Adeno-associated virus (AAV)-mediated antioxidant gene therapy for the prevention, amelioration, and / or treatment of hearing loss

Abstract

The present disclosure relates to compositions and methods, including recombinant AAV vectors that comprise polynucleotide sequences encoding superoxide dismutase (SOD) proteins. Also included are methods for treating or preventing acute hearing loss, progressive hearing loss, age-related hearing loss, and ototoxic hearing loss, including the methods and compositions.Also included are methods for treating or preventing acute vestibular hair cell loss, progressive vestibular hair cell loss, age-related vestibular hair cell loss, and ototoxic vestibular hair cell loss, including the methods and compositions. TIFF2025532136000028.tif107141

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63 / 409,568, filed September 23, 2022, which is incorporated herein by reference in its entirety. [Background technology]

[0002] background Hearing loss is the third most common chronic health problem in the United States, with noise-induced hearing loss affecting nearly one in four adults. According to the World Health Organization (WHO), more than 500 million people suffer from some form of hearing loss. Only a small proportion of these cases fall into the congenital category, where genetic disorders cause unilateral or bilateral hearing loss at birth or progressive hearing loss that leads to moderate to severe hearing loss as the child develops. These syndromic and nonsyndromic genetic disorders involve hundreds of genes with complex upstream and downstream interactions, limiting gene therapy approaches. The majority of hearing loss results from occupational or recreational noise, natural aging, and exposure to ototoxic drugs, such as chemotherapy or certain types of antibiotics. These disorders share a common etiology: oxidative processes produce free radicals that must be eliminated by hair cells, supporting cells, and ganglion cells in the cochlea and vestibular peripheral organs of the inner ear before causing damage that can lead to apoptosis and necrosis.

[0003] Noise-induced hearing loss is a function of the duration of exposure and the intensity / amplitude of the sound. Although the majority (over 70%) of people exposed to hazardous levels of noise do not wear hearing protection, noise-induced hearing loss (NIHL) can occur even in individuals who use hearing protection. Commercially available hearing protection devices can only reduce sound intensity by 20 dB (foam earplugs) or at most 37 dB (earmuffs). Therefore, prolonged exposure to hazardous levels of sound (120 + 15 dB) will eventually lead to inner ear damage and hearing loss, even with adequate protection.

[0004] Thus, both recreational and occupational noise exposure pose a wide range of health problems. Virtually all active-duty military personnel are exposed to hazardous noise levels, and most face lifelong hearing loss and hearing impairments (e.g., tinnitus, speech comprehension disorders). Patients with untreated hearing loss also experience higher rates of dementia. Over the past few decades, much of the research into treating NIHL has focused on regenerating lost hair cells, but this has not led to any breakthroughs. While small molecule research targeting inner ear hair cells has shown promise, limitations in delivery and sustained action / efficacy have prevented clinically useful, FDA-approved treatments. Much of the noise-induced damage stems from the secondary effects of oxidative stress following intense activation (prolonged exposure to loud noise), leading to apoptosis and necrosis of inner ear hair cells.

[0005] In the elderly, the probability of hearing loss increases with age. At age 65, one in three adults has some level of hearing loss, but this percentage increases significantly as individuals approach their 80s. This natural by-product of aging is largely related to oxidative damage over time. While it is impossible to avoid all noise exposure, a lifetime of experience and daily use accumulates, ultimately leading to the death of cochlear hair cells, supporting cells, and ganglion cells. In the elderly, when these levels reach a critical threshold, the appearance of hearing loss and balance disorders is observed. Superoxide dismutase, which is highly neuroprotective against oxidative damage processes, naturally declines with age. Therefore, it is likely that the age-related loss of these protective enzymes contributes to increased sensitivity and susceptibility in the elderly population.

[0006] Exposure to ototoxic drugs is another common factor in the development of hearing loss in both children and adults. For example, approximately 20–40% of individuals who undergo chemotherapy to treat cancer develop mild to moderate / severe permanent hearing loss. Compounds used to treat cancer are not easily eliminated by inner ear cells, leading to cell damage and death through oxidative pathways. Another type of ototoxic inner ear cell damage results from certain types of antibiotics. While no longer widely used in the United States, many countries continue to administer inexpensive antibiotics, such as gentamicin, to their populations, which damage cochlear and vestibular cells, ultimately leading to hearing loss and balance disorders. Gentamicin is more vestibular than cochleotoxic, but the toxicity profile induced by antibiotics varies depending on the antibiotic.

[0007] The majority of hearing loss currently experienced by humans worldwide is etiologically linked to oxidative processes that damage cells in the inner ear. Thus, there is a need for treatment strategies that can prevent the onset of hearing loss and halt its progression by targeting the overproduction of ROS in inner ear cells. The present invention addresses these needs. Summary of the Invention

[0008] Disclosure Overview As described herein, the present disclosure relates to compositions and methods comprising recombinant AAV vectors comprising polynucleotide sequences encoding superoxide dismutase (SOD) proteins. Also included are methods of treating, ameliorating, and / or preventing progressive hearing loss comprising such methods and compositions.

[0009] Thus, in one aspect, the present disclosure includes a polynucleotide encoding an adeno-associated virus (AAV) vector. In certain embodiments, the AAV vector comprises a first AAV inverted terminal repeat (ITR) nucleic acid sequence. In certain embodiments, the AAV vector comprises a nucleic acid encoding a superoxide dismutase (SOD) protein operably linked to a promoter. In certain embodiments, the AAV vector comprises a second AAV inverted terminal repeat (ITR) nucleic acid sequence. In certain embodiments, the AAV vector targets cells of the inner ear.

[0010] The present disclosure further includes a recombinant adeno-associated virus (AAV) vector. In certain embodiments, the AAV vector comprises a first AAV inverted terminal repeat (ITR) nucleic acid sequence. In certain embodiments, the AAV vector comprises a second AAV inverted terminal repeat (ITR) nucleic acid sequence. In certain embodiments, the AAV vector comprises a polynucleotide sequence encoding a superoxide dismutase (SOD) protein, operably linked to a promoter, sandwiched between the first AAV inverted terminal repeat (ITR) sequence and the second AAV ITR. In certain embodiments, the AAV vector has specificity for cells of the inner ear.

[0011] The present disclosure further includes a cell comprising an adeno-associated virus (AAV) vector.In certain embodiments, the AAV vector comprises a first AAV inverted terminal repeat (ITR) nucleic acid sequence.In certain embodiments, the AAV vector comprises a second AAV inverted terminal repeat (ITR) nucleic acid sequence.In certain embodiments, the AAV vector comprises a nucleic acid encoding a superoxide dismutase (SOD) protein, which is operably linked to a promoter, sandwiched between the first AAV inverted terminal repeat (ITR) sequence and the second AAV ITR.

[0012] The present disclosure further includes a method for treating, ameliorating, and / or preventing progressive hearing loss in a subject in need thereof.In certain embodiments, the method comprises administering to the subject an effective amount of an adeno-associated virus (AAV) vector comprising a nucleic acid encoding a superoxide dismutase (SOD) protein operably linked to a promoter.In certain embodiments, the SOD protein is expressed in inner ear cells.In certain embodiments, the administration treats, ameliorate, and / or prevents progressive hearing loss in the subject.

[0013] The present disclosure further includes a method for reversing, minimizing, and / or preventing progressive hearing loss in a subject in need thereof.In certain embodiments, the method comprises administering to the subject an effective amount of an adeno-associated virus (AAV) vector comprising a nucleic acid encoding a superoxide dismutase (SOD) protein operably linked to a promoter.In certain embodiments, the SOD protein is expressed in the subject's inner ear cells.In certain embodiments, the administration reverses, minimizes, and / or prevents progressive hearing loss in the subject.

[0014] The present disclosure further includes a method for reversing, minimizing, and / or preventing chemotherapy-related hearing loss in a subject in need thereof.In certain embodiments, the method comprises administering to the subject an effective amount of an adeno-associated virus (AAV) vector comprising a nucleic acid encoding a superoxide dismutase (SOD) protein operably linked to a promoter.In certain embodiments, the SOD protein is expressed in the subject's inner ear cells.In certain embodiments, the administration reverses, minimizes, and / or prevents chemotherapy-related hearing loss in the subject. [Brief explanation of the drawings]

[0015] The following detailed description of selected embodiments of the invention will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the invention, non-limiting embodiments are shown in the drawings. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

