

[0023] In some embodiments, the drug or formulation of the present invention is administered by injection through the round window, oval window, semicircular canals, or common canal of the cochlea; and may be administered once or multiple times throughout life, with a total dose of 1×10⁻⁶. 9 -1×10 13 Viral genome. Attached Figure Description

[0024] Figure 1 shows Sox9 CreER / + / tdTomato loxp / loxp Expression range of Sox9 in the mouse cochlea. Inset A of Figure 1 shows the expression of Sox9. CreER / + / tdTomato loxp / loxp Mice were injected intraperitoneally with tamoxifen on day 6 after birth to induce tdTomato expression, and protein expression was analyzed on day 13 after birth. Figure B shows the widespread expression of Sox2 and Sox9 (marked with red tdTomato) in the developing mouse inner ear. "HeC" represents Hensen's cells; "DC" represents Deiters cells; "PC" represents pillar cells; "IPhC" represents inner phalangeal cells; and "IBC" represents inner border cells. Figure C shows the expression of Sox9 (marked with red tdTomato) in cochlear epithelial supporting cells, the spiral rim, and the lateral wall of the cochlea, essentially covering the expression range of GJB2. "LW" represents the lateral wall; "OC" represents the organ of Corti. DAPI staining was used to label cell nuclei. This invention utilizes Sox9-CreER transgenic mice to knock out the GJB2 gene in the aforementioned tissues and cells, constructing a GJB2-deficient mouse model. Sox2 is a marker of cochlear epithelial supporting cells; the use of a Sox2 antibody aims to validate the Sox9 gene. CreER / + / tdTomato loxp / loxp Whether the range of Sox9 tdTomato-tagged cells in mice covers the range of cochlear support cells expressing GJB2, thereby ensuring that the GJB2 gene is knocked out in all cochlear support cells.

[0025] Figure 2 shows Sox9 CreER / + / GJB2 loxp / loxp After tamoxifen injection to induce GJB2 gene knockout in mice, GJB2 protein expression was significantly reduced. The small images at the top of column A in Figure 2 show GJB2 expression in the cochlear support cell region of C57BL / 6J wild-type mice. The small images at the bottom of column A show Sox9... CreER / + / GJB2 loxp / loxp The staining image shows the GJB2 gene knocked out in the cochlear support cell region of a mouse. The top small image in column B shows GJB2 expression stained on the lateral cochlear wall of a C57BL / 6J wild-type mouse control. The bottom small image in column B shows an overlay of GJB2 and DAPI staining in the control mouse. The top small image in the far right column shows Sox9. CreER / + / GJB2 loxp / loxp A staining image of the GJB2 gene knocked out in the lateral wall of the mouse cochlea; the small image at the bottom right shows Sox9. CreER / + / GJB2 loxp / loxp Overlay of GJB2 and DAPI staining on the lateral wall of the mouse cochlea. "Control" in the figure represents the C57BL / 6J wild-type mouse control. "LW" represents the lateral wall; "OC" represents the organ of Corti: DAPI staining was used to label the cell nuclei.

[0026] Figure 3 shows the conditional knockout of the GJB2 gene into Sox9. CreER / + / GJB2 loxp / loxp The mice had a significantly elevated hearing threshold and experienced hearing loss. The WT mice were C57BL / 6J wild-type mice.

[0027] Figure 4 shows Sox9 CreER / + / GJB2 loxp / loxp The endocochlear potential (EP) value in mice was significantly reduced.

[0028] Figure 5 shows Sox9 CreER / + / GJB2 loxp / loxp In mice, after GJB2 gene knockout, the number of outer hair cells in the apical and middle turns of the cochlea was significantly reduced, while no damage was found in the inner hair cells. In the figure, "Apex" represents the apical turn; "MID" or "Middle" represents the middle turn; "BASE" or "Base" represents the basal turn; "Control" represents C57BL / 6J wild-type mice; "OHC" represents outer hair cells; ns indicates no significant difference, p>0.05; * indicates P<0.05; ** indicates P<0.01; *** indicates P<0.001; **** indicates P<0.0001.

[0029] Figure 6 shows that equal amounts of AAV-ie-CAG-mNeonGreen, AAV1-CAG-mNeonGreen, and AAV-DJ-CAG-mNeonGreen viruses (1.5E10 GCs) were injected into the cochlea of ​​wild-type C57BL / 6J mice on day 1 (P1) after birth via a round window. The cochlea was harvested at P7 to detect the expression rate of green fluorescent protein in the cochlear supporting cells.

[0030] Figure 7 shows that equal amounts of AAV-ie-CAG-mNeonGreen, AAV1-CAG-mNeonGreen, and AAV-DJ-CAG-mNeonGreen viruses (1.5E10 GCs) were injected into the cochlea of ​​P1 wild-type C57BL / 6J mice through a round window. The cochlea was harvested at P7 to detect the expression rate of green fluorescent protein in the lateral wall of the cochlea.

[0031] Figure 8 shows the hearing threshold results 4 weeks after injecting two viruses, AAV-ie-CAG-EGFP (represented as "CAG-EGFP" in the figure) and AAV-ie-CAG-GJB2 (represented as "CAG-GJB2" in the figure), into the left cochlea of ​​C56 / BL6J wild-type mice on day 1 (P1) after birth.

[0032] Figure 9 shows the immunofluorescence results 4 weeks after AAV-ie-CAG-EGFP and AAV-ie-CAG-GJB2 viruses were injected into the left cochlea of ​​C56 / BL6J wild-type mice on day 1 (P1) after birth.

[0033] Figure 10 shows the immunofluorescence results in the hair cell region (“HClayer”) and supporting cells (“SC layer”) 7 days after AAV-ie-S4-mNeonGreen, AAV-ie-S7-mNeonGreen, and AAV-ie-S47-mNeonGreen viruses were injected into the left cochlea of ​​C56 / BL6J wild-type mice on day 1 (P1). Red indicates the expression of “Myo7a”, and green indicates the expression of “mNeonGreen”. Myo7a is a marker of hair cells.

[0034] Figure 11 shows the immunofluorescence results in the hair cell region (“HClayer”) and supporting cells (“SC layer”) of the left cochlea of ​​C57BL / 6J wild-type mice injected with AAV-ie-S47-mNeonGreen on day 1 (P1) after 7 days. In the figure, “HeC” represents Hensen’s cells; “DC” represents Deiters cells; “IPC” represents inner pillar cells; “OPC” represents outer pillar cells; “IPhC” represents inner phalangeal cells; and “IBC” represents inner border cells.

[0035] Figure 12 shows that AAV-ie-S47-EGFP (referred to as "S47-EGFP" in the figure) and AAV-ie-S47-GJB2 (referred to as "S47-GJB2" in the figure) were injected into the left cochlea of ​​C57BL / 6J wild-type mice on day 1 (P1) after birth. ABR audiometry was performed at one month (1M) and two months (2M). The results showed that, compared with WT and the control group injected with AAV-ie-S47-EGFP, GJB2 driven by the S47 promoter had no effect on hearing after cochlear expression.

[0036] Figure 13 shows the immunofluorescence results of AAV-ie-S47-EGFP and AAV-ie-S47-GJB2 injected into the left cochlea of ​​C57BL / 6J wild-type mice on day 1 (P1) after birth, at one month (1M). AAV-ie-S47-EGFP served as the control group.

[0037] Figure 14 shows the immunofluorescence results 7 days after AAV-ie-S47-mNeonGreen and AAV1-S47-mNeonGreen were injected into the left cochlea of ​​C57BL / 6J wild-type mice on day 1 (P1) after birth. Type I, II, III, and V represent different regions of the lateral wall of the cochlea indicated by the arrows in the figure.

[0038] Figure 15 shows the injection of two viruses, AAV-ie-S47-GJB2 and AAV1-S47-GJB2, into Sox9. CreER / + / GJB2 loxp / loxp Immunofluorescence results in the left cochlea of ​​mice after 4 weeks, with the right ear serving as a control without AAV virus injection.

[0039] Figure 16 shows the injection of a 1:1 mixture of AAV-ie-S47-GJB2 and AAV1-S47-GJB2 viruses into Sox9 via a round window. CreER / + / GJB2 loxp / loxp The ABR results were analyzed in the left ear of a mouse unilateral cochlea after 4 weeks. The right ear served as a control ear that was not injected with AAV virus.

[0040] Figure 17 shows the results of endothelial prophylaxis (EP) after a 1:1 mixture of AAV-ie-S47-GJB2 and AAV1-S47-GJB2 viruses was injected into the left ear of a mouse through a round window. The right ear served as a control without AAV virus injection. The three lines in Figure 16 represent the results of the three mice.

[0041] Figure 18 shows that only AAV-ie-S47-GJB2 virus and only AAV1-S47-GJB2 virus were injected into Sox9 via a round window. CreER / + / GJB2 loxp / loxp The ABR results were analyzed in the left ear of a mouse unilateral cochlea after 4 weeks. The right ear served as a control ear that was not injected with AAV virus. Detailed Implementation

[0042] Unless otherwise defined below, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. Furthermore, the materials, methods, and examples described herein are illustrative only and are not intended to be limiting. Other features, objects, and advantages of the invention will become apparent from this specification and the accompanying drawings, and from the appended claims.

[0043] I. Definition

[0044] In this document, the term "about" when used in conjunction with a numeric value means to cover a range of numeric values ​​having a lower limit of 5% less than the specified numeric value and an upper limit of 5% greater than the specified numeric value. The term is also intended to cover values ​​within ±1%, ±0.5%, or ±0.1% of the specified numeric value.

[0045] In this document, the expression “and / or” is used to refer to any one of the listed related items, or any and all possible combinations of multiple listed related items.

[0046] In this document, the terms "comprising" or "including" mean including the stated elements, integers, or steps, or groups of elements, integers, or steps, but do not exclude any other elements, integers, or steps, or other groups of elements, integers, or steps. When the terms "comprising" or "including" are used herein, unless otherwise specified, they also cover situations consisting of the stated elements, integers, or steps. For example, when referring to a polypeptide / protein that "comprising" a specific sequence, it is also intended to cover polypeptides / proteins consisting of that specific sequence.

[0047] In this document, "hearing loss" refers to hearing below the normal hearing threshold level as determined by audiometry, including mild, moderate, severe, and profound hearing loss, as well as deafness. Hearing loss can be described as a percentage of hearing loss, such as 30%, 60%, 80%, or even 100% hearing loss, or by a hearing loss grading. The hearing loss can be caused by or associated with a genetic defect, such as congenital deafness and prelingual deafness caused by genetic factors, or hearing loss associated with genetic factors but induced by environmental factors (e.g., aging, noise, drugs, or infection). Hearing loss can be asymptomatic (i.e., without associated visible abnormalities of the outer ear or other organs) or symptomatic. In some embodiments, the hearing loss is sensorineural hearing loss.

[0048] In this article, "hearing loss-related genes" refers to genes whose variations can cause hearing loss or create susceptibility to hearing loss by altering the inner ear's ability to function normally. Such genes are also referred to as "hearing loss genes" in this article. More than 100 genes have been identified as being associated with hearing loss (see Hereditary Hearing Loss Homepage, https: / / hereditaryhearingloss.org / , which lists the known locations and identification data of single-gene asymptomatic hearing loss). In cases of susceptibility to hearing loss, individuals carrying variations in these hearing loss genes are more likely to experience hearing loss due to environmental factors such as aging, noise, medications, or infections compared to healthy individuals.

[0049] In this article, "cochlear hair cells" refers to isolated or in vitro cochlear hair cells, cell lines, or cell populations from mammals, or hair cells in the cochlea of ​​mammals.

[0050] In this article, "cochlear outer hair cells" refers to cochlear outer hair cells, cell lines, or cell populations derived from mammals, either isolated or in vitro, or to outer hair cells in the cochlea of ​​mammals.

[0051] The organ of Corti, also known as the spiral organ, is the auditory receptor located in the cochlea of ​​mammals, within the cochlear duct, between the scala vestibulae and scala tympani. The epithelial cells of the organ of Corti convert auditory signals into action potentials that act as nerve impulses. Specifically, the ossicles in the middle ear transmit mechanical vibrations to the inner ear, causing pressure on the cochlear fluid (endolymph), which in turn generates corresponding fluctuations, prompting the hair cells within the organ of Corti to produce corresponding electrochemical signals. The basilar membrane of the organ of Corti consists of three rows of outer hair cells and one row of inner hair cells, surrounded by supporting / protective cells. The outer hair cells amplify low-level sound frequencies through mechanical movement of hair cell bundles or electrically driven movement of the hair cell bodies. The inner hair cells convert vibrations in the cochlear fluid into electrical signals that are transmitted to the brain by the auditory nerve.

[0052] The term "support cell" or "inner ear support cell" refers to the inner ear cells that maintain the structure of the inner ear and the sensory epithelial environment of the inner ear. In some respects, inner ear support cells include, but are not limited to, cell types such as inner finger cells, inner column cells (IPC), outer column cells (OPC), Deuter cells, and Hensen cells.

[0053] The cochlear lateral wall plays a crucial role in regulating ion and fluid transport in the inner ear to maintain the volume and unique composition of the endolymph (e.g., high potassium [K]) necessary for normal inner ear function. + ] and low sodium [Na + [Ion concentration]. The lateral wall of the cochlea is located at the lateral boundary of the scala media. It consists of the stria vascularis, spiral prominence, and spiral ligament. Cells in the lateral wall of the cochlea are interconnected through an extensive network of gap junctions.

[0054] The term "gap junction," also known as a gap junction, is a type of cell junction. Except for fully developed skeletal muscle cells and non-fixated cells such as erythrocytes, gap junctions are widely present in various animal tissues and are a specialized type of animal cell-cell connection. The cell membranes of the two cells forming a gap junction are often parallel and tightly packed, leaving a nanoscale gap. Two connexons located on adjacent cell membranes align and connect, forming a narrow intercellular channel that allows various small molecules, ions, and electrical signals to pass directly. This process is selective, and the opening and closing of gap junctions is often regulated. In vertebrates, connexons are isomeric or isomeric hexamers composed of connexins. Genes encoding gap junctions are broadly classified into three categories based on sequence similarity: A, B, and C (e.g., GJA1, GJB2, GJC1). Another common method for classifying connexins is based on their molecular weight (e.g., connexin26, connexin43, connexin30.3, etc.).

[0055] As used herein, the term “functional GJB2 protein” refers to a full-length wild-type (natural) GJB2 protein (having the amino acid sequence shown in SEQ ID NO:2), its variants (e.g., variants with conserved amino acid substitutions), or its fragments, said variants or fragments providing at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, or about the same, or greater than 100% of the biological activity level of the full-length wild-type (natural) GJB2 protein.

[0056] As used herein, the term “conservative amino acid substitution” or “conservative amino acid replacement” refers to the alteration, substitution, or replacement of an amino acid with a different amino acid having similar biochemical properties (e.g., charge, hydrophobicity, and size), as is known to those skilled in the art.

[0057] There are various methods available for measuring the expression and activity levels of GJB2 protein using conventional techniques. See, for example, Examples 3 and 4 of this specification.

[0058] The term "effective connection" means that the specified components are in a relationship that allows them to function in the intended way.

[0059] The following is a calculation of sequence identity between sequences.

[0060] To determine the percentage of identity between two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., vacancies may be introduced in one or both of the first and second amino acid sequences or nucleic acid sequences for optimal alignment, or non-homologous sequences may be discarded for comparison purposes). In a preferred embodiment, for comparison purposes, the length of the reference sequence being aligned is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 100% of the reference sequence length. The amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, the molecules are identical at that position.