[0016] [Figure 1A] Figures 1A-1D show a hypothetical mechanism of SOD1 neuroprotection during noise exposure. Figure 1A is a diagram of a hair cell showing the locations of SOD1 (intracellular), SOD2 (mitochondrial), and SOD3 (extracellular). All three are viable candidates for neuroprotection from various forms of oxidative stress. Figure 1B is a schematic diagram showing how chronic noise exposure overwhelms the intracellular SOD1 pathway with reactive oxygen species (ROS). Figure 1C is a schematic diagram showing a hypothetical mechanism of targeted AAV delivery to hair cells, which induces upregulation of SOD1 transcription within the cells. Figure 1D is a diagram of the hypothetical mechanism of noise neuroprotection in AAV-protected hair cells. [Figure 1B] See legend to Figure 1A. [Figure 1C] See legend to Figure 1A. [Figure 1D] See legend to Figure 1A. [Figure 2A]Figures 2A-2H show targeted delivery of AAV to the cochlea via direct round window injection and global cisternal injection. Figure 2A is a diagram of the ear showing the location of the round window (RW) within the cochlea, the target injection site. Figure 2B is a diagram of the surgical site (Sx) for round window injection using entry through the ear bulla. Figure 2C is a photomicrograph taken with a surgical camera showing the round window through the bulla opening. Figure 2D shows successful penetration of the round window for AAV injection. Figure 2E is a photomicrograph taken with a surgical camera showing the exposed cisterna magna. Figure 2F is a higher-resolution image showing the needle successfully inserted into the cisterna magna for AAV injection. Figure 2G illustrates one route for how injection of AAV through the cisterna magna allows delivery of AAV through the perilymphatic duct and expression in target cell populations (e.g., inner hair cells [IHCs] / outer hair cells [OHCs]). FIG. 2H illustrates a second hypothetical pathway for AAV migration from the cisterna magna through cerebrospinal fluid (CSF) flowing along the internal auditory canal that holds the auditory nerve. [Figure 2B] See legend to Figure 2A. [Figure 2C] See legend to Figure 2A. [Figure 2D] See legend to Figure 2A. [Figure 2E] See legend to Figure 2A. [Figure 2F] See legend to Figure 2A. [Figure 2G] See legend to Figure 2A. [Figure 2H] See legend to Figure 2A. [Figure 3A]Figures 3A-3E show the cisternal injection of targeted AAV to express SOD1 protein. Figure 3A is a brightfield micrograph (2x magnification) showing a horizontal section (5 mm) of the cochlea. The line indicates the ROI hair cell complex, which retains two of the patent target cell types (inner and outer hair cells). The dashed box indicates the area shown in B. Figure 3B is a brightfield micrograph (10x magnification) showing a cross-section of a hair cell complex in the high-frequency region of the cochlea. Figure 3C is a brightfield micrograph (40x magnification) showing the hair cell complex within the box in Figure 3B. Here, the three outer hair cells connected to the inner hair cells and innervated by the primary afferent processes of the auditory ganglion, as well as their surrounding supporting cells, are clearly visible. Figure 3D is a fluorescent image (FITC, 40x magnification) of a hair cell complex from an animal that received 1 µL of AAV2.7m8.SOD1.GFP two weeks earlier. Here, moderate levels of GFP expression are seen by IHC and OHC, suggesting adequate expression of the SOD1 enzyme at this dose. Figure 3E shows a fluorescent image (FITC, 40x magnification) of a hair cell complex from an animal that received 2 µL of AAV2.7m8.SOD1.GFP two weeks prior. Here, higher levels of GFP expression are seen by IHC and OHC, indicating better expression of the SOD1 enzyme at this dose. [Figure 3B] See legend to Figure 3A. [Figure 3C] See legend to Figure 3A. [Figure 3D] See legend to Figure 3A. [Figure 3E] See legend to Figure 3A. [Figure 4A]Figures 4A-4D show that SOD1 expression protects against noise-induced hearing loss (increased hearing threshold). Figure 4A is a diagram illustrating the auditory brainstem response technique. The diagram (top left) shows the auditory pathways activated by sound stimuli at various frequencies and how these map to the signal resulting in the seven peaks of the ABR wave (bottom left). A diagram of subcutaneous electrode placement for performing the ABR (top right) and a representative example of the typical effect of noise exposure on ABR thresholds (deterioration of 10-30 dB SPL) (bottom right) are shown. Figure 4B is a scatter plot showing the ABR threshold shifts before and after noise exposure at each frequency (1, 2, 4, 8, and 16 kHz) for saline control animals (left) and SOD1 animals (right). The dashed lines indicate the typical thresholds at which mild to moderate hearing loss is diagnosed in humans (deterioration of 20-40 dB SPL). Figure 4C is a graph showing a direct comparison of pre- and post-noise threshold difference scores (post-minus pre-noise) between saline control and SOD1 animals. Arrows indicate data points where there was no threshold deterioration before or after noise at that frequency. Figure 4D is a representative example of threshold deterioration before and after noise for saline CTL animals (left) and SOD1 animals (right). ***p<0.001. [Figure 4B] See legend to Figure 4A. [Figure 4C] See legend to Figure 4A. [Figure 4D] See legend to Figure 4A. [Figure 5A]Figures 5A-5D show that SOD1 expression protects against noise-induced hearing loss and changes in ABR amplitude and latency. Figure 5A is a scatterplot showing a paired comparison of pre-noise amplitude at each frequency (1, 2, 4, 8, and 16 kHz) with post-noise amplitude at each frequency for saline control animals (left) and SOD1 animals (right). The dashed lines highlight the number of cases where amplitude was recorded before the noise but no sound-induced response was observed after the noise. Figure 5B is a graph showing a direct comparison of pre- and post-noise amplitude difference scores (post-pre) between saline control and SOD1 animals. Figure 5C is a scatterplot showing a paired comparison of pre-noise latency at each frequency (1, 2, 4, 8, and 16 kHz) with post-noise latency at each frequency for saline control animals (left) and SOD1 animals (right). The dashed lines highlight the number of cases where latency was recorded before the noise but no sound-induced response was observed after the noise. Figure 5D is a graph showing a direct comparison of pre- and post-noise latency difference scores (post-pre) between saline control and SOD1 animals. ***p<0.001. [Figure 5B] See legend to Figure 5A. [Figure 5C] See legend to Figure 5A. [Figure 5D] See legend to Figure 5A. [Figure 6] 1 is a map of the AAV2-7m8 vector used to target hair cells. [Figure 7]Figures 7A-7B show long-term safety and efficacy data for SOD1 neuroprotection against noise exposure. Figure 7A is a schematic diagram showing the normal effects of noise-induced oxidative stress in normal cells (left) and cells treated with SOD gene therapy (right). Increased SOD bioavailability provides neuroprotection from free radicals overproduced by noise exposure. Figure 7B is a line graph showing data from a group of animals (N8) that received an SOD1 injection (5 μl) and were monitored for hearing health for 5 months (left). These animals were then exposed to 110 dB sound for 2 weeks (2 hours). Here, SOD1 treatment provided strong neuroprotection even 5 months after transgene expression (center). This suggests that transgene expression is persistent and neuroprotection is stable over time. Finally, after monitoring hearing status for an additional 3 months, the animals were exposed to 110 dB noise for an additional 2 weeks (2 hours). Again, SOD1 treatment provided sustained neuroprotection from noise exposure, demonstrating robust efficacy. [Figure 8A] Figures 8A-8F show SOD1 neuroprotection against acute traumatic noise exposure. Figure 8A is a schematic diagram showing the effects of acoustic trauma (120 dB, 30 minutes) on the cochlear complex of the inner ear. Figure 8B is a line graph showing data for control animals exposed to 120 dB SPL noise for 30 minutes, 1 hour, and 2 hours. These animals developed significant persistent hearing loss over 4 weeks. Animals receiving SOD1 treatment before exposure had significant neuroprotection over time. Animals receiving SOD1 treatment after noise exposure (1 hour) had significant rescue from hearing loss over time (peaking approximately 2 weeks), corresponding to expression of the SOD1 transgene. Figure 8C is a scatter plot showing significant neuroprotection for pre-treated and post-noise-exposure treated animals. Figures 8D-8F are line graphs showing the data from Figure 8B broken down by frequency and group. [Figure 8B] See legend to Figure 8A. [Figure 8C] See legend to Figure 8A. [Figure 8D]See legend to Figure 8A. [Figure 8E] See legend to Figure 8A. [Figure 8F] See legend to Figure 8A. [Figure 9] Figures 9A-9C show neuroprotection by SOD1 from cisplatin-induced hearing loss over time. Figure 9A is a line graph showing the gradual accumulation of cisplatin (2 mg / kg IP weekly) leading to hearing loss in the control group and the neuroprotection provided by SOD1 gene therapy. Figure 9B shows data for the control group showing that hearing loss occurred at all frequencies when injury occurred, and Figure 9C shows that SOD1 pretreatment protected these animals at all frequencies throughout the cisplatin treatment regimen. [Figure 10] Figures 10A-10B show neuroprotection by SOD1 from cisplatin-induced hearing loss at low, mid, and high frequencies. Figure 10A is a line graph showing that in the control group, all frequency ranges are affected by cisplatin. SOD1 pretreatment provided nearly 100% neuroprotection across the entire frequency range. Figure 10B is a scatter plot showing significance in the SOD1 gene therapy group compared to the control group. DETAILED DESCRIPTION OF THE INVENTION

[0017] Detailed Description definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains. Although any methods and materials similar or equivalent to those described herein can be used in practicing the present invention, exemplary materials and methods are described herein. In describing and claiming the present invention, the following terminology is used:

[0018] It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting.

[0019] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. For example, "an element" means one element or more than one element.

[0020] "About," as used herein, when referring to a measurable value, e.g., an amount, a length of time, etc., is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and even more preferably ±0.1% from the specified value, as such variations are appropriate for practicing the disclosed methods.

[0021] As used herein, the term "AAV vector" refers to a polynucleotide vector that contains one or more genes of interest (or transgenes) flanked by AAV terminal repeats (ITRs).When AAV vectors are present in host cells transfected with one or more helper plasmids that encode and express rep protein and cap protein, and one or more proteins derived from adenovirus open reading frame E4 open reading frame 6, they can be produced and packaged into infectious viral particles.AAV vectors can be operably linked to promoter and enhancer sequences that can control the expression of the proteins encoded by AAV vectors.

[0022] As used herein, the term "AAV virion" or "AAV virus particle" or "AAV vector particle" refers to a viral particle composed of capsid proteins from at least one AAV serotype, enclosing a polynucleotide AAV vector.When a particle contains a heterologous polynucleotide (i.e., a polynucleotide other than the wild-type AAV genome, for example, a transgene to be delivered to mammalian cells), it is typically referred to as an "AAV vector particle" or simply an "AAV vector."Therefore, the production of AAV vector particles necessarily includes the production of such vectors, and therefore the vectors are contained within AAV vector particles.

[0023] The term "packaging," as used herein, refers to the intracellular process by which viral virions or particles (e.g., AAV virions or particles), specifically viral vector particles or virions, are assembled in a host cell. The "packaging" cell contains the polynucleotide (e.g., helper plasmid) and protein components necessary to assemble a functional viral virion.

[0024] By "agent" is meant any nucleic acid molecule, small molecule compound, antibody, or polypeptide, or fragment thereof.

[0025] "Alteration" or "change" means an increase or decrease. The alteration may be as little as 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, or as much as 40%, 50%, 60%, or even as much as 70%, 75%, 80%, 90%, or 100%.

[0026] As used herein, the term "autologous" is intended to refer to any material derived from the same individual that is subsequently reintroduced into that individual.

[0027] "Allogeneic" refers to a graft derived from a different animal of the same species.

[0028] "Xenogeneic" refers to a graft derived from an animal of a different species.

[0029] By "biological sample" is meant any tissue, cell, fluid, or other material derived from an organism.

[0030] As used herein, the term "cassette" or "expression cassette" or "regulatory cassette" refers to a separate nucleic acid vector consisting of a payload transgene and regulatory sequences (i.e., promoters, enhancers, terminators, etc.) that regulate its expression. Upon successful insertion into a host cell, the regulatory sequences allow for the transcription and translation of the payload transgene.

[0031] As used herein, the term "conservative sequence modification" refers to an amino acid modification that does not significantly affect or alter the binding characteristics of an antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibodies of the present invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are those in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0032] A "disease" is a state of health in an animal in which the animal is unable to maintain homeostasis and, if the disease does not go into remission, the animal's health continues to deteriorate. In contrast, an animal "disorder" is a state of health in which the animal is able to maintain homeostasis, but the animal's health is poorer than it would be in the absence of the disorder. If left untreated, the disorder does not necessarily cause a further decline in the animal's health.

[0033] "Effective amount" or "therapeutically effective amount," as used interchangeably herein, refers to an amount of a compound, formulation, material, or composition described herein that is effective to achieve a particular biological result or to provide a therapeutic or prophylactic benefit. Such results include, but are not limited to, anti-tumor activity as determined by any suitable means in the art.

[0034] "Encoding" refers to the inherent property of a particular nucleotide sequence of a polynucleotide, e.g., a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes that have either a distinct sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a distinct sequence of amino acids, and the biological properties resulting therefrom. Thus, a gene encodes a protein when transcription and translation of mRNA corresponding to that gene produces that protein in a cell or other biological system. Both the coding strand, which has a nucleotide sequence identical to the mRNA sequence and is generally provided in a sequence listing, and the non-coding strand, which is used as a template for transcription of the gene or cDNA, can be said to encode the protein or other product of that gene or cDNA.

[0035] As used herein, "endogenous" refers to any material that originates or is produced within an organism, cell, tissue, or system.

[0036] As used herein, the term "exogenous" refers to any material introduced from outside or produced outside an organism, cell, tissue, or system.

[0037] The term "expression," as used herein, is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter.

[0038] An "expression vector" refers to a vector containing a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art into which a recombinant polynucleotide has been incorporated, such as cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., Sendai virus, lentivirus, retrovirus, adenovirus, and adeno-associated virus).

[0039] "Homologous," as used herein, refers to the subunit sequence identity between two polymer molecules, e.g., between two nucleic acid molecules, e.g., two DNA molecules or two RNA molecules, or between two polypeptide molecules. When a subunit position in both molecules is occupied by the same monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, they are homologous at that position. The homology between two sequences is a direct function of the number of matching or homologous positions; for example, if half of the positions in two sequences (e.g., five positions in a polymer 10 subunits long) are homologous, the two sequences are 50% homologous; if 90% of the positions (e.g., 9 out of 10) are matched or homologous, the two sequences are 90% homologous.

[0040] As used herein, "identity" refers to the subunit sequence identity between two polymer molecules, specifically between two amino acid molecules, for example, between two polypeptide molecules. When two amino acid sequences have the same residue at the same position, for example, if a position in each of the two polypeptide molecules is occupied by arginine, they are identical at that position. Identity, or the degree to which two amino acid sequences have the same residue at the same position in an alignment, is often expressed as a percentage. The identity between two amino acid sequences is a direct function of the number of matching or identical positions; for example, if half of the positions in two sequences (e.g., five positions in a 10-amino acid-long polymer) are identical, the two sequences are 50% identical; if 90% of the positions (e.g., 9 out of 10) are matched or identical, the two amino acid sequences are 90% identical.

[0041] As used herein, "instructional material" includes publications, records, diagrams, or any other medium of expression that can be used to communicate the usefulness of the compositions and methods of the invention. The instructional material of the kits of the invention may, for example, be attached to a container containing the nucleic acids, peptides, and / or compositions of the invention, or may be shipped together with a container containing the nucleic acids, peptides, and / or compositions. Alternatively, the instructional material may be shipped separately from the container, with the intention that the instructional material and the compound will be used cooperatively by the recipient.

[0042] "Isolated" means altered or removed from the natural state. For example, a nucleic acid or peptide that is naturally present in a living animal is not "isolated," but the same nucleic acid or peptide that is partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or protein may exist in a substantially purified form, or may exist in a non-native environment, such as a host cell.

[0043] The term "modified," as used herein, means that the state or structure of a molecule or cell of the invention has been changed. Molecules can be modified in many ways, e.g., chemically, structurally, or functionally. Cells can be modified through the introduction of nucleic acids.

[0044] The term "modulate," as used herein, means to mediate a detectable increase or decrease in the level of a subject's response compared to the level of the subject's response in the absence of treatment or compound, and / or compared to the level of the response in an otherwise identical, untreated subject. This term encompasses interfering with and / or affecting a native signal or response in a subject, preferably a human, thereby mediating a beneficial therapeutic response.

[0045] In the context of the present invention, the following abbreviations for commonly occurring nucleobases are used: "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

[0046] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence encoding a protein or RNA can also include introns, to the extent that a nucleotide sequence that encodes a protein may, in some versions, contain introns.

[0047] The term "operably linked" refers to a functional linkage between a control sequence and a heterologous nucleic acid sequence that confers expression of the heterologous nucleic acid sequence. For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if it affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame.