[0061] Mathematical algorithms can be used to compare sequences and calculate the percentage of identity between two sequences. In a preferred embodiment, the Needlema and Wunsch ((1970) J. Mol. Biol. 48: 444-453) algorithm (available at http: / / www.gcg.com) is used in the GAP program integrated into the GCG software package, employing a Blossum 62 matrix or a PAM250 matrix and vacancy weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6, to determine the percentage of identity between two amino acid sequences. In yet another preferred embodiment, the GAP program in the GCG software package (available at http: / / www.gcg.com) is used, employing an NWSgapdna.CMP matrix and vacancy weights of 40, 50, 60, 70, or 80, and length weights of 1, 2, 3, 4, 5, or 6, to determine the percentage of identity between two nucleotide sequences. The particularly preferred set of parameters (and a set of parameters that should be used unless otherwise specified) is a Blossum 62 scoring matrix with a vacancy penalty of 12, a vacancy extension penalty of 4, and a shift vacancy penalty of 5.

[0062] Alternatively, the PAM120 weighted remainder table, gap length penalty of 12, and gap penalty of 4 can be used to determine the percentage of identity between two amino acid sequences or nucleotide sequences using the E. Meyers and W. Miller algorithm ((1989) CABIOS, 4:11-17) which has been incorporated into the ALIGN program (version 2.0).

[0063] Additionally or alternatively, the nucleic acid and protein sequences described herein may be further used as “query sequences” to perform searches against public databases, for example, to identify other family member sequences or related sequences.

[0064] The term "regulatory sequence" or "expression control sequence" refers to a nucleic acid sequence that induces, inhibits, or otherwise controls the transcription of a protein encoding a nucleic acid sequence that is effectively linked to it. Regulatory sequences can be, for example, initiation sequences, enhancer sequences, intron sequences, and promoter sequences.

[0065] As used in this article, the term "adeno-associated virus (AAV)" is named after its discovery in adenovirus products. AAV is a member of the Parvovirus family, which includes multiple serotypes, and its genome is single-stranded DNA.

[0066] AAV is a dependent virus, requiring other viruses such as adenovirus, herpes simplex virus, human papillomavirus, or cofactors to provide auxiliary functional proteins in order to replicate.

[0067] The earliest isolated AAV virus was serotype 2 AAV (AAV2). The AAV2 genome is approximately 4.7 kb long, with 145 bp inverted terminal repeats (ITRs) at both ends, exhibiting a palindromic-hairpin structure. The genome contains two large open reading frames (ORFs), encoding the rep and cap genes, respectively. The full-length AAV2 genome has been cloned into an E. coli plasmid (Samulski RJ et al., Proc Natl Acad Sci USA. 1982; 79:2077-2081. Laughlin CA et al., Gene. 1983; 23:65-73).

[0068] ITRs are cis-acting elements of the AAV vector genome, playing a crucial role in AAV virus integration, rescue, replication, and genome packaging. ITR sequences contain a Rep protein binding site (RBS) and a terminal resolution site (trs), enabling them to be recognized by Rep protein binding and creating a nick at the trs. ITR sequences can also form a unique "T"-shaped secondary structure, playing a vital role in the AAV virus life cycle.

[0069] The rest of the AAV2 genome can be divided into two functional regions: the rep gene region and the cap gene region.

[0070] The rep gene region encodes four Rep proteins: Rep78, Rep68, Rep52, and Rep40. Rep proteins play important roles in the replication, integration, rescue, and packaging of AAV viruses.

[0071] The cap gene encodes the capsid proteins VP1, VP2, and VP3 of the AAV virus. VP3 has the smallest molecular weight but is the most abundant; in mature AAV particles, the ratio of VP1, VP2, and VP3 is approximately 1:1:10. VP1 is essential for the formation of infectious AAV; VP2 assists VP3 in entering the cell nucleus; VP3 is the main protein constituting the AAV particle.

[0072] As used in this article, the term "AAV vector" refers to a highly efficient exogenous gene transfer tool created by modifying wild-type AAV viruses, based on our understanding of the AAV virus life cycle and related molecular biological mechanisms. The modified AAV vector genome contains only the AAV virus's ITR sequence and an expression cassette carrying the exogenous gene to be transferred. The Rep and Cap proteins required for AAV virus packaging are provided trans-associated through other exogenous plasmids, thereby reducing the potential harm caused by packaging the Rep and Cap genes into the AAV vector. Furthermore, the AAV virus itself is non-pathogenic, making AAV vectors one of the most recognized safest viral vectors. Deleting the D and trs sequences from one side of the ITR sequence of an AAV virus can also enable the genome carried by the packaged recombinant AAV viral vector to self-complement, forming a double strand, which significantly improves the in vivo and in vitro transduction efficiency of the AAV vector (Wang Z et al., Gene Ther. 2003; 10(26):2105-2111; McCarty DM et al., Gene Ther. 2003; 10(26):2112-2118). The packaged virus is called scAAV (self-complementary AAV) virus, also known as double-stranded AAV virus. It is different from ssAAV (single-stranded AAV), which is a traditional AAV virus, where neither side of the ITR is mutated.

[0073] The scAAV viral vector has a smaller packaging capacity, only half that of the ssAAV viral vector, approximately 2.2kb-2.5kb, but it has a higher transduction efficiency after infecting cells.

[0074] AAV virus has many serotypes, and different serotypes have different tissue tropisms. Therefore, AAV vectors can be used to transport exogenous genes to specific organs and tissues (Wu Z et al., Mol Ther. 2006; 14(3):316-327).

[0075] In addition, AAV vectors have stable physicochemical properties and exhibit strong tolerance to acids, alkalis and high temperatures (Gruntman AM et al., Hum Gene Ther Methods. 2015; 26(2):71-76), making it easy to develop bioproducts with high stability.

[0076] Existing technologies have relatively mature packaging systems for AAV carriers, which facilitates the large-scale production of AAV carriers.

[0077] Currently, commonly used AAV vector packaging systems mainly include three-plasmid co-transfection systems, systems using adenovirus as a helper virus, packaging systems using herpes simplex virus type 1 (HSV1) as a helper virus, and baculovirus-based packaging systems. Each packaging system has its own characteristics, and those skilled in the art can make an appropriate selection according to their needs.

[0078] The three-plasmid transfection packaging system is the most widely used AAV vector packaging system due to its high safety profile and the lack of auxiliary viruses required, and it is also the mainstream production system internationally. A slight drawback is that the lack of efficient, large-scale transfection methods limits the application of the three-plasmid transfection system in the large-scale preparation of AAV vectors.

[0079] The term "vector genome (vg)" refers to the nucleic acid sequence packaged within the rAAV capsid to form an rAAV vector. In one embodiment, the vector genome contains at least a 5' to 3' AAV2 5' ITR, a nucleic acid sequence encoding a functional GJB2 protein, and an AAV2 3' ITR. ITRs from different sources of AAV besides AAV2 may also be selected. Furthermore, the vector genome may contain regulatory sequences that guide the expression of the functional GJB2 protein.

[0080] The term "treatment" refers to a clinical intervention intended to alter the natural course of a disease in an individual undergoing treatment. Desired therapeutic effects include, but are not limited to, preventing the onset or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, slowing the rate of disease progression, improving or mitigating the disease state, and alleviating or improving prognosis.

[0081] II. Gap-joint β-2 (GJB2) gene

[0082] The GJB2 gene encodes connexin 26 (also known as "Cx26 protein" or GJB2 protein). GJB2 protein is a member of the connexin family. The human GJB2 gene is located on chromosome 13q12. It contains two transcriptional isoforms starting from a variable transcription origin. These two transcriptional isoforms are approximately 5469 nucleotides and 4675 nucleotides respectively (NCBI gene ID 2706, NCBI reference sequence: NG_008358.1). Each isoform contains two exons and a single intron, with the intron separating the two exons. The coding region is entirely contained within exon 2, and the coding sequence is approximately 681 nucleotides long, encoding the 226-amino acid Cx26 protein (also referred to as "GJB2 protein" in this paper).

[0083] Within the inner ear, the GJB2 protein is synthesized by all supporting cell types within the organ of Corti, including inner finger cells, inner column cells (IPC), outer column cells (OPC), Deuter cells, and Hensen cells. Additionally, the GJB2 protein is also present on the lateral wall of the cochlea.

[0084] More than 200 mutations in the GJB2 gene have been identified that are associated with hearing loss. In some implementations, GJB2 gene mutations are associated with nonsyndromic hearing loss, which can be inherited in a dominant (e.g., DFNA3) or recessive (DFNB1) manner. In this paper, each type of deafness is sometimes also described according to the naming conventions for deafness genes; for example, DFNA3 is the third autosomal dominant type of deafness discovered. DFNB1 is the first autosomal recessive nonsyndromic deafness described.

[0085] In some embodiments, the GJB2 gene is a mammalian GJB2 gene. In some embodiments, the GJB2 gene is a mouse GJB2 gene, for example, a mouse or rat GJB2 gene. In some embodiments, the GJB2 gene is a porcine GJB2 gene. In some embodiments, the GJB2 gene is a non-human primate GJB2 gene. In some embodiments, the GJB2 gene is a human GJB2 gene.

[0086] III. Polypeptides encoded by the GJB2 gene

[0087] This invention provides polypeptides encoded by the GJB2 gene. In some embodiments, the polypeptide encoded by the GJB2 gene is a polypeptide encoded by the mammalian GJB2 gene. In some embodiments, the polypeptide encoded by the GJB2 gene is a polypeptide encoded by the mouse GJB2 gene. In some embodiments, the polypeptide encoded by the GJB2 gene is a polypeptide encoded by the non-human primate GJB2 gene. In some embodiments, the polypeptide encoded by the GJB2 gene is a polypeptide encoded by the human GJB2 gene. The amino acid sequence of an exemplary human GJB2 protein is as follows:

[0088] In some embodiments, the present invention provides GJB2 protein variants comprising one or more (e.g., 1, 2, 3, 4, 5, 7, 8, 9, or 10) amino acid residue substitutions, deletions, and / or additions (preferably conserved amino acid substitutions) of the wild-type GJB2 protein, and substantially without impairing the function of the wild-type GJB2 protein, e.g., without impairing gap junctions and cochlear homeostasis. In some embodiments, the GJB2 protein variants comprise an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence shown in SEQ ID NO:2.

[0089] IV. Expression Box

[0090] In one aspect, the present invention provides an expression box comprising a nucleotide sequence encoding a functional GJB2 protein and a regulatory sequence guiding its expression.

[0091] In one embodiment, the expression cassette comprises a nucleotide sequence encoding a functional GJB2 protein as described herein and a regulatory sequence guiding its expression. The functional GJB2 protein comprises an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence shown in SEQ ID NO:2. In some embodiments, the nucleotide sequence encoding the functional GJB2 protein is a nucleotide sequence encoding an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence shown in SEQ ID NO:2. In some embodiments, the nucleotide sequence encoding the functional GJB2 protein is selected from nucleotide sequences encoding the amino acid sequence shown in SEQ ID NO:2.

[0092] In some embodiments, the regulatory sequence guiding the expression of functional GJB2 protein comprises the promoter sequences shown in SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, or variant promoter sequences having at least about 90% identity with them (e.g., at least 95%, 96%, 97%, 98%, 99%, or higher). Compared to using the broad-spectrum CAG promoter (SEQ ID NO:1), using promoters with sequences of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or variant sequences thereof avoids hair cell damage caused by ectopic expression of functional GJB2 protein in hair cells due to the broad-spectrum CAG promoter.

[0093] In some preferred embodiments, the regulatory sequence guiding the expression of the functional GJB2 protein comprises the promoter sequence shown in SEQ ID NO:5 or a variant promoter sequence having at least about 90% identity with it (e.g., at least 95%, 96%, 97%, 98%, 99% or higher identity). Compared to using promoters using SEQ ID NO:3, SEQ ID NO:4 or variants thereof, using SEQ ID NO:5 or a variant thereof as the promoter sequence enables the target gene effectively linked to it to be expressed at a higher intensity in supporting cells.

[0094] In addition to the promoter and the polynucleotide encoding the functional GJB2 protein, the expression cassette may contain other regulatory sequences as needed. The terms "regulatory sequence" and "expression control sequence" are used interchangeably herein, referring to a nucleic acid sequence that induces, inhibits, or otherwise controls the transcription of a protein encoding a nucleic acid sequence to which it is effectively linked. Regulatory sequences can be, for example, initiation sequences, intron sequences, post-transcriptional regulatory elements (WPREs) of marmot hepatitis or variants thereof, transcription termination sequences, etc.

[0095] In some embodiments, the expression cassette of the present invention comprises, in a transcriptional direction, elements functionally connected to each other:

[0096] -The promoter sequence element according to the present invention is a promoter sequence comprising the promoter sequences shown in SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or a variant promoter sequence having at least about 90% identity with them (e.g., having at least 95%, 96%, 97%, 98%, 99% or higher identity);

[0097] - The nucleic acid encoding the functional GJB2 protein is a nucleotide sequence encoding an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence shown in SEQ ID NO:2;

[0098] - One or more transcription terminators, and

[0099] -polyA signal sequence.

[0100] In some embodiments, the expression cassette also includes a Kozak sequence located upstream (i.e., 5') of the polynucleotide encoding the transgene.

[0101] To achieve the expression of a target gene in mammalian cells, various methods can be used to introduce an expression cassette containing a transgene operatively linked to the promoter of this invention, or a vector containing the expression cassette, into target cells. These methods include, but are not limited to, transformation, transfection, transduction, direct uptake, and encapsulation in liposomes. Suitable methods for transfecting or transforming cells are known in the art, including calcium phosphate precipitation, electroporation, microinjection, viral infection, and lipid transfection.

[0102] In some implementations, the expression box is inserted between the two ITR sequences of the AAV.

[0103] V. Viral vector

[0104] In another aspect, the present invention provides a viral vector, which is an artificial recombinant viral particle, wherein a replication-defective viral genome sequence containing an expression cassette encoding a functional GJB2 protein is packaged in a viral capsid or envelope, such that the recombinant viral particle cannot produce progeny virions but retains the ability to infect target cells.

[0105] In one implementation, the genome sequence of the viral vector does not contain genes encoding enzymes required for viral replication. Therefore, the use of viral vectors in gene therapy is considered safe because replication and infection of progeny viruses will not occur in the absence of enzymes required for viral replication.

[0106] The recombinant viral vector of the present invention may be a recombinant adeno-associated virus (AAV), adenovirus, bocavirus, AAV / bocavirus hybrid, herpes simplex virus, or lentivirus.

[0107] Packaging cell lines for producing recombinant viral vectors (e.g., recombinant AAV vectors) can be prokaryotic or eukaryotic cells (e.g., human cells, insect cells, or yeast cells) containing exogenous DNA introduced into the cells by any means (e.g., electroporation, calcium phosphate precipitation, microinjection, transformation, viral infection, transfection, and protoplast fusion). Packaging cell lines include, but are not limited to, E. coli cells, yeast cells, human cells, non-human cells, mammalian cells, insect cells, HEK293 cells, hepatocytes, kidney cells, glial cells, or stem cells.

[0108] As used herein, the term "target cell" refers to a target cell in which the functional GJB2 protein is intended to be expressed. Examples of target cells include, but are not limited to, inner ear supporting cells and lateral cochlear wall cells. In some embodiments, a recombinant viral vector is delivered in vivo to the target cell.