[0048] "Parenteral" administration of the immunogenic compositions includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im), or intrasternal injection or infusion techniques.

[0049] The term "polynucleotide," as used herein, is defined as a chain of nucleotides. Furthermore, a nucleic acid is a polymer of nucleotides. Therefore, nucleic acid and polynucleotide, as used herein, are interchangeable. It is generally known to those skilled in the art that a nucleic acid is a polynucleotide that can be hydrolyzed into monomeric "nucleotides." Monomeric nucleotides can be hydrolyzed into nucleosides. As used herein, polynucleotides include, but are not limited to, any nucleic acid sequence obtained by any means available in the art, including, but not limited to, recombinant means, i.e., cloning a nucleic acid sequence from a recombinant library or a cellular genome, using conventional cloning technology and PCR™, and synthetic means.

[0050] As used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to compounds composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that may comprise a protein or peptide sequence. Polypeptides include any peptide or protein containing two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to, for example, short chains, commonly referred to in the art as peptides, oligopeptides, and oligomers, as well as longer chains, commonly referred to in the art as proteins, of which many types exist. "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, and fusion proteins, among others. Polypeptides include natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0051] The term "promoter," as used herein, is defined as a DNA sequence recognized by the synthetic machinery of a cell or introduced synthetic machinery necessary to initiate the specific transcription of a polynucleotide sequence.

[0052] As used herein, the term "promoter / regulatory sequence" refers to a nucleic acid sequence required for expression of a gene product operably linked to that promoter / regulatory sequence. In some cases, this sequence may be a core promoter sequence, and in other cases, this sequence may also include enhancer sequences and other control elements required for expression of the gene product. The promoter / regulatory sequence may, for example, be one that confers tissue-specific expression of the gene product.

[0053] A "constitutive" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes the production of the gene product in a cell under most or all physiological conditions of the cell.

[0054] An "inducible" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes production of the gene product in a cell substantially only when an inducer corresponding to that promoter is present in the cell.

[0055] A "tissue-specific" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoded or specified by a gene, causes production of a gene product in a cell substantially only if the cell is a cell of the tissue type corresponding to the promoter.

[0056] The term "epigenetic," as used herein, refers to genetic influences on gene expression that do not involve alterations in the DNA nucleotide sequence. Epigenetic regulation can enhance or inhibit the expression of affected genes and can involve chemical modifications of the deoxyribose backbone of DNA, or the association of DNA / histone protein complexes, or both.

[0057] The term "epigenetic regulator," as used herein, refers to a factor, enzyme, compound, or composition that acts to alter the epigenetic state of a particular DNA locus. Epigenetic regulators can induce or catalyze modifications of DNA-associated proteins or the chemical structure of the DNA itself.

[0058] The term "epigenetic tag" or "epigenetic marker" or "epigenetic mark" is used interchangeably herein to refer to specific chemical modifications made to DNA and DNA-associated proteins that result in epigenetic control of gene expression. Examples of epigenetic marks or epigenetic tags include, but are not limited to, the addition or removal of methyl or acetyl groups from CpG dinucleotides and histone proteins. The number and density of epigenetic tags or epigenetic marks can be correlated with the degree of epigenetic control that a particular DNA locus undergoes.

[0059] A "signal transduction pathway" refers to the biochemical relationships between various signaling molecules that play a role in transmitting a signal from one part of a cell to another part of the cell. The phrase "cell surface receptor" includes molecules and molecular complexes that can receive a signal and transmit the signal across the plasma membrane of a cell.

[0060] The term "specifically binds," as used herein with respect to antibodies, refers to an antibody that recognizes a specific antigen but does not substantially recognize or bind to other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more species. However, such cross-species reactivity does not, in itself, change the classification of the antibody as specific. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross-reactivity does not, in itself, change the classification of the antibody as specific. In some cases, the terms "specific binding" or "specifically binds" are used with respect to the interaction of an antibody, protein, or peptide with a second chemical species to mean that the interaction is dependent on the presence of a specific structure (e.g., an antigenic determinant or epitope) on the chemical species, e.g., the antibody recognizes and binds to a specific protein structure rather than proteins in general. If an antibody is specific for epitope "A," the presence of a molecule containing epitope A (or free, unlabeled A) in a reaction containing labeled "A" and the antibody will reduce the amount of labeled A that binds to the antibody.

[0061] The term "subject" is intended to include a living organism (e.g., a mammal) in which an immune response can be elicited. A "subject" or "patient," as used herein, can be a human or a non-human mammal. Non-human mammals include, for example, livestock and pets, such as non-human primates, ovine, bovine, porcine, canine, feline, and murine mammals. Preferably, the subject is a human.

[0062] "Target site" or "target sequence" refers to a genomic nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule can specifically bind, under conditions sufficient for binding to occur.

[0063] The term "therapeutic" as used herein means treatment and / or prophylaxis. A therapeutic effect is achieved by suppressing, ameliorating, or eradicating the disease state.

[0064] The terms "transfected" or "transformed" or "transduced," as used herein, refer to the process by which exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" cell is one that has been transfected, transformed, or transduced with exogenous nucleic acid. The cell includes the original host cell and its progeny.

[0065] The term "transgene" refers to genetic material that has been or is to be artificially inserted into the genome of an animal, particularly a mammal, more particularly a mammalian cell of a living animal.

[0066] The term "transgenic animal" refers to a non-human animal, generally a mammal, e.g., a transgenic mouse, in which a non-endogenous (i.e., heterologous) nucleic acid sequence is present in some of its cells as an extrachromosomal element or stably integrated into its germline DNA (i.e., the genomic sequence of most or all of its cells). The heterologous nucleic acid is introduced into the germline of such a transgenic animal, e.g., by genetic manipulation of the host animal's embryo or embryonic stem cells.

[0067] The term "knockout mouse" refers to a mouse in which an existing gene has been inactivated (i.e., "knocked out"). In some embodiments, the gene is inactivated by homologous recombination. In some embodiments, the gene is inactivated by replacement or disruption with an artificial nucleic acid sequence.

[0068] The term "treating" a disease, as used herein, means reducing the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject.

[0069] The phrases "under transcriptional control" or "operably linked," as used herein, mean that the promoter is in the correct location and orientation relative to the polynucleotide to regulate initiation of transcription by RNA polymerase and expression of the polynucleotide.

[0070] A "vector" is a composition that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid into a cell. Numerous vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes autonomously replicating plasmids or viruses. This term should also be interpreted to include non-plasmid, non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds, liposomes, etc. Examples of viral vectors include, but are not limited to, Sendai virus vectors, adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, lentivirus vectors, etc.

[0071] Ranges: Throughout this disclosure, various aspects of the invention may be presented in a range format. It should be understood that the description in range format is for convenience and brevity only and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all possible subranges as well as individual numerical values ​​within that range. For example, description of a range such as 1 to 6 should be considered to have specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numbers within that range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This is true regardless of the breadth of the range.

[0072] explanation In one aspect, the present invention is based on the unexpected observation that expression of the superoxide dismutase (SOD) gene in certain cells of the inner ear, such as hair cells, can protect these cells from damage and death caused by stress, such as the production of reactive oxygen species (ROS) due to overstimulation. In certain embodiments, the SOD gene is delivered to cells of the inner ear via a recombinant adeno-associated virus (AAV) vector that efficiently delivers a nucleic acid encoding the SOD gene operably linked to a promoter that drives its expression. In certain embodiments, the present invention includes a method for treating, ameliorating, and / or preventing progressive hearing loss, comprising expression of the SOD gene in cells of the inner ear (e.g., hair cells). Thus, expression of the SOD gene acts to protect cells from damage caused by ROS production.

[0073] Reactive oxygen species in progressive hearing loss As used herein, reactive oxygen species (ROS) refer to a variety of molecules that are derivatives of molecular oxygen, including hydroxyl radicals, superoxide anions, hydrogen peroxide, and singlet oxygen. In eukaryotic cells, ROS species are typically generated by mitochondria as a by-product of normal cellular processes, such as the production of adenosine triphosphate (ATP) via respiration. Moderate amounts of ROS produced by normal processes play an essential role in cell signaling and are rapidly metabolized or removed by endogenous antioxidant mechanisms. However, abnormally elevated ROS production due to high metabolic activity or in response to various cellular stresses can lead to molecular damage. Due to their unstable nature, ROS species easily react with other molecules and can damage proteins, lipids, and nucleic acids. Accumulation of ROS-related damage can lead to a state of oxidative distress, which can induce cell death pathways, such as apoptosis. The accumulation of ROS above normal levels and the subsequent induction of apoptosis are major causes of several diseases and aging, including numerous hearing loss pathologies.

[0074] Within the inner ear is the spiral cavity of the cochlea, which contains the organ of Corti, the core auditory component that generates nerve impulses in response to sound vibrations. The organ of Corti contains two types of sensory hair cells, inner and outer hair cells, which possess mechanosensory organs called stereocilia. Mechanical displacement of the stereocilia opens transducing ion channels, resulting in nerve impulses.

[0075] Hearing loss is a decrease in sound sensitivity and can be broadly divided into two types: acquired hearing loss and hereditary hearing loss. Both types of hearing loss typically involve the loss of inner and / or outer hair cells in the inner ear due to a dramatic reduction in the number of cells capable of converting mechanical vibrations (e.g., sound waves) into nerve impulses. There are many well-known types of acquired hearing loss, including, but not limited to, ototoxic drug-induced hearing loss, age-related hearing loss, trauma-induced hearing loss, inflammatory autoimmune-induced hearing loss, and noise-induced hearing loss. The two most common classes of drugs known to cause toxicity in inner ear cells (cochlea and vestibule) are aminoglycoside antibiotics and platinum-based anticancer chemotherapy drugs (e.g., cisplatin). Both classes induce hair cell damage by inducing excessive ROS production, which leads to apoptosis. Hair cell death by apoptosis leads to irreversible hearing loss. Recent studies have identified aminoglycosides as having a tendency to accumulate in hair cell mitochondria, where they inhibit mitochondrial ribosomal function and induce ion permeability. Platinum-based chemotherapy drugs induce short-term acute effects on transduction and voltage-dependent calcium currents, as well as long-lasting changes in potassium conductance within hair cell mitochondria that induce ROS production and apoptosis. Age-related hearing loss is typically associated with the accumulation of mutations in mitochondrial DNA, which encodes components of oxidative phosphorylation, leading to increased ROS production. Noise-induced hearing loss is a common cause of progressive hearing impairment and is often associated with loud environments, such as the military, concert venues, and industrial activities. Here, mechanical damage to the stereocilia triggers the production of ROS and the release of other apoptosis-inducing factors, which leads to cumulative hair cell loss.

[0076] In certain embodiments, the present disclosure includes compositions and methods that act to reduce or prevent increased ROS accumulation through the expression of superoxide dismutase proteins, which absorb and decompose ROS molecules. In certain embodiments, the present disclosure includes vectors, e.g., viral vectors, that can induce the expression of superoxide dismutase proteins in inner ear cells (e.g., inner hair cells and outer hair cells). In certain other embodiments, the present disclosure includes vectors, e.g., viral vectors, that can induce the expression of superoxide dismutase proteins in other cochlear cells (e.g., Deiters cells, Hansen cells, Claudius cells, primary afferent neurons, supporting cells, limbus cells, spiral ligament cells, stria vascularis cells), and type I and type II vestibular hair cells within the vestibular peripheral organs. Thus, the expression of superoxide dismutase protein absorbs excess ROS molecules and prevents the induction of apoptosis, thus preventing both hearing loss associated with inner ear cell death and loss of vestibular hair cells due to aging or ototoxicity.

[0077] Superoxide dismutase protein Superoxide dismutase (SOD) is a family of enzymes whose primary function is to catalyze the dismutation of superoxide anion radicals, a common component of ROS, into the more inactive forms molecular oxygen and hydrogen peroxide (which are decomposed by other enzymes, e.g., catalase). Three members of the SOD family have been described in mammalian cells. SOD1, or CuZn-SOD, was the first to be characterized and is a copper- and zinc-containing homodimer typically expressed in the cytoplasmic space within cells. SOD2, or Mn-SOD2, is a manganese-containing enzyme that exists as a tetramer and is targeted to mitochondria. SOD3, or EC-SOD, is a copper- and zinc-containing tetramer typically secreted into the extracellular environment. SOD proteins are widely expressed in both prokaryotic and eukaryotic cells due to their potent antioxidant properties. Loss of SOD protein function is associated with numerous genetic disorders, such as familial amyotrophic lateral sclerosis caused by mutations in SOD1, and certain cardiomyopathies.