[0109] In a preferred embodiment, the viral vector is a recombinant adeno-associated virus (rAAV) vector containing an AAV capsid and a vector genome packaged therein. The rAAV vector is used to treat sensorineural hearing loss caused by a GJB2 gene defect. The vector genome contains an AAV 5' inverted terminal repeat (ITR) or AAV 5'ΔITR, a nucleic acid sequence encoding a functional GJB2 protein, a regulatory sequence guiding the expression of the GJB2 protein in target cells, and an AAV 3'ITR or AAV 3'ΔITR. The ΔITR is an ITR with the D sequence and terminal unwinding site trs deleted. The ITR is the genetic element responsible for genome replication and packaging during vector production and is the only viral cis-element required for rAAV production. ITRs from different serotypes of AAV can be selected. In one embodiment, the ITR is from an AAV different from the capsid of the viral particle.

[0110] Apart from the AAV capsid, unless otherwise stated, the AAV ITR and other AAV components described herein can be readily selected from any AAV, including but not limited to serotypes commonly identified as AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh.10, or combinations thereof. As will be understood by those skilled in the art, the capsid and ITR of the AAV viral vector can be derived from the same or different AAV serotypes.

[0111] AAV proteins VP1, VP2, and VP3 are capsid proteins that interact to form the AAV capsid. Different serotypes of AAV viruses have different tissue tropisms, and exogenous genes can be transported to specific organs and tissues by selecting the serotype from which the AAV virus vector capsid originates (Wu Z et al., Mol Ther. 2006; 14(3):316-327).

[0112] In a preferred embodiment, the AAV viral vector of the present invention comprises an AAV-ie serotype capsid for targeting all supporting cell types within the organelle of Corti. A viral vector having an AAV-ie serotype is described in NATURE COMMUNICATIONS, (2019) 10:3733, https: / / doi.org / 10.1038 / s41467-019-11687-8, wherein a CPP-like short peptide DGTLAVPFK (SEQ ID NO:6) is inserted between amino acids 589 and 590 of the VP1 capsid protein of the AAV-DJ virus.

[0113] In a preferred embodiment, the AAV viral vector of the present invention comprises an AAV1 serotype capsid for targeting the lateral wall of the cochlea.

[0114] VI. Methods for preparing viral vectors

[0115] The recombinant adeno-associated virus (AAV) vector of the present invention can be produced using known techniques. Such methods involve culturing packaging cells containing a nucleic acid sequence encoding an AAV capsid; a functional rep gene; an expression cassette as described herein, flanked by an AAV inverted terminal repeat (ITR) or ΔITR; and sufficient auxiliary functions to allow the expression cassette to be packaged into an AAV capsid protein.

[0116] This document also provides a packaging cell containing a nucleic acid sequence encoding an AAV capsid; a functional rep gene; an expression cassette as described herein, flanked by an AAV inverted terminal repeat (ITR) or ΔITR; and sufficient auxiliary functions to allow the expression cassette to be packaged into the AAV capsid protein. In one embodiment, the host cell is a HEK 293 cell.

[0117] Other methods known in the art for producing rAAV can be utilized. Suitable methods may include, but are not limited to, baculovirus expression systems or production via yeast.

[0118] VII. Pharmaceutical Composition

[0119] In some aspects, the present invention provides pharmaceutical compositions comprising the viral vector of the present invention formulated together with pharmaceutically acceptable excipients. Furthermore, the compositions may also contain one or more other therapeutic agents suitable for treating or preventing, for example, hearing loss. Pharmaceutically acceptable excipients include pharmaceutically acceptable substances that can enhance or stabilize the composition, or pharmaceutically acceptable substances that can be used to facilitate the preparation of the composition. For example, pharmaceutically acceptable excipients may include physiologically compatible solvents, surfactants, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delay agents, etc.

[0120] The pharmaceutical compositions of the present invention can be administered by a variety of methods known in the art. The route and / or mode of administration varies depending on the desired outcome. Round window injection is preferred. The pharmaceutically acceptable excipients should be suitable for the route of administration, such as cochlear, intravenous, subcutaneous, or topical administration.

[0121] The composition should be sterile and fluid. Appropriate flowability can be maintained, for example, by using coatings (such as lecithin), by maintaining the desired particle size in the case of dispersants, and by using surfactants. In many cases, it is preferred to include isotonic agents in the composition, such as sugars, polyols (e.g., mannitol or sorbitol), and sodium chloride.

[0122] The pharmaceutical compositions of the present invention can be prepared according to methods well known and conventionally practiced in the art. See, for example, Remington: The Science and Practice of Pharmacy, Mack Publishing Co., 20th edition, 2000; and Sustained and Controlled Release Drug Delivery Systems, JR Robinson, editor, Marcel Dekker, Inc., New York, 1978. The pharmaceutical compositions are preferably manufactured under GMP conditions. A therapeutically effective dose of the viral vector of the present invention is typically used in the pharmaceutical compositions of the present invention. The viral vector can be formulated into a pharmaceutically usable dosage form using conventional methods known to those skilled in the art. Furthermore, the dosing regimen can be adjusted to provide the optimal desired response (e.g., therapeutic response). For example, it can be administered as a single dose, administered in several fractions over time, or the dose can be proportionally reduced or increased as indicated by the urgency of the treatment situation. Particularly advantageous is to formulate the pharmaceutical compositions into unit dosage forms to facilitate administration and dosage uniformity. As used herein, a unit dosage form refers to a physically discrete unit suitable as a unit dose for the treated object; each unit contains a predetermined amount of an active compound calculated to produce the desired therapeutic effect in combination with the desired pharmaceutical carrier.

[0123] The actual dose level of the active ingredient in the pharmaceutical composition of the present invention can be modified to obtain an amount of active ingredient that effectively achieves the desired therapeutic response for a specific patient, composition, and administration mode without toxicity to the patient. The selected dose level depends on a variety of pharmacokinetic factors, including the activity of the specific composition of the present invention used, route of administration, time of administration, excretion rate of the specific compound used, duration of treatment, other drugs, compounds, and / or materials used in combination with the specific composition used, the age, sex, weight, condition, general health status, and medical history of the patient to be treated, and similar factors.

[0124] Physicians or veterinarians may begin with a dose lower than the level required to achieve the desired therapeutic effect, using the viral vector of the present invention in the pharmaceutical composition, and gradually increase the dose until the desired effect is achieved. Generally, the effective dose of the present invention's compositions for treating hereditary hearing loss as described herein varies depending on many different factors, including the route of administration, target site, patient physiological state, whether the patient is human or animal, other drugs administered, and whether the treatment is prophylactic or therapeutic. Treatment dose titration is necessary to optimize safety and efficacy.

[0125] The viral vector described herein can be administered once per ear, or repeatedly as needed. The dosage may vary depending on whether the treatment is prophylactic or therapeutic.

[0126] If desired, the pharmaceutical compositions of the present invention may be presented in a packaging or dispensing device, which may contain one or more unit dosage forms comprising the active ingredient. The packaging may, for example, contain metal or plastic foil, such as blister packs. The dispensing device may be an injection device, such as an injection pen. Instructions for use may be included with the packaging or dispensing device.

[0127] Therefore, in some aspects, the present invention also provides a kit for carrying out the treatment methods of the present invention. Such a kit contains a therapeutically effective amount of the pharmaceutical composition according to the invention in one or more containers. The composition in the vial of the kit may be in the form of a pharmaceutically acceptable solution, for example, in combination with sterile saline, glucose solution, or buffer solution or other pharmaceutically acceptable sterile fluid. Alternatively, the pharmaceutical composition may be in lyophilized or dehydrated form; in this case, the kit optionally further contains, in the container, a preferably sterile, pharmaceutically acceptable solution (e.g., saline, glucose solution, etc.) to reconstitute the composition to form a solution for injection purposes. In some cases, the kit may further include a needle or syringe preferably packaged in a sterile form for injecting the composition, and optionally include instructions for administering the composition to a clinician or patient.