[0078] In certain embodiments, the present disclosure includes compositions and methods for preventing progressive hearing loss and arresting the progression of hearing loss through the expression of SOD proteins in cells of the inner ear (e.g., inner and outer hair cells), which act to degrade elevated levels of ROS caused by chemical factors, overstimulation, or other environmental or genetic factors. Furthermore, in certain embodiments, the present disclosure includes compositions and methods for preventing progressive peripheral vestibular loss and arresting the progression of vestibular loss through the expression of SOD proteins in cells of the vestibular peripheral organs of the inner ear (e.g., type I and type II vestibular hair cells), which act to degrade elevated levels of ROS caused by chemical factors, overstimulation, or other environmental or genetic factors. In certain embodiments, expression of SOD proteins is induced by transducing inner ear cells with nucleic acids encoding SOD proteins through the use of recombinant AAV viral vectors. In certain embodiments, the SOD protein is SOD1 protein. In certain embodiments, the SOD protein is SOD2 protein. In certain embodiments, the SOD protein is SOD3 protein. It is also contemplated that the methods and compositions of the present invention may include any combination of SOD1, SOD2, and SOD3 proteins, and variants thereof, to optimize protection of inner ear cells from oxidative damage.

[0079] AAV vectors AAV is a relatively small, non-enveloped virus with a genome of approximately 4 kb flanked by inverted terminal repeats (ITRs). The genome contains two open reading frames, one of which provides proteins necessary for replication, and the other provides components required for viral capsid assembly. Because adenovirus provides helper proteins essential for packaging the AAV genome into virions, wild-type AAV is typically found in the presence of adenovirus. Therefore, AAV production requires co-infection with adenovirus and relies on three main elements: the genome flanked by ITRs, the open reading frame, and adenovirus helper genes. Due to its non-pathogenic ability to easily infect human cells, AAV has been extensively studied as a vector for gene delivery. AAV is readily available, and its use as a vector for gene delivery has been described, for example, in Muzyczka, 1992; U.S. Patent No. 4,797,368; and PCT Publication WO 91 / 18088. The construction of AAV vectors has been described in numerous publications, for example, Lebkowski et al., 1988; Tratschin et al., 1985; Hermonat and Muzyczka, 1984.

[0080] AAV-based vector systems typically separate the viral AAV genes, adenovirus-derived helper genes, and transgene payload into two or three separate plasmids. The three-plasmid system consists of an AAV helper plasmid containing the rep (replication) and cap (capsid) genes, an adenovirus helper plasmid containing at least the E2a, E4, and VA (virus-associated) RNAs, and a payload plasmid containing the transgene and associated promoter and enhancer sequences flanked by ITR sequences. The helper plasmid does not contain ITRs to prevent packaging of a functional infectious viral genome.

[0081] The two-plasmid system simplifies viral vector production by combining the AAV rep and cap genes with adenovirus helper genes in a single plasmid, reducing the number of transfected plasmids. Dedicated packaging cell lines engineered to express the AAV / helper genes prior to introduction of the payload plasmid are often used.

[0082] Successful gene therapy requires efficient infection of target tissues and the establishment of long-term gene expression. AAV vectors can successfully infect and transduce a wide variety of cell and tissue types, including the brain, liver, and muscle, among others, and have the ability to infect both dividing and quiescent cells. Furthermore, AAV-mediated tissue transduction has been shown to result in long-term transgene expression for more than 1.5 years in animal models, such as dogs, mice, and hamsters.

[0083] The tissue tropism of AAV vector particles is influenced by the serotype of the capsid protein, but the receptors and co-receptors to which the capsid protein bind are often poorly understood and may be expressed by multiple tissue types. For example, one of the most well-studied serotypes, AAV2, has a binding affinity primarily for heparan sulfate proteoglycans (HSPGs), and therefore, in humans, has tropism for eye, brain, lung, liver, muscle, and joint tissues. Similarly, AAV1, 4, 5, and 6 have a binding affinity primarily for sialic acid and a tropism for neural tissues, while AAV5 and 8 share a tropism for skeletal muscle. Thus, the serotype of the AAV capsid protein can be selected to target the payload nucleic acid (e.g., regulatory cassette) of the AAV vector to a specific tissue or cell type. Alterations or modifications to the capsid protein structure can also alter the tissue or cell tropism and affinity of the resulting AAV vector particle.

[0084] In certain embodiments, the present disclosure includes AAV vectors comprising capsid proteins derived from AAV9 and its variants. AAV9 and capsid proteins based on AAV9 can transduce muscle, liver, and lung tissues approximately 100 times more efficiently than AAV2, and can also cross the blood-brain barrier.

[0085] In certain embodiments, the present disclosure includes an AAV vector comprising a capsid protein derived from AAV2. One non-limiting example of a derivative of AAV2 capsid protein is AAV2-7m8. AAV2-7m8 contains a 10-amino acid peptide inserted at position 588 of the AAV2 capsid protein sequence, which is involved in the binding of AAV2 to its main receptor, heparan sulfate proteoglycan, and was first characterized as having increased infection efficiency of mouse photoreceptors. AAV2-7m8 has the ability to highly efficiently infect both inner and outer hair cells of the cochlea and type I and type II vestibular hair cells of the vestibular peripheral organs.

[0086] The AAV vectors and compositions expressing the SOD protein of the present disclosure may be any naturally occurring, modified, hybrid, or engineered AAV capsid protein, including, but not limited to, among others, that provides the desired tissue tropism (e.g., inner and outer hair cells of the inner ear, supporting cells, stria vascularis cells, other cochlear cells, and type I and type II vestibular hair cells of the vestibular peripheral apparatus). It is contemplated that capsid proteins may be used with, but not limited to, AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV12, AAV-B1, AAV-DJ, AAV-Retro, AAVrh8, AAVrh10, AAVrh25, Anc80L65, LK03, AAVrh18, AAVrh74, AAVrh32.33, AAVrh39, AAVrh43, Oligo001, PHP-B, and Spark100. One of skill in the art will be able to select an appropriate capsid protein for use in connection with the present disclosure based on the desired target tissue or cell type.

[0087] AAV2-7m8 (SEQ ID NO: 4) TIFF2025532136000002.tif130146TIFF2025532136000003.tif224146TIFF20255321360 00004.tif224146TIFF2025532136000005.tif224146TIFF2025532136000006.tif210146

[0088] CAG (SEQ ID NO: 5) TIFF2025532136000007.tif137146

[0089] Meriones unguiculatus superoxide dismutase 1 (SOD1) mRNA (SEQ ID NO: 6) TIFF2025532136000008.tif83146

[0090] eGFP (SEQ ID NO: 7) TIFF2025532136000009.tif97146

[0091] Woodchuck hepatitis virus (WHV) posttranscriptional regulatory element (WPRE) (SEQ ID NO:8) TIFF2025532136000010.tif70146

[0092] SV40 promoter (SEQ ID NO:9) TIFF2025532136000011.tif77146

[0093] Nucleic acids and vectors The present disclosure provides a polynucleic acid encoding an AAV vector. In certain embodiments, the AAV vector comprises a nucleic acid encoding the SOD1 protein (also known as ALS and ALS1). Examples of sequences encoding the SOD1 protein include, but are not limited to, NCBI Gene, 6647; HGNC, 11179; OMIM, 147450; RefSeq, NM_000454; and UniProt, P00441. In certain embodiments, the nucleic acid encoding the SOD1 protein comprises Homo sapiens chromosome 21, GRCh38.p14 primary assembly NCBI reference sequence: NC_000021.9>NC_000021.9:31659693-31668931. In certain embodiments, the nucleic acid encoding the SOD1 protein is derived from Meriones unguiculatus. In certain embodiments, the SOD1 protein is encoded by a nucleic acid comprising a polynucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:1 or SEQ ID NO:6. In certain embodiments, the SOD1 protein is encoded by a nucleic acid comprising the nucleotide sequence set forth in SEQ ID NO:1 or SEQ ID NO:6.

[0094] SOD1 DNA(NM_000454)(SEQ ID NO:1) TIFF2025532136000012.tif110146

[0095] In certain embodiments, the present invention also provides AAV vectors comprising a nucleic acid encoding the SOD2 protein (also known as Mn-SOD, IPO-B, and MVCD) or an isoform or fragment thereof. Examples of sequences encoding the SOD2 protein or an isoform or fragment thereof include, but are not limited to, NCBI Gene 6648, HGNC 11180, OMIM 147460, RefSeq NM_000636, and UniProt P04179. In certain embodiments, the nucleic acid encoding the SOD2 protein or an isoform or fragment thereof comprises Homo sapiens chromosome 6, GRCh38.p14 primary assembly NCBI Reference Sequence: NC_000006.12>NC_000021.9:31659693-31668931. In certain embodiments, the SOD2 protein is encoded by a nucleic acid comprising a polynucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:2. In certain embodiments, the SOD2 protein is encoded by a nucleic acid comprising the nucleotide sequence set forth in SEQ ID NO:2. In certain embodiments, the SOD2 protein is an isoform or fragment of the SOD2 protein that retains some or all of the function of the full-length SOD2 protein. Non-limiting examples of SOD2 isoforms or fragments encompassed by the present invention include sequences encoding SOD2 proteins with truncations at either the N- or C-terminus. In certain embodiments, these truncations are made to reduce the overall size of the sequence encoding the SOD2 protein, for example, to enable packaging into a size-limited vector (e.g., an AAV vector).

[0096] SOD2 DNA(NM_000636)(SEQ ID NO:2) TIFF2025532136000013.tif224146TIFF2025532136000014.tif224146TIFF2025532136000015.tif224146TIFF2025532136000016.tif224146 TIFF2025532136000017.tif224146TIFF2025532136000018.tif224146TIFF2025532136000019.tif224146TIFF2025532136000020.tif117146

[0097] SOD2 gerbil sequence (SEQ ID NO:10) TIFF2025532136000021.tif90146TIFF2025532136000022.tif224146TIFF2025532136000023.tif130146

[0098] In certain embodiments, the present invention also provides AAV vectors comprising a nucleic acid encoding an SOD3 protein (also known as MEC-SOD and MGC20077). Examples of sequences encoding an SOD3 protein include, but are not limited to, NCBI Gene 6649, HGNC 11181, OMIM 185490, RefSeq NM_003102, and UniProt P08294. In certain embodiments, the nucleic acid encoding an SOD2 protein comprises Homo sapiens chromosome 4, GRCh38.p14 primary assembly NCBI Reference Sequence: NC_000004.12>NC_000004.12:24795573-24800842. In certain embodiments, the SOD3 protein is encoded by a nucleic acid comprising a polynucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 3. In certain embodiments, the SOD3 protein is encoded by a nucleic acid comprising the nucleotide sequence set forth in SEQ ID NO: 3.

[0099] SOD3 DNA(NM_003102)(SEQ ID NO:3) TIFF2025532136000024.tif170146

[0100] SOD3 variant 1 Mongolian gerbil (SEQ ID NO: 11) TIFF2025532136000025.tif37145TIFF2025532136000026.tif210146

[0101] SOD3 variant 2 Mongolian gerbil (SEQ ID NO: 12) TIFF2025532136000027.tif204146

[0102] Gene transfer systems and adeno-associated viruses (AAV) Gene transfer systems, such as those described in this disclosure, rely on vectors or vector systems to deliver gene constructs to target cells. Methods for introducing nucleic acids into cells, such as cells of the inner ear, include physical, biological, and chemical methods. Physical methods for introducing polynucleotides, such as RNA, into host cells include calcium phosphate precipitation, lipofection, biolistics, microinjection, electroporation, and the like. RNA can be introduced into target cells using commercially available methods, such as electroporation (Amaxa Nucleofector-II [Amaxa Biosystems, Cologne, Germany]), (ECM 830 (BTX) [Harvard Instruments, Boston, Mass.]), or Gene Pulser II (BioRad, Denver, Colo.), Multiporator (Eppendort, Hamburg, Germany). RNA may be introduced into cells using cationic liposome-mediated transfection using lipofection, using polymer encapsulation, using peptide-mediated transfection, or using a biolistic particle delivery system, e.g., a "gene gun" (see, e.g., Nishikawa, et al. Hum Gene Ther., 12(8):861-70 (2001)).

[0103] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, such as oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., artificial membrane vesicle).

[0104] Suitable lipids for use are available from commercial sources. For example, dimyristyl phosphatidylcholine ("DMPC") is available from Sigma (St. Louis, MO), dicetyl phosphate ("DCP") is available from K&K Laboratories (Plainview, NY), cholesterol ("Chol") is available from Calbiochem-Behring, and dimyristyl phosphatidylglycerol ("DMPG") and other lipids are available from Avanti Polar Lipids, Inc. (Birmingham, AL). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at approximately -20°C. Chloroform is used as the only solvent because it evaporates more readily than methanol.

[0105] "Liposome" is a generic term encompassing a variety of unilamellar and multilamellar lipid vesicles formed by the formation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as vesicular structures with a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-reorganization before forming a closed structure, trapping water and dissolved solutes between the lipid bilayers (Ghosh et al., (1991) Glycobiology 5:505-10). However, compositions with structures in solution that differ from the typical vesicular structure are also encompassed. For example, lipids may adopt a micellar structure or simply exist as heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also contemplated.

[0106] Biological methods for introducing a polynucleotide of interest into host cells include the use of DNA vectors and RNA vectors.Viral vectors, particularly retroviral vectors, have become the most widely used method for inserting genes into mammalian, for example, human cells.Other viral vectors can be derived from lentivirus, poxvirus, herpes simplex virus I, adenovirus, adeno-associated virus, etc.See, for example, U.S. Patent No. 5,350,674 and 5,585,362.

[0107] Currently, the most efficient and effective way to achieve the transfer of gene constructs into living cells is through the use of vector systems based on replication-defective viruses.Some of the most effective vectors known in the art are based on adeno-associated virus (AAV).AAV is a small virus of the Parvoviridae family, which is replication-defective, is known not to cause human disease, only causes very mild immune response, can infect both actively dividing cells and quiescent cells, and stably persists in an extrachromosomal state without being integrated into the genome of target cells, making it an attractive vector for gene transfer.In certain embodiments, the present disclosure provides an AAV vector comprising the dCas9-based CRISPRi system of the present disclosure.

[0108] Regardless of the method used to introduce nucleic acid into cell, various assays can be carried out to confirm the presence of nucleic acid in cell.Such assays include, for example, "molecular biological" assays well known to those skilled in the art, such as Southern blotting and Northern blotting, RT-PCR and PCR; "biochemical" assays, for example, detecting the presence or absence of specific peptides, for example, by immunological means (ELISA and Western blot), or by the assays described herein to identify agents within the scope of the present disclosure.

[0109] method In one aspect, the present disclosure includes a method for treating, ameliorating, and / or preventing progressive hearing loss in a subject in need thereof. In certain embodiments, the method includes administering to the subject an effective amount of an adeno-associated virus (AAV) vector comprising a nucleic acid encoding a superoxide dismutase (SOD) protein operably linked to a promoter, wherein the SOD protein is expressed in cells of the inner ear, thereby treating, ameliorating, and / or preventing progressive hearing loss. In certain embodiments, the AAV vector of the present disclosure targets inner ear cells (e.g., hair cells). In certain embodiments, the AAV vector comprises a capsid protein derived from AAV2. In certain embodiments, the capsid protein is AAV2-7m8. In certain embodiments, the AAV vector comprises a capsid protein derived from AAV9. In certain embodiments, AAV vector is administered via lumbar puncture, intravenous injection, nasal / olfactory delivery, pulmonary delivery, middle ear injection, round window diffusion, round window injection, oval window injection, intracochlear electrode or drug delivery system, and / or labyrinth disruption injection.In certain embodiments, AAV vector is administered together with an effective amount of the agent that destroys blood-brain barrier.

[0110] In another aspect, the present disclosure includes a method for preventing, reversing, and / or minimizing progressive hearing loss in a subject in need thereof. In certain embodiments, the method includes administering to the subject an effective amount of an adeno-associated virus (AAV) vector comprising a nucleic acid encoding a superoxide dismutase (SOD) protein operably linked to a promoter, wherein the SOD protein is expressed in cells of the inner ear, thereby preventing, reversing, and / or minimizing progressive hearing loss.

[0111] In certain embodiments, the inner ear cells are hair cells, and expression of an SOD protein protects the cells from oxidative damage. In certain embodiments, the SOD protein is selected from the group consisting of SOD1, SOD2, and SOD3.

[0112] In certain embodiments, the cochlear cells are Deiters' cells, Hansen's cells, Claudius' cells, primary afferent neurons, supporting cells, limbal cells, spiral ligament cells, and / or stria vascular cells, and the expression of SOD protein protects the cells from oxidative damage. In certain embodiments, the vestibular cells are type I and type II vestibular hair cells of the vestibular peripheral organ, and the expression of SOD protein protects the cells from oxidative damage. In certain embodiments, the SOD protein is selected from the group consisting of SOD1, SOD2, and SOD3.

[0113] In certain embodiments, the genetic disease or disorder treated by the method of the present disclosure is a disease or disorder related to hearing loss.Non-limiting examples of hearing loss diseases or disorders that can be treated by the method of the present disclosure include, but are not limited to, ototoxic drug-induced hearing loss (ODIHL), age-related hearing loss (ARHL), trauma-induced hearing loss, inflammatory autoimmune-induced hearing loss, and noise-induced hearing loss (NIHL).Non-limiting examples of vestibular loss diseases or disorders that can be treated by the method of the present disclosure include, but are not limited to, ototoxic drug-induced vestibular loss, age-related vestibular loss (age-related balance disorder), inflammatory autoimmune vestibular loss, and overstimulation-induced vestibular loss.Among these, noise-induced hearing loss is the main cause of hearing loss and is associated with overstimulation or physical damage to inner ear cells (e.g., inner hair cells and outer hair cells), which leads to oxidative damage and death of these cells. Ototoxic drug-induced hearing loss and vestibular loss can occur as a side effect of treatment with aminoglycoside antibiotics, such as gentamicin, tobramycin, amikacin, plazomicin, streptomycin, neomycin, and paromomycin, among others. ODIHL can also be an unwanted side effect of anticancer treatment with certain platinum-based drugs, such as cisplatin, carboplatin, and oxaliplatin, among others. Age-related hearing loss and vestibular loss can be attributed to a number of factors, including certain environmental exposures, hereditary genetics, and other medical disease-related factors.

[0114] In certain embodiments, the AAV vector is administered with an effective amount of an agent that disrupts the blood-brain barrier. Such agents are used to facilitate the access of therapeutic agents, such as small molecules, viral vectors (e.g., AAV vectors), and other biological molecules, such as proteins, antibodies, signaling molecules, etc., to tissues of the central nervous system (CNS), including the inner ear, which are otherwise separated from the circulatory system by the blood-brain barrier (BBB). The BBB is located at the interface between blood and CNS tissues and comprises a semipermeable membrane barrier composed of a complex system of endothelial cells, astrocytes, pericytes, and perivascular mast cells. Reversible methods for permeabilizing the BBB sufficiently to allow the AAV vector of the present disclosure to access inner ear tissues include, but are not limited to, chemical methods using mannitol treatment to induce osmotic changes that allow greater BBB permeability, physical methods using focused ultrasound, or modification of AAV capsid proteins to allow better diffusion through the BBB.

[0115] In another aspect, the present disclosure includes a method for reversing, minimizing, and / or preventing chemotherapy treatment-associated hearing loss in a subject in need thereof, the method comprising administering to the subject an effective amount of an adeno-associated virus (AAV) vector comprising a nucleic acid encoding a superoxide dismutase (SOD) protein operably linked to a promoter, wherein the SOD protein is expressed in inner ear cells of the subject, thereby reversing, minimizing, and / or preventing chemotherapy treatment-associated hearing loss in the subject.

[0116] In certain embodiments, the chemotherapy-induced hair cell loss is a platinum drug or a platinum-based drug. Platinum drugs or platinum-based drugs are antitumor chemotherapy drugs commonly used to treat various types of cancer, including cisplatin, carboplatin, oxaliplatin, nedaplatin, and lobaplatin, among others. Malignant tumors commonly treated with platinum drugs include ovarian, endometrial, bladder, testicular, and head and neck cancer, among others. They can also increase survival in some cases of squamous cell carcinoma and osteosarcoma. Platinum drugs form DNA adducts, which ultimately lead to the activation of p53-dependent and p53-independent apoptotic pathways, resulting in cell death. In the context of the present disclosure, cochlear cells are sensitive to platinum drug treatment, and progressive, permanent hearing loss is a common serious side effect of anticancer therapy with these drugs. In certain embodiments, the AAV vector of the present disclosure that expresses SOD protein is administered to subject before treatment with one or more platinum drugs to combat or prevent hearing loss.In certain embodiments, the platinum drug is cisplatin.It is also expected that the AAV vector of the present disclosure that expresses SOD protein can be used to treat chemotherapy-related hearing loss caused by treatment with any platinum-based chemotherapy drug, which causes increased oxidative damage in inner ear cells.

[0117] In certain embodiments, the inner ear cells are hair cells. In certain embodiments, the inner ear cells are Deiters cells, Hansen cells, Claudius cells, primary afferent neurons, supporting cells, limbal cells, spiral ligament cells, and / or stria vascularis cells. In certain embodiments, the vestibular cells are type I hair cells, type II hair cells, primary afferent neurons, and / or supporting cells. In certain embodiments, the expression of SOD protein protects cells from oxidative damage.

[0118] In certain embodiments, the AAV vector targets inner ear cells. In certain embodiments, the AAV vector comprises a capsid protein derived from AAV2. In certain embodiments, the capsid protein is AAV2-7m8. In certain embodiments, the AAV vector comprises a capsid protein derived from AAV9. In certain embodiments, the AAV vector is administered via middle ear injection, round window diffusion, round window injection, oval window injection, labyrinthectomy injection, intracochlear electrode or drug delivery system, oral administration, inhalation, nasal administration, nebulization, intravenous injection, intramuscular injection, intrathecal injection, intrapleural delivery, intracisternal injection, subcutaneous injection, and / or transdermal injection. In certain embodiments, the method further comprises administering to the subject an effective amount of an agent that disrupts the blood-brain barrier. In certain embodiments, the SOD protein is selected from the group consisting of SOD1, SOD2, and SOD3.

[0119] Pharmaceutical Compositions The pharmaceutical composition of the present disclosure can be combined with one or more pharmaceutically or physiologically acceptable carriers, diluents, adjuvants or excipients as described herein.Such compositions can include buffer solutions, such as neutral buffered saline, phosphate buffered saline, etc.; carbohydrates, such as glucose, mannose, sucrose, or dextran, mannitol; proteins; polypeptides or amino acids, such as glycine; antioxidants; chelating agents, such as EDTA or glutathione; adjuvants (such as aluminum hydroxide); and preservatives.The composition of the present disclosure is preferably formulated for intravenous administration.

[0120] The pharmaceutical compositions of the present disclosure may contain the AAV vector particles described herein in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may include buffers, such as neutral buffered saline, phosphate buffered saline (PBS), etc.; carbohydrates, such as glucose, mannose, sucrose, or dextran, mannitol; proteins; polypeptides or amino acids, such as glycine; antioxidants; chelating agents, such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The compositions of the present disclosure may be formulated for multiple administration routes, including middle ear injection, round window diffusion, round window injection, oval window injection, labyrinthectomy injection, intracochlear electrode or drug delivery system, oral, inhalation, nasal, nebulized, intravenous, intramuscular, intrathecal, intrapleural, cisternal, subcutaneous, and / or transdermal injection. The pharmaceutical compositions of the present disclosure may be administered in a manner appropriate for the disease to be treated (or prevented). The amount and frequency of administration will be determined by factors such as the patient's condition, the type and severity of the patient's disease, and the type and functional nature of the patient's immune response to the phage particles, but appropriate dosages can be determined through clinical trials.

[0121] The AAV vector particles of the present disclosure can be administered at dosages, routes, and times determined in appropriate preclinical and clinical experiments and tests. Administration of the AAV vector particles of the present disclosure can be combined with other methods useful for treating the desired disease or condition, as determined by one of skill in the art.

[0122] In certain embodiments, the effective dose range is measured in units known to those skilled in the art as appropriate for describing AAV vector particle doses. In some embodiments, the effective dose range of the vaccine or therapeutic compound of the present disclosure is measured in transducing units (TU) / kg / dose or genome copies (GC) / kg / dose or particles / kg / dose. In some embodiments, the dose provided to a patient is about 10 6 ~10 14In some embodiments, the dosage provided to the patient is about 10 6 ~10 14 GC / kg. In some embodiments, the effective dose range is based on colony forming units (CFU), 50% tissue culture infectious dose (TCID 50 ), and combinations thereof.

[0123] Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present disclosure may be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration without toxicity to the patient.

[0124] The therapeutically effective amount or dose of a compound of the present disclosure will depend on the age, sex, and weight of the patient, the patient's current medical condition, and the progression of the disease or disorder contemplated in the present disclosure.