[0128] VIII. Treatment Methods

[0129] In one aspect, the present invention provides a method for treating hearing loss in an individual, comprising administering the pharmaceutical composition of the present invention to the individual. The individual suitable for the method of the present invention may be an individual suffering from hearing loss. In some embodiments, the hearing loss is hereditary hearing loss. In some embodiments, the hereditary hearing loss is autosomal dominant hearing loss DFNA3 or autosomal recessive hearing loss DFNB1.

[0130] In some embodiments, the pharmaceutical composition is administered in a therapeutically effective amount to reduce hearing loss and improve hearing.

[0131] In some embodiments, the individual is a human. In other embodiments, the individual is a non-human mammal, such as a rodent.

[0132] Example

[0133] The technical solution of the present invention will be clearly and completely described below through embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, all reaction reagents involved in the embodiments can be purchased through commercial channels.

[0134] Example 1. Construction of a GJB2 hearing loss mouse model

[0135] 1.1 Sox9 was built CreER / + / GJB2 loxp / loxp Conditional knockout mice

[0136] First, administer Tamoxifen intraperitoneally on day 6 after birth (also referred to as "P6" in this article). CreER / + / tdTomato loxp / loxp In mice (obtained from Southeast University, Nanjing), Cre enzyme was activated and tdTomato expression was induced, and the expression range of Sox9 was determined to cover the cell types in the cochlea that express GJB2 (Sox9 is expressed in cochlear epithelial supporting cells, spiral rim, lateral cochlear wall and other cell tissues, basically covering the expression range of GJB2).

[0137] Sox2 is a marker of cochlear epithelial supporting cells. Sox2 antibodies were used to validate Sox9. CreER / + / tdTomato loxp / loxp Does the extent of tdTomato-tagged Sox9 in mice cover the extent of GJB2-expressing cochlear support cells, so that GJB2 can be knocked out in all cochlear support cells (Figure 1)?

[0138] Furthermore, by crossing Sox9-CreER transgenic mice (obtained from the National Institute of Biological Sciences, Beijing) with mice flanked by the GJB2 gene carrying loxP, Sox9 was obtained. CreER / + / GJB2 loxp / loxp Mice. In Sox9 CreER / + / GJB2 loxp / loxpMice (obtained from Southeast University, Nanjing) were intraperitoneally injected with tamoxifen on day 6 (P6) after birth. GJB2 protein expression was then detected in the cochlear epithelium and lateral cochlear wall regions. The results showed that tamoxifen injection induced Sox9... CreER / + / GJB2 loxp / loxp After the GJB2 gene was knocked out in mice, the expression of GJB2 protein was significantly reduced (Figure 2).

[0139] 1.2 Sox9 CreER / + / GJB2 loxp / loxp Conditional knockout mice exhibited hearing impairment, decreased cochlear potential, and hair cell damage.

[0140] The cochlear structure and function of mice after GJB2 gene knockout were examined.

[0141] Auditory brainstem response (ABR) results showed that the hearing threshold of GJB2 conditional knockout mice was significantly increased, resulting in hearing loss (Figure 3).

[0142] Studies have shown that GJB2 function is related to potassium ion circulation in the endolymph, and is key to maintaining high potassium ion concentration in the endolymph and cochlear potential (EP), which is maintained by the stria vascularis. Therefore, Sox9 was tested. CreER / + / GJB2 loxp / loxp EP in mice revealed Sox9 CreER / + / GJB2 loxp / loxp The EP value of mice was significantly reduced (Figure 4), and the change in the potential of the inner ear lymph fluid may have caused damage to hair cells.

[0143] Furthermore, the cochlear hair cells of GJB2 gene knockout mice were statistically analyzed. Compared with the control group, the number of outer hair cells in the apical and mid-rotation of the cochlea was significantly reduced after GJB2 gene knockout, while no damage was found in the inner hair cells (Figure 5). The above data indicate that the GJB2 hearing loss model was successfully constructed.

[0144] Example 2. Screening for AAV serotypes for GJB2 gene therapy

[0145] 2.1 Construction of pAAV-CAG-mNeonGreen plasmid and viral packaging

[0146] Construction of pAAV-CAG-mNeonGreen plasmid (construction method: the nucleic acid encoding green fluorescent protein mNeonGreen was cloned into a pAAV plasmid containing cytomegalovirus enhancer / chicken β-actin promoter (CAG promoter) and marmot hepatitis virus post-transcriptional regulatory element (WPRE), with AAV2 inverted terminal repeat sequences flanking the target gene expression cassette) (Xiao X et al., Production of High-Tier Recombinant Adeno-Associated Virus Vectors in the Absence of Helper Adenovirus. J Virol. 1998; 72(3):2224-2232)) and AAV-ie capsid protein expression plasmid (Fangzhi Tan et al., “AAV-ie enables safe and efficient gene transfer to inner ear cells”, Nature Communications, Vol. 10, Article (Number: 3733(2019), DOI: 10.1038 / s41467-019-11687-8), AAV1 capsid protein expression plasmid (Yilai Shu et al., “Identification of Adeno-Associated Viral Vectors That Target Neonatal and Adult Mammalian Inner Ear Cell Subtypes”, Hum Gene Ther, 2016 Sep; 27(9): 687-699. DOI: 10.1089 / hum.2016.053) or AAV-DJ capsid protein expression plasmid (Min-A Kim et al., “Targeted Gene Delivery into the Mammalian Inner Ear Using Synthetic Serotypes of Adeno-Associated Virus Vectors”, Methods & Clinical) Development, 2019, 13: 197-204. DOI:10.1016 / j.omtm.2019.01.002), and pHelper plasmid (NCBI: AF369965).1) AAV viruses were obtained by co-transformation into HEK-293T cells using a three-plasmid method, resulting in AAV-ie-CAG-mNeonGreen, AAV1-CAG-mNeonGreen, and AAV-DJ-CAG-mNeonGreen. The AAV viruses were purified by gradient centrifugation with iodixanol, and viral titers were measured to be between 1E12 and 1E13 GC / mL. In this study, AAV viruses were named according to their serotype, promoter, and target protein gene. For example, AAV-ie-CAG-mNeonGreen refers to the AAV virus obtained by co-transformation into HEK-293T cells using the pAAV-CAG-mNeonGreen plasmid, the AAV-ie capsid protein expression plasmid, and the pHelper plasmid (NCBI: AF369965.1) using a three-plasmid method. These are also referred to below as serotypes AAV-ie, AAV1, or AAV-DJ viruses carrying the mNeonGreen gene.

[0147] CAG promoter sequence:

[0148] 2.2 AAV-ie efficiently infects cochlear supporting cells

[0149] Equal amounts of 1.5E10 GCs carrying the mNeonGreen (green fluorescent protein) gene of three serotypes (AAV-ie / AAV1 / AAV-DJ) prepared in Example 2.1 were injected into the left cochlea of ​​wild-type mice on day 1 (P1) after birth through a round window. The cochlea was harvested on day 7 (P7) after birth to detect the expression rate of green fluorescent protein in the cochlear supporting cells.