[0125] In humans, a medical practitioner, e.g., a physician or delegated advanced practice provider, having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or delegated advanced practice provider can start doses of a compound of the present disclosure utilized in a pharmaceutical composition at a level lower than that required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

[0126] In the case of animals, a medical practitioner, e.g., a physician, or a designated advanced practice provider, or a veterinarian, having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician, or a designated advanced practice provider, or a veterinarian can start doses of a compound of the present disclosure utilized in a pharmaceutical composition at a level lower than that required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

[0127] In certain embodiments, the compositions of the present disclosure are administered to patients in a range of doses ranging from 1 to 5 or more times daily. In other embodiments, the compositions of the present disclosure are administered to patients in a range of doses, including, but not limited to, once daily, every two days, once every three days to once a week, and once every two weeks. It will be readily apparent to those skilled in the art that the frequency of administration of the various combination compositions of the present disclosure will vary from individual to individual depending on many factors, including, but not limited to, age, disease or disorder being treated, gender, overall health, and other factors. Therefore, the present disclosure should not be construed as limited to any particular dosing regimen, and the exact dosage and composition to be administered to any patient will be determined by the attending physician, taking into account all other factors related to the patient.

[0128] The dosage size can be adjusted according to the weight, age and stage of the patient to be treated.AAV vector particles can be administered multiple times at these dosages.AAV vector particles can be administered by using injection techniques that are generally known in the field of immunotherapy or vaccinology.The optimal dosage and treatment plan for specific patient can be easily determined by those skilled in the medical field by monitoring the patient for signs of disease and adjusting treatment accordingly.

[0129] The administration of the AAV vector particle composition of the present disclosure can be carried out in any convenient manner known to those skilled in the art.The AAV vector particle of the present disclosure can be administered to a subject by aerosol inhalation, injection, oral ingestion, infusion, implantation, or transplantation.The compositions described herein can be administered to a subject or patient via middle ear injection, round window diffusion, round window injection, oval window injection, labyrinthectomy injection, intracochlear electrode or drug delivery system, oral, inhalation, nasal, nebulization, intravenous injection, intramuscular injection, intrathecal injection, intrapleural injection, cisternal injection, subcutaneous injection, and / or transdermal injection.It should be understood that the methods and compositions useful in the present disclosure are not limited to the specific formulations described in the examples.

[0130] In certain embodiments, the compositions of the present disclosure are formulated using one or more pharmaceutically acceptable excipients or carriers.In certain embodiments, the pharmaceutical compositions of the present disclosure comprise a therapeutically effective amount of the compounds of the present disclosure and pharmaceutically acceptable carriers.

[0131] The carrier can be a solvent or dispersion medium containing, for example, saline, buffered saline, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by using a coating such as lecithin, maintaining the required particle size in the case of dispersion, and by using surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it is advisable to include isotonic agents, for example, sugars, sodium chloride, or polyalcohols such as mannitol and sorbitol, in the composition.

[0132] The formulations may be utilized in admixture with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for any suitable mode of administration known to those skilled in the art. Pharmaceutical preparations may be sterilized and, if desired, may be mixed with auxiliary substances, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorants, flavors, and / or aromatic substances. If desired, they may be combined with other active agents, such as analgesics.

[0133] The practice of the present disclosure will utilize, unless otherwise indicated, conventional techniques of molecular biology, microbiology, cell biology, biochemistry, and immunology (including recombinant techniques) that are well within the skill of the art. Such techniques are fully explained in the literature, e.g., "Molecular Cloning: A Laboratory Manual," fourth edition (Sambrook, 2012); "Oligonucleotide Synthesis" (Gait, 1984); "Culture of Animal Cells" (Freshney, 2010); "Methods in Enzymology" and "Handbook of Experimental Immunology" (Weir, 1997); "Gene Transfer Vectors for Mammalian Cells" (Miller and Calos, 1987); "Short Protocols in Molecular Biology" (Ausubel, 2002); "Polymerase Chain Reaction: Principles, Applications and Troubleshooting" (Babar, 2011); and "Current Protocols in Immunology" (Coligan, 2002). These techniques are applicable to the production of the polynucleotides and AAV particles of the present disclosure and, therefore, may be considered in the manufacture and practice of the present disclosure.

[0134] It should be understood that the methods and compositions that may be useful in this disclosure are not limited to the specific formulations described in the examples. The following examples are provided so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the cells, methods of expansion and culture, and treatment methods of the present disclosure, and are not intended to limit the scope of what the inventors regard as the present disclosure. [Example]

[0135] Experimental example The present disclosure will be further described in detail by reference to the following experimental examples. These examples are provided for illustrative purposes only and, unless otherwise specified, are not intended to be limiting. Therefore, the present disclosure should in no way be construed as being limited to the following examples, but should be construed to encompass any and all variations that become evident as a result of the teachings provided herein.

[0136] Without further description, it is believed that one of ordinary skill in the art can, using the foregoing description and the following illustrative examples, make and utilize the disclosed compounds and practice the claimed methods. The following examples therefore specifically illustrate preferred embodiments of the disclosure, and are not to be construed as limiting in any way the remainder of the disclosure.

[0137] Example 1: Expression of SOD1 before noise exposure preserves hair cell proliferation One major cause of both progressive and noise-induced hearing loss involves hair cell death and damage due to oxidative stress. Prolonged exposure to high or moderate levels of noise can lead to the overproduction of oxidative species known as free radicals. These consist primarily of reactive oxygen species (ROS, e.g., peroxidases), which can damage various intracellular components and cell membranes. Hair cells possess a natural protective enzyme against ROS called superoxide dismutase (SOD). There are three versions of SOD that function in different regions of the cell: SOD1 functions internally, SOD2 functions within mitochondria, and SOD3 functions extracellularly. However, under normal conditions, all three protect cellular components from oxidative damage (see, for example, Figure 1A). Figure 1B shows an example of how noise increases oxidative stress. This leads to an increase in ROS, which overwhelms the natural SOD1 enzyme levels. The disclosed approach uses AAV technology targeted to inner and outer hair cells to upregulate the production of SOD enzymes (here, SOD1) to address increased oxidative stress (Figure 1B). Figure 1D provides a hypothetical example of how this gene therapy would work in vivo during a period of noise exposure.

[0138] To deliver AAV to its intended target within the inner ear (inner and outer hair cells), promoters highly selective for these cell types are used (e.g., 7m8). Two delivery methods into the cerebrospinal fluid have been developed that achieve this goal (Figure 2). In gerbils, access to the round window of the cochlea allows for direct injection of AAV into the inner ear (Figure 2A). In humans, this can also be achieved through the middle ear. In gerbils, this is easily accessed through the bulla (Figure 2B). The bulla above the cochlea is removed (Figure 2C), and a gastight Hamilton syringe is gently inserted into the round window, after which a few microliters of AAV can be injected (Figure 2D). A second, lower-risk approach to the inner ear involves injection through the cisterna magna (Figure 2E). In humans, this is achieved by delivery via lumbar puncture (spinal tap). After exposing the cisterna magna, a small needle is inserted into the dura mater between the skull and spinal cord, allowing access to the cerebrospinal fluid (Figure 2F). This exposes the larger brain to AAV, but the use of targeted AAV reduces or prevents transfection of non-target cells. There are at least two routes by which AAV can reach the inner ear from the CSF. In the first, AAV enters the cochlea through the perilymphatic duct (Figure 2G). The second route of transfection occurs through the CSF surrounding the auditory nerve, which accesses and innervates the organ of Corti. The organ of Corti contains inner and outer hair cells, as well as primary afferent processes that form synapses on the hair cells.

[0139] Regardless of the route of administration, AAV transfects both inner and outer hair cells after cisternal injection, even in small volumes (1 µL) (Figure 3). In these proof-of-principle experiments, a histological approach was used to horizontally section the cochlea (5 µm), thereby allowing visualization of the organ of Corti, which contains inner and outer hair cells, respectively (Figure 3A-C). Fluorescent imaging of these regions in animals injected 2 weeks prior with AAV.7m8.GFP demonstrated successful transfection of inner and outer hair cells at both 1 µL and 2 µL doses of high-titer AAV (10–13). The presence of GFP fluorescence confirmed successful transfection, thereby enabling progress toward in vivo pilot studies.

[0140] Studies to test the effectiveness of the viral technology of the present invention exposed animals to two hours of daily noise exposure at 110 dB SPL, which has previously been shown to induce hair cell damage, neuronal damage, and synaptic damage in the inner and outer hair cells of the cochlea (Kujawa et al. (2009) J Neurosci. 29:14077-14085; Liberman MC. (2015) Sci Am. 313:48-53; Kurabi et al. (2017) Hear Res. 349:129-137).

[0141] To test the hypothesis that upregulation of SOD1 in inner and outer hair cells prevents or reduces hair cell injury, neuronal damage, and synaptic damage, we used 14 Mongolian gerbils with human-like audiograms. For this experiment, baseline ABR recordings were performed, and then animals were randomly selected to receive a cisternal injection of saline (N = 8) or 3 μL of AAV.7m8.SOD1.GFP (N = 6). The animals were then allowed 2 weeks of recovery and targeted AAV expression before noise exposure. The animals were then exposed to 110 dB SPL noise for 2 hours per day for 5 days, and then returned to their home cages for 1 week before final ABR recordings and perfusion for histology.

[0142] Figure 4 shows the results of threshold measurements after noise exposure between both groups. The series of figures in Figure 4A illustrates the ABR technique and how it corresponds to brain physiology. Wave 1 (a compound action potential generated at the distal portion of the cochlear nerve exiting the cochlea) was measured, along with the amplitude, latency, and overall hearing threshold for each frequency (1, 2, 4, 8, or 16 kHz). Figure 4B shows paired comparison tests for threshold difference scores. These scores represent the post-noise threshold minus the pre-noise value, indicating an improvement (decrease) or deterioration (increase) in hearing threshold. These data showed that for the saline control group, thresholds were highly significantly worsened (increased) compared to before noise exposure, with the majority exceeding the mild hearing loss threshold (t = 12.38, p < 0.001). The group receiving SOD1 had a small but significant threshold deterioration (increase) (t = 4.1, p < 0.05) and relatively little hearing loss in the mild HL range. A direct comparison of the threshold difference scores between saline controls and SOD1 in Figure 4C shows a highly significant deterioration (increase) for the saline control group (Tukey's Honestly Significant Difference; q = 1.99, p < 0.0001). ABR threshold is a good measure of neuronal damage and IHC / OHC cell death, and therefore, these results indicate that AAV.SOD1 expression had a good protective effect during noise exposure.

[0143] Figure 5 shows the results of ABR amplitude and latency measurements of wave 1 after noise exposure between both groups. Figure 5A shows a paired comparison test for amplitude difference scores. These scores represent the pre-noise amplitude minus the post-noise amplitude. These data indicate that the saline control group experienced a highly significant decrease (worsening) in amplitude from the pre-noise amplitude (t=14.21, p<0.001). The group receiving SOD1 had a smaller but significant decrease (worsening) in amplitude (t=5.9, p<0.05). A direct comparison of the amplitude difference scores between the saline control and SOD1 in Figure 5B shows a highly significant decrease (worsening) in amplitude for the saline control group compared to the SOD1 group (Tukey's Honestly Significant Difference; q=1.96, p<0.0001). Figure 5C shows a paired comparison test for ABR latency difference scores (post-noise latency minus pre-noise latency). Here, a highly significant increase (worsening) in ABR latency from pre-noise amplitude is observed for the saline control group (t = 10.11, p < 0.001). The group receiving SOD1 did not have a significant increase (worsening) in ABR latency (t = 1.4, p = 0.22). Direct comparison of the latency difference scores between the saline control and SOD1 groups in Figure 5D shows a highly significant increase (worsening) in ABR latency for the saline control group compared to the SOD1 group (Tukey's Honestly Significant Difference; q = 1.96, p < 0.0001). The decrease (worsening) in ABR amplitude measurements and the increase in ABR latency measurements are indicative of synaptic damage between auditory hair cells and auditory nerve fibers. Therefore, these results indicate that AAV.SOD1 expression protected and minimized loss of auditory hair cell-fiber synapses during noise exposure.

[0144] Example 2: Clinical Development of AAV-Mediated Antioxidant Therapy for Hearing Loss In certain embodiments, the gene therapy of the present disclosure comprises the following sequenced portion: AAV2-7m8.CAG.SOD1.IRES.eGFP.WPRE.SV40. Here, AAV2.7m8 is a serotype with a seven-residue insertion in the AAV2 cap. This AAV serotype has a high transfection rate in the inner and outer hair cells of the cochlea and type I and II hair cells of the vestibular peripheral organs. The CAG promoter is a strong synthetic promoter frequently used to drive high-level gene expression in mammalian expression vectors. The SOD1 sequence is a gerbil-specific nucleotide sequence for superoxide dismutase 1. An IRES (internal ribosome entry site) is an RNA element that allows for cap-independent translation initiation as part of a larger process of protein synthesis, allowing for simultaneous expression of several genes under the control of the same promoter. eGFP, or green fluorescent protein (GFP), is a protein that exhibits bright green fluorescence when exposed to light ranging from blue to ultraviolet, making it easy for researchers to identify transfected cells. WPRE (Woodchuck Hepatitis Virus (WHP) Posttranscriptional Regulatory Element (WPRE)) is a DNA sequence that enhances the expression of genes delivered by viral vectors. SV40 is the abbreviation for simian vacuolating virus 40 or simian virus 40, which enhances gene expression.

[0145] While this constructed AAV demonstrated significant efficacy in transfection and treatment in a gerbil model, it is important to note that the sequences of successful FDA-approved human variants of AAV differ in many potential ways. First, the human sequence of the SOD enzyme differs from that of the Mongolian gerbil. Thus, in certain embodiments, AAV constructed for use in non-human primates contains a nucleotide sequence specific to that species (e.g., macaque). Thus, the SOD nucleotide sequence used in certain embodiments is matched to the species of the subject receiving treatment. In certain embodiments, the disclosed invention includes human variants suitable for clinical use. In certain embodiments, the work disclosed in this example includes not only the SOD1 sequence, but also SOD2 and SOD3 variants 1 and 2. It should be understood that in certain embodiments, the isoforms of the SOD gene used in the present invention can be one or all of these sequences. Furthermore, there are many different serotypes that have demonstrated effective transfection of cochlear hair cells, including AAV1.RPG, AAV2, AAV2.HDG, AAV2.7m8, AAV8, AAV8.BP2, AAV.Anc80.L65, AAV9, and AAV9.KGG, among others. In certain embodiments, the CAG promoter is one of many types of promoters that can be used in AAV construction. Those skilled in the art can select an appropriate promoter for use in a desired subject, including non-human primates and humans, according to safety and efficacy. Thus, in certain embodiments, a different promoter that expresses at a higher level in human cochlear tissue is used. In certain embodiments, the therapeutic AAV of the present invention does not contain GFP. Thus, for clinically useful embodiments of the present invention, since only a single transgene is present, an IRES sequence, which is used to enhance the expression of multiple transgenes (SOD1, GFP), may not be necessary. Furthermore, similar to promoters, there are many types of enhancers that can be selected for use in certain embodiments of the present invention.

[0146] Figure 7A shows the normal effects of noise-induced oxidative stress in normal cells (left) and cells treated with SOD gene therapy (right). Increased SOD bioavailability provides neuroprotection from free radicals overproduced by noise exposure. Next, we conducted an animal study to investigate the effects of SOD1 AAV treatment on long-term exposure to moderately loud noise. Here, a group of animals (N8) received an SOD1 injection (5 ml) and subsequently monitored their hearing health for 5 months (Figure 7B). There was no change from baseline. In the next phase, SOD1 and control animals were exposed to 110 dB SPL for 2 hours per day for 5 days. Post-exposure monitoring was then conducted for 3 months. Results showed significant neuroprotection in the SOD1 gene therapy group, while CTL animals developed persistent moderate hearing loss. Finally, the animals were exposed to a second noise exposure (110 dB SPL, 2 hours per day, 5 days). Again, there was a sustained neuroprotective effect of SOD1 gene therapy, although control animals developed severe hearing loss.

[0147] Next, a series of studies were conducted to investigate the effects of AAV-SOD treatment on hearing impairment caused by short-term exposure to high-level noise. Figure 8A illustrates the effects of acoustic trauma (120 dB, 30 min) on the cochlear complex of the inner ear. Here, control animals were exposed to 120 dB SPL noise for 30 min, 1 h, and 2 h. These animals developed significant, persistent hearing loss over 4 weeks. Animals receiving SOD1 treatment before exposure had significant neuroprotection over time. Animals receiving SOD1 treatment after noise exposure (1 h) received significant protection from hearing loss over time (peaking approximately 2 weeks), corresponding to expression of the SOD1 transgene. See Figures 8B-8F.

[0148] Cisplatin is a platinum-based chemotherapy drug commonly used to treat many cancers, including bladder, ovarian, and testicular cancer, affecting approximately 20% of cancer patients in the United States. A common side effect of cisplatin treatment is progressive hearing loss due to damage to the cochlea, leaving 40%–80% of adults and at least 50% of children with significant, permanent hearing loss, a condition that can significantly impact quality of life. To determine whether treatment with AAV SOD therapy could prevent cisplatin-associated hearing loss, we conducted studies in which animals were treated weekly with 2 mg / kg cisplatin, administered intravenously, for 8 weeks after administration of AAV SOD treatment or vehicle control. Results from these studies are illustrated in Figures 9A–9C. While control animals experienced progressive hearing loss at all frequencies, animals treated with SOD1 were consistently protected. Further analysis of these data in Figures 10A and 10B found that SOD1 pretreatment conferred nearly 100% neuroprotection across the entire frequency range. Without wishing to be bound by theory, these data suggest that AAV SOD therapy may be useful in protecting patients from this common side effect of cisplatin treatment.

[0149] Enumerated Aspects Enumerated aspects are provided below, the numbering of which should not be construed to imply any level of importance.

[0150] Aspect 1 provides a polynucleotide encoding an adeno-associated virus (AAV) vector. In certain embodiments, the AAV vector comprises a first AAV inverted terminal repeat (ITR) nucleic acid sequence. In certain embodiments, the AAV vector comprises a nucleic acid encoding a superoxide dismutase (SOD) protein operably linked to a promoter. In certain embodiments, the AAV vector comprises a second AAV inverted terminal repeat (ITR) nucleic acid sequence. In certain embodiments, the AAV vector targets cells of the inner ear.

[0151] Embodiment 2 provides: the polynucleotide of embodiment 1, wherein the AAV vector further comprises at least one AAV capsid protein.

[0152] Embodiment 3 provides: the polynucleotide of embodiment 2, wherein the capsid protein is AAV2.

[0153] Embodiment 4 provides: the polynucleotide of embodiment 3, wherein the capsid protein is AAV2-7M8.

[0154] Embodiment 5 provides: the polynucleotide of embodiment 2, wherein the capsid protein is AAV9.

[0155] Embodiment 6 provides: the polynucleotide of any one of embodiments 1 to 5, wherein the inner ear cell is a hair cell.

[0156] Embodiment 7 provides: the polynucleotide of any one of embodiments 1-6, wherein the inner ear cell is a Deiters cell, a Hansen cell, a Claudius cell, a primary afferent neuron, a supporting cell, a limbal cell, a spiral ligament cell, a stria vascularis cell, a type I vestibular hair cell, and / or a type II vestibular hair cell.

[0157] Embodiment 8 provides: the polynucleotide of any one of embodiments 1 to 7, wherein the vector expresses the SOD protein in an inner ear cell.

[0158] Embodiment 9 provides: the polynucleotide of any one of embodiments 1 to 8, wherein the SOD protein is selected from the group consisting of SOD1, SOD2, and SOD3.

[0159] Embodiment 10 provides a recombinant adeno-associated virus (AAV) vector. In certain embodiments, the AAV vector comprises a first AAV inverted terminal repeat (ITR) nucleic acid sequence. In certain embodiments, the AAV vector comprises a second AAV inverted terminal repeat (ITR) nucleic acid sequence. In certain embodiments, the AAV vector comprises a polynucleotide sequence encoding a superoxide dismutase (SOD) protein operably linked to a promoter, flanked by the first AAV inverted terminal repeat (ITR) sequence and the second AAV ITR. In certain embodiments, the AAV vector has specificity for cells of the inner ear.

[0160] Embodiment 11 provides the recombinant AAV of embodiment 10, wherein said AAV vector further comprises at least one AAV capsid protein.

[0161] Embodiment 12 provides: the recombinant AAV of embodiment 11, wherein the at least one AAV capsid protein is AAV2 or a derivative thereof.

[0162] Embodiment 13 provides: the recombinant AAV of any one of embodiments 11 to 12, wherein the at least one capsid protein is AAV2-7M8.

[0163] Embodiment 14 provides the recombinant AAV of embodiment 11, wherein the at least one AAV capsid protein is AAV9 or a derivative thereof.

[0164] Embodiment 15 provides: the recombinant AAV of any one of embodiments 10 to 14, which expresses said SOD protein in an inner ear cell.

[0165] Embodiment 16 provides: the recombinant AAV of any one of embodiments 10 to 15, wherein the inner ear cell is a hair cell.

[0166] Embodiment 17 provides: the recombinant AAV of any one of embodiments 10 to 16, wherein the inner ear cell is a Deiters cell, a Hansen cell, a Claudius cell, a primary afferent neuron, a supporting cell, a limbal cell, a spiral ligament cell, a type I vestibular hair cell, and / or a type II vestibular hair cell.

[0167] Embodiment 18 provides: the recombinant AAV of any one of embodiments 10 to 17, wherein the SOD protein is selected from the group consisting of SOD1, SOD2, and SOD3.

[0168] Embodiment 19 provides: a cell comprising an adeno-associated virus (AAV) vector comprising a first AAV inverted terminal repeat (ITR) nucleic acid sequence, a second AAV inverted terminal repeat (ITR) nucleic acid sequence, and a nucleic acid encoding a superoxide dismutase (SOD) protein operably linked to a promoter, the nucleic acid being flanked by the first AAV inverted terminal repeat (ITR) sequence and the second AAV ITR.

[0169] Embodiment 20 provides: the cell of embodiment 19, which is an inner ear cell.

[0170] Embodiment 21 provides: the cell of any one of embodiments 19-20, which is a hair cell.

[0171] Embodiment 22 provides: the cell of any one of embodiments 19-21, wherein the cell is a Deiters cell, a Hansen cell, a Claudius cell, a primary afferent neuron, a supporting cell, a limbal cell, a spiral ligament cell, a type I vestibular hair cell, and / or a type II vestibular hair cell.

[0172] Embodiment 23 provides: a method of treating, ameliorating, and / or preventing progressive hearing loss in a subject in need thereof, comprising: Administering to the subject an effective amount of an adeno-associated virus (AAV) vector comprising a nucleic acid encoding a superoxide dismutase (SOD) protein operably linked to a promoter, wherein the SOD protein is expressed in inner ear cells, thereby treating, ameliorating, and / or preventing progressive hearing loss in the subject.

[0173] Embodiment 24 provides the method of embodiment 23, wherein the inner ear cells are hair cells.

[0174] Example 25 provides: the method of any one of Examples 23-24, wherein the inner ear cell is a Deiters cell, a Hansen cell, a Claudius cell, a primary afferent neuron, a supporting cell, a limbal cell, a spiral ligament cell, a type I vestibular hair cell, and / or a type II vestibular hair cell.

[0175] Embodiment 26 provides: the method of any one of embodiments 23 to 25, wherein expression of the SOD protein protects the cell from oxidative damage.

[0176] Embodiment 27 provides: the method of any one of embodiments 23 to 26, wherein the AAV vector targets the inner ear cells.

[0177] Embodiment 28 provides: the method of any one of embodiments 23 to 27, wherein the AAV vector comprises capsid proteins derived from AAV2.

[0178] Embodiment 29 provides the method of embodiment 28, wherein the capsid protein is AAV2-7m8.

[0179] Embodiment 30 provides: the method of any one of embodiments 23 to 27, wherein the AAV vector comprises capsid proteins derived from AAV9.

[0180] Embodiment 31 provides: the method of any one of embodiments 23 to 30, wherein the AAV vector is administered via middle ear injection, round window diffusion, round window injection, oval window injection, labyrinthectomy injection, intracochlear electrode or drug delivery system, orally, inhalation, nasally, nebulized, intravenous injection, intramuscular injection, intrathecal injection, intrapleural injection, cisternal injection, subcutaneous injection, and / or transdermal injection.

[0181] Embodiment 32 provides: the method of any one of embodiments 23 to 31, further comprising administering the AAV vector together with an effective amount of an agent that disrupts the blood-brain barrier.

[0182] Embodiment 33 provides: the method of any one of embodiments 23 to 32, wherein the SOD protein is selected from the group consisting of SOD1, SOD2, and SOD3.

[0183] Embodiment 34 provides: a method of reversing, minimizing, and / or preventing progressive hearing loss in a subject in need thereof, comprising: Administering to the subject an effective amount of an adeno-associated virus (AAV) vector comprising a nucleic acid encoding a superoxide dismutase (SOD) protein operably linked to a promoter, wherein the SOD protein is expressed in the subject's inner ear cells, thereby reversing, minimizing, and / or preventing progressive hearing loss in the subject.

[0184] Embodiment 35 provides the method of embodiment 34, wherein the inner ear cells are hair cells.

[0185] Example 36 provides: the method of any one of Examples 34-35, wherein the inner ear cell is a Deiters cell, a Hansen cell, a Claudius cell, a primary afferent neuron, a supporting cell, a limbal cell, a spiral ligament cell, a type I vestibular hair cell, and / or a type II vestibular hair cell.