[0150] Specifically, the three different serotypes of the virus were injected into the cochlea of ​​P1 wild-type mice via a round-window administration method. Mice were anesthetized with hypothermia in ice for 1-2 minutes. After anesthesia, an incision was made behind the ear and in the neck to expose the round window, and the virus was manually injected using a glass microsyringe. The wound was sealed with surgical tissue adhesive after injection, and the mice were placed in a 37°C warming pad for resuscitation before being returned to their mothers. One week later, the infection efficiency of the three serotypes on cochlear supporting cells was studied, and the data were statistically analyzed. Immunofluorescence staining of the mouse cochlea was performed using antibodies against the hair cell marker Myo7a, the supporting cell marker gene Sox2, and the nuclear marker DAPI. Before cochlear slide preparation, the cochlea was fixed and decalcified. After dissecting the cochlea and removing the tectorial membrane, the tissue was washed three times with PBS, blocked with Block solution for 1 hour, and then incubated overnight at 4°C with the primary antibody (Myo7a, Proteus Biosciences, #25-6790, 1:1000 dilution; Sox2, Santa, #SC-17320, 1:400 dilution). The tissue was washed three times with PBS and counterstained for 1 hour at room temperature with the secondary antibody (Donkey anti-rabbit Alexa Fluor 555, Abcam, #ab150074, 1:400 dilution; Donkey anti-goat Alexa Fluor 647, Abcam, #ab150131, 1:400 dilution). The tissue was then washed three times with PBS. Remove the slide, label the mouse information and cochlear processing procedure, add an anti-quenching agent to the slide, and gently cover it with a coverslip, avoiding air bubbles during the process. Place the slide in a cool, dark place. Confocal images were taken using a Zeiss LSM700 laser confocal microscope. The complete cochlear images were processed using ImageJ software. The acquired images were projected along the Z-axis using ImageJ (small figure A in Figure 6). The positive supporting cells double-labeled with Sox2 and mNeonGreen were counted. Immunofluorescence results from cochlear smears showed that the AAV-ie serotype virus had a transduction efficiency of approximately 90% for different types of supporting cells, including Henson cells (HeC), Deuter cells (DCs), column cells (PCs), inner finger cells (IPhCs), and inner marginal cells (IBCs). The AAV-DJ serotype virus had an infection efficiency of 70%–90% for supporting cells (DCs, PCs, IPhCs / IBCs), while the AAV1 serotype virus had a higher infection efficiency for IPhCs / IBCs and IHCs, but a lower infection rate for other supporting cells in the inner ear (small figure B in Figure 6). Therefore, the AAV-ie serotype virus is the preferred serotype virus for efficient delivery of GJB2 to sensory epithelial supporting cells.

[0151] 2.3 AAV1 efficiently infects the lateral wall of the cochlea

[0152] Equal amounts of 1.5E10 GCs carrying the mNeonGreen (green fluorescent protein) gene, prepared in Example 2.1, were injected into the left cochlea of ​​P1 wild-type mice via a round window. The cochleas were harvested at P7 to detect the expression rate of green fluorescent protein in the lateral wall of the cochlea. The cochleas were dehydrated with different concentrations of sucrose, embedded in OCT embedding medium, and then sectioned using a cryostat to a thickness of 13 μm. The sections were incubated overnight at -20°C and subjected to immunofluorescence treatment the following day. The tissue was washed three times with 0.1% PBST, blocked with Block solution for 1 hour, and then incubated overnight at 4°C with the primary antibody (Connexin 26 Polyclonal Antibody, Thermo, #512800, 1:200 dilution). The tissue was washed three times with 0.1% PBST and counterstained with a secondary antibody (Donkey anti-rabbit Alexa Fluor 555, Abcam, #ab150074, 1:400 dilution) for 1 hour at room temperature. It was then washed three times with 0.1% PBST. The slides were removed, and mouse information and cochlear processing procedures were labeled. Anti-quenching agent was added to the slides, and coverslips were gently placed on top, avoiding air bubbles. The slides were then placed in a cool, dark place. Infection of the lateral wall of the cochlear wall by three serotypes of AAV was observed in the frozen cochlear sections.

[0153] Immunofluorescence staining results showed that AAV1 had a higher infection efficiency on the lateral wall of the cochlea compared to AAV-ie and AAV-DJ (Figure 7), indicating that AAV1 serotype is the dominant serotype for transducing the lateral wall of the cochlea.

[0154] Based on the experimental results of Examples 2.2 and 2.3, the combined use of AAV-ie and AAV1 may enable the re-expression of GJB2 in both the supporting cells of the cochlear sensory epithelium and the lateral wall of the cochlea.

[0155] Example 3. Highly efficient and specific expression of mNeonGreen in cochlear supporting cells driven by a cochlear supporting cell-specific promoter.

[0156] 3.1 Construction and viral packaging of pAAV-CAG-mNeonGreen, pAAV-S47-mNeonGreen, pAAV-CAG-EGFP, and pAAV-CAG-GJB2 plasmids

[0157] The construction and viral packaging of the pAAV-CAG-mNeonGreen plasmid are the same as in Example 2.1.

[0158] Similarly, the CAG promoter in the pAAV-CAG-mNeonGreen plasmid was replaced with the S47 promoter to construct the pAAV-S47-mNeonGreen plasmid and then packaged into a virus; the mNeonGreen coding sequence in the pAAV-CAG-mNeonGreen plasmid was replaced with the EGFP coding sequence to construct the pAAV-CAG-EGFP plasmid and then packaged into a virus to obtain the virus AAV-ie-CAG-EGFP; the mNeonGreen coding sequence in the pAAV-CAG-mNeonGreen plasmid was replaced with the GJB2 coding sequence to construct the pAAV-CAG-GJB2 plasmid and then packaged into a virus to obtain the virus AAV-ie-CAG-GJB2.

[0159] 3.2 The broad-spectrum CAG promoter drives the ectopic expression of GJB2 protein in cochlear hair cells, causing hearing loss and damage to cochlear hair cells.

[0160] Two viruses, AAV-ie-CAG-EGFP (1.5E10 GCs) and AAV-ie-CAG-GJB2 (1.5E10 GCs), were injected into the left cochlea of ​​C56 / BL6J wild-type mice on day 1 (P1) after birth, and analyzed after 4 weeks. ABR results showed that, compared with the control group, GJB2 expression driven by the CAG promoter significantly increased the hearing threshold (Figure 8), and immunofluorescence results showed that ectopic expression of GJB2 in hair cells led to significant hair cell damage (Figure 9).

[0161] 3.3 Highly efficient and specific expression of mNeonGreen in cochlear Sertoli cells driven by the AAV-ie serotype-mediated S47 promoter.

[0162] To avoid ectopic expression of GJB2 in cochlear hair cells, the S4, S7, and S47 promoters were screened to identify promoters specific to cochlear supporting cells. The nucleotide sequences of the S4, S7, and S47 promoters are as follows.

[0163] The nucleotide sequence of the S4 promoter:

[0164] The nucleotide sequence of the S7 promoter:

[0165] The nucleotide sequence of the S47 promoter:

[0166] Similar to Example 2.1, pAAV-S4-mNeonGreen, pAAV-S7-mNeonGreen, and pAAV-S47-mNeonGreen were constructed and packaged with AAV-ie virus and AAV1 virus, respectively.

[0167] AAV-ie virus 1.5E10 GCs expressing mNeonGreen under the three different promoters were injected into the left cochlea of ​​C57BL / 6J wild-type mice on day 1 (P1). Immunofluorescence results on day 7 showed that mNeonGreen, driven by the S4, S7, and S47 promoters, was not expressed in the hair cell region (“HClayer” in Figure 10), but mNeonGreen driven by the S47 promoter showed the strongest expression intensity in supporting cells (“SC layer” in Figure 10). Figure 11 shows the immunofluorescence results of AAV-ie-S47-mNeonGreen in the hair cell region (“HClayer”) and supporting cells (“SC layer”).

[0168] Therefore, the S47-specific promoter was selected for further research.

[0169] 3.4 Cochlear Support Cell Specific Promoter S47 Drives Safe Expression of GJB2 in the Cochlea

[0170] Similar to Example 2.1, pAAV-S47-EGFP and pAAV-S47-GJB2 were constructed and packaged with AAV-ie virus.

[0171] To verify the safety of the cochlear support cell-specific promoter S47, 1.5E10 GCs of AAV-ie-S47-EGFP and 1.5E10 GCs of AAV-ie-S47-GJB2 were injected into the left cochlea of ​​C57BL / 6J wild-type mice on day 1 (P1). ABR (auditory response) tests were performed at one month and two months. The results showed that, compared with C57BL / 6J wild-type mice (WT) and the control group injected with AAV-ie-S47-EGFP, S47 promoter-driven GJB2 expression in the cochlea of ​​C57BL / 6J wild-type mice had no effect on hearing (Figure 12). In addition, immunofluorescence results showed that S47 promoter-driven GJB2 was specifically expressed in cochlear support cells and did not damage hair cells (Figure 13).