[0186] Embodiment 37 provides: the method of any one of embodiments 34 to 36, wherein expression of the SOD protein protects the cell from oxidative damage.

[0187] Embodiment 38 provides: the method of any one of embodiments 34 to 37, wherein the AAV vector targets the inner ear cells.

[0188] Embodiment 39 provides: the method of any one of embodiments 34 to 38, wherein the AAV vector comprises capsid proteins derived from AAV2.

[0189] Embodiment 40 provides the method of embodiment 39, wherein the capsid protein is AAV2-7m8.

[0190] Embodiment 41 provides: the method of any one of embodiments 34 to 38, wherein the AAV vector comprises capsid proteins derived from AAV9.

[0191] Embodiment 42 provides: the method of any one of embodiments 34 to 41, wherein the AAV vector is administered via middle ear injection, round window diffusion, round window injection, oval window injection, labyrinthectomy injection, intracochlear electrode or drug delivery system, orally, inhalation, nasally, nebulized, intravenous injection, intramuscular injection, intrathecal injection, intrapleural injection, cisternal injection, subcutaneous injection, and / or transdermal injection.

[0192] Embodiment 43 provides: the method of any one of embodiments 34 to 42, further comprising administering to said subject an effective amount of an agent that disrupts the blood-brain barrier.

[0193] Embodiment 44 provides: the method of any one of embodiments 34 to 43, wherein the SOD protein is selected from the group consisting of SOD1, SOD2, and SOD3.

[0194] Embodiment 45 provides: a method of reversing, minimizing, and / or preventing chemotherapy treatment-associated hearing loss in a subject in need thereof, comprising: Administering to the subject an effective amount of an adeno-associated virus (AAV) vector comprising a nucleic acid encoding a superoxide dismutase (SOD) protein operably linked to a promoter, wherein the SOD protein is expressed in the subject's inner ear cells, thereby reversing, minimizing, and / or preventing chemotherapy treatment-related hearing loss in the subject.

[0195] Embodiment 46 provides the method of embodiment 45, wherein the chemotherapy is a platinum drug.

[0196] Embodiment 47 provides the method of embodiment 46, wherein the platinum drug is cisplatin.

[0197] Embodiment 48 provides: the method of any one of embodiments 45 to 47, wherein the inner ear cell is a hair cell.

[0198] Embodiment 49 provides: the method of any one of embodiments 45-48, wherein the inner ear cell is a Deiters cell, a Hansen cell, a Claudius cell, a primary afferent neuron, a supporting cell, a limbal cell, a spiral ligament cell, a type I vestibular hair cell, and / or a type II vestibular hair cell.

[0199] Embodiment 50 provides: the method of any one of embodiments 45 to 49, wherein expression of the SOD protein protects the cell from oxidative damage.

[0200] Embodiment 51 provides: the method of any one of embodiments 45 to 50, wherein the AAV vector targets the inner ear cells.

[0201] Embodiment 52 provides: the method of any one of embodiments 45 to 51, wherein the AAV vector comprises capsid proteins derived from AAV2.

[0202] Embodiment 53 provides the method of embodiment 52, wherein the capsid protein is AAV2-7m8.

[0203] Embodiment 54 provides: the method of any one of embodiments 45 to 51, wherein the AAV vector comprises a capsid protein derived from AAV9.

[0204] Embodiment 55 provides: the method of any one of embodiments 45 to 54, wherein the AAV vector is administered via middle ear injection, round window diffusion, round window injection, oval window injection, labyrinthectomy injection, intracochlear electrode or drug delivery system, orally, inhalation, nasally, nebulization, intravenous injection, intramuscular injection, intrathecal injection, intrapleural delivery, cisternal injection, subcutaneous injection, and / or transdermal injection.

[0205] Embodiment 56 provides: the method of any one of embodiments 45 to 55, further comprising administering to said subject an effective amount of an agent that disrupts the blood-brain barrier.

[0206] Embodiment 57 provides: the method of any one of embodiments 45 to 56, wherein the SOD protein is selected from the group consisting of SOD1, SOD2, and SOD3.

[0207] Other Aspects The recitation of a list of elements in a definition of a variable herein includes definitions of that variable as a single element or as a combination (or subcombination) of the listed elements. The recitation of an embodiment herein includes that embodiment as a single embodiment or in combination with other embodiments or portions thereof.

[0208] The disclosures of each and every patent, patent application, and publication cited herein are incorporated herein by reference in their entirety. Although the present disclosure has been disclosed with respect to specific embodiments, it is apparent that other embodiments and variations of the present disclosure may be devised by those skilled in the art without departing from the true spirit and scope of the present disclosure. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

1. The first adeno-associated virus (AAV) inverted terminal repeat (ITR) nucleic acid sequence, A nucleic acid encoding a superoxide dismutase (SOD) protein functionally linked to a promoter, The second AAV inverted terminal repeat (ITR) nucleic acid sequence and A polynucleotide encoding an AAV vector containing, The vector is a polynucleotide that targets cells of the inner ear.

2. The AAV vector further comprises at least one AAV capsid protein, Optional, (a) The capsid protein is AAV2, and optionally the AAV2 capsid protein is AAV2-7M8, or (b) The capsid protein is AAV9, The polynucleotide according to claim 1.

3. The inner ear cells, (a) hair cells; or (b) Deiters cells, Hansen cells, Claudius cells, primary afferent neurons, supporting cells, limbus cells, spiral ligament cells, type I vestibular hair cells, and / or type II vestibular hair cells The polynucleotide according to claim 1, which is at least one of the following.

4. The polynucleotide according to claim 1, wherein the vector expresses the SOD protein in the inner ear cells, and optionally the SOD protein is selected from the group consisting of SOD1, SOD2, and SOD3.

5. The first adeno-associated virus (AAV) inverted terminal repeat (ITR) nucleic acid sequence, The second AAV inverted terminal repeat (ITR) nucleic acid sequence, A polynucleotide sequence encoding a promoter-linked superoxide dismutase (SOD) protein, flanked by a first AAV inverted terminal repeat (ITR) sequence and a second AAV ITR, and A recombinant AAV vector containing, The recombinant AAV vector having specificity for cells of the inner ear.

6. The AAV vector further comprises at least one AAV capsid protein, Optional, (a) The capsid protein is AAV2 or a derivative thereof, and optionally the AAV2 capsid protein is AAV2-7M8, or (b) The capsid protein is AAV9 or a derivative thereof. Recombinant AAV according to claim 5.

7. The recombinant AAV according to claim 5, wherein the AAV expresses the SOD protein in the inner ear cells, and optionally the SOD protein is selected from the group consisting of SOD1, SOD2, and SOD3.

8. The aforementioned inner ear cells, (a) hair cells; or (b) Deiters cells, Hansen cells, Claudius cells, primary afferent neurons, supporting cells, margin cells, spiral ligament cells, type I vestibular hair cells, and / or type II vestibular hair cells The recombinant AAV according to claim 5, which is at least one of the following.

9. A cell comprising an AAV vector comprising a first adeno-associated virus (AAV) inverted terminal repeat (ITR) nucleic acid sequence, a second AAV inverted terminal repeat (ITR) nucleic acid sequence, and a nucleic acid encoding a promoter-functionally linked superoxide dismutase (SOD) protein, flanked by the first AAV inverted terminal repeat (ITR) sequence and the second AAV ITR, Optionally, the cell is an inner ear cell.

10. The inner ear cells (a) hair cells; or (b) Deiters cells, Hansen cells, Claudius cells, primary afferent neurons, supporting cells, margin cells, spiral ligament cells, type I vestibular hair cells, and / or type II vestibular hair cells The cell according to claim 9, which is at least one of the following.

11. A pharmaceutical composition for administration to subjects in need of treating, relieving, and / or preventing progressive hearing loss; The pharmaceutical composition comprises an adeno-associated virus (AAV) vector containing a nucleic acid encoding a superoxide dismutase (SOD) protein functionally linked to a promoter; The SOD protein is expressed in inner ear cells, thereby treating, relieving, and / or preventing progressive hearing loss in the subject; The expression of the SOD protein protects the cells from oxidative damage; The AAV vector targets the inner ear cells; Optionally, the SOD protein is selected from the group consisting of SOD1, SOD2, and SOD3. The aforementioned pharmaceutical composition.

12. The aforementioned inner ear cells, (a) hair cells; or (b) Deiters cells, Hansen cells, Claudius cells, primary afferent neurons, supporting cells, margin cells, spiral ligament cells, type I vestibular hair cells, and / or type II vestibular hair cells The pharmaceutical composition according to claim 11, wherein at least one of the following.

13. The pharmaceutical composition according to claim 11, wherein the AAV vector comprises a capsid protein derived from AAV2 or AAV9, and optionally the AAV2 capsid protein is AAV2-7M8.

14. The pharmaceutical composition according to claim 11, wherein the administration is by middle ear injection, round window diffusion, round window injection, oval window injection, labyrinth disruption injection, intracochlear electrode or drug delivery system, oral, inhalation, nasal, spray, intravenous injection, intramuscular injection, subarachnoid injection, intrapleural delivery, cisterna magna injection, subcutaneous injection, and / or transdermal injection.

15. The pharmaceutical composition according to claim 11, used in combination with an effective amount of a blood-brain barrier disrupting agent.

16. A pharmaceutical composition for administration to subjects in need of reversing, minimizing, and / or preventing progressive hearing loss; The pharmaceutical composition comprises an adeno-associated virus (AAV) vector containing a nucleic acid encoding a superoxide dismutase (SOD) protein functionally linked to a promoter; The SOD protein is expressed in the inner ear cells of the subject, thereby reversing, minimizing, and / or preventing progressive hearing loss in the subject; The expression of the SOD protein protects the cells from oxidative damage; The AAV vector targets the inner ear cells; Optionally, the SOD protein is selected from the group consisting of SOD1, SOD2, and SOD3. The aforementioned pharmaceutical composition.

17. The aforementioned inner ear cells, (a) hair cells; or (b) Deiters cells, Hansen cells, Claudius cells, primary afferent neurons, supporting cells, margin cells, spiral ligament cells, type I vestibular hair cells, and / or type II vestibular hair cells The pharmaceutical composition according to claim 16, wherein at least one of the following:

18. (a) The AAV vector comprises a capsid protein derived from AAV2, optionally the AAV2 capsid protein being AAV2-7m8; or (b) The AAV vector contains a capsid protein derived from AAV9, The pharmaceutical composition according to claim 16.

19. The pharmaceutical composition according to claim 16, wherein the administration is by middle ear injection, round window diffusion, round window injection, oval window injection, labyrinth disruption injection, intracochlear electrode or drug delivery system, oral, inhalation, nasal, spray, intravenous injection, intramuscular injection, subarachnoid injection, intrapleural delivery, cisterna magna injection, subcutaneous injection, and / or transdermal injection.

20. The pharmaceutical composition according to claim 16, used in combination with an effective amount of a blood-brain barrier-destroying agent.

21. A pharmaceutical composition for administration to subjects in need of reversing, minimizing, and / or preventing chemotherapy-related hearing loss; The pharmaceutical composition comprises an adeno-associated virus (AAV) vector containing a nucleic acid encoding a superoxide dismutase (SOD) protein functionally linked to a promoter; The SOD protein is expressed in the inner ear cells of the subject, thereby reversing, minimizing, and / or preventing chemotherapy-related hearing loss in the subject; The AAV vector targets the inner ear cells; The expression of the SOD protein protects the cells from oxidative damage; Optionally, the SOD protein is selected from the group consisting of SOD1, SOD2, and SOD3. The aforementioned pharmaceutical composition.

22. The pharmaceutical composition according to claim 21, wherein the chemotherapy is a platinum drug, and optionally the platinum drug is cisplatin.

23. The aforementioned inner ear cells, (a) hair cells; or (b) Deiters cells, Hansen cells, Claudius cells, primary afferent neurons, supporting cells, margin cells, spiral ligament cells, type I vestibular hair cells, and / or type II vestibular hair cells The pharmaceutical composition according to claim 21, which is at least one of the following.

24. (a) The AAV vector comprises a capsid protein derived from AAV2, and optionally the AAV2 capsid protein is AAV2-7m8; (b) The AAV vector contains a capsid protein derived from AAV9; or (c) The administration is via middle ear injection, round window diffusion, round window injection, oval window injection, labyrinth disruption injection, intracochlear electrode or drug delivery system, oral, inhalation, nasal, spray, intravenous injection, intramuscular injection, subarachnoid injection, intrapleural delivery, cisterna magna injection, subcutaneous injection, and / or transdermal injection. The pharmaceutical composition according to claim 21.

25. The pharmaceutical composition according to claim 21, used in combination with an effective amount of a blood-brain barrier disrupting agent.

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