[0172] 3.5 Highly efficient expression of mNeonGreen on the lateral wall of the cochlea, mediated by the AAV1 serotype-mediated S47 cochlear support cell-specific promoter.

[0173] AAV-ie-S47-mNeonGreen from 1.5E10 GCs and AAV1-S47-mNeonGreen from 1.5E10 GCs were injected into the unilateral (left) cochlea of ​​C57BL / 6J wild-type mice on day 1 (P1). Immunofluorescence results showed that the expression efficiency of mNeonGreen driven by the S47 promoter mediated by AAV1 in the lateral wall of the cochlea was significantly higher than that of AAV-ie (Figure 14).

[0174] Therefore, the inventors chose to use serum AAV-ie and AAV1 in combination to mediate the safe and efficient expression of GJB2 in cochlear supporting cells and the lateral wall driven by the cochlear supporting cell-specific promoter S47.

[0175] Example 4. Dual AAV-mediated GJB2 reexpression effectively restores hearing in GJB2-deficient mice.

[0176] 4.1 Construction of pAAV-S47-GJB2 plasmid and viral packaging

[0177] Similar to Example 2.1, the pAAV-S47-GJB2 plasmid was constructed and packaged with two viruses, AAV-ie-S47-GJB2 and AAV1-S47-GJB2.

[0178] 4.2 Dual AAV-mediated GJB2 in Sox9 CreER / + / GJB2 loxp / loxp Reexpression in mice

[0179] Sox9 CreER / + / GJB2 loxp / loxp Mice were injected into one cochlea (left ear) at P3-4 with a mixture of AAV-ie-S47-GJB2 and AAV1-S47-GJB2 viruses from 1.5E10 GCs. At P6, tamoxifen was injected to knock out the GJB2 gene. Analysis was performed 4 weeks later. Immunofluorescence results showed that the treated ear (right ear) expressed GJB2 compared to the contralateral ear (right ear) that had not been injected with either AAV-ie-S47-GJB2 or AAV1-S47-GJB2 (Figure 15).

[0180] 4.3 Dual AAV-mediated GJB2 in Sox9 CreER / + / GJB2 loxp / loxp Reexpression in mice resulted in partial recovery of hearing.

[0181] Sox9 CreER / + / GJB2 loxp / loxpMice were injected with a 1:1 mixture of two viruses (AAV1-ie-S47-GJB2 from 1.5E10 GCs and AAV1-S47-GJB2 from 1.5E10 GCs) into the left cochlea via a round window at P3-4. At P6, tamoxifen was injected to knock out the GJB2 gene. Analysis was performed 4 weeks later. ABR results showed that, compared to the untreated ear of the same mouse, the treated ear with the two mixed AAVs (i.e., dual AAVs) showed some degree of hearing recovery across all frequencies, especially significant hearing recovery in the 8K-24K frequency range, with a 20dB recovery in hearing threshold at the optimal frequency (Figure 16).

[0182] As a control, single AAV treatment with 1.5E10 GCs AAV-ie-S47-GJB2 and 1.5E10 GCs AAV1-S47-GJB2 respectively showed significantly worse hearing recovery than dual AAV treatment (Figure 18).

[0183] EP was further measured in the treated ears of the three mice with dual AAV. Compared with the control ears of the three mice, the EP of the treated ears with dual AAV showed a recovery of 10-15 mV, indicating a significant improvement (Figure 17).

[0184] The foregoing describes exemplary embodiments of the present invention. Those skilled in the art should understand that these disclosures are merely exemplary, and various other substitutions, adaptations, and modifications can be made within the scope of the present invention. Therefore, the present invention is not limited to the specific embodiments listed herein.

Claims

1. An isolated promoter sequence comprising the nucleotide sequence set forth in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or a variant having at least about 90% identity thereto (e.g., at least 95%, 96%, 97%, 98%, 99% or more identity thereto), respectively. for driving expression of an exogenous nucleic acid of interest in inner ear supporting cells and cochlear lateral wall cells.

2. An expression cassette comprising, in the 5’-3’ direction, operably linked: (a) a promoter sequence element that is a promoter sequence comprising SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or a variant promoter sequence having at least about 90% identity thereto (e.g., at least 95%, 96%, 97%, 98%, 99% or more identity thereto), respectively; and (b) a nucleic acid encoding a functional GJB2 protein of a mammal, e.g., a human, non-human primate, mouse, pig, or rat, e.g., the nucleic acid encoding the GJB2 protein has at least 85%, e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the sequence of SEQ ID NO: 2; optionally, the expression cassette is inserted between two ITR sequences.

3. The expression cassette of claim 2, further comprising an additional expression regulatory element, e.g., the expression regulatory element is a woodchuck hepatitis post-transcriptional regulatory element (WPRE) or a variant thereof.

4. A nucleic acid vector, e.g., a nucleic acid expression vector, comprising the expression cassette of claim 2 or 3, e.g., the two ITR sequences are the same or different, e.g., the ITR sequences are derived from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9.

5. Use of the expression cassette of claim 2 or 3 or the nucleic acid vector of claim 5 for the manufacture of a recombinant adeno-associated viral (rAAV) vector, preferably a recombinant AAV-ie vector or a recombinant AAV1 vector.

6. A recombinant adeno-associated viral (rAAV) vector, preferably a recombinant AAV-ie vector or a recombinant AAV1 vector, comprising in its genome the expression cassette of claim 2 or 3 inserted between two ITR sequences, e.g., the two ITR sequences are the same or different, e.g., the ITR sequences are derived from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV 8, or AAV9, preferably the genome of the recombinant adeno-associated viral vector can self-complement to form a double-stranded DNA molecule.

7. A combination of recombinant adeno-associated viral (rAAV) vectors, which is a combination of at least two recombinant adeno-associated viral (rAAV) vectors of claim 6, preferably it is a combination of two recombinant adeno-associated viral (rAAV) vectors of claim 6, e.g., it is a combination of a recombinant AAV-ie vector and a recombinant AAV1 vector.

8. A pharmaceutical composition comprising the nucleic acid vector of claim 4, the recombinant AAV viral vector of claim 6, or a combination of the recombinant AAV viral vector of claim 7, and a pharmaceutically acceptable excipient.

9. Use of the nucleic acid vector according to claim 4, the recombinant AAV viral vector according to claim 6, or a combination of the recombinant AAV viral vector according to claim 7, for the preparation of a medicament for the treatment or prevention of hearing loss associated with defects in the GJB2 gene.

10. A medicament or preparation for treating or preventing hearing loss associated with a defect in the GJB2 gene, prepared from the recombinant AAV viral vector according to claim 6 or the combination of the recombinant AAV viral vectors according to claim 7, wherein, The adeno-associated virus is obtained by packaging an adeno-associated virus packaging vector system into a host cell. The adeno-associated virus packaging vector system includes the nucleic acid vector of claim 4 that expresses the GJB2 protein, a vector carrying the AAV rep and cap genes, and an auxiliary viral vector.

11. The medicament or preparation according to claim 10, wherein, The drug or preparation may also contain neutral salt buffers, acidic salt buffers, basic salt buffers, glucose, mannose, mannitol, proteins, peptides, amino acids, antibiotics, chelating agents, adjuvants, preservatives, nanoparticles, liposomes, and positively charged lipid particles.

12. The medicament or preparation according to claim 10 or 11, wherein, Injection administration through the round window, oval window, semicircular canal, common canal of the cochlea; and single or multiple administration for life, total dose is 1 x 10 9 -1 x 10 13 viral genome.

